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Supplementary protocol S1
Supplementary Material and Methods
RNA Hybridization
Total RNA was extracted from 15 ml of H. pylori liquid culture grown to an optical density at 600 nm (OD600) of 1.0 using Trizol (Gibco) according to the manufacturer's instructions. RNA concentration and purity were determined by A260 and A280 measurements, and the quality of the preparation was assessed rapidly by electrophoresis in an agarose gel. RNA was denatured in RNA dilution buffer [1 з SSC (0.15 M NaCl plus 0.015 M sodium citrate), 50% formamide and 6.7% formaldehyde] at 68АC for 15 min and put on ice. RNA was applied to nylon membranes (Roche, Basel, Switzerland) using a Bio-Dot microfiltration apparatus (Bio-Rad, Hercules, CA). Following transfer, RNA was covalently bound to the membrane by cross-linking with 0.120 J/cm2 UV light of 254 nm wavelengths. Northern blot hybridization were performed according to a standard protocol described earlier ADDIN EN.CITE Bereswill20001117Bereswill, S.Greiner, S.van Vliet, A. H.Waidner, B.Fassbinder, F.Schiltz, E.Kusters, J. G.Kist, M.Department of Microbiology and Hygiene, Institute of Medical Microbiology and Hygiene, University of Freiburg, D-79104 Freiburg, Germany. bereswil@ukl.uni-freiburg.deJ. Bacteriol.5948-595318221Bacterial Proteins/*metabolismCopper/pharmacologyDown-RegulationFerritins/biosynthesis/genetics/*metabolismHelicobacter pylori/genetics/growth & development/*metabolismIron/pharmacologyManganese/pharmacologyMutationNickel/pharmacologyRNA, Messenger/geneticsRepressor Proteins/*metabolismZinc/pharmacology2000Nov11029412http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11029412 [1] QUOTE "(8;18)" ADDIN REFMAN џ\11\05\19\01\00\00\00\06(8;18)\00\06\00*D:\5Canwender\5Csb\5Csb-publi\5Chp\5CHP1326\5C1326-lit\03\00\0514183,14183 /id Fassbinder, van Vliet, et al. 2000\00,\00 QUOTE "" ADDIN REFMAN џ\11\05\19\01\00\00\00\00\01\00\00*D:\5Canwender\5Csb\5Csb-publi\5Chp\5CHP1326\5C1326-lit\03\00\0514231)14231 /id Bereswill, Greiner, et al. 2000\00)\00 . PCR products carrying parts of the target genes were amplified with primers listed in suppl. table 3 and were used for the production of antisense RNA probes labeled with digoxigenin by in vitro transcription using T7 RNA polymerase (Roche) ADDIN EN.CITE van Vliet20013317van Vliet, A. H.Kuipers, E. J.Waidner, B.Davies, B. J.de Vries, N.Penn, C. W.Vandenbroucke-Grauls, C. M.Kist, M.Bereswill, S.Kusters, J. G.Department of Medical Microbiology, Faculty of Medicine, Vrije Universiteit, Amsterdam, The Netherlands. vanvliet@mdl.azr.nlInfect. Immun.4891-4897698Bacterial Proteins/geneticsCulture Media*Gene Expression Regulation, Bacterial*Gene Expression Regulation, EnzymologicHelicobacter pylori/*enzymology/genetics/growth & developmentNickel/*pharmacologyPromoter Regions (Genetics)Repressor Proteins/genetics*Transcription, GeneticUrease/*genetics/metabolism2001Aug11447165http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=11447165 [2]. Northern hybridization and stringency washes were performed at 68АC, and bound probe was visualized with the DIG-Detection Kit (Roche) and the chemiluminescent substrate CPD-Star (Amersham Pharmacia, Roosendaal, NL).
