The Helicobacter pylori Urease Virulence Factor Is Required for the Induction of Hypoxia-Induced Factor-1α in Gastric Cells

Chronic Helicobacter pylori infection increases the risk of gastric cancer and induction of hypoxia-induced factor (HIF), which is frequently associated with the development and progression of several types of cancer. We recently showed that H. pylori activation of the PI3K-AKT-mTOR pathway in gastric cells increased HIF-1α expression. Here, we identified the H. pylori virulence factor responsible for HIF-1α induction. A mutant of the H. pylori 84-183 strain was identified with reduced ability to induce HIF-1α. Coomassie blue staining of extracts from these bacteria separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) revealed poor expression of urease subunits that correlated with reduced urease activity. This finding was confirmed in the 26695 strain, where urease mutants were unable to induce HIF-1α expression. Of note, HIF-1α induction was also observed in the presence of the urease inhibitor acetohydroxamic acid at concentrations (of 20 mM) that abrogated urease activity in bacterial culture supernatants, suggesting that enzymatic activity of the urease is not required for HIF-1α induction. Finally, the pre-incubation of the human gastric adenocarcinoma cell line AGS with blocking antibodies against Toll-like receptor-2 (TLR2), but not TLR4, prevented HIF-1α induction. In summary, these results reveal a hitherto unexpected role for the urease protein in HIF-1α induction via TLR2 activation following H. pylori infection of gastric cells.

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Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽

10.1182/blood.v64.6.1212.1212 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1212-1219 ◽

Cited By ~ 56

Author(s):

N Kieffer ◽

B Boizard ◽

D Didry ◽

JL Wautier ◽

AT Nurden

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Blue Staining ◽

Igg Antibody ◽

Immune Disorder ◽

Immunochemical Characterization ◽

Coomassie Blue Staining ◽

Coomassie Blue

Abstract We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

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Oviductal fluid protein patterns in the rhesus monkey (Macaca mulatta) during the menstrual cycle

Reproduction Fertility and Development ◽

10.1071/rd9920249 ◽

1992 ◽

Vol 4 (2) ◽

pp. 249 ◽

Cited By ~ 2

Author(s):

A Paliwal ◽

B Malaviya ◽

VP Kamboj

Keyword(s):

Menstrual Cycle ◽

Protein Profile ◽

Periodic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Blue Staining ◽

Protein Patterns ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Oviductal Fluid

Oviducts were obtained from monkeys on Days 8, 14, 19 and 25 of the menstrual cycle and changes in the pattern of luminal fluid proteins were examined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). Densitometric analysis after periodic acid Schiff's reagent (PAS) and coomassie blue staining of the gels revealed 85 and 95 kDa proteins only up to Day 14 whereas a 130 kDa glycoprotein persisted up to Day 19 and reached a nadir at mid-menstrual cycle (Day 14). The absence of the 130 kDa glycoprotein in the serum and its presence in cytosolic preparations up to Day 19 suggest that it is of oviductal origin. The 130 kDa glycoprotein is of particular interest since it was present in the oviductal fluid during mid cycle, a period when the oviduct participates in gamete transport, fertilization and embryo development. The conclusion drawn from this study is that the protein profile of monkey oviductal fluid changes during the menstrual cycle.

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Simplified sodium dodecyl sulfate-polyacrylamide gel electrophoresis of unconcentrated urine with enhanced resolution and detection sensitivity.

Clinical Chemistry ◽

10.1093/clinchem/33.10.1886 ◽

1987 ◽

Vol 33 (10) ◽

pp. 1886-1887 ◽

Cited By ~ 11

Author(s):

T Marshall ◽

K M Williams

Keyword(s):

Sodium Dodecyl Sulfate ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Polyacrylamide Gel ◽

Detection Sensitivity ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Dodecyl Sulfate

Abstract We applied a simple sodium dodecyl sulfate-polyacrylamide gel electrophoresis method to urine. The method, developed for serum protein analysis (Clin Chem 1984;30:475-9), has a high sample throughput and gives excellent resolution with unconcentrated urine. It clearly distinguishes and characterizes proteinuric urine (7.5 microL) by Coomassie Blue staining and gives complex silver-stained patterns with nonproteinuric urine (2 microL). The former is recommended for routine clinical screening, the latter for research purposes.

