Urinary proteomics and the role of orosomucoid
(ORM) in vascularization of bladder cancer
ISO Coated v2 and Fogra39/F39 profiles should describe exactly the same reference print condition. For example, Kodak ship F39 or Fogra39 icc profiles with their software - however as they are not supplied by the ECI, they are created by Kodak from F39/F39L data and are not called ISO Coated v2 etc.
Dissertation Inaugural-
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Zur Erlangung des
aften hDoktorgrads der Naturwissensc
(Dr. rer. nat.)
am Fachbereich Chemie
ersität Duisburg-Essenan der Univ
vorgelegt von
Ster Irmak
aus Mardin
Essen 2007
Die der vorliegenden Arbei
t zugrunde liegenden Experiment
e wurden am Institut
-Essen und des Universitätsklinikum Duisburgfür Anatomie der Universität
Hamburg-Eppendorf durchgeführt.
Gutachter: Prof. Dr. R. Sustmann 1.
Gutachter: Prof. Dr. Dr2. t o. H. de Gro
Gutachter: Prof. Dr. S. Ergün 3.
r chönbuchehusses: Prof. Dr. A. SVorsitzender des Prüfungsaussc
hen PrTag der mündlicüfung: 03.07.2007
s
To my Family and
nephew Yusuf Heja
Ever tried, ever failed. Try again, fail again. Fail better Samuel Beckett
Acknowledgement
Acknowledgements
tween March 2003 - August 2006 at the The present study has been carried out be
spital Hamburg-Eppendorf, Germany and Department of Anatomy, University Ho
Anatomy, University 2007 at the Department ofbetween August 2006 March
Hospital Essen, Germany.
ted andto everyone who aided, suppor my sincere gratitude I wish to express
, in one way to another, throughout this study. einspired m
thank my supervisor Professor Dr. Süleyman I would especially like to First of all,
ting field of research, for his constant Ergün for introducing me to work in an exci
e study and for the friendly atmosphere interest and support in the progress of th
within the department.
Hamburg-Eppendorf, I would ogy, University Hospital From the Department of Urol
support and the opportunity to work in the like to thank Professor Dr. Huland for his
e to thank PD Dr. Martin y likiallogical Department. I would speclaboratory of the Urol
Friedrich for his encouragement, optimism and for giving the clinicians point of view. I
laboratory of the Urologyformer and present, at the thank all the lab members,
Hospital Hamburg -Eppendorf. Department at the University
ra-e to thank Dr. Leticia OliveiI would likerrer for her help and scientific and F
in the first months of my life in sometimes non-scientific support especially
uschild for her support, always having a ssica HaeGermany. I would like to thank J
breakfasts and coffe breaks. smile and the happy times, daily
I thank Dr. Nerbil Kilic, Dr. Ergin Kilic and Dr. Derya Tilki for their warm support and
fellowship. I would like to thank Kiersten Miete, Elvin Zengin, Ege Erenler, Biranda
ly atmosphere in lab.doviding a frienKocaoglu, Mehmet Varol, Deniz Tilki for pr
al Essen, I would firstly like to omy, University HospitWithin the Department of Anat
-Yasar for her help anthank Dr. Ferya Banazd support during the writing of my thesis.
Prof. Dr. Ergün. Among them, I speciallyI thank all members of the research group of
thank Dorothee Schünke for her help.
for his help in editing of figures. I would like to thank Oguzhan A. Polat
support encouragement and their continuousFinally, I want to thank my parents for
during all the years. Without them this work would not have been possible. I would
en for her deep friendship and motivation. like to thank my best friend Elmas Yurtsev
Index
I
Index 1 Introduction.......................................................................................................1
1.2 1.1 ProteomicsTechnology of proteomics: 2-Dim...................................................................................................ensional Gelelectrophoresis (2-DE).........3 2
1.2.2 Protein 1.2.1 Sample extractionpreparation.................................................................................................................................................................4 4
1.2.2.2 De1.2.2.1 Chaottergentsropic agent........................................................................................................................................................................5 5
6 ................................................................................gentsing a1.2.2.3 Reduc1.2.3 1.2.4 Second First Dimension: Isoeldimension: SDectric focuS-PAGEsing (IEF)......................................................................................................10 7
1.2.5 Equi1.2.6 Identificatiolibrationn of proteins..............................................................................................................................................................11 11
1.3.1 1.3 Urinary proteomicsFormation of urine........................................................................................12 12
1.4 Bladder1.3.2 Sources cancerof urinary proteins............................................................................................................................................................15 12
1.4.2 Cellular 1.4.1 Staging of classifibladder cationcancer..............................................................................................................................................16 15
1.5 Clinical 1.6 Urinary proteombiomarkersics and biomarkers.................................................................................................................................................18 18
19 ...................................................................................isangiogenes1.7 Tumor 1.8 1.9 The One of urinplasminogen ary proteiactivatins: Orosomon systemucoid (ORM).......................................................................................................2220
2 The aim of study..............................................................................................26
3 Material and Methods......................................................................................27
3.1 Mate3.1.1 Chemical rialsand Consumables....................................................................................................................................................................27 27
28 ......................................................................................................Kits3.1.2 3.1.3 Stock Solutions and buffers................................................................29
3.1.5 Anti3.1.4 Equipment bodiesand applications...........................................................................................................................................................31 32
3.1.6 Cell lines and medium for cultivation of cell lines................................32
3.1.8 Primers3.1.7 Bacterial strains...................................................................................................................................................................................33 32
34 .....................................................................................................thods3.2 Me3.2.1 Protei3.2.1.1 Determinatn analysesion of total protein..........................................................................................................................................34 34
3.2.1.3 3.2.1.2 Preparation of Two dimensional polyacrylurine Samples for 2-DEamide gel electrophoresis (2D-PAGE)...........................................35 35
3.2.1.4 3.2.1.5 Rehydration First dimensional separation: Isoelectric ...................................................................................focusing (IEF)................36 36
3.2.1.7 3.2.1.6 E Second dimquilibrationensional separation........................................................................................................................................37 37
3.2.1.9 3.2.1.8 Silv Proter staiein detecningtion............................................................................................................................................................38 38
3.2.1.10 Coomassie staining.......................................................................39
Index
II
39 ...............................................................analysisage and data 3.2.1.11 Im3.2.1.13 SDS-polyacry3.2.1.12 Mass spectrometrylamide gelele and bioictrophornformaticsesis.................................................................................41 39
3.2.1.14 We3.2.2 Molecularbiostern bllogical ottingmethods...........................................................................................................................................42 41
42 ..................................................E. coli Cultivation and storage of 3.2.2.1 3.2.2.3 3.2.2.2 Bacter Preparatiial transfoon of comprmaettionent cells.....................................................................................................................43 43
3.2.2.5 3.2.2.4 Miniprepar Purification of ation of plDNA from soluasmid-DNAtion and gel bands.............................................................................44 44
3.2.2.6 3.2.2.7 Max Restriictpreparion digesation of plt of DNAasmid-DNA................................................................................................................45 44
3.2.2.9 3.2.2.8 S Agarose equencing ofgel electr DNAophoresis...............................................................................................................................45 45
3.2.2.11 3.2.2.10 Determination Construction of pcDNA3.of DNA conc1(-)/ORMentration expression vector..............................................................46 46
3.2.2.13 Ligation 3.2.2.12 Polymerase of Pchain reactiCR product into pCRon (PCR)® 2.1-TOPO®................................................ Vector................46 47
3.2.2.15 siRNA 3.2.2.14 Subcloning construcinto pcDNtionA3.1(-)..............................................................................................................................48 48
3.2.3.1 3.2.3 Cellbiolog Culturing ofical me cellsthods..................................................................................................................................................49 49
3.2.3.3 3.2.3.2 Freezi Determination of ng and thawing cell numof ceberlls.............................................................................................................50 49
3.2.3.4 Cell extraction..............................................................................50
3.2.3.6 3.2.3.5 Overex Transfection pression/ of HDMECs via gene silencing of NucleofeORM in ctorHDMECs....................................................51 51
3.2.5 Histologi3.2.4 Endothelial tube cal methodsformation assay...................................................................................................................................53 52
3.2.5.2 3.2.5.1 I Fixammunohistochemtion and HE staiistryning for tissue samples an.................................................................d cells.................53 53
3.2.5.3 Immunocytochemistry..................................................................54
4 4.1 Profiling Resultsof urinary proteins by 2D gel electr.............................................................................................................ophoresis...............................5555
preparation method for 2D gel Optimization of the sample 4.1.1 4.1.2 electrophoresis Determination of urinary protein patteon human urinern in rela..........................................................tion to bladder cancer by 55
56 .................................................................................................... 2-DE4.1.3 Protein pattern of normal urine versus urine of patients with pTa.......57
4.1.4 4.1.5 Protein pattern of normProtein pattern of normaal urine l urine versus urine of patients with pT2versus urine of patients with pT1..............60 58
of follow-up versus urine samples of Protein pattern of urine samples 4.1.6 4.1.7 bladder cancProtein pattern of urine samples er pateints.......................................................................of patients on follow-up versus that of 61
patients of tumor stage pTa.................................................................64
4.1.8 patients of tumor sProtein pattern of urine samples tage pT1.................................................................of patients on follow-up versus that of 64
of patients on follow-up versus that of Protein pattern of urine samples 4.1.9 patients of bladder cancer stage pT2..................................................64
Index
III
cancer4.1.10 Identification of protein spots in urine samp.................................................................................................les of patients with bladder 66
Human serum albumin in normal urine versus in urine samples of 4.1.11 4.1.12 patients with bladder Mass spectrums and matched peptcancer and follow-upides for identified proteins.....................................................70 68
4.2 Idendification of two urinary proteins using Western blot analyses............75
4.2.1 patients with bladder cancerDetection of orosomucoid (ORM) in and patients on follow-up urine samples of healthy persons, .......................75
urine samples of noprotein (ZAG) iDetection of zinc-alpha-2-glyc4.2.2 healthy persons, patients with bladder cancer and patients on ..............
4.3 follow-upExpression pattern of ORM and ZAG ..............................................................................................in bladder cancer versus normal 76
4.4 bladder tissueLocalization of ORM on endothelial cells (HDMECs) by .............................................................................................76
79 .................................................................................hemistry immunocytoc4.5 ORM overexpression versus ORM gene silencing in HDMECs.................82
4.6 Mechanistic studies via in-vitro angiogenesis assays.................................83
ng ORM-overexpressing versus endothelial tube formation usiIn-vitro4.6.1 4.6.2 ORMsilencThe interaction between ORed HDMECsM and PAI-1 in endot.....................................................................helial tube .................83
84 ............................................................................................. formation4.7 Immunolocalization of ORM in in-vitro induced endothelial tubes..............87
5 5.1 DiscussionThe impact of 2-DE for characterization of ur......................................................................................................inary proteins.......................9494
5.2 Optimized powerful tool for identificseparation ofation of uri urinary proteins for ne proteinsperforming of 2-DE as a ..........................................95
the identification of proteins related to bladder Urine proteomics enables 5.3 5.4 cancerNovel protein spots in urine samples ........................................................................................................of patients with bladder cancer using 96
5.5 2-DE may be of diagnostic relevanceORM is increased in urine samples of patients with bladder cancer...................................................................97 97
5.6 Endothelial ORM support the VEoverexpressiGF-inducedon of ORM as well as endothelia endothelial tube formationl stimulation by ...................100
5.7 formationORM interaction with PAI-1 influen..................................................................................................ces the VEGF-induced endothelial tube 101
5.8 bladder cancer particularlZinc-alpha-2-glycoprotein is increasy in the invasive stagesed in urine samples.................................... of patients with 103
6 Conclusion.....................................................................................................105
7 Literature........................................................................................................106
8 8.1 AppendiMap of 3.x1(-) vector..........................................................................................................................................................................................117117
118 .......................................................................................m Vitae8.2 Curriculu
Figure Index
Figure Index
Figure 1:
Figure 2:
Figure 3:
Figure 4:
Figure 5:
Figure 6:
Figure 7:
Figure 8:
Figure 9:
Figure 10:
Figure 11:
Figure 12:
Figure 13:
Figure 14:
Figure 15:
Figure 16:
Figure 17:
Figure 18:
Figure 19:
Figure 20:
Figure 21:
Figure 22:
Figure 23:
Figure 24:
IV
formation to protein products2 The flow of genomic in
Proteomics and .2 genomics integration
5 Urea and ThioureafoStructure
Structure of detergents used in solubilisation of proteins.6
dithiothreitol (DTT)6 fStructure o
.7 tributylphosphine (TBP) a reducing agentfoStructure
s, isoelectric point of protein as amphoteric moleculeStructures of
proteins.8
yacrylamide gel matrix9 polfStructure o
ome formation and excretion..14 os of urinary exMechanism
bladder cancer stages.16 ation of Represent
..20 and initiation of angiogenesisVascular destabilization
for the inhibition ofHypothesis leukocyte extravasation by ORM.22
Plasminogen activation system..23
ns of plasminogen, idomaentation of the protein Graphical repres
tPA and uPA..24
teracting partners..25 entation of uPA and its inSchematic pres
Schematical representation of two dimensional separation..35
Experimental flow diagram..40
..48 of gene silencing via siRNAMechanism
le of a patient with bladder cancer Protein pattern in urine samp
of the sage pTa...............56 t
in normal urine (A) and in urine of Comparison of protein pattern
e stage pTa,GI (B).58 ancer of thpatients with bladder c
Comparison of protein pattern in normal urine (A) and in urine of
e stage pT1, GIII (B).59 thancer of patients with bladder c
in normal urine (A) and in urine of Comparison of protein pattern
patients with bladder c (B).61 ancer of the stage pT2, GIII
in urine of patients on follow-up (A) Comparison of protein pattern
of the stage pTa, GI (B)62 s with bladder cancer and patient
s on follow-up (A) in urine of patientComparison of protein pattern
pT1, GIII (B)..63 cancer of the stage s with bladder and patient
Figure Index
s on follow-up (A) in urine of patientComparison of protein pattern Figure 25:
V
pT2, GIII (B)..65 cancer of the stage s with bladderand patient
ne of a patient with DE image of uriProtein identification from 2-Figure 26:
...66 bladder cancer of the stage pTa/GI
pots indicating HSA in all urine Comparison of protein sFigure 27:
sample groups...69
..70 ion of UromodulinMS identificatFigure 28:
ing glycoprotein and Ribozomal Protein71 MS identification of DNA bindFigure 29:
..72 MS identification of HSAFigure 30:
on of Orosomucoid 1...73 MS identificatiFigure 31:
glycoprotein and Zinc-alpha-2-MS identification ofFigure 32:
Complex forming glycoprotein74
Detection of ORM and ZAG in urine samples..75 Figure 33:
adder tissue..77 normal and tumor blLocalization of ORM inFigure 34:
M in bladder tumor cells..78 Localization of ORFigure 35:
bladder tumor tissueG inLocalization of ZAFigure 36: .79
ation of ORM and ZAG ng for localizImmunocytochemical stainiFigure 37:
HDMECs80 in
ation of ORM and ZAG in r localizImmnucytochemical staining foFigure 38:
bladder cancer cell line81
HDMECs and RT4 cell line81 Detection of ORM inFigure 39:
for ORM in HDMECs.82 and gene silencing via siRNAOverexpression Figure 40:
Endothelial tFigure 41: ube formation assay...83
on85 antibody on endothelial tube formatiEffect of ORM and anti-PAI-1 Figure 42:
Figure 43: Effect ORM and anti-PAI-1 at VEGF-induced endothelial tubes..86
iPAI-1 combination on endothelial Effect of VEGF, ORM and antFigure 44:
..................87 ...formation tube
type endothelial cells after n of ORM on wildLocalizatioFigure 45: tube
ssay.88 a formation
on VEGF-induced endothelial tubes89 ining for ORM ImmunostaFigure 46:
on endothelial tubes treated with Localization of ORM Figure 47:
antibody...90 anti-PAI-1
helial tubes treated with ORM in Localization of ORM on endotFigure 48:
91 low concentration and anti-PAI-1antibody
Figure Index
Figure 49:
Figure 50:
helial tubes treated with ORM in Localization of ORM on endot
VI
.92 h anti-PAI-1 antibodyhigh concentration wit
hin the uPAR-uPA-PAI-1 systems and Important interactions wit
the role of ORM..102
Table Index
Table Index
Table 1:
Table 2:
Table 3:
Table 4:
Table 5:
Table 6:
Table 7:
Table 8:
Table 9:
Table 10:
Table 11:
Table 12:
Table 13:
Table 14:
Table 15:
Table 16:
Table 17:
Table 18:
Table 19:
Table 20:
VII
Sources of urinary proteins.13
Staging of bladder cancer17
AJCC stage groupings.18
Primary antibodies32
Secondary HRP antibodies.32
Cell lines.32
edium for cell lines.32Cultivation m
Bacterial strains.33
leotides used for in PCR..33Oligonuc
leotides used Oligonuc .33for in siRNA construction
Urine samples34
es.36 IPG strips used for 2-DE analys
Applied voltage steps for IEF..37
riction digest reaction.45 Composition of rest
of PCR reaction.46 Composition
M.47 PCR conditions for OR
.52 The stimulating factors used for tube assay
Proteins identified by MS analyses....67
of protein levels of identified Semiquantitative determination
proteins in urine samples by 2-DE.....68
Determination of tube lengths.93
Summary
ySummar is currently the method of choice for 2-dimensional gel electrophoresis (2-DE)
VIII
separation of complex protein as in cell and tissue extracts or body hmixtures suc
s a central role in clinical urine playuding urine or serum. Human lfluids inc
anc of diseases such as cdiagnosticser and inflammation. Researchers and
of a map of the human proteome but in scientists are working on the development
urinary proteomics and theiraboutare only a few investigations the literature there
first aim of this study was to view the tumor. The behaviour in the case of bladder
t their functions. For this aim, fourtyfive whole proteins present in urine and to predic
cancer of different stages, patients on les from patients with bladderpurine sam
ed by 2-DE. At the beginning of this volunteers were analysfollow-up and healthy
study the 2-DE protein patte unknown because of lackrn of human urine was almost
s prior to the 2-DE. Thus, the studyof appropriate preparation of urine sample
samples providing the best preservationfocused on methods for preparation of urine
optimization of 2-DEof urinary proteins. After experimental , usable 2-D protein
oteins related to bladder cancer using patterns of urine were analysed to identify pr
and/or conventional immunoblotting and subsequent mass spectrometric analyses
to other cancer types, the bladder ochemical methods. In comparison immunohist
and ninth in womausing death in man carcinoma is the seventh camong malignant en
n- as the transformation of superficial notumors. The growth and metastasis as well
invasive tumors to an invasive tumor phenotype are closely associated with
tion of tumor tissue. Thisaovascularisactivation of angiogenesis resulting in ne
process is regulated by a net balance between angiogenic activators and inhibitors.
RM) and human zinc-alpha-2-glycoprotein In the present study, orosomucoid (O
2-DE of urine sed in(ZAG) have been identified to be increasmples of patients with a
e urine samples of healthy volunteers.bladder cancer in comparison to th
a part of oin addition to cancer cells alsImmunohistochemical results let assume that
ndothelial cthe tissue resident inflammatory cells and e lls of tumor associated bloode
vessels may serve as source for this increase of ORM in urine samples of patient
ein and increased in acute infection, with bladder cancer. ORM is an acute phase prot
inflammation, and cancer. Recent studies show that ORM forms a complex with the
active form of plasminogen activator inhibitor-1 (PAI-1) in thymosin β4 (Tβ4)-
it was aimed to determine a lls. Therefore e endothelial cactivated but not in quiescent
is. The findings presented in this giogenespotential role of ORM up-regulation in an
Summary
IX
ition to the cancer cells,rensson showed, that, in addstudy and results reported by So
ORM endogenously. For cells (HDMECs) produce human vascular endothelial
al cells, ORM-gene in vascular endothelifunctional characterization of ORM
ng were perfromed. Employing the sion and ORM-gene silencioverexpres
supernatants of HDMECs-ORM and HDMECs-ORM-siRNA in in-vitro angiogenesis
ed endothelial tube upports the VEGF-inducassays, it was found that ORM s
ORM was potentiated by co-treatment of formation. This supportive effect of
and anti-PAI-1 antibody. HDMECs with VEGF, ORM
hat ORM is increased demonstrates for the first time tThis study in urine of patients
c supporting the tube forming effects of with bladder cancer and acts pro-angiogeni
icantly increased by additive blockage of VEGF. This supportative effect was signif
PAI-1.
seems mM and PAI-1 and anti-PAI-1 systeIn summary, the interaction between OR
in the -mediated angiogenesis and probablyto be essentially involved in the VEGF
cancer. vascularisation of urinary bladder
sung smmenfaZusa
X
Zusammenfassung Bei der 2D-Elektrophorese (2-DE) handelt es sich zurzeit um die Methode der Wahl
usammensetzungen von Zell- und für die Trennung komplexer Proteinzn wie Urin oder Serum. Die Untersuchung rakten sowie KörperflüssigkeiteGewebeextankheiten wie Krebs gnostik von Krentrale Rolle in der Dia eine zvon Urinproben stellt ung eines humanenten an der Entwicklund Entzündungen dar. Wissenschaftler arbei h nur wenige Untersuchungentur sind jedocGesamtproteomenmusters, in der Litera beschrieben. Die Harnblasenkarzinomzu Urinproteomics und ihrem Muster beim von nuchung des Proteinmusters im UriZielsetzung dieser Arbeit war die Unterszu normalen Patienten. Dazu wurden 45 enkarzinompatienten im Vergleich HarnblasPatienten und gesunden en, Follow-up-arzinompatientUrinproben von Harnblasenkmente i Beginn dieser Experysiert. ZuProbanden mittels der 2D-Elektrophorese analder angemessener und fehlennproben aufgreinmuster in Uriwar das 2-DE ProtVorbehandlung der Urinproben für die 2D-Elektrophorese kaum bekannt. Daher
bereitung der Urinproben, chst auf Methoden zur Aufwaren die Untersuchungen zunä fokussiert. Nach gewährleisten,ung oteinerhaltdie die bestmögliche Pr rophorese wurden 2D-Proteinmuster dererung der 2D-Elektiexperimenteller Optimektrometrischer Analysen, Western Blot-Urinproben analysiert, um mittels massenspr die mit ne zu identifizieren,Analysen und Immunhistochemie Protei es sich Beim Harnblasenkarzinom handeltenkarzinomen assoziiert sind. Harnblasum die siebthäufigste Krebstodesursache beim Mann und die neunthäufigste
asentumorwachstum- und metastasierung ursache bei der Frau. HarnblKrebstodesinvasiven in einen invasiven - nichtsowie der Übergang von einem oberflächlichen,Aktivierung der Angiogenese und somit ark verknüpft mit der tTumorphänotyp sind sss wird geregelt durch ein Gleichgewicht erung. Dieser Prozeider Tumorvaskularis toren und Inhibitoren.achen Aktivzwischen angiogenetislpha-2-aomucoid (ORM) und humanes Zink-In der vorliegenden Arbeit wurden Orosktrophorese als im Urin von Glykoprotein (ZAG) anhand der 2D-Eleleich zu normalen Urinproben erhöhte enkarzinompatienten im VergHarnblasche Untersuchungen lassen stochemisMarkerproteine identifiziert. Immunhivermuten, dass zusätzlich zu Tumorzellen auch ein Teil der gewebaansässigen
er Blutgefäße die QuelleEndothelzellen tumorassoziiertinflammatorischen Zellen und enten darstellenenkarzinompatinblasfür den ORM-Anstieg in Urinproben von Har ute-Phase-Protein, welches bei akuten könnten. Bei ORM handelt es sich um ein Ak
smmenfaZusasung
XI
rankungen erhöht ist. In neuerenInfektionen, Entzündungen und Tumorerk
aktivens ORM einen Komplex mit derdasPublikationen konnte gezeigt werden,
Form von Plasminogen-Aktivator-Inhibitor-1 (PAI-1) in Thymosin- β4 (T β4)-
aktivierten, aber nicht in ruhenden Endothelzellen eingeht. Unsere weitergehenden
ner möglichen Rolle von ORM in der Untersuchungen zielten auf die Ermittlung ei
ierten Ergebnisse als auch Daten derAngiogenese. Sowohl die hier präsent
neben Tumorzellen auch humane vaskuläre son zeigen, dass Arbeitsgruppe Sorens
ORM endogen exprimierEndothelzellen (HDMECs) en. Zur funktionellen
Charakterisierung von ORM in Endothelzellen wurde ORM überexprimiert und
in-vitro-gesilenct. Anhand von ys mit Überständen ORM-aAngiogeneseass
e gezeigt werden, dass ORM encter HDMECs konntüberexprimierender und gesil
eigert. Diese unterstützendermierung stierte endotheliale Tubefodie VEGF-induz
r eden durch simultane Behandlung dWirkung von ORM konnte potentziert wer
und PAI-1-Antikörper. HDMECS mit VEGF, ORM
ist in Urinproben vonmalig, dass ORM erhöht Die Daten zeigen erst
Harnblasenkarzinompatienten und proangiogenetisch wirkt durch die Verstärkung
terstützende Effekt konntenDieser udes tubeformierenden Effektes von VEGF.
signifikch zusätzliche Blockade von PAI-1. ant gesteigert werden dur
zwischen ORM und PAI-1 und dem Anti-Zusammenfassend scheint die Interaktion
PAI-1-System entscheidend involviert zu sein in die VEGF-vermittelte Angiogenese
sierung des Harnblasenkarzinoms. herweise in die Vaskulariund möglic
s nAbbreviatio
Table of Abbreviations 2 dimensional gel elec 2-DE complex Avidin-biotin ABC American Joint Committee on Cancer AJCC Ammonium per sulphate APS lignment search tool l aBasic loca BLASTmembrane Basement BM Coomassie blue brillant CBB ntary Compleme cDNA3-[(3-Cholamidopropyl)diCHAPS 3,3-Diaminobenzid DAB modified eagle medium Dulbeccos DMEM lfoxide uDimethylesDMSO ucleic DNA Deoxyribon
uclease DNase Deoxyribon
leosid DeoxynucdNTP ol Dithiothreit DTTcells Endothelial EC EscherichE.coli coli a iEthylendiamine EDTA MoleculaEuropean EMBL Fetal calf serum FCS Fibroblast growth factor FGF minute,second Hour, h,min,sec Human dermal microvascular endothelial cells HDMECsHuman umbilical cord vein endot HUVEC focusing Isoelectric IEF pH gradient dImmobilize IPG Kilobasepairs kb Kilodalton kDa Liter l broth Luria LB Liter Mol/ M Miliamper mA
trophoresis 2 dimensional gel eleccomplex Avidin-biotin American Joint Committee on Cancer Ammonium per sulphate lignment search tool l aBasic locamembrane Basement Coomassie blue brillant DNA ntary Complememethylammonio]-1-propanesulfonate 3-[(3-Cholamidopropyl)diin-tetrahydrochloride 3,3-Diaminobenzid modified eagle medium Dulbeccoslfoxide uDimethylesDeoxyribonucleic acid
uclease nyriboxDeotriphosphate leosid Deoxynucol Dithiothreitcells Endothelial Escherichcoli a itetraacetate Ethylendiamine r Biology Laboratory MoleculaEuropean Fetal calf serum Fibroblast growth factor minute,second Hour, Human dermal microvascular endothelial cells helial cellsHuman umbilical cord vein endotfocusing Isoelectric pH gradient dImmobilizeKilobasepairs Kilodalton Liter broth Luria Liter Mol/ Miliamper
XII
s nAbbreviatio
MALDI MALDI-TOF MEM mg ml µg µl
mM mmol mRNA
MS
Mu m/z
MVB NCBI
ng OD
ORM PAI PAP PB
PBS PCR pI RISC RNA
rpm RT
RTK
r desorpsion/ ionization Matrix assisted lasetime of flight ion/ ionization-Matrix-assisted laser desorps Minimum essential medium -3Milliliter Milligram (=10(=10-3l) g)
-6Microliter Microgram (=10(=10-6l) g)
Liter / lMillimoMillimol (=10-3mol)
RNA Messenger
spectrometry Mass
unit Mass Mass to charge ratio
body Multivesicular National Center for Biotechnology Information
Nanogramdensity Optical
Orosomucoid Plasminogen activator inhibitor e-antiperoxidase Peroxidasbuffer Phosphate
Phosphate buffered saline chain reaction ePolymeraspoint Isoelectric ing complex ed silencRNA-inducRiboacid ic nucle
Revolutions per minute temperature Room
Receptor tyrosine kinase
Sodium dodecyl sulphate SDS gelelectrophoresis SDS-PAGE SDS-polyacrylamide siRNA Small interfering RNA
Tris-acetate-EDTA TAE
phosphin Tributyle TBP
XIII
s nAbbreviatio
TBS-T
TE
TEMED
TFA
TFB
TRIS
U
UV
V
VEGF
VEGFR
Vh
WHO
Wt
ZAG
Tris buffered saline with tween
Tris-EDTA
Tetramethylethylendiamin
etic acid buffer Trifluoroac
Buffer tion Transforma
Tris(hydroxymethyl)aminomethane
Units
e violatUltra
Volt
Vascular endothelial growth factor
growth factor receptor endothelial Vascular
Volt-hour
World Health Organization
Wild-type
zinc-alpha-2-gylcoprotein Human
XIV
ction Introdu
1
on i1 Introduct ingly important part of life sciences, The field of proteomics has become an increas
especially after completion of the human genome sequence. Proteome analysis
includes separation, identification, and quantitation of proteins from biological
from prognosis of many types of cancer samples with the purpose of revealing the function of living cells. over drug development to monitoring ofApplications range
environmental pollution. The study of genes cannot provide much information on the properties of proteins,
e because the molecules responsible for cellular functions (e.g. signal transduction) ar
post-translational 200 different types of proteins. Proteins may impose more than phorylation, glycosylation, modification, including phosacetylation, deamination,
farnesylation, myristolation, palmitoylation, and proteolysis (Krishna and Wold, 1993).
quences. e be predicted purely from DNA sSuch a wide range of modifications cannot
eins themselves their characteristics and functions can Only through the study of protbe expounded. From the data obtained from studies of the genome project, an
wever, it isthe genome are known. Ho estimated number of proteins encoded bydifficult to predict the actual numbers of a number of reason (Eisenberg et al., 2000)proteins enc. Firstly, the exonoded based on-intron cannot be genomic data, for
(Dunham et al., 1999) i.e. genomic accurately predicted from genomic DNA
ained from protein studies to confirm information needs to be integrated with data obtlicing of a transcript can , alternative spgene. Secondlythe presence of a specific yield more than one protein product (Newman, 1998). Therefore, the direct analysis
Thirdly, as a result of coof mRNA or genome does not rempartmentalization and translocaflect the exact number of prtion, the same protein can otein products in a cell.
(Fig. 1) ons in different locationsunctibe found with different properties and f999). (Colledge and Scott, 1
These problems can only be solved by proteomics, which can directly identify the
ation of he appropriate integron by tproteins and provide the genomic informati
technology to solve problems which cannotgenomic and proteomic data (Fig. 2). Scientists worldwide are be resolved by traditional methoapplyids. ng proteomic
ction Introdu
2
leadinFigure 1g: Th to physically ande flow of ge/or functionomic infornalmly variable ation to prformotein prs of protoducts. ein isofoDifferermnst epigen from a sinetic procgle genessees
sequence. The concept of one-gene to one-protein is over-simplified since an RNA can be
than 20differentially spli0 different typeced and can s ofpr post-tranoduce slatvariouional modis proteifin producations. cts.TComphe prartmentaliotein zcaatin beon an expod transesld to moocatioren
(Lau et al., 20result in the same p03). rotein can be found with different properties and functions in different locations
1.1 Proteomics a genome, and thus ed by eins expressThe term proteome refers to all the protproteomics involves the identification of proteins in the body and the determination of
ocesses. their role in physiological and pathological pr
appliFigure 2: cations of proteoProteomicsmics, and th and genoe bmics integrenefits of integratination. g Reprotpreseneomic and gtation ofenomi categoc riesdata (Lau et , potential., al
).2003
ction Introdu
3
the characterization of all or selected The studies of proteomics give a chance for can outline the flow of information withinproteins expressed within a given cell and It is estimated that more than 500,000 .etricoin et al., 2004)that protein network (Pproteins comprise the human proteome, derived from ~35,000 genes in the human
Proteomics offer more complexity but.genome (Banks et al., 2000; Stein, 2004) of proteomics canThe studythan examining genes alone. potentially more specificity generally be divided into two categories: (i) the characterization of protein expression
) the characterization of protein function. iiand (Expression proteomics evaluate cellular protein production encoded by those genes
gan. It exposes the differential expression esent in the target oractive in a cell and prstates. The technologies applied to of proteins between healthy and diseased expression proteomics allow investigation of protein expression in multiple sources
including tissue as well as serum and urine. and/or interactions between he activation state oft Functional proteomics evaluatensive network of signaling pathways in a proteins and can be used to map the extecell. Mapping protein-protein interactions can be useful due to discovery of new
binding interactions which may give insight into new proteins that are involved in
provide evidence of common downstream cancer as well as novel oncogenes or information of how varioussignaling networks. Further events shared by two distinct ns directed againsttherapeutic interventiopathways intersect can help in developing these pathway targets (Cai et al., 2004). in proteomics. The first technology isThere are several major technologies used l l polyacrylamide geher. Two-dimensionaotnecessary to separate proteins from each thod of choice for separation of complex esis (2-DE) is currently the meelectrophornd tissue extracts or body fluids. protein mixtures such as in cell a 1.2 Technology of proteomics: 2-Dimensional Gelelectrophoresis (2-DE)
itative analyses of r qualitative and quantotein databases fo2-DE can provide prochemical, biological and biomedical protein expression in a wide range of bins in two steps, chnique separates proteiinvestigations (Celis et al., 1989). This terst-dimension is isoelectric focusing operties: the fi two independent proaccording tto their isoelectric points (pI); the second-(IEF), which separates proteins according parates eectrophoresis (SDS-PAGE), which sacrylamide gel eldimension is SDS-polyproteins according to their molecular weights (MW). In this way, complex mixtures
ction Introdu
4
and the relative amount rent proteins can be resolvedconsisted of thousands of diffeof each protein can be determined.
preparation 1.2.1 Sample
Appropriateprocedure must be determined empirically sample preparation is ess foential for good 2DE results. The optimalr each sample type. An ideal process
should result in complete solubilisation, disaggregation, denaturation, and reduction
the final 2-DE resultof the proteins in the sample. It i when developing s important to have a cleara sample preparation strategy. Different idea of what is desired in
samples; some proteins are naturally ftreatments and conditions are required to ound in complexessolubilise different types of protein with membranes, nucleic
acids, or other proteins; some proteisome proteins precipitate when remons form various non-specifved from their natural environment. The ic aggregates; and
the choice of cell disruption method, effectiveness of solubilisation depends on protein concentration and discomposition of the sample solution. The solution methods, choice ofcomposition of the sample s detergents, and olution is
bilisation treatments for the first-dimension critical for 2-DE, because soluparticularlyconductive solution. In general, cseparation must not effect isoelectric pointoncentrated (pI), nor leave the surea as well as one aor more detergents mple in a highly
is used.
extraction 1.2.2 Protein
equires the disruption of the cell membraneThe extraction of proteins from a cell rained in the cell and the cell membrane. An and the solubilization of the proteins contids ure them, and exclude nucleic acact all proteins, denatextraction buffer should extrd widely used solubilising buffers contain and cell debris. The most successful anchaotropic agents that will disrupt the hydrogen bonding, a combination of several
de chainsiions between hydrophobic sdetergents that prevents hydrophobic interactand helps solubilize the proteins, and a reducing agent that breaks the disulfide
residues (Chevallet et al., 1998; Tastetbridges initially formed between two cysteine uption, removal of interfering ll disre ceet al., 2003). Such detergents will achievagents, and solubilization and denaturing of proteins (Gorg et al., 2004; Herbert,
1999).
ction Introdu
5
agent 1.2.2.1 Chaotropic disrupting the hydrogen bonding (RabilloThe use of urea and thiourea (Fig. 3A,B) increase the solubilityud et al., 1997). Urea solubilises and of proteins by
denaturates proteins, unfolding them to expose internal ionisable amino acids.
ration can be increased to 9 or 9.8 M. 8 M urea is used, but the concentCommonlyUsing thiourea in addition to urea improves solubilisation, particularly of hydrophobic
Rabilloud, 1998). membrane proteins (Molloy, 2000;
for the proteiFigure 3: Structurn extractioe of Urn. The presenea and Thiourea.ce of the Two sche aotropitwo rec reageagents, urents in the lysis buffer alloa (A) and thiourea (B)ws for th usede
solubilizatiooxygen of the carbn of non-soluonyl groble proteiup of an amins by no aciddisrupting the with the hydro hydrogegen n boof an amino nding that argroue formed bp of anotetweeher amin the no
acid. 1.2.2.2 Detergents hains from side c between hydrophobic interactionsDetergents prevent hydrophobicproteins. SDS is considered the most fility ooccurring and allow for better solub ie et al., 1995; Harder etbilisation of proteins (Boucherefficient detergent for the soluc nature, it may interfere with the first dimension of al., 1999), but because of its ionireaks on the gel (Gorg et al., zontal st2-DE and it has been reported to cause hori ylglucopyranoside) and zwitterionic2004). Non-ionic detergents like OG (Octfonate) are lholamidopropyl)dimethylamino]-1-propanesureagents like CHAPS (3-[(3-cbeing favoured over SDS use (Kersten et al., 2002; Rabilloud et al., 1997; Rabilloud
et al., 1999) (Fig. 4A-C). A combination of several non-ionic and zwitterionic
bilization ct for the solue no one detergent is perfedetergents should be used becausoteins (Schuck et al., 2003). of most of the hydrophobic pr
ction Introdu
6
(SDS), an aFigure 4: Strnion ucturdee of detergent; terge(B) Onts uctyl seglucopyranod in solubilisation oside (OfG) a prnon-ioteins. oni(Ac d) etergeSodium Dodent; and (C) cyl Sulfate 3-[(3-
cholamidopropyl)dimethylamino]1 propanesulfonate (CHAPS) a zwitterionic detergent.
agents 1.2.2.3 Reducing
To obtain well resolved spots, disulfide bridges should be cleaved to achieve total
protein. This can be done using a reducing agdenaturation of the
l (DTT) (Fig. 5). oDithiothreit
ent like
Figure 5: Structure of dithiothreitol (DTT). A reducing agent used to cleave the disulfide bridges
that can form between two cysteine residues to achieve complete denaturation of proteins and
r solubility. increase thei
ate to the basic region of the IPG strip r, DTT is a weak acid which will migreHowev
when the sample is loaded for isoelectric focusing (IEF) and will only reduce proteins
ction Introdu
7
al., 2002). Tributyl phosphine in that region, necessitating the introduction(TBP) (Fig. 6) was reported of excess DTT at the cathode (Hoving et as an alternative reducing
agent (Herbert et al., 1998).
Figure 6: Structure of tributylphosphine (TBP). A reducing agent used in 2-DE process.
oelectric focusing (IEF) First dimension: Is1.2.3
proteins according to their isoelectric IEF is an electrophoretic method that separateseither positive, negative, or eric molecules; they carry points (pI). Proteins are amphotthe pH of their surroundings (Fig. 7). The net charge zero net charge, depending on ges of its amino acid side harall the negative and positive cof a protein is the sum of chains and aminoand carboxyl-termini. The isoelectric point (pI) is the specific pH at
e positively charged at pH otein is zero. Proteins arwhich the net charge of the pre their pI. If the net at pH values abovively charged values below their pI and negatpH of its environment, the resulting curve charge of a protein is plotted versus the of a pH gradient is (Fig. 7). The presence intersects the x-axis at the isoelectric pointcritical to the IEF technique. In a pH gradient, under the influence of an electric field,
ent where its net charge is zero. A protein a protein moves to the position in the gradi ly lessethe cathode, becoming progressivwith a positive net charge migrates toward reaches its pI. A gradient until itpositively charged as it moves through the pH the anode, becoming lesscharge migrates toward h a negative net protein wit If a protein should diffuse reaches zero net charge. negatively charged until it alsoy gains charge and migrates back. s pI, it immediateltaway from iThis is the focusing effect of IEF, which concentrates proteins at their isoelectric
ed on the basis of very small charge oteins to be separatpoints and allows prdifferences.
ction Introdu
Figure 7:
Str es of pructurhoteric moleotein as ampctric point ocules, isoele proteins.f
8
the electric field the pH gradient and The resolution is determined by the slope of
ically in excformed at high voltages (typstrength. IEF is therefore per ess of 1000 V).
When the proteins have reached their final positions in the pH gradient, there is very
little ionic movement in the system, resulting in a very low final current (typically
a given electrophoresis system is generallybelow 1 mA). IEF of a given sample in
of Volt-hours (Volt-hour (Vperformed for a constant number h) being the integral of
the volts applied over the time).
ghest resolution and the conditions gives the hi denaturing IEF performed under
achiesolubilization isuration and cleanest results. Complete denatved with a mixture
of urea and detergent, ensuring that each protein is present in only one configuration
and aggregation and intermolecular interaction is minimized.
covalenAn immobilized pH gradient (IPG) is created byating a gradient of ly incorport
ylamide gel at the time it is cast. The cra into a polyacidic and basic buffering groups
Immobiline reagents is: general structure of
ction Introdu
CH2 = CHCNHRO
R = weakly acidic or basic buffering group.
9
Figure 8:matrix with attac Structurhee of pd bufferiolyng groacrylupsamid. e gel matrix. Graphical representation of the polyacrylamide
Immobilized pH gradients are formed using two solutions, one containing a relatively
acidic mixture of acrylamido buffers and the other containing a relatively basic
mixture. The concentrations of the various buffers in the two solutions define the
range and shape of the pH gradient produced. Both solutions contain acrylamide
on, the acrylamide portion of the buffers is. During polymerizattmonomers and catalys
bisacrylamide monomers to form a copolymerizes with the acrylamide and
polyacrylamide gel. The graphical representation of polyacrylamide matrix with
attached buffering groups is shown in figure 8.
he 2-DE process. The proteins should be The IEF is the most critical step of t
solubilised without charged detergents, usually in high concentrated urea solution,
reducing agents and chaotrophs. To obtain high quality data it is essential to achieve
it self. Since different types of samples rength conditions before the IEF low ionic st
to adjust the IEF buffer and the electrical ntent, it is necessaryodiffer in their ion c
profile to each type of sample.
ction Introdu
SDS-PAGE mension: di1.2.4 Second
10
ed vertically according to protein molecules are separatIn the second dimension, the proteins (that is, ate treatment denatures their molecular weight. Sodium dodecyl sulfit unfolds them into long, straight molecules) and coats all proteins essentially in
proportion to their mass. ft proteins according to size because of the second dimension gel siThe poresoportion to their mass. The ially in prlphate coats all proteins essentudodecyl sellipsoids with a uncoating of the protein in negativiform negative charge-ely-charged SDS to-mass ratio, with mobilityallows the proteins to migrate as related
logarithmically to mass. Proteins are therefore separated horizontally based on their
isoelectric point and vertically based on their molecular mass. r, forms globular micelleshat, when in solution in wateergent, tSDS is an anionic detcomposed of 7080 molecules with the dodecyl hydrocarbon moiety in the core and
proteins form complexes the hydrophilic shell. SDS andthe sulfate head groups in with a necklace-likshort flexible polypepte structuride segments (Ibel et al., 1990)e composed of protein-decor. The result of the necklace ated micelles connected by
structure is that large amin a ratio of approximately ounts of SDS are inco1.4 g SDS/g protein. SDS rporated in the SDmasks the chargS-protein complex e of the
c complexes have a roughly constant net proteins themselves and the formed anioninegative charge per unit mass. Besides SDS a reducing agent such as dithiothreitol
inkages present in the proteins. When (DTT) is also added to break any -S-S-lelectrophorproteins are treated witetic separation with bothin a polyacrylamide gel h SDS and a reducing agent, the degree of depends largely on the
y linear relationship there is an approximatel In fact, molecular weight of the protein.lative distance of migration the re the molecular weight andbetween the logarithm of of the SDS-polypeptide complex.second-dimension SDS-PAGE is the tris-r The most commonly used buffer system foli, 1970). This buffer system separates tem described by Laemmli (Laemmglycine sysion and advantage of minimal protein aggregats the proteins at high pH, which confer. The Laemmli buffer system vy protein loadsclean separation even at relatively healimited gel shelflife. has the disadvantage of a
ction Introdu
1.2.5 Equilibration
11
quired for p with the SDS buffer system reriThe equilibration step saturates the IPG st contains buffer, urea, n. The equilibration solutionthe second-dimension separatio tional equilibration step replaces theeductant, SDS, and dye. An addiglycerol, ron introduces reagents essential for the reductant with iodoacetamide. Equilibratisecond-dimension separation. Urea (6 M) together with glycerol reduces the effects of electroendosmosis by
increasing the viscosity of the buffer (Gorg et al., 1985). Electroendosmosis is due to
ectric field and can interfere l on the IPG strip in the ethe presence of fixed chargessecond-dimension gel. the IPG strip to the with protein transfer fromGlycerol (30%) together with urea reduces electroendosmosis and improves transfer
second-dimension (Gorg et al., 1985). of protein from the first to the oteins. of denatured, unalkylated prpreserves the fully reduced state DTT Sodium dodecyl sulfate (SDS) denatures proteins and forms negatively charged
protein-SDS complexes. The amount of SDS bound to a protein, and therefore the
n. Thus, mass of the proteidirectly proportional to the harge, is additional negative celectrophorproteins on the basis of molecular mass. esis of proteins through a sifting gel in the presence of SDS separates
preventing their reoxidation during alkylates thiol groups on proteins, Iodoacetamide an result in streaking and dation during electrophoresis cesis. Protein reoxielectrophorto prevent point streaking also alkylates residual DTT other artifacts. Iodoacetamide and otherequilibration step. The second equilibration silver-staining artifacts. Iodoacetamide is intr with iodoacetamide is also used tooduced in a second
dues (i.e. when mass spectrometry is tominimize unwanted reactions of cysteine resibe performed on the separated proteins). esis. allows monitoring of electrophor(bromophenol blue)Tracking dye
Identification of proteins 1.2.6
,ns are detected by color, immunoanalysisAfter first and second dimension the protei staining intensity.e quantified by their or prebound fluorescent dyes, unique spots arto protease digestion, and sed from gel and subjected Spots of interest are exciMass Spectrometry. Mass spectrometry ing ined uspeptide fragments can be exameins as they migrate through ionized protmeasures the mass-to-charge ratio (m/z) of are analyzed and identified based on unique an electric or a magnetic field. Proteins
ction Introdu
12
structural features on offeatures allow identificatispectrometric signatures. These such as phosphorylation or methylation. Trypsin digestion of proteins yields peptides
another method byng MS with MALDI isthat are amenable to MS sequencing. Coupliwhich proteins can be identified. 1.3 Urinary proteomics
1.3.1 Formation of urine
plasma to eliminate waste by ultrafiltration from the Urine is formed in the kidney . Although the kidney accounts for onlyproducts, for instance urea and metabolites of plasma (350-400 ml/100 g tissue/min) 0.5% of total body mass, a large volume of ultrafiltrate (150-180 l/day) under flows into the kidney, generating a large amountultrafiltrate such as water, glucose, in the normal physiologic conditions. Componentsamino acids, and inorganic salts are selectively reabsorbed, and less than 1% of
ultrafiltrate is excreted as charges at the glomeruli (Haraldsurine. Serum proteins are filtson and Sorensson, 2004). ered based on their sizes and
After passing through glomerimmunoglobulin light chain, transferrin, vitamin D binding proteiuli, abundant serum proteins such as albumin,n, myoglobin, and
receptor-associated protein are reabsorbed, mainly by endocytic receptors, megalin,
d Gburek, 2004; Maunsbach,enal tubules (Christensen anand cubilin in proximal rurine is1997). Thus, protein concentration in normal (less than 100 mg/l very low s than 150 mg/day. etion is lesrprotein excwhen urine output is 1.5 l/day), and normal with other body fluids such as plasma. s compared This is about a factor 1000 lesday protein is defined as proteinuria and is indicative Excretion of more than 150 mg/eabsorption dysfunction. of glomerular or r
1.3.2 Sources of urinary proteins
components of solid phase luble proteins and protein Urinary proteins include soents consist of sediments that can be elements of urine (Tab. 1). Solid phase elemprecipitated at low centrifugation speeds and exosomes that are of very low density
and sediment only with ultracentrifugation. be useful as a means of enriching for markers of partiPrefractionation of thescular types of disease. A study e components can
that, of the total urinary l human adult subjects indicated of urine collected from normaremaining 3% was in exosprotein excreted, 48% was omes (Zhou et al., 2006).contained in sediment s, 49% was soluble, and the
ction Introdu
Comments
13
Normally present (<150 mg/day)
Defects in glomerular filter increase high
molecular weight protein (e.g. albumin)
n excretio Defects in proximal tubule reabsorption or
abnoplasma prmal proteirons iductioncrease ln of low moleow moleculacular r weiweigght ht
light chainprotein (e.g. sβ2, retinol-microgl-binobding ulin, immunoprotein,globulin and
n ) excretioscidaminoa
glycosVia exocytoylphosissphatidylino (e.g. episitol-andermal gcrhoowthred fa proteinctor) or
detachment (e.g. Tamm-Horsfall protein)
Increased cell number compatable with several
diserenalases in tubulcle celluding ac shediute tubulang) andr ne glcrosis omerular (e.g.
diseases (e.g. podocyte shedding).
Coulapoptoticd be du procese to nonses. specific, nephrotoxic, or
Sources of urinary proteins Comments
ns iSoluble Prote • Glomerular filtration of plasma proteins Normally present (<150 mg/day)
Defemoleculacts in r weigglomerularht protein (e.g. filter increasealbumin high)
n excretio abnoDefects inrmal p roductioproximal tubn of low moleule reabsocularpr weigtion or ht
plasma proteins increase low molecular weight
light chainprotein (e.g. sβ2, retinol-microgl-binobding ulin, immunoprotein, and globulin
aminoacids) excretion
• Epithelial cell secretion of soluble proteins. glycosVia exocytoylphosissphatidylino (e.g. episitol-andermal gcrhoowthred fa proteinctor) or
detachment (e.g. Tamm-Horsfall protein)
Solid phase components
• Epithelial cells Increased cell number compatable with several
a. Whole cell sheding diserenalases in tubulcle celluding ac shediute tubulang) andr ne glcrosis omerular (e.g.
diseases (e.g. podocyte shedding).
b. compoPlasma menent sheddimbraneng and intracellular Could be due to nonspecific, nephrotoxic, or
apoptotic processes.
c. Exosome secretion Normal process
• Other cells cellIn certain dis, or tumor cellseases, red bls (e.g.ood bladdecells, whir cantcer ae bloodnd
lymhome) can be present in urine.
Table 1: Sources of urinary proteins. (Pisitkun et al., 2004).
glomerular filtration. Thee are derived largely from in urinThe soluble proteins proteins. ssage of high molecular weight filter effectively retards paglomerularHowever, even with very low sieving coefficients, proteins that are abundant in the
erular filter in he glomlins can pass tma such as albumin and various globublood plas the nephron. Beyond this, peptides andsubstantial amounts to enter the lumen ofsmall proteins (10 kDa) are freely filtered by the glomerulus. Most of the proteins and
ed in the are scavenged and proteolyzular filterhat pass the glomerpeptides tproximal tubule by highly specialized apical uptake processes that involves receptor-
Normal process
cellIn certain dis, or tumor cellseases, red bls (e.g.ood bladdecells, whir cantcer ae bloodnd
lymhome) can be present in urine.
ction Introdu
14
, 2001; ntensen and Birypeptide molecules (Chrislike recognition of the polotein that ount of a given soluble prChristensen, 2002). Thus, a change in the am a change in its concentration in the bloodreaches the final urine can result from , or an alteration in the ilteron of the glomerular fplasma, a change in the functiproximal tubule scavenging system (Fig. 9).
Figure 9: Mechanism of urinary exosome formation and excretion. Apical membrane proteins
undergo endocytosis followed by targeting to the.multivesicular body (MVB). The membrane proteins
are segreinvagination, encapgated initially isulatingn cytosoli the MVB c proteins in the outer membprorane acess. After nd thenaccum are internulatioalizen of numerod by muse internmbrane al
vesicles, the outer membrane of the MVB fuses with the apical plasma membrane releasing its
internal vesicles, called exosomes, into the urinary space. Exosomes contain both membrane and
cytosolic proteins (Hoorn et al., 2005).
