Roles of Helicobacter pylori infection and cyclooxygenase-2 expression in gastric carcinogenesis

AIM: Cyclooxygenase (COX)-2 is over expressed ingastrointestinal neoplasm. Helicobacter pylori ( H pylon)infection is causally linked to gastric cancer. However, theexpression of COX-2 in various stages of Hpylori-associatedgastric carcinogenesis pathway has not been elucidated.Therefore, the aim of this study was to clarify the role ofH pyloriinduced COX-2 expression during carcinogenesisin the stomach.METHODS: Gastric biopsies from 138 subjects [30 casesof chronic superficial gastritis (CSG), 28 cases of gastricglandular atrophy (GA), 45 cases of gastric mucosal intestinalmetaplasia (IM), 12 cases of moderate gastric epithelialdysplasia and 23 cases of gastric cancer] were enrolled.Hpyloriinfection was assessed by a rapid urease test andhistological examination (modified Giemsa staining). Theexpression of COX-1 and COX-2 in human gastric mucosawas detected by immunohistochemical staining.I^7~I_~: Hpyloriinfeddon rate was 64.3% in GA and 69.5%in gastric cancer, which was significantly higher than that(36.7%) in CSG (P<0.05). The positive expression rates ofCOX-2 were 10.0%, 35.7%, 37.8%, 41.7% and 69.5% inCSG, GA, IM, dysplasia and gastric cancer, respectively.From CSG to GA, IM, dysplasia and finally to gastric cancer,expression of COX-2 showed an ascending tendency, whereasCOX-1 expression did not change significantly in the gastricmucosa. The level of COX-2 expression in IM and dysplasiawas significantly higher in H pylon:positive than in H pylor/-negative subjects (P<0.01).

Response of human REV3gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-N-nitrosoguanidine and its role in mutagenesis

AIM: To understand the response of human REV3gene to gastric cancer inducing carcinogen N-methyl-N'-nitro-Nnitrosoguanidine (MNNG) and its role in human mutagenesis.METHODS: The response of the human REV3 gene to MNNG was measured in human 293 cells and FL cells by RT-PCR. By using antisense technology, mutation analysis at HPRTlocus (on which lesion-targeted mutation usually occurs) was conducted in human transgenic cell line FLREV3- by 8-azaguanine screening, and mutation occurred on undamaged DNA template was detected by using a shuttle plasmid pZ189 as the probe in human transgenic cell lines 293-REV3- and FL-REV3-. The blockage effect of REV3 was measured by combination of reverse transcriptionpolymerase chain reaction to detect the expression of antisense REV3 RNA and Western blotting to detect the REV3protein level.RESULTS: The human REV3 gene was significantly activated by MNNG treatment, as indicated by the upregulation of REV3 gene expression at the transcriptional level in MNNG-treated human cells, with significant increase of REV3 expression level by 0.38 fold, 0.33 fold and 0.27fold respectively at 6 h, 12 h and 24 h in MNNG-treated 293 cells (P＜0.05); and to 0.77 fold and 0.65 fold at 12 h and 24 h respectively in MNNG-treated FL cells (P＜0.05).In transgenic cell line (in which REV3 was blocked by antisense REV3 RNA), high level of antisense REV3 RNA was detected, with a decreased level of REV3 protein.MNNG treatment significantly increased the mutation frequencies on undamaged DNA template (untargeted mutation), and also at HPRT locus (lesion-targeted mutation). However, when REV3 gene was blocked by antisense REV3 RNA, the MNNG-induced mutation frequency on undamaged DNA templates was significantly decreased by 3.8 fold (P＜0.05) and 5.8 fold (P＜0.01)respectively both in MNNG-pretreated transgenic 293 cells and FL cells in which REV3was blocked by antisense RNA,and almost recovered to their spontaneous mutation levels.The spontaneous HPRT mutation was disappeared in REV3disrupted cells, and induced mutation frequency at HPRT locus significantly decreased from 8.66×10-6 in FL cells to 0.14×10-6 in transgenic cells as well (P＜0.01).CONCLUSION: The expression of the human REV3 can be upregulated at the transcriptional level in response to MNNG. The human REV3 gene plays a role not only in lesion-targeted DNA mutagenesis, but also in mutagenesis on undamaged DNA templates that is called untargeted mutation.