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Analysis of proteins synthesizedin vitroby the erythrocytic stages ofPlasmodium knowlesi

Parasitology ◽

10.1017/s0031182000055141 ◽

1980 ◽

Vol 81 (1) ◽

pp. 177-198 ◽

Cited By ~ 5

Author(s):

A. A. McColm ◽

P. G. Shakespeare ◽

P. I. Trigg

Keyword(s):

Developmental Stages ◽

Plasmodium Knowlesi ◽

Protein Pattern ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Electrophoretic Analysis ◽

The Other ◽

Blue Staining ◽

Coomassie Blue Staining

SUMMARYStudies were performed to identify specific parasite proteins synthesized withinPlasmodium knowlesi-infected rhesus erythrocytes. Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) of whole parasites freed from the host erythrocyte by immune lysis, of membranous and cytoplasmic parasite fractions, and of isolated merozoites, detected several parasite-specific components after Coomassie Blue staining of the separated proteins. However, significant contamination with host erythrocyte material generally occurred, particularly in the whole parasite and parasite membrane preparations. Improved identification of plasmodial proteins was subsequently afforded by a radioisotope labelling technique in which parasitized erythrocytes were cultivatedin vitrowith [3H] isoleucine prior to electrophoretic analysis. Of 11 principal labelled peaks ranging in molecular weight from approximately 17000 to 145000 which were detected upon electrophoresis of whole parasites harvested from culture, all were observed in the cytoplasmic fraction while at least 5 were also associated with the membranous cell fraction. Analysis of different developmental stages of the intra-erythrocytic parasite revealed no significant stage-specific qualitative variations in the electrophoretic profiles. Quantitatively, however, the middle to late trophozoites incorporated more [3H] isoleucine into protein than the other intra-erythrocytic stages. Analysis of merozoites purified from labelled schizonts showed a protein pattern similar to the other stages. This confirmed that host components did not contribute to the labelling pattern and that none of the labelled proteins were specific to the residual cytoplasm remaining after merozoite formation.

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Isolation and electrophoretic characterization of the plasma membrane of sea-urchin sperm

Journal of Cell Science ◽

10.1242/jcs.59.1.13 ◽

1983 ◽

Vol 59 (1) ◽

pp. 13-25

Author(s):

N.L. Cross

Keyword(s):

Plasma Membrane ◽

Plasma Membranes ◽

Specific Activity ◽

Periodic Acid ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Blue Staining ◽

Coomassie Blue Staining ◽

Electrophoretic Patterns ◽

Sperm Plasma Membrane

A subcellular fraction containing plasma membranes was isolated from flagella of the sperm of Strongylocentrotus purpuratus by differential centrifugation, and analysed by sodium dodecyl sulphate/polyacrylamide gel electrophoresis. Coomassie Blue staining revealed nine major bands and 14 minor species. Five bands of apparent molecular weights approximately 200 X 10(3), 149 X 10(3), 120 X 10(3), 75 X 10(3) and 59 X 10(3) also stained with periodic acid-Schiff's reagent and so are probably glycoproteins. These five components are externally exposed, as determined by lactoperoxidase-catalysed radio-iodination. Isolation of membranes from radio-iodinated sperm results in an enrichment of about tenfold in the specific activity of 125I. Comparison of the electrophoretic patterns of labelled sperm and of the membranes isolated from 125I-labelled sperm suggests that no major labelled proteins are lost during the isolation procedure, and so to this extent the membrane fraction is representative of the entire sperm plasma membrane.

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Cyclic GMP phosphodiesterase from bovine retina. Evidence for interspecies conservation

Biochemical Journal ◽

10.1042/bj2170129 ◽

1984 ◽

Vol 217 (1) ◽

pp. 129-133 ◽

Cited By ~ 4

Author(s):

L J Takemoto ◽

J Hansen ◽

D B Farber ◽

D Souza ◽

D J Takemoto

Keyword(s):

Peptide Mapping ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Enzymic Activity ◽

Blue Staining ◽

Partial Purification ◽

Visual Response ◽

Coomassie Blue Staining ◽

Coomassie Blue ◽

Protein Components

A light-activated phosphodiesterase (PDE) from retinal rod outer segments (ROS) has been strongly implicated as a possible mediator in the propagation of the visual response. In view of the probable importance of this enzyme in the visual system, a comparison of the PDE proteins from ROS of different evolutionary species was made. Partial purification of the PDE, as measured by enzymic activity, followed by resolution of the protein components on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis, indicated that the major Coomassie Blue-staining species in the ROS of all species studied is a doublet of 84000-88000 Da. After radioiodination, this doublet was converted in all but the frog proteins into a single band of 85000 Da. Two-dimensional tryptic peptide mapping of the radioiodinated peptides indicated that at least six major peptides of the putative PDE have been conserved in all of the species studies. If this protein is indeed associated with PDE activity, such conserved peptides may play an important role in the catalytic and/or regulatory functions of the enzyme.