Based on t ion rate of specific urinary proteinshese mechanisms, changes in excretproximal tubule, rescan be indicative of systemic disease, glomerpectively. Some of the soluble proteins in urine originate asular disease, or diseases affecting the
ically cleaved from their membrane membrane-bound proteins that are proteolytattachments.
ction Introdu
15
cancer Bladder 1.4 ccuring worldwide. The rate he most common cancer type oBladder cancer is one of ttries, where they rank as the sixth most loped counefor these tumors is highest in dev rrently ranks as the fourth most common cause ofufrequent neoplasm. The disease cdeaths from bladdercancer death in men and the eighth in cancer are 13,180 in women. Estimated new casthe United States ein 2005 (American s are 63,210 and
bladder cancer increases with age 2005). The incidence ofCancer Society, Atlanta, before age fourty. The xty and is uncommon high after age of si particularlyand isurinary bladder has a flat, smooth, shiny, watertight lining consisting of layers of cells
ng of the bladder can be imagined to be h each other. The linitightly connected witsimilar to the lining in the oral cavity (mouth). Underneath this lining is the muscle
the time out the urine ate is responsible for pushing tissue of the bladder. The muscl in The bladder liesound 350-400 cc in adults.of voiding. The capacity of bladder is arthe pelvis anterior and inferior to the peritoneal cavity, and posterior to the pubic
bones. In the female, it rests directly on the muscular pelvic floor, while in the male
der and separates it the base of the blad attached directly tothe prostate gland isfrom its muscular support. The cells grow abnorBladder cancer is an abnormal growth or tumomally fast causing a tumor r arising from the linto sprout up from the flat lining into a ing of the bladder.
he bladder cavity. Bladder cancer occurs within growth projecting into the interior of tent types of etiological factors such the domain of human neoplasms by many differ -naphtylamine, benzydine, 4-nitro diphenyl),βas some aromatics (i.e. xylamine, coffee, artificial sweetWynder and Stellman, 1977). Most eners and smoking of bladder cancers present(Cohen et al., 1979; Morrison et al., 1982; as superficial disease
The rate of recurrence on by endoscopic resection.iand are amenable to local excisogression in stage is from 15% to 25%. ranges from 50% to 75% and the rate of prwith biopsyCystoscopy is the gold standard for routine of suspicious lesions and histsurveillancopathologic evaluation e for bladder tumor recurrence, required to confirm
the diagnosis (Tsihlias and Grossman, 2000).
Staging of bladder cancer 1.4.1
the bladder is determined by the depth of The clinical staging of carcinoma of aluation (Fig. 10). Based on morphologic ev the bladder wall by the tumor invasion ofing assified into two groups havl been cand natural history, urothelial neoplasms have
ction Introdu
16
) low-grade tumors (always papillary and usually idistinct behavior and prognosis; (superficial) and (ii) high-grade tumors (either papillary or non-papillary and often
mors (stages Ta, Tis, and T1) account for). Clinically, superficial bladder tueinvasiv75% to 85% of neoplasms, whereas the remain (T2, T3, eing 15% to 25% are invasivT4) or metastatic (N+, M+) lesions at the time of initial presentation (Rabbani and
CC) haserican Joint Committee on Cancer (AJAmCordon-Cardo, 2000). The er (American Joint define bladder cancodesignated staging by TNM classification tAJCC stage grouping is shown in table 3. (Tab. 2). Committee on Cancer, 2002)
Figure 10based-on the: Repr depth of tuesentamor invation of bladdsioner canc into the bler satages. dder wall (FScherom Hame for blautmanddn er tuand Hulamor clnad, ssifUrolicatiogy, on
r). isheer-PublSpring
classification 1.4.2 Cellular
transitional cell carcinomas derived from More than 90% of bladder carcinomas are the uroepithelium. About 6% to 8% are squamous cell carcinomas and 2% are
arcinomas may be either of urachal adenocarcinomas (Mostofi et al., 1988). Adenoctype is generally thought to arise from origin or of nonurachal origin; the latter metaplasia of chronically iPathologic grade, which is based on cellurritated transitional epitheliumlar atypia, nuclear abnormalities, (Wilson et al., 1991). and the
ic importance. Resection of the tumor in number of mitotic figures, is of great prognosthis entirety, with inclusion of the lamina propria and muscularis propria, and biopsy of
17 ction Introdued erytematous areas) are us areas (i.e., nontraumatizopically suspicioall endosc ensure proper staging.generally performed to ons TNM DefinitiTX Primary tumor cannot be assessed
mor ce of primary tunNo evideT0 noma illary carcipapnvasive Ta NoniTis Carcinoma in situ (i.e., flat tumor)
T1 Tumor invades subepithelial connective tissue
T2 Tumor invades muscle
pT2a Tumor invades superficial muscle (inner half)
pT2b Tumor invades deep muscle (outer half)
T3 Tumor invades perivesical tissue
PT3a Microscopically
PT3b MacrTumor invaosdecopically (extras any of the followinvesical masgs) ; prostate, uterus, vagina, pelvic wall, or
T4 nal wall abdomi PT4a Tumor invades the prostate, uterus, vagina
PT4b Tumor invades the pelvic wall, abdominal wall
mph nodes (N) al lyRegionNX Regional lymph nodes cannot be assessed
NO No regional lymph node metastasis
N1 Metastasis in a single lymph node, =2 cm in greatest dimension
N2 Metastamultiple lympsis in h nodea singls, =5 cm in greatee lymph node, >2 cm but =5 cm ist dimension n greatest dimension; or
N3 Metastasis in a lymph node, >5 cm in greatest dimension
Distant Metastasis (M)
MX Distant metastasis cannot be assessed
is sstant metastaNo diMO metastasis M1 Distant Table 2: StaJoint Committee on Canging of bladcder caer (AJCC) ncer.using TNM cl Definition of bladdassification. (Ler cancamer whim, 1994).ch wa s designed by American
e adjacent or distant mucosa if urineDirected biopsies may be performed to evaluatt in the emenurethra to evaluate its involv positive. Biopsy of the prostatic cytology isoverall tumor diathesis (especially in the setting of CIS) is also important (Lee and
Droller, 2000).
ction Introdu
e groupings agtCC sAJ
18
Ta, N0, M0 Stage 0a N0, M0 ,TisStage 0is T1, N0, M0 Stage I T2a, N0, M0; T2b,N0, M0 Stage II T3a, N0, M0; T3b, N0, M0; T4a, N0, M0 Stage III Stage IV T4b, N0, M0; Any T, N1,M0; Any T, N2,M0; Any T, N3,M0; Any T, any N,M1
CancTable 3: AJerCC s (AJCC) (Lamm, 19tage groupings. 94). Classification of bladder cancer by American Joint Committee on
Clinical proteomics and biomarkers 1.5 iology, sough severe changes in human phyMany diseases manifest themselves thrwhich forms the basis for clinical chemistry and confers its value in diagnoses and
subsequent therapeutic interventions (Bischoff and Luider, 2004). Clinical proteomics
eins expressed by the genominclude the analysis of prot e of an organism, with thetypical aim being the evaluation of quantitative changes that occur as a function of
e . Proteomics strategies aronment (Somiari et al., 2003)disease, treatment, or envirbeing used to identify disease-specific protein markers called biomarkers that could
new diagnosis methodologies, treatments, provide the basis for the development of 000; Somiari et al., of cancer (Hanash, 2and early detection of diseases particularly identification using followed by protein 2003). Already for a number of years, 2-DEtechnique for biomarker discovery in mass spectrometry has been the primary org et al., 2000; Hanash, 2000).conventional proteomic analyses (G 1.6 Urinary biomarkers
Because urine can be collected noninvasively in large amounts, it provides an
(Thongboonkerd et al.attractive alternative to blood plasma as , 2004). The use of urinary biomarkers a potential source of disease bto diagnose disease is a iomarkers
omarkers of disease in urine have beenlong-standing practice. Studies to identify bith century and igative medicine throughout the 20ing component of investan underlyearly 21st century. These studies have been based on knowledge of the
al ould be tested in clinicidentify biomarkers that cpathophysiology of disease to mples has revealednormal human urine sag of trials. Large scale proteomics profilinducts and many more peptide otein gene prothe presence of at least 1000 different pr(Castagna et al., 2005; Jurgens et al., 2005; Oh et al., fragments of larger proteins 2004; Pisitkun et al., 2004; Smith et al., 2007; Sun et al., 2005). There is hope,
ction Introdu
19
therefore, for discovery of urinary protein excretion profiles that can be used clinically
tion of diseases particularly of cancer, caifor tasks such as early detection and classif and monitoring of a particularsessment of prognosis, agents, aschoice of therapeuticbeen identtherapeutic regimen. Several ified, including nuclear matrix potential diagnostic markers for blprotein 22, bladder tuadder cancmor antigen, and er have
telomerase. angiogenesis 1.7 Tumor ancer growth is the development of a newOne of the critical events required for cvasculogenesis (Folknetwork of blood vesmsels. This is provian and D'Amore, 1996)ded by angiogenesis as . Angiogenesis is defined as the well as postnatal
sprouting of new vessels from pre-exisCarmeliet, 2004), while postnatal vasculogting blood vessenesis desels (Folkman, 2003; Luttun and cribes the formation of new
d or derived from ooating in peripheral blvessels by endothelial precursor cells circul Gehling et al., 2000; Rafii et al., the bone marrow (Asahara and Kawamoto, 2004;ly demonstrated (Zengin et al., 2006). 2002) from the vessels wall as recent ated by angiogenicis are regulsculogenesAngiogenesis as well as postnatal vaman, 1996). The structural formation and activators and inhibitors (Hanahan and Folk a veryis and angiogenesis issculogenesmaturation of blood vessels during va iferation and tube steps including prolcomplex process that runs in successive the basement membrane, integration of formation of endothelial cells, construction of, and embedding of blood vessels into the peri-endothelial cells into the vascular wall1996). Numerous angiogenic fperi-vascular tissue (Ergun et al., 2006; Foactors including vasclkman et al., 1989; Foular lkendothelial growth factor man and D'Amore,
(VEGF), fibroblast growth factor-2 (FGF-2), angiopoietins (Ang1, Ang2), and their
receptors, which belong to the receptor tyrosine kinase family, are involved in several
aisonpierre et al., 1997). steps of this process (M and acts asnesisand pathological angiogeVEGF is the key regulator of physiological , both in vitro and in vivo (Ferrara, 2005). a survival factor for endothelial cells (EC)The biological effects of VEGF(RTKs), vascular endothelial growth fact are mediated by two receptor receptor-1 (VEGFR-1) and VEGFR-2 or tyrosine kinases
F with its receptor VEGFR-2 , the interaction of VEGularly(Ferrara et al., 2003). Partic
ction Introdu
AnAngg22
VEGFVEGF
eicice
fnfn
VEGFVEGFFGFGFF--22
20
pgapgaAABBCC
with a deFigure 11: Vascular nse BM (gray) enclodestabilization sing both endothand initiationelial cell of angios (gregenesis.en) and peri A normal stabilicyte (red) (Azed capillary ) will be
destaendothebilized lial by action of fenestrationpro (fn), o-angiogepennic factoing of inters as r-eexemplandothrelial contactsily shown by VEGF and An (iec), develg2opment leading toof
endothtransendelial lothelayer (Bial gaps (g). Thaese mp), degorpradhogenetiation of BM ac events are nd finally daccoempanietachmd byent of an abn pericyteormsal vascula from ther
and ileakinnestabss. Thle bloode furthe vesselr durats (C), aion of pro- process dangiogeefinednic a acsti angioon wogeneuld finasis (Adolly lead to sppted from Ergürouting of nen et al., Canw nascentcer
Encyclopedia, 2007, in press).
(known also as KDR in human or Flk-1 in mouse) intiates the angiogenic activity by
causing the structural destabilization of the vascular wall with subsequently increased
vascular leakeness (Ergun et al., 2006; Thurston et al., 2000). Also angiopoietin-2
and both Ang1 and Ang2 act in-1 (Ang1)of the angiopoiet(Ang2), a partial antagonist e in the-2, is essentially involvedvia the same tyrosin kinase receptor Tidestabiliazion of the vascular wall as shown in figure 11. enzymes degrading he extravasation of mal vascular leakeness results in tThe abnore enzymes is the uPA, the urokinase the vascular basement membrane. Among thes volved in the remodelling extracellularnogen activator, a key enzyme initype plasmthe angiogenic activity of on of endothelial cells and thus matrix supporting the migratihe action of this enzyme is ., 2003). T (Agirbasli, 2005; Rakic et althese cellsr-1) which has been shown to otor inhibitamodulated by the PAI-1 (plasminogen activinder et Bor the receptor of uPA, uPAR (have a dual role in the interaction with uPA al., 2007). The concerted action of the mentioned factors above howeever results in
the complete disintegration of endothelial cells of pre-existing blood vessels with
and finally in the of endothelial cells,oliferation subsequent migration and prels. outgrowth of new vessels from pre-existing blood vess proteins: Orosomucoid (ORM) yOne of urinar1.8 ha1-acid glycoprotein (AGP), was firstly alpOrosomucoid (ORM), also known as Schmid, 1953). It belongs to a group of described in 1950 by Schmid (Schmid, 1950;
ction Introdu
21
acute phase proteins (APPs) and plays a role in the modulation of the immune
functions (Schmid K, 1975; Bennet M, response to stress, along with many other ORM in the serum of healthy humans is 1980). The relatively high concentration of conditions such as acutee to differentfold in responsknown to rise two- to fiveer (Schmid, 1975). s and cancive disorderinfection, inflammatory and lymphoproliferata polypeptide chain ed and is composed of The structure of ORM is well characteriz of fucosylic and sialice including a large amount containing about 45% carbohydratacid. Thus, its proposed immunomodulatory activities have been attributed to its
fucosylated and sialylated ORM glycoforms glycosylation pattern, since the strongly inhibit complement activation (De Graaf ethave the ability to bind E-selectin and to al., 1993). ent component 3, serum amyloid A, C-Type 1 APPs, including ORM, complemegulated by IL-1, IL-6 and in and hemopexin, are rreactive protein, haptoglobglucocorticoids and type 2 APP, including the three chains of fibrinogen and several
proteases inhibitors, are regulated by IL-6- type cytokines and glucocorticoids
types e.g. granulocyt(Baumann et al., 1989).es (Fournier T, 2000) ORM is synthesiz. ed in livIL-1, IL-6 and glucer and various extrahepatic cell-ocorticoids are the
et al., 1989; Baumann and Gaulmajor modulators of ORM gene expression in liver cdie, 1990). In most instells ances, a strong synergistic(Alam et al., 1993; Baumann
action is achieved by the combination ofBaumann et al., 1987). An induced expression of sialyl the three factors (Alam et al., 1993; Lewis X (sLeX) on ORM
influencduring acute the E- or P-selectin-e inflammation has been reported, mediated inflleading tux of sLeX-expressing leukoco the speculation thatytes into it might
inflamed areas. It has been suggested that ORM could have a feedback inhibitory effect an increaon the extravasation of leukocsed level of sLeX-expressing ytes, by
competition for the E-selectmodify the permeability of the vascular endin adhesion molecules (Lasky,othelium, possibly by interacting with the 1992). ORM is thought to
endothelial glycocalyx (Curry and Michel, 1980). Thus far it has been shown that
ORM binds to the vascular endothelial cell surface (Fig. 12) and then causes
intercellular junction (Predescu et al., e cell without passing the transcytosis across th1998).
Introduction
22
Figure 1transmigratio2: Hyn of leukopothesis focr thytes to a site oe inhibitif inflaon of leukmmation are ocyte extravsummariseasationd. ORM e byx ORM.pressin Events lg sialeadinyl Lewisxg to
interacts with E-selectin expressed at the surface of endothelial cells and compete with leukocytes
expreleukocytessisng ES may then bL-1, the ligeand of E-sel inhibited. (From E.C. ectin. ConseHavenquently, rolliaar, Thesisng, ad, Vrije hesion and extravaUniversiteit, Amsterdam,sation of
Netherlands.).
stem The plasminogen activation sy1.9 e the formation of the serine proteasThe plasminogen activation system regulates
arts with s of fibrinolysis sthe procesplasmin and subsequent fibrinolysis. T
plasminogen, a 92-kDa single-chain oenzyme, conversion of an inactive pr
to the active enzyme, plasmin (Forsgren glycoprotein consisting of 791 amino acids in
net al., 1987). Plasmin is, in turdegrade fibronectin, laminin, vitronectin, , able to
proteoglycans, as well as fibrin and activate latent collagenases. Angiogenic growth
factors induce the expression of tissue-type plasminogen activator (tPA) and
PA) on the surface of endothelial cellsvator (uiurokinase-type plasminogen act
e serine-Pepper et al., 1991). Both tPA and uPA ar(Flaumenhaft et al., 1992;
ymogen plasmin by proteolytic cleavage of its zproteases that can generate
plasminogen. Plasminogen, like fibrinogen and other plasma components of the
ction Introdu
23
provisional matrix, is synthesized in th response to e liver and deposited in is essential for invasion and migration ofhyperpermeability. The formation of plasmin minogen activation scularized. The plasendothelial cells into the tissue to be vasystem is not limited to endothelial cells. While tPA is almost exclusively expressed
by endothelial cells (Mandriota and Pepper, 1997), uPA also facilitates migration of
ts and tumor cells (Del et al., 1990; fibroblas like epithelial cells,other cellsMacDonald et al., 1998). A variety of cell types can bind components of the
fibrinolytic system, including plasminogen (Miles and Plow, 1985), plasmin (Correc et
PA (Hajjar et al., 1987). There are two lli et al., 1985) and taal., 1990), uPA (VassPAI-1 and PAI-2. Plasminogen activator minogen activator inhibitors, main plastor of plasminogen activators (Carmeliet et inhibitor type 1 (PAI-1) is the main inhibiminogen-ents 60% of plasThe PAI in blood repres al., 1993; Fay et al., 1992).(Fig. 13). PAI-1 inhibits both tPA and uPA, activation inhibitor activity in plasma only uPA.swhereas PAI-2 inhibit
PAI-1
Plasminogen
t-PA /u-PA
Fibrin degradation
Plasmin
Cellular ECM migration remodelling Angiogenesis
moleculeFigure 13: s is the maiPlasminogen plasn activminogen-actiation syvator inhistem. bitor in The pla blood asmind nogenit is impo-artantctivator inhibito for fibrin der 1gradatio (PAI-1) n,
remodelling of ECM and angiogenesis (Agirbasli, 2005).
which only a signaling peptide and a short Unlike the simple digestive proteases, inytic proteases bear c region, the fibrinolactivation domain are attached to the catalytiatalytic regions (Patthy, 1985). large nonc ch as 'kringle', 'growthonally autonomous modules, suThese regions contain functi outside the serine ns, which also occure' and 'finger' domaifactorlike' or 'EGFlikprotease family. A graphic representation of the domain arrangement in plasminogen,
uPA and tPA is shown in Fig. 14.
Introduction
24
Figure 14: Graphical representation of the protein domains of plasminogen, tissuetype
EGFlike plasminoge(EGF) and n activator (tPA) ancatalytic (CD) domaid urokinase ns are marke(ud. PA) proThe activatenzyimes.on site The krins are igle (Kndicated by a), Fingen arrow. r (F),
lfide bond-linked polypeptide uof two dise plasmin consists The serine proteinas
The C-five so called kringle domains. chains. The N-terminal A chain contains
terminal B chain contains a typical serine proteinase domain, which is responsible for
olysis of peptide bonds on the s the hydrelyzcatalytic activity (Fig. 15). Plasmin cata
and ArC-terminal side of Lys asminogen to plasmin cang residues. Conversion of pl
her uPA or tPA. be catalyzed by eit
an exposed center loop that is availableThe active form of PAI-1 is thought to have
renc (Lawfor interaction with proteinasesrence, 1997). The e et al., 1995; Law
ating this surface-res incorpo form involvconversion from active PAI-1 to its latent
-sheet of the protein βve center into the central exposed loop containing the reacti
1992). ORM, one of the major acute phase (Lawrence et al., 1994; Mottonen et al.,
ct with PAI-1 and stabilizeproteins, can intera
tory activity towardi its inhib
Introduction
25
plasminogen activators (Boncela et al., 2001). The ORM-PAI-1 co-localization was
found in thymosin β4 (Tβ4)-activated but not in quiescent HUVECs.
Figure 15: Schematic presentation of uPA and its interacting partners. uPA consists of a serine
proteinase domain (SPD), a linker, a kringle (K), and a uPAR-binding epidermal growth factor-like
and 3 domain m(E). uake contacPAR has thret with uPA. e domainPlasmins an had is ans a sechorinered proteinase do to the membrane by a GPmain (SPD) andI ancho five krir. Dongles (Kmains 1)
and binsite of uPA. Vitroneds to pericellular prctin, in the suboteins stratwith C-terminal lysium, has anne N-termins. The al PAI-1- aRCL of PAI-1 is abnd uPAR-binding sole to bind to the active matomedin
B domain (SomB) next to an integrin-binding RGD sequence. α and β integrin subunits have
transmembrane helices (TM) and are anchored to the cytoskeleton. Integrins can interact with uPAR
(Andreasen et al., 2000).
udy The aim of st
The aim of study2
26
urine proteins present in) to analyse the whole pattern ofiThe aim of this study was: (
urine samples of healthy persons in comparison to that of patients with bladder
r etion between urine proteomics and bladdcancer, and to find out the potential rela
ORM, as one of urinary proteins ) to explore the potential effect ofiicancer stages. (
found as an increase amount in cancer, in vascular and urine of patients with bladder
the interaction between ORM ) to study potential effect ofiiicapillary morphogenesis. (
. and capillary morphogenesisand PAI-1 in angiogenesis
s Methodnd Material a
Methods and 3 Material 3.1 Materials
Chemical and Consumables 3.1.1
Sigma
27
Sigma Acetonitril Serva Acrylamide PeqLab Agarose Sigma Ampicillin Merck APS SiTrypsin Bovine LadderBench Mark Prestained Protein CHAPS PureCol Collagen, Coomassie Brillant Blue G-250 Gibco DMEM Merck DMSO BioRad DTT Ethidiumbromide
Gibco FCS ThermoShandon, Histomount Iodacetamide Sigma AgarLB Gibco L-Glutamin (NP-40) Nonidet Gibco MEM Normal Swine Serum (NSS) Normal Rabbit Serum (NRS) Nitrocellulose-Membrane otides (Primer,siRNA) leOligonuc Seromed cco lbeDuPBS Penicillin/Streptomycin
ktail Protease Inhibitor Coc KodakX-Röntgenfilms Sigma Solution Silvernitrate
(Taufkirchen) Sigma (Heilderberg) (Erlangen) (Taufkirchen) Sigma (Darmstadt)gma-Aldrich (Steinheim) Holland) Invitrogen (Groningen, LadderSigma (Taufkirchen) Inamed (Nutacon,Finnland) BioRad (München) (Karlsruhe) BRL (Darmstadt) (München) Serva (Heidelberg) (Karlsruhe) BRL USA) (Pittsburg, Sigma (Taufkirchen) (Taufkirchen) (Karlsruhe) BRL tadt) Calbiochem (Darms (Karlsruhe) BRL Dako, (Hamburg) Dako, (Hamburg) Schleicher & Schuell BioScience (Dassel)
MWG Biotech (Ebersberg) (Berlin) Gibco BRL (Karlsruhe) Sigma (Taufkirchen) AR5 Kodak (Stuttgart) (Taufkirchen)
28s Methodnd Material aSDS-PAGE marker wide range Sigma (Taufkirchen)
T4 DNA Ligase New England Biolabs (Schwalbach/Taunus)
(München) BioRad TBP (Taufkirchen) Sigma TEMED (Taufkirchen) Sigma Thiourea Sigma (Taufkirchen) X-100 Triton Trypsin/EDTA (1x) Gibco BRL (Karlsruhe)
(Taufkirchen) Sigma Urea Mowiol Calbiochem (Darmstadt)
Falcon (Heidelberg) Cell culture flasks, plates, and tubes (Neustadt) C.C.pro edium) Cryo-Save1 (conservation m were purchased from following umablesals and consUnless otherwise stated chemicreiburg), BioRad (München), Biozym ences (Fi Amersham Biosccompanies:nnheim), Merck (Darmstadt), Roche (Ma Holland), (Hameln), Invitrogen (Groningen, unus), Promega (Heidelberg), RocheNew England Biolab (Schwalbach/Taberg) and Sigma-Aldrich (Taufkirchen). rva (Heidele(Mannheim), Roth (Karlsruhe), S3.1.2 Kits Vector (USA) x (ABC) kit Avidin-Biotin Comple Pierce (USA) Protein Assay Kit BCAQiagen (Hilden) RNeasy Mini Kit(Freiburg) Biosciences t Amersham ECL Plus Western Blotting Detection KiAmbion (Darmstadt) on Kit nstructincer siRNA CoSileSA) Amaxa-Biosystem (UHMVEL-L Nucleofector Kit Holland) trogen (Groningen,Invi TOPO TA Cloning kit®Macherey-Nagel (Düren) Plasmid Kit NucleospinQiagen (Hilden) ation Kit EndoFree Plasmid Maxi PreparQiagen (Hilden) Gel Extraction Kit(Freiburg) Bioscience t Amersham el Band Purification KiGFX PCR DNA and GMillipore/Amicon (Bedford, USA) it Microcon YM-10 Centrifugal Filter UnMillipore/Amicon (Bedford, USA) Unit Centricon YM-10 Centrifugal Filter BioRad (München) ReadyPrep Sequential Extraction Kit BioRad (München) 3-10 IPG Strips (11,17 cm) pHBioRad (München) Criterion Precast Gel
s Methodnd Material a
Stock Solutions and Buffers 3.1.3
29
epared according to standard proceduresStandard media and stock solutions were prusing deionised water. Solutions were sterilised by autoclaving (25 min/ 121ºC/ 2
ock solutions, filter-sterilised tpared as ss were prebars). Heat sensitive componentm) and added to the medium/buffer after cooling to 50ºC. μ(0.2 Tris-HCl 5x SDS-PAGE Running 125 mM Buffer (TGS Buffer)
SDS Glycin 0.5% 960 mM (w/v)
2D-PAGE Rehydration Buffers: equential Extraction Kit)Reagent 3 (ReadyPrep SUrea Thiourea 5 2 M M
CHAPS SB 3-10 2% 2% (w/v) (w/v)
Tris Biolyte 3-10 40 2% (v/v) mM
TBP(fresh) DTT(fresh) 2 0.5% mM (w/v)
Bromphenolblue trace Urea 8 Reagent 2 (ReadyPrep SM equential Extraction Kit)
SB 3-10 CHAPS 4% 2% (w/v) (w/v)
Tris Biolyte3-10 40 0.2% mM (v/v)
DTT (fresh) TBP (fresh) 2 mM 0.5% (w/v)
Bromphenolblue trace Urea 5 2D-PAGE EquilibrationM Buffer I Urea 5 2D-PAGE EquilibrationM Buffer II
SDS Tris-HCl (pH 8.8) 20% 1.5 M (w/v) SDS Tris-HCl (pH 8.8) 20% 1.5 M (w/v)
Glycerol DTT(fresh) 0.5% 50% (w/v)(w/v) I Glycerol odacetamid(fresh) 2.5% 50% (w/v) (w/v)
Fixer Solution I for gel staining Fixer solution II for gel staining
Methanol Acetic acid 50% 10% (v/v) (v/v) Methanol Acetic acid 5% 7.5% (v/v) (v/v)
s Methodnd Material a
M M
(v/v)
Sodiumcarbonate Developer Solution 6% (v/v) Coomassie BB G-250 Coomassie Staining Solution 0.1% (w/v)
HFormaldehyde 6 mM (NH3PO4)42 SO4 2% 8% (v/v(w/v) )
5x Laemlie Protein Loading Buffer RIPA Lysis Buffer
Glycerol Tris-HCl (pH 6.8) 30% 50 mM (v/v) NaCl 150 Tris-HCl (pH 7.4) 50 mM mM
SDS 4% (w/v) NP-40 1% (v/v)
mM 1 (w/v) EDTA 0.5% Bromphenolblue DTT (fresh) 0.1 M SDS (fresh) 0.1% (w/v)
nhibitor cocktail (fresh) I Protease Triton X-100- SDS Lysis Buffer 10x Blotting Buffer
SDS Triton X-100 1% 1% (v/v) (w/v) Glycin Tris-HCl 1 1.93 M M
10x TTris-HCl 200 BS Buffer, pH 7.6 mM TBS-T Buffer1x TBS (pH 7.6)
NaCl 1.37 M Tween-20 0.1% (v/v)
(w/v) 0.5% SDS rylamide GelsclyaSDS Po Tris; pH 6.80 5 M Running Gel (12%):1.5 M Tris; pH 8.8 Stacking Gel (4%):SDS 4% SDS 4%
Stripping Buffer RNA/DNA-Loading Buffer
βTris-HCl (pH 6.7) -Mercapthoethanol 100 62.5 mM mM XyBromphenlencyanol olblue 0.4% 0.4% (w/v) (w/v)
SDS 2% (w/v) EDTA 1 mM
Glycerin 50% (v/v)
RbCl TFB1 (pH 5.8) 100 mM MOPS TFB2 (pH 6.8 by KOH) 10 mM
MnCl2 Potassium acetate 50 30 mM mM RbCl CaCl2 10 15 mM mM
CaCl2 10 mM Glycerol 15% (v/v)
(v/v) 15% Glycerol 0.1 M PBS (pH 7.4) 0.1 M PB (pH 7.4)
NaNaCl 2HPO 0.2 4 0.2 M M Na KH22POHPO4 4.2H2 0.018 O 0.082 MM
Antibody dilution buffer (PBS/ BSA/ NaN3) 50x TAE Buffer (pH 7.5)
BSA 0.1 M PBS (pH 7.4) 0.2% (w/v) Tris-BaseGlacial acetic acid 2 M 5.7% (v/v)
NaN3 0.1% (w/v) EDTA 50 mM
30
s Methodnd Material a
Glucoseoxidase solution (for 50 ml)DAB 0.063 PB 0.1 M (pH 7.4) M 1 45 ml ml
NHNiSO44 Cl 3.35 M 0.05 M 900 100 µl µl
Glucose (10%) Glucose-oxidase 179 U/ml 900 150 µl µl
l Mixture (for 10 ml)eCollagen GSterile wat10x MEM Medium er 3.95 1 ml ml
L-Glutamin 200 mM Sodiumpyruvate 100 mM 100 µl 100 µl
Collagen 4 Sodium bicarbonate (7.5%) 500 µl ml
40 µl Sodium hydroxyle 0.1 M
Equipment and Applications 3.1.4
: Amaxa., BioSystems NucleofectorCameras: ProgResTMC10plus, Jenoptik
: Phase contrast microscope (Zeiss, Jena); Axiovert 25 (Zeiss, Jena); Microscopes
SM-Lux (Leitz)
: 5415D Centrifuge (Eppendorf) Centrifuges
: Hera Cell 240 (Thermo) Incubator
mocyclerPCR-Ther iometra): T3000 Thermocyler (B
artSpec 3000 (BioRad) m: UV-Spectrometer, SPhotometer
Axima CFR (Shimadzu Biotech) :MALDI-TOF spectrometer
Criterion Dodeca Cell, Protean :electroblotting equipmentGel electrophoresis and
Equilibration Tray (BioRad), Mini protean and ion/IEF Cell, Disposable Rehydrat
Trans-Blot-System (BioRad).
31
s Methodnd Material a
3.1.5 Antibodies
32
Antibody Source Working dilution Supplier
Anti-Alpha1 acid glycoprotein Rabbit 1:200 Dako, Hamburg
GP) (AAnti-Zinc Alpha Glycoprotein(ZAG) Goat 1:200 Santa Cruz, USA
Vimentin Mouse 1:1000 Dako, Hamburg
Table 4: Primary antibodies used for western blot and Immunohistochemistry
Antibody Working dilution
Anti-mouse IgG 1:8000 Sigma,
1:30,000 Sigma, t IgG -rabbiAnti1:30,000 Sigma, t IgG -goaAntiTable 5: Secondary HRP antibodies used for western blot
3.1.6 Cell lines and medium for cultivation of cell lines
Supplier en rchTaufki en rchTaufkien rchTaufki
ons and supplements used in cell culturechased sterile, all media, solutiUnless purwork were sterilised with a 0.2 µm filter, strored at 4°C and prewarmed to 37°C prior
to use. Source cs acteristiCharCell Lines HDMECs CellHumas (HDMECn Dermal Microvas) desrived from dermicular Endots helial PromoCell, Heildelberg
Obtained from Department of RT4 Human Bladder Cancer Cells UrolHamburg-Eppendogy, University Hospitalorf
d cell lines UseTable 6:
Medium Cell LinesSupplemEndothelial entCell GMix, the conrowthcentration Medium s ofMV grow (Promth faoctCeors are; ll): After ad0.4% ECGS/H; ding the
HDMECs Hydrocortison; 1ng/mL ba5% Fetal Calf Serum; 0.1 ng/mL Epidermsic Fibroblast Factor al Growth Factor; 1µg/mL
RT4 peniMcCoys Mecillin/ streptomycin dium (Gibco,USA): 10% Fetal Calf Serum; 1% Glutamine; 1%
ation medium for cell lines CultivTable 7:
strains 3.1.7 Bacterial
and XL1-Blue were used. Ligation α DH5ations, the E. coli strainsFor cloning applicstrain; plasmids requiring digestion with reactions were transformed into XL1-Blue strain. PCR αstriction enzymes were transformed into DH5emethylation sensitive rproducts were cloned using the TOPO TA cloning kit (Invitrogen) with the supplied
Top10F' bacteria.
s Methodnd Material a
33
Strains Genotype Source
DH5α F' endφA1 80hslacdZ∆R17(rM15 k-∆(,mklac+) ZYphA-oA argsupF)U16E44 9ג - deothi-R 1 regyrcAA1 96 Invitrogen (Groningen,
Holland)A1 rel XL1-Blue recA1, endA1, gyrqA96, thi-1, hsdR17, RsupE44, relA1, Stratagene(Heildelberg)
lac {F', proAB, lacIZΔM15, Tn10(Tet)}
F- {lacIq Tn10 (TetR)} mcrA ∆(mrr-hsdRMS-mcrBC) Invitrogen (Groningen,
Top10F' φgal80U lacgaZl∆K rpM15 s∆L (StrlacR) X74 endrecA1A1 nuparaG D139 ∆(ara, leu)7697 Holland)
Table 8: Bacterial Strains. Names, genotypes and suppliers of the bacterial strains used.
3.1.8 Primers
leotides used for PCR and contructOligonucion of siRNA were purchased from MWG Biotech.
NamPrimer e Line Sequence ( 5→3 ) NumGenbaber nk Tm (°C) PCR cnumbers ycle
ORM ForReversware d ACGTGCCTTTAGCTGTTCCAAACACCTGGTCTCCAGTA AGAAGNM_00067 56.5 62.1 35
GAPDH RevForwarderse TTTCTTACTCCTTGATGACATCAAGAAGGTGG 55.3 TGGAGGCC NM_00206457,3 35
Table 9: Oligonucleotides used for in PCR
Primer Name Line Sequence ( 5→3 )
siRNA ORM sense AAGACTTATTGTACTCCTCGTCCTGTCTC
GTCCCTGTCTC Aense AAACGAGGAGTACAATAantisTS231 siRNA ORM sense AATGAAGTAAAAGAAGGTTGCCCTGTCTC 63.9
TS266 antisense AAGCAACCTTCTTTTACTTCACCTGTCTC
siRNA sense AATCGAAGTATTCCGCGTACGCCTGTCTC 55,3
Luciferase antisense AACGTACGCGGAATACTTCGACCTGTCTC 55,3
Table 10: Oligonucleotides used for in siRNA construction
) (°CTm65.3 65.3 63.9
s Methodnd Material a
34
samples 3.1.9 Urine h bladder cancer of different stages witSpontaneous urine samples of 45 patientss were lthy volunteer and heaGIII), patients on follow-up(pTa, pT1, pT2-3, GI, GII, collected and after centrifugation at 5000 rpm for 5 min stored at 20°C (for a longer 80°C) until their use. time than 2 months at
(n=45) ts cer Patienr CanBladdePT1(n=8) PT2-3(n=8)
Age, 60-80 Age, 60-80
Female, n=2 Female, n=1
Male, n=7 Male, n=6 GI, n=GII, n=1 3 GIII, n=8
4GIII, n=
(n=45) ts cer Patienr CanBladdepTa (n=12) PT1(n=8) PT2-3(n=8)
Age, 60-80 Age, 60-80 Age, 60-80
Female, n=7 Female, n=2 Female, n=1
Male, n=7 Male, n=6 Male, n=5 GI, n=4 GI, n=1 GIII, n=8
GII, n=8 GII, n=GIII, n=4 3
Patients on follow-up (n=10) Healthy Volunteers (n=7)
0 30-50 Age 60-8n=3 Female n=4 n=7 Male n=6 Table 11: Urine samples used in 2-DE analyses and western blotting
Tissue samples 3.1.10 Tissue samples were provided from the Department of Pathology of the University
esamples from human bladder werHospital Hamburg-Eppendorf. Normal tissue d tumor cancer, anobtained by biopsy or by cystoprostatectomy because of prostate undergone surgical om patients who hadtissues of bladder cancer were obtained frment ofage was made by the Departtherapy. The determination of tumor st classification. Theealth Organization) Pathology according to the WHO (World Htissue blocks were fixed in formaldehyde and embedded in paraffin and subsequently
sectioned for use in immunohistochemistry. 3.2 Methods es syanal3.2.1 Protein Determination of total protein 3.2.1.1 assay kit which ismined by BCA protein The concentration of proteins was deteration of lorimetric detection and quantithoninic acid (BCA) for the cobased on bicinc
s Methodnd Material a
35
total protein. The complex giving colour exhibits a strong absorbance at 562 nm. BSA
andard. 2 ml of the diluted and used as st serially o(Bovine Serum Albumin) was als standard tubes. After aadded to each sample and to mix was BCA working reagent 37°C for 30 min. For each dilution, short vortex the tubes were incubated aterage was taken for the ate and the avd in duplicmeasurements were performeotein concentration. calculation of the pr 3.2.1.2 Two dimensional polyacrylamide gel electrophoresis (2D-PAGE)
protein mixtures due to differences in Two dimensional gelelectrophoresis separates cular edimension and subsequently by their molrst their isoelectric point (pI), in the fiesented in figure 16. ension reprweight (MW) in the second dim
two dimenFigure 16sions ho: Schematicrizontallayl repres by iso-eleentatioctric poin of twnto (pI) a dimend vnsional sepertically byaration. moleculaProtr weins aeight (Mre sepaW). rated in
urine samples for 2-DE of 3.2.1.3 Preparation l protein was applied containing 200 µg totaAfter protein determination, urine volume to remove molecules smaller than 10 kDaonto a Centrifugal Filter Column (Amicon) such as urea, electrolytes, salts and to increase the protein concentration of the urine
was centrifugated at speed mn containing urine volumehe centrifugal colusamples. T at 4°C until complete urine volumeseased from 3000 rpm to 6000 rpm rlinearly incwere filtered through twith 50 µl distilled water he filter membrane. Remained protand centrifugated at 6000 rpm foein molecules were washed r 30 min. Filtered urine
samples were diluted in rehyration buffer 2 (3.1.3). After fresh adding of TBP, DTTaotein lysand trace of bromophenolblue, pr tes were centrifugated at 15,000 rpm for10 min at 4°C to remove insoluble particles. Supernatant containing urinary proteins
was used for sample loading step of 2-DE procedure.
Methodnd Material as
nge IPG Strip Length / pH Ra11 cm, pH 3-10, nonlinear (NL)
) r (L 3-10, LineaH17 cm, p
otein amount Loaded pr µl volume) 00200 µg (in 2 µl volume) 00300 µg (in 3
36
Table 12: IPG strips used for 2-DE analyses
Additionallysamples to precipitate all proteins present , TCA (tricholoroaceticacid) precipitation was pein urine. Shortly, an equal volumerformed on the urine of 20%
TCA were added to the urine samples centrifugation at 13,000 rpm for 10 min at 4°C, the sand incubated on icupernatant was remoe for 30 min. After ved and
effective for 2-DE analysis; it led to vertprotein pellets were diluted in rehydration buffer2-3 (3.1.3). But,ical streaks due to remained T thiCs method was notA particles
protein present in urine, To precipitate a specific ).(data was not shown r, the 2-DE results ofed on urine samples. Howeveimmunoprecipitation was perform informative due to remained IgGimmunoprecipitated samples were notwas not shown). Therefore ata antibody (dom secondary (Immunglobulin G) coming frheir use in 2-DE process. r all urine samples prior tultracentrifugation was used fo dration y3.2.1.4 Reh The PROTEAN IEF cell with integrated power supply was used for rehydration and
IEF protocols. Diluted protein samples were loaded onto IPG strips in IEF focusing
strip gels were placed side t dimensional separation. IPG tray for rehydration and firsrehydration solution. Fdown in the channel of a focusior urine samples mostng or rehydration tray thatly 11 cm nonlinear IPG strips with 3-10 contains the sample in
pH range were used and for a large scale used (Tab. 12). Strips were covered with 2 mlof gel the strips wit of mineral oil to prh 17 cm length wereevent evaporation
which causwith sample in a focusing tray were activees the urea to precipitate as it becomes more rehydrated by running concentrated. IPG strips of IEF cell under
low voltage as 50 V overnight. ion: Isoelectric focusing (IEF) First dimensional separat3.2.1.5 After the strips have rehydrated the endswicks were inserted between the strip and the of each strip was lifted and wet electrode electrodes. Strips were covered with
mineral oil before starting the focusing run to prevent evaporation and carbon dioxide
s Methodnd Material a
37
increased fabsorpsion during focrusing. Isoelectric foom 250 to 5000 V during the first 5 h, followed by 8000 V for a total of cusing was performed at voltage linearly
70000 Vh/h (Tab. 13). C ûTemperature 20Current max. 0.05 mA per strip strip, 300 µl for 17 cm strip Sample volume 200 µl for 11 cm
Time / Volt-hour d30 min. rapi w60 min. slo w60 min. slo w60 min. slo w60 min. slo w60 min. slo w60 min. slo w4 h sloear 70,000 Vh lin3 h linear
Voltage Step Time / Volt-hour d30 min. rapi150 V 1 3 2 1000 V 300 V 60 min. slo60 min. sloww
w60 min. slo1500 V 4 w60 min. slo2000 V 5 w60 min. slo3000 V 6 8 7 8000 V 5000 V 4 h slo60 min. slow w
ear 70,000 Vh lin8000 V 9 3 h linear 500 V 10 Table 13: Applied voltage steps for IEF
3.2.1.6 Equilibration lfhydryl groups of ates the resultant suThis process reduces disulfide bonds and alkylthe cysteine residues. Rehydration/equilibration trays sized for each size strip were
used for equilibration. Each strip was moved to equilibration tray and the channels
re first incubated in DTT e. Strips w with the equilibration bufferswere filledoups for 15 min at RT, than the hat reduces sulphydryl grequilibration buffer-I (3.1.3) tchannels were refilled with iodacetamide equilibration buffer-II (3.1.3) which alkylates
sulfhdryl groups and incubated for 15 min at RT. For the strips with 11 cm length, 3
rips with 17 cm length 5 ml of each ml of each equilibration buffer and for the stbration, IPG strips were removed and equilibration buffer were used. After equilidimension gel. embed into the prepared second- mensional separation Second di3.2.1.7 rried outaoelectric focused proteins was cThe second dimension electrophoresis of isfrom BioRad (Criterion Precast Gel crylamide gels obtained aon pre-prepared poly were placed on top of the The equilibrated IPG gel strips 10% Tris-HCl, 1.0 mm).e prepared in SDS-ed agaroslaid with 2 ml 0.5% meltpolyacrylamide gel and over
s Methodnd Material a
38
s added to the agarosel amount of Bromophenolblue waPAGE running buffer. A smaloverlay to track the ion front during the run. The SDS gel running was performed with
BioRad Dodeca Cell 150 V for 20 min. a voltage setting of 120 V for 45 min and r proteomics analyses. To r run was mostly used fowhich accommodate 12 gels peestimate the molecular mass of each spot, marker proteins (SigmaMarkerTM, wide
molecular weight) were placed on a filter strip left to the strip on the gel.
ant Blue (BioRad) and/or Coomassie BrillerPolyacrylamide gels were stained with silvaccording to the procedures described previously (Merril et al., 1981). detection 3.2.1.8 Protein To visualize proteins after separation in 2-DE gels it is necessary to use an adequate
e of the in characteristics and abundanc variation staining method. The enormousstaining technique. The most important dual proteins put high demands on the iindivgh reproducibility and dynamic range, hinearrequirements are high sensitivity, high li s of protein stainingtrometry. Major developmentcompatibility with mass specs. Since silver staining is the most he last few yearmethods have occurred during tly e and the costs for reagents are relativeve detection techniquisensitive non-radioactlow when compared to fluorescence techniques, it is still widely used. There are
staining protocol of the original silverpresently more than 100 different modifications In this study silver staining and .that was introduced by (Merril et al., 1981)used to visualize the protein spots. coomassie blue staining methods were staining 3.2.1.9 Silver s were taken out and incubated first with ion the gelAfter second dimensional separat 10% acetic acid for 30 min methanol andfixer solution-I (3.1.3) containing 50% following second incubation with fixer solution-II (3.1.3) containing 5% methanol and
fixation, the gels were washed twice with 7.5% acetic acid for 10 min at RT. After distilled water for 5 min at RT on a shaker. Gels were then incubated with 0.002%
led water for 1 min. Forn and washed twice with distilsodiumthiosulphate for 1 mi eed. The gels werate solution was usstaining, fresh prepared 0.2% silver nitrat RT. After a washing step as for 30 min trate solution in darkincubated with silver nideveloper solution (3.1.3) containing 6% incubated with fresh e2x1 min, the gels werr 5-20 min until the protein spots de fosodium carbonate and 6 mM formaldehy
s Methodnd Material a
39
acid particles were added to stop the tric ied. A few solid cbecame visualizage analysis. m gels were scanned for the idevelopment of the gels. After washing, staining e 3.2.1.10 Coomassi For coomassie staining, the gels were incubated with fixer solution-I (3.1.3) for 30
with distilled water for 5 min, gels weree min at RT on a shaker. After washing twicstained by incubation using coomassie solution (3.1.3) for overnight. Stained gels
ed water to remove remained coomassie were washed several times with distillcrystals and excess dye from gel. Image and data analysis 3.2.1.11 d using PDQuest software programmeThe resulting 2-DE images were analyse(BioRad). Gel analysis includes spot detection, spot quantitation, gel comparison,
d the protein spotsee automatically detectand statistical analysis. Before the softwar corrected and background was subtracted.of a 2-DE gel, the raw image data were and bioinformatics ryspectromet3.2.1.12 Mass Some of the visible protein spots on the stained gels have been identified by mass
titute of Hamburg University. Coomassie -Pette-Insh in Heinricspectrometric analysisBrilliant Blue-stained protein spots (Coomassie G250) were excised, cut in 1 mm3
r and destained with 50% v/v acetonitrile pieces, washed twice with distilled wate15 min, shrunk by dehydration in e for 2x(MeCN) in 25 mM ammonium bicarbonat uum centrifuge for 10 min. The gel piecesacetonitrile for 15 min, and dried in a vacwere reswollen in 10-20 µl of 25 mM ammonium bicarbonate, containing 200-400 ng
of bovine trypsin (proteomics sequencing grade; Sigma-Aldrich) at 4°C. After 60 min,
sary to keep the gel es added if nece were10-20 µl of 25 mM ammonium bicarbonatovernight). The peptides were extracted during tryptic cleavage (37ºC, pieces wetTFA, 20% MeCN/ 0.1% TFA 1% TFA, 10% MeCN/0.1% r, 0.esequentially with watThe separated liquids were combined h. and 40% MeCN/0, 1% TFA for 30 min eacredissolved in 4 µl matrix solution uum. The peptides were and dried under vaccontaining 10 mg dihydroxybenzoic acid, 5 mg diammonium hydrogen citrate, 0,1%
TFA, and 0,05% phosphoric acid in 50% MeCN. For MS-analysis 1 µl were spotted
MS measurements were performed on an onto the target plate and air-dried. MALDI-
s Methodnd Material a
40
in positive mode with ometer (Shimadzu Biotech)R MALDI-TOF spectrAxima CFized for 2500 mu. Dependent on spectra reflectron, delayed extraction was optimquality 200-400 lasspectra processing was done with the Axer shots were accumulatima CFR soed. Control of the ftware V2.2.1. Mass spectraspectrometer and
Angiotensine III andagments of trypsin, were calibrated using known autolytic fracetylated Insulin B chain as internal standards. The peptide masses were measured
as monoisotopic masses.
