Construction and preservation of a stable and highly expressed recombinant Helicobacter pylori vacuolating cytotoxin A with apoptotic activity

Abstract Background H. pylori is closely related to the occurrence and development of various digestive gastritis, peptic ulcer and mucosa-associated lymphoid tissue (MALT) lymphoma. H. pylori is also a class I carcinogen of gastric cancer. VacA is the only exocrine toxin of H. pylori, which plays a very important role in the pathogenesis of H. pylori. The production of VacA in natural circumstances is complex with heavy workload and low yield. Therefore, it is very important to obtain recombinant VacA protein which is stable and biologically active. This study therefore aims to explore the expression, purification and stable storage of VacA toxin of H. pylori in E.coli, and to provide experimental basis for further exploration of the role of VacA in H. pylori -induced inflammation of cancer. Results A 2502-bp fragment and VacA gene were identified. An 89.7-kDa VacA34–854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay. Conclusions A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

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Construction and Preservation of a Stable and Highly Expressed Recombinant Helicobacter pylori Vacuolating Cytotoxin A with Apoptotic Activity

10.21203/rs.3.rs-94497/v1 ◽

2020 ◽

Author(s):

Ling-Zhi Yuan ◽

Xiao Shi ◽

Dan Tang ◽

Shao-Peng Zheng ◽

Zhi-Ming Xiao ◽

...

Keyword(s):

Acetic Acid ◽

Recombinant Protein ◽

Efficient Production ◽

Acid Buffer ◽

Acetic Acid Buffer ◽

Apoptotic Activity ◽

H Pylori ◽

Induced Apoptosis ◽

And Function ◽

Cancer Studies

Abstract Background: VacA is the only exocrine toxin of H. pylori, which plays a very important role in the customization of H. pylori in the gastric mucosa, contributing to the pathogenesis of gastritis-cancer. Studies about vacA structure and function was hindered by the lack of efficient production system. In this study, we developed methodology for expression, purification and stable storage of a functionally active vacA toxin in E.coli.Results: A 2502-bp fragment and vacA gene were identified. An 89.7-kDa VacA34-854 recombinant protein was expressed and purified from the recombinant engineering bacteria and was preserved stably in 50 mM acetic acid buffer (pH 2.9). The amount of the recombinant protein was larger in the inclusion bodies than in the supernatant. In addition, after a 24-h culture with VacA recombinant protein, GES-1 cells demonstrated evidence of apoptosis including early nuclear immobilization and clustering under inverted microscope and TEM. It was found that VacA recombinant protein induced apoptosis by TUNEL assay.Conclusions: A VacA recombinant protein that is stably and highly expressed and possesses pro-apoptotic activity is successfully constructed. The protein is stably preserved in 50 mM acetic acid buffer (pH 2.9).

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pH Measurement Problems Affecting Assay of Carcinoembryonic Antigen

Clinical Chemistry ◽

10.1093/clinchem/21.2.255 ◽

1975 ◽

Vol 21 (2) ◽

pp. 255-257 ◽

Cited By ~ 4

Author(s):

Jack H Ladenson ◽

C Elliott Bell

Keyword(s):

Acetic Acid ◽

Carcinoembryonic Antigen ◽

Ammonium Acetate ◽

Electrode System ◽

Acid Buffer ◽

Ph Values ◽

Blood Ph ◽

Acetic Acid Buffer ◽

Ph Electrodes ◽

Commercial Kit

Abstract Measurement with combination pH electrodes of the pH of the dilute buffers used in a commercial kit (CEA-Roche) for assay of carcinoembryonic antigen resulted in pH values 0.1 to 0.3 unit lower than pH values measured on an electrode system with a capillary junction. If the pH values of these buffers were adjusted, based on such measurements, an error in the assay of 0.2 to 0.6 ng/ml in the 1.5-3.0 ng/ml range would result. We recommend that the pH of dialyzed samples and of the working ethylenediaminetetraacetate and ammonium acetate-acetic acid buffers be monitored with pH electrodes that have a capillary junction between sample and saturated KCl, as is true of most blood-pH instruments. We also recommend use of a 1 mol/liter rather than 2.5 mol/liter stock ammonium acetate-acetic acid buffer, because of the closer similarity of the pH of buffers at this molarity to those at 0.01 mol/liter.

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2-Methoxyestradiol Sensitizes B-NHL Cells to TRAIL-Mediated Apoptosis Via Inhibition of HIF-1α, NF-κ B, YY1 and Induction of DR5 Expression.

Blood ◽

10.1182/blood.v108.11.4642.4642 ◽

2006 ◽

Vol 108 (11) ◽

pp. 4642-4642

Author(s):

Sara Huerta-Yepez ◽

Mario I. Vega ◽

Stavroula Baritaki ◽

Eriko Suzuki ◽

Theresa La Vallee ◽

...