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Immunochemical characterization of the platelet-specific alloantigen Leka: a comparative study with the PlA1 alloantigen

Blood ◽

10.1182/blood.v64.6.1212.bloodjournal6461212 ◽

1984 ◽

Vol 64 (6) ◽

pp. 1212-1219

Author(s):

N Kieffer ◽

B Boizard ◽

D Didry ◽

JL Wautier ◽

AT Nurden

Keyword(s):

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Human Platelets ◽

Blue Staining ◽

Igg Antibody ◽

Immune Disorder ◽

Immunochemical Characterization ◽

Coomassie Blue Staining ◽

Coomassie Blue

We report the immunochemical characterization of a new platelet- specific alloantigen detected using an IgG antibody isolated from the serum of a patient with posttransfusion purpura (PTP). In indirect immunoprecipitation experiments, the antibody, termed anti-Leka, predominantly precipitated glycoprotein (GP) IIb from Triton X-100 lysates of normal human platelets. In an immunoblot procedure, which involved the transfer of platelet polypeptides separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to nitrocellulose membrane, anti-Leka bound exclusively to GP IIb. Under identical conditions, four anti-PlA1 antibodies each reacted with GP IIIa. No binding of anti-Leka IgG occurred to Leka (-) platelets or to their separated polypeptides although GP IIb was normally detected by Coomassie blue staining. After electrophoresis of reduced platelet proteins, the Leka determinant was localized to the IIb alpha chain. Thus, unlike the PlA1 antigen, the Leka determinant was not destroyed by disulfide reduction. Analysis of platelets from a patient with Glanzmann's thrombasthenia revealed little or no binding in the GP IIb position. Anti-Leka permitted the identification of 76,000 and 60,000 dalton fragments of GP IIb retained by the platelet following chymotrypsin treatment. Our results further highlight the immunogenicity of the GP IIb-IIIa complex. They also suggest that antibodies against GP IIb can cause the thrombocytopenia observed in PTP and that anti-PlA1 antibodies do not account exclusively for the pathophysiology of this immune disorder.

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A Novel Role for Helicobacter pylori Gamma-Glutamyltranspeptidase in Regulating Autophagy and Bacterial Internalization in Human Gastric Cells

Cancers ◽

10.3390/cancers11060801 ◽

2019 ◽

Vol 11 (6) ◽

pp. 801 ◽

Cited By ~ 2

Author(s):

Jimena Bravo ◽

Paula Díaz ◽

Alejandro H. Corvalán ◽

Andrew F.G. Quest

Keyword(s):

Gastric Cancer ◽

Helicobacter Pylori ◽

Virulence Factor ◽

Bacterial Infections ◽

Membrane Integrity ◽

Autophagic Flux ◽

Ags Cells ◽

Gastric Cells ◽

H Pylori

The risk of developing gastric cancer is strongly linked to Helicobacter pylori (H. pylori) infection. Alternatively, autophagy is a conserved response that is important in cellular homeostasis and provides protection against bacterial infections. Although H. pylori is typically considered an extracellular bacterium, several reports indicate that it internalizes, possibly to avoid exposure to antibiotics. Mechanisms by which H. pylori manipulates host cell autophagic processes remain unclear and, importantly, none of the available studies consider a role for the secreted H. pylori virulence factor gamma-glutamyltranspeptidase (HpGGT) in this context. Here, we identify HpGGT as a novel autophagy inhibitor in gastric cells. Our experiments revealed that deletion of HpGGT increased autophagic flux following H. pylori infection of AGS and GES-1 gastric cells. In AGS cells, HpGGT disrupted the late stages of autophagy by preventing degradation in lysosomes without affecting lysosomal acidification. Specifically, HpGGT impaired autophagic flux by disrupting lysosomal membrane integrity, which leads to a decrease in lysosomal cathepsin B activity. Moreover, HpGGT was necessary for efficient internalization of the bacteria into gastric cells. This important role of HpGGT in internalization together with the ability to inhibit autophagy posits HpGGT as a key virulence factor in the development of gastric cancer.