NaNattiivvee U Urrineine
CeCeFFiltiltntrifugationntrifugationrarattiion on aandnd
SeSepapararationtion of u of urrinainaryrypprrootteeinsins
2-2-DDEE
DeDetteeccttioionnoof prf proteinoteinssppootsts
((CCoooomassmassieie/ Sil/ Silvveerrststaiainniinngg))
IdentificaIdentificationtionof pof prroteinsoteins
MMaassssSpSpeectctrroommetetricricananaalylysesses
urineFigure 17: E samplexs. Native uperimental florinwe containi diagrang m. a certain amThis diagram shoount total wprots the experimeein was centntal steprifugated ans of analysed filtrated. s of
silver After firstand/o anr d secoomcondassie dime blue nsional stainingse prparation uotocols to visuing 2-alizeDE techni protein quespot, the 2s. The-DE sp gels ots of iwere nterestainst weed ure sincutg
out from gel and identified by mass spectrometric analyses.
using the programng the NCBI database Proteins were identified by searchiMASCOT (Matrix Science; http://www.matrixscience.com/serach_form_select.html).
ons acetamide on ifollows: the modificateters for the search were as The param
Material as Methodnd
41
re considered as a partial modification cysteine residues and methionine oxidation we .and three was used as the maximum missed tryptic cleavage sites 3.2.1.13 SDS-polyacrylamide gelelectrophoresis
li, 1970) under denaturing conditions (in One dimensional gel electrophoresis (Laemme of their ns on the basr separation of the proteiopresence of SDS) was used fs prepared as a separating gel (sometimes molecular size. The polyacrylamide gel wa gel) topped by a stacking gel and secured in ancalled resolving or running r sample proteins are solubilised by atus from BioRad. Afteesis apparelectrophorlution is applied to a gel an aliquot of the protein soe of SDS,boiling in the presenc y of theed electrophoretically. The mobilitl proteins are separatline, and the individua heir molecular mass. SDS isonal to the logarithm of t inversely proportiproteins is deins, to dissociate protein complexes anemployed to effect denaturation of the proties proportional to negative charge densitto impart upon the polypeptide chains net existing as DTT was used to reduce anythe length of the molecule. A reducing agent oteins was detected usingmensional separation the prdisulphide bond. After one di immunoblotting for and byable to detect all proteinsstaining methods which is ific protein. detection of a spec blotting 3.2.1.14 Western ylamide gels onto retentive membranes wasElectroblotting of proteins from polyacron of accumulated proteins using a ectiperformed for immunoblotting following det electrophoreticallyed by SDS-PAGE and specific antibody. Proteins were separattransferred from the polyacrylamide gel to a nitrocellulose membrane at 350 mA
constant current for 1 hour. lamide gel which was prepared according SDS-AcryProteins were separated on 12% om BioRad. For preparation of gels 30%ruction frtto the Mini Protean Manual Insused. 25 µl of different protein samples acrylamide solution obtained from Serva was of sample loading buffern were denaturated by adding containing 50 µg total protei95ºC for 5 min. Protein samples were (3.1.3) including DTT and following heating at lt for 90 min. Prestainedrunning was performed at 120 voo the gel and loaded ont size of separated ogen was used to estimate molecularprotein marker from invitr om SDS gel to nitrocellulose membrane,proteins. For transferring of proteins frrding to oained from BioRad was used accequipment as Mini Trans-Blot-System obt
s Methodnd Material a
42
blotting nitrocellulose membranesthe manufacturers recommendations. For (Schleicher & Schuell, BioScience) were cut in size as 7x9 cm and wetted by transfer
pre-wetted with 1x and filter papers werebuffer shortly before placing. The fiber padsTransfer buffer. Gel containing proteins and nitrocellulose membranes were allowed
ber pad, a filter paper, gel, ing in order as a fil placto contact by a sandwich mode rom cathode plate to anodea filter paper, a fiber pad (fnitrocellulose membrane, beside pack was puthe chamber and a colded in tplate). The cassettes were placophoretically transfer was er buffer (3.1.3). Electrfilled with pre-chilled 1xTransf andellulose, transfer onto nitroc on a stir plate. After the performed 1 h at 350 mA currentthe blots were taken out and washed with distilled water. To prevent unspecific
bindings, blots were incubated with blocking buffer containing 5% non-fat milk
day the blots were incubated r (3.1.3) overnight at 4ºC. NextBS-T buffesolution in 1xT min washing RT. After 2x10BS-T solution for 1 h at in 1xTwith the primary antibodyubated for 1 h at RT buffer the membranes were incBS-Tin 1xTwith the horseradish blocking buffer. Finally, the membranes e-coupled secondary antibody in peroxidis BS-T buffer. The antigen-antibody1xTwere washed 2x10 min and 2x20 min with complex was detected by enhanced chemiluminescence using ECL reagents
(Amersham-Pharmacia) and visualized by auroradiography. The X-ray films were
the bands using thec quantification of digitalized for subsequent densitometriTMSA). (Seattle, Umorphometric program Optimas
methods 3.2.2 Molecularbiological
3.2.2.1 Cultivation and storage of E. coli
)-broth or on LB-agar plates at 37°C. ForBacteria were cultivated in Luria-Bertani (LBlied with 50 mg/l re suppteria, broth and plates weselection of transformed bacampicillin. hen stored at 4°C. ne month woximately oFreshly plated bacteria were viable for apprFor long-term storage, glycerol stocks were prepared by mixing 500 µl of an
l. The stocks were stored at e with 500 µl of 15% sterile glyceroovernight liquid cultur80°C.
s Methodnd Material a
43
Preparation of competent cells 3.2.2.2 DH5-α bacterial were obtained from Invitrogen and competent E. coli cell stocks were
cellsαiously (Sambrook, 1989) 200 µl of DH5-prepared using protocol described prevedium without antibiotic. After m LB-e and were mixed with 10 mlwere thawed on icat 225-250 rpm, 200 µl of mixture were overnight incubation at 37°C with shaking plated on an agar plate without antibiotic and incubated overnight at 37°C. A single
After overnight .hcolony was picked and 10 ml LB medium was inoculated wited to 100 ml pre-warmed LB overnight culture was addincubation at 37°C, 1 ml ofed at 37°C until an and shak incubated The culture wasmedium without antibiotic.OD600 OF 0.5 was reached (approximately 90-120 min). The culture was cooled on
ound-bottom centrifuge tube. The cells were ice for 5 min and transferred to a sterile rm, 4°C). After supernatant min, 1500 rp5 low speed (collected by centrifugation atwas removed, the cells were resuspended in cold TFB1 buffer (3.1.3) (30 ml for a 100
ml culture) and suspension was incubated on ice for 90 min., followed centrifugation
were resuspended in 4 ml ice-cold TFB2 for 5 min at 1500 rpm at 4°C. The cells ed andile microcentrifuge tubes were preparbuffer (3.1.3). Aliquots of 100 µl in ster were stored at -80°C. ent cellsfrozen in liquid nitrogen. The compet transformation 3.2.2.3 Bacterial plication was performed using needed for cloning apBacterial transformation which is competent cells were thawed on and XL1-Blue (Tab. 8). 50 µl ofα strains DH5E. coli of cellseaction was added to one aliquotion rice. 1-5 ng purified DNA or 1-5 µl of ligat e incubated on iceng cells and DNA werand mixed by pipetting. The tubes containi bath for 45 seconds, followed 42°C waterfor 30 min, and then were heat-pulsed in a h to eac 500 µl pre-warmed LB mediumincubation on ice for 2 min. After addingg at 225-250 rpm. for 1 hour with shakinsample, the tubes were incubated at 37°C ead on LB agar plateson mixture were sprAfter incubation 50-500 µl of transformatire incubated overnight at 37°C. Single containing 50 mg/l ampicillin. The plates we medium containing ampicillin at 37°C colonies were picked and incubated with LB using either Plasmid Maxi Kitisolatedmid DNA was overnight with shaking. The plasion, EndoFree Plasmid Maxi Kit (Qiagen). at(Qiagen) or for further transfection applicl stock at 80°C. oure with sterile glycerxtTransformed bacteria were stored as 1:1 mi
s Methodnd Material a
44
Purification of DNA from solution and gel bands 3.2.2.4 ct and/or agarose gel bands, ion of DNA from PCR produicatFor isolation and purif ciences) was used. Kit (Amersham BiosGFX PCR DNA and Gel Band Purification ates proteins, dissolves agarose, and that denaturThis kit uses a chaotropic agentilobase-pairs) pairs to 48 kof double-stranded DNA (100 base-promotes the binding ovided DNA was performed using the protocol pr fiber matrix. Purification of to a glassband purification, denatured and for gel by GFX purification kit. First DNA was re passed through the GFX column ed. DNA samples weagarose was dissolvprovided with kit to capture the DNA onto the glass fiber matrix. Then Matrix-bound
remove salts and other contaminants. h an ethanolic buffer to DNA was washed witth buffer (TE pH 8.0, a low ionic streng in nPurified DNA was eluted from GFX colum10 mM Tris-HCl pH 8.0, or water). ion of plasmid-DNA 3.2.2.5 Minipreparat For small-scale preparation of plasmid DNA, Nucleospin® Plasmid Kit (Macherey-
protocol fromwas performed according to used. Isolation of plasmid DNA Nagel) wasNucleospin® Plasmid kit. The pelleted bacteria were resuspended and plasmid DNA
was liberated from the E.coli host cells by SDS/alkaline lysis. SDS precipitate and
onto a upernatant was loadeda centrifugation step, the sed by cell debris were pelletNucleospin® Plasmid column. Pure plasmid DNA was finally eluted under low ionic
-Cl, pH 8.5) which provided slightly alkaline buffer (5 mM Trish strength conditions witmeasured by UV-spectrophotometer. with kit. Concentration of DNA was ion of plasmid-DNA 3.2.2.6 Maxipreparat mid Maxi Kit (Qiagen) was in high culture volume, PlasFor isolation of plasmid DNA used. The purification protocol of kit is based on a modified alkaline lysis procedure,
n under ito Qiagen Anion-Exchange Resfollowed by binding of plasmid DNA ning Ampicillin LB-Medium containd pH conditions. 100 ml of appropriate low-salt agar plate and then from a selective angle colony iwas inoculated by a picked sC on a rotation shaker. Plasmid DNA was over night incubation at 37°cultured byted in 200 µl TE buffer or in ol from Plasmid Maxi Kit and diluisolated using the protoc determined by UV entration wasDNA concbuffer containing 10 mM Tris-Cl, pH 8.5. on an agarose gel. Plasmid DNA which isspectrophotometry and quantitative analys
s Methodnd Material a
45
was further transfected into the cells was isolated using EndoFree Plasmid Maxi Kit
(Qiagen) due to endotoxin effects efficiency of gene transfer. Restriction digest of DNA 3.2.2.7 were obtained from NEB. Restriction Restriction enzymes and appropriate buffersDNA were performed according to the striction digest of edigest and double r ction digestion of DNA isgeneral protocol for restrimanufacturers' protocol. The shown in table 14. Components Concentration / Volume
Restriction DNA Enzym e 3 1-3 U/ µgµ g DNA
10x Distilled Buffer wat er 2 ad µl 20 µl
Table 14: Composition of restriction digests reaction
After incubation of the reaction mixture at 37°C for 1 h per 5U of restriction enzyme,
incubation at 75°C for 20 min. the reaction was inactivated by l electrophoresis eAgarose g3.2.2.8 separated on 1-2% allyDNA were electrophoreticPCR fragments and/or digested AE buffer containing 0.1 g/ml ethidiumepared in T(w/v) analytical agarose gels prbromide. 1x TAE buffer (3.1.3) was used as running buffer. DNA samples were mixed with 6x
gel. 500 ng of DNA molecular markerloading buffer (3.1.3) and loaded onto the egrity. The DNA was and intsample size(Invitrogen) were used for evaluation of at 302 nm and documented by a gelvisualised on an UV transilluminatorre cut out from agarose as). Interested DNA bands wedocumentation instrument (Int ed from cut gel using GFX PCR DNA kit oring a sterile scalpel. DNA was isolatgel usion and purification, DNA on Kit (Qiagen). After isolatin some cases Gel Extractisamples were electrophoretically separated on agarose gel for controlling. After that,
DNA concentration was measured by UV-spectrophotometry. ing of DNA cSequen3.2.2.9 he sequence data d by MWG Biotech. T performeAll sequencing of plasmids wasl (Basic Locapared using sequence comparing programme, BLASTwere com
s Methodnd Material a
46
Laboratory)-Databank n Molecular Biology Alignment Search Tool) in EMBL (Europea). http://www.ncbi.nlm.nih/gov/Blast/( ation rDetermination of DNA concent3.2.2.10 SmartSpec3000 UV-Spectrometer (BioRad)entration For determination of DNA conc o1:100 with distilled water, which used alsles were diluted as was used. DNA samps were placed into micro-quartz cuvetes. as blank and 100 µl of diluted DNA sample and DNA using UV-light by 260 nmre was measured for double stAbsorbancwavelength. DNA3.1(-)/ORM expression vector Construction of pc3.2.2.11 s via nucleofection technology, the ECFor overexpresssion of ORM in HDMtructed. For this, the full-length cDNA DNA3.1(-)/ORM was conson vector pciexpressencoding ORM was cloned into the XhoI and HindIII restriction sites of the
Hygro(-) (Clontech). Firstly the cDNA encoding for ORM pression vector pcDNA3.1/ex using gene specific human granulocytes gene was amplified by PCR from cDNA of® vector. he PCR product was ligated into the 2.1-TOPOprimers. Subsequently t merase chain reaction (PCR) yPol3.2.2.12 Polymerase chain reaction (PCR), a procedure for rapid in vitro enzymatic
, was used for the amplification of cDNA amplification of a specific segment of DNApartment eom the Dobtained as a gift frencoding ORM. cDNA of granulocytes which of Clinical Chemistry of University Hospital Hamburg-Eppendorf, was used directly as
ytes. e ORM genes present in granuloca template for PCR to amplificat Components Concentration / Volume
dNTP DNA 1 10 µl mM
M Primer gCl2 20 50 mM pmol each
Taq DNA polymerase 1 U
Distilled 10xPCR watBuffer er 1x ad 25 µl
Table 15: Composition of PCR reaction
s Methodnd Material a
sec 45 sec 45 min 1 10 min
47
Step Temperature [°C] Time
Initial denaturation 95 10 min
35 Cycles:
Denaturation 95 45 sec
Primer annealing 60 45 sec
Elongation 72 1 min
Final elongation 72 10 min
for ORMns: PCR conditioTable 16 ubes (Biozym), using a PCR ml PCR tThe reactions were performed in 0.2 imers annealing to the vector backbone thermocycler (MWG Biotech). Specific prwere used for PCR reaction (Tab. 15). GAPDH was used as control reaction.
The PCR products scribed in table 16. Thermocycler conditions were used as deied using GFX PCR DNA and agarose gel (3.2.2.8) and purif sed on a 1%were analyham Biosciences) (3.2.2.4). Gel Band Purification Kit (Amers 3.2.2.13 Ligation of PCR product into pCR® 2.1-TOPO® Vector
purified using GFX PCR DNA and Gel The PCR product coding for ORM gene was Taqences). For direct insertion of Band Purification Kit (Amersham Biosci plasmid vector, TOPO TA cloning Kite-amplified PCR product into a polymeras(Invitrogen) was used. The pCR® 2.1-TOPO® vector provided with kit was used as
) residueshanging 3´-deoxythymidine (Tlinearized with single 3´-thymidine (T) overto ligate with the vector. rts ws PCR insellowhich a o vector was performed according tInsertion of PCR product into the TOPO PCR product and TOPO vector were t.manufacturers protocol provided with kimixed and incubated at RT for 5 min. TOPO cloning reaction was transformed into
and single colonies were selected to isolate DNA. chemically competent cellsThe selected single colonies were cultured in LB-medium overnight and DNA was
® DNA concentration Plasmid kit. Afterisolated using protocol from Nucleospin restriction site and XhoI and HindIIImeasurement, DNA samples were digested at elected ones was s One of positive cletically separated on agarose gel.electrophorinto the expression vector as next step. O/ORMTOPand used for subcloning of
s Methodnd Material a
Subcloning into pcDNA3.1(-) 3.2.2.14
48
ansient rned for high-level stable and tThe pcDNA3.1(-) is 5.4 kb vector desig
expression in mammalian cells. The pcDNA3.1(-) expression vector and TOPO/ORM
plasmid vector were digested at HindIII and XhoI restriction site and after extraction
om agaroof digested pcDNA3.1(-) and ORM insert frse gel ligation reaction were
ernight at 16 °C tion was incubated oveac Ligation rperformed using the T4-Ligase.
and directly transformed into E. coli strains DH5α or XL1. Single colonies on LB-agar
plates were selected and analysed for the presence of insert by HindIII and XhoI
ect restriction pattern wasrh has correstriction digestion. A transformant whic
M gene was cloned in confirm that ORselected and sequenced by MWG Biotech to
the proper orientation.
truction scon3.2.2.15 siRNA
mcing via sFor ORM gene silenall interfering RNA (siRNA) th
Kit (Ambion) was used. Construction
Figure 18: Mechanism of gene silencing via siRNA
e Silencer
iRNA s™
s Methodnd Material a
49
post-ele-stranded RNAs, which inducThe siRNAs are 21-23 nucleotides doubng the siRNAs into the cells, they were lencing. After introduciitranscriptional gene sg complex (RISC). ed silencina RNA-inducassembled with protein components into vates the RISC, which in turn binds to An ATP-generated unwinding of the siRNA acties the mRNA. This interaction and cleavthe homologous transcripts by base pairing sults in gene silencing (Fig. 18).adation of mRNA repecific degrsequence scted from the cDNA of the siRNA sequences are seleFollowing target regions for hir et al. (Elbashir et al., 2001). Elbasaccording to the guidelines described by ORMferase gene was chosen as a control for Target sequence (cDNA) from the firefly luciORM silencing studies.
methods 3.2.3 Cellbiological
General cell culture work was performed under sterile conditions under a laminar flow
hood using disposable plastic ware. cells of 3.2.3.1 Culturing Cell lines (Tab. 6) were placed in polystyrene culture flasks 25 cm2 (T25) and 75 cm2
ophobic vent caps and maintained in a(T75) (Falcon) provided with 0.2 µm hydrCell 240) at 37°C. All culture atmosphere in an incubator (Hera d 5% COhumidifie2 ed by heating at Glass wares were steriliz filtration.media (Tab. 7) were sterilized bylaved at 120°C and 1.2 bar for 20 min. 180°C for 8 h, and plastic wares were autocluence of 70-90%. Medium was removed lls were cultured until a confeAdherent ccubated with trypsin for one lls were ining, the ceand after two times PBS washtion of double used trypsin addihe reaction was stopped byminute at 37°C and tn was harvested by centrifugation (1200 volume of culture medium. Cell suspensio were washed bywas removed and cellsrpm /5 min/ RT). The supernatant equent centrifugation. For subculturing cells wereubsresuspension in PBS with sfold with fresh medium. diluted approximately 1:5 ing of cells Freezing and thaw3.2.3.2 500,000 viable human dermal microvascular endothelial cells (HDMECs) (Tab. 6),
freezing medium, after eserved in 1ml serum-free derived from dermis and cryoprcultured and passaged until re PromoCell. HDMECs wethawing were obtained from
Material as Methodnd
50
HDMECs were not used after passage passage four, and then used for experiments.ents thawed and viable ozen and stored. For all experim not froeight and were alsfrom PromoCell. ed HDMECs were shipp6 cells/ml, washed with ed at a concentration of 0,5x10RT4 cells (Tab. 6) were harvestPBS and resuspended in 1 ml ice cold cryo-conservation medium. The cryovials
placed in a -80°C freezer and transferred (Nunc) with 1 ml cell suspension each were to liquid nitrogen for long-time storage. bath, transferred to 20 ml r eed rapidly in a 37°C watCryopreserved cells were thawSupernatant was discarded rifuged (1200 rpm/ 5 min). prewarmed medium and centand cells were seeded at high density in culture flasks (25 cm2 and 75 cm2,
respectively). Determination of cell number 3.2.3.3 For determination of cell number 10 µl of the cell suspension was mixed with 90 µl
trypan blue solutions. After 3 minutes of staining, only dead cells turn blue, while
were counted in a Neubauer chamber. The tained. The latter living cells remain unsnumbers of cells were calculated according to the equation below. Number of cells/ml = number of cells over a large square x dilution factor x 104
extraction 3.2.3.4 Cell Cell lysates of HDMECs and RT4 cells were prepared by chemical lysis. The cells in
culture were washed two times with cold-PBS and after third adding of cold-PBS the
cells were scraped using a scraper on ice. Cell suspensions were collected into a
upernatant was removed andr 6 min. Sfalcon tube and centrifuged at 1200 rpm focell pellets were washed three times more with ice-cold PBS. RT4 cell pellets were
s pellets were lysed in Triton X-100-SDS PA-Lysis buffer (3.1.3). HDMEClysed in RIspensions uadding lysis buffer onto the cell pellets, cell sLysis buffer (3.1.3). After ing. Cell lysates were incubated on ice were mixed first by pipetting followed vortexmin. After that, the cell ed at RT for 10 rtex, incubatfor 30 min and after a short vo ant were collectedmin at 4ºC. Supernatlysates were centrifuged at 15,000 rpm for 15 and stored at -80ºC until further analyses. Protein concentration was determined
using BCA protein assay kit (Pierce).
s Methodnd Material a
51
HDMECs via Nucleofector of 3.2.3.5 Transfection the Nucleofector technology (Amaxa ansfection was performed using rHDMECs t a non-viral alternative to primary cell Biosystems). Nucleofector technology offersal parameters and cell-ination of electrictransfection, which bases on a unique combtype specific solutions. HDMECs were transfected using HMVEC-L Amaxa
Nucleofection Kit along with program S-05 on the Nucleofector device according to
cy, the neaching 70% conflueanufacturer (Amaxa). After rthe optimized protocol of mtion. The cells after passage ten were cells were passaged 3-4 days before nucleofecfer efficiencies and gene transad to lowertion because it may lenot used for nucleofecy damage the cells. For transfection, amtrypsin treatment is more difficult and nization and count to determine cell density.cultivated cells were harvested by trypsi5x105-1x106 cells per nucleofection were centrifuged at 1200 rpm for 10 min and
resuspended in 100 µl Nucleofector solution and mixed with 2 µg DNA or siRNA. Cell
suspension was transferred into an Amaxa certified cuvette and inserted into the in the Nucleofector, 500 µl of full cuvette holder. After running the adequate program could be transferred into a preparedmedium was added to the cells and the sampleculture flask. in HDMECs g of ORMsilencingene 3.2.3.6 Overexpression/ To overexpress ORM in HDMECs the cells were transfected with the expression
bp full-length cDNA of ORM was cloned vector pcDNA3.1(-) ORM, in which the 770into the Xhol and HindIII restriction sites of the expression vector pcDNA3.1/Hygro(-)
(-) was used as a negative control. (Clontech). Empty vector pcDNA3.1.2.15). The siRNA-ed siRNA-ORM (3.2ing construct silenced usORM gene was e 2 µg of plasmid DNA or siRNA were control.luciferase was used as negativ.3.5). Transfected ector technology (3.2transfected into the HDMECs using nucleofHDMECs were cultured for 1-2 days and used for further studies. After 2 days llected and stored at -20°C and for later were cosupernatant of transfected cellswere later used for tube formation assay Supernatants which .western blot analyses6 min to remove dead cells. Supernatant (3.2.4) were centrifuged at 1200 rpm for was taken and sterilised using 0.2 μm filter. To see if overexpression and gene
ormed for were perfn level WB analysesing are effective at the proteisilenctransfection, cells were after Two daysfected cells.supernatant and extract of trans
s Methodnd Material a
52
ses (3.2.1.14). After protein analylysed (3.2.3.4) and used for western blottotal protein were loaded onto SDS-concentration measurement, same amount of PAGE to compare ORM level for each sample. The same blot was then striped by ibody as control for same bated with anti vimentin antfer (3.1.3) and incustriping bufoaded protein. amount of l
3.2.4 Endothelial ube formation assayt
was carried out using three-diThis assay mensional type I collagen gel (PureCol,r tissue culture dishes as described ustelInamed) which was prepared in 48-well cpreviously (Ergun et al., 2000; Oliveira-Ferrer et al., 2004; Pepper et al., 1990).
ice-cold collagen gel mixture (3.1.3) and 48-well plates were coated with 190 µl of ymerization of the gel, wild type ORM- at 37°C. After polincubated over nightoverexpressolidified gels at a csing and ORM-silenced endothelial oncentration of 4x104cells cells /well in 300 µl of growth medium (HDMECs) were seeded onto
luence, the medium was replaced by basal medium containing 5% FCS. At confcontaining 2-4% FCS without further supplements. After 24 hours VEGF-A was
3 days after taking photographs with ery added to the wells and was renewed evphase conttubes of interest were fixed rast microscopy (Zeiss, Jena). overnight with Bouin´s fixativeThe collagen gels containing endothelia solution following overnightl
incubation with 70%left out from 48-well using a scalpel ethanol and two times ethanol incubation and embedded in paraffin to use for for 1 h. The gels were
ochemical studies. immunohist
Stimulating Factors Description Working concentration Supplier
ORM Orosomucoid , human serum 2. 300 ng/ml 1. 100 ng/ml Calbiochem
Oxford AntiPAI-1 Anti-humaantibody (fron PAI-1 monom mouse) clonal 15 µg/ml Biomedical
Re hrcseaVEGF-A Recombinant Human VEGF-A 50 ng/ml Immunotools
VEGF-A Recombinant Human VEGF-A 50 ng/ml
Table 17: The stimulating factors used for tube assays
unction modulating and the ORM-PAI-1 complex has any fTo find out if PAI-1 or/ was seeded intobes, endothelial cellseffects on VEGF-A induced endothelial tucollagen gel and incubated until confluent. Afterwards cells were left untreated or
stimulated with; (i) VEGF-A (positive control), (ii) ORM, (iii) VEGF-A together with
ORM, (iv) VEGF-A and anti-PAI-1 antibody, (v) VEGF-A, ORM and anti-PAI-1
s Methodnd Material a
53
antibody, (vi) anti-PAI-1 antibody alone (Tab. 17). Endothelial cells and VEGF-A
by day aphed days examined and photogrube formation wainduced endothelial tJena) equipped with a digital camera using phase contrast microscope (Zeiss,(ProgResTMC10plus, Jenoptik).
methods 3.2.5 Histological
ples and cells ng for tissue samFixation and HE staini3.2.5.1 Paraffin-embedded bladder tissue sections obtained from patients who have clinically
cancer as well as normal bladder tissue, obtained been diagnosed to have bladder The bladder tissue unohistochemical analyses.by biopsy, sections were used for immsections used in this study were obtained from department of Pathology of University
ctionseunohistochemical staning the sHospital Hamburg-Eppendorf. Before imm on tissue sections. Eosin to confirm presence of cellsmatoxylin-ed with Hewere stainSlides containing paraffin sections were deparaffinised by incubation in order as
% ethanol, and lately deionized water for lene, isopropanol, 96% and 70three times xyxylin after a short washing ons were stained with Hemato5 min. After that, the sectiwith water, destained with acid ethanol and washed again with water. For
treated with Eosin and dehydrated by 96%cytoplasmatic staining the sections were isopropanol incubation. ethanol and subsequently y3.2.5.2 Immunohistochemistr Each section was deparaffinised (rehydrated) in descending alcohol row and
ed to immunohistochemistry. he sections were subjectincubated in 1xPBS. Then tmal swine serum to block the unspecific After treatment with normal rabbit or norubated with the corresponding were incbackground for 30 min at RT, the sectionsprimary antibodies against ORM and/or ZAG diluted in PBS-BSA-NaN3 buffer (3.1.3)
10 min and exposed to shed with 1xPBS for 3x waefor 24 h at 4°C. The sections werthe secondary antibodies for 1 h at RT. Then washed again and treated with the
eat RT. Subsequently, the sections werperoxidase-anti-peroxidase (PAP) for 30 min treated with ABC-complex (Vector) for 30 min at RT, washed again for 2x10 min with
1xPBS and transferred to the solution for the immunostaining development. For the
tibody. nubated only with secondary a were inccontrol reaction, same samples
s Methodnd Material a
54
the modified nickel-oped by means of ed and devele activity was enhancPeroxidasglucose oxidase technique (Ergun et al., 1997). Glucoseoxidase solution was
prepared freshly in dark according to the receipt described in section 3.1.3. After
washing with 1xPB buffer (3.1.3) for 10 min, the sections were incubated with
at RT. Developing was followed by glucoseoxidase solution for 10-30 min ear, the reaction was stopped by removingmicroscope. After the staining became clthe developing solution followed washing 3x5 min with PBS.
hed 2x5 sec at RT and was0m Red for 2Sections were counterstained with Calciumin with distilled water. The sections were dehydrated by incubation in order as
three times xylene for 5 min. After deionized water, 70%, 96%, 100% ethanol and don). In some cases Mowiol hermoshanomount (Tthat, were embedded using histm red staining to embed the sections. after Calciu(Calbiochem) was used directlyUsing the microscope system equipped with a digital camera the immunostaining
were studied and photographed. ochemistryt3.2.5.3 Immunocy Wild type cells were seeded anparaformaldehyde for 15 min and washing 3 d cultured in chamber slidestimes with 1xPBS, the cells were . After fixation with 4%
eunspecific background. The cells werincubated in normal serum to block the incubated with biotinincubated with primary ylated secantibodyondary ant for 24 h. After washiibody. Afterwng in 1xPBS the cells were ards, they were washed in
PBS and incubated with peroxidase (PAP). After washing the slides were incubated
or). The immunostaining were developed (ABC Kit, Vectin avidin-biotin complexusing Nickin immunohistochemistry section. If cel enhanced-glucoseoxidase technique (Davounterstaining is needed the sections were idoff et al., 1990) as described
stained in Calcium Red for 1-2 min. Using a microscope system equipped with a
method we have studied the digital camera the immunostaining werelocalization of ORM and know studied and photographed.n angiogenic fac Using tthe sameors and
o after ORM overexpressiontheir receptors on endothelial tubes induced in vitrusing Bouin´s fixative overversus ORM silencing in HDMECs. Endot nighthelial tubes on colla and embedded in paraffin. On 5-7 µm thick tissue gen gel were fixed
e. performed as mentioned abovsections immunhistochemistry were
s sultRe
4 Results 2D gel electrophoresis proteins byProfiling of urinary4.1
4.1.1 Optimization of the sample electrophoresis on human urine preparation method for 2D gel
55
cancer, 2-DE analysIn order to determine the ces were performed on urhanges in urinary protein patteine samples of fourtyfive patients with rns by presence of bladder
(n=8), GI, GII, GIII), 12), pT1 (n=8), pT2-3 bladder cancer of different stages (pTa (n=lunteers (n=7). The details of 2-DE patients on follow-up (n=10) and healthy voanalyses were described in the section of material and methods. Protein spots were
silver staining. Obtained 2-DE images visualized using Coomassie Brillant Blue or (BioRad). Several protein spots in sed by PDQuest software programmewere analyadder cancer were identified using a patient with blDE gel ofcoomassie stained 2-It is well kmass spectrometric analyses, which were nown that urine contains trace amounts of protein orignot present in normal urine samplesinating from blood.
and amounts of body filtrates such asplasma, the kidneys and the urogenital tract, waste products (Anderson and Anderson,water, salts, electrolytes and nitrogenous mple preparation of urine samples is2002; Beetham and Cattell, 1993). Thus, the sapreliminary screening of biomarkers. very important for protein analysis and les several techniques including protein In order to optimize 2-DE for urine sampimmunoprecipitation, ultracentrifugation and precipitation with TCA or in some cases ecipitation led to the vertical TCA prfiltration were used for sample preparation.streaks on 2-DE gel probably due to residual TCA in protein sample and difficulties in
resolubilization of proteins (data was not shown). Immunoprecipitation was used for
ose proteins whicermine changes in thdetection of a specific protein and to deth suppose to be related to bladder cancer. However, immunoprecipitated proteins
caused horpresence of remained immunoglobulin G leadiizontal streaks and dark background on 2-ng to ovDE geer loading of protein amount. ls probably due to the
Ultrafiltration was performed on native urine samples using Centrifugal Filter Column
rfering molecules containing salts, inte(Amicon) to remove high abundance ofwaste products, which disturb the electrolytes and/or some nitrogenous t dimension of 2-DE. After that, different esis process especially in the firselectrophorrehydration buffers were used to solubilise filtrated and concentrated urine proteins.
methods and different rehydration buffersThe efficiency of each buffer was compared to were applie each other. Ultrafiltrad on urine samples and tion, precipitation
s sultRe
56
urine hod for sample preparation of to find the best metcompared each otheree 3.2.1.3) revealed preparation methods (ssamples. The use of optimized sample and reduced background and streaks.clear detectable protein spots ficial bladder cancerof a patient with superThe 2-DE protein pattern of urine sample length and 3-10 pH range wasp with 17 cm IPG striis exemplary shown in figure 19. staining ing silverpots were visualized usused for this urine sample and the protein s in thens and focused particularlying different proteiprotocol. Many spots representstained 2-DE gel. seen in the silver pH-spectrum ranging from pH 5 to 8 were pHpHpHMWMWMW333444555676767888999101010
66 k66 kDDaa
55 k55 kDDaa
45 k45 kDDaa
36 k36 kDDaa
29 k29 kDDaa
24 k24 kDDaa
UrinFigure 19e sampl: e contaiProtein paning 2tter00 µg n in urine sample of a total proteins were apatiennalyset d wby ith 2-DE ubladder casncer of ing 17 cm strip with pH ranthe stage pTa.ge
3-10. Silver staining revealed numerous spots, particularly focused in the pH range 5-8.
4.1.2 Determination of urinary protein pattern in relation to bladder cancer by
2-DE Urine samples were subdivided into three groups: urine samples of (i) healthy
persons, (ii) patients with bladder cancer of different stages, and (iii) follow-up
patients.
s sultRe
57
hanges in whole protein pattern of urine cFirstly, it was aimed to find out possible
. To this aim the urinary ncerbe related to bladder casamples, which thought to
hat of patients with bladdterns of healthy volunteers to tprotein patr cancer of e
pT3, as well as of patients on follow-up different stages such as pTa, pT1, pT2 and
which were present in urine samples ofwere compared to eachother. Several spots,
urine up, were not present in follow-patients with bladder cancer and in those of
ted with a were indichese protein groupssamples of healthy persons (Fig. 20-25). T
circles and arrows.
th pTa iversus urine of patients wProtein pattern of normal urine 4.1.3
persons (n=7) and patients with bladder om healthyNative urine samples obtained fr
ed by 2-DE technique. Urine samplesre analysecancer of stage pTa (n=12) w
on IPG strips with 11 cm length and 3-10 containing 200 µg total protein were loaded
ed protocol. Protein spots were visualiz(NL) pH range for the first dimension of 2-DE
using silver staining (3.2.1.9).
healthy volunteers (Fig. 20A) and patients Comparison of urinary protein pattern of
n20B) revealed big differences in patterwith bladder cancer of stage pTa, GI (Fig.
and intensity of protein spots. 2-DE images of urinary protein of different healthy
shown in figure 20A, 21A,he same protein pattern as volunteers revealed almost t
sizes of 55 and 66 kDa. Using the same 22A. The strongest spots were seen at the
sulted ea patients rDE analyses on a urine sample of pTprotocols and conditions, 2-
protein spots (Fig. 20B). Compared to in protein patterns showing many additional
healthy urine, the number and the density of detectable protein spots were higher in
urine samples of pTa patients. The proteins which were seen as faint spots in normal
patients but in significurine were also present in urine sample ofantly higher amount.
s sultRe
pHpH3434MWMW
6666 k kDDaa
5555 k kDDaa
pHpHMWMW333444
6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa22
55
565656
66
797988
797979888
11
334455
66
0011
AA
111000
58
BB Figure 20: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pTa,GI (B). Urine samples containing 200 µg protein were loaded on 11
cm IPG strips with pH range 3-10 non linear (NL). Protein spots were visualized by silver staining. 2-
DE image of urine sample of a healthy volunteers (A) and urine of a patient with bladder cancer of the
stage pTa, GI (B) are shown. The protein spots at 55 and 66 kDa indicated by circles are similar in
both urine samples, but significantly stronger in urine samples of bladder cancer (B). In urine samples
of bladder cancer patients there are numerous additional spots as numbered (1-6).
th pT1 iversus urine of patients wProtein pattern of normal urine 4.1.4 urine, containing eange, nativNL pH rUsing IPG strips with 11 cm length and 3-10 adder cancer of pT1 stages (n=8) were of patients with blg total protein,μ200 ng protocol. Protein ned using silver stainianalysed by 2-DE. The 2-DE gels were stai a healthy person revealed two different mple obtained fromapattern of the urine s
s sultRe
59
ilar to healthy urine which was shown in ig. 21A) simFprotein spots at 55 and 66 kDa (pT1,GIII refigure 20A. The urinary protein pattern ofvealed several additional protei a patient with bladder cancer of stage n spots, compared to normal urine (Fig.
21B).
pHpH3434MWMW
6666 k kDDaa
5555 k kDDaa
pHpH3434MWMW6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa22
55
55
33
66
66
879879
7987981166
774455
0101
AA
0101
55BB Figure 21: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pT1, GIII (B). Urine samples containing 200 µg total proteins were
loaded on 11 cm IPG strips with pH range 3-10 NL. Protein spots were visualized by silver staining.
The protein spots indicated by cycles in healthy urine (A) and in urine of patient with bladder cancer of
the stage pT1, GIII (B) are similar. There are additional spots which were numbered and indicated by
doted sequares (1-7) in urine samples of patients. Among marked protein spots, especially the protein
groups at 55 kDa are stronger in urine samples of patients (B).
s sultRe
60
in normal urine, were seen in ent kDa, which were also presThe spots at 55 and 66 urine of pT1 patients too. But the protein group at 55 kDa was present in urine of pT1
patients in a significant higher amount than that in normal urine. Spot 1 and spot 2,
detected in urine of pTa patients, were not present in urine of pT1 patients and also
6 were detected in urine samples of both were not present in normal urine. Spot 3- much stronger present in bladder cancer but they werepatients, either pTa and pT1 y pattern of pT1 patients, inarin figure 20B. In ururine of pTa patients shown pot 7 representing severaled as 7 were detected. Sadditional protein spots numbera patients. esent in the urine of samples pTdifferent proteins, at 35-45 kDa, was not pr
th pT2 iversus urine of patients wProtein pattern of normal urine 4.1.5
from patients with ein were obtained Urine samples containing 200 µg total protbladder cancer of invasive stages as pT2-3 (n=8) and were separated using 2-DE and 3-10 NL pH range ps with 11 cm length technique. For the first dimension IPG strisualized using silver staining i were vwere used. The protein spots on 2-DE gels thy urine (Fig. 22A) revealed the similarprotocol (3.2.1.9). Protein pattern of healurine samples of healthy volunteers in protein spots at 55 and 66 kDa as shown for of urine sample of a image obtained from analyses figure 20A and 21A. The 2-DEprotein spots at 55 and 66 age pT2, GIII revealed tncer with spatient of bladder caa well as in urine of pT present in normal urine, askDa (Fig. 22B) which were alsoand pT1 patients, but in comparison to the stages pTa and PT1, these protein spots
were weaker in the urine samples of pT2 patients. The protein spot at 55 kDa was
rmal urine. Spot 1, which patients with pT2 than the nostronger in urine samples ofs, was present in the urine pT1 patientwas not present in the urine samples of tients. Spot 2 was not present in urinary samples of pT2 patients as well as in pTa pa ents. Spot 3 and 5, with that of pT1 patiprotein pattern of pT2 patients, in similarity detected were also,ine samples of pTa and pT1 patientswhich were present in the urble in urine of pT2 4 and 6 were not detectain the urine samples of pT2 patients. Spot detection of these spots in urine samples of pTa and pT1patients in contrast to the patients. Spot 7, which was seen only in the urine samples of pT1 patients (Fig. 21B),
8,spots numbered as2 patient. Additional was not present in the urine samples of pT 9 and 10 were present only in the urine samples of pT2 patients.
s sultRe
pHpHpH3434MWMWMW
6666 k kDDaa
5555 k kDDaa
pHpH3434MWMW6666 k kDDaa
5555 k kDDaa
4545 k kDDaa
3636 k kDDaa
2929 k kDDaa
22
33
55
55
66
66
44
798798
79798810101166778855
0101
AA
0101
61
3636 k kDDaa99
22882929 k kDDaa33
4545BB Figure 22: Comparison of protein pattern in normal urine (A) and in urine of patients with
bladder cancer of the stage pT2, GIII (B). 200 µg urinary proteins were loaded on 11 cm IPG strips
with pH range 3-10 NL. Protein spots were visualized by silver staining. The protein spots at 55 and 66
kDa indicated by circles are similar in both urine samples, healthy urine (A) and urine of a patients with
bladder cancer of the stage pT2,GIII (B). In urine samples of patients with bladder cancer of the stage
were detected. 1, 3, 5, 8, 9,10 s and numbered asspotpT2,GIII additional protein es of follow-up versus urine samples of Protein pattern of urine sampl4.1.6 bladder cancer pateints patients on follow-up (n=10) rn of the urine samples of In the next step, protein patte 3) were compared., pT1, pT2-der cancer of different stage (pTaand patients with blads on follow-up, IPG strips samples obtained from patientFor 2-DE analyses of urine
s sultRe
62
length and 3-10 NL pH range were used. Native urine containing 200 µg with 11 cmsualiztotal proteins were loaded and the gels were vied using silver staining protocol.
pHpHpH3434MWMWMW66 kDa66 kDa
55 kDa55 kDa
29 kDa29 kDa
pHpHpH333MWMWMW
66 kDa66 kDa
55 kDa55 kDa45 kDa45 kDa36 kDa36 kDa29 kDa29 kDa
33
55
444565656
33
66
7979
797979
88
888
0011
AA
111000
BB bladder cFigure 23ancer of: Comparison o the stfage pTa, GI (B). protein pattern in urine 200 µg of paproteins, involvtients oed in un follorinw-e saup (A) anmples, wed patiere loadents wd onith
spot11 cm IPG s, at 55 strips and 6with p6 kDa,H whirange ch a3-1re marked 0 NL. Protein with spciotrcles were vis, are seen in sualized by silvurine er stsamples aining. Tof patients ohe proteinn
follow-up (A) and patients with bladder cancer of the stage pTa, GI (B). Additionally, the protein spot
numbere21B, 22B, 23B) is also pred as 3, which wass deteent in uricted inne sampl urine samplee of patients on folloof patients with bladdw-up. er cancer (shown in figure
ed with doted circles were detected in 2-DE spots groups at 55 and 66 kDa markurine of follow-up patients with similarity to all urine samples used including urine
h bladder cancer. However, the density samples of healthy persons and patients wit
Res sult
63
of these proteins wasdifferent stages and patients on follow-up t stronger in urine sahan those in urine of healthy persons. mples of patients with bladder cancer of
pHpHpH3434MWMWMW
kDa66 kDa66
kDa55 kDa55
kDa29 kDa29
pHpHpH3434MWMWMW
kDa kDa6666
kDa55 kDa55 kDa45 kDa45 kDa36 kDa3629 kDa29 kDa
55
44
55
44
66
66
7979
7979
88
88
0101
AA
0011
BB bladder Figure 24cancer of the : Comparison ofstage pT1, GIII (B). protein pattern in urine Urine sampleof pas contatients oining n follo2w00 -uµgp (A) an proteins wed patiere nts usewd ithfor
2-DE staininanalyg wases uses. 11 d. The spcm IPG stripots grosup with spH ra, at 55 and 66 kDnge 3-10 NL were ua, indicatesed. For d by circldetees care sition of proteimilar in urinans, silveryr
protein pattern of patients on follow-up (A) and patients with bladder cancer of the stage pT1,GIII (B).
22B), is alThe protein spots numso present in uberine samred as 4, plewhis ocfh was see patients on follow-up. n in urine sample of pTa and pT1 patients (Fig. 21B,
ients on follow-up (Fig. 23A, 24A, 25A) and In comparison of urinary pattern of pat(Fig. 23B), pT1,GIII stage such as pTa,GI patients with bladder cancer of different different spots in urinary revealed similar but also ig. 25B) F(Fig. 24B), pT2,GIII (
s sultRe
64
mples of patients on follow-up, there are few protein patterns. In 2-DE gels of urine saein spots in addition to protein spot groups marked detectable and faint stained protby doted circles.
4.1.7 patients of tumor staProtein pattern of urine samples ofge pTa patients on follow-up versus that of
As mentioned above the protein spots at 55 and 66 kDa were present in urine
doted circles (Fig. 23A). The densities of sample of follow-up patients as marked by these protein groups seen in urine samples of patients on follow-up were as strong
onal protein spot numbered as ing bladder cancer. The additias in that of patients havs on follow-up, which was not present in 3 was detected in urine samples of patientients with bladder cancer ent in urine samples of patesnormal urine but prominently pr pots representing different proteins, asof stage pTa (Fig. 23B). Many additional sshown and numbered for the same image in figure 20B, were detected in urine
to urine samples of patients on follow- patients with pTa, in comparisonsamples ofup.
4.1.8 patients of tumor staProtein pattern of urine samples ofge pT1 patients on follow-up versus that of
urine samples of another kDa were present also in The protein spots at 55 and 66 patient on follow-up as shown in figure 24A. These spots were marked as doted rked as number 4 was detected in urine pot at about 30 kDa macircles. The protein spatients with bladder cancer of pT1 (Fig. sample of pateints on follow-up and also 24A,B). The same protein spot was also present in urine sample of pTa patients and
higher ed with the same number in figure 20B. The density of spot 4 waswas indicatage pT1 (Fig. 24B) then the ients with bladder cancer stin urine sample of the patein spots, which were numbered for the follow-up patients. But the additional protine samples of patients with y in the ursame image in figure 21B, were seen onl bladder cancer.
4.1.9 Protein pattern of urine samples ofpatients of bladder cancer stage pT2 patients on follow-up versus that of
The protein spot at 66 kDa as marked by doted circle was not present in urine
in figure 25A. But the spot at 55 kDa wassample of this follow-up patient as shown present in urine sample of a patient on follow-up (Fig. 25A) in a higher intensity as in
65s sultRethe urine sample of a pT2 patient (Fig. 25B). The protein spots numbered as 3, 4, s on follow-up and those with th patient present in urine samples of boeand 5 werbladder cancer stage pT2/GIII.
pHpHpH333
MWMWMW
kDa66 kDa66 kDa55 kDa55
pHpHpHMWMWMW333
kDa66 kDa66 kDa55 kDa55 kDa45 kDa45 kDa36 kDa36 kDa29 kDa29
444565656
33
44
444565656
33
44
797979888
55
797979888
55
111000
AA
111000
BB Figure 25: Comparison of protein pattern in urine of patients on follow-up (A) and patients with
11 cm stbladder caripnscer of with pHthe range 3stage pT2, GIII-10 NL. (B). Silver staini Urinne sampleg was contas used for ining 20detec0 µg tion protein of proteiwen re loadespots. Thd one
protein spots seen at 55 kDa which marked by circles are similar in both sample, urine of follow-up
patients (A) and and patients with bladder cancer of the stage pT2, GIII (B). The protein spot at 66
kDa, which was seen in other 2-DE images is not visible in urine sample of this follow-up patient (A).
The pAnother protein rotein spspot marked byot in figure B marked by fat s a fat square in figure A isquare i not pres not psernt in urine esent in urinsamplee sampls of pT2 es ofpatie follow-unts (B). p
patients. The protein spots numbered as 3, 4, and 5 are present in both urine samples.
sultRes
66
up (Fig. urine sample of a patient on follow-The spot marked as fat square in 2-DE of er stage pT2 (Fig. patient with bladder cancmple of a25A) was not present in urine s t square, present in urineprotein spot ,marked by fa25B). On the other hand, another ig. 25B) was not seen in urine sample of sample of a patient with tumor stage pT2 (F at 55 erent groups of spotsfmmary, two difa patient on follow-up (Fig. 25A). In suand 66 kDa were present in all urine samples (Fig. 20-25), but they were particularly
tients on follow-up. h bladder cancer and paprominent in urine samples of patients witNumerous additional proteins were detected in urine samples of patients with bladder
persons urine samples, including healthycancer in comparison to other groups ofre analysed in this study. and follow-up patients, which we
th bladder iurine samples of patients wIdentification of protein spots in 4.1.10 cancer GI were stage PTa/s with bladder cancer patientProteins from urine samples ofh both silver and CBB (Fig. 26). separated on a 2-DE gel and stained witpHpHpHMWMWMW333444565656797979888111000
6666 k kDDaa
5555 k kDDaa
3636 k kDDaa
2929 k kDDaa
2424 k kDDaa
1122
6677
565656
88
99
797979888
3355
44
111000
the staFigure 26ge : PpTa/GI. rotein ideThe ntiurinarfication y patternfrom of a p 2-DE imaatientge with of blurine addof a per caancer tient of with bladdthe stage pTer canca/GI ewar of s
chosen for protein identification. Nine protein spots indicated by quadrates in Coomassie stained 2-
DE gel were cut out and subsequently underwent mass spectrometric analyses.
s sultRe
67
Several protein spots observed in urine samples of patients with bladder cancer of
and underwent mass ained gel oomassie stthe stage PTa/GI were cut out from the cspectrometric analys , several proteins called as uromodulin,es. After these studiesalbumin (HSA), orosomucoid1 (ORM), DNA-binding glycoprotein, human serum been en (HC) havx-forming glycoproteihuman zinc-alpha-2-glycoprotein and compleidentified (Tab. 18).
NumSpot ber Match to Score NCreferBI datencabae se CovSequencerage (e %)
1 Urourommoducoid (Homo saulin; Tamm-Hopienrsfas) ll glcoprotein; 38 gi | 4507833 11
2 Nuclear DNA-binding protein (Homo 36 gi | 37550855 -
s) ensapi3 Ribosomal Protein S19 (Homo sapiens) 39 gi | 3164200 -
4 Human Serum Albumin (Homo sapiens) 164 gi | 4389275
63
5 Human Serum Albumin (Homo sapiens) 229 gi | 4389275 74
6,7 Orosomucoid 1 precursor (Homo 152 gi | 20070760 38
s) ensapi8 Human Zinc-Alpha-2-Glycoprotein 212 gi | 4699583 72
ns) o sapiem(Ho9 Complex-forming glycoprotein HC 94 gi | 223373 41
ns) o sapiem(Ho Table 18: Proteins idendified by MS analyses. For spot 2 and 3 no sequence coverage was
determined. These protein spots were predicted to be Nuclear DNA-binding protein (spot2) and
Ribosomal protein S19 (spot3).
uromodulin, is one ofOne of these proteins abundant , tamm-horsfall glycoproteiprotein in normal urine. n (Fig. 26, spot 1), also referred to asIn 2-D images the spot
kDa in figure 20-25) wasmentioned as the spot at 66 (belonging to uromodulin cancer but almost patients with bladderprominently present in the urine sample ofvolunteers and patients on follow-up. DNA-equally present in the urine of healthy as shown in figure spot group at 66 kDa binding protein (Fig. 26, spot 2) included inalmost equally20-25 was particularly pres prominentent in urine sample in urine samples of patis of patients on follow-up and healthyents with bladder cancer but
volunteers. The spots 4 and 5 (Fig. 26) were identified as human serum albumin,
which were present in all urine ssimilarity with spots marked by doted circleamples ans for the protein at alysed in this study. These spot55 kDa as shs were in own in
ticularlyman serum albumin were parpots representing hufigures 20-25. The sThe proteins indicated as prominent in urine samples of patients witspot 6 and 7 in figure 26 wereh bladder cancer and pa identified as orosomtients on follow-up. ucoid 1
s sultRe
68
s with bladder cancer (showns of patientand they were present only in urine samplein figure 20B as spot 2), but were absent in urine samples of healthy persons and h the number 8 in figure 26 was identified patients on follow- up. The spot marked witominently present in the urine samples as human zinc-alpha-glycoprotein and was prof patients with bladder cancer (Fig. 20-25, included in spot 3) and in those of
patients on follow-up.spot 9 in figure 26 was identified as But it was absent complex-forming glyin urine samples of healthy persons. The coprotein (alpha-1-
with bladder cancer, particularly smicroglobulin, also called HC). This protein trong in the was found in urine sstage of pTa, and visiable as a faint spot mples of patients
23-25). llow-up (Fig.mples of patients on fo(Fig. 20-25, included in spot 4) in urine saIn contrast, it was not detectable in urine samples of healthy persons. The spot 3 in
figure 26, which was identifieurine, but no considerable changes were sd as ribosomal protein S19,een for this protein in urine samples of was not present in normal
patients witmarked by doted circle at 55 kDa (Fig. 20-h bladder cancer and patients on follow-up. It is 25). The changes in densincluded in the spot group ity of identified
proteins in urine samples were summarized in table 19.
pT1
pT2-3 -/+
number Spot Idendified Proteins Healthy Follow-up pTa pT1 pT2-3
1 Uromodulin -/+ -/+
2 DNA-binding protein -/+ -/+
3 Ribosomal Protein S19 -/+
4,5 Human Serum Albumin -/+
6,7 Orosomucoid 1 --- ---
8 HumaGlycopn Zinroteinc -Alpha-2---- ---
9 gylcopComplex-frorotein ming --- -/+ -/+
Table 1samples b9: Sy e2-DE. miquantitative determination of protein levels of identified proteins in urine
4.1.11 Human serum albumin in normapatients with bladder cancer and follow-up l urine versus in urine samples of
The 2-DE results showed that most abundant proteins in urine samples, which were
ese protein spots, a. One of thD and 66 kanalysed in this study, were detected at 55albumin using mass spectrometry (Tab. at 55 kDa, was identified as human serum the amount of human serum albumin in normal urine 18). To view changes in
s sultRe
69
samples and urine samples of patients with bladder cancer the urinary protein
mples were compared to each other. patterns of all groups of urine sa
pHpHMWMW333444
kDa55 kDa55
kDa55 kDa55
kDa55 kDa55
565656
797979888
111000
AA
BB
CC
HuHumaman Sn Seerruum m AAllbumbuminin
HuHumaman Sn Seerruum m AAllbumbuminin
HuHuman Seruman Serumm AAllbubummiinn
CC Figure 27: Comparison of protein spots indicating human serum albumin (HSA) in all urine
as Humsample grouan Seps.rum The prot Albumin. The ein spotproteis at 55 kDa, whichn pattern of heal are prthy uriesne en(A), urint in all urine sae of patients mples, were ion follow-up dentified (B),
shoand pwna as tients exemplawith blardder y) (C) were comcancer with dpared ito eafferent stach otges (uriher for thnary e sppatots tern of showina patig HSA. Theent with propTa,tein spot GI was s
compof HSA are arisonstronger to healthy person in urine s. samples of patients with bladder cancer and patients on follow-up in
s sultRe
70
Human serum albumin was present in all urinary protein patterns (Fig. 27 A-C), but
urine of patients witthe strongest protein spots which belong toh bladder cancer (Fig. 27C) and human serum albumin were detpatients on follow-up (Feig. 27B)cted in
when compared to those in healthy urine (Fig. 27A).
ptides for identified proteins Mass spectrums and matched pe4.1.12
he NCBI database as shown for all identified Proteins were identified by searching t patients with bladder cancer proteins in urine samples of(Fig. 26). Spectrum of mass given for spot 1 which wase werspectrometric analyses and matched sequencesilar to and 3 which were identified as sim; spot 2 identified as uromodulin (Fig. 28)ein (Fig. 29A-B); spot 4 and 5 which ein and ribosomal protDNA-binding glycoprot(Fig. 30A,B); spot 6 and 7 which were were identified as human serum albumin 8 which was identified as zinc-alpha-2- 31); spotidentified as orosomucoid1 (Fig.mplex-forming glycoprotein (Fig. 32A,B). glycoprotein, and spot 9 identified as Co
%I%I%Innnttt4215.6424215.15.66
100100100909090808080692.69842.69842.84
70707060606046637.4646637.637.
50505060791.6060791.791.
407230.7297230.930.9
404030303041.6534141..65365329.11192299..11119119
202020720.14377220.0.143143111.17811111.1.178178
60932.60932.60932.