Neural cell injury microenvironment induces neural differentiation of human umbilical cord mesenchymal stem cells

This study aimed to investigate the neural differentiation of human umbilical cord mesenchymal stem cells （hUCMSCs） under the induction of injured neural cells. After in vitro isolation and culture, passage 5 hUCMSCs were used for experimentation, hUCMSCs were co-cultured with normal or AI31.4o-injured PC12 cells, PC12 cell supernatant or PC12 cell lysate in a Transwell co-culture system. Western blot analysis and flow cytometry results showed that choline acetyltransferase and microtubule-associated protein 2, a specific marker for neural cells, were expressed in hUCMSCs under various culture conditions, and highest expression was observed in the hUCMSCs co-cultured with injured PC12 cells. Choline acetyltransferase and microtubule-associated protein 2 were not expressed in hUCMSCs cultured alone （no treatment）. Cell Counting Kit-8 assay results showed that hUCMSCs under co-culture conditions promoted the proliferation of injured PC12 cells. These findings suggest that the microenvironment during neural tissue injury can effectively induce neural cell differentiation of hUCMSCs. These differentiated hUCMSCs likely accelerate the repair of injured neural ceils.

Effects of bone marrow-derived mesenchymal stemcells engraftment on vascular endothelial cell growthfactor in lung tissue and plasma at early stage of smoke inhalation injury

BACKGROUND： This study was undertaken to determine the effect of mesenchymal stem cells （MSCs） engraftment on vascular endothelial cell growth factor （VEGF） in lung tissue, plasma and extravascular lung water at early stage of smoke inhalation injury.METHODS： A rabbit smoke inhalation injury model was established using a home-made smoke inhalation injury generator, and rabbits were divided into two groups randomly： a control group （S group, n=32） and a MSCs treatment group （M group, n=32）. 10 ml PBS was injected via the ear marginal vein immediately at injury into the S group. Third generation MSCs with a concentration of 1×107/10 ml PBS were injected via the ear marginal vein immediately at injury into the M group. VEGF in peripheral blood and lung tissue were measured at 0 （baseline）, 2, 4 and 6 hours after injection respectively and analyzed. The right lungs of rabbits were taken to measure lung water mass fraction.RESULTS： In the lung tissue, VEGF decreased gradually in the S group （P〈0.05） and signi？ cantly decreased in the M group （P〈0.05）, but it increased more signi？ cantly than the values at the corresponding time points （P〈0.05）. In peripheral blood, VEGF increased gradually in the S group （P〈0.05） and markedly increased in the M group （P〈0.05）, but it decreased more signi？ cantly than the values at corresponding time points （P〈0.05）.CONCLUSION： MSCs engraftment to smoke inhalation injury could increase VEGF in lung tissue, decrease VEGF in plasma and reduce extravascular lung water, indicating its protective effect on smoke inhalation injury.

Efficacy of omeprazole and amoxicillin with either clarithromycin or metronidazole on eradication of Helicobacter pylori in Chinese peptic ulcer patients