Generation of fluorescent protein fusions
To create a C-terminal fusion of Ccrp59 and of Ccrp1143 with GFP (mut1), a 507 bp 3'-fragment or the entire 1544 bp gene were amplified by PCR using primer pairs 0059_up/0059_dw or HP1143gfp_up/HP1143gfp_dw, (suppl. table HYPERLINK "http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=2216020&rendertype=table&id=T2" 2), respectively, and were cloned into ApaI and EcoRI restriction sites on pSG1164 vector ADDIN EN.CITE Lewis1999212382124317Lewis, P. J.Marston, A. L.Sir William Dunn School of Pathology, University of Oxford, South Parks Road, Oxford OX1 3RE, UK. plewis@molbiol.ox.ac.ukGFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilisGeneGene101-102271*Artificial Gene FusionBacillus subtilis/*geneticsBase SequenceGene Expression Regulation*Genetic VectorsGreen Fluorescent ProteinsLuminescent Proteins/*geneticsMolecular Sequence DataPromoter Regions (Genetics)Transcription, Genetic1999Feb 40378-1119 (Print)9931458http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=9931458 eng[3]. All fluorescent tag vectors were integrated into the H. pylori chromosome via single crossover integration, which was verified by PCR.
To create a C-terminal fusion of Ccrp59 with YFP (eYFP) for transfection of the S2 cells, the coding sequence of HP0059 was amplified by PCR, using primer pair SS0059_up/SS0059_dw and was cloned into KpnI and EcoRV restriction sites on pRmHa-3YFP containing eYFP ADDIN EN.CITE Kidane2004212512125117Kidane, A. G.Salacinski, H.Tiwari, A.Bruckdorfer, K. R.Seifalian, A. M.University Department of Surgery, Royal Free and University College Medical School, University College London, Royal Free Hospital, London NW3 2QG, United Kingdom.Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfacesBiomacromoleculesBiomacromoleculesBiomacromoleculesBiomacromoleculesBiomacromoleculesBiomacromolecules798-81353Anticoagulants/chemistry/*pharmacologyPlatelet Aggregation Inhibitors/chemistry/*pharmacologyTissue Engineering2004May-Jun1525-7797 (Print)15132664http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&dopt=Citation&list_uids=15132664 eng[4]. Additionally, HP0059 was amplified by PCR using primer pair SS0059_up2/SS0059_dw2 and cloned into KpnI and EcoRV restriction sites on pRmHa-3 ADDIN EN.CITE Bunch1988212521650517Savary-Bataille, K. C.Bunch, S. E.Spaulding, K. A.Jackson, M. W.Law, J. M.Stebbins, M. E.Percutaneous ultrasound-guided cholecystocentesis in healthy catsJ.Vet.Intern.Med.298-303173Abdominal Painadverse effectsAnimalsBileBiliary Tract Surgical ProceduresbloodCat DiseasesCatschemistryCholecystitiscomplicationsCulturecytologyFemaleGallbladderGallbladder DiseasesGrowthHealthHelicobacterHumanisolation & purificationmicrobiologyPainPeritonitisRiskSafetyScienceSupport,Non-U.S.Gov'tsurgeryultrasonographyUniversitiesveterinary20030511698[5].
Western Blotting
Serum raised against H. pylori urease B subunit (SE744 kindly provided by K. Melchers, ALTANA) was used for detection after gel electrophoresis (equal numbers of cells were applied, and equal amounts of protein were verified by Bradford tests) and blotting onto nitrocellulose membranes. Bound rabbit antibodies were detected with a protein A-alkaline-phosphatase-conjugate followed by incubation with Nitroblue-tetrazolium as substrate.
References
ADDIN EN.REFLIST 1. Bereswill S, Greiner S, van Vliet AH, Waidner B, Fassbinder F, et al. (2000) Regulation of ferritin-mediated cytoplasmic iron storage by the ferric uptake regulator homolog (Fur) of Helicobacter pylori. J Bacteriol 182: 5948-5953.
2. van Vliet AH, Kuipers EJ, Waidner B, Davies BJ, de Vries N, et al. (2001) Nickel-responsive induction of urease expression in Helicobacter pylori is mediated at the transcriptional level. Infect Immun 69: 4891-4897.
3. Lewis PJ, Marston AL (1999) GFP vectors for controlled expression and dual labelling of protein fusions in Bacillus subtilis. Gene 227: 101-110.
4. Kidane AG, Salacinski H, Tiwari A, Bruckdorfer KR, Seifalian AM (2004) Anticoagulant and antiplatelet agents: their clinical and device application(s) together with usages to engineer surfaces. Biomacromolecules 5: 798-813.
5. Savary-Bataille KC, Bunch SE, Spaulding KA, Jackson MW, Law JM, et al. (2003) Percutaneous ultrasound-guided cholecystocentesis in healthy cats. JVetInternMed 17: 298-303.
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