101010.991233.991233.99123356.161556.161556.161582.041682.041682.041658.251958.251958.2519535.221535.221535.221
000
100100100000150150150000MMMasasass20ss2020///C0CC00000hargehargeharge250250250000300300300000
11 M MGGQQPPSLSLTTWWMMLL M MVVVVVVAASSWWFFIITT TAATDTAATDTTSEASEARR WWCCSSECECHHSNAT CSNAT CTTEEDDEEAVTAVTTCTC
51 51 TTCQECQEGGFFTTGGDGDG LT LTCVCVDLDLDEDECCAA IIPPGAGAHNCSAHNCSAN SSN SSCCVVNTNTPPGGSSFF SCV SCVCPCPEEGGFFRLRL
101 101 S SPPGGLGLGCTCTDVDVDD E ECCAAEEPPGGLSLSHHCC HA HALALATTCCVVNNVVVV GS GSYYLCVLCVCPCPAAGG Y YRRGGDDGGWWHHCCEECC
151 151 S SPPGGSSCCGPGPGLDGLD CV CVPPEEGGDDAALVLVCC A ADDPPCCQQAAHRHRTTL DL DEEYYWWRSRSTTEEYYGG E EGGYYAACCDDTTDLRDLR
201 201 G GWWYYRRFVFVGGQQGGGGAARRMMAAEETCTCVVPPVVLLRRCNCNTAAPTAAPMMWW L LNNGGTTHHPSPSSDSDE E GIVSR GIVSRKAKACCAHAH
251 251 WWSSGHGHCCLCCLWWDDAA S SVVQQVVKKAACACAGGGG Y YYYVVYYNNLTLTAAPPPP E ECCHLAHLAYYCCTTDDPP S SSSVVEEGTGTCECEEECC
301 301 SI SIDDEEDCDCKKSSNNN GRN GRWWHHCCQQCCKKQQDD FN FNIITTDDIISLLESLLE H HRRLLECGAECGANNDDMM KVSLGKC KVSLGKCQQLKLK
351 351 SLGSLGFFDKDKVFVFMMYY LSD LSDSSRRCCSSGFGFN DRDNN DRDNRDRDWWVVSSVV VT VTPPAARDRDGPGPCG TCG TVVLTLTRNETRNETHAHA
401 401 T TYYSSNNTTLLYYLLAADD E EIIIIIIRDLRDLNINIKK I INNFFAACSCSYYPPLD MLD MKKVVSSLLKKTTAALLQQPMPMVSVSAALLNNIIRVRV
451 451 GGTGGTGMGMFTFTVVRRMM AL ALFQTPFQTPSYTOSYTO P PYQYQGGSSVTLS SSVTLS TEA TEAFFLLYVYVGGTMTM L LDDGGGGDDLLSRFASRFA
501 501 LLM LLMTNCTNCYATPYATP S SSSNNAATTDDPLKYPLKY FI FIIQDIQDRRCCPPHHTT R RDDSSTIQTIQVVVVEENN GESSQ GESSQGRGRFSVFSV
551 551 QM QMFRFRFFAAGNGNYDYD LVYLDLVYLDCCEVYL CEVYL CDDTTMNMNEKEKCCKKP P TCTCSGSGTTRRFRFRSSGG S SVVIDQSRIDQSRVLVLNN
601 601 LGPITLGPITRRKGKGVQ AVQ ATTVSRVSRAAFFSSL GSSL GLLLLKKVVWWLLPPLLLL L LSATLTLSATLTLTTFQFQ
Figure 28: MS identification of Uromodulin (spot 1). Matched peptides are in red.
353535000000
s sultRe
AAA
35035035035035000000BBB
71
AAA%I%I%I%I%Innnnnttttt.....83615.83615.83615.83615.83615.
9090909090808080808094601.94601.94601.94601.94601.
7070707070606060606044.71144.71144.71144.71144.711
5050505050989.122989.122989.122989.912289.122
404040404031.73531.73531.73531.73531.735
303030303061930.61930.61930.61930.61930.
2020202020671.119671.119671.119671.119671.119404.134404.134404.134404.134404.134066.151066.151066.151066.151066.151214.163214.163214.163214.163214.163
141.180141.180141.180141.180141.180788.187788.187788.187788.187788.187244.199244.199244.199244.199244.199926.215926.215926.215926.215926.215286.226286.226286.226286.226286.226510.235510.235510.235510.235510.235671.243671.243671.243671.243671.24366.432566.432566.432566.432566.432532.352732.352732.352732.352732.352703.782803.782803.782803.782803.782895.433095.433095.433095.433095.433086.503286.503286.503286.503286.503295.563395.563395.563395.563395.5633
1010101010000001001001001001000000015151515150000000000200200200200200000002502502502502500000030303030300000000000350350350350350
MMMMMasasasasassssss/////CCCCChargehargehargehargeharge00000
BBB%I%I%I%I%Innnnnttttt.....
5237.52637.65237.65237.65237.631.82931.98231.98231.98231.982
9090909090808080808047653.47653.47653.47653.47653.
7070707070321.6432321.1.6464321.64321.64763.35177663.3.351351763.351763.351
75903.75903.75903.75903.75903.89933.89933.89933.89933.89933.
606060606050505050504040404040164730.164730.1647370.31640.164730.
30303030307975.91175.117795.117795.117795.1179004.4.125125904.125904.125904.125933117.7.146146317.146317.146317.146
2020202020305.617305.617305.6173035.05.661717265.335265.335265.33522665.5.333535
1010101010575.74.279427957.427957.427957.4279
00000100100100100100000001501501501501500000020020020020020000000250250250250250000003030303030000000000035035035035035000000
MasMasMasMasMassssss/////ChChChChChargeargeargeargearge
Figure 29: MS identification of DNA binding glycoprotein and ribosomal protein. Spot 2 was
idendified as DNA binding glycoprotein (A) and spot 3 was identified as Ribosomal protein (B).
Matched peptides were not given for this two proteins.
77777666663.3.3.3.3.351351351351351
Res sult
732.341732.734132.341
350350350000
AA
BB
72
AA%In%In%Inttt...45.61545.61545.615
90909080808070707047637.47637.47637.
606060505050404040673.49673.49673.49
73875.73875.73875.64880.64880.64880.528.146528.146528.146420.164420.164420.164
303030199.411199.411199.411
20202005617.05617.05617.18960.18960.18960.16.912216.912216.912263.918963.918963.91894.520404.520404.52040
698.135698.135698.135893.144893.144893.144917.154917.154917.154033.166033.166033.1665.258335.258335.25833430.269430.269430.269544.279544.279544.279610.299610.299610.299
101010352.214352.214352.214341732.341732.341732.
000100100100000150150150000202020000000252525000000300300300000350350350000
MMMass/Chargeass/Chargeass/Charge
11 M MKKWWVTFVTFIISSLLLL F FLLFFSSSAYSAYSRG VFSRG VFRRRRDDAAHKHKSESEVAVAHRFKDLHRFKDLGEGEEENFNFKKALALVVLLIIAA
5511 F FAAQQYLYLQQQQCCPFPF EDH EDHVKLVKLVNVNEEVV TEF TEFAAKKTTCCVAVADD ESA ESAENENCDKSLCDKSLHHTTLLFFGGDDKKLLCCTT
11001 1 VAVATTLLRETRETYYGGEE M MAADDCCACCAKKQQEP ERNEP ERNEECFCFLLQQHHKK DDNP DDNPNLNLPRLPRLVV RPEVD RPEVDVMVMCTCTAA
11551 1 FFHHDNEETDNEETFLFLK K KYKYLLYYEIEIAARRRRH PYH PYFYFYAAPELPELLFLFFAFAKKRRYYKKAAFAAFT ECT ECCQCQAADAADKAKAAA
22001 1 CCLLLLPPKKLLDDEELLR DEGKR DEGKASSAKASSAKQQ RL RLKCKCASLASLQQKKF F GGEERRAFAFKKAAWWAVAVARARLLSSQQRRFFPPKKAA
252511 EFAEVSKEFAEVSKLLVVTTDLDLTTKKVVHTHTECCECC HGDL HGDLLLEECACADDDD RA RADLDLAAKKYYIICE CE N NQQDSISSDSISSKLKLKK
33001 1 ECECCEKCEKPPLLLLEK SHCEK SHCIIAAEVENDEVEND EM EMPAPADLDLPSLPSLAA AADDFFVVEESSKDKDVCVCKKNYNYAEAAEAKDKDVFVF
33551 1 LGLGMMFFLLYYEEYYAARR RHPRHPDYDYSVVLLSVVLLLLRRLALAKKTTYYETETTTLLEKEKCCACCAAADAADP HP HEECCYYAKAKVFVFDDEE
44001 1 FFKKPLPLVEEPVEEPQQNN LI LIKQNCELKQNCELFFEE Q QLLGGEEYYKKFQFQNANALLLLVRVRYYTTKKKKVP QVSTPTLVVP QVSTPTLVEVEV
55001 1 CTESLCTESLVNRVNRRRP CFP CFSSAALLEVDEEVDETTYYVPKVPKEFEFNANAETET F FTTFFHHADADIICCTLTL SE SEKEKERQRQIIKKKKQQ
55551 1 TATALLVELVELVKVKHK PKAHK PKATTKEQKEQLLKKAA VMVMDDDDFFAAAFAFVEVE K KCCKACCKADDDKDKETETCFCFAEEGKAEEGKKKTCTC
66001 1 CCCCKKSSSSCCLRLRLI TSLI TSHHLLKKASQASQPTPT MMRRIIRREERRKK
BB%In%In%Inttt....84277.84277.84277
1001001009090905454673.673.54673.
808080707070927.927.79927.7979
606060628.14666228.8.146146
50505050.63750.63750.637599.114599.114599.114030.401630.416030.416
4040403030305189.65189.65189.65834.85834.85834.889960.89960.89960.
202020681.112681.112681.112596.512296.122596.12299.813599.891359.8135214404.144204.144204.59.515359.551539.5153442.193442.193442.193
318.162318.162318.16255.420455.420455.4204825.258825.258825.258544.279544.279544.279
101010431.184431.418431.184045.212045.021245.212922.225922.225922.225232.244232.244232.244430.269430.269430.269315.296315.296315.296323.310323.310323.310398.319398.319398.319430.337430.337430.337913.352913.352913.352
000100100100000150150150000MMMasasas202020sss///000CCC000hargehargeharge252525000000300300300000350350350000
11 KKSEVASEVAHRHRFKFKDD LG LGEEEENNFKFKAALLV V LLIIAFAAFAQQYYLLQQQQ CPF CPFEEDDHHVKVKLLVVNEVTNEVTEFAEFAKKTTCC
5511 VVAADESADESAENCD KENCD KSSLLHHTTLLFFGDKGDKLLCTVACTVATTLLRREETT YGE YGEMMADCCAKADCCAK QEQEPERPERNECFNECFLL
101101 QHKDDNQHKDDNPPNLNLPP R RLLVRPEVRPEVVDVDVMM CT CTAAFFHDNEHDNEETET FFLLKKKYKYLLYYEIEIAA RRHPY RRHPYFFYYAAPEPE
151151 LLLLFFFFAAKRKRYYKA KA AFT AFTEECCQCCQAAAD KAAAD KAACLCLLLPPKLD ELKLD ELRDRDEGKAEGKASSSSAAKKQRQRLKLKCACASLSL
201201 QKFGQKFGERERAAFFKA KA W WAAVVAARLRLSSQQRRFF PK PKAAEEFFAAEVEVSK LSK LVVTDLTTDLTKVHKVHTT EC ECCHCHGGDDLLLLEECC
251251 AADDRADDRADDLLAAKKY Y ICICENQDSISS KLENQDSISS KLKKEECCECCEKPKPLLLLEKEKSSHCIHCIAEV AEV ENDEENDEMMPPADLADLPP
330011 S SLLAADFAADFVVEESK DVSK DVCKCKNNYYAEAEAKAK DVFLDVFLGGMMFFLLYYEE Y YAARRRRHHPDPDYSYSVV V VLLLLLLRRLALAKKTYTY
351351 EETTTLTLEKCEKCCCAAAA AADDPHECYPHECYAAKKVV F FDDEEFFKPLKPLVVEEEE PQ PQNLNLIIKKQQNCENCE LLFFEEQQLLGGEEYYKKFF
401401 QNAQNALLLLVVRRYTYTKK KVKVPQPQVSTVSTPPTTLLVEVVEVSRSRNLNLGGKKV GV GSKCSKCCKHCKHPPEEAA KRM KRMPPCCAEDYAEDYLL
451451 SSVVLVVLNQNQLCVLLCVL HEHEKTKTPVSPVSDDRRVVTTKKCCCCTETESSLLVNVN R RRPCRPCFFSASALLEEVVDDETETYYVVPKEFPKEFNN
501501 AEAETFTTFTFHFHAADDII CCTTLSLSEEKKEERRQIQI K KKQKQTALTALVELVELVV K KHKPKHKPKATATKEQKEQ LLKKAVAVMMDDDDFFAAAA
551551 FVEKFVEKCCKCCKADD KADD KETETCCFFAAEEEGEGKKKLKLVVAASQAASQAAAALL G G
Figure 30: MS identification of Human Serum Albumin (HSA). Spot 4 (A) and spot 5 (B) were
identified as human serum albumin. Matched peptides are in red.
s sultRe
AA
350350350000
BB
73
%I%Inntt..AA
1001001009090908080807171842.842.71842.
70707060606050505049.69649.69649.6961269.041269.041269.04
40404042615.4242615.615.
30303020202045662.45662.45662.65832.65832.65832.98.011698.011698.0116247.209247.209247.209
378.101378.101378.101126.182126.182126.182
101010847.92847.92847.92007.125007.125007.12541541.141541.141541.1980.222980.222980.222433.269433.269433.269
000100100100000151515000000200200200000250250250000303030000000350350350000
MMMass/Chargeass/Chargeass/Charge
11 M MAALLSSWWVVLLTTVLVL SLSLLLPPLLLLEAQEAQI I PL PLCCAANLNLVPVVPVP P ITN ITNAATLTLDDQQIITT GGKKWEWEYYIIAASSAAFF
51 51 RRNEEYNNEEYNKSVQKSVQ EIQEIQAATTFFYFFYFT PFT PNNKTEDKTEDTIFL TIFL RREYEYQTQTRRQQDDQQCC IY IYNNTTYLNTTYLNVQVQ
111151 T51 T01 RE01 RETTKKNGTNGTEQEQIISSRRLGEFYLGEFYYYVVGGEEAALGQELGQEDDCCHFHFLRLRAAHIPHIPKKLLLL SSIILDLDVVVVRDTRDTKYTDKYTDTYMWTYMWLL AKK DK AKK DKFFDVNDVNCECEDPLDPLEEEEKKKKNNQQWWHH GL GLEEKKEESVYSVYRRKKADQADQEEEEKPEKPEGGEE
2201 01 SS
%I%I%I%Innnntttt....BB
1001001001009090909039931.39931.3399931.931.
80808080707070706060606037.66637.6666637.37.6666
91842.9191842.842.91842.
50505050404040403030303069733.69733.69733.69733.76826.76826.76826.76826.311.124311.124311.124311.12413.617513.617513.617513.6175
2020202039903.39903.39903.39903.8.101298.101298.101298.10129901.116901.116901.116901.11607.212507.212507.212507.2125
967.17999667.7.179179967.179
10101010231.154231.154231.154231.154777.209777.209777.209777.209790.218790.218790.218790.218383.226383.226383.226383.226460.247460.247460.247460.247671.260671.260671.260671.260804.271804.271804.271804.271937.283937.283937.283937.283983.298983.298983.298983.298324.306324.306324.306324.306944.315944.315944.315944.315356.323356.323356.323356.323471.337471.337471.337471.337167.345167.345167.345167.345380.354380.354380.354380.354
000010010010010000001515151500000000MasMasMasMasssss////200200200200ChChChCh0000argeargeargearge250250250250000030303030000000003503503503500000
555111111 M M MRRRNNNEEALSALSEALSEYNEYNWWEYNWVLVLVLKSKSKSTTTVQVQVLVLVQVL E E E SLL SLL SLLIIIQQQAAPLPLAPLTFTFLLTFLEEEFFFYYYAQAQAQFT FT FT I PI PI PPPPLLLNNNCCCKTKTKTANANANLLLEDTIEDTIEDTIVPVPVPVPVPVPFLFLFL RRRITITITEYEYNNEYNQQQTTATLDATLDTATLDRRRQQQQQQDDDITITIT QCQCQC G G GKKK IYNT IYNT IYNTWEYWEYWEYIIITYLTYLAATYLASSSNNAANAFFFVQVQVQ
101 101 101 RE RE RENNNGGGTTTIIISSSRRRYYYVVVGGGGGGQEQEQEHHHFFFAAAHHHLLLLLLIIILLLRDTKRDTKRDTKTYMTYMTYMLLL AFD AFD AFDVVVNNNDDDEEEKNKNKNWWW G G GLLLSVYSVYSVYADKPEADKPEADKPE
201 201 151 201 151 151 S S T S T TTTT KKK EQ EQEQ LGLGLGEEEFFFYYYEEEAAALLLDDDCCCLLLRIPKRIPKRIPKSSSDDDVVYVVYVVYTTTDDDWWWKKKKKKDDDKKKCCCEPEPEPLLLEEEKQKQKQHHHEEEKERKERKERKQEEGKQEEGKQEEGEEE
Figure 31: MS identification of orosomucoid 1. Spot 6 (A) and spot 7 (B) were identified as
orosomucoid. Matched peptides are in red.
s sultRe
AA
71.134871.134871.134871.1348
3503503503500000
BB
74
%I%I%I%Innnntttt....AA
10010010010090909090808080807070707054615.54615.54615.54615.73926.73926.73926.73926.
60606060562.83562.83562.56832.83
5050505048662.48662.48662.48662.73990.73990.73990.73990.903.153903.153903.153903.153
4040404054.66354.66354.66354.66358834.58834.58834.58834.47928.47928.47928.47928.68.811168.811168.811168.811193.324093.324093.324093.3240
30303030610.158610.158610.158610.158
20202020486.112486.112486.112486.112802.129802.129802.129802.12940.251440.251440.251440.251432.971732.971732.971732.9717815.619815.619815.619815.619.142152.142152.142152.142152139.122139.122139.122139.122
75.724175.724175.724175.724106.625106.625106.625106.625126933.426933.426933.426933.471.134871.134871.134871.1348
10101010909.278909.278909.278909.278
0000100100100100000015015015015000002002002002000000250250250250000030303030000000003503503503500000
MMMMass/Chargeass/Chargeass/Chargeass/Charge
1 1 1 D D DGGGRRRYSYSYSLLLTTTYIYIYIYYYTTTGGGLLLSSSKHVKHVKHVEEEDVDVDVPPPAAAFFFQQQAAALLLGGGSSSLLLNNN DL DL DLQQQFFFFFFRYNRYNRYNSSSK DRK DRK DRKSKSKSQPMQPMQPMGGGLLLWWW
101510151015111 IIIRRREEEQQQNNNNNNVEVEVERSRSRSGGGSMSMSMGGGEDEDEDAAAFFFWKWKWK WWW Q Q QKKKDDDSSSYYYDYYYDYYYDQQQLQLQLQGGGKAKAKAKDY KDY KDY RRR IE IE IEEEEFDFDFDIIINNNFFFKKKMEMEMEEEEIPTIPTIPTLLLAAAKKKWWW DI DI DI V V VPPPFVEYFVEYFVEYDDDPPPYNYNYNAAAADADADQSQSQSINININGGGT KQT KQT KQS HVLS HVLS HVLKKKWWWEQEQEQAAAGRGRGREEEPPPVVVFGCFGCFGCYYYEEE
151151151 VQVQVQRRRAAAKKKAAAYYYLLLEEEEEEECPECPECPAAATTTLLLRKRKRKYLYLYL KYSKKYSKKYSKNININILDLDLDRRRQQQDDDPPPPPPSSSVVVVVTVVTVVTSHSHSH Q Q QAAAPGPGPGEKKEKKEKKKKKLKLKLK
222201520152015111 C TAPC TAPC TAPLLLAAAYYYDYSCDYSCDYSCFFFYHYHYHVPVPVPQGQGQGHKHKHK S S SIDVHIDVHIDVHSLSLSLAQAQAQWWWPTPTPTLRLRLRAAAGGGVVP VVP VVP EEEWWWVQVQVQEEEASASASEPEEPEEPELRLRLRGGGDDDV V V L L LHHHNGNGNGNNNGGGTTTYQYQYQS S S WWWVVVVVAVVAVVAVVVPPQPPQPPQDDD
%I%I%I%Innnntttt....BB
10010010010045615.45615.45615.45615.78.84278.84278.84278.842
909090908080808070707070481.102481.410281.102481.102
60606060319.65319.65319.65319.65
5050505060761.60761.60761.60761.102183.102183.102183.102183.
404040403030303020202020483.1244843.81243.124483.124712.1357172.11352.135712.135237.1582327.31587.158237.158
514.173514.173514.173514.173636.182636.182636.182636.182068.195068.195068.195068.195350.207350.207350.207350.20792.352192.352192.352192.352167.642267.642267.642267.642243.612443.612443.612443.6124346.924346.924346.924346.92442.532642.532642.532642.532651.362751.362751.362751.3627
10101010007.28700007.7.287287007.287743.29877443.3.298298743.298
000010010010010000001515151500000000MasMasMasMasssss//200/200/200200CCCC0000hargehargehargeharge250250250250000030303030000000003503503503500000
5151511 1 1 G G GEGEGEGPPPAVAAVVTTTEEEPTPPPTPPPTPPAAADDDEISM TNEISM TEISM TNNI I I QQQSTSTSTVQVQVQRRRWRENFWRWRENFENFKGVKGVKGVBISR BISR BISR CCCEIYGEEIYGIYGEEETKTTKKSWSSWWGGGYYYAAANNNYLYYLLEAEEAAKKKI GI GI GTTT DS D DSSTTCTTTCTCPLPLPLDGKFDGKFDGKFKIKIKILMLLMMYDYYDDHK RHKHK R RMMMTTTSKSKSKWVSTWWVSTVSTBBBITMITMITMLVLGLVLGLVLGEEESSSYYY
111111505500111111 VV VV VV VGIPED VGIPED VGIPEDHHHTTTNNNYYYSSSDDDIIIEEEFT FT FT YA YA YA M M MADIFLADADIFLIFLRTRRTTGGGKKKKKKEEEFFFCCCSSSRHRHRHVPG EQVPG EQVPG EQHGPHGPHGPEPTEPEPTTIIITTTAAAEPILEPILEPILKKKLLLIPIPIPYYY R R R GR GR GR AAAPPPQLQLQLRERERETTTLLLLLLQQQDFDFDFRRRVVVVAQVAQVAQGGG
Figure 32: MS identification of Zinc-alpha-2-glycoprotein (ZAG) and Complex forming
glycoprotein. (spot 8, 9). Spot 8 (A) was identified as Human zinc-alpha-2-glycoprotein and spot 8
(B), as Complex forming glycoprotein (HC). Matched peptides are in red.
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4.2 Idendification of two urinary proteins using Western blot analyses
4.2.1 Detection of orosomucoid (ORM) patients with bladder cancer and patients on followin urine samples of healthy-up persons,
r stages classified as pTa, pT1, pT2, pT3 In urine samples (n=45) of all bladder tumoORM was detected at the size of 41 kDa by Western blot analyses using the
M was also found in the urine samples ig. 33A). ORpolyclonal anti-ORM antibody (F, but prominently in the urine samples y volunteershof patients on follow-up and healtof patients with bladder cancer, particularly in the invasive tumor stages as pT2-3.
y three times in 85 % of urineeased b volunteers ORM was incrCompared to healthysamples of bladder cancer patients on follow-up, by seven times in 83 % of patients
pT1, and twenty times in 71 % of patients with pTa, 16 times in 85 % of patients with with pT2-3.
4141 k kDDaa
4545 k kDDaa
AA
BB
HHHealtealtealthhhyyyFFFooollowllowllow---upupuppTpTpTaaa
HHHealtealtealthhhyyyFFFooollowllowllow---upupuppTpTpTaaa
pTpTpT111
pTpTpT111
pTpTpT222
pTpTpT222
pTpTpT333
pTpTpT333
Figure 33: Detection of ORM and ZAG in urine samples. Western blot analyses using polyclonal
anti-Ofollow-up RM antand in higheibody revealst ameount id ORM (An urin) ate sampl the referrees of d patients withsize of 41 invasive kDa in uribladdene sampler cancer stas of pages pTtients of 2-
ZAG at the expected 3. In similar pattern of ORsize of 45 kDM, westerna in urine blot analsamyseples usis (B) of patieng a polyclonal antints on follo-ZAw-up and pG antibody revealeatients withd
er canbladder. c
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4.2.2 healthyDetection of zinc-alpha-2-gly persons, patients with blcoprotein (ZAG) in urine samples of adder cancer and patients on follow-up
he size of 45 kDa in urine samples-glycoprotein (ZAG) was detected at tZinc-alpha2(n=45) of all bladder tumor stages classified as pTa, pT1, pT2, pT3 by western blot
anti-ZAG antibody (Fig. 33B). ZAG was also found in analyses using the monoclonal but prominently in eerson follow-up and healthy volunturine samples of patients er, particularly in invasive tumor stages h bladder cancurine sample of patients wit mples of healthy volunteers, ZAG wasasuch as pT2-3. Compared to urine ssample of bladder cancer patients on follow-increased by four times in 85 % of urine five times in 85 % of those s with pTa, by those patientup, by sixteen times in 75 % ofof patients with pT1, and twenty times in ients with pT2-3. 71 % of those of pat
4.3 bladder Expressiotisnsue pattern of ORM and ZAG in bladder cancer versus normal
an urinary bladder tissue comparing tumor e ORM and ZAG proteins in humTo localizes were performed on paraffin analysand normal tissue areas immunohistochemicalsections of bladder tissue. onsiderable staining in normal areas ofImmunohistochemistry for ORM revealed no c ge and small blood vessels larhereas endothelial cells ofbladder tissue (Fig. 34A) wy of tumor tissue (Fig. 35A) e proximitwithin tumor tissue (Fig. 34B) or in closexhibited ORM. In addition to the blood vessels, also some single cells, probably
inflammatory cells (Fig. 35B), within or around the tumor tissue were strongly positive
cularly those of invasive r cells exhibited ORM, partifor ORM. Also a part of tumo ZAG immunostaining was found at the tumor stages as pT2 and pT3 (Fig. 35A). 36A) which disappeared in superficial .luminal surface of normal urothelium (Figurrently, one or two cell rows at the basal bladder tumors as pTa (Fig. 36A). Concterestingly, the ZAG immunostaining was d ZAG (Fig. 36A). Inea expressside of pTmost prominent in tumor cells located at the invasive front (Fig. 36B) where tumor
ll marking the transition of tumor from de the lamina propria of bladder waacells invpTa to pT1 stage.
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there is nFigure 34: Lo consiocalizaderable tstaining for ORion of ORM in normal and bladder tumoM, neither in transitional r tissue. epithelium (TIn normE) nor in bloal bladdod vesseler tissues
(A). In contrast, blood vessels (arrows) of tumor tissue are strongly positive for ORM (B).
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Figure 35: Localization of ORM in bladder tumor cells. Vascular endothelial cells (arrows) and
exhibit ORMpositive for OR immunoM in closstaie ning (Aproximity of bladde). Inflammatory r tumor cellstissu (arrows) e, but also some tumwithin the tuor cellmor tissue s (arroalwso sho heads)w
). staining (BMRpositive O
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Figure 36: Localization of ZAG in bladder tumor tissue. ZAG immunostaining is localized at the
luminal suepithelium rfa(acrroe w headof normal transitios) when supenal eprficial blithelium (aadder trrowumor s) whisucch ah switchs pTes a is to thseee ban (A). Thsal side of e strongebladder st
staining of ZAG is found in tumor cells located at the invasive front (B).
4.4 immunocytochemistry Localization of ORM on endothelial cells (HDMECs) b
y
To determine whether the ORM staining in vascular endothelial cells due to
helial binding of ORM produced byendogenous production or only due to the endot
ltivated human primary microvascular immunostaining for ORM on cuother cells
These studies confirmed the resultsendothelial cells (HDMECs) were performed.
er tissue and revealed a strong stainingfrom immunohistochemistry on bladder canc
lls were mostly negative for ZAG (Fig. for ORM in HDMECs (Fig. 37A) while the ce
37B). ORM immunostaining was also present in cultivated bladder cancer cell line
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t, ZAG was not detectable in ECs (Fig. 38A). In contrasRT4 but weaker than in HDM
RT4 cells (ig. 38B). The presence of ORF
western blot analyses (Fig. 39A) whereas
extract of RT4 nor in that of HDMECs.
AA
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med by was confirM in endothelial cells
ZAG was neither detectable in protein
Figure 37Cultivated hu: Immunocyman dermal microvastochemical staining for cular endothlelial cellocalizats (HDMEion Cof Os) are RM anstrod ZAG in ngly positiveHDME for ORMCs.
(arrows, A), but are mostly negative for ZAG (B).
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line. Figure 38ORM staining is al: Immnucytso preochemical staisent in bladder ning for locacancerliz ceation oll line RT4, f ORM and but its expreZAGssion i in bladder cs weakaerncer c than inell
HDMECs (arrows, A). ZAG is not detectable in RT4 cells (B).
HDMHDMEECC
4141 kD kDaa
RT4RT4
kDa55 kDa55
HDMHDMEECC
4RT4RT
BABA Figure antibody 39: coDetecnfirm thet immuion of ORMnohi in HDMstochemical ECs and RTdata and4 shcell ow Oline. RM Western at the blot aexpenacted lyses usize ofsing 41anti O kDaR iMn
perfoHDMECrmed bys (A). immunobl ORM was notting usiot seen in blng adder caanti-vimentin antibody (B). ncer cell line RT4. The control of protein loading was
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versus ORM gene silencing in HDMECs nORM overexpressio4.5 us ORM esis, ORM overexpression verss the role of ORM in angiogenTo addres The effeciency was confirmed by gene silencing were performed in HDMECs. and protein extracts of were performed on supernatantwestern blot analyses which at 41 kDa in lysate of . ORM protein was detectable (Fig. 40)transfected HDMECs . 40A) and their supernatant (Fig. 40C)ng endothelial cells (FigiORM overexpresscted endothelial cells (Fig. ibody. Empty vector transfeonal anti-ORM antlusing polycin the literature hed data publiseviously40A, Lane1) produce ORM as described in pr the gher amount of ORM was detected inicantly hi(Sorensson et al., 1999). A signifprotein extract of ORM-overexpressing HDMECs in comparison to empty vector
transfected HDMECs (Fig. 40A, Lane2). But ORM expression was almost completely
or plus texpressing vec simultaneously with ORM reduced in HDMECs transfected gmed usinM siRNA was confirORM-siRNA (Fig. 40A, Lane3). The specificity of ORsiRNA for luciferase, which did not cause any significant reduction of ORM in
On the other hand, as shown in figure contrast to ORM siRNA (Fig. 40A, Lane4). HDMECs was detected inafter its overexpression in40C, the majority of ORM (Fig. 40C, Lane2). ORM was detectable ng HDMECsisupernatant of ORM expressctor transfected cells (Fig. 40C, Lane1). as a faint band in supernatant of empty ve
121212
4141 k kDDaa
AA
a55 kDa55 kD
333444
111232323
a41 kDa41 kD
C C
444
B B Figure 40cells (HDME: OvCs). Therexpression and gee ORM amount in necell extra silencing vcit of ORM-a siRNA overexfor pressiORM in humng HDMECs (A, Laan dermal ene2) indothse onlylial
slightly higher as in the control. The use of ORM-siRNA reduces the ORM amount significantly below
the endogenous level (A, Lane1). No change is seen by application of luciferase siRNA used as
negative control (A, Lane4). Control of protein loading for cell lysates was performed by
immunoblotting using anti-vimentin antibody (B). Immunoblotting using anti-ORM antibody reveals
high amount of ORM at the size of 41 kDa in the supernatnats (C) of ORM-overexpressing HDMECs
(C, Lane2), which is sucsessfully reduced by additional transfection with ORM-siRNA (C, Lane3) while
luciferase siRNA used as control did not change the ORM amount (C, Lane4).
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ed ORM protein amount (Fig. siRNA reducThe transfection of HDMECs with ORM 40C, Lane3) while luciferase siRNA which used as a negative control did not change
the ORM amount (Fig. 40C, Lane 4). in-vitro angiogenesis assays via studies 4.6 Mechanistic
4.6.1 ORMsilenced In-vitroHDMECs endothelial tube formation using ORM-overexpressing versus
M in angiogenesis and capillary morhogenesis,To find out the potential effects of ORin-vitro endothelial tube formation assay was performed.
AA
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enhaFigure 4nced by 1: combiEndothelial ned aptubeplicatio formatin of VEGF-A anon assay. d VEGF-inthe supernataducedn endt of ORothelial M-overexpressitubes (arrows; A) areng HDMECs
(arrows; B). The treatment with VEGF-A and supernatant of HDMECs transfected with ORM
expressing vector plus ORM-siRNA reduced the tube formation (C) in comparison to VEGF-A alone
(A) and VEGF-A plus supernatant of ORM overexpressing HDMECs (B). Untreated endothelial cells
a negative control (D). swere used a nd ORM-silenced HDMECs were used in The supernatant of ORM-overexpressing are transfected with empty vector, ORM endothelial tube formation assay. HDMECs weexpressing vector, and ORM siRNA using nucleofector technology as described in
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itive control which was used as a posmaterial and methods (3.2.3.5). VEGF-A as expected. The number and the ube formation (Fig. 41A)induced endothelial ted endothelial tubes were increased by combined network of VEGF-A inducapplication of VEGF-A and the supernatant of ORM-overexpressing HDMECs (Fig.
ECs transfected with supernatant of HDM41B). Combined application of VEGF-A,ORM expressing vector and ORM siRNA simultaneously did not affect VEGF-
ed as a treated endothelial cells were usinduced endothelial tubes (Fig. 41C). Un negative control and did almost not induce tubes (Fig. 41D).
nd PAI-1 in endothelial tube formation The interaction between ORM a4.6.2
l -1 and ORM on the endotheliaed of PAI a complex composSince it was reported that endothelial cells, it was aimed to study es the behaviour ofcells surface influency between ORM and PAI-1 in capillarpotential effect of the interaction re cultured and at weendothelial cellsmorphogenesis. To this aim, untreated ed until confluence. llagen gel and culturpassage 4 the cells were seeded onto coAfterwards, endothelial cells were stimulated with: (i) ORM, (ii) VEGF-A, (iii) anti-PAI-
ion of these factors. Two different alone and with different combinat1 antibodyre used for stimulation of HDMECs. concentration of ORM as 100 and 300ng/ml wedium were used as negative control and Endothelial cells exposed only to basal meused as a positive control and induced did not form tubes (Fig. 42A).VEGF-A was endothelial tube formation as also mentioned in tube assay studies above (Fig. 42B).
most not induce tube formation as also ORM, at 100 ng/ml concentration, alone did al h anti-PAI-1 antibody. 42C). Treatment witobserved in cases of untreated cells (Figk PAI-1 protein, induced the formation of a few ed to blocalone, which was usig. 42D). To test the effects of the endothelial tubes in contrast to ORM alone (F stimulated with these nd anti-PAI-1 the cells werecombined application of ORM afactors alone or in combination with VEGF-A. The application of ORM at 100 (Fig.
eased the VEGF-A A simultaneously incr43A) and 300 ng/ml (Fig. 43B) plus VEGF-depending on the applied concentration of ORM. induced endothelial tubes PAI-1 increased the network and the Combined application of VEGF-A and anti-number of VEGF-induced tubes too (Fig. 43C). Treatment of endothelial cells with
few tubes (Fig. 43D). The combined ORM at 300 ng/ml and anti-PAI-1 induced a lyM, anti-PAI-1, and VEGF simultaneousapplication of all three factors as ORtube formation. The application of anti-exhibited the strongest effect on endothelial
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in combination with VEGF-A increased PAI-1 and ORM at 300 ng/ml (Fig. 44B,C)
n of VEGF-A and 100 ng/ml of ORM with than the combinatiomore endothelial tubes
anti-PAI-1 (Fig. 44A).
AAA
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DD
to untreFigure 42ated : EffeHDMEct oCf Os, as RMnegative and ancontti-PAIrol (A-1 an), andtibody VEGF-on endoA treatetheliad celll tube s, as poformsitive coation. ntrol In com(B), OparisRonM
(C) did not effect endothelial tubes, while treatment of antibody against PAI-1 formed several
endothelial tubes (D), but it is not a considerable effect. Wild type HDMECs were stimulated with 100
ng/ml ORM protein and 15 ng/ml of anti-PAI-1 antibody.
Res sult
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appliFigure 4cation of3: Effect o VEGF-A andf ORM a ORM proteind an,nti-PAI-1 at two diffeat VEGF-indurent concceentrad endotions asthelial tubes. 100 (A)and 30 By c0 ngomb/ml (B), ined
the numbers of VEGF-induced endothelial tubes are significantly increased in correlation with applied
ORM VEGF-indconcuecentration. Thd endotheliae treatment with l tubes in comparisVEGF-A pluon to OsRM appli anti-PAI-1 acation (arrontibody induws, C). Combinces a higheed ar increpplicationase of
of 300 ng/ml of ORM and anti-PAI-1 antibody does not effect endothelial tube formation (D).
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Figure 44VEGF-indu: ceEffecd endotht ofelial VEGF, ORM tubes are eaxnd antiteremly incr-PAI-1 combinaeased by cotmbined ion on endappliocthelial tubation of Oe RformatioM, as 10n. 0
ng/ml (aVEGF-indrroucews, A) and 3d endothelial 0tubes 0 ng/ml cocorrelncentration ates with OR(aM rrowconcens, B,C) witration. th anti-PAI-1 antibody. Increase of
dothelial tubes -vitro induced enImmunolocalization of ORM in in4.7 Endothelial tubes on collagen gel which were formed by wild type HDMECs after
re fixed eing factors such as ORM, VEGF-A and anti-PAI-1, wtreatment with stimulatOn paraffin sections of 5-night and embedded in paraffin. using Bouin´s fixative over 7 μm thickness obtained from paraffin embedded tissue block of in-vitro induced
performed using polyclonal anti-ORM endothelial tubes immunhistochemistry was a weakcells and tubes on collagen gel revealedantibody. Untreated endothelial staining for ORM (Fig. 45A) in comparison to endothelial tubes induced by VEGF-A
(Fig. 46A). There is no immunostaining in control sections exposed only to secondary
1ubes induced by treatment with anti-PAI-antibody (Fig. 45B; Fig. 46B). Endothelial tORM (Fig. 47A) in comparison to tubes alone exhibited stronger immunostaining for VEGF and anti-PAI-1 (Fig. 47B). induced by combined application of
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UnFigure 45stimulated: Localiza wild type tionHDMECs of ORM o(arron wwis) in ld tcollypeag endothen gel erevealed a lial cells afteweak str tube formatiaining for ORM (Aon assay). In.
control section, no staining is seen (B).
induced by treatment ion of endothelial tubes ed in control sectNo staining was detectlication of ORM at by app Endothelial tubes induced with anti-PAI-1 alone (Fig. 47C).taining (Fig. 48A) in comparison to mmunos100 ng/ml alone exhibited stronger ORM ium (Fig. 45A). But the treatment of ediendothelial cells treated only with basal m and anti-PAI-1 revealed strongest ORM endothelial cells with ORM at 100 ng/mlimmunostaining on endothelial cells (Fig. 48B,C). Interestingly, in the case of
entrationh the same combination but a higher concwittreatment of endothelial cells on endothelial tubes was almost weakerof ORM as 300 ng/ml ORM immunostaining ,B). The control section of endothelial than the use of ORM at 100 ng/ml (Fig. 49Aed only to the PAI-1 which was expostubes treated with ORM at 300 ng/ml and anti-unostaining (Fig. 49C). Immunostaining secondary antibody did not show ORM imm hl tubes treated witobtained from endotheliafor ORM was most prominent in sections PAI-1 antiobody and VEGF-A simultaneously (Fig. 49D). ORM at 300 ng/ml, anti-network of endothelial tubes which were To define exact changing in number and g on -1 plus VEGF-A, during their growin treatment with ORM plus anti-PAIinduced byeasured. After the cells have endothelial tubes were mcollagen gel, the length of been stimulated with the factors ment of wellsioned above the photographse contrast taken every day using phascontaining endothelial collagen gel were microscope
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immunoFigure 46staini: Ing for mmunostainORM on ening for OdothelRial cellM on VEGFs form-induceing VEGF-id ennduced tubdothelial tues,bes. whi
positive control (arrows, A). Control section is negative for ORM (B).
89
cTheh rwee ires po used asitives
th of each (Zeiss, Jena). The endothelial tubes were marked with arrows and the leng
ed by a factor or a of all tubes inducendothelial tube was measured. Total lengths
combination of factors were determined
summarized in table 20.
for each s
mple and the values werea
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Figure 47: Localization of ORM on endothelial tubes treated with anti-PAI-1 antibody. ORM is
strongly positive in endothelial cells forming tubes by blocking of PAI-1 (arrows, A) in comparison to
combined application of VEGF-A and anti-PAI-1 antibody (arrows, B). The control section shows no
ng (C). stainiific cspe
The quantification of mentioned above revealed that the endothelial tubes as
ount as ml tubes in considerable aapplication of VEGF alone induces endothelia
al medium as al cells treated with basexpected (Tab. 20, column2) while endothelia
lial tubes (Tab. 20, column1).negative control did not form endothe
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anti-PFigure 4AI-18: L anocalizatibodyt. ion ofEndothelial ORM oncell ens formingdothelial tubetubs byes tr ORMea ted stwimuilth ORation (100M in lo ngw c/ml) aonrcene sigtratnion anificantlyd
againspositive for Ot PAI-1 shoRM (aws stronrrows, A), whileger immuno the applistaining for Ocation ofRM (arro the same Ows, B, C).R M concentration with the antibody
was capable to form a few alone to endothelial cellsation of anti-PAI-1The applic
while application of ual addition of ORM, tubes (Tab. 20, column4) but not the individ
ORM at 100 ng/ml plus VEGF enhanced tnduced endothelial he length of VEGF-i cantly (Tab. 20, column5).itubes signif
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CCC
BBB
DDD
92
Figure 49: Localization of ORM on endothelial tubes treated with ORM in high concentration
wleadith ans to a weti-PAIake-1 anr stainitibodyng . Interefor ORM ostingly, applin endothcelial tation ubes of ORM compatare 3d00 ng/ml to the samwiteh combi anti PAI-1 nation with low antibody
constroncegenstrationt stai of Oning for ORM atR 1M00 ng in endothelia/ml (arrows, A,B). l tubes is seTenhe control se by treatmection isnt of endothe negative lial cellfor ORs with M (C). VEGF, The
i PAI-1 antibody (arrows, D). 0 ng/ml and antORM at 30
bes was achieved when VEGFlength of endothelial cells tuAdditional increase of the
re applied to endotheliaplus ORM (100 ng/ml) plus anti-PAI-1 weollagen l cells on c
othelial tubes wasease of VEGF-induced endgel (Tab. 20, column9). The most incr
I-1 and ORM in 300 ng/ml AVEGF, anti-Pseen by the combined application of
concentration (Tab. 20, column8). Also tof ORM (300 ng/ml) application he combined
s sultRe
with anti-PAI-1 was capable to induccolumn10).
m)m)mm((ngthngth Le LeubeubeTT
e endothelial tube formation (Tab. 20,
1 1 2 2 3 3 4 4 5 5 6 6
93
1 1 2 2 3 3 4 4 5 5 6 6 7 7 8 8 9 9 1 100
Table 20: Graphical representation of determination of tube lengths. Representation of tube
lengths formed by endothelial cells treated only with basal medium (1), with VEGF-A alone (2), with
100 ng/ml of ORM alone (3), with anti-PAI-1 (15 ng/ml) antibody alone (column4), with 100 ng/ml of
ORM and VEGF-A simultaneously (5), with 15 ng/ml of anti-PAI-1 antibody and VEGF-A
simultaneously (6), with ORM at 300 ng/ml and VEGF-A simultaneously (7), the application of ORM at
300 ng/ml and anti-PAI-1 antibody in combination with VEGF-A (8), with 100 ng/ml of ORM, anti-PAI-1
antibody and VEGF-A simultaneously (9), with 300 ng/ml of ORM and anti-PAI-1 antibody
y (10). sleousimultan
sion sDiscu
5. Discussion
94
e urine lycoproteins in thification of new proteins/g provides the identThis studysample of patients with cancer of urinary bladder by combined application of
th subsequent mass spectrometric analyses, proteomic technology such as 2-DE wi chniques such as immunoblotting,conventional protein research te of bladder tissue, geneticsectionsochemical studies on paraffin immunohist in-vitro silencing via siRNA technique and finallyapproaches including gene ons assay on collagen gel. In tube formatimechanistic studies such as endothelial established for appropriate separation ofthis study, the optimum conditions were obtained y on urine samplesication of 2-DE technologurinary proteins for the applcancer of different stage as well as s with bladder from healthy persons and patientamong y, two urinary proteins, ORM and ZAG,patients on follow-up. Going this waother proteins found in the urine samples, which were not known to have a functional
relation to the stages of bladder cancer and particularly to angiogenesis until now,
focussed on the functions of ORM in rlywere identified. This work was particulaangiogenesis, genetic and he potential role of ORM in endothelial cells. To explore tapillary formation assay were studies such as endothelilal cin-vitromechanistical performed.