AIM: One-week triple therapy with proton pump inhibitors, clarithromycin and amoxicillin has recently been proposed as the first-line treatment for Helicobacter pylori (H pylori) infection; however, data regarding the effects of this regimen in China are scarce. The aim of this prospective and randomized study was to compare the efficacy of clarithromycin and metronidazole when they were combined with omeprazole and amoxicillin on eradication of H pylori and ulcer healing in Chinese peptic ulcer patients. METHODS: A total of 103 subjects with Hpylori-positive peptic ulcer were randomly divided into two groups, and accepted triple therapy with omeprazole 20 mg, amoxicillin 1 000 mg and either clarithromycin 500 mg (OAC group, n = 58) or metronidazole 400 mg (0AM group, n - 45). All drugs were given twice daily for 7 d. Patients with active peptic ulcer were treated with omeprazole 20 mg daily for 2-4 wk after anti-H pylori therapy. Six to eight weeks after omeprazole therapy, all patients underwent endoscopies and four biopsies (two from the antrum and two others from the corpus of stomach) were taken for rapid urease test and histological analysis (with modified Giemsa staining) to examine H pylori. Successful eradication was defined as negative results from both examination methods. RESULTS: One hundred patients completed the entire course of therapy and returned for follow-up. The eradication rate of H pylori for the per-protocol analysis was 89.3% (50/56) in OAC group and 84.1% (37/44) in 0AM group. Based on the intention-to-treat analysis, the eradication rate of H pylori was 86.2% (50/58) in OAC group and 82.2% (37/45) in 0AM group. There were no significant differences in eradication rates between the two groups on either analysis. The active ulcer-healing rate was 96.7% (29/30) in OAC group and 100% (21/21) in 0AM group (per-protocol analysis, P>0.05). Six patients in OAC group (10.3%) and five in OAM group (11.1%) reported adverse events (P>0.05). CONCLUSION: One-week triple therapy with omeprazole and amoxicillin in combination with either clarithromycin or metronidazole is effective for the eradication of H pylori. The therapeutic regimen comprising metronidazole with low cost, good compliance and mild adverse events may offer a good choice for the treatment of peptic ulcers associated with H pylori infection in China.

DNA polymerase ξ:new insight into eukaryotic mutagenesis and mammalian embryonic development

Information about the mechanisms that generate mutationsin eukaryotes is likely to be useful for understanding humanhealth concerns, such as genotoxicity and cancer.Eukaryotic mutagenesis is largely the outcome of attacksby' endogenous and environmental agents. Except for DNArepair, cell cycle checkpoints and DNA damage avoidance,cells have also evolved DNA damage tolerance mechanism,by which lesion-targeted mutation might occur in thegenome during replication by specific DNA polymerases tobypass the lesions (translesion DNA synthesis, TLS), ormutation on undamaged DNA templates (untargetedmutation) might be induced. DNA polymerase ζ (poiζ),which was found firstly in budding yeast Saccharomycescerevisiae and consists of catalytic subunit scRev3 and stimulating subunit scRev7, has Received more attention in recent years. Poi ζ is a member of DNA polymerase δsubfamily, which belongs to DNA polymerase B family, and exists in almost all eukaryotes. Human homolog of the scRev3gene is located in chromosome region 6q21, and the mouse equivalent maps to chromosome 10, distal to the c-myb gene and close to the Macs gene. Alternative splicing, upstream out-of frame ATG can be found in yeast scRev3, mouse and human homologs. Furthermore, the sequence from 253-323 immediate upstream of the AUG initiator codon has the potential to form a stem-loop hairpin secondary structure in REV3 mRNA, suggesting that human REV3 protein may be expressed at low levels in human cells under normal growth conditions. The functional domain analysis showed that yeast Rev3-980 tyrosine in conserved region II is at the polymerase active site. Human REV3 amino acid residues 1 776-2 195 provide a REV7binding domain, and REV7 amino acid residues 1-211provide a bind domain for REV1, REV3 and REV7 itself.More interestingly, REV7 interacts with hMAD2 and therefore might function in the cell cycle control by affecting the activation of APC (anaphase promoting complex).Currently it has been known that poi ζ is involved in most spontaneous mutation, lesion-targeted mutation via TLS,chemical carcinogen induced untargeted mutation and somatic hypermutation of antibody genes in mammalian.In TLS pathway, polζ acts as a "mismatch extender" with combination of other DNA polymerases, such as pol t. Unlike in yeast, it was found that pol ζ also functioned in mouse embryonic development more recently. It was hypothesized that the roles of pol ζ in TLS and cell cycle control might contribute to mouse embryonic lethality.