Keyword(s):

Annexin V ◽

Nuclear Accumulation ◽

Microtubule Disruption ◽

Low Concentrations ◽

Combination Studies ◽

Apoptotic Activity ◽

Induced Apoptosis ◽

Patients Experience ◽

First Time

Abstract There have been many advances in the treatment of B-NHL by both chemotherapy and immunotherapy. However, many patients experience recurrences and relapses and develop resistance to further treatments. Therefore, there is a need for new therapies. TRAIL and mAbs directed against DR4 or DR5 are currently being examined clinically, either alone or in combination with other therapies, for the treatment of resistant cancers. The objective of our study is to examine whether B-NHL can be sensitized to respond to TRAIL. 2-Methoxyestradiol (2ME2), a naturally occurring metabolite of estradiol, is known to have apoptotic activity and anti-angiogenic activity and has been examined for its therapeutic efficacy, both preclinically and in humans. Further, we and others have shown that 2ME2 can sensitize solid tumors to TRAIL-induced apoptosis. Thus, we hypothesized that 2ME2 may also sensitize B-NHL cells to TRAIL-induced apoptosis and that 2ME2-induced microtubule disruption and inhibition of NF-κ B activity may be involved in 2ME2-induced sensitization. The present study examined the role of 2ME2 in TRAIL-sensitization of the B-NHL cell line, Ramos, as a model for B-NHL. Ramos cells were treated with 2ME2 (0.1, 1.0 μ m for 5 h) and then treated with TRAIL (2.5–10 ng/ml for 18 h). The cells were harvested and examined for apoptosis by Annexin V/PI and for activation of caspase-3. The findings demonstrate that, while single agents were not cytotoxic, the combination treatment resulted in significant cytotoxicity demonstrating synergy in apoptosis. The synergy was obtained with very low concentrations of 2ME2 (0.1μ m) and TRAIL (2.5 ng/ml), concentrations that were not effective in other tumor cell lines studied. We examined the potential mechanism involved in synergy induced by 2ME2 and TRAIL. Surface DR-5 expression was upregulated following treatment with 2ME2. In addition, the transcription repressor YY1 protein expression was inhibited as assessed by Western and immunohistochemistry (IHC). Microtubule disruption by 2ME2 results in inhibition of HIF-1α transcriptional activity through impairment of HIF-1α nuclear accumulation. Additionally, we have found that 2ME2 inhibits HIF-1α accumulation in the nucleus as assessed by IHC. Inhibition of HIF-1α has been shown to regulate apoptosis via upregulation of BID and phosphorylyzation of Bcl-2. The present findings demonstrate, for the first time, that 2ME2 sensitizes B-NHL cells to TRAIL-induced apoptosis via inhibition of both HIF-1α and YY1 and upregulation of DR5. The findings support potential therapeutic combination studies of 2ME2 with TRAIL for the treatment of resistant B-NHL In addition, we suggest that HIF-1α, YY1, and DR5 may serve as targets for therapeutic intervention and potentially as biomarkers for activity.

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Apoptotic Signaling Pathway Activated byHelicobacter pylori Infection and Increase of Apoptosis-Inducing Activity under Serum-Starved Conditions

Infection and Immunity ◽

10.1128/iai.69.5.3181-3189.2001 ◽

2001 ◽

Vol 69 (5) ◽

pp. 3181-3189 ◽

Cited By ~ 37

Author(s):

Keigo Shibayama ◽

Yohei Doi ◽

Naohiro Shibata ◽

Tetsuya Yagi ◽

Toshi Nada ◽

...

Keyword(s):

Membrane Fraction ◽

Proteinase K ◽

Caspase 8 ◽

Peptic Ulcers ◽

Caspase 9 ◽

Effector Caspase ◽

Epithelial Cell Apoptosis ◽

Apoptotic Activity ◽

H Pylori ◽

Induced Apoptosis

ABSTRACT The enhanced gastric epithelial cell apoptosis observed during infection with Helicobacter pylori has been suggested to be of significance in the etiology of gastritis, peptic ulcers, and neoplasia. To investigate the cell death signaling induced by H. pylori infection, human gastric epithelial cells were incubated with H. pylori for up to 72 h. H. pyloriinfection induced the activation of caspase -8, -9, and -3 and the expression of the proapoptotic Bcl-2 family proteins Bad and Bid. The peak of the activity of the caspases occurred at 24 h. At this time, the inhibition of caspase-8 or -9 almost completely suppressedH. pylori-induced apoptosis. Inhibition of caspase-8 suppressed the expression of Bad and Bid and the subsequent activation of caspase-9 and -3. These observations indicate that H. pylori induces apoptosis through a pathway involving the sequential induction of apical caspase-8 activity, the proapoptotic proteins Bad and Bid, caspase-9 activity, and effector caspase-3 activity. Activation of the pathway was independent of CagA or vacuolating toxin. A membrane fraction of H. pylori was sufficient to activate this pathway, and treatment with proteinase K eliminated the activity. Apoptotic activity of the membrane fraction was significantly increased by incubating the bacteria under serum-starved conditions for 24 h. These observations suggest that environmental conditions in the human stomach could induce H. pylori-mediated pathogenesis, leading to a variety of clinical outcomes.

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