5.1 The impact of 2-DE for characterization of urinary proteins
fluids including in urine. In the last few years, new technologies Among them a widely used metwere developed to identhod is the technique of theify proteins in body
coupled with MS/MS (Spahr entire protein followed by LCproteolytic digestion of the et al., 2001). Surface-enhanced laser desorpsion/ionization (SELDI)-MS has been
utilized to profile urinary proteins as an alternative high-throughput approach (Rogers
Among these techniques 2-DE with subsequent et al., 2003; Schaub et al., 2004).MS analysis is the more effective method for generating reproducible results in
human proteome pattern and protein identification in body fluids including urine. This
method has an advantage over the technique of proteolysis with subsequent MS/MS
analysis due to a 2-DE which can show the relative protein expression profiles with
molecular weights and pI.
sion sDiscu
95
5.2 powerful tool for identiOptimized separation of urinary prfication of urine proteins oteins for performing of 2-DE as a
was to view complete protein pattern of At the beginning of this study the first aim address this aim this studyurine samples and to predict the function of proteins whicfocused on developing an effective sample preparation h are present in urine. To
strategy for 2-DE. Additional samDE images, but each additional step can resuple preparation stepslts in the selectiv can improve the quality of 2-e loss of protein
Malasit, 2005).species, which is disc To avussed in seoid loss of proteins, a sampleveral studies (Oh et al., 2004; preparation strategy was Thongboonkerd and chosen
e this preparation methods and of the 2-DE arples of ias simple as possible. The princdescribed in section of material and methods. e been of protein spots havlarge numberUntil now, through extensive studies, a specific breakdown products ofof them are charge-isoforms oridentified, but many abundant proteins swhether the pattern of these breakdowuch as albumin, fibrinogen, immunoglobulin, n products shows a diseasetce specific. However,
stion but needs future analyses. In the composition or not is an interesting queliterature there are,abundance of proteins and the co however, limited proteomic datmplex nature of urine. Ta on human urine due to low he majority of proteins
ed from cytoplasm and have functional urine are originatwhich are present in normal 2002; Oh et al., nderson and Anderson, oteins (Aproperties similar to plasma pr of the urine samples are:ations of proteomic analysis 2004). The current certain limit(i) the total protein amount is low, (ii) few proteins present in urine samples in a
urinary proteinss in identification ofrelatively high amount may lead to difficultie(iii) urine cpresent only in very low concentrationsontains high amounts of body filtra and the locations of tes such as water, salts, electrolytes expressed proteins and
ich disturb 2-DE process. and nitrogenous waste products whdes and techniques to achieve a higher There are several studies describing metho ch as dye- or solvent-precipitation,protein concentration in urine samples suultracentrifugation, lyophilisation and the removal of the proteins abundantly present
nkerd et 1998; Pang et al., 2002; Thongbood Williams, mples (Marshall anin urine saultracentrifugation technique was applied al., 2004). To overcome these difficulties the salt, electrolytes, and small moleculesusing a centrifugal filter column to removete urine samples. Furthermore, the ra(<10 kDa) and as well as to concent mples prior to theirar prepare the urine sdevelopment of novel methodologies to bette
sion sDiscu
96
able studies were Although, consider was a necessary step.esuse in 2-DE analysoaches st of these appran urinary proteome, mopublished on mapping the hum r to its use for 2-DE analyses such asrequire many steps for urine preparation prioprecipitation methods, ultracentrifugation, removal of gylcosaminoglycans, dialysis
the most abundanten immunosubtraction ofsteps, lyophilisation, gel filtration, evoonkerd and Malasit, 2005). e-fractionation tools (Thongbproteins and/or other pr Recently, after publishing the data presented in this work, a novel method of solidphase bas ty ligands under controlledon of proteins for affinied, selective adsorptioncentration of low-abundance ease the ccrsaturation conditions was described to inon of high-abundance proteins from same proteins and to decrease the concentratiher improvement in protein hulasiraman et al., 2005). A furtep (Tmixture in a single st recently described using beads coated concentration/equalization technique was alsoastagna et al., 2005) and in that study with hexameric peptide ligand libraries (Cproteins has been reported. additional 251 hidden urinary ed in literature at that methodological knowledge reportUnder consideration of the several techniques were performed at the time of the study presented in this work, ion methods (TCA, tsuch as different protein precipitabeginning of the experiments rehydration buffers. We applied by testing the use of different acetone, etc.) followedultracentrifugation as a simple method to avoid loss of proteins and alteration in
for urine samples this sample preparation method usednature of urine. Efficiency ofnd high-resolution human urine proteome was enough to obtain a reproducible apattern in 2-D gels. However, for further extended studies on urinary proteins,
ques mentioned above tohe novel technimbined with tultracentrifugation can be coimprove the protein separation in 2-D gel. 5.3 bladder cancer Urine proteomics enables the identification of proteins related to
ive and opic evaluation, which is expens cystoscBladder cancer is diagnosed byinvasive. Urine cytology is the current non-invasive gold standard but it has low
tzenberg, 2001; Murphy et al., 1984). The search for sensitivity (Konety and Ge e cystoscopy as a diagnosticentially replacbladder cancer biomarkers that could poterogeneity of this by the molecular hetand surveillance tool has been complicated on urine proteomics as a source for a e focused disease. Numerous studies havd SELDI-TOF-MS to urine lieVlahou (Vlahou et al., 2001) apppotential biomarker.
sion sDiscu
97
e several carcinoma (TCC). Up to datll bladderesamples to detect transitional c related to bladder cancer, are described h are supposed to beurinary proteins, whic urinary calreticulin (Kageyama et al.,arker for bladder cancer such asas a biomear matrix protein lis et al., 2004), nucl2004), calcium-binding protein psoriasin (Ce tumor antigen, and telomerase (Ramakumar et al., 1999). 22, bladder 2-D gel ation and of protein preparAfter optimization of the techniquesses on a we performed extensive analyesis on human urine samples electrophor er, andpersons, patient with bladder canclarge number of urine samples of healthy oteomic map of normal human urine with on of pr Comparispatients on follow-up.w and patients on follow-up revealed fe cancer h bladderurine of patients witThe major differences were seen especially but significant differences. similaritiesients and healthy persons. The similarities concerned between urinary pattern of patsome protein spots at 55 kDa, which were later identified by mass spectrometric
a which were present in all urine analyses as human serum albumin, and 66 kDsamples. 5.4 using 2-DE mayNovel protein spots in urine sampl be of diagnostic relevance es of patients with bladder cancer
this study with already the proteins identified inhe list ofThe comparison of t et al., 1996; Grover lises (Castagna et al., 2005; Cepublished urinary protein databasal., 2004; Pang et al., 2002; Pieper et al., 2004; and Resnick, 1993; Oh et garding erevealed similarities r Rasmussen et al., 1996; Thongboonkerd et al., 2002)in the urine abundantly and knowingly being present the detection of few proteins etc. Among a high min, immunolglobulin,deriving from blood plasma such as albu of the 2-DE gel only a small ilver stainingbeing identified in snumber of protein spots ning. These protein spots which have been part was also detectable in the CBB staiesent in the urine samplesentially up-regulated or only pridentified to be either differ of patients with bladder cancer were cut out from the gel and analyzed in mass
ation and ible identifice analyses led to the reproducspectrometric analyses. Thesns such as ORM and ZAG. characterization of protei 5.5 ORM is increased in urine samples of patients with bladder cancer
Prior to the discussion of the impact of ORM in bladder cancer it is necessary to give
scribed in 1950 by Schmid (Schmid, 1950;a brief survey about ORM. It was firstly de
sion sDiscu
98
e phase proteins playing a role in the Schmid, 1953) and belongs to a group of acut to stress, along with many othermodulation of the immune response The relatively high . id, 1980; Schmid, 1975)functions(Bennett and Schmmans is known to rise two- to concentration of ORM in the serum of healthy hu such as acute infection, inflammatory andfivefold in response to different conditions .s and cancer (Schmid, 1975)lymphoproliferative disordera polypeptide chain ed and is composed of The structure of ORM is well characterizacid as mentioned above. containing about 45 % carbohydratThus, its proposed immunomodulate including a large amount of fucosylicory activities have been and sialic
e the strongly fucosylated and sialylatedattributed to its glycosylation pattern, sincE-selectin and to inhibit complement ORM glycoforms have the ability to bind activation (De Graaf et al., 1993). An induced expression of sialyl Lewis X (sLeX) on
might influence the E- or ORM during acute inflammation has been rP-selectin-mediated influx oeported, leading tof sLeX-expressing leukocytes the speculation that it
expressiinto inflamed areas.ng ORM could have a feedback inhibi It has been suggested that an increastory effect on the extravased level of sLeX-ation of
ORM is thought to modify the pleukocytes, by competition for thee E-selermeability ctin adhesion moleculesof the vascular endot (Lasky, 19helium, possibly by 92). So
interacting with the endotheliabeen shown that ORM binds to the vasculal glycocalyx (Curry and Michel, 1980). Thus far it has r endothelial cell surface and then causes
intercellular junction (Predescu et al., e cell without passing the transcytosis across th1998). is elevated in different types of ORMIt has been shown that the serum concentration al., 2000) leading to the lung and ovary cancer (Duche etof cancers such as breast, cancer cells themselves. The present assumption that ORM might be produced by tumors, sion in urinary bladderM expresdata here show a significantly increased ORy, the detection of er cancer. Consequentl in the invasive stages of bladdparticularlyadder cancer patients, as eins in the urine of bllarge amount of ORM protcreased production and secretion of ORM. tes an inademonstrated in this work, indicnumber of studies report about ORM glycoform determination In the literature, a high such as colorectal (Hansen et al., 1987)in serum diagnosis of different cancer types, However, only a few studies are and ovarian cancer (Dobryszycka W., 1992). available concerning the changes of ORM levels in the urine of patients. It has been
sion sDiscu
99
ute inflammation (Pang et al.,nts with acobserved to be increased in urine of patie .betic patients (Narita et al., 2004)2002) and of normoalbuminuric type2 diaAfter being identified in 2-DE of the urine analyses performed in this study also
vel of ORM in the urine samples ofed leimmunoblotting studies revealed an increaspatients with bladder cancer and patients on follow-up. To my knowledge this is the
first report showing that ORM is samples of patientsantly increased in urine significwith urinary bladder cancer, particularly in those with invasive tumor stages. The
patients with acute ved to be increased in urine of level of ORM has been obser patients 2 diabetic inflammation (Pang et al., 2002) and of normoalbuminuric typehe results presented in 2-DE and t(Narita et al., 2004). Correspondinglyimmunoblotting studies here, the present immunohistochemical results suggest that,
ent inflammatory cells and, interestingly,in addition to cancer cells, some tissue residendothelial cells of angiogenicly activated blood vessels which are to find in close
increase of ORM in the urine ofas source for thisssue serve association to tumor tit increase in the mean protein would explain the highesbladder cancer patients. This1, which marks the switch from a non-amount of ORM at tumor stage pTa to pT invading the lamina proria where the tumor to a tumor typevascularized superficial tumor with activation of to blood vesselsget direct accestumor cells son et al. the previous results presented by Soerensneovascularization. Confirmingngs show that human vascular endothelial (Sorensson et al., 1999), the present findie of ORM in endothelial cells esencly. Thus, the prcells produce ORM endogenous obably forming new tumor vessels asactivated by tumor angiogenesis or prthe endothelial-bound ORM but also ORM demonstrated here does not only reflect haracteristic phenomenons of clls. One of the essential originating from endothelial ce is the abnormal vascular leakage orlly activated blood vessels aangiogenic ORM has been reported to modify the permeability (Hashizume et al., 2000). Indeed,x (Curry ing with the endothelial glycocaly by interactendothelial permeability, possibly al., 1998). Serum level of ORM is increased in and Michel, 1980; Predescu etinflammatory and lymphoproliferative disorders and cancer, such as breast, lung and
lved the id, 1975) which might be invoovary cancer (Duche et al., 2000; Schmmodulation of vascular leakage. ajor carrier of sialyl Lewis X a mructure is that ORM is A further aspect of ORM st acute inflammation. This finding led to the speculation that particularly during(sLeX), via this carbohydrate moiety ORM might be able to bind to E- or P-selectin and might
sion sDiscu
100
influence the binding and rolling as well as extravasation of leukocytes into inflamed
ed that CEACAM1, as recently demonstratky, 1992). Similar to ORM, it waareas (Las carrying sLeX and Lewis X residues andosylated cell adhesion moleculehighly glyc ndothelial cells of new immature blood is up-regulated in eacting pro-angiogenic, eira-Ferrer et al., 2004). Thisr stages (Olivly tumovessels of bladder cancer in earicly les, which are present in angiogenstructural similarity between both molecumilar to CEACAM1 also ORM ition that sactivated blood vessels, leads to the assumption. To aegulation of angiogenesis and tumor vascularizmight be involved in the rogy and angiogenesis, ORM ORM in endothelial cell bioladdress the potential role of a siRNA in HDMECs were performed. sion and ORM gene silencing vioverexpres ental manipulations, genetically modifiedimAfter testing the efficiency of these exper (endothelial tube formation). rmation assayHDMECs were used in in vitro capillary fo 5.6 ORM support the VEEndothelial overexpression of ORM as GF-induced endothelial tube formation well as endothelial stimulation by
Until now it has been shown that ORM binds to endothelial cell surface (Boncela et
M plays a role in capillary ether ORal., 2001) but it is so far unclear whcells. The results presented here morphogenesis provided by endothelial the overexpressed ORM in HDMECs isdemonstrate clearly, that the majority ofing suggests that probably ORM produced secreted into the supernatant. This find as might also be mainly secreted into the extrendogenously by endothelial cells and in endothelial d in cultured HDMECcellular space and thus, the ORM detectein ORM r blood vessels might mainly result activated tumocells of angiogeniclybinding on endothelial cell surface. Also data obtained by endothelial tube assay as
While the use of HDMECs transfected for presented here support this interpretation. significant differences in comparison toORM in endothelial tube assay did not lead to the simultaneous,stimulated by VEGFthe wild type HDMECs when they were stimulation of wild type HDMECs by the supernatant of ORM overexpressing
and network of endothelial gnificantly increased length i and VEGF led to a sHDMECstubes in comparison to the stimulation of HDMECs by VEGF alone. This effect was
partly reversed when endothelial cells on collagen gel were stimulated
simultaneously with supernatant of ORM silenced HDMECs and VEGF indicating that
the ORM-siRNA used here is to some extent effective in the in-vitro mechanistic
ECs transfected for ORM and ORM siRNAstudies. Since the supernatant of HDM
sion sDiscu
101
by application ofdothelial tubes induced simultaneously was not able to block enVEGF completely one can assume that either the ORM-siRNA was not effective
enough to block the endogenous production of ORM totally or there are other factors
ORM in endothelial tube formation. replacing the functional effects of 5.7 ORM interaction with PAI-1 influences the VEGF-induced endothelial tube
formation To gain more insight into the function of ORM in capillary morphogenesis, further in
vitro endothelial tube formation studies were performed to investigate the role of the
M and PAI-ly it has been shown that ORinteraction between ORM and PAI-1. Recent ly influences thel cells which obvious1 form a complex on the surface of endotheliamigration of endothelial cells (Boncela et al., 2001). These studies show that it is the
r-1 (PAI-1) which constitutes the complex otor inhibitaactive form of plasminogen activtive form of PAI-1 suggesting complex stabilizes the acwith ORM. The building of thisy effect of PAI-1. The ORM-he inhibitorthat ORM-PAI-1 complex would increase tPAI-1 co-localization was found in thymosin β4 (Tβ4)-activated but not in quiescent
nce the helial cells). ORM may influebilical cord vein endothuman umHUVECs (ll as their ability to generate plasmin e properties of endothelial cells as weadhesivinase type plasminogen activator (uPA) in al., 2001). The role of the urok(Boncela et rature (Andreasen et al., 2000; Binder etangiogenesis is widely described in the litesapillai, 2003; Chorostowska-Nade al., 2001; Choong and al., 2007; Boncela etko et al., 2004b). While the generation Wynimko et al., 2004a; Chorostowska-Wyniml for the regulation of ssentia action of uPA or tPA is eof plasmin from plasminogen byented that uPA plays a crucial stem, it is well documion sythe activity of the coagulatuding the vascular basement the extra cellular matrix inclrole in the remodelling of membrane normally underlying the endothelial cell layer and enclosing the pericytes
of the basementA well construction in capillaries (Andreasen et al., 1997). integrity of the vascular wall and also formembrane is of importance for the structural ep during the new tich is an essential smigration activity of endothelial cells whformation of blood vessels by angiogenesis (Folkman and D'Amore, 1996). The pro-
cked by PAI-1 (Andreasen et al., 2000; effects mediated by uPA are bloangiogenicn to interact with uPA and uPAR howBinder et al., 2007) which has been sharacter This interaction shows a dual c(Andreasen et al., 1997; Binder et al., 2007).as demonstrated in figure 50: (i) The interaction of PAI-1 with uPA and uPAR leads
102sion sDiscuto the internalization of uPAR and its subsequent recycling and translocation to the
e of endothelial migration and in an increasmembrane at the cellular tips which resultangiogenic activity. (ii) The presence of ORM results in the building of a complex with
PAI-1 at the endothelial cells surface which stabilizes the activity of PAI-1 leading to
cycling which consequently result in the ation and rethe blockage of uPAR internalizand angiogenesis (Andreasen et al., 1997). suppression of endothelial migration
EndotheEndothelliiaallCCeellslls
AAnormal procnormal proceessssBB+ORM+ORMCC+O+ORMRM
uPuPAA--PAPAII--11-uP-uPAARRuuPPA-A-uuPPARAR--PPAIAI--11--ORORMMPAPAII--11--ORORM M
vvlevileviaaeell LDL LDLooff P P re reAAccII--ee1 1 ptptoror
dependedependennttStabStabililiizzaattioionnooff P PAAI-I-111-IAP-itn+a1-IAP-itn+a
uPuPAARRinterninternalalizatizationionFrFree ORMee ORM
uPuPAARRiinnterternnalalizizatationion
uPuPAARRrereccyyclingcling+VEGF+VEGF
uPuPAARRrereccyyclingclingtutuMiMibegbegrraattforforioiommnnat aat aininooddnn
MiMiggrraattioionn
MiMiggrraattioionn
aaccttiivvaattiioonn
ssuupppprreessiioonn
aaccttiivvaattiioo
nnANGIANGIOOGGEENNESESISIS
Figure 50: Important interactions within the uPAR-uPA-PAI-1 systems and the role of ORM.
Normally uPresulting in acA andtivation of endotheli uPAR interaaction ll migration aeads to ind antengiogenernalization of uPAR sis (A). In the presenand then recyce of ORcling of M there is auPAR
recycomplecling. Thx of ORM ais results nd PinAI-1 whic inhibition of endothh stabilizes PAI-1 and elial migratiosuppn anressed ansgioge then unesis (B). PAR internThealiz additionation anald
applicof free ORM acation ofting anti-PAI-1 which blowith VEGF-A togetcks the comher proangipleogenicx building of PAI-1 and O and increases the RM increaseendothelial migration s the amount and
capillary formation, thereby, promotes angiogenesis (C).
sion sDiscu
103
antibody as used here in ication of the anti-PAI-1 r, the additional appleHowevbuilding of ORM-PAI-1 complex with he endothelial tube formation assay blocks te the angiogenic activity of endothelial probably two consequences which increasM and PAI-1 results in lex building between OR) The blockage of compicells: () the inhibition of ORM-iion as described above, and (atienhanced uPAR internalizly act pro- free ORM which obviousPAI-1-complex increases the amount ofangiogenicly when applied in combination with VEGF and enhances the VEGF-
. Thus, it is conceivable to postulate a role ofin-vitro induced capillary morphogenesis-1 in the vascular morphogenesis. the interaction between ORM and PAI 5.8 Zinc-alpha-2-glycoprbladder cancer particularlyotein is incr in the invasive stages eased in urine samples of patients with
that ZAG is detectable in an increased demonstrates for the first timeThis study e tumors ofwell as invasiv patients with superficial as amount in the urine samples of demonstrate that unohistochemical studiesurinary bladder. Interestingly, our immpresence of ZAG at the luminal surface of normal transitional epithelium of urinary
the stage pTa ise a papillary tumor ofbladder switches to the basal side onc est immunostaining for ZAG was found inobserved. Since in our studies the strongtumor cells invading the lamina propria of the bladder wall, we suggest a relationship
rther studies. In contrast to ORM, between tumor invasion and ZAG, which needs fu the superficial tumor stage pT1 to thee of ZAG is seen from the strongest increasassumption that the ZAG enhancement is tumor stage pT2, confirming our einvasiv ent of an invasive tumor and not with angiogenesis,associated with the developmwhereas ORM increase seems to be related to angiogenic activ tion as discussedae need further studies on a higher ed her involvabove. The exact mechanismse protein present in the lbine samples. ZAG is a solunumber of tumor tissues and urserum, and has also been found in the cytoplasm of normal secretory epithelial cells,
, as well as in salivary, bronchial, east, prostate, and liverincluding those of br gi and Schmid, 1961; Tada et al., 1991).gastrointestinal, and sweat glands (Burral types of malignant tumors (Brysk et Increased expression of ZAG occurs in seve vel of ZAG has been used as a canceral., 1997; ez-Itza et al., 1993) and the serum lemarker. Although the biological functions of ZAG remain largely unclarified, a
noma, MAC16, leading to purified from murine adenocarciing factorlipidmobilizcachexia, and from the urine of patients with cancer cachexia has been shown to be
sion sDiscu
identical to ZAG (Todorov et al., 1998).
sion of ZAG in adipocytes enhancesoverexpres
(Bing et al., 2004).
More recently, it was shown that
tumor-associated
104
cachexia in mice
Consion luc
6 Conclusion In summary, the present results demonstrat
105
e that the combined use of proteomics
ology approach and mechanistic with molecular cell bitechnology assays in-vitro
der as actors related to disoreins and fool to detect new protserve as a powerful t
er and to clarify their role in the bladder cancshown here for human urinary
M plays aSpecifically, the findings here show that ORpathogenesis of the disorders.
morphogenesis which might be of relevanceclear role in the VEGF-induced capillary
for tumor vascularization and anti-angiogenic tumor therapy.
Literature
Literature 7
106
Agirbasdisease. Int. J. Clin. Pract. li,M. (2005). Pivotal role of pl59, 102-106. asminogen-activator inhibitor in vascular
tantinou,J. (1993). ., Ge,X., and PapaconsAlam,T., An,M.R., Mifflin,R.C., Hsieh,C.Celement bytrans-activation of the multiple isoformsalpha 1-acid gly of C/EBP ancoprotein d glucocortigene accoid receptute phase reor J. Biol. Chem. sponsive
, 15681-15688. 268
Anderson,character, and diagnostN.L. and Anderson,ic prospects. Mol. CN.G. (2002). Tell Proteomics., 845-867. he human plasma proteome: history,
Andreasen,system in tumor growth, invasion,P.A., Egelund,R., and Petersen, and metastasis. Cell Mol. Life Sci. H.H. (2000). The plas57, 25-4minogen activation0.
Andreasen,plasminogen activator system in cancer P.A., Kjoller,L., Christensen,L., metastasis: a reand Duffy,M.J. (1997). The urokinase-type view. Int. J. Cancer 72, 1-22.
helial progenitor cells for postnatal Asahara,T. and Kawamoto,A. (2004). Endot, C572-C579. 287vasculogenesis Am. J. Physiol Cell Physiol
Pappin,D.J., and Selby,P.J. Banks,R.E., Dunn,M.J., Hochstrasser,(2000). Proteomics: new persD.F., Sanchez,J.C., Blackstpectives, new biomedical ock,W.,
, 1749-1756. 356opportunities., Lancet
Baumann,protein genes by hepatocyte H. and Gauldie,J. (1990). Regulatstimulating factors and other ion of hepatic acute phase plasmamediators of inflammation.
, 147-159. 7Mol. Biol. Med.
Baumann,Stimulation of hepaticH., Prowse,K.R., Marinkovic,S acute phase response ., Won,K.A., and Jahreis,G.P. (1989). by cytokines and glucocorticoids. Ann.
, 280-95, discussion. 557N. Y. Acad. Sci.
Baumann,stimulating factors, interleukin 1,H., Richards,C., and Gauldie,J. and glucocorticoids for regulation of acute phase (1987). Interaction among hepatocyte-
, 4122-4128. 139oma (HepG2) cells J. Immunol. plasma proteins in human hepat
recommendations for measurement in clinBeetham,R. and Cattell,W.R. (1993). Proteinuria: pathical practice. Ann. Clin. Biocophysiology, significance and hem. 30 ( Pt
, 425-434. 5)
Bennett,M. and Schmacid glycoprotein: importance id,K. (1980). Immunosupprof the carbohydrate moiety. Pression by human plasma alpha 1-oc. Natl. Acad. Sci. U. S.
, 6109-6113. 77A
signaling: a vascular biologisBinder,B.R., Mihaly,J., and Prager,G.W. (2t's view. Thromb. Haemost. 007). uPAR-uPA-PAI97, 336-342. -1 interactions and
Literature
107
Tisdale,M.J., and Manieri,M., Cinti,S., Bing,C., Bao,Y., Jenkins,J., Sanders,P., in adipocytes and isTrayhurn,P. (2004). Zinc-alpha2- up-regulated in mice witglycoprotein, a lipid mobilizh cancer cachexia Pring factor, is expresseoc. Natl. Acad. Sci.d
, 2500-2505. 101U. S. A
Bischoff,R. and Luider,T.M. (2protein and peptide disease markers J. Chro004). Methodologmatogr. B Analyt. Teical advances in the discovery of chnol. Biomed. Life
, 27-40. 803Sci.
ewski,C.S. (2001). lkowiak,B., and CierniBoncela,J., Papiewska,I., Fijalkowska,I., WaAcute phase protein inhibitor type 1 and stabilizes its inhialpha 1-acid glycoprotein intebitory activity J. Biol. Chem. racts with plas276, 35305-35311. minogen activator
(1995). Two-dimensional proteiBoucherie,H., Dujardin,G., Kermorgant,M., Monribot,C., Slonimskn map of Saccharomyces cerevisiae: construction of a i,P., and Perrot,M.
, 601-613. 11gene-protein index Yeast
anayagam,P., Rassekh,C.H., Brysk,H., Brysk,M.M., Lei,G., Rajaraman,S., Selvnormal human epiderTyring,S.K., and Arany,I. (1997). Gene exmal and buccal epithelia In Vivo pre11, 271-274. ssion of zinc-alpha 2-glycoprotein in
Burgi,W. and Schmglycoprotein of normalid,K. (1961). Prepar human plasma J. Biol. Chem. 236ation and properties of Zn-alpha 2-, 1066-1074.
metastasis Genomics ProtCai,Z., Chiu,J.F., and He,Q.Y. (2eomics. Bioinformatics. 004). Applicati2on of pr, 152-166. oteomics in the study of tumor
Ream,B., van,N.A., Prendergast,G., Carmeliet,P., Kieckens,L., Schoonjans,L., igan,R.C. (1993). Plasminogen activatorCole,M., Bronson,R., Collen,D., and Mullcharacterization J. Clin. Invest inhibitor-1 gene-deficient mice. I. Gener92, 2746-2755. ation by homologous recombination and
ilber,J., Guerrier,L., Fortis,F., A., Cecconi,D., Sennels,L., RappsCastagna,Boschetti,E., Lomas,L., and Rigproteome via ligand library beadshetti,P.G. (2005). Ex., J. Proteome. Res. 4, 1917-1930. ploring the hidden human urinary
en,P., Andersen,A., e,B., Leffers,H., MadsCelis,J.E., Gesser,B., Dejgaard,K., HonordatabasesBasse,B., Celis,A., Laurids offer a systematic approach to en,J.B., and . (1989). Two dimethe study of cell prolifernsional gel human proteination and
, 407-416. 33differentiation. Int. J. Dev. Biol.
B., Dejgaard,K., Olsen,E., rgaard,M., Madsen,P., Honore,Celis,J.E., Gromov,P., OsteVandekVorum,H., Kristenserckhove,J., and Rasmussen,H.H. (en,D.B., Gromova,I., Haunso,A., Van,D.J., P1996). Human 2-D PAGE databases foruype,M.,
http://biobase.dk/cgi-bin/celis FEBS Lett. health and disease:proteome analysis in , 129-134. 398
proteomics on bladderCelis,J.E., Gromova,I., Moreira,J.M., C cancer research Pharmacogenomics. abez5on,T., and Gromov,P. (2004). Impact of , 381-394.
Literature
108
D., Fuchs,A., Kieffer,S., Rossignol,M., Chevallet,M., Santoni,V., Poinas,A., Rouquie,analysis ofLunardi,J., Garin,J., and Rabill membrane proteins by two-dimoud,T. (1998). New zwitterionic detensional electrophoresis. Electrophoresis ergents improve the
, 1901-1909. 19
asminogen activator system: i,A.P. (2003). Urokinase plChoong, P.F. and Nadesapillaon and metastasis. Clin. Orthop. Relat Res. a multifunctional role in tumor progressiS46-S58.
k-Jankun,E., and Jankun,J. (2004a). Chorostowska-Wynimko,J., Skrzypczals the process of the in vitro sprout Plasminogen activator inhibitor type-1 contro, 49-56. 55 Suppl 3formation. J. Physiol Pharmacol.
Chorostowska-Wynimko,J., Swiercz,R., Jankun,J. (2004b). Plasminogen activatoSr inhibitor type-1 mutants regulate krzypczak-Jankun,E., Selman,S.H., and
1155-1162. angiogenesis of human umbilical and lung vascular endothelial cells. Oncol. Rep. 12,
Christensen,E.I. (200proximal tubule. Nephrol. Dial. Transplant. 2). Pathophysiology of17 Suppl 9, 57-58. protein and vitamin handling in the
Christensen,E.I. andreceptors in renal proximal tubul Birn,H. (2001). Megaline. Am. J. Physiol Renal Physiol and cubilin: sy280, F562-F573. nergistic endocytic
function and dysfunction in kidney patChristensen,E.I. and Gburek,J. (2004). Protein hophysiology. Pediatr. Nephrol. reabsorption in renal prox19, 714-721. imal tubule-
saccharin and DL-tryptophan inCohen,S.M., Arai,M., Jacobs,J.B., and Fri urinary bladder carcinogenesis.edell,G.H. (1979). Promoting effect of Cancer Res. 39,
1207-1217.
Biol. Colledge,M9., 216-221. and Scott,J.D. (1999). AKAPs: from structure to function. Trends Cell
Correc,P., Fondanecplasmin receptors on three mammary carcihe,M.C., Bracke,M., and Burtin,P. (noma MCF-7 sublines1990). The presence of . Int. J. Cancer 46,
745-750.
Microvasc. Res. Curry,F.E. and Michel,C.C. (20, 96-99. 1980). A fiber matrix model of capillary permeability.
yes,C., Ward,M., as,K., Wardner,J., HDavidoff,G., Schultz,J.S., Lieb,T., Andrewacute spinal cord injuKarunas,R., and Maynard,F. (1990). Rehosry: incidence and pitalizrisk factors. Arch. Phys. Med. Ration after initial rehabilitatehabil. ion for 71,
121-124.
InflammatiDe Graaf,T.W., Van der Stelt,M.E.,on-induced expression of sialyl Lewis X-contai Anbergen,M.G., and ning glycan structures on van,D.W. (1993).
, 657-666. 177id) in human sera. J. Exp. Med. alpha 1-acid glycoprotein (orosomuco
Literature
109
,G., Grappone,C., Pucci,M., Caldini,R., Magnelli,L.,Del,R.M., Fibbi,G., Diniurokinase-Fimiani,M., Lotti,T., and Panconesi,E. (1990). Rodependent migration of human keratinocytes. J. Invest Dermatol. le of specific membrane receptors in 94, 310-
316.
ElectrophorDobryszycka W., Katnik, I., (1992). in: Breborowiesis: Principles and Application, CRC Press,cz J., Mackiewicz, Boca Raton, 211-225. A. (Eds.), Affinity
Expressi.on of the genetic vaDuche,J.C., Urien,S., Simon,N., Mariants of human alpha-1-acidlaurie,E., Monnet,I., and Barre,J. (2000). glycoprotein in cancer.
, 197-202. 33Clin. Biochem.
ssoe,S., Hunt,A.R., Collins,J.E., Dunham,I., Shimizu,N., Roe,B.A., ChiBruskiewich,R., Beare,D.M.Babbage,A., Bagguley,C., Bailey,J., Barlow,K, Clamp,M., Smink,L.J., Ai., Bates,K.N., Benscough,asR., Almeida,J.Pley,O., Bird,C.P., .,
Blakey,S., Bridgeman,(1999). The DNA sequence of human chromosome 22. Nature A.M., Buck,D., Burgess,J., Burrill,W.D., O'Brien,K.P., and . 402, 489-495.
nd Yeates,T.O. (2000). Protein function in Eisenberg,D., Marcotte,E.M., Xenarios,I., a, 823-826. 405the post-genomic era. Nature
Yalcin,A., Weber,K., and Tuschl,T. (2001). Elbashir,S.M., Harborth,J., Lendeckel,W., RNAs mediate RNA interference in cultured mammalian Duplexes of 21-nucleotide 411cells. Nature , 494-498.
adhyay,A.K. (1997). Vascular endothelial Ergun,S., Kilic,N., Fiedler,W., and Mukhopgrowth factor and its receptors in normal human testicular tissue. Mol. Cell
, 9-20. 131 Endocrinol.
Ergun,S., Kilik,N., Ziegeler,G., Hansen,Horst,A., Weil,J., Fernando,M., and WagenerA., Nollau,C. (2000). CEA-related cell adhesP., Gotze,J., Wurmbach,J.H., ion
growth factor. Mol. Cell molecule 1: a potent angiogeni5, 311-320. c factor and a major effector of vascular endothelial
vascular stabilization fErgun,S., Tilki,D., Oliveira-Ferrer,L., Schucor tumor growth and metastasis. Cancer Lett. h,G., and Kilic,N. (2006). Sign238, 180-187. ificance of
ez-Itza,I., Sanchez,L.(1993). Zn-alpha 2-glyMcoprotein levels in ., Allende,M.T., Vizoso,F., Ruibal,A., and Lopez-Otin,C. breast cancer cytosols and correlation with
, 1256-1260. 29Aal parameters. Eur. J. Cancer ological and biochemicclinical, hist
,J.L., Schleef,R.R., and Ginsburg,D. (1992). Brief report: Fay,W.P., Shapiro,A.D., Shihtor inhibitor type 1 due to a frame-shift complete deficiency of plasminogen-activamutation. N. Engl. J. Med. , 1729-1733. 327
Ferrara,N. (2005). VEGF as a therapeutic target in cancer. Oncology 69 Suppl 3, 11-
16.
receptors. Nat. Med. Ferrara,N., Gerber,H.P., and LeCouter,J9, 669-676. . (2003). The biology of VEGF and its
Literature
110
Flaumenhaft,R., Abe,M., Mignafactor-induced activation of latent transforming tti,P., and Rifkin,D.B. (1992). growth factor beta in endothelial cells:Basic fibroblast growth
, 901-909. 118tor activity. J. Cell Biol. regulation of plasminogen activa
Med. 3, 643-651. Folkman,J. (2003). Fundamental concepts of the angiogenic process. Curr. Mol.
basis? Cell Folkman,J. and D'Am87, 1153-1155. ore,P.A. (1996). Blood vessel formation: what is its molecular
Folkman,J., Weisz,P.B., Joullie,angiogenesis with synthetic heparM.M., Li,Win substitutes. Science 243.W., and Ewing,W.R. (1989). Control of , 1490-1493.
cloning and characterization of a full-lForsgren,M., Raden,B., Israelsson,M., Larengtssh cDNA clon,K., and Heden,L.O. (1987). Molecular one for human plasminogen.
, 254-260. 213FEBS Lett.
.Biophy. AcFournier T., Medjoubi-N N., Porquet D., ta. 1482 (1-2), 157-71. (2000).Alpha-1-acid glycoprotein. Biochim
Gehling,U.Schafhausen,P., Mende,T., Kilic,N., Kluge,M., Ergun,S., Schumacher,U., Wagener,C., Pantel,K., OtK., Schafer,B., Hossfeld,D.K., and te,M., Schuch,G.,
differentiation of endothelial cells from AC133-positive Fiedler,W. (2000). In vitro , 3106-3112. 95progenitor cells. Blood
., and Roguth,G., Harder,A., Scheibe,B., Wildgruber,Gorg,A., Obermaier,C., BimmobilizedWeiss,W. (2000). The current state of pH gradients. Electrophoresis 21, 1037-1053. two-dimensional electrophoresis with
Gorg,A., Poresolution of PI (alpha 1-antstel,W., Weser,J., Patutschnick,W., and itrypsin) phenotypes by a laCleve,H.rge-scale immobilized pH (1985). Improved
, 922-930. 37gradient. Am. J. Hum. Genet.
technology for proteomGorg,A., Weiss,W., and Dunn,M.J. (2004). Cuics. Proteomics. 4, 3665-3685. rrent two-dimensional electrophoresis
unprocessGrover,P.K. and Resnick,M.I. (1993). Two-ed human urine using double stain. J. Urol. dimensional analy150, 1069-1072. sis of proteins in
plasminogen activator to cultured Hajjar,K.A., Hamel,N.M., Harpel,P.C., and Nahuman endothelial cells. chman,R.L. (1987). Binding of tissJ. Clin. Invest 80, 1712-ue
1719.
Hanahan,D. and Folkangiogenic switch during tumorigenesis. Cell man,J. 86(1996). Patterns and emerging m, 353-364. echanisms of the
Hanash,S.using immobilized pH gradients: M. (2000). Biomedical applications of two-dimcurrent status. Electrophoresis 21ensional electrophoresis, 1202-1209.
reaction, heterogeneitHansen,J.E., Iversen,J., Lihme,A., and y, and microheterogeneity Bog-Hansen,T.C. (1987). Acute phaseof serum proteins as nonspecific
, 1630-1635. 60tumor markers in lung cancer. Cancer
Literature
111
update of our current Haraldsson,B. and Sorensson,J. (2004). Whunderstanding of the glomerulary do we not all hav barrier. News Physiol Sce proteinuria? Ani. 19,
7-10.
Comparison of yeasHarder,A., Wildgruber,Rt cell protein s., Nawrocki,A., Fey,olubilization procedures for two-dimensional S.J., Larsen,P.M., and Gorg,A. (1999).
esis. Electrophoresis electrophor, 826-829. 20
Hashizume,H., Baluk,P., Jain,R.K., and McDonald,D.M. (2000). OpeningsMorikawa,S., McLean,J.W., T between defective endothelial cells hurston,G., Roberge,S.,
explain tumor vessel leakiness. Am. J. Pathol. 156, 1363-1380.
electrophorHerbert,B. (1999). Advanceesis. Electrophoresis s20 in protei, 660-663. n solubilisation for two-dimensional
(1998). Improved protein solubility in two-diHerbert,B.R., Molloy,M.P., Gooley,A.A., Walsh,B.J., Bryson,W.G.,mensional electrophores and Williams,K.L. is using tributyl
, 845-851. 19agent. Electrophoresis phosphine as reducing
Hoorn,E.J., Pisitkun,T.Star,R.A., and Knepper,M.A. (2, Zietse,R., Gross,P005). Prospects for uri., Frokiaer,J., Wang,N.nary proteomics: exosomes as S., Gonzales,P.A.,
, 283-290. 10kers. Nephrology. (Carlton.) a source of urinary biomar
. (2002). shol,H., Muller,D., Roberts,R.C., and van,O.J,B., VoHoving,S., GerritsimmobilizedPreparative two-dimensional pH gradients. Proteomics. gel electrophor2, 127-134. esis at alkaline pH using narrow range
Protein-decIbel,K., May,R.P., Kirschner,K., Szadkoworated micelle struskcture of sodium-dodecyl-sulfai,H., Mascher,E., and Lundahl,P. (1990). te--protein complexes as
, 311-318. 190determined by neutron scattering. Eur. J. Biochem.
Jurgens,M., Appel,A., Heine,G., Neitz,S., Menzel,C., Tammen,H., and Zucht,H.D. human urinary peptidome. Comb. Chem. (2005). Towards characterization of the , 757-765. 8High Throughput. Screen.
Y., Kontani,K., kabayashi,Y., Okada,S., Isono,T., Iwaki,H., WaKageyama,analysis ofYoshimura,K., Terai,A., Arai,Y., and Yos calreticulin as a marker fohiki,T. (2004). Identificr bladder cancer and evaluation of the ation by proteomic
, 857-866. 50tion in urine. Clin. Chem. diagnostic accuracy of its detec
Kersten,B., Burkle,L., Kuhn,E.J., GiavalisEickhoff,H., and Schneider,U. (2002). Large-scaleco,P., Konthur,Z., Luek plant proteomics. Plant Mol. Biol. ing,A., Walter,G.,
, 133-141. 48
malignancy.Konety,B.R. and Getzenberg, J. Urol. 165, 600-611. R.H. (2001). Urine based markers of urological
Krishna,R.Enzymol. Relat Areas Mol. Biol. G. and Wold,F. (1993). Post-transla67, 265-298. tional modification of proteins. Adv.
Laemmli,U.K. (1970). Cleavage ofhead of bacteriophage T4. Nature 227, 680-685. structural proteins during the assembly of the
Literature
, 1213. 152 bladder cancer. J. Urol. Lamm,D.L. (1994). RE: Editorial:
112
during inflammation. SLasky,L.A. (1992). Selectins: incience 258, 964-969. terpreters of cell-specific carbohydrate information
Lau,A.T., He,Q.Y., aapplications. Sheng Wu Hua Xue. Yu Shnd Chiu,J.F. (2003). Preng Wu Wuoteomic technology and its biomedical Li Xue. Bao. (Shanghai) 35,
965-975.
339-341. Lawrence,D.A. (1997). The serpin-proteinase complex revealed. Nat. Struct. Biol. 4,
Lawrence,D.A., GiKvassman,J.O., and Shore,J.D. (1995). Snsburg,D., Day,D.erpE., Berkenpas,M.B., Verhamme,I.M.,in-protease complexes are trapped as
, 25309-25312. 270stable acyl-enzyme intermediates. J. Biol. Chem.
S., and Ginsburg,D. (1994). EngineeringD.A., Olson,S.T., Palaniappan,Lawrence,plasminogen activator inhibitor 1 mutants with increased functional stability
, 3643-3648. 331. Biochemistry
therapy. Urol. Clin. NoLee,R. and Droller,M.J. rth Am. 27(2000). The natural hist, 1-13, vii. ory of bladder cancer. Implications for
of the past year. Curr. Opin. Hematol. Luttun,A. and Carmeliet,P. (2004). Angi11, 262-271. ogenesis and lymphangiogenesis: highlights
MacDonald,T.J., DeClerck,Y.Amediated brain tumor cell migrat., and Laug,ion and invaW.E. (1998). Uroksion. J. Neurooncol. 40inase induces, 215-226. receptor
RadziejewsMaisonpierre,P.C., Suri,C., Jones,Pki,C., Compton,D., McClain,J., .F., Bartunkova,S., WiAldrich,T.H., Papadopoulos,N., Daly,T.J., egand,S.J.,
Davis,S., Sato,T.N., and Yancopoulos,G.Dantagonist for Tie2 that disrupts in vivo angiogenesis. Science . (1997). Angiopoi277, 55-60. etin-2, a natural
lar endothelial growth factor-induced Mandriota,S.J. and Pepper,M.S. (1997). Vascuin vitro angiogenesisendogenous basic fibroblast grow and plasminogen acth factor. J. Cell Sci. 110 ( Pt 18)tivator expression are dependent on, 2293-2302.
using high resolution electrophorMarshall,T. and Williams,K.M. (1998). Clinicetic methods. Electrophoresis al analysis of human19 urinary, 1752-1770. proteins
Maunsbackidney proximal tubule cells. h,A.B. (1997). Absorption of IA study of microperfused si125-labeled homologous albumin by rat ngle proximal tubules by
ochemistry. 1966. J. Am. Soc. Nephrol. electron microscopic autoradiography and hist, 323-351. 8
for proteins in polyaMerril,C.R., Goldman,D., Sedman,S.A., and crylamide gels shows regional variation in cerebrospinal fluid Ebert,M.H. (1981). Ultrasensitive stain
211proteins. Science 1437-1438. ,
Miles,L.A. and Plow,platelet surface. J. Biol. Chem. E.F. (1985). Binding260, 4303-4311. and activation of plasminogen on the
Literature
113
immobilizedMolloy,M.P. (2000). Two-dimensional elec pH gradients. Anal. Biochem. 280, 1-10. trophoresis of membrane proteins using
Morrison,A.S., Buring,J.E., (1982). Coffee drinking and cancVerher of the looek,W.G., Aoki,K., Leck,I.wer urinary tract. J. Natl. Cancer, Ohno,Y., and Obata,K. Inst. 68,
91-94.
Mostofi,F.Ktumors of the urinary trac., Davis,C.J., Jr., and Sesterhent. Prog. Clin. Biol. Res. 277, 1-20. n,I.A. (1988). Progress in pathology of
Gerard,R.D., and GolMottonen,J., Strand,A., Symersky,J., Sweedsmith,E.J. (1992). Structural basis of latency in plast,R.M., Danley,D.E., Geoghegan,K.F., minogen
, 270-273. 355activator inhibitor-1. Nature
Urinary cytology and bladder Murphy,W.M., Soloway,M.S., Jukkola,A.Fcancer. The cellular feat., Crabtree,W.N., and Ford,K.S. (1984). ures of transitional cell
, 1555-1565. 53neoplasms. Cancer
Koshimura,J., Fujita,H., KaNarita,T., Sasaki,H., Hosobakei,M., and Ito,S. (2004). Para,M., Miura,T., Yoshioka,N., Morllel increase in urinary ii,T., Shimotomai,T.,
excretion ranormoalbuminuric type 2 diabetictes of immunoglobulin G, cerul patients. Diabetes Care 27oplasmin, transferrin, and orosomucoid in , 1176-1181.
, R903-R905. 8ing. Curr. Biol. Newman,A. (1998). RNA splic
Oh,J., Pyo,J.H., Jo,E.H., Choi,J.Y., and Lim,J. (2004). EstablishmenHwang,S.I., Kang,S.C., Jung,J.H., Pt of a near-standard two-dimensional ark,E.K., Kim,S.Y.,
, 3485-3497.4map. Proteomics. human urine proteomic
Oliveira-Ferrer,LHuland,H., Friedrich,M., and ., Tilki,D., Ziegeler,G., HErgun,S. (2004). Dual role auschild,J., Loges,S., Irmak,S., Kilic,E.,of carcinoembryonic antigen-
genesis and invasion of human urinary related cell adhesion molecule 1 in angio, 8932-8938. 64bladder cancer. Cancer Res.
discovery in urine by protPang,J.X., Ginanni,N., Dongreeomics. J. Proteome. Res. 1,A.R., Hefta,S.A., and Opitek,G.J. (2002). Biomarker , 161-169.
assembly from modules. Cell Patthy,L. (1985). Evolution of the proteas41, 657-663. es of blood coagulation and fibrinolysis by
.D. (1990). Transformingalli,Jano,R., Orci,L., and VassPepper,M.S., Belin,D., Montesangiogenic properties of growth factor-beta 1 modulates basic fibrendothelial cells in vioblast growttro. J. Cell Biol. h111, 743-755. factor-induced proteolytic and
Pepper,M.S., Ferrara,N., Orcil ,L., and Montesano,R. (1991). Vascular endotheliainhibitor-1 in microvascular endotgrowth factor (VEGF) induces plasminoghelial ceen activators and plaslls. Biochem. Biophys. Res. Commun. minogen activator 181,
902-906.
Petricoin,E.for prostate cancer biomarker diF., Ornstein,D.K., and Liotta,L.A. (2004). Clinicascovery and detection. Urol. Oncol. 22l proteomics: Applications, 322-328.
Literature
114
Pieper,R., Gatlin,C.L., McGraField,E., Schatz,C.R., Estock,M.A., Ahmed,th,A.M., Makusky,A.J., Mondal,M., Seonarain,M., N., Anderson,N.G., and Steiner,S. (2004).
Characterizof urinary proteins on twation of the human urinary proto-dimensional electrophoresis eome: a method for higels with a yield of nearlygh-resolution display
, 1159-1174. 4spots. Proteomics. 1400 distinct protein profiling of exosomesPisitkun,T., Shen,R.F., and Knepper,M.A. in human urine. Proc. Natl. Acad. Sci. U. S. A (2004). Identification and pr101, 13368-oteomic
13373. alpha1-acidic glycoprPredescu,D., Predescu,S., Mcotein in Quistan,T., the continuous microvasculaand Palade,G.E. (1998). Transcytosis of r endothelium. Proc. Natl.
, 6175-6180.95Acad. Sci. U. S. A impact on superficial bRabbani,F. and Cordon-Cardo,C.ladder cancer. Urol. Clin. North Am. (2000). Mutation of cell cycle regulators and their27, 83-102, ix.
Rabilloud,Tin two-dimensional elec. (1998). Use of thiourea to incrtrophoresis. Electrophoresis ease the solub19, 758-760. ility of membrane proteins
solubRabilloud,Tilization of proteins ., Adessi,C., Giraudel,A., and in two-dimensioLunardi,J.nal electrophor (1997). Improvement of the esis with immobilized pH
, 307-316. 18esis gradients. ElectrophorRabilloud,TBreton,C. (1999). Analysis., Blisnick,T., Heller, of membrM., Luche,S., Aebersoane proteins by two-dimensionald,R., Lunardi,J., and Braun-l
electrophorfalciparum-infected erythroesis: comparison of the proteinscyte ghosts. Electrophoresis 20 extracted from normal or Plasmodium , 3603-3610.
and Heissig,B. (2002). Vascular and Rafii,S., Lyden,D., Benezra,R., Hattori,K.,Cancer 2, 826-835. haematopoietic stem cells: novel targets for anti-angiogenesis therapy?. Nat. Rev.
Rakic,J.M., Maillard,Foidart,J.M., and Noel,A. (2003). Role of plC., Jost,M., Bajou,K., Masson,V., Devy,asminogen activator-plasmin syL., Lambert,V., stem in
, 463-473. 60is. Cell Mol. Life Sci. tumor angiogenesRamakumar,S., BhuiyO'Kane,D.J. (1999). Comparisan,J., Besse,J.A., Ron of screening methods in the detoberts,S.G., Wollan,P.C., Blute,M.L., and ection of bladder
, 388-394. 161cancer. J. Urol. T.F., Wolf,H., and Celis,J.E. (1996). Towards aRasmussen,H.H., Orntoft,Urol. 155, 2113-2119.comprehensive database of prot eins from the urine of patients with bladder cancer. J.
P., Paul,A., Selby,P.J., and Banks,R.E. .., Munro,NRogers,M.A., Clarke,P., Noble,Jlaser desor(2003). Proteomic profilinption ionizag of tion and neural-neturinary proteins in renwork analyal cancer by surface enhancesis: identification of key issues d
, 6971-6983. 63 utility. Cancer Res. affecting potential clinicalr,K., Rush,D., and Nickerson,P. (2004). Schaub,S., Wilkins,J., Weiler,T., SangsteUrine protein profiling mass spectrometry. Kidney Int. 65with surface-enhanced laser-des, 323-332. orption/ionization time-of-flight
Literature
Chem. Soc. 72, 2816. Schmid,K. (1950). J. Am.
115
genetic control, Academic PreSchmid,K. (1975). inPutman, F.W.Y (Ed.), Plss, NewYork. asma prvol.1. 183-288. oteins, structure, function and
. Chem. Soc.75. 68-60. Schmid,K. (1953). J. Am
A., and Simons,K. (2003). Schuck,S., Honsho,M., Ekroos,K., Shevchenko,ad. Sci. U. S. A ergents. Proc. Natl. AcResistance of cell membranes to different det, 5795-5800. 100
inter,M., Porter,A., and court,S., Heaps,M., Pa., HarSmith,G., Hippisley-Cox,JPringle,M. (2007). Developifor health protection--a surveillance tool for ng a national primary care-the future? Analysis of routinely cobased early warning systellectedm
, 75-82. 29data. J. Public Health (Oxf)
dan,R., George,A., ari,S., Hu,H., JorSomiari,R.I., Sullivan,A., Russell,S., Somi(2003). High-throughput prKatenhusen,R., Buchowieckoteomic analysa,A., Arciero,Cis of human infilt., Brzeski,H., Hooke,rating ductal carcinoma ofJ., and Shriver,C.
, 1863-1873. 3the breast. Proteomics.
Sorensson,endothelial cells produce orosJ., Matejkomucoid, an a,G.L., Ohlson,M., and Haraldsson,important component B. (1999)of the capillary. Human
H534. , H530-276barrier. Am. J. Physiol
Bures,E.J., Beierle,J., nley,M.D., Robinson,J.H., Spahr,C.S., Davis,M.T., McGi,R.C., Yu,W., Luethy,R., and Patterson,S.D. Mort,J., Courchesne,P.L., Chen,K., Wahlmass spectrometry. I. Profilin(2001). Towards defining the urg aninary pr unfraoteome using liquid ctionated tryptic digest. Proteomics. chromatography-tandem 1, 93-
107.
, 915-916. 431end of the beginning. Nature Stein,L.D. (2004). Human genome:
Sun,W., Li,F., Wu,S., Wang,Xproteome analysis by three separ., Zheng,D., Wang,J., and Gao,ation approaches. Proteomics. 5, 4994-5001.Y. (2005). Human urine
., Tateyama,H., and Eimoto,T. (1991). Tada,T., Ohkubo,I., Niwa,M., Sasaki,Mtissues. J. Histochem. Cytochem. Immunohistochemical localization of Zn39, 1221-1226. -alpha 2-glycoprotein in normal human
(2003). Structure-.lloud,Tallet,M., Luche,S., and RabiTastet,C., Charmont,S., Chevefficiency relationships of zwitterionic detergents as protein solubilizers in two-
, 111-121. 3oresis. Proteomics. dimensional electroph
Thongboonkerd,V., Kproteomics and biomarker discovlein,J.B., Jevans,Aery for glomerular dis.W., and McLeish,K.R. (2004). Urinaryeases.. Contrib. Nephrol. 141,
292-307.
applications and challenges. Proteomics. Thongboonkerd,V. and Malasit,P. (2005). Renal5, 1033-1042. and urinary proteomics: current
Literature
116
analysis ofThongboonkerd,V., McLeish,K.R. normal human urinary proteins, Arthur,J isolated by ac.M., and Klein,J.B. etone precipit(2002). Proteomic ation or
, 1461-1469. 62ultracentrifugation. Kidney Int.
Lathrop,J., Lomas,L., Hammond,D., and iraman,V., Lin,S., Gheorghiu,L., Thulason difference of proteins in biological Boschetti,E. (2005). Reduction of the concentrati, 3561-3571. 26liquids using a library of combinatorial ligands. Electrophoresis
McDonald,D.M., andThurston,G., Rudge,J.S., Ioffe,E., Zhou,H., Ro Yancopoulos,G.D. (2000). Angiopoietin-1 protects thss,L., Croll,S.D., Glazer,N., Holash,J., e adult
, 460-463. 6 leakage. Nat. Med. vasculature against plasma
Todorov,P.T., McDevitt,T(1998). Purification and characterization of .M., Meyer,D.J., Uea tumor liyama,H., Ohkubo,I., and Tisdale,M.Jpid-mobilizing factor. Cancer.
Res. , 2353-2358. 58
products in superficial bladder Tsihlias,J. and Grossman,H.B. (2000). The cancer. Urol. Clin. North Am. utility of27 fibrin/fibrinogen d, 39-46. egradation
Vassalli,J.D., Baccino,D., and Belin,D. (55,000 form of the human plasminogen activa1985). A cellular binding stor, urokinase. J. Cell Biol. i100te for the Mr , 86-92.
Vlahou,A., Schellhammer,P.Nasim,S., and Wright,J.G., JrF.. (2001). Dev, Mendrinos,S., Patel,Kelopment of ., Kondylis,F.I., Gong,L., a novel proteomic approach
158, 1491-1502. for the detection of transitional cell carcinoma of the bladder in urine. Am. J. Pathol.
Wilson,T.G., Pritchett,T.R., Lieskovsky,G.Primary adenocarcinoma of bladder. Urology 38, 223-226. , Warner,N.E., and Skinner,D.G. (1991).
ative epidemiology of tobacco-related Wynder,E.L. and Stellman,S.D. (1977). Compar, 4608-4622. 37cancers. Cancer Res.
ReichZengin,E.,enspurner,H., K Chalajour,F., ilic,N., and Ergun,S. (Gehling,U.M., Ito,W.D., 2006). Vascular wall resident proTreede,H., Lauke,H., Weil,J., genitor
, 1543-1551. 133cells: a source for postnatal vasculogenesis. Development
., GonzalesZhou,H., Yuen,P.S., Pisitkun,T,J.W., Gross,P., ,P.A., Yasuda,H., Dearnormalization of human urinary exosKnepper,M.A., and Star,R.A. (2006). Colleomes ction, storage, for biomarker discovery. Kidney Int. preservation, and 69,
1471-1476.
Appendix
8 Appendi
8.1
x
Map of 3.1(-) vector
117
tae ulum VicCurri
118
Vitae 8.2 Curriculum Irmak Surname : First Name: Ster 25.02.1978 : Birthdayrkey uBirthplace : Mardin/ TSingle : FamilyTurkish : lityNationa: Raumerstr. 1 Adress 45144 Essen, Deutschland ster.irmak@uk-essen.de Education Background:chool in Konya, Turkey : Primary S1982-1990 a, Turkey : High School in Kony1990-1993 epartment, Selcuk University, Turkey : Undergraduate in Chemistry D1994-1998
itute of ochemistry Division, Inst: Graduate (MSc.) in Chem. Dept., Bi1999-2001 ersity, Turkey Pure and Applied Science, Hacettepe Univy istry Division, Hacettepe Universit: PhD in Chem. Dept., Biochem2001-2002 and Applied Science, Turkey (not University, Institute of Pure completed)
: Scientific research in Department2003-2006 of Urology, University Hospital of Hamburg-Eppendorf (for doctoral thesis)
ent of Anatomy, University Hospital of 2006-present : Scientific research in DepartmEssen (for doctoral thesis) Employment: 2006-present of Essen : Research Scientist in Department of Anatomy, University Hospital
2002-2006 : Research Scientist in Department of Urology, University Hospital of
Hamburg-Eppendorf
tment of Chemistry, Biochemistry : Research Scientist in Depar 1999-2002 Division, Hacettepe University, Turkey
tae ulum VicCurri
Publications:
119
1. Tilki D, IrHammerer P, Friedricmak S, Oh MG, Schuch G, Galaliveira-Ferrer L, Hauschildlae R, Stief CG, K J, Miethe K,ilic Atakaya H, E, Huland H,
switch in prostate cancerErgun S. CEA-related cell adhesion mo, Oncogene, lecule-1 is25(36) (2006),4965-74. involved in angiogenic
2. Ster IrmakFriedrich, Hartwig Huland, Suleyman , Derya Tilki, Jochen Heukeshoven, Ergun, Stage-DependenLeticia Oliveira-Ferrer, t Increase of Martin
Proteomics 5(16)Orosomucoid and Zi (2005),4296- 304. nc alpha-2 Glycoprotein in Urinary Bladder Cancer,
Hauschild, Sonja Gudrun Ziegeler, JessicaL.Oliveira-Ferrer, Derya Tilki, 3. Loges, Ster IrmakErgun, Dual Role of CEACAM1 in A, Ergin Kilic, Hartwig Huland, ngiogenesis and Invasion of HumanMartin Friedrich, Suleyman
Reseach 64 (2004), 8932-8938. er, CancerUrinary Bladder Canc
4. Eroğlu M., Irmak S., Acar A., Denkbaş E.B., Design and Evaluation of a
Chemotherapy in SuperficiMucoadhesive Therapeutic al Bladder Cancer, InteAgent Delivery System for Postoperative rnational Journal of
Pharmaceutics, 235 (2002) 5159. Presentations (Abstracts Available):1. Irmak S, Tilki D, Olivorosomucoid (ORM) in vascular endoteira-Ferrer L, Ergün S,helial cells and Urinary proteomics and the role oangiogenesis.102nd Annual f
ft, 30 March-2 April, 2007, Giessen, Meeting of the Anatomische Gesellscharal) OGermany. (2. S. Irmakdetection of orosomucoid in , D. Tilki, J. Heukeshoven, L. Oliveiraurinary bladder cancer, 17-Ferrer, , M. Friedrich, S. Ergün., The th Symposium
h 2006, Essen, Germany.(Poster) Experimentelle Urologie, 17-18 Marc3. S. Irmak, L. Oliveira-Ferrer, J. Heukeshoven, M. Friedrich, H. Huland, S. Ergün.,
Determination of urinary protein patternanalyses., European Association of Urology in bladder(EAU), XIXth Congres carcinoma by proteomic s, 24-27 March
2004, Vienna, Austria.(Poster) 4. Sevinc, E. Kilic, M. FriM.Fernando, L. Oliveira-Ferrer, D.edrich, C. Wagener, S. Ergün, Tilki, G. ZeigelerDual role, J. Hauschild, of CEACAM1 in S. Irmak, S.
inary bladder cancer., European angiogenesis and invasion of human ur h Congress, 24-27 March 2004, Vienna,Association of Urology(EAU), XIXtAustria.(Poster) 5. S. Irmak, L. Oliveira-Determination of urinary protein patternFerrer, J. Heukeshoven, in bladderM. Friedrich, H. Huland, S. E carcinoma by proteomic rgün.,
th analyses., 16Germany.(Poster) Experimental Urology Congress, 11-13 March 2004, Lübeck,
6. Denkbaş E.B., Irmak S.Mucoadhesive Polymeric Carriers to Use , Özdemir N., Eroğin Postlu M., Acar M., Mitomycin-C Loaded operative Chemotherapy in
Studies, IX. International Symposium on In-vitro Superficial Bladder Cancer: chnology, Antalya, Turkey, 19-22 Eylül 2002. (Poster) Biomedical Science and Te
cCurritae ulum Vi
120
7. Denkbaş E. B.., Kõlõçay E., Birlikseven C., Irmak S., Öztürk E., Preparation of
pheres and determination of their magnetical magnetic chitosan microsndproperties, 2June, 2001, Kõrõkkale. (Poster) National Congress of Chromatography, Kõrõkkale University, 6-8
8. Kõlõçay E., Irmak S., Baysal M.Y., Denkbaş E.B., Preparation and
CharacterizImmobilization, 3ardtion of Magnetic Ch Mediterranean Basin Conferencitosan Microspheres for Enzyme e on Analytical Chemistry,
a, Turkey. (Poster) June 4-9, 2000, Antaly Courses, Congress and Summer Schools:1. .102nd2007; Giessen, Germany. Annual Meeting of the Anatomische Gesellschaft, 30 March-2 April,
2. 17th Symposium Experimentelle Urologie, 17-18 March 2006, Essen,
Germany. 3. 15th Annual International CEA Symposium, 21-24 July 2005, Berlin,
Germany. 4. Vienna, Austria. European Association of Urology(EAU), XIXth Congress, 24-27 March 2004,
5. 16th Experimental Urology Congress, 11-13 March 2004, Lübeck, Germany.