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Effect of testicular hormones on synthesis of soluble proteins by dog prostate slices

Canadian Journal of Biochemistry and Cell Biology ◽

10.1139/o83-095 ◽

1983 ◽

Vol 61 (7) ◽

pp. 756-763 ◽

Cited By ~ 13

Author(s):

Jean Y. Dubé ◽

Pierre Chapdelaine ◽

Roland R. Tremblay

Keyword(s):

Seminal Plasma ◽

Gel Electrophoresis ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Blue Staining ◽

Secretory Proteins ◽

Molecular Weights ◽

Coomassie Blue Staining ◽

Coomassie Blue

We have submitted adult mongrel dogs to various endocrine manipulations. Prostate slices from these animals were then incubated in vitro in the presence of [3H]leucine or [35S]methionine. We have analyzed the cytosolic proteins by polyacrylamide gel electrophoresis. In intact adult uncastrated dogs, the radioactive amino acids were incorporated into three major bands having respective molecular weights (MW) of 32 000,16 000, and 15 000 in one-dimensional gels in presence of sodium dodecyl sulfate and mercaptoethanol. Two-dimensional electrophoresis revealed heterogeneity of each of these bands, both in isoelectric focussing (IEF) or nonequilibrium pH gel electrophoresis (NEpHGE) conditions. The 32 000 MW proteins showed five to six major radioactive spots and the 15 000 – 16 000 MW proteins showed six to seven spots by IEF. However, the highest incorporation of radioactivity occurred in a 16 000 MW protein seen only in NEpHGE. The lower MW proteins corresponded to some of the major proteins of dog seminal plasma as observed by immunoprecipitation of prostate proteins with antibodies against whole seminal plasma. By contrast, the 32 000 MW proteins were minor proteins of prostate cytosol and seminal plasma by Coomassie blue staining. Castration for 2 weeks completely abolished the synthesis of all these proteins. When castrated animals were treated with 5α-androstane-3α-17β-diol (10 mg/day for 2 weeks), the pattern of protein synthesis returned to the one observed in intact uncastrated animals. These observations show that testicular androgens control the synthesis of dog prostate major secretory proteins.

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Conserved Low-Affinity Nickel-Binding Amino Acids Are Essential for the Function of the Nickel Permease NixA of Helicobacter pylori

Journal of Bacteriology ◽

10.1128/jb.184.5.1438-1443.2002 ◽

2002 ◽

Vol 184 (5) ◽

pp. 1438-1443 ◽

Cited By ~ 23

Author(s):

Lutz Wolfram ◽

Peter Bauerfeind

Keyword(s):

Amino Acids ◽

Helicobacter Pylori ◽

Urease Activity ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Sequence Motif ◽

Nickel Binding ◽

Transporter Family ◽

Nickel Uptake ◽

Conserved Amino Acids

ABSTRACT Nickel acquisition is necessary for urease activity, a major virulence factor of the human gastric pathogen Helicobacter pylori. The nickel permease NixA of H. pylori is a member of the single-component nickel-cobalt transporter family. To identify functionally relevant amino acids of NixA, single-site exchanges were introduced into NixA via PCR-based mutagenesis. This study investigated one of the recognition motifs for this family in transmembrane segment III and other conserved amino acids, mostly with possible nickel-binding capacities. The mutant alleles were expressed in Escherichia coli, and activity of the altered permeases was analyzed by measuring nickel accumulation and urease activity. Expression was checked by immunoblotting after sodium dodecyl sulfate-polyacrylamide gel electrophoresis with a NixA-specific antibody. Replacement of Phe-75 and His-79—both part of the characteristic sequence motif—and of Asn-127, Thr-195, and Ser-197 with alanine abolished nickel uptake in the E. coli system. The results were unchanged if these amino acids were replaced with residues more similar to the original amino acid. The phenotype of the null mutants was independent of the culture medium. Mutation of Val-82, Tyr-242, Thr-260, His-181, and His-15 strongly affected uptake activity under nickel limitation on complex Luria-Bertani medium but had little effect in minimal medium. Eight other conserved amino acids (Ser-80, Ser-81, Phe-119, Trp-180, Tyr-183, Trp-244, Pro-249, and Asn-256) were found to be dispensable for the function of NixA. These results show that atypical nickel-binding amino acids play an important function in nickel uptake and that most of the essential amino acids are clustered in conserved motifs.

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