6. 2nd2001, Kõrõkkale, Turkey. National Congress of Chromatography, Kõrõkkale University, 6-8 June,
7. Controlled Turkey. Release Systems, Marmara University, 11 May, 2001, Istanbul,
8. 7th National Symposium on Biomedical Science and Technology, Hacettepe
University, 25-27 September, 2000, Ankara, Turkey. 9. Modern and ClassiUniversity, 23 August - 2 September 2000, Kucal Biochemistşadasõ, İry Methods, Summer School, Agean zmir, Turkey.
10. 3rdJune, 2000, Antalya, Turkey. Mediterranean Basin Conference on Analytical Chemistry, MBCAC III, 4-9
11 .'1st National Chromatography Congress, 16-18 June, 1999, Kõrõkkale
kkale, Turkey. õrõUniversity, K12 Selçuklu, Konya, Turkey. .Certificate of Education, Selçuk University, Education Faculty, 1997,
sity, Education Faculty, 1997, rUnive
g unrErklä
Ergün leyman üSDr.
121
Erklärung eiche 6 ionsordnung der FachberAbs. 2, Nr. 8 der PromotHiermit erkläre ich, gem § 6 ss ich das Arbeitsgebiet, dem das Thema bis 9 zur Erlangung des Dr. rer. nat., da oid (ORM) in vascularization of bladderUrinary proteomics and the role of orosomucLehre vertrete und den Antrag von Ster cancer zuzuordnen ist, in Forschung und worte. rIrmak befü Essen, 13. April 2007 Prof. Dr. Süleyman Ergün
Erklärung g der Fachbereiche . 8 der PromotionsordnunHiermit erkläre ich, gem. § 6 Abs. 2, Nr6 bis 9 zur Erlangung des Dr. rer. nat., dass ich die vorliegende Dissertation
ebenen Hilfsmittel anderen als der angegselbständig verfasst und mich keinerbedient habe. Essen, 13. April 2007 Ster Irmak
Erklärung g der Fachbereiche. 8 der PromotionsordnunHiermit erkläre ich, gem. § 6 Abs. 2, Nrdass ich keine anderen Promotionen bzw. r Erlangung des Dr. rer. nat., u6 bis 9 z durchgeführt habe und dass diese ArbeitPromotionsversuche in der Vergangenheit von keiner anderen Fakultät abgelehnt worden ist. Essen, 13. April 2007 Ster Irmak
ter Irmak
ter Irmak
Cleanzine: your weekly cleaning and hygiene industry newsletter8th August 2019Issue no. 882
Your industry news -first Number 1 for Recruitment
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...rs have criticised the law for targeting the wrong substances. The REACH regulation was initially designed to protect consumers from exposure to hazardous chemicals, but the bureaucracy it created ended up encompassing metals such as cobalt, which hardly comes into contact with consumers at all. David Weight, from the Cobalt Development Institut... ...e EU-Flower certified too.' We liked the way everything is colour coded and that the handles are designed to make the bottles easy to hold. Another product that offers controlled dosing, but which is shortlisted in the chemicals category, is the Gynius box, from Ar-Co Chimica. This was another item we'd seen the day before on the Innovations stan... ...ully for ease of cleaning... it has stainless steel squeegee holder and a HEPA filter... 'It was designed to strike a real balance between cost and productivity,' said Stefano Abelli. 'We believe we've succeeded and this is just the beginning.' See next week's issue of Cleanzine for the results of the Awards, together with more of what we saw at ... ...nals and ensure all of their workers have union contracts. We have to organise.' UNI has already signed Global Agreements in the Property Services sector with G4S, ISS and Securitas. With these companies, UNI is focused on enforcing the agreement and organising the companies' workers around the world. UNI Property Services also presented an a... ... use with 'Velcro' microfibre mop heads or 'Pocket' microfibre mop heads. The 'Velcro' version is designed for use in commercial premises whilst the 'pocketed- Blik' within the professional healthcare sector. The high quality full coloured looped microfibre mop heads provide high levels of cleaning; removing dirt, dust and bacteria from the surface... ... and durable, all washers in the 6 Series range offer spin speeds of up to 1,000rpm and have been designed to provide excellent washing performance with reduced cycle times. Girbau's patented GDRIVE system uses microprocessor control to optimise the high speed spin cycle to suit different types of fabric as well as saving energy and water, reducing... ...ew voluntary labelling system for the recycling of packaging later this month. This initiative is designed to help consumers better understand how to recycle various packaging components and to provide a harmonised approach to consumer communication on recycling. T: +1-202-249-6619 E: [email protected] W: www.plastics.a... ...safe and hygienic areas for the user and protection against misuse and theft. The trolleys can be designed to use traditional mops or microfibre 'wet' or 'pre-soaking' systems. Designed to be used with a complete range of high quality microfibre products and additional cleaning equipment for floor, wall and glass cleaning and high level dust con... ...ercial upright vacuum cleaner. Boasting a 'Flex' neck and an on-board hose, the vacuum cleaner is designed to meet the demands of commercial cleaning. The Dart is available in two brush widths - 31cm and 37cm. Sebo's UHS polisher, which is an add-on to the Dart, is the perfect way to keep hard floors looking great with a brilliant gloss shine. S... ...lls-Royce. The facility comprises six floors with over 249,615 square feet of surface area and is designed to accommodate 700 - 900 people, including delegates from all over the world who are there to attend training courses. NMK awarded MITIE the £ multi-million contract because of its innovative approach and willingness to work in partne... ...ames and Microfibre Mini Mops offer simple and professional cleaning in just one single movement. Designed for use in any area where polished or gloss surfaces require routine maintenance. With its ergonomic grip, Glass Clean System 'Hand and Swivel' is the new revolution in cleaning in just one third of the time (compared to using cloths). Usin... ..., EMEA, Rubbermaid Commercial Products. 'The system's integrated water filter was specifically designed with the bottom line and end user in mind. It improves productivity, reduces both water and chemical usage and creates healthier, safer environments.' Designed to promote green cleaning and improve cost efficiencies, Hygen provides several ... ...ding recycling system, which is supported by WRAP (the Waste & Resources Action Programme) is designed to produce the best quality material for recycling and has already produced dramatic results, while delivering £500,000 of savings. These figures and the award prove that Newcastle-under-Lyme residents have embraced recycling with its be... ...t savings with customers. Vernacare is also operating an exclusive discount scheme on its British designed and manufactured macerators. 'Vernacare is a top 30 supplier to the NHS - supplying the health service for almost 50 years with the innovative pulp products and the VernacareSystem of patient toileting we invented,' said Lesley Webster, UK ... ...arly suitable for the industrial market; Libera - a top-quality, professional cold water pressure designed for the industrial sector; Double Life, the nebuliser for detergent, with its plastic-coated 24-litre tank that isolates the tank from the effects of any chemicals; the Red 25 and Red 50 Lines that feature Club dei Riparatori guns and lances, ... The UK Driving Standards Agency has signed a four year total facilities management contract with Interserve, the services, maintenance and building group, which will save it some £9m over the contract lifetime. The contract, worth around £12 million over its lifetime, has been signed as part of a collaborative procurement process by... ...developed, tested, and confirmed by independent testing that a small UVC dispenser - specifically designed for sterilising bare or gloved hands safely and quickly - achieves -6log10 inactivation of all relevant pathogens, including tough-to-kill Clostridium difficile spores. It has developed a simple, proprietary means to protect bare skin from UVC... ...C) in Geneva, Switzerland, last Friday. The study, funded by the US Department of Defense, was designed to determine the efficacy of antimicrobial copper in reducing the level of pathogens in hospital rooms, and whether such a reduction would translate into a lower risk of infection. Researchers at the three hospitals involved in the trial, M... ... Award for Exelerate ZTF, which is thought to be the industry's first and only cleaning programme designed to address zero trans fat oil build-up in food processing environments. Presented to Ecolab by the Institute of Food Technologists at its Annual Meeting & Food Expo, the award recognises outstanding innovation in products, ingredients, app... ...as bestowed the quality award 'Top Performance 2010' on Fimap. This award, founded in 2005, is assigned each year to the organisation which, having been certified for at least three years, has demonstrated at each review, consistent quality performance at a level that meets the judges' strict criteria. 'We are very proud to have received this... ...ominations. The decisions of the judges will be final. Each award winner will receive a trophy designed specifically for the BTA Innovation Awards and a framed BTA Innovation Award certificate. The awards will be announced and presented to the winners by a celebrity guest during the 2011 Loo of the Year Awards presentation event on Friday 2nd... ...the cleaning and facilities management industries under one roof. The conference and seminars are designed to cater to the different segments of the cleaning industry and end users. Organised by Virtual Info Systems and Afidamp Servizi, the show is expected to attract almost double the number of visitors that attended the 2009 event. T: +91-2... ... basis for the new Joint Venture company that was to become Devon Norse. The deal was officially signed on May 23rd in Exeter by Devon County Council's solicitor, Jan Shadbolt, and Norse MD Peter Hawes. Devon County Council leader John Hart says 'We are delighted to be partners in this venture with Norse Commercial Services. It is very import... ...eper (Master CEH) and Master Registered Executive Housekeeper (Master REH). These credentials are designed to raise awareness of the knowledge, professionalism and skill levels achieved by designation holders, and to highlight their premier role as leaders and educators within IEHA and the housekeeping field. 'Just as a master's degree is an aca... ...lifting and wringing of heavy buckets and mops. The Vikan ErgoClean ultra microfibre system is designed and developed to be versatile and meet the demands of modern cleaning from floor to ceiling. In addition, one touch cleaning tools also reduce the risk of repetitive strain on muscles and joints. Four Vikan trolleys, each with their set of ... ... work. This is precisely the same reason why only the best high rise cleaning companies should be assigned the job. These companies are empowered with the most modern and updated equipment that are capable of scrubbing the mire from the concerned facade, leaving them sparkling clean. They will have prior experience in cleaning tall constructions... ...sued yesterday, Byotrol, the developer of antimicrobial hygiene technologies, announced that it has signed a potentially £multi-million contract with Rentokil Initial. The four year contract covers multiple countries and a range of products. This potentially transformational deal represents a landmark commercial milestone for Byotrol, whos... ...ir Healthcare Environmental Decontamination is an innovative new disinfection technology that was designed specifically for the healthcare environment to deliver thorough environmental disinfection in a way that is safe for patients, healthcare staff and medical equipment. Studies have shown hydrogen peroxide vapour decontamination devices, such... ...ion Flex trolley-bucket * Heavy-duty handle with built-in storage tray for tools * Wet/dry vacuum designed for commercial and industrial floor maintenance * 150 psi 'Black Box' pump for indoor pressure rinsing and chemical injection (used in the spray-and-squeegee and spray-and-vac configurations) * Components that can be configured into complete s... ...e Pod is the most compact reserve paper hand towel dispenser available on the market and has been designed to accommodate a large variety of paper types: different lengths, 1ply, 2 ply the choice is yours! 'With its sleek and contemporary design, a full transparent cover to allow quick paper level inspection, the Pod is an excellent choice for a... ...ne's Summer break, with an important new speaker added to the line-up and the final sponsors having signed on the dotted line. Latest addition to the impressive list of speakers is broadcaster and political commentator Daniel Finkelstein, who will be discussing his experiences at the heart of the coalition government. He will join: - Norman B... ...or has finally reached an agreement on wages with the industry's employer associations. The parties signed a three year agreement, which as well as an 8% increase across the board, includes the following:[TRIMTO] * The wage increase from 1st December 2011 will be 8% for area A and 8.5% for area C * Second year Consumer Price Inflation (2% both a... ...ederation of Building Service Contractors (WFBSC) has revealed that over 600 delegates have already signed up for their 2012 Congress, due to be held in Curitiba, southern Brazil, from 10-14 October 2012.[TRIMTO] Hosted in partnership with chief sponsors Diversey and Karcher, the Congress will focus on 'Cleaning for Health', as it also combines ... ...n.co.uk Polythene film and bag broker Polythene UK , has launched a free consultancy service designed to help users save money by putting them in touch with the most suitable supplier for their needs. E: [email protected] Kimberly-Clark Professional has launched a new range of Kleenex branded hand sanitisers. www.kcprofessional.c... ...o added a secret ingredient that will leave you wanting more!' Airoma Xtreme refills have been designed for use in the Airoma automatic aerosol air-freshener which automatically delivers small bursts of fragrance at specific intervals in order to keep malodours at bay. The dispenser provides flexible and easy to use programming options, and can ... ...estrian and animal care centres is now available as part of a new 'Complete Laundry Care' service designed to make running an on-premise laundry simple, hassle-free and affordable.[TRIMTO] Complete Laundry Care offers the latest Girbau energy efficient washers, dryers and ironers with a fixed monthly rental that includes all service and maintena... Trojan Battery Co., the well known manufacturer of deep-cycle batteries, has signed a distribution agreement with Accutrade expanding Trojan's presence in Russia. As Trojan's authorised master distributor in the region, Accutrade is responsible for distributing and servicing Trojan products throughout the country.[TRIMTO] 'The addition of Acc... ...ged the system to have, in particular, 'outstanding handling features', and to be especially well designed for use in sanitary environments, kitchens and workshops. Winner in the category Washroom Equipment: _SCA Hygiene Products AFH Sales GmbH _Dietmar Mühr, Member of the Jury; Judith Rapp, Product & Market Manager, SCA Hygiene Produ... ...uk Polythene film and bag broker Polythene UK , has launched a free consultancy service designed to help users save money by putting them in touch with the most suitable supplier for their needs. E: [email protected] Kimberly-Clark Professional has launched a new range of Kleenex branded hand sanitisers. www.k... ...ull, worn floors. The new 400grit Storm Pad is coarser than the existing pads in the range and is designed to effectively refurbish surfaces by removing the build up of grime. This can then be followed by working up through the finer grit sizes to achieve a deep clean and high shine polish. One company that has experienced the benefits of the n... ... the Automotive Aftermarket Industry Association. The portfolio, online and in printed format, is designed to build awareness of the industry's extensive contributions to a cleaner environment and is part of AAIA's initiative, 'Driving Toward a Cleaner Environment: The Automotive Aftermarket's Green Story'. The automotive aftermarket's common en... ...uk Polythene film and bag broker Polythene UK , has launched a free consultancy service designed to help users save money by putting them in touch with the most suitable supplier for their needs. E: [email protected] Kimberly-Clark Professional has launched a new range of Kleenex branded hand sanitisers. www.k... ...Hyatt Regency and Armani Hotel among others, use our products.' The Arab Achievement Awards is designed to recognise companies and institutions that own, manage and create projects which have had a significant impact on the development of the Arab region. Being committed to sustainability and being customer-centric, Dhofar Global, two years ago,... ...n 1991, the family-run business has flourished over the years. Today it employs 18 people and has designed and installed around 2,500 quality bathrooms across Hampshire, West Sussex and Surrey. Bill Gosney now hands over the reigns of Managing Director to his son, Simon Gosney, (who has been with the company for 15 years) and daughter, Sarah Fas... ...n scrubber drier range: the Hakomatic B 115 R series.[TRIMTO] These ride-on machines have been designed for the efficient wet cleaning of large areas. They combine the manoeuvrability and comfort of the previous generation, the Hakomatic B 650R/B 750 R, with numerous improvements and additional functions. According to the type of floor surfac... ... Noble Biomaterials develops, manufactures, sells and markets advanced antimicrobial technologies designed to manage all forms of bacterial contamination. Noble is a strategic partner of APIC and works closely with the infection prevention community toward the common goal of reducing healthcare associated infections. It has more than 200 global lic... ...thin the new HiFlo Indoor Window Cleaning System. The Starter Kit (RRP £48.70 excl. VAT) is designed for those already in possession of the basic cleaning equipment and consists of the Padholder, 5 Polish Pads and a thread Adapter - which means that the Padholder fits onto any existing Unger pole. A complete Master Kit (RRP £98.42 ex... ...0 Watts output with AutoSave energy conservation, large trolley wheels, a telescopic handle, well designed storage for the suction tube and hose, and a large, swivel-mounted drum. Other outstanding features are the excellent filtration (HEPA Filter Category H10) and a volume of 15 litres. T: + 44 7793 439900 E: [email protected] W: ... ... a filter bucket & wringer and eight specially engineered double-sided microfibre mops and is designed to improve productivity and reduce water consumption and chemical usage. This system, launched in Geneva in June and one which has just won an ISSA/Interclean Innovation Award, generates cleaner water, cleaner mops and cleaner floors.[TRIMTO] ... ...da Professional in the UK and around the world, as well as a useful 'Consult the Experts' section designed to help users identify the best cleaning system for them. Vileda Professional aims to inform and assist users by providing useful and informative cleaning guides, as well as information on how to cut costs without losing efficiency. In a... ...d an industry-first web programme at Greenbuild that year in Denver. Working together, they also designed and introduced what is believed to be the first recycling kit for small quantities of CFLs generated by consumers. RecyclePak is Veolia Environmental Services' complete pre-paid lamp and ballast recycling solution. One price includes everyt... ... fly-tipping and graffiti. Over 400 Network Rail staff worked tirelessly, filling their specially designed 'wombling' [waste collection] bags with around 1,000 tonnes of unwelcome rubbish. 1,000 tonnes!! Naturally, because the railway and its embankments can be dangerous, it's not always possible to involve other people in clean-up operations bu... ...ation, to join with their medical professionals to prevent HAIs. In addition, the programme is designed to engage more patients in HAI prevention by increasing awareness of the problem, helping patients put into context the hospital HAI data that is becoming increasingly accessible to the public and providing guidance on prevention strategies. ... ...on. Also being launched this month - initially in the US - is 'Transpare', and online platform designed to help purchasers easily evaluate cleaning products using a comprehensive and transparent range of environmental, safety and health attributes. Users will be able to easily identify what comprises a product's overall environmental profile and... ...lleges and council buildings.' Tork foam soaps are housed in a dispenser that has been purpose-designed to allow children and people with reduced hand strength to easily access the soap inside. The dispenser has been given the stamp of approval by the Swedish Rheumatism Association. Tork Mild Foam Soap contains natural skin lipids to prevent ... ...ms. 'It expands our range substantially and introduces an improved choice of equipment that is designed with health & safety legislation in mind - particularly important for the healthcare industry. It's a step forward for us and we are excited to work with Filmop and introduce an expanded set of products to our valued UK clients.' Filmop... Designed specifically for everyone involved in the window cleaning industry and the cleaning, maintenance and restoration of carpets, upholstery and flooring, Carpex & Windex 2012 is being staged at the Ricoh Arena, Coventry, on 8th and 9th March. The organisers report that 60% of stand space is already sold. T: 01737 855086 E: [email protected] W: www.quartzltd.com ... ... Vacuum, Landscape and Irrigation. He says that cleaning specialists work together as a team with assigned equipment and tasks. But there's plenty of scope for personal development... 'We provide a two hour 'orientation training' and then monthly training done on-site,' he says. 'The orientation training is followed by a simple test to assess i... ...er than as a liquid. Supplies of disinfectant are made up in-situ and on demand using a specially designed diluter. Two different strengths can be made, for general environmental cleaning and for more serious terminal and barrier cleaning, both to the correct level of dilution. SoChlor tablets have been developed to achieve the optimum balance betw... ...ers experience its efficacy at wiping out pathogens from their water systems. Absulox has been designed for companies in the healthcare, food, engineering and leisure industries - indeed anywhere that legionella and other waterborne microbes are likely to thrive. The company is in late stage talks with a number of Primary Care Trusts and the ... The SkySiren, an innovative safety solution designed by BlueSky to reduce the risk of injury through entrapment when operating a boom type MEWP, has been unveiled by powered access provider, Lavendon Access Services.[TRIMTO] Entrapment can occur when an operator is pinned between an object (from behind or above) and the MEWP's control panel. S... ..., chemicals and cytotoxins are used throughout the NHS under a comprehensive framework agreement. Designed to keep healthcare staff fully protected against dangerous spillages while providing 100% effective clean-up, they now include SoChlor. T: +44 (0) 1920 463098 F: +44 (0)1920 484664 E: [email protected] W: www.gvhealth.com ... ...whenever the need arises. Like all Taski by Diversey scrubber driers these efficient machines are designed to clean the surface, remove dirt and leave the floor dry so that it can be walked on almost immediately with minimal risk of slips or trips. The machines are quiet during operation and, if one of the many battery powered model models is us... ...ves will offer customers an exclusive line of RCP microfibre cleaning wipes and mops specifically designed for the rigors of the rental market. These products, along with other Rubbermaid cleaning tools, will be made available to all Cintas rental locations. Cintas' full-service microfibre programme helps customers maximise the removal of challe... ...kinetic energy created when vehicles are driven over those awful sleeping policemen, (road humps) designed to slow traffic in built up areas, is being harnessed and put to good use. This week I read that studies are underway to see if the fat which gets vacuumed out of overweight people's bellies and thighs during liposuction, could be transformed ... ...dues producing a finer finish. Fro dry use, the technology of the KGS Flexis FM diamond pad is designed to withstand high speed without generating high heat, although a vacuum is required to remove and control any dust. The pads work well under propane burnishers at 2,400 rpm with no adverse effects on the pad. The open weave of the pad allows f... ..., and believe that the independent accreditation provided by membership of schemes and programmes designed to ensure product quality gives customers additional confidence. 'Joining the CHSA's Scheme was a very simple decision for us; we knew our products would pass the auditing process and believe the Scheme benefits buyers of these products. As... ...rs.' Headquartered in Hamilton, Ohio, Kaivac, delivers complete science-based cleaning systems designed to produce healthy results and outcomes while raising the value of cleaning operations and the professionalism of the worker. The originator of No-Touch Cleaning, Kaivac offers an integrated portfolio of environmentally friendly cleaning produ... ...ts by the commercial property company's 'People into Action' initiative.[TRIMTO] The scheme is designed to reward those people who go above and beyond the call of duty. OCS staff all received quarterly awards including Kathleen Healy from Leeds Shopping Plaza in the 'Excellence' category for her work in improving cleaning standards at the centre... MITIE Group, has signed a 10-year contract with Essex County Council. The FTSE 250 company will work with the Council to deliver a transformational outsourcing contract which includes facilities and property management as well as energy services. MITIE will be working in partnership with Lambert Smith Hampton to provide estate management serv... ...re allows the old roll to be fitted onto a spindle and used right up to the end. The system is designed to cut consumption and costs in use while also reducing waste and maximising hygiene. The dispenser presents the washroom user with only one sheet of paper at a time, and each towel is touched only by the user. The Tork Premium and Tork Adv... ...a product offer meeting specific problems with individual solutions.[TRIMTO] Prochem's newly redesigned 40-page catalogue stands as testimony to the Surrey-based company's remit to remain at the forefront in cleaning chemicals and technology development. Its solutions enjoy a worldwide reputation for outstanding performance in the professiona... ...ce, charters are often highly elaborate manuscripts. The Royal Charter is printed on vellum and was signed by Her Majesty. The official document was formally presented to the Master of the Worshipful Company, Peter Holt, by the Windsor Herald of Arms, Mr William Hunt, at the Ladies Banquet held in Drapers' Hall, in the company of some 200 member... Thanks to everyone who has kindly signed and publicised the e-petition to make it compulsory for Local Authorities to provide public toilets. Please could you make it a new year's resoLOOtion to circulate the link as widely as possible to friends, family and colleagues and see if we can get that signature count climbing! If you have the time /... ...l financial performance over the last four years is presented in the publication. The analysis is designed for the busy manager who needs to spot trends in the market, identify and understand threatening competitors. For those looking for quick growth, the analysis can also be used to seek out acquisition prospects.Copies of the full 714 paged anal... ...ity, in addition to providing exceptional customer service. 'Lotus Professional dispensers are designed to be a long term cost and waste efficient solution for all sectors and the extension of our leasing policy reflects this,' explains Emma Nourry, Trade Marketing Manager at Georgia Pacific for the Lotus Professional brand. 'By providing custom... ...roduct manager for CFR, explains that responsible cleaning refers to cleaning strategies that are designed to help protect the environment as well as natural resources, specifically water. 'We want cleaning professionals and carpet cleaning technicians to be more aware of the need to use water responsibly. Historically, it has just not been much... A leading hand hygiene specialist has designed a new compliance system in a bid to stop foodborne outbreaks which caused 4,356 hospitalisations and 46 deaths in 2009.*[TRIMTO] Gojo Industries-Europe is launching a range of colour coded dispensers created to simplify hand hygiene compliance and help reduce cross contamination. Colours and vi... ...uch right away.[TRIMTO] As well as the exhibition, there's a comprehensive education programme designed to cover all areas of cleaning and hygiene, so there will be plenty for visitors to do over the two days.[TRIMTO] T: + 61 2 8586 6106 E: [email protected] W: www.cleannzexpo.co.nz ... ...logy to deliver extra power, higher reliability and prolonged service life. The new batteries are designed for use in electric vehicles such as floor cleaning machines, materials handling equipment and access platforms as well as golf carts and mobility vehicles.[TRIMTO] The Pro-Spec range has been specifically designed to provide durability and... ...d economically responsible manner. The Sustainability Dashboard is an advance reporting system designed for everyone who is involved in sustainability programmes. It is specifically designed to be easy to use, reduce environmental impacts and save money. E: [email protected] W: www.green2sustainable.com ... ...l be ready for shipment in early March 2012. The wipes are offered in multiple packaging options designed for clinical surfaces (6.75' x 5.75' wipes, 155 ct.), multipurpose use (6.75' x 9' wipes, 95 ct.), terminal cleaning (12' x 11' wipes, 185 ct. and refill) as well as in a 32 oz. spray and a 128 oz. economical refill. For more information or... ...ion, is developing a transparent and reliable assessment scheme for commercial cleaning machines, designed to enable customers to compare different machines based on their resource efficiency.[TRIMTO] To be able to evaluate machines adequately, a measurement of cleaning results must first be established. Thus, a working group - WG4 - has been es... ...re Washer and Rotowash Professional floor cleaner, already manufactured by Demon. The Combi is designed for cleaning flat surfaces, including car parks, walkways, roads, pontoons and sports grounds etc. It comes complete with twin spinning arms which rotate at 2000rpm and a 50mm heavy duty skirt that eliminates overspray to anything nearby, whic... ...rvice has been introduced in the city of Chennai. According to The Hindu, a route map has been designed, taking in all 200 wards of the Chennai Corporation, which will facilitate better accountability in as much as any streets not cleaned by 10.00 each day will be listed.[TRIMTO] An online route map covering waste collection will be completed... ...rovides a sweet smelling 'touch of class' to any washroom, office or retail area.[TRIMTO] Well designed and hand constructed with smart curved edges, the new Scentronic Stainless air freshener is available in either a high gloss mirror or grained finish. A choice of four scents is currently provided including the increasingly popular 'baby po... ...professional cleaners have job-related illnesses or injuries each year. As carpet extractors are designed more ergonomically, the number of job-related injuries in the carpet and professional cleaning industry likely will be reduced considerably.' US Products is a leading manufacturer of carpet, upholstery, drapery, stone, concrete, and tile cl... ...consultations with appropriate works councils. As a result, a sales and purchase agreement has been signed by both parties.[TRIMTO] The process will continue towards finalising the acquisition, including the approvals by relevant competition authorities and further local consultation processes.[TRIMTO] It was announced in November that SCA ha... ... addition, a federal BioPreferred programme created by the US Department of Agriculture (USDA) is designed to help stimulate 'Green' jobs, assist rural economies, and help wean the US off of foreign oil. As part of the programme, the USDA unveiled a BioPreferred product label last year to help specifiers identify products that meet specific criteri... ...ts on show, which covered every need and thus will encourage visitors to return. This fair was designed to cover two specific sectors which are most active in the area: laundry and urban cleaning. For this reason, the exhibition hall included an area known as Zona ADV (the ADV Zone) where several firms gave demonstrations of specialist street... ...st brand of washroom dispensing systems (which are installed within the SECC washrooms!) that are designed to offer an end to wastage, over-use, abuse, pilferage and run-out. The hand towel, toilet tissue and foam soap dispensers offer cost savings in use and the tissue, towels and soaps are manufactured at our base in West Yorkshire. 'We are ce... Hospital superbugs could become a thing of the past thanks to a new, long lasting disinfectant designed, tested and manufactured in the UK. The Genie Miracle sanitisation range has been proven to kill prominent the superbugs that are currently wreaking havoc throughout the world.[TRIMTO] Unlike some conventional sanitisers and disinfectants th... ...her chemicals can be dispensed safely and reliably with our fluid transfer pumps. The cleverly designed, patented, double action pumping principle with no critical parts ensures: - fast, continuous flow - no wear as there are no conventional pistons or O-rings and no critical fitting parts - the pumping of viscous liquids up to 8,800 cps F... Kennedy Hygiene Products has announced the introduction of the Ellipse range, its completely redesigned washroom dispenser line that defines Kennedy`s new approach to integrated product design.[TRIMTO] The Ellipse range brings a highly modern and attractive look to Kennedy`s tried-and-tested robust and best-in-class, service-friendly, washroom... The American Cleaning Institute (ACI - formerly The Soap and Detergent Association) today expressed disappointment with research which wrongly raises unfounded safety concerns over cleaning products and ignores enhanced efforts to communicate with consumers about product ingredients.[TRIMTO] 'An enormous amount of research, development and test... ... world. This impressive range of products of surface disinfectants, hand gels, soaps and wipes is designed to be effective in any environment including the domestic, commercial, medical, healthcare, professional, commercial cleaning, infection, disease control and transport sectors. Genie Miracle is a safe, non-alcohol product range whose effica... ...ne of Brainwave's centres or clinics where each child is assessed and a Programme of exercises is designed by the therapy team according to the child's individual needs. Parents and carers are shown how to carry out the programme so that they can undertake the exercises and activities at home and work towards achieving the child's goals. This co... ...f the cleaning team, which will use 32 new trolley systems introduced by Jigsaw Cleaning Systems, designed for the optimum use of space, with a variety of sized buckets and storage compartments for the cleaning products and liquids. The team at the hospital is among the first healthcare teams in the UK to introduce the new innovative trolley sys... ...ble grip handle makes pickup painless and easy. Also available in the range is the TrashStik - designed for safety and productivity. The 105cm long tool is ergonomically engineered so bending and stretching isn't necessary. The red locking button releases the protective sleeve to expose dual stainless steel pins. Press the red locking button ... ... skincare systems, all carrying the same coordinated look and sleek, contemporary styling that is designed to deliver an enhanced washroom experience for every user.[TRIMTO] The Clean Look family comprises different products drawn from the Air Care, Surface Care, and the newly launched Skin Care system ranges. Each dispenser is available in blac... Vileda Professional has announced the launch of its new VoleoFlex tailor-made compact trolley, designed to be used with the company's microfibre flat mopping systems - Swep and UltraSpeed..[TRIMTO] 'At Vileda Professional we understand the demands of operating an effective, efficient cleaning service,' comments Karen Barker, Marketing Manager ... ...y, earning the IFT Food Expo Innovation Award as the industry's first and only cleaning programme designed to address low trans fat oil build-up in food processing environments. www.ecolab.com ... ...l, first introduced in 2011, will be back again this year, with a special focus on 'Four Nations' designed to highlight issues and successes across England, Ireland, Scotland and Wales. With the coalition government and the CQC (Care Quality Commission) focussed as strongly as ever on the need to maintain the highest standards of cleanliness and... ... world. This impressive range of products of surface disinfectants, hand gels, soaps and wipes is designed to be effective in any environment including the medical, healthcare, sectors and are designed with the reduction of HCAIs in mind. Genie Miracle disinfectants contain EnviroCair as the active ingredient. This has been proven to destroy the... ...ion of recyclables from Newcastle-under-Lyme's 15 bring sites.[TRIMTO] Acumen Distribution has assigned two compacter trucks for this additional recycling work to provide weekly collections at the bring sites for a wide range of recyclable materials including plastic bottles, paper and glass and metals. For the remaining four days, it will main... ...eaning Services is one of the latest group of successful companies to join a leading edge scheme, designed to help industry improve its safety record. The West Sussex based firm recently received accreditation from SAFEcontractor, a programme which recognises very high standards of health & safety practise amongst UK contractors. Employin... ...ionise the way in which feminine hygiene units are serviced.[TRIMTO] The new Femcare system is designed mainly for customers servicing feminine hygiene units with liner exchange rather than bin exchange. It allows these individuals and organisations to overcome some of the inherent drawbacks to an otherwise convenient, low cost and flexible way ... ...nsible for engaging new and existing customers across the UK and Europe with a training programme designed to showcase the company's range of products and their unique benefits. 'We are over the moon to have Linda joining the team,' said Managing Director, Angela Gill. 'I've known her personally for many years and her vast experience, infectious... ...oup has expanded its floor finishing and refurbishment product range even further with a cleverly designed mobile system that polishes and renovates terrazzo; marble; concrete and natural stones with ease.[TRIMTO] KGS Rocky is a revolutionary 'all-in-one' system designed with cleaning contractors and facilities managers in mind. It is light an... Sodexo and UMF have signed a multiyear agreement whereby UMF, innovator and specialist in environmental hygiene, will provide its infection prevention products to Sodexo. Under the terms of the agreement, UMF will provide its advanced antimicrobial technology with PerfectClean products, education, training and support to the more than 6,000 cli... ... not used properly, can sometimes spread contaminants rather than remove them. The systems are designed to thoroughly remove contaminants to eliminate odours and reduce the risk of disease. Plus, workers are no longer forced to crawl around washroom floors, wiping contaminated fixtures and surfaces by hand. No-Touch Cleaning dramatically incr... ...-cycle batteries around the globe, has this week launched 'Trojan Tips' - a video tutorial series designed to expand awareness of deep-cycle battery technology. It provides in-depth information focusing on a variety of battery topics such as deep-cycle battery technologies, maintenance practices, charging procedures and safety when handling batteri... ...olleys represents the evolution of Italian manufacturer VDM's MX project...[TRIMTO] This line, designed to cope with the diverse demands of the contract cleaning market, includes all the features needed to clean in every type of environment, efficiently and with controlled timing. VDM tells us it was the first to realise the market need to wo... The Scott Pro Shop Towels brand, a professional-grade clean up solution designed for automotive DIYers, yesterday announced a new partnership with AJ Allmendinger, driver of the No. 22 Penske Racing Dodge Challenger in the NASCAR Sprint Cup Series.[TRIMTO] AJ Allmendinger will serve as the official spokesperson for the Scott Pro Shop Towels 'T... ...cility's specific hand-drying needs, thereby reducing excess waste. The MOD Dispensing System was designed to reduce consumption across the entire product life cycle, further reflecting the K-CP Reduce Today, Respect Tomorrow approach to sustainability. www.kcpreducetoday.com ... ...and a suite of health & safety forms is provided to help get started. 'Up & coming' is designed for businesses that need in-depth feedback on their current health & safety management system and are looking for a comprehensive bespoke approach for advice, feedback, documentation to suit the needs of their business. An employer handboo... ... provider, and has traded in the UK since 1963 with an increasingly diverse portfolio of services designed to improve work and leisure spaces, while making life a whole lot easier for facilities, building and people managers. In June 2001, PHS Group plc began trading on the London Stock Exchange in what was one of the biggest flotations of the y... ... leading the way in reducing the noise of dryers without reducing functionality. Quality designed with the environment in mind, the Classic+ MkII is a fast drying, energy efficient hand dryer, which has the lowest noise levels in its class. Today’s hand dryers are much faster than previous generations. With an average dry time of ... ...given the go ahead to start rehab now on my chest, shoulders, neck, back and ribs, I have not been 'signed off' yet as I still have issues with my diaphragm and I may even have to have part, if not all, of the metal clamps removed from my 9th rib via surgery later in the year, due to this becoming detached from the rib. I could go on however; th... ...ons and our industry. In total contrast, this week the press reported on a £10million deal signed by EU chiefs to fly them around the world in private jets. Yes - while we hard-pressed taxpayers throughout Europe are struggling to pay our bills, the European foreign affairs supremo Baroness Ashton and other unelected eurocrats will be g... ...his could help kick-start the process.' The Forum is one of 17 leading industry bodies that have signed the Governmentís ëProcurement Pledgeí. Its aims are to help deliver a more streamlined procurement process that will ensure all but the most complex procurements are completed within 120 working days, thereby making it easie... ... system that’s better for the professional to use. Every part of the new system has been designed specifically with the user in mind. The clamps, which adjust pole height or attach the extensions, are simple to operate – no fiddly allen key-type devices needed – and easily adjustable to ensure the right tension; plus the goosen... ...dles can resolve these hazards and reduces injuries.' Strap-n-Go Ladder Handle is specifically designed to facilitate a safe and ergonomic solution for lifting and transporting ladders. The simple design evenly distributes the weight of any ladder while maintaining balance, comfort and control. This inexpensive solution can prevent injuries and ... ...essionals in 148 countries worldwide! Welcome to the first of our ISSA/Interclean Dailies; designed to give you a flavour of the show and what you can expect to see and do here if you’re coming along… and what you’re missing out on if you’re not coming! Amsterdam is a wonderful city – and with over 170 diffe... ...ion? There will also be a Miniskills competition that will be worth watching and four teams have signed up to take part: [TRIMTO] ... ...unced the overall winner. The winning team were clearly delighted and so were we as this cleverly designed mopping system, which uses magnets to attach the mophead to the mop. is unlike anything we've ever seen before. The jury explained that it felt that the Mag system brings an entirely new way of attaching mop covers to the mop holder. In an ... The B 80 W from Karcher is an 80-litre walk-behind scrubber dryer designed for areas between 1500 and 3000 square metres. Simplicity and time saving are the key criteria for any user when purchasing a machine like this, and the B 80 W meets those needs. One of the features that impressed the judges most was the intelligent RFID key system. This c... ...redited with the EU Ecolabel, the range includes toilet tissue, hand towels and foam soap systems designed to put an end to wastage, over-use, abuse pilferage and run-out as well as offering cost savings. T: +441484854434 W: www.baywesteurope.com ... ...ss and building facades, stainless steel, solar panels, signage, and vehicles. Further a cleverly designed hose management system allows you to have the hose inside or outside the pole. The four section master pole cleans up to a height of 6.63m/22ft, but taller locations are easily reached by adding two-stage extensions to the base of... The Snappy is the latest machine to join the range of well designed and innovative machines from Italian manufacturer Macro. While step-on scrubber-driers are not completely new, its the little ideas that go to make this one so neat. First of all, the operator space is in the centre of the machine, not only good for balance, but also for support for the operator's back, to reduce fatigue during the 3.5 hours battery run time. And then there's the easy access for maintenance simply by removing the front panel as shown. ... ... quick and easy to ue for both the occasional user as well as for professional service providers. Designed for warehouse aisles, however an area coverage of up to 6,660 square metres per hour and 3.5 hours operation one one battery charge, means that this compact machine is nevertheless more than capable of large area cleaning. Conveniently, the tw... ...unced the overall winner. The winning team were clearly delighted and so were we as this cleverly designed mopping system, which uses magnets to attach the mophead to the mop is unlike anything we've ever seen before. [TRIMTO] The jury explained that it felt that the Mag system brings an entirely new way of attaching mop covers to the mop hol... Tork products are 'Designed for your convenience' - that was the key message from SCA at ISSA/Interclean. The Tork stand incorporated a number of convenient features for visitors - including water bottles, protein bars and even a baker on hand to provide freshly-baked cinnamon buns. There was also a wash-station allowing visitors to freshen up... ...tion from the British Institute of Cleaning Science.[TRIMTO] The BICSc accreditation scheme is designed to provide the industry with an independent assurance of field-tested excellence. The Hygen system was submitted to the Institute and thorough trials were performed to test the reliability, efficiency and safety of the system, in its intended ... ...ning Professionals, Healthcare Cleaner of the Year 2012 awards has been announced. The awards are designed to encourage excellence in healthcare cleaning by highlighting and recognising achievement, raising awareness of the profession and the vital work its members perform in hospitals, clinics and residential care homes throughout the UK and Irela... ...n of its company website which has recently been given a 'new look' to bring it up to 2012 and is designed with the user in mind. With innovative, moving pictures on the interactive home page, it enables users to download even more information such as the latest press coverage, corporate brochure and the most recent newsletter as well as view the ... ...Cloud technology to store and access information. Costing from just £14.99 per month, it is designed to help businesses of any size manage employees' and sub-contractors' attendance more accurately, more flexibly and more affordably than ever before. For KCC Cleaning which has contracts for regular cleaning of homes, offices, public servic... ... The Healthy Workplace Project The Healthy Workplace Project is a multi-faceted programme designed by Kimberly-Clark Professional to help companies provide their employees with a healthier and more productive office environment. It offers a unique approach to hand and surface hygiene that helps employees understand how germs are transmitted... ...suming - and, as we all know, time is money. Morclean's efficient system has been specifically designed to keep these steps to a minimum. There is no need to have to manoeuvre a wheelie bin inside a van, drag it onto a wash tray and manually removing it again, then times that by 150! With the trailer-mounted lift, it's all so easy. The Morcle... ...nched two new air fresheners. Effective in spaces between 25m? and 60m? in size, Neo breeze is designed to create a pleasant and constantly perfumed environment, whilst removing undesirable odours. Equipped with a timer with three frequencies of activation: 7, 5, 15, 30 minutes (approximately 3000 sprays) and with a sensor which allows a choi... ...rox chlorine wipes showed an 85% reduction of transmission of C. difficile spores. The wipes - designed specifically for healthcare environments - are only available in the UK. Available in canisters with 70 wipes measuring 230 x 170mm, the wipes are designed for daily, multi-surface disinfection by environmental services and nursing staff. T... ...ard water areas, a 5 litre DI (de-ionising, ion exchanger) vessel is added to the system. This is designed to remove all minerals (salts) from the water, achieving a water purity of approximately 100%. Water quality is measured with a TDS meter (Total Dissolved Solids) in 'parts per million' of TDS. In practice, low single digits (0-6 ppm) water... ...nd staff, especially those with respiratory sensitivities. EPA-Registered Sani-Cloth Bleach is designed to kill the most clinically relevant pathogens in healthcare. The stabilised 1:10 dilution of sodium hypochlorite is now tested effective against 50 microorganisms, including 14 MDROs with an overall contact time of four minutes. It is ideal f... ...ye cannot see. * Clean remotes or wraps for the remote control device - These cleverly-designed, seamless remote controls are designed specifically to make it easy to clean and disinfect before each guest stay. * Pillow and blanket wraps - Extra pillows, blankets and towels are wrapped in 100% recyclable and biodegradable sing... ... accessories and waste bins. The company was formed in early 2010 and has since then successfully designed and developed over 30 new products. Specialising in stainless and mild steel dispensers, JDS offers its Synergise range in both brushed satin and a highly polished steel finish as well as a white powder coated steel range. To complement ... ...ailblazing authorities settled more than 90% of their bills in ten days or less. Others proactively signed up to the Government's Prompt Payment Code and made concerted efforts to speed up payments to aid local companies. However, others appeared oblivious to the 10-day target. Some admitted that none of their suppliers received payment within 1... ...ctories and workshops. 'Demon pressure washers are built to the highest standards and quality, designed to withstand the most rigorous environments,' says Says Emma Hancock Sales Manager, Clemas & Co. 'The range includes hot and cold water machines, petrol and diesel, bowser and mini bowser pressure washers - you can find a machine suitab... In a key strategic move designed to further reduce costs and boost productivity, UK contract cleaning specialist LPM Cleaning & Support Services has agreed a new partnership with HSS Hire's Outsource service for the supply, management and maintenance of high performance Reintec cleaning equipment.[TRIMTO] Underlining LPM's ongoing commitmen... ...nsortium.' Ecolab's Global Healthcare business offers a complete line of hand hygiene products designed to meet the unique efficacy and skincare needs of healthcare professionals, including soaps, sanitisers, lotions and surgical scrubs. www.ecolab.com ... ...hip and overcoming unthinking reliance on old habits at an event in Chester recently. The day was designed to help public service managers engineer change in their business models and develop their managerial skills.[TRIMTO] Facilitators Rob Walker and Lloyd Ansermoz of Fedelis Group, which offers management consultancy services across the UK, s... Kimberly-Clark Professional has launched a groundbreaking new initiative designed to address poor access to water and sanitation in the communities where it operates. The Water for Life programme, which is being rolled out to the entire Kimberly-Clark Corporation, will act as a platform to enable the organisation to collaborate with non-gove... Rubbermaid Commercial Products, has introduced two new products, designed specifically to work in conjunction with one another for a superior cleaning experience for the end-user - the Pulse 2-Sided Mop and the Flow Backpack.[TRIMTO] Exceptionally lightweight and durable, the innovative new PulseMop and FlowBackpack allow the operator to move ... ... surface protection. SteriTouch is a range of antimicrobial masterbatches, additives and coatings designed to reduce the growth of harmful organisms such as bacteria, mould and fungi while remaining safe for even the most sensitive applications. This uses patented antibacterial technology that harnesses the natural sterilising properties of silv... ...peripherals, has launched what it believes is the industry's first rechargeable wireless keyboard designed for infection control. To date, the SBW-97-TP is iKey's most innovative and flexible user-friendly design.[TRIMTO] The SBW-97-TP is said to be the first fully sealed, rechargeable, wireless keyboard on the market that uses QuickPair technol... ...d is used for a wide variety of events and concerts. Liverpool Town Hall (pictured right) was designed by the famous architect, John Wood from Bristol, and was opened in 1754. In 1795 fire destroyed much of the building, but restoration work began almost immediately, under the supervision of James Wyatt, a London architect. The rebuilding took ... ...e incidents relate to deaths occurring offshore or on railways, where a region cannot be reliably assigned. The average rate of fatal injury over the last five years has been 0.7 per 100, 000 workers. In each of the last five years, the number of fatal injuries has been: 2010/11 - 175 workers died: finalised figures (previous figure was 171) ... ...s now developed a range of own label domestic and light commercial dehumidifiers, which have been designed to alleviate damp issues in a variety of situations. Designing and manufacturing to the high standards that the company's background demands has allowed Meaco to expand rapidly into other markets and to become a major player not just in the... ...ntly completed a major refurbishment and re-organisation of its Leeds facility: 'The catalogue is designed to provide professionals and the general public with an at-a-glance guide to the full range of products and equipment available over the counter,' he explains. 'There is also expert advice readily available from our teams to talk through any o... ...ving a dedicated Women's Hospital. The cleaning systems used at the facility have been custom designed to suit requirements. Simon Baldock, Sales Director at Ramon says: 'The TTS modular system enables customers to design a bespoke system to fit around their individual requirements. Unlike many other trolleys in the market, all the space is ... ...aying and hygiene solutions, offers clients throughout Europe a choice of over 2000 products -all designed and manufactured to withstand the rigours of everyday intensive commercial cleaning. Probably the largest available range of cleaning products from any one manufacturer in the UK, Contico's portfolio of equipment has been specifically desig... ...hly qualified workers to enter the country and take up well paid positions. The EU has also just signed an addendum to the current visa facilitation agreement with Ukraine which extends the categories of individuals qualifying for the simplified procedure for obtaining visas to travel to EU member states. The amendment covers those visiting rela... The Sanicom 2 grey water pump is a commercial unit designed for 'heavy use' in demanding commercial and professional situations.[TRIMTO] With its twin 1500W motors and a maximum operating temperature of up to 90 degrees C, the Sanicom 2 means additional dishwashers, two minute glass washers, coffee stations, extra sinks and basins, even laundr... Stop-painting.com has introduced a new line of removable floor marking tapes, specially designed to be used on carpeted floors. Made with durable patent pending Superior Mark material, these new Velcro backed floor marking tapes are perfect for classroom floors, municipal buildings and any carpeted floor that needs to be visually organised to dir... ...leaning machine also incorporates a chemical delivery system. The aptly named machine has been designed on a heavy duty chassis and is fitted with all terrain wheels, which enable it to be manoeuvred over very large areas and uneven surfaces with the greatest of ease. 'As the UK's leading steam cleaning manufacturer we are in the enviable pos... ...he compromise agreement on WEEE garnered broad support across political groups and national leaders signed off on the recast directive in December 2011. Still, retailers expressed fears they would bear the brunt of responsibility for collections and transport. Advocates say the new directive will help businesses by setting common EU standards ai... ... removes water from human waste and vaporizes it using a hand-operated vacuum pump and a cleverly-designed membrane system. The remaining solids are turned into fuel that can also be used as fertilizer. The water vapour is condensed and can be used for washing, or irrigation. Eram Scientific Solutions' grant of more than $450,000 will make publi... ...w Slimliner trolley from Vikan, renowned for its advanced hygienic cleaning solutions, is purpose designed for professional and efficient cleaning in busy environments with limited space.[TRIMTO] Flexible and easily manoeuvrable, the Slimliner is ideal for use in shops, restaurants, offices, schools and health clinics. Specially developed to im... ...ed with Energy Solutions and Building Services. A programme of conference quality education is designed especially to assist in the challenges experienced by a high caliber audience and senior professionals and will sit along side a plethora of networking areas. To register you interest, please visit www.thefmevent.com ... ...d to previous electrically-heated models. The HDS-E's heating tank is insulated by a specially-designed boiler jacket which keeps heated water hotter for longer, and therefore reduces power consumption and shortens heating times to as little as five minutes. The 36KW model offers a class-leading continuous working temperature of 58°C and a m... ...ions. The new postgraduate programme is closed to those who are not an FMA member and has been designed only for the member organisations. The distance learning course will be provided by Sheffield Business School at Sheffield Hallam, allowing independent study and completion of the programme in just three years, with a total of 20 campus days i... The British Safety Council has this week launched its 2013 International Safety Awards scheme designed to recognise good health & safety management over the past year. 2013 will mark the 55th year of the International Safety Awards which have made a significant contribution over many years in promoting the benefits that well managed workplace ... ...er, Marketing Manager at Vileda Professional. 'We are constantly developing new products that are designed with the user in mind and ClickSpeed demonstrates this perfectly by offering a simple to use, fast and hygienic way to mop floors.' ClickSpeed is a smart solution when hygiene is paramount, despite tight cleaning budgets and sites with limi... ...e proper maintenance cannot be guaranteed. [TRIMTO] 'The 12-AGM, 12-volt battery is designed to support a wide range of applications including renewable energy and backup power for industrial and residential applications, especially those found in remote areas where there is limited access to clean water which is required to maintain ... ...ector, Paul Wonnacott. 'As the most crucial component of the system, the integral pumps have been designed to withstand thousands of activations and have been subjected to a series of automated robotic tests before being printed with a unique identification number.' Robust and reliable, the dispensers are available in a choice of finishes and ha... ...reen and sustainable initiatives.' The Sustainability Dashboard is an advance reporting system designed for everyone who is involved in sustainability programmes. It is specifically designed to be easy to use, reduce environmental impacts, save money, and create a culture of sustainability. E: [email protected] W: www.green2sustainable.... ... as work had to be completed before furniture was delivered for the open day. Regular cleaning was signed up in time for the school's first operational day and Head Teacher, Tina Jackson, reports: 'We were delighted by both the reactive response and the high standard of cleaning from Cresswell and look forward to working in partnership with the co... ...tered Building Service Manager workshops in Northern and Southern California. These workshops are designed to help qualified individuals involved in custodial contracts become RBSM-certified.[TRIMTO] RBSM Workshops are designed for contract cleaning supervisors, project managers, area managers and professionals with a minimum of one year's exper... ...afer and more productive in extreme conditions.' The innovative product bundle is specifically designed for the oil and gas industry. It includes head-to-toe protection - such as disposable apparel, eye protection, head and face protection, hearing protection, respiratory protection and hand protection, as well as welding blankets and contour la... ...ntenance for this versatile machine. High output is also a key characteristic, with a cleverly designed heat exchanger producing more usable heat (121 C / 250 F max) with minimal heat loss to the environment, and a last-step chemical injection maximising pump performance. Additional features include thermostatic temperature control allowing t... ...plaint Investigation course. The Fibre Care Specialist course consists of a variety of modules designed to give trainees the skills and confidence to effectively and safely clean wool carpets or rugs. With sections dedicated to the science behind carpet cleaning and preventative measures, this is ideal for those who want to enhance their existin... ...nd steam/detergent to make short work of cleaning walls, pavements and other hard surfaces. While designed to be tough on graffiti, this model is also excellent at sanitation tasks. Combining the key features of both the Battery and Graffiti models, Edge's Dual steam cleaner is designed for high performance and comes with two standard 6m hoses, ... ...o the lowest common denominator, where he/she didn't have to think about anything. The system was designed more for a robot than a thinking person. No decisions were required about anything. It was a case of, 'Just do your job which we have laid out for you and have done with'. This is a reactive system - and they always fail. The company health &a... ...eneral Manager and Vice President. 'Our newest dispensers, the Purell ADX and LTX systems, are designed to deliver 1.2 ml of hand sanitiser, providing users with the assurance that they are getting the right amount of product for effective germ kill every time.' Purell Advanced Instant hand sanitiser formulations contain a patent-pending blen... ...s embracing 'individualisation'. 'BSCs will not accept anything other than an individual machine, designed for specific sites and tasks,' he said. That Pulire CEO Toni D'Andrea delivered the final 'wrap up' presentation was so appropriate, reports Alan, who says that, 'His almost academic dissertation 'Cleaning as an absolute value' was ac... ...ble for use with the manufacturer's infra-red taps, the new self-sustaining power supply has been designed for use in washrooms with a large volume of users per day, helping commercial premises such as pubs, restaurants and hospitals effortlessly fulfill their green credentials. Mounted directly on the angle stop valve of the fresh water pipe, a... ...o the largest possible audience. With the SkySiren we have a system that's both comprehensive and designed entirely with the needs and the behaviours of end-user in mind. 'We're incredibly proud of our latest success which recognises years of hard work in developing the system.' Mick Ledden, Executive Director - Business Development and Key A... ... floor. The OmniFlex Crossover cleaning system already includes a variety of add-on components designed for different cleaning situations, including Kaivac's Dispense-and-Vac and Spray-and-Vac systems. The AutoVac can be used with either of these add-ons, or users may also select the 'AutoVac Complete' - a preconfigured system that includes t... ...-embossed in an attractive design and laminated, giving a soft touch and luxury feel, yet each is designed to suit the varied requirements of different types of user. OptiSilk Gold is a three ply, micro-embossed, laminated pure pulp white tissue, which offers ultimate luxury and a silky feel. Thick to touch and comprising 160 sheets per roll, wi... ...nd) The company was a finalist in a total of seven categories. These hard-fought awards are designed to promote continuous improvement within the industry and to recognise the hard work and dedication of the cleaning personnel and the standards set are incredibly high. 'We are delighted to have been recognised by the ICCA and our peers as ... ...mberly-Clark Professional is delighted to announce the launch of the Golden Service Awards 2013. Designed to recognise service excellence in the cleaning & support services industry, the awards provide a benchmark for quality and are the ideal opportunity for contract cleaning companies and in-house cleaning teams to promote the excellent work... ...inars and networking events, the association continued to earn attention with exclusive new tools designed to help its members prove their value to discerning facility executives. Meanwhile, the ISSA Resource Centre was buzzing with attendees taking tours to discover many of the association's valuable services and watching the live feed broadcastin... ...0 billion.[TRIMTO] The contract makes the full line of Sani-Cloth Germicidal Disposable Wipes, designed to service all areas of the healthcare facility, available to HealthTrust members. 'This new agreement will afford PDI the opportunity to work with HealthTrust's diverse membership, aid them in reducing healthcare-associated infections and ... CRL's new surface protection system has been specifically designed to protect both glass and stainless steel, making them look cleaner for longer and cutting the time taken to clean them. [TRIMTO] 6K by CRL is a two-step surface protection treatment with a three year protection lifespan. It uses nanotechnology and provides a hydrophobic, ... ...ne on the market that is manufactured from stainless steel.[TRIMTO] The compact Omega has been designed to offer user friendliness, reliability and robustness, but at an affordable price that it is said to compete on price with the domestic machines that are currently available, whilst providing all the attributes one would expect from a profess... ...ards demonstrate a janitorial services provider's ability to deliver consistent, quality services designed to meet customers' needs and expectations. It sets forth processes, procedures, and supporting documentation proven to be characteristic of customer-driven organisations. The evaluation process focused on six areas of management and best pr... ... for use in cold weather; they are perfect for all-year-round protection.[TRIMTO] Specifically designed to enhance grip, whilst protecting the hands from fibre dust abrasion, grit, weather conditions and the painful chaffing associated with prolonged pole use, the gloves are made from a Teflon coated fabric for superior water resistance and dura... ...rol and help reduce healthcare associated infections whilst being gentle on the skin. It has been designed as an 'entire hospital' formulation to optimise the effectiveness of ethanol and demonstrate that formulation matters. It incorporates patent-pending technology that sensitises microbial cells to alcohol. In this way it achieves a level of eff... ...ng system were also important factors in the winning of the Museum of London contract. CCW is designed to streamline the monitoring process whilst allowing a more efficient means of sharing information, and was developed from the C4C system originally put in place for the Department of Health. CK Group with its many years experience in healthca... ...ows ergonomic cleaning, without any bending, thanks to the double pedal and to the mop's cleverly designed fastening system. Key features include: · shockproof and non-marking rubber edge · quick opening and closing system, suitable for both right and left-handed o... ...Ganga Action Plan (GAP) under the leadership of Swami Chidanand Saraswati and an agreement has been signed by GAP, the Federation of Indian Chambers of Commerce & Industry and the Defence Research & Development Organisation - the designer of the toilets. 'Most of the people living in the Ganga river basin have no sanitary facilities,' Sw... ...art to invest again, in part due to the European Stability Mechanism, a new European Union agency designed to safeguard financial stability in Europe - reducing fears of 'Eurogeddon.' That excessive boardroom and banker remuneration has to end, was the message from former Greggs CEO, Sir Michael Darrington, who is the founder of 'Pro Business Ag... ...ris James, CEO of WAMITAB and Professor Kamal Bechkoum, Dean of the University of Northampton, have signed a Memorandum of Understanding to collaborate in the development and delivery of facilities, waste and resource management training overseas - and particularly in the developing world. The agreement, initially for three years, sets out... ...esting when used as a system in order to qualify for this certification. The AutoVac system is designed to provide high-speed cleaning in hard-surface areas (such as hallways, cafeterias, lobbies, warehouses, and fitness areas) without the cost, bulk, or complexity of a standard automatic floor scrubber. This makes it ideal for a wide range o... ...t which eliminates overspray to anything nearby - essential when cleaning in urban areas - and is designed for cleaning flat surfaces, including pontoons, slipways, walkways, roads, car parks and sports grounds etc. It is manufactured with an integral pressure washer, either a Hurricane P2 providing 2200psi at 13 litres per minute or with the opti... ...poration of self-dimming, self-repairing and photovoltaic capabilities into a single glazing unit designed for maximal energy efficiency. And the firm also believes self-cleaning windows as being a perfect fit with such combinations. The report projects that by 2018, advanced multifunctional window systems of this kind will generate around $100 m... ...ioneering step when it introduced its first web-based system, Service Smart, back in 2008. It was designed to give customers instant updates on repairs and servicing at the touch of a button, and included a comprehensive asset management tool displaying real time updates on inventory status; from individual machines to specific locations, even acro... ...stops large particles such as rust from entering the building. The particle filter has a cleverly designed back flush facility that clears the filter either manually or automatically via a solenoid and timer. The technology behind the ContraCalc non chemical hard water scale removal system lies in its in-flow electrode design to ensure the energ... ...esign, whilst providing maximum hygiene. With the majority of feminine sanitary units having been designed many years ago, Vectair Systems' Femcare MVP range is a refreshing take on a mature concept. It is thought to be the first of its kind to incorporate a revolving carousel, rather than the usual modesty flap, which hides waste from view. Whilst... ...eeps mats as clean and healthy as possible. Experts recommend using disinfectants specifically designed for gym mats. These will disinfectant effectively without leaving a chemical residue on the mat. Never apply previously used disinfectant. Always use a fresh solution. While it is not necessary to clean under mats after each use, moisture a... ...raining provider in Wales to train and find work for 50 unemployed people.[TRIMTO] The agreement signed between n-ergy group and the skills body's new social enterprise company Asset Skills for Employment, is part of a UK-wide programme called 'Routes to Employment', which is working to place 200 long-term unemployed candidates into jobs. The... ...he beginning of our developments in the field of protective clothing.' The collection has been designed in close cooperation with some of the company's largest customers. Together, they have carried out a review of needs and functional requirements such as pockets, materials and fits, but also of what protection is required to ensure safer work.... ...vices to most of Telenor's operations throughout the Nordic countries.[TRIMTO] The new contract, signed with Telenor Real Estate, includes delivery of property and support services, cleaning, catering and security, as well as facility management to more than 5,000 offices, buildings and telecommunications installations in the Nordic countries. ... ...ax in collecting and disposing of garbage will not be given a permit unless they produce a contract signed with a cleaning company. He revealed that the mayoralty is set to launch a campaign very soon that will aim to raise awareness amongst students about public health principles and cleanliness. www.saudigazette.com.sa ... ...blem is that it works but a revolving door is created with accounts lost almost as fast as they are signed. Some rapidly growing franchises take this approach, as do some national multi-location cleaning management companies. It is not a new strategy and it never worked to build a profitable, healthy operation. Janitorial sales famines are custo... ...iatives can have in driving sustainable production and consumption. With more than 190 companies signed up, the A.I.S.E. Charter for Sustainable Cleaning is recognised as a mark of continual commitment from the sector, to drive high sustainability standards throughout the life cycle for the detergent and maintenance products sector. European Com... ...ld damage the mat. Conveniently, the Aquatread dries quickly and easily. The Scraper II mat is designed for outdoor use, but works great inside where frequent spills may occur. It is constructed of 100% durable nitrile rubber, so the cold weather, snow and even ice-melting salt won't damage the mat. Cleats act as scrapers to remove dirt, soil, i... ...nd TVH UK. Visitors to the show can expect to find a wealth of products, information and ideas designed to help them to lower costs, improve standards, tackle problems such as pests and chewing gum, improve hygiene and cleanliness to stamp out MRSA, as well as advice on issues concerning insurance, employment legislation and the latest environme... Many properties use ice melt compounds this time of year. These are minerals designed to reduce the freezing point of water, helping to keep surfaces such as pavements and pathways safer to walk on during cold, icy weather. However, these compounds are often tracked into buildings where they dry, leaving a white haze on interior hard-surface fl... ...ingent standards demonstrate a service provider's ability to deliver consistent, quality services designed to meet and exceed customer needs and expectations while performing all the services in a sustainable environment. With over 2,500 employees in 43 states and over $110 million in annual sales, Triangle Services is an industry leader in faci... ...iculous in our demands (and no doubt still are, by some!) Many of us believe though that what we signed up to has now changed beyond all recognition and it's not just the Brits I'm talking about either - it's millions of citizens from other parts of the EU who feel we've been duped by our politicians who are no longer listening to us. We all wan... ...se who have to pay for the products in the first place. Manufacturers produce lockable dispensers designed to prevent waste and make things difficult for thieves, but a story shared with me by a reader shows that in some cases this can be taken to another level entirely! In what has been dubbed ‘Operation Klopapier’, Germany's police... ...se fragrances were created in response to the growing global market trend of aromatherapy and are designed to rejuvenate the mind and body while neutralising odours in any environment, naturally improving health and wellness for users,' says Nicoletta Pagani, Senior EMEA Product Marketing Manager, RCP. 'Key decision-makers can easily update curr... ...ing together to produce the best results - both on and off the pitch.' The Truvox Multiwash is designed to be highly effective on 'difficult' floors, which can include non-slip safety flooring, low pile carpets, escalators and entrance matting. It optimises cleaning time by being able to wash, mop, scrub and dry on both hard and soft floors in o... ...field, leaders on both sides of the Atlantic are to start negotiations for a free trade agreement designed to establish the largest economic alliance in the world, which already accounts for some €455bn in trade and millions of jobs. I’m wondering though whether the talks might be scuppered by the EU’s plans for a Financial Transac... ...asy glide Microfibre Cleaning Pad.[TRIMTO] Easy to use and durable, the aluminium Padholder is designed to facilitate the application of perfect pressure for thorough and precise cleaning. The curved, non-slip rubber coated handle comfortably fits into the operator's hand, reducing fatigue and improving the control of movement, while the practic... ISS and Kalmar County Council in Sweden have signed an agreement valued at more than SEK 50 million, which means ISS will deliver cleaning services to the County Hospital in Kalmar for the coming years.[TRIMTO] Kalmar County Council covers the entire county and is responsible for healthcare, dental care, as well as some education. Kalmar Count... ...l their records in order for these programs to work.' The Business of the Business Advisory is designed to help building service contractors operate their businesses more efficiently and profitably. Tornado Industries has been engineering quality cleaning equipment for more than 80 years. The company has a long history of developing innovativ... ...or online training globally.[TRIMTO] The WoolSafe Academy offers a range of specialist courses designed for both cleaning industry professionals and flooring retailers; amongst them the Fibre Care Specialist course, which is specifically suitable for carpet and rug cleaners that want to extend their knowledge. It is also the course that must b... ...embers have partnered with All Hands Volunteers to create The Sandy Solution, a cleaning solution designed to remediate houses with mould growth as a result of flood damage from Hurricane Sandy. The clean-up can be carried out by volunteers working with the property owner.[TRIMTO] Michael A. Pinto, CSP, CMP, CEO Wonder Makers Environmental, and ... ...ne of people, planet and profit.[TRIMTO] 'The IFMA Service Center of Excellence was conceived, designed and built around the core concepts of sustainability, usability and productivity,' says Marina Badoian-Kriticos, the sustainability director at IFMA. 'By knowing exactly what we wanted to achieve from the beginning, IFMA found ingenious ways t... ...Action Programme), the Environment Agency and the Environmental Services Association (ESA). It is designed to meet a recognised need for cost-effective training for businesses across a wide range of sectors to help them implement more sustainable practices around waste and resource use. 'In these tough economic times, waste is becoming an increa... ...lth operates a Food Hygiene Rating Scheme in partnership with the Food Standards Agency, which is designed to help consumers choose where to eat out or shop for food. More information on the ratings can be found at: www.food.gov.uk ... ...fresh air and its electric panel is simplified, with only one on/off switch.[TRIMTO] Libera is designed for heavy duty use and is ideal, for example, in sandblasting applications. Its main components are a tubular frame set, an industrial pump set (1450 rpm), a simplified electric panel and a motor-pump direct drive. It is available in three mod... ...d into a strategic alliance with Versus Technology, to introduce the SafeHaven monitoring system, designed to help healthcare workers improve their hand hygiene participation rates. [TRIMTO] Healthcare associated infections are a growing challenge affecting the quality of healthcare, and according to the Centers for Disease Control & Pr... ...look at today's product sets and to look forward to the innovation of the future. The content was designed to be very practical, memorable and fun - and along the way, attendees absorb lots of facts, figures and product detail as well as having the chance to practice using this information in a live, but non-customer environment. 'Within our com... ...areas to destroy viruses, bacteria and bacterial spores in just 5 - 10 minutes per room. Uniquely designed for ease of use and portability, the device can be operated by a hospital's environmental services staff without disrupting hospital operations or requiring the use of expensive chemicals. It can disinfect more than 30 rooms per day, so hospit... ...products used is the company's popular VoleoFlex trolley, which is compact and easy to manoeuvre, designed to transport cleaning utensils, waste collection, and the UltraSpeed 25 litre Microfibre mopping system around the mall. In smaller areas the UltraSpeed Mini Microfibre mopping system is used and also facilitates the cleaning of safety floors ... ...pment and fine-tuning, the WoolSafe Organisation is set to launch its Stain Guide App this month. Designed especially for carpet owners, housekeepers, facilities managers and building maintenance managers, the app is set to become an essential tool across the board and proves WoolSafe's commitment to finding new and innovative ways of sharing its k... ...rocess easier and more effective. The surface of hygienic wall cladding includes a finish that is designed to be especially easy to keep clean. Usually, a PVC finish is added to insulated panels. The joints of ordinary cladding are, unfortunately, ideal homes for bacteria. Hygienic cladding has joints that are designed not to be homes for bacter... ...ntry into the awards programme is restricted to those exhibiting at the show and the programme is designed to encourage and reward the research and innovation carried out by companies operating in this specific market. This year the judges are looking for projects that will have proven to increase competitiveness, improve profitability, improve ... ... launched a new, high-performance Melamine Floor Pad tailormade for the job.[TRIMTO] Specially designed for daily, deep and spot cleaning of ceramic, polished concrete, terracotta, VCT and other modern micro-porous floors, 3M Melamine Floor Pads are ideal for use on large surface areas such as shopping centres, warehouses and swimming pools. Not... ...dy and – importantly – is National Health Service approved. Mark explained that it is designed to take NHS Supply Chain approved plastic bags, has colour-coding on the lids, offers hands-free operation and integrated silent closure. Further, it is leak-free. T: 0121 772 4491 E: [email protected] W: www.ehpl.net ... ...loors.[TRIMTO] A special pad was being launched at the show for the retail sector. It has been designed specifically for areas of heavy footfall and will quickly take away the dirt and produce a really shiny floor, quickly and efficiently. T: +46 (0)121 261 200 / 0845 460 2500 W: www.htc-twister.com ... ...een-print industry, we have developed a unique range of polyurethane squeegee blades specifically designed for the scrubber dryer market. [TRIMTO] 'Our range of Viper high performance squeegee blades has been developed from a bespoke polymer elastomer, which is highly chemical and wear resistant.' T: 01282 772 000 W: www.bmpworldw... ...roWipers. Emma Wood (pictured) told us that Optimum ProWipers is a range of seven non-woven wipes designed to meet the needs of those working in industrial workplaces where fast and effective cleaning solutions are needed. OptiSilk is a range of micro-embossed, luxury silky-soft toilet tissue available in three varieties, OptiSilk Gold, Silver and ... ...ctrostatic charge so surfaces such as metal, glass and plastic stay dust-free for longer. They're designed for dusting naturally, so say goodbye to the aerosols and chemicals that polute the environment and hello to dusting in a fast and effective, eco-friendly way!'[TRIMTO] Roy explained that under normal usse, the dusters will last for years, ... ...g feedback, data and ideas from people he knows around the world and as a result he has totally redesigned the system and produced what we consider to be the most advanced, effective, efficient, versatile and user-friendly system for stone restoration ever created.[TRIMTO] 'There hasn't been any official announcement yet and sales are already th... ...ike the Green Gorilla Advanced Spray System! 'Our two series, Professional, and Commercial are designed to meet the needs of a wide range of applications and users. Whether you're working around the house, tackling the big projects, or equipping your business, we have a system for you. Start with one of our high quality spray tanks and add the a... ...y would give the show a boost. So what about the exhibits on the stand? 'Our dispensers are designed with the service specialist in mind and are prized for reliability, quality, cost control, ease of service and hygiene,' explained Jade. 'Every new dispenser is designed to fit seamlessly with previous product generations - it is this gradual ... ...vel and patented features to bring to market what it thought to be the first commode specifically designed to help improve cleaning compliance. With only 10 components and not a single screw, nut or bolt to harbour any grime, the commodes are incredibly easy to dismantle and reassemble. The design is ultra-hygienic, multifunctional, simple and e... ... Thanks for you positive comment about the backpack vacuum, hopefully now ProTeam has made a well designed product available in the UK, with ergonomics and comfort in mind for the user, things will change for the benefit of all. Kind regards, Des Fulcher T: +1 208 3777 9555 W: www.pro-team.com ... ...ironments). 'These healthcare machines are the result of in-depth consultations with the NHS - designed to not only meet, but exceed the requirements of deep clean sanitising,' says Mike. 'They are compact, easily transportable, highly manoeuvrable and so durable that they can be used consistently. 'Patient and operator friendly, they are quiet ... ...eisure centres if they had improved facilities. Each of the three steps in the report has been designed to show how organisations can develop a more strategic approach. Step 1 is to recognise that members care about cleanliness; step 2 involves upholding the operator's brand image to retain members; and step 3 is to use a club's cleanliness as a... ...s a high-capacity three-roll holding toilet tissue dispenser, which features a carousel mechanism designed to deliver continuous supply. The system reduces waste and manpower and the toilet rolls are kept locked and protected, making them secure and hygienic. Bay West Optiserv hands-free is a controlled usage dispenser, which delivers pre-measur... ...ing.[TRIMTO] But what really made the product stand out to us is its packaging... The cleverly designed patented container with its integrated spray bottle and stillage tap provides easy dispensing and compact storage. The sprayer allows simple application and pre-wetting prior to brushing or wiping and the stillage tap offers controlled dilutio... ...nable Exhibitors Program, Facility Engineering Associates. This innovative, voluntary programe is designed to encourage and highlight environmentally conscious exhibition practices.[TRIMTO] 'This is an opportunity for IFMA to recognise the environmentally responsible initiatives and achievements of our members and strategic partners,' said Marin... ...for the London Olympics, the ISO 20121: Event Sustainability Management System standard, has been designed to help organisations in the events industry improve the sustainability of event-related products and services. The new international standard makes Ryans one of only a handful of organisations in Europe to hold all four certificates. Other... Independent facilities management company Incentive FM has signed a three-year deal to provide a fully integrated TFM solution at the Legal & General owned Grosvenor Centre in Northampton.[TRIMTO] Under the terms of the contract, Incentive FM will deliver a full range of services required for the retail environment, including cleaning, sec... ...will be at the Facilities Show 2013 in May to launch Waste Smart. This one-day training course is designed to help businesses cut waste costs, improve their environmental performance and fulfil legal requirements. The course has been developed with support from the Department of Environment, Food & Rural Affairs and other key expert bodies incl... ...incorporates two single phase machines and three, 3-phase machines. These have been specifically designed to fulfil all heavy duty cleaning applications. Each machine features a continuous stainless steel boiler with steam temperatures at source between 165 - 180 degrees deployed at pressures from eight to 10 bar.[TRIMTO] All Matrix machines a... ...gh its paces at Travelodge's Head Office in Thame and its hotels in Swansea and Swanleigh, the UK designed product came out the clear winner.[TRIMTO] Travelodge has recently announced a £223 million brand investment to further grow its business, with £57 million of that going into a new room concept. To ensure this new product offeri... The Madrid-based Eulen group and the Berlin-based Dussmann Group, have signed a an agreement for the joint provision of integrated facilities management services in 35 countries. The partners are both said to be leaders in their home markets for facilities services, reports Reinigungs Markt .[TRIMTO] The agreement will enable companies with gl... ...ead of the messy process of mixing manually, DyeGone is provided in a dual bottle with a cleverly designed trigger that accurately mixes just the right proportions of the two bottles together at the point of application. Consequently the product is always fresh and full strength, there is no wastage of any unused mixture and the container, with th... ...an benefit from our donations.' Assure is an alcohol -free hand sanitiser that is specifically designed for hygienic hand disinfection. It also helps prevent dry skin and does not require water or rinsing, leaving hands soft and moisturised. Its antimicrobial technology conforms to EN1500 and EN1276 standards and is effective against a range o... ...oducts and for end-customers to choose the right one for them.' All Tork paper wiping products designed for mopping up spills will now incorporate Tork Wiping into their naming structure. The new, logical names will denote their strength and application: for example Tork Wiping Paper, Tork Heavy-Duty Wiping Paper and Tork Industrial Heavy-Duty W... ...Each is aligned to meet the participant's individual and market specific needs. Programmes are designed to be affordable by all and group discounts are available. Courses can be held at the SOS training suite or at client premises. SOS offers a complete end-to-end consultancy service to provide all Health & Safety needs including creati... ...: one for exhibitors and one for visitors. We understand that the entire catalogue is online with assigned QR codes, which will facilitate what has been described as a match-making system for managing appointment books and agendas as well as the real time monitoring of visitors arriving at the show, so interested parties will know when others have entered the halls and be able to make contact. ... ...mine the shortlisted products first hand, try them out for ourselves and speak to the people that designed and built them. Look out for our reports on each of these over the coming days as well as news of the winners once they've been chosen. 'The aim of these awards is to pay tribute to the introduction of creative solutions and entrepreneurial... ...s by up to 20%. That is why Karcher has launched the iSolar, a new accessory for pressure washers designed for the thorough cleaning of solar panels,” explained Ulrich Engenhardt. “We launched this two weeks ago and we have already sold out of the first batch!” Not surprising perhaps, when you consider that Germany alone has 2.1 m... ...cleaning machine has been built to simplify cleaning for women. Its beauty is in the pivot system designed to make emptying easy; it uses the weight of the head to provide the gravitational pull required to tilt the drum to the angle needed to drain the contents. It makes cleaning faster, safer and less tiring for the operator.[TRIMTO] “Pr... ...rt on europallets and provided with back wheels that facilitate the climbing of stairs. The newly designed ergonomic handle makes for easier lifting and carrying too. www.elsea.net ... ...am! It was clear as we looked around that this 360 Electrica from Isal Power Sweepers was Italian designed - it was so sleek and stylish. However, it has many other attributes as Leonardo Bernasconi explained:[TRIMTO] 'This electrically driven urban sweeper boasts zero emissions and is very quiet in operation and technologically advanced,' he sa... ... Dr Sara Bagassi told us that this was for extra stability and strength in a machine which was designed to be more compact but quite a bit taller than traditional machines.[TRIMTO] 'The tubular painted steel structure gives high rigidity, the stainless steel hood is 1mm thick and all tanks are made by rotational moulding (average thickness 5m... ...some potential buyers, we thought we’d stop and listen. [TRIMTO] The Gaia has a cleverly designed brush system, a steel frame and large 250mm wheels, which enable it to work efficiently on different types of floor. “Its simple concept ensures that it’s user friendly and the main components are fully accessible for ease of ma... Considering that industrial kitchens are equipped with exhaust systems designed to evacuate smoke and odours, the extracted vapours contain a high degree of fat that accumulates in the filters, hoods, ducts and extractors. The fat is highly flammable and is responsible for most fires in restaurants. Furthermore, dirty systems force the system&rsq... ...ontains a small LPG canister and a bottle of gum solution. At the touch of a button, the cleverly designed pump will inject 3ml per second of the solution into the lance - which is the exact amount required to provide a constant source of the vaporised chemical that is needed for the job. At the point of contact there is a small brass brush that wh... ...ny showcased a wide range of Discover Clean & Green EV Traction Dry Cell Batteries at Pulire. Designed and manufactured specifically for the floor machine and cleaning industry and recognised as the ideal green alternative to traditional hazardous batteries, these batteries are completely sealed, non-spillable and provide consistent and clean p... ...owing graffiti removal systems and a hand aerosol that cleans and disinfects seating and which is designed for public transport systems.[TRIMTO] John and I arrived in time as the team were cutting a birthday cake make to mark the company's birthday celebrations. A slice of cake was just what we needed to recharge our dying batteries and we were ... ...s that impressed us was the brush head, pictured here in the hands of Gianluca Bignotti, which is designed for use in food industry applications. In case you cannot see properly from the image, Gianluca is holding a small magnet that is pulling the bristles - which contain a magnetic component - towards it. The idea behind this is that the food ... ...airly and being less reliant on benefits and having dignity and respect for the work they do. 'Designed by economists, the living wage is £7.45 an hour and £ 8.55 in London - over £1 an hour more than the minimum wage. It is set so as to be a reflection of the true cost of living, which at the lowest levels of pay, is more than... ...doubling the amount of space they are taking, compared to recent years. Contracts have already been signed by many exhibitors and I’m taking new enquiries every day. There has been great interest from the large machine manufacturers too, who are saying, “This is right up our street!” Seven machine companies have already booked and... ...ronments. These new products are manufactured from the highest quality stainless steel and are designed to be integrated discreetly into whichever environment they're destined for. Of attractive appearance, easy to use and very sturdy, the new line consists of: * Double roll toilet tissue dispenser - designed to be fitted to the wall... ...010, at a cost of around Rs 50 lakh each (almost £50,000). One of the machines, which is designed for use on narrow roads, has a tractor base, while the other, which is designed for use on wider roads, has a truck base. TOI reports that only one of the machines has ever been used in the city and then only two or three times, and that it is... ...nt and skills development programme that truly changes lives.[TRIMTO] The two organisations have signed a partnership agreement that focuses on supporting ex-offenders by helping them to achieve meaningful qualifications and skills whilst being employed by Amaryllis within UK prisons. 'This programme represents the significant steps we are ta... ...ne of 19 Ecolab research centres around the world, the Campinas facility has several laboratories designed to meet the technological demands of the markets Ecolab serves, including a lab for the development of solutions to increase the efficiency and sustainability of oil and gas operations. The centre also has facilities to research new industrial... ...municate the brand change and product ranges will be renamed according to the task the product is designed to aid - Wiping, Cleaning or Polishing. - From 1st October SCA distributors will start to receive Tork branded products for Washroom and Healthcare. Further details will be available early June, however, key systems to include in the ... ...rigger spray bottles that can be purchased as part of a contract, reducing the amount of plastic consigned to landfill. The team knows that supplying products in powdered form has saved clients up to 50% on their commercial cleaning budgets. This, as well as being fantastic news for existing clients, is, however, the team's biggest hurdle for a... ...tainability and the environment, the new site has undergone a complete refurbishment and has been designed to be eco-friendly. The 7,500 sq ft modern office facilities will include a state of the art reception, offices, training room and product display areas. The manufacturing facility has been fully refitted with energy efficient lighting in an ... ...increasingly using auto scrubbers for their efficiency, and these products have been specifically designed for these machines,' added Goldman. The floorcare products in the new line work together to prolong floor life. When floors are properly maintained with PortionPac, they do not need to be stripped and recoated as frequently. This reduces th... ...d users only via a website. Over 170 major nationwide businesses, from several key sectors, have signed up to use the scheme when selecting contractors for services such as building, cleaning, maintenance, refurbishment or electrical and mechanical work. T: 01923 204020 E: [email protected] W: www.thejpmgroup.com T: 029 2026 67... ...ks to its small size, Violetta is ideal for continuous work of up to three hours' duration. It is designed and made in Italy and even though it is small, it has all the characteristics which are common to all the pressure washers that are manufactured by the Idrobase Group. * The axial pump (2800 rpm, brass head ,three ceramic plungers) i... ...e six other shows and a two-day cleaning conference, along with free-to-attend seminars specially designed for each show,' promises Steven Lee, Project Manager at Shanghai UBM Sinoexpo International Exhibition Co. 'In addition to that, we will also be holding a China Clean Expo Innovation Awards gala dinner. These new features of the expo are: W... ...ions. It is virtually maintenance free and no water topping-up is necessary. The batteries are designed for optimum performance when used with a Hawker high-frequency charger. This combination is more efficient than traditional equipment and ensures correct charging whatever the battery's state of discharge. This reduces overall power consumptio... UBM Live, organiser of the Facilities Show, recently


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signed to confirm whether such hazards or problems exist in that workplace or not. If necessary, you can leave a question open and go back and answer it later. * Rating: Here you can set the tool for each level of risk. * Action Plan: At the fourth a... ...w operation is expected to create approximately 120 new local jobs.[TRIMTO] 'This expansion is designed to drive innovation and improve the speed of service for our growing cleanroom service customer base in the Southwest,' says Brad Drummond, chief operating officer for Aramark's Uniform business. 'The McKinney processing facility is the second... ...e customised to satisfy the demands of the nuclear industry and incorporate site specific options designed to improve safety and driver comfort while also promoting equipment longevity. Maintenance support covers not only the Konecranes equipment but LLWR's entire rolling fleet totalling 30 assets. To ensure the right level of support, Cooper So... ...e used frequently to cleanse whilst being kind to hands. The formulation is fragrance free and is designed to fit inside the Sanitex MVP Dispenser, which is available in white, white and chrome and black and chrome. Durable, sealed soap cartridges ensure optimum hygiene at all times. T: Tel: +44 (0) 1256 319 500 E: [email protected] ... ...arehouse, making sure 'what was on the box is in the box'. On joining DJB Cleaning Supplies also signed a declaration that it will only stock and offer for sale CHSA Accredited products or products that conform to the same Standards as required by the relevant CHSA Manufacturing Standards Accreditation Scheme. To maintain its Accredited statu... ...e launched from Monday at ProMAT 2017 in Chicago, USA. The new ICL1200 is a 1200 Watt charger, designed to optimally charge lithium battery systems with any lithium-ion chemistry (e.g. LCO, NCA, NMC, LMO, LFP, LTO, etc). Delta-Q's new lithium charger is suitable for use on any electric machine including floorcare machines, automated guided vehic... ...the global adoption of new products and solutions. 'Sealed Air's advanced product portfolio is designed to reduce waste, conserve resources and provide product security, and deliver measurable value to customers and the planet.' Ken Hanau, a managing director at Bain Capital Private Equity, added: 'Diversey has a long track record of leadersh... ... to be placed through Remploy almost 10 years ago, when the partnership started in 2006. TCFM re-signed its partnership with Remploy last year, committing to place the 1000th candidate who has found work through their relationship which now spans over 10 years. During this time, Remploy has been able to repeatedly find superb candidates to work ... ... yellow card, and those that are judged to have failed are shown a red card. The initiative is designed to encourage and celebrate good standards of health & safety, while constructively challenging teams if their working procedures fall short of what is expected. Lanes Utilities, part of Lanes Group, the UK's largest independent drainage... ...al 360 System is a cost-effective solution for efficient, comprehensive surface treatment that is designed to help keep facilities healthier while saving them time, money and labour. From absenteeism and facility closures to negative press, illnesses and infections can have serious impacts on public and commercial settings - and not just during ... ...ce as the foundation, SCA has developed a well-invested, resource-efficient industrial operation, designed to generate the highest possible value from the forest using all parts of the tree. The forest products business produces solid-wood products, pulp, kraftliner, publication papers and renewable energy. Following the split, Essity will be a ... ...of last year, The Restroom Association (Singapore) unveiled plans for SaniGives, a new initiative designed to recognise cleaning attendants. RAS asked its existing partners - Adkel Solutions, Adsec Global, and Viso - to support SaniGives by raising donations. The donations will be used to organise tribute meals and present grocery vouchers to cl... ... our smartphones to help reduce water consumption. CEO Klaus Reichardt, says there are now 'apps' designed specifically to 'help us realise how much water we use and suggest ways to reduce consumption'. 'Take time to explore the following water apps,' he says. 'While we cannot mention manufacturers, a little googling should uncover them.'[TRIMTO... ... JPC brand to provide horizontal and vertical cleaning services to 88 Wood Street in the City. Designed by architects Richard Rogers Partnership, the 18-story building was constructed between 1993 and 2001 and has an office space of 33,000sq.m (355,209 sq ft). Tenants include National Australia Bank, Hewlett Packard, Bryan Cave, IP Soft, Collins... ...s: 'We are thrilled to have been chosen from hundreds of applicants. Our hand dryers have been designed to transform the washroom market by delivering unrivalled levels of sustainability, energy savings and operational resource savings. We look forward to showcasing them to people who share our vision and values for a sustainable future.' Ala... .... 'The award pays tribute to the groundbreaking design quality of Unger Stingray. The product was designed in such a way that it guarantees optimal performance and simple operation.[TRIMTO] 'This in turn allows us to provide building cleaners with considerable added value in their daily work. Thanks to Unger Stingray, they are now able to clean ... ...that engineers and plant managers get the best possible experience. The content of the aerosol is designed to make the maintenance process more efficient for engineers and now the packaging itself also does its part.' T: +44 (0)1902 510 200 W: www.ncheurope.com ... ...urer of innovative medical devices. The portfolio acquired includes UVC disinfectant technologies designed to disinfect rooms, surfaces, non-critical equipment and devices. Daylight's solutions are noted for their highly innovative, portable and affordable design, allowing safe and targeted dosing of UVC light to quickly and effectively kill har... ...ation, useful hints and tips, FAQs and details about the judging process. 'The 2018 Awards are designed to have a stronger focus on raising standards in the industry though innovation, development and investment,' adds Peter Oliver. 'We look forward to receiving the entries and wish all entrants best of luck.' Sponsorship opportunities remain... ...tion,' he says. 'Our comprehensive portfolio is attractive to our customers, with sacks specially designed for specific markets; crucially, they can produce UN Approved clinical waste sacks, Cleaning & Hygiene Suppliers Association accredited sacks and meet customer derived specifications such as those from NHS Supply Chain. 'We actively bri... ...cal's UVC technologies are highly innovative, ultra-portable and proven effective, yet affordably designed. Diversey Care acquired Daylight Medical's UVC solutions, including the MoonBeam3 disinfection system, SKY 6Xi and 7Xi devices, as well as a robust, ground-breaking pipeline of futures. Designed to disinfect patient rooms, surfaces, non-cri... C.A-L. Italy is using Pulire to showcase some new machinery for the first time. Designed and manufactured in house, one is a waste water biodegradation system and the other is a cloud fleet management system that can be retrofitted onto all kinds of industrial machines..[TRIMTO] Also on show will be the range of Hydrobay stations, Twister disk... ...selling machines, but on providing solutions; its machines, accessories, detergents (specifically designed to optimise the performance of the machine) and after sales service all combine to deliver ideal cleaning solutions. It's why the new building includes a training Academy three times larger than its predecessor and the premises include the ... ...enex brand manager, Kimberly-Clark Professional. 'The new proprietary Kleenex Branded towel is designed to provide exceptional comfort and care for users, without compromising on efficiencies in the washroom.' Kleenex Ultra Rolled Hand Towels work together with the compact Aquarius Dispenser with an improved never-run-out feature. The stub ro... ...l floors and all hard floor surfaces.' Kaivac delivers complete science-based cleaning systems designed to produce healthy results and outcomes while raising the value of cleaning operations and the professionalism of the worker. Kaivac offers an integrated portfolio of environmentally friendly cleaning products designed to remove the maximum am... ...e affected area of most vomiting incidents. The absorbency of these pads can vary, with some pads designed to be as much as 8.5 times more absorbent than other brands, making product selection due diligence a must. * Have an Environmental Protection Agency-registered disinfectant effective against norovirus included in the cleanup supplies. '... ... urgency, and to include a focus on improved safety in the forthcoming Parliament. The letter is signed by the Institution of Occupational Safety & Health (IOSH), Park Health & Safety, the Royal Society for the Prevention of Accidents (RoSPA) and the British Safety Council. The Chartered Institute of Environmental Health (CIEH), Civil... ...New build care home, St John's House offers a new style of premium luxury care in Norwich. It was designed and built with the passion and belief in creating a place where people can feel at home, receive exceptional care and experience a great quality of life. Attention to detail and providing an exceptional care service is at the core of St Joh... GV Health has announced a new end-to-end approach for public health and social care, which is designed to transform safety and hygiene. The new approach addresses four key areas where leading public health and social care providers are seeking transformation. These are the management of waste - in particular infectious waste - to reduce enviro... ...perations at ARL, says: 'This contract showcases our ability to deliver bespoke services that are designed to deliver the highest level of cleanliness to very different kinds of buildings.' T: 0800 856 8800 W: www.arlsupportservices.co.uk ... More than 700 organisations and individuals have now signed an open letter to UK Prime Minister Theresa May which challenges arbitrary deregulation of health & safety. In the wake of the Grenfell Tower blaze, global health & safety professionals, leading academics and some MPs have joined leading organisations in signing the letter. Sen... ... and operators to participate in the Happy Toilet Programme, the Restroom Association Singapore redesigned the HTP certificate with the 'Happy Toilet' wordings omitted to enhance its professional branding image.[TRIMTO] The newly-designed certificate aims to shift the focus from the HTP branding to RAS branding, with higher visibility of the ass... ...tic grill-cleaning robot, uses advanced technology to take the grunt work out of cleaning grills. Designed for use on any-style grill, users simply place it on the grill, press a button and the Grillbot does all the cleaning. Driven by a powerful CPU that controls movement, speed and direction of brushes, the Grillbot is equipped with three high... ...om bacteria, viruses, pathogens and even medicines. 'As a result our new I-Con system has been designed to protect the worker from both risk factors whilst at the same time reducing the threat of cross contamination. 'By reflecting the BICSc recommended colour code, workers and managers can quickly and easily ensure that the right gloves are ... ...d the barriers and context of hygiene behaviour amongst the workers. Ultimately, the programme is designed to target improved behavioru through promotional events and a supply of adequate liquid hand soap at the factory level. With exports of $25.5 billion in 2014-2015, Bangladesh has become the second largest apparel manufacturer in the world, ... ...ties that top your list. If you've ever visited a restroom not to use it, but to see its uniquely-designed stalls or charming chandeliers, then Cintas wants to hear about it. 'Public restrooms don't often receive a lot of praise, so we want to recognise the most talked about toilets that are known for their super clean stalls and innovative desi... ...d include Car Valeting, On Premises Laundry, Train the Trainer and a Health & Safety workshop designed specifically for individuals working in the cleaning industry.[TRIMTO] 'As the leading professional and educational body for the cleaning industry, it is our responsibility to ensure the training courses we offer reflect not only the highes... ...Upright (VBU) - now available from Truvox International - is a light yet powerful upright vacuum, designed for maximum productivity. Powered by a lithium ion battery, the VBU provides more than 60 minutes' continuous runtime. In the Cleaning Show 2017 Innovation Awards, the machine won in the carpet cleaning category. Built to Truvox Intern... ...onal, were UL 410 Certified for Slip Coefficient, and introduced a new line of floorcare products designed to help prevent slip and fall accidents * Floorcare products were developed specifically to help clean and maintain rubber and sheet flooring * New encapsulation and dehydration technologies - designed specifically for carpet spotting, b... ...ned the line-up of innovative cleaning products provided by Denis Rawlins. SpaceVac's cleverly designed package of lightweight carbon fibre poles, cleaning accessories and wireless cameras has revolutionised cleaning at height. The system allows operators to clean up to 15m from ground level in a safe and simple operation with no need for expens... ...r laundry room.' So, what's the solution? 1. Make sure the drier and ventilation points are designed and fitted properly by a qualified engineer. 2. Don't block off the ventilation points, under any circumstances. 3. Regularly service all driers as you would all commercial equipment. 4. Schedule in regular gas-safe checks as well as servicing... ...rol, hand washing, surface care, feminine hygiene disposal, infant care and products specifically designed for the 'away from home' washroom. With European head offices in Basingstoke, England, and North American Headquarters in Memphis, US, along with manufacturing facilities based strategically around the world, Vectair truly has a global reach. ... ...se cleaning robots which can clean smooth surfaces and windows without an operator; ergonomically designed mops; paper towel dispensers for highly frequented public washrooms; fast-acting all-purpose cleaners for coated and uncoated surfaces and floor coverings; easy-care cleaning cloths and gloves for removing stains without chemical agents; easy-... ...gth, waterborne cleaner concentrate formulated for unwaxed, polyurethane finished wood floors, is designed for gym floor cleaning and auto scrubbers. It leaves no residue and will not dull the floor's finish * The Bona SuperCourt Winter Formula Cleaner - Athletic Wood Floor Cleaner Concentrate, a professional strength, waterborne cleaner concent... ...se cleaning robots which can clean smooth surfaces and windows without an operator; ergonomically designed mops; paper towel dispensers for highly frequented public washrooms; fast-acting all-purpose cleaners for coated and uncoated surfaces and floor coverings; easy-care cleaning cloths and gloves for removing stains without chemical agents; easy-... ...luding LEV (local exhaust ventilation) testing. The acquisition by Telford-based Filtermist is designed to ensure Dustcheck retains the same ethos and values instilled in the company by previous owners Ed and Di Spires, as Ed explains: 'As we are a family-run business, we wanted to be sure that whoever took Dustcheck on would continue to oper... ...t the primary purpose of the fair to finalise deals, nevertheless just under half of the exhibitors signed sales contracts during the event. At the time they were questioned, four fifths of the exhibitors expressed their satisfaction with the commercial success of their participation. Over 90% of all exhibitors would also recommend attendance at... The Lindhaus LB4 L-ion Superleggera Backpack vacuum cleaner is superlight and multifunctional. Designed to be light for ease of use, but without compromise to performance, the LB4 L-ion weighs only 4.9 kg battery included, but offers a 5-stage filtration system and air volume lift of 30 litres/sec. The electric version weighs only 3.8 kg and ... ...ent 2) Ensure full implementation of international environmental agreements that the UK and EU have signed up to 3) Ensure that sound science underpins the evidence base upon which decisions about the environment are made The Core Principles also call for a 'progressive approach to improvement'; specifically there must be full transposition of t... ...business development at Roma Systems, writes... 'Workforce Management is a series of processes designed to help in the management of your staff. These processes are linked to HR, and are largely to do with the people who provide your service. This means your cleaning operatives. There are many ways to manage these processes but the most effic... ...identified a number of places where the pipes were blocked, and where the drainage system was not designed for optimum flow of water down though the building. 'This is not unusual in buildings that have been developed over time. Building use changes over time. New developments, including to adjoining properties, can influence the way water drain... ...ed training even more user-friendly and versatile.' The Jangro Learning Management Solution is designed to give Jangro customers the tools to get their teams fully trained quickly, so that they can offer the very best cleaning service possible. www.jangro.net ... ...e's flushing-reducing technology to its pre-commissioning cleaning services; innovative solutions designed to extend system life and cut costs spent on chemicals, consultants and laboratory sampling.[TRIMTO] In line with Guardian's commitment to providing sustainable, whole-life support, the combined expertise and innovation offered by these new... ...rol, hand washing, surface care, feminine hygiene disposal, infant care and products specifically designed for the 'away from home' washroom. With European head offices in Basingstoke, UK and North American Headquarters in Memphis, US, along with manufacturing facilities based strategically around the world, Vectair truly has a global reach. T: ... ...re for nursing home residents. Essity's Tena Identifi was the first incontinence care product designed to electronically track a patient's voiding patterns. The data is graphically converted into actionable, evidence-based reports to help nursing home staff optimise individualised continence care by ensuring that the right products are being us... ...ment. One of the most impactful innovations introduced to clients is a contract management app designed specifically to meet the needs of the cleaning sector. The many benefits and efficiencies this has brought, include an increase of 11% in the contractor's recycling rates and a reduced need for time consuming paperwork, meaning less time in th... ...family of lithium battery chargers to create the ICL Series. The new ICL900 is a 900W charger, designed to optimally charge lithium battery systems of any lithium-ion chemistry from 9 to 15 cells in series. Delta-Q's new lithium charger is suitable for use on any electric machine including floorcare machines, lift trucks, scooters, sports and ut... ...orking methods to suit the requirements of a busy and high-profile events space.' Work.Life is designed for modern working life. Individuals and businesses can book desk spaces or meeting rooms with stylish, contemporary interiors by award winning designers, across four sites in London and one in Reading. Elliot Gold, co-founder of Work.Life,... ...nziclean is a safe, very effective bioactive cleaner, organic waste digester and odour eliminator designed especially for porous floors & walls. Formulated to impart a pleasant scent, Enziclean is recommended for use at food service and food processing facilities, as well as in institutional washrooms and shower areas. Enziclean's bioacti... ...ework, so cleaning can be achieved without the need to dismantle the installation or pipework. Designed, developed and manufactured in the UK, Puracator's multi-function process is simple, rapid and avoids high dismantling and rebuilding costs, making it extremely efficient. T: (+44) 01908 511079 W: www.puracator.co.uk ... ...l also allow for good continuity in the service delivery. A new element is that this agreement is designed as a partnership agreement, which focuses on our and the supplier's mutual interests in finding good, innovative solutions that ensure user satisfaction whilst achieving our purpose as effectively as possible.' Flemming Bendt, CEO of ISS De... ...ot knowing if it is available is a time-consuming problem. The new Heads Up Lock is specifically designed to increase traffic flow in restaurants, coffee shops, office spaces, retirement homes, and retail stores to name a few. T: : 408-966-4365 W: www.headsuplock.com ... ...kind to be certified as safe for ATEX use in explosive atmospheres and hazardous environments. Designed and engineered in Northampton, SpaceVac is highly flexible due to its wide array of brushes and accessories. From gutter cleaning heads and specialist outdoor tools, to a specially designed selection of indoor brushes, the system has a solutio... ...urved riser surface and reach into the crevice adjoining the treads. Wiping with the specially designed REN Riser Pad then removes the remaining grime, leaving a restored, gleaming riser surface. The deep cleaning solution can then be used on the treads. Their restorative clean is completed with the RENclean pad used for routine cleaning. Bot... ...usinesses, including many in the event, hospitality, restaurant and hotel sectors. 'Our carefully designed collections are natural fits for these aspirational institutions,' she explains. 'We're constantly trying to improve our products and we're proud that we didn't cut any corners in developing this new range. We're very passionate about being... ...group facilities manager at Wonga. Alicia joined Incentive earlier this month and was immediately assigned to Camelot, where she will be based at the company's head office in Watford. Her remit includes overall responsibility for maintenance, cleaning, security, M&E, reception, print room, and office services at six Camelot properties across... ...Vendors will demonstrate innovative features, like simple programming systems and USB interfaces, designed to make the laundry process more efficient. This will show that organisations looking to bring their laundry services in-house can calibrate their machines to meet their exact requirements - driving improvements in the quality of laundry ca... ...or the Environment and Water Resources, Masagos Zulkifli, unveiled the strategies and initiatives designed to create a vibrant, sustainable and professional environmental services industry, able to provide the services and solutions required to achieve Zero Waste and a clean environment. More than 78,000 professionals and 1,700 companies in the ... ...ngine and its eDiscovery Platform that will help customers quickly scan and tag data, using a pre-designed set of classification policies, to ensure that sensitive or risky information is properly managed and protected. Veritas commissioned independent technology market research specialist Vanson Bourne to undertake the research upon which this ... ...rethink their value preposition. In light of rising conscious consumption, new products should be designed with sustainable materials and for long-term use. The importance of recycling packaging materials was expressed by TerraCycle. The organisation is helping develop a circular economy by collecting packaging waste to design new products. D... ...ully adjustable backrest with integrated accessory holder, for easy and comfortable operation. Designed to be used in tough environments, where it can combine fast cleanup with a high level of performance in spite of furnishings and narrow spaces, such as cinemas, theatres, buses, trains, aircraft, conference rooms, stairs etc, the machine boast... ...t measure is being announced by the Prime Minister later today; this one part of several measures designed to eliminate avoidable plastic waste by the end of 2042. Building on Tuesday's world-leading ban on the manufacture of products containing microbeads, the Government is to extend the 5p carrier bag charge to all retailers in England, injec... Sodexo has signed a five-year partnership agreement with Chinese global information & communications technology solutions provider, Huawei, which opens the door to Sodexo providing Huawei with integrated facilities management services on a global scale. Huawei has upcoming IFM projects totaling US$400 million. The partnership represents a ... ...al disciplines, Bureau Veritas and Sky-Futures have joined forces in a pioneering new partnership designed to take inspection strategy to, quite literally, a whole new level, throughout the UK and continental Europe.[TRIMTO] Drawing on Bureau Veritas' vast repertoire as a world-leading inspection company and Sky-Futures' cutting-edge, drone-base... ...can deliver the best washroom experience with no compromise.' The system has been specifically designed to help control usage in the washroom whilst providing the highest standards of hygiene. The centrefeed format brings controlled single sheet dispensing, helping to reduce usage of toilet tissue. For the facilities manager, this has a positive... ...inviting visitors to explore its recently refreshed website. The refreshed look and feel has been designed to offer a more engaging experience with improved navigation and functionality. All this to allow both existing and prospective customers to see the full range of solutions TC Facilities Management can offer.[TRIMTO] Created with the us... Cleanlogic™ is a collection of courses and workshops that have been designed by the technical minds at The British Institute of Cleaning Science. This training will provide you with the specialist knowledge to ultimately manage an entire cleaning operation. All Cleanlogic™ courses and workshops are designed for: C... ...ere are violations where conciliation is strictly prohibited.' He said that the legislation is designed to assist inspectors in their work and is aimed at achieving set hygiene objectives for Qatar's residential areas. He added that it is the result of hard work and a long study by specialists at the Ministry of Municipality & Environment in... ...s in 110v, 240v or 380v, single and 3-phase versions, operating between 8 and 10 bar pressure. Designed specifically for the industrial market where reliability and robustness is essential in allowing users to achieve their desired results in improving cleanliness and hygiene standards, the machines boast an efficient dry steam function that red... An investigation is being held into the progress made by an initiative designed to modernise the Disclosure & Barring Service, which provides services that help employers check people's backgrounds against police databases such as criminal records and government lists of those considered unsuitable to work with or in the vicinity of children v... ...ing innovative solutions to their customers.' The SC Johnson Professional Floor Care System is designed to simplify and improve the way maintenance professionals work. It includes one stripper, one finish and one cleaner that seamlessly integrate into existing workflows and requires minimal training. The TruFill Dispensing System offers easy-... ...n from the ventilation shafts of the majestic passenger liners of a bygone era. Says Laura: 'It's designed so people don't just place or drop plastic waste in the bin, they throw it in, so it becomes fun, something children and adults can enjoy, like playing basketball.' The students, all in the first year of their product design course, showcas... ...nformation brings us to the product we offer, appropriately named Foot The Ladder. This device is designed to prevent ladders from slipping away from underneath you. The Foot The Ladder is strong, effective, and very easy to use. How easy? It takes just two seconds to attach to any ladder, and the same amount of time, two seconds, to remove it! ... ...oduction of a new Floor Pad Guide. Available to download from the Jangro website, the guide is designed to help choose the correct pad for the floor type and the required job - whether that is stripping, cleaning or polishing - and includes a handy colour-coding system for easy identification. Jangro floor pads come in a variety of sizes from... Avidbots has this week announced that it has signed a distribution agreement with Macnica, supplier of speciality technology products in Japan and worldwide. Macnica will distribute Neo self-driving floor scrubber across Japan to customers challenged by the largest labour shortage in the country in over 40 years.[TRIMTO] An aging and declining... ...ment.' Visionstate specialises in analytics, interactive customer service and IoT applications designed to streamline operations. The company recently acquired Montreal-based Chatbot Incubator, which specialises in applications for artificial intelligence. Visionstate CEO, John Putters, says: 'Visionstate is extremely pleased to be working wi... ...ailable in South Africa, which is suffering its worst drought in more than a century. Xeros has signed a partnership with local Cape Town company, Fanute, which will distribute and service its award-winning laundry systems in South Africa. Fanute will be opening its doors to the public for a demonstration day today, showcasing this revolutio... ...cial Offer 25% off Cleanlogic™ is a collection of courses and workshops that have been designed by the technical minds at The British Institute of Cleaning Science . This training will provide you with the specialist knowledge to ultimately manage an entire cleaning operation. Cleanlogic™ Courses and Works... ...inspection, because the judge had wiped a clean white tissue around the curves of the intricately designed wrought iron staircase and discovered dust on it. High standards indeed… I know which companies have made this year’s Awards shortlist - and, knowing the judging process, am fully aware of how hard they’ve worked to get t... ...ursday 13th September, is moving to a new venue - the stunning Rockliffe Hall near Darlington. Designed by Hawtree, the world-renowned course architect, Rockliffe Hall's 18-hole championship course is one of the most challenging in Europe. While it presents a stern test for even the best professional golfer, the five individual tee boxes on each... ... to be trained and would welcome new courses. 'Fleet Source's driver courses are interactively designed to engage delegates and ensure the experience is interesting, thought-provoking and enjoyable, whilst achieving the training objectives. We believe that employees who are trained and are able to demonstrate their competency will be an asset to... ...hemical regulation in yesterday's debate. REACH, adopted in 2007, is a world-leading framework designed to provide a high level of protection of human health and the environment from the use of chemicals and to make those who place chemicals on the market responsible for understanding and managing the risks associated with their use. It also see... ...d products will be on display throughout Interclean Amsterdam 2018 in the InnovationLAB (Hall 8). Designed to showcase the latest innovations in cleaning products, services, concepts and ideas, it is an appropriate setting in which to highlight this year's nominees. 'We are excited to once again share the most innovative products and solutions w... ...ks to small-scale projects. The first piece of joint work under the memorandum of understanding, signed this week by ICE and BIFM chiefs, will be to identify the contract-related needs and challenges of those working in FM through a discrete piece of research. The insight resulting from the research - to be announced at the NEC User's Group Conf... ...cilities, enabling business owners to offer their clients great experience. The dispensers are designed with the service specialist in mind and are prized for high quality, elegance, ease of service and ultimate hygiene. The satin chrome gives the dispensers stainless-steel appearance and elegance. The durable lacquer finish eliminates visibl... ...funding gap that prevents many women from starting their own businesses. The Telegraph's letter, signed by Samantha, can be read here: www.telegraph.co.uk www.franchise.domestic-angels.com / www.domestic-angels.com ... ... than have your team sit in a stationary position for the majority of the working day, Mats4U has designed an alternative to promote employee wellbeing. Anti-fatigue mats offer a cushioned high-quality surface underfoot for employees who prefer to work from a standing desk rather than sit for several hours at a time. Standing for three additiona... ...xtensive wastewater treatment plant (pictured) with two steel tanks (Ø15.52m, h=8.78m) was designed and installed in Bavaria as a wastewater collection tank. In addition, a corrugated steel tank (Ø2.75m, h=8.78m) serves as an equalising tank for the paper industry. Larger wastewater pre-treatment plants have also been installed for... ...-built floor scrubbing robot that integrates state-of-the-art navigation technology with hardware designed for ease of use, longevity, serviceability, safety, and high productivity. Deploying the Neo allows innovative facilities and building service contractors to reduce costs, improve the productivity of their cleaning teams,- and position them... ...iting thing is that this is just the start of a whole raft of new product launches this year, all designed to help our customers and strengthen our standing in this marketplace'. T: 01506 430309 E: [email protected] W: www.empteezy.co.uk ... ...r hour, works up to four times faster than traditional cleaners and uses 65% less solution. It is designed to be easily incorporated into a facility's workflow and with the end user in mind. 'The system provides superior surface coverage to help keep facilities, like Fenway Park, healthier while also saving time, money and labour.' www.cloro... ...eraemia cases began in April 2013. Between April 2013 and March 2014, the incidence rate of trust-assigned cases remained stable at 1.2 cases per 100,000 bed days while the incidence rate of CCG-assigned cases decreased by 22% from 1.0 to 0.7 cases per 100,000 population. Following the introduction of third-party assignment category in April 201... ...d risk management, has extended its training provision with the launch of a new training facility designed to provide businesses with the insight, support and practical learnings needed to ensure workplace best practice. Officially launched this week, the Bureau Veritas Learning Academy offers learning solutions, covering health & safety, fi... ...cilities, enabling business owners to offer their clients great experience. The dispensers are designed with the service specialist in mind and are prized for high quality, elegance, ease of service and ultimate hygiene. The satin chrome gives the dispensers stainless-steel appearance and elegance. The durable lacquer finish eliminates visibl... ...such a success, this year it promises to be bigger and better than ever, with almost 100 exhibitors signed up and a full and varied conference programme.[TRIMTO] Find out more at: www.cleaningshow.co.uk ... ... which operates land owned by the Queen across the UK, is one of 1,000 or so employers who’ve signed up to pay it. According to the article, the Palace housekeeping role is a full-time one and pays £16,995 annually, which works out at £326 a week for a 40-hour week and means that the cleaners – working at either Buckingha... ...using plants inside coconuts that sit on a tank, cleaning the water that flows through the roots. Designed to be a zen garden on top of the aquarium, the product is inspired by the Japanese gardening technique called Kokedama. 'We developed an old technique for a device that is adaptable to existing aquariums,' he explains. 'Then we found the na... ....' Nina Purcell, Director of the FSA's ROF programme said: 'This was a valuable study that was designed to see whether accessing data remotely from a food business could make inspections by local authorities more efficient and more effective. 'The businesses involved liked this type of technology and found it more efficient than the current p... ...ing the current OSHAS 18001, the new standard is intended to offer one single set of requirements designed to help organisations of all types and sizes create and implement systems that proactively prevent work-related injury and ill health. This major safety shakeup comes as it is estimated that over 2.78 million people still die each year as a... ... mesh filter with low impact on airflow and static pressure, this weather-resistant pre-filter is designed specifically for high velocity airflow applications such as chillers, dry air coolers, air handling units, cooling towers and air conditioning units. 'As well as allowing pollen to enter a building, fouling caused by airborne debris restric... ... implementing this innovative tool, which has enhanced the patient experience.' The FM Awards, designed to recognise and celebrate the most innovative and ground-breaking projects in facility management over the past year, have been judged by a panel of experts from the industry. Northern Health is one of ISS' largest health contracts in Aust... ...nds every year improving water and sewerage services in this country, but our sewers are just not designed to handle these new wipes which clog up the system.[TRIMTO] 'Sewer blockages end up costing the country about £100 million every year so it's clear that something needs to change. 'We can all take action to combat the problem - wat... ...stand on promoting bathroom equality by launching 'Pants down for equality' - an all-day Looathon designed to highlight the conditions many disabled people face when using public toilets. The campaign is launched in partnership with Sarah Brisdion, who helped bring the issue to public attention in December 2017 through her #ChangingPlaces toilet... ...s a plant holder; look closer and you can see that it's a beautiful and sustainable air purifier, designed and crafted in Italy. It also comes with its own stand, in pure fine wood. The name Natede comes from the combination of nature, technology and design, the three key elements of this innovative, smart and sustainable air purifier which revo... ...these needs by creating an innovative online application tool called myRentokil. This tool was designed to be user-friendly and complements Rentokil's Integrated Pest Management programmes. It enables users to obtain pest management information and recommendations on-the-go. In short, myRentokil allows better traceability and easy access to all ... ...s is partly due to the great focus on the efficacy, efficiency and sustainability of each product designed at Vectair HQ. 'The focus on providing the customer with a high-quality system that makes the customer stand out in its own channel was and is the driving force behind the company ethos. 'We take pride in the quality, design and innovati... ... equipment manufacturer OspreyDeepclean has continued to spearhead the industry with innovatively designed, cutting edge technology. From general cleaning and sanitation to heavy duty cleaning and degreasing, the company offers an unrivalled range of dry steam cleaning equipment capable of tackling the toughest and most difficult cleaning tasks ... ...ciple. The human demonstrates how the room is to be cleaned, and Marvin then imitates them. It is designed for use in small areas and the company is aiming to make robotic technology more affordable for a wider range of applications - while not replacing cleaning personnel. Equipment/tools for cleaning, care and safety - winner - Powr-Mop Lite f... ...shrooms * waste bin, strong, durable and suitable for any environment * Multiflex soap dispenser, designed for environments that require the highest level in hygiene 'We believe in guiding our customers through every step of finding the right dispenser solution,' said Nicole. No wonder the company was able to celebrated 70 successful years tradi... ...quo;t checked what precedes the boxes I’ve ended up ticking. Goodness knows what I might have signed up to! I’ve also received a huge number of these emails from companies that are completely alien to me… I’ve come to the conclusion that some clever (but unscrupulous) marketeers have used GDPR to expand their ‘full... ...r, is moving to a stunning new venue this year - Rockliffe Hall, Hurworth-on-Tees, Darlington. Designed by Hawtree, the world-renowned course architect, Rockliffe Hall's 18-hole championship course is one of the most challenging in Europe. While it presents a stern test for even the best professional golfer, the five individual tee boxes on each... ...inspire a generation to rethink, redesign and build a positive future circular economy. It has also signed up to Operation Clean Sweep, an international initiative to keep plastic litter out of the marine environment, and set up by the plastics industry led in the UK by the British Plastics Federation. RPC bpi recycled products is part of Britis... ...rkers - this one to evaluate the influence of using green-certified cleaning solutions, which are designed to protect IAQ. 'We found that workers in buildings that used green certified [cleaning solutions] scored higher on the tests,' the scientists reported. Says Mike Watt, Avmor's director of training: 'The takeaway is simple. Better indoor... ...ces by installing specialist Forklift Matting within your warehouse or factory. Forklift mats are designed to wipe excess dirt and water from the wheels of forklift trucks and prevent accidents at work. 2 Have specific, designated areas for pedestrians to walk and move around the factory or warehouse, to ensure they are not walking into or cross... ... VBUII can easily be carried from one vacuuming task to the next. But this is a robust machine designed to produce excellent cleaning results. The 43.8V lithium ion battery delivers 312W of vacuum motor power and a 3,000rpm brush speed. Filtration performance is similarly high thanks to its HEPA 13 media bag. A squeegee improves the pick-u... ... It's another case of 'great idea in principle, but how will it actually work?' - new legislation designed by people trying to do the right thing, that just hasn't been thought through properly. I haven't seen similar reviews from the other EU member states (also instructed to meet these gender goals) but I'm hoping that companies in other countrie... ...the public, on users who don't outwardly appear to be eligible to use these facilities, which are designed for people with disabilities... 'Unfortunately, the use of the accessible toilet by those of us with 'invisible' conditions is still continuing to cause abuse. There also continues to be a lack of awareness of why the accessible toilet is s... ...dmissions it was simply incorrect to describe ISS as a 'third party', if the use of that word was designed to minimise or reduce ISS's responsibility for its involvement in the contraventions. The Ombudsman alleged that First Group of Companies hired the cleaners - most of whom were international students from India, the Philippines, Colombia an... ... of a single standard bacteria type for every application, the Evogen range has been specifically designed with tailored microbial strains which help to prolong cleaning efficacy. As the beneficial bacteria continue to break down soiling after application, the frequency of cleaning required is also significantly reduced. This ensures the Evogen ... ...ay in placing dignity and respect at the heart of their organisation. Over 4,300 employers have now signed up to the movement, and their leadership is making a profound difference to the lives of families and communities across the UK. 'Enhance's work in celebrating and championing the Living Wage has been vital to its success.' The awards we... ...anding option is available across the Skanwear workwear range, incorporating hundreds of protects designed to safeguard against workplace hazards. Skanwear has built up an enviable reputation in the UK and Europe as one of the leading suppliers of personal protective equipment for the industrial, manufacturing, energy and utility sectors. It des... ...firms: PI Polymer Recycling along with Recycling Technologies and Impact Recycling, with Binn Group signed on as supply chain partner. The system they're developing uses new state-of-the-art separation systems to support mechanical recycling, fused with a game-changing chemical feedstock recycling process. This includes a patented process based ... ...assed away on Sunday after a short illness.[TRIMTO] At Mulberry, over the years, Chris helped, designed, or ran the communications campaigns for a diverse portfolio of clients including ISSA, Diversey (in several of its many forms), Truvox International, Hydro Systems, Unger Enterprises, Metsa Tissue, Deb and Rubbermaid Commercial Products. Our ... ...lth specifications. They are concentrated, non-toxic, non-flammable, fully biodegradable products designed for today's industrial environments. www.nanoteragroup ... ...eraemia cases began in April 2013. Between April 2013 and March 2014, the incidence rate of trust-assigned cases remained stable at 1.2 cases per 100,000 bed days while the incidence rate of CCG-assigned cases decreased by 23% from 1.0 to 0.7 cases per 100,000 population. Following the introduction of third-party assignment category in April 201... ...s indicated on the label. Upon joining the CHSA Accredited Distributors Scheme last year, Jangro signed a declaration to only stock and offer for sale CHSA Accredited products - or products that conform to the same Standards as required by the relevant CHSA Manufacturing Standards Accreditation Scheme. Purchasing from an Accredited member, like ... ...ange, Caretex Pro, is now handled by Cole & Wilson alongside its Wet and Dry Cleaning ranges. Designed for use in small hard-working laundries, Caretex Pro provides outstanding levels of cleanliness and hygiene whilst also offering excellent value. Like the original Caretex Pro range, each powder has a specific function and is effective with... ...et storage capacity to 9,500. The new 3,767sq.ft laboratory is state of the art, with purpose designed equipment, a microbiological research facility and a virus research facility; further developing the core of Selden; research and development of new products and finding new ways to hygienically clean hard surfaces. Part of the lab w... ...ement Standard (CIMS) and CIMS for Green Buildings have been thought “best practices” designed to boost worker productivity and clean more efficiently * Automate. Manual cleaning methods, for instance, traditional floor mopping, can be automated, significantly speeding up the floor cleaning process * Cleaning solutions. High-perf... ... Kaivac's different cleaning machines. It provides a variety of videos that can be updated. It is designed to help cleaning workers excel at their cleaning tasks by delivering critical operational and educational information, when and wherever it's needed. 'We refer to this as 'just-in-time' skill building,' says Tom Morrison, vice president of ... ...ofessionals and property developers all appreciate knowing in advance how buildings perform. Well-designed spaces and smooth people flows translate into better user experiences, higher rents and faster ROIs.[TRIMTO] However, it can be difficult to strike the right balance between higher occupancy rates - and better income - and efficiency. A bui... ...IMTO] The Avidbots Neo integrates state of the art navigation technology with hardware that is designed for ease of use, longevity, serviceability, safety, and high productivity. 'The Neo has proven to be a cost-effective and productive tool for our operations team, allowing us to maintain our commitment to continuous improvement and innovati... ...intenance and a 20% improvement in asset availability throughout the facility. The agreement was signed by Sanjay Shah (left of picture), executive vice president, DSFH, and Ahmad El Houri, general manager, Serco Saudi Services (third left of picture), in the presence of the Rt Hon Philip Hammond MP, Chancellor of the Exchequer of the United Kin... ...re local people - by supporting local charities to provide the help required, we're all for it. I'm signed up for the run and can't wait to be a part of it.' While Ideal Response is leading the way with returning homes back to normal following a devastation such as a fire or flood, its workforce fully recognises that true healing from the event ... ...oper and provider of water saving technologies with multiple commercial applications, has this week signed a licensing contract with Jiangsu SeaLion Technology Development Co, a subsidiary of Chinese commercial washing machine manufacturer Jiangsu SeaLion Machinery Co (Sea-Lion) at the annual China International Laundry Industry Exhibition. Sea-... ...anaging work-related stress. It also promotes HSE's new Stress Indicator Tool survey software, designed to give you a scientifically robust insight into the six key areas of work design that, if not properly managed, are associated with poor health, lower productivity and increased accident and sickness absence rates. www.hsl.gov.uk ... ...one lucky team on the course. This year the Golf Day has moved to a new venue near Darlington. Designed by Hawtree, the world-renowned course architect, Rockliffe Hall's 18-hole championship course is one of the most challenging in Europe. While it presents a stern test for even the best professional golfer, the five individual tee boxes on each... ... October 2018 and held across three locations - Milton Keynes, Dartford and Liverpool - the newly-designed events will provide attendees with information on the latest regulatory changes and obligations affecting international haulage, including the ban on drivers taking weekly rests within their vehicles, and recent updates to minimum wage require... ... positive impact on the world around them through their consumer choices. The Becọ range was designed by Forpeople as part of its 'forgood' pro bono programme. Forpeople is a multi-disciplinary creative agency, designing distinctly human solutions for some of the world's most interesting and innovative brands. The Becọ identity takes strong ... ...tion facility after a complete handwash using the CleanTech handwashing systems.' Hygienically designed for easy maintenance and cleanability, the ergonomic walk-through design of the ProTech XV employs two hand sanitising troughs, each with sets of spray nozzles that apply a no-rinse quat sanitiser directly onto hands or gloved hands while empl... ... for the domestic cleaning sector. The first is a pilot staff retention workshop which will be designed to help owners of small domestic cleaning companies manage, incentivise and ultimately retain cleaning staff. The second project is the production of a series of best practice videos which will help cleaning company owners understand the ke... ...ere able to prove they had the means to clean up after their pet. More than 250 people have also signed up to the council's Green Dog Walkers scheme which is a non-confrontational, friendly way of helping to change attitudes towards dog fouling. Those who sign up pledge to always clean up after their dog, carry extra dog poo bags, be happy to... ..., enabling business owners to offer their clients a great washroom experience. The dispensers are designed with the service specialist in mind and are prized for high quality, elegance, ease of service and ultimate hygiene. The satin chrome finish gives the dispensers stainless-steel appearance and elegance. The durable lacquer finish eliminates... ... is taken into consideration when at work. Mats4U has an innovative range of mats specifically designed to optimise foot care for standing workers. The collection consists that are primarily designed to alleviate stress to the back and legs by cushioning the areas where people have to constantly stand to work. Anti-fatigue mats work by allowi... ...tion,' explains Paul Wonnacott, managing director at Vectair Systems. 'This combination of a well-designed, fragrant and flexible screen, with an active central core, really does present a unique solution and can provide supreme protection to urinals in any setting, all over the world.' www.vectairsystems.com ... ...er dryers, with numerous models consisting of walk behind or ride-on machines that were carefully designed with the basic TSM principles: Simple, Mechanical, Affordable, Reliable & Tough (SMART). Extremely simple to use and maintain, the Grande Brio machines are light and manoeuvrable and between them are able to meet any cleaning requirement f... Honeywell has published the second in its two-part series of eGuides designed to help health & safety professionals confidently select the right chemical protective gloves. 'A Guide to Selecting Chemical Protective Gloves - Part 2' looks specifically at three key considerations for glove selection - task, environment and fit - and outlines st... ...to invest in new technology as part of the contract using its Live Landscapes web portal which is designed to provide live data and management information across the estate. This will facilitate performance monitoring by site whilst providing downloadable reports which highlight issues, trends and other key data. Mitie's new responsibilities for... KaiTutor is an onboard training system designed to help custodial workers excel at job-related activities, delivering critical operational and educational information when and where it is needed. The result is 'just-in-time' skill building for improved performance and productivity.[TRIMTO] Effective immediately, any No-Touch Cleaning system... ...and organising cupboards - Guide to mopping a floor The Jangro Learning Management Solution is designed to give their customers the tools to get their teams fully trained quickly, so that they can offer the very best cleaning service possible. www.jangrolms.net ... ...network of trained engineers and include the use of genuine original equipment manufacturer parts designed for peak performance in the equipment. 'Faced with a major equipment purchase, it's easy to be overwhelmed by the options available and it can be hard to make an apples-to-apples comparison. We help our customers make an equipment sourcing ... ...ductions' purpose-built facility on the Victoria Business Park in Burgess Hill. The IBC, which is designed to store an impressive 1,000 litres of liquid, was the perfect size for a stay-at-home hydrotherapy pool and Eric wasted no time by getting in touch. 'I spoke with Phil Karn, a director at Bio-Productions, and explained why I wanted to the tu... Tennant Company announced this week that it has signed a definitive agreement to acquire Gaomei Cleaning Equipment Company, a privately held designer and manufacturer of commercial cleaning solutions based in China. Gaomei, a family-run business, offers a range of cleaning machines and equipment, including single disc scrubbing machines, vacuu... ...any other water operated piece of equipment. The high flow capability of this unit at 96 GPM, was designed with gasketed & brazed plate heat exchangers as well as large condenser barrel cleanings in mind. 'The development of scale and other deposits can have a detrimental effect on your equipment-from increasing energy costs to causing equip... ...ion, reduce work orders, and have more control over building management. The Onvation platform is designed to integrate an entire ecosystem of technology to help facilities managers work more efficiently. Steve Schultz, president and COO, Gojo North American and International, says: 'This type of smart solution - the integration of two trusted b... ...pany's online channels like email, social media, website, intranet and digital signage. 'We've designed the Scott Clean toolkit to help facilities managers and employers show they really care for their guests and employees by promoting Scott single use paper hand towels and foam hand cleansers in the washroom as an essential step before every me... ...of the leading marketers of a unique pair of technologies, Biomor and Ecopure. These products are designed to digest organic soils, grease, and oil, remove odors, as well as promote sustainability in the professional cleaning industry. - Avmor is the only company to be fortunate enough to have had two staff members serve as president of ISSA, th... ... this industry was the skill set of employees who provided the actual cleaning services... 'We designed these skins to be peel-and-stick for easy installation,' he reveals. 'We also made sure our products could hold up to regular cleaning cycles as well as cleaning chemicals and disinfectants, so cleaning staff can continue their normal routine ... ...a, Reflex Vans, Arnold Clark and Venson Automotive Solutions, which are the first companies to have signed up to the scheme. Mark Cartwright, head of vans and light commercial vehicles at the Freight Transport Association (FTA), which administers the scheme, explains why he launched the campaign: 'When I heard suicide is now the biggest killer o... ...hin the waste pile for signs of temperature increase and automatically operated turrets (cannons) designed to target the hotspot (rather than a sprinkler system which will deluge a larger area). 'The turrets discharge water with an environmentally-friendly additive which penetrates the waste pile to cool the affected material and seal off the po... ...rooms, pest control, window cleaning and consumable supplies. Dedicated cleaning teams will be assigned at each of the six UK sites and both planned and reactive maintenance delivered through the national mobile engineering team. Around 40 staff will transfer under TUPE and will undertake additional training. A single dedicated account manager w... ... using Pacvac, you can save time by creating a more productive workforce. Pacvac has also been designed to be ultra-light and work with the shape of your body with huge ergonomic benefits. You do not need to bend or stretch as much as you do with a canister vacuum cleaner as the weight is actually balanced on your back. This reduces strain on yo... ...le for delivery of the QI - the contract to provide the post-build clean prior to its opening. Designed by the Quadram Institute Bioscience (formerly the Institute of Food Research), and the Norfolk & Norwich University Hospital, the QI is an £81 million-pound medical facility that will capitalise on the world-class bioscience expertis... ...dispensers, along with the Elements range - a high quality but lower cost entry-level range. Designed-in features benefit washroom service personnel in other ways. For example, they are able to convert Twin Roll toilet paper dispensers to a Mini Jumbo in moments, without even having to take the dispenser off the wall, simply by changing the co... ...eplenish the stocks of disposables. Running costs are extremely low because the dispensers are designed with low dispensing dosage and everlasting batteries. The dispensers come in a selection of colour inserts and in attractive premium finishes: designer grey, chrome and satin. T: 01825 768141 E: [email protected] W: www.kenned... ...nt, utilities, recycling and environmental health. A number of major industry names have already signed up to attend and these include - after a 10-year absence - Diversey, which has agreed to sponsor of the show's VIP lounge. Other leading brands and businesses returning next year include Karcher, Vileda, Northwood Hygiene, Numatic, Makita, Eva... ...es users to review washrooms in any venue, and Waypoint - supplier of P Wave washroom deodorisers designed to keep urinals clean by minimising splash and bad smelling bacteria, leaving a pleasant fragrance. Mike Bone, Loo of the Year Awards managing director says of the sponsors' commitment: 'Sponsorship is an integral element of the annual Loo ... ...g will be recyclable, reusable or compostable by 2025. Currently, 90% of its plastic packaging is designed to be recyclable, reusable or compostable. * Triple the amount of post-consumer recycled (PCR) plastic content in its packaging by 2025. The company will increase its use of PCR plastic in its packaging from 10 million kilograms to more than 3... ...by the Employers' Liability Insurance Association that contract cleaners should use ergonomically-designed equipment. The aim is to provide solutions to the industry to avoid physical injuries to staff and consequent downtime, due to excessive physical strain from cleaning routines. The new erGO! cleaning system with S-shaped telescopic handle a... ...also offers Controller Area Network (CAN bus) communications for seamless machine integration. Designed with floorcare and maintenance original equipment manufacturers in mind, the RC1000 is suitable for use in battery-powered scrubbers, sweepers, and burnishers. Similar to its family series (the RC900 and the RC1200), the RC1000 has an IP66... ...s launched its product to the UK market - and has plans to take it worldwide OneSpray has been designed specifically as the first line of defence against everyday bacteria. Its specially developed formula is not only effective at cleaning screens and surfaces but also guarantees to keep them bacteria-free for up to two days afterwards. Create... ...ment for use in high level hygiene environments more time efficient and sanitary. The cover is designed to prevent contamination of castor wheels within; food, pharmaceutical, biological, medical, automotive, aerospace, optical, nanotechnology and military industries, as well as laboratory and research facilities. Dishwasher and autoclave saf... ...e for the company and its customers. The new 380,000sq.ft facility will be Kärcher owned and designed. Wendy Mitchell, president and CEO of AEDC, says of the move: 'We're thrilled to be welcoming Kärcher into the community as a partner. Aurora is a prime location for global companies to grow because of the connectivity that the City ca... ... robots, announced this week that it is powering autonomous capabilities in a new vacuum cleaner, designed specifically by SoftBank Robotics for use in commercial spaces such as retail and office environments. Available early 2019 and focused initially on the Japanese market, the machine will be powered by BrainOS, Brain Corp's proprietary oper... ...ed telescopic with auto-drive handles; rotatable hand grips allow a greater and swifter mobility. Designed to reduce the stresses and strains placed on a user's wrists, shoulder and back in comparison to conventional floor cleaning tools with a straight handle, the system impressed the Professor and his team. The reduced physical strain caused b... ...ircuit boards increases reliability while the control electronics and new power output stages are designed with energy-saving operation in mind. Assessing performance and planning maintenance cycles couldn't be simpler, since information about all battery charging cycles and their energy consumption is saved over the lifetime of the chargers. Th... ... Stock Exchange Group's international business support and capital raising programme, Elite, too. Designed to help ambitious and fast-growing companies, Elite's international platform is deeply rooted in domestic markets thanks to partnerships with local institutions. 'Elite provides the opportunity to access international support and advice,' a... ...n is known for converting unconventional yet attractive premises into pubs which are individually designed. Part of this design is the provision of washrooms that are spacious and meet the needs of all types of users and the organisation was the UK's first pub chain to introduce Changing Places toilets. Wetherspoon gained Platinum Award grade fo... ...ld lead to higher insurance premiums. Gutters and downpipes when full cannot do what they were designed to do properly, which is to take away large volumes of water away from your building when it rains. When full guttering becomes extremely heavy, it is no wonder they start leaking and sometimes break totally and need replacing when neglected. ... ...nd non-discrimination are key pillars of the council's working practices. 'The work to rule is designed to be proportionate and to allow the council time to do the right thing. It will be disruptive, but the council should listen to the message from their workforce and take immediate remedial action. 'How the council responds will dictate whe... ...n we started getting calls about using the security bin mat in other facilities,' he reports. 'We designed the mat specifically for airports, but soon after the launch we were contacted by nuclear power plants and government facilities since they have similar security screening processes.' NanoSeptic products are positively disrupting the travel... ...1.5 million annually, the company will handle all internal cleaning requirements at the specially-designed accommodation that will be used by construction workers at the site of the EDF Energy Hinkley Point C nuclear power station near Bridgwater in Somerset. The accommodation will be located on two campuses providing total of 1,496 bedrooms and... ...ductions' portfolio of environmentally-sympathetic, pH neutral and chemically innovative products designed to clean environments from healthcare to housekeeping, kitchen to carpets, washrooms to laundry and more. The manufacturer also produces a range of renowned biological products, which uses enzymes to naturally lift dirt. 'I really feel that... ...h the launch of the Window Cleaning World Cup (see more below). The Carpet Cleaning Village is designed for anyone connected to the carpet cleaning industry and carpet cleaning supply chain. Dave Wheadon from The National Carpet Cleaners Association, says: 'It's fantastic that The Cleaning Show has recognised the significance of carpet cleaning ... ...ries' largest agreements for facilities management services, a flexible outcome-based partnership designed to deliver on the parties' mutually developed vision and objectives.[TRIMTO] After working together since 2006, the two signed an official 'Vested' agreement across the Nordic countries just prior to Christmas, formalising the outcome-based... ...d improve performance,' it says. 'The WSWP is currently running a number of campaigns that are designed to reduce waste. One of these initiatives is our Fight Against Food Waste! Residents and businesses can find out more about how to reduce the amount of food they throw away with our #FightAgainstFoodWaste campaign. 'Campaigns like this will... ...with the launch of the Window Cleaning World Cup (see more below). The Carpet Cleaning Village is designed for anyone connected to the carpet cleaning industry and carpet cleaning supply chain. Dave Wheadon from The National Carpet Cleaners Association, says: 'It's fantastic that The Cleaning Show has recognised the significance of carpet cleaning ... ...sing some sewer blockages. These wipes - which included a high proportion of baby wipes - are not designed to be flushed. Commenting on the new 'Fine to Flush' standard Water UK Chief Executive Michael Roberts said: 'This is an important step in the battle against blockages. We've all seen the impact of fatbergs recently, and we want to see f... ...y refused until a tribunal forced its disclosure in 2017, by which time a revised contract had been signed. This was only released on 20th December 2018. 'It was a very difficult decision to take this course of action when so much taxpayer money has already been spent on legal battles,' says Patricia Watson, a waste consultant and volunteer dire... ...nable you to prepare for a 'no-deal' scenario. For the UK market, there will be a phased approach designed to minimise disruption and ensure continuity. www.hse.gov.uk ... ...s finest minds, while the second day will offer visitors the chance to take part in two workshops designed to help organisations gain a better practical understanding of how healthcare cleaning impacts them. Day one of the MENA Regional Conference will see five expert speakers take to the floor to discuss areas of importance for healthcare clean... ...is: - Good for you and the planet. Made with fully biodegradable cellulose fibres specifically designed to be flushed without causing septic or sewer issues, Hyge leaves you 80% cleaner than conventional toilet paper and cuts back on waste. One Hyge roll is equivalent to seven traditional toilet paper rolls. One tree provides some 288 more Hyge ... ...rer's Centenary celebrations and present some long service awards. Pland products are largely designed for the washroom, healthcare, sanitaryware, laboratory, education, catering, janitorial and secure accommodation marketplaces, although a growing proportion of the business involves bespoke products to both commercial and retail markets. Pl... ...xecutive vice president of operations. The new facility in Aurora is Kärcher-owned and is designed to operate more effectively and efficiently with regard to production, distribution, quality and purchasing. The new facility will be equipped with a digital warehouse management system designed to optimise intralogistic processes. Along with ... ...st information on apps, online monitoring tools, e-learning platforms and workflow software - all designed to improve the efficiency of their operations. Talking to experts in the field from providers such as Facility Apps, Haltian, and Progiclean, visitors to the pavilion will have a unique opportunity to understand more about the E-cleaning ch... ...ne train operator identified that newspapers constituted around 80% of its recyclable waste and redesigned its recycling to segregate them more effectively. Now, instead of paying a more expensive mixed recycling cost, it's enjoying a steady revenue stream by claiming the rebate value on discarded tabloids. Stop 2: Map your approach, site by sit... ... the contract. Its Direct Audits system will further enhance the delivery of engineering service. Designed to keep a close check on the condition of assets, it also enables easy day to day monitoring, as well as supporting long term investment decisions. Connect Group will also be taking advantage of Mitie's remote security centre MiTec, to ensu... ...engagement which follows that of the IFM Group in embracing 'freedom within a framework' which is designed to empower our people. Moving Forward This year we have a number of great opportunities in the pipeline with both new and existing clients. We have set ourselves ambitious targets and are committed to meeting them. We are also aware that... ...ff a sheet. Some contractors I've spoken with have said that although the dispensers they use are designed for the roll to go up and over, they deliberately put the roll in upside down to reduce waste, as it's more difficult to let the roll run free that way - clever that! Please get in touch either by emailing me or posting a comment on our Fa... ...ature for the first time a Carpet Cleaning Village dedicated to the sector. The space is being designed for anyone connected to the carpet cleaning industry and will give them the chance to try out the latest products, attend workshops and meet with fellow carpet cleaners and carpet cleaner equipment providers. The National Carpet Cleaning As... ...own industry names such as Kimberly Clark, Rubbermaid, Seko, Supermax, Essity and Vaclensa have all signed-up as exhibitors. Further, The Cleaning Show continues to be used as a platform to announce breakthrough products and cutting-edge services. For example, cleaning machine maker MotorScrubber has confirmed it will be launching a new model at... ...ff a sheet. Some contractors I've spoken with have said that although the dispensers they use are designed for the roll to go up and over, they deliberately put the roll in upside down to reduce waste, as it's more difficult to let the roll run free that way - clever that! ... ...ctors worldwide - will launch its new range of scent diffusers with advanced atomiser technology, designed to bring the magic of scent to interior spaces. Scent is an important emotional influencer, and in addition to fragrancing an environment as a solution to mask unpleasant smells and odours, nowadays it is common to experience fragrance as a... ... What is a DIN? A Drug Identification Number (DIN) is a computer-generated, eight-digit number assigned by Health Canada to a drug product before being marketed in Canada. So, it is just for drugs? No. Most chemicals used in Canada as disinfectants or antimicrobials are regulated as drugs. So, that is the connection. What does the DIN m... ...range of products and services to determine their impact on improving indoor air quality. ASL has designed a series of unique testing protocols and suitability specifications for products to achieve in order to be certified asthma & allergy friendly. ASL's mission is to help people create the healthiest possible indoor environment through sc... ISS and T-Systems, a subsidiary of Deutsche Telekom, have signed a three-year strategic partnership on a platform for Internet of Things (IoT). During the next six-months, ISS will collaborate with T-Systems to build a strategic roadmap of IoT services to be implemented for a three-year contractual term. In 2018, ISS successfully deployed IoT ... ...o being a 'stand out' company or individual deserves recognition, and the 11 award categories are designed to recognise these achievements. Last year's winner of the 'Outstanding Candidate of the Year' award, Fern Soo, ISS Facility Services (pictured here with Samantha West of Vinci Facilities), spoke about the impact winning an award had for th... ...at there are so many reasons why the Excelerator 2.0 will be an absolute winner...[TRIMTO] * Redesigned gripping jaw allowing for deeper reach when closing out a window. * Extreme angle range: -20° | -5° | +10° | +25°| +40° * The negative angles and thinner jaw allow for easier closing out. * New channel locking mechanism, mo... ...ies also come with a fast charger which charges both batteries simultaneously in 1hour 45mins. Designed with Lithium-Ion battery technology and brushless motor technology, the machine delivers a superior combination of HEPA rated filtration, efficiency and productivity. 'We believe that when you move fast, your business moves fast!' says the ... OspreyDeepclean is producing the steam behind the cleverly-designed Puracator unit - an effective, innovative and simple solution to blocked drains. And while the strapline is: 'To the P-Trap and beyond!' the machine can also clean, clear and pasteurise the drains of sinks and multi-deck refrigerated display cabinets without the need for you to d... ...Stafford Tuesday 23rd April - Southampton Friday 26th April - Swindon[TRIMTO] The sessions are designed to be interactive with the opportunity to speak with key staff involved in Policy & Evidence and the following areas will be covered: * Minimum Service Standards proposal * Organics collections * Business waste proposal and LA roles * I... ...s. Headquartered in Hamilton, Ohio, Kaivac, Inc. delivers complete science-based cleaning systems designed to produce healthy results and outcomes while raising the value of cleaning operations and the professionalism of the worker. Kaivac offers an integrated portfolio of environmentally friendly cleaning products designed to remove the maximum am... ... its new Elefantino Lavaggio Fog Maker at the Pulire and Autopromotec 2019 Trade Fairs in May. Designed for the prevention of dust pollution (PMI 10) and the washing of polluted surfaces, the new Elefantino Lavaggio Fog Maker is a 2-in-1 cannon with nebuliser and pressure washer function. Pulire is taking place in Verona, from 21st - 2rd May ... ...siness service contractor members.' The ISSA Consumable Product Cost & Usage Calculator is designed to help members effectively anticipate inventory quantities as well as the costs associated with purchasing consumable items such as toilet tissue, hand towels, hand soap, and bin liners. Members can use this tool to analyse how much product i... ...ducts manufactured from virgin polymers can improve their environmental credentials when they are designed - or redesigned - using Sustane polymers. Sustane polymers have been proven to perform in a wide range of non-food contact applications where previously prime polymer was used. An early adopter of Sustane is the recently launched sustainabl... ...e vision for businesses, people, communities, and our planet.' This year's conference has been designed to align its focus with the United Nation's Sustainability Development Goals in Health, Sanitation, and Innovation and will help attendees examine technologies, innovations, ideas, and solutions that go 'beyond clean' to support businesses, or... ...ublic outsourcing agreements, there is a stand-still period of 10 days, allowing the contract to be signed no sooner than 16th April 2019. www.issworld.com ... ...r dishwasher and a pass-through, the new Nelson Advantage Plus AD51 'Ergo' has been ergonomically designed to avoid the need for the operator to bend. 'Back problems are the most common occupational ailment in the catering workplace,' explains John Nelson, managing director of Nelson Dish & Glasswashers. 'The Ergo provides an affordable solu... ...xury The Soap Co. range is perfect for environments where you want to make a real statement. It's designed for good - with 100% of the revenue from sales being reinvested into employing people with disabilities. Our soap empowers our customers to have a positive impact through their procurement choices. The Citrus hand wash features Pro-Vitamin ... ...embership related projects as needed Collaborate with other staff as needed Other duties as assigned by management as needed Key Competencies Excellent communication skills both written and verbal Committed to continuously educating yourself by attending professional workshops, industry eve... ...embership related projects as needed Collaborate with other staff as needed Other duties as assigned by management as needed Key Competencies Excellent communication skills both written and verbal Committed to continuously educating yourself by attending professional wor... ...ive safety training from Crain Walnut Shelling or Fresno-based Cal North Farm Labor, before being assigned to clean concrete and machinery at the facility. 'Working near large moving parts of equipment and machinery can be deadly,' said Cal/OSHA Chief Juliann Sum. 'Employers must identify and evaluate workplace hazards and unsafe conditions and ... ...his week launched Moviemento, a fully adaptable Learning Management System and e-learning package designed to meet the stringent hygiene requirements of food and beverage production environments. The platform is a result of the collaboration between government agencies, food and beverage safety experts and online training specialists and provide... ...ys: 'I remember when the then Council chair, Doug Cooke, talked about how the change of venue was designed to boost visitor and exhibitor numbers, with a particular emphasis on attracting more global brands and international delegates. Fast-forward to 2019 and ExCel now seems like The Cleaning Show's natural home. It is also now firmly established ... ...ility Management Association, and MediaEdge's Real Estate Management Industry (REMI) Network. Designed to provide a platform to connect like-minded industry professionals focused on keeping buildings clean, green, and operating in a sustainable and energy-efficient manner, the show will be held in tandem with the REMI Show, whose attendees incl... SC Johnson Professional has relaunched its Refresh foam soap line, with new formulations designed to elevate the handwashing experience. Available in a range of new signature fragrances and fragrance-free options, just one pump of product removes over 99% of dirt and germs, when used properly. 'Refresh products are designed to deliver a pleasa... ...aunch a 100% Social Plastic Windex bottle with partner Plastic Bank by Autumn 2019; the bottle is designed to help the environment and address poverty at the same time. Five Southeast Asian countries - China, Indonesia, the Philippines, Vietnam and Thailand - accounted for up to 60% of the plastic waste leaking into the ocean, according to a 201... ...each individual application. Brian Allen, SVP & global ECaSP brand manager, says:'Our team designed the PCS 60 to be extremely efficient by utilising next-generation technology to maximise our customers' return on investment while reducing their overall total cost of ownership. It cleans more effectively and requires significantly less dry i... Designed for kitchens that produce more than 600lbs of food waste daily, the new SBT-140 safely and efficiently unloads full bins into a biodigester or dumpster. Powered by a low-pressure hydraulic mechanism that assures worker safety and quiet operation, the stainless-steel bin tipper lifts bins weighing up to 150lbs (70Kg) and deposits the c... ... as well as numerous newcomers. 'Much of the appeal of CMS 2019 can be attributed to a concept designed to meet the demands of this market: a combination of a quality trade fair, the CMS World Summit as a premium conference for the leading representatives of the industry, and numerous networking events. 'All these factors make CMS Berlin 2019... ...he parade this Saturday - ahead of it being painted in Pride rainbow colours. Around 40 specially designed Pride hearts have also been installed in bus shelters across the city centre with Pride rainbow vinyls decorating bus totems. Mitie is also showing its own Pride, by adding Pride hearts to its Birmingham fleet and volunteering time to suppo... ...nered with AutoNation to introduce PrecisionCare powered by Clorox Total 360, a sanitising system designed to ensure that vehicles are cleaner and healthier. The system uses patented electrostatic technology combined with Clorox disinfectant and sanitiser to quickly and easily kill the most common illness-causing germs. Drivers can also keep their ... ...pioneering industry event and has held an Innovation Award for the last 10 editions and 20 years, designed to recognise the efforts and progress made by manufacturers in the area of innovation. www.afidampservizi.com / www.afidamp.it / www.pulire-it.com ... ...nham (Cordant Cleaning), Kelly Kendall (Cordant People) and Jamie Reynolds (PMP Recruitment) - have signed up to the daunting event on Saturday 15th June. The Yorkshire Three Peaks are situated in the Yorkshire Dales and the challenge consists of climbing the summit of Pen-y-ghent (694m; 2,227 ft), Whernside (736 m; 2415 ft) and Ingleborough (72... ...t line, one family company that includes the same trusted DebMed skincare and monitoring products designed to help reduce infections, increase patient safety and decrease costs.' The transition to the parent brand is expected to be completed throughout the next year. The new brand will be shown for the first time at the APIC 2019 conference, Jun... ...e screens each day. The anti-microbial and anti-fingerprint overlay film has been specifically designed to eliminate germs and protect the display surface in interactive environments. Its anti-microbial property has shown a reduction rate of 99% over a 24-hour period in laboratory tests using colon bacillus and Staphylococcus aureus bacteria. Th... ...his week as its new corrosion-preventative, dewatering and light lubricant solution and has already signed up a number of customers from across the automotive, aerospace, rail and oil and gas sectors. Significant investment has been channeled into updating the firm's hugely popular and approved Shield solution, with the new formula importantly n... ...es including: * Responsive 5in. shatter-proof touch screen that works while wearing gloves * Redesigned user interface that functions like a smartphone and configures to fit any facility or network of facilities * Wi-fi capabilities and wireless sync. technology for secure data transfer to new cloud-based software * Collection and storage of imp... ...telligent air purifier to keep the air we breathe while inside our vehicles as clean as possible. DeIso Coated V2 300%
signed for safety and tested to global standards, the new app connected Blueair Cabin P2i rids the vehicle of the harmful gases, particles and germs that we are exposed to when on the road. Research by UK Parliament shows that the air in our vehic... ...e with four new designs including the Felix Wild, Felix Heart and Felix Oriental. The Felix is designed to be suitable for all floor types - even the most delicate floor surfaces. A swivel neck lets you manoeuvre around obstacles with ease and the handle height can be adjusted for comfort. High performance motors and efficient air flow design co... ...given a helping hand to clean up after messy guests. Aroma Care Solutions' Little Accident Box is designed especially for those occasions when guests have unfortunate accidents that lead to unpleasant smells which make the room or property unready to let again. The Little Accident Box has everything you need for to clean up and when used with th... ...als. Manual sorting tasks can be conducted at the kerbside during waste collection and, if poorly designed, can create risks - hence the publication by the Health & Safety Executive of an in-depth analysis of the many types of equipment used and procedures followed.[TRIMTO] This report describes research to better understand the kerbside col... ...ch Easy-Click pole makes working comfortable and eliminates the need for manual spraying. All are designed to eliminate the need to use ladders and thus keep operators safely on the ground. Able to quickly and safely remove fingerprints, grease, etc. from all smooth surfaces, the Unger Stingray is particularly suitable for cleaning interior glas... ... on the agenda. The latest catalogue offers a wide choice of products and supplies, which are designed to have a minimum impact on the environment, such as compostable waste sacks and Jangro's popular and expanding Enviro collection. As well as innovative soluble sachets, the Enviro range now has options for buckets and eco-mops for mopping ... ...rs of the mains socket brush. * TIM duty roster planner - An online duty roster planner specially designed for the cleaning industry. The development programme of the Federal Ministry of Economics and Energy (BMWi) enables Germany companies to take part in leading international trade fairs on favourable terms. These trade fairs feature a high nu... ...Suppliers Association conference on plastics. First, products, packaging and processes need to be designed with recycling in mind. Second, an integrated approach, from the start to the finish of the supply chain, is crucial if recycling is to be increased. CHSA members, who are working hard to build environmental sustainability into the heart of... ...arlier this year as part of Green Month - a County Council initiative with radio stationSpirit FM designed to raise awareness of environmental issues. The key stage 2 pupils got to head out to the viewing platform which sits above the specialist sorting machinery to see first-hand how recycling is sorted and baled ready to be made into new produ... ... Auditing Professional certification program for the Canadian marketplace. The CMI AAP program is designed for industry professionals looking to enhance their knowledge and skill with regard to quality assurance and building audits. Approximately 30 professionals participated in the day-long session. ISSA Show Canada kicked off on 12th June, wit... ...uietly, generating a low operating sound as quiet as just 56 decibels. The Whisper XL has been designed with hygiene and efficiency at the forefront, equipped with sensors that start or stop the airflow automatically as the hands are fully inserted or removed, eliminating the need to touch surfaces and reducing the risk of scattering airborne pa... ...ll. Together with Mitie's supply partners, the contract will establish community partnerships, designed to support local charities and foodbanks, underpinning the College's commitment to sustainability. 'We are proud to be expanding our long-term partnership with Fife College, to incorporate a range of eco-initiatives which support Fife Colle... ...tion franchise which operates in 20 countries and has over 160 franchise territories worldwide, has signed on as official launch partner for Coulson Ice Blast's latest innovation - the IceStorm45. Coulson Ice Blast just released its latest innovation in its IceStorm product line, and with the new IceStorm45, delivers its first mass-market offeri... ... are suited for hospitals, nursing homes, leisure facilities, schools and hotels, the brushes are designed to be used in accordance with industry standards as follows: - Red brushes for washrooms - Yellow for isolation zones - Green for catering departments, ward kitchen sections and food service at ward level - Blue for general areas including ... ...ne Systems displayed its new electric vehicle, with a high-pressure washing, skid mounted system, designed to clean at height in areas with emission zones, such as central London. The event helped connect visitors with ideas and developments shaping the profession, with 100 continuing professional development (CPD) accredited seminars, presentat... ...upport for four modes of operation and multiple standard lumen packages. SpectraClean has been designed to provide continuous environmental disinfection and complement cleaning and disinfection strategies. One of the biggest advantages of SpectraClean technology is what it doesn't do - harm humans, materials, plants and any other items that are ... ... safety and wellbeing of their organisations' workers. This year, there are several new awards designed to recognise specific health, safety and wellbeing achievements, including the James Tye Award, the Health & Safety Transformation Award and the CEO Award. The new James Tye Award recognises an organisation or team of individuals who ha... ...n of the machine. Just imagine using spurious or pattern parts in one of Numatic's H type vacuums deIso Coated V2 300 Eci
signed for asbestos and carcinogenic dusts - well would you want to use it if its repaired with spurious parts? I wouldn't even join the queue behind you.[TRIMTO] When you use original Numatic manufactured parts you have the knowledge and security... ...elps to store spare consumables making the refill process quick and easy. The Modulo system is designed and manufactured in a specialist engineering facility in Lancashire. Dudley Industries has years of experience in the washroom accessories market, having been designing and marketing washroom dispensers since 1942. Constructed from stainles... ...unique brand whose eco and ethical values aligned with its own,' she adds. 'The Soap Co. is truly designedMagenta Shift When Converting To Iso Coated V2
for good and is a response to growing interest in clean beauty and the call for 100% transparency throughout the supply chain. We place great emphasis on creating award-winning products - lessening any negative impact on our planet and continu... ... Design Chemistry. The Cradle-To-Cradle (C2C) design approach enables eco-friendly products to be designed and materials / nutrients to have infinite cycles. Method cleaning products and C&A jeans are examples of products made according to the C2C design approach. * Indoor pollution and allergens. According to Dr John Ryan from Allergy Stand...Subscribe | Unsubscribe | Cookies | Sitemap