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Background N6-methyladenosine (m6A) RNA methylation may play an important role in the process of malignant transformation of cells induced by environmental carcinogens. However, the specific roles and mechanisms need to be further explored. Objective To explore the role and mechanism of m6A binding protein insulin-like growth factor 2 mRNA-binding protein 3 (IGF2BP3) in the malignant transformation of human gastric mucosal epithelial cells GES-1 induced by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG). Methods Based on the GES-1 malignant transformation cells MC-30, a stable knockdown IGF2BP3 MC-30 cell line (MC30-shIGF2BP3, abbreviated as MC30-shI3) was constructed by lentiviral transfection technology, and a negative control group (MC30-NC) was also prepared. Real-time quantitative polymerase chain reaction (qRT-PCR) and Western blotting were applied to detect the mRNA expression and protein levels of IGF2BP3. RNA binding protein immunoprecipitation (RIP-qPCR) was used to examine the combination between IGF2BP3 protein and MYC mRNA in malignant cells MC-30. Furthermore, the stability of MYC mRNA was detected by actinomycin D assay. CCK-8 and Transwell respectively were employed to detect cell proliferation, migration, and invasion. Western blotting was applied to detect the expression of EMT markers (N-cadherin, Vimentin, α-SMA, and Snail). The role of the downstream target gene MYC was further elucidated by a rescue assay in MC30-shI3 cells transfected with a plasmid overexpressing MYC to observe changes in cellular phenotypes (proliferation, migration, invasion) and expression of key EMT proteins. Results Compared with the control group, the expression of IGF2BP3 mRNA was up-regulated after 5, 10, 20, and 40 μmol·L−1 MNNG infection of GES-1 cells (P<0.05). After 20 μmol·L−1 MNNG infection, the expression level of IGF2BP3 mRNA increased with prolongation of exposure time (P<0.05). Compared with the control group, the mRNA and protein expression levels of IGF2BP3 were up-regulated in the 10th, 20th, and 30th generations of 5 μmol·L−1 MNNG malignant transformation (P<0.05). The results of qRT-PCR and Western blotting showed that, compared with the MC30-NC group, the IGF2BP3 and MYC mRNA expression and protein expression decreased in the MC30-shI3 group (P<0.01). The CCK8 and transwell assay results showed that, compared with the MC30-NC group, the cell proliferation, migration, and invasion abilities significantly reduced in the MC30-shI3 group (P<0.01). The results of the Western blotting showed that, compared with the MC30-NC group, the protein levels of EMT markers N-cadherin, Vimentin, α-SMA, and Snail decreased in the MC30-shI3 group (P<0.01). The results of RIP-qPCR showed that, compared with the IgG group, the mRNA level was higher for the enriched MYC in the IGF2BP3 group (P<0.01); the results of the actinomycin D assay showed that, compared with the MC30-NC group, the stability of MYC mRNA significantly reduced in the MC30-shI3 group (P<0.01). While the rescue experiment showed that, compared with the IGF2BP3 knock-down+vector group, the MYC protein level significantly increased in the IGF2BP3 knock-down + MYC over-expression group (P<0.01), the proliferation, migration, and invasion abilities significantly enhanced (P<0.01), and the EMT key proteins (N-cadherin, Vimentin, α-SMA, Snail) increased in the MC30-shI3+MYC group (P<0.01). Conclusion Exposure to MNNG could result in up-regulation of IGF2BP3 expression in GES-1 cells. IGF2BP3 may enhance the proliferation, migration, and invasion of malignantly transformed human gastric epithelial cells by binding to MYC mRNA and increasing its stability and expression level and thus promoting the EMT process, which in turn affects the progression of malignant transformation.
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Aim: To develop a mutant strain with high endoglucanase productivity and optimization of some cultivation parameters. Place and Duration of Study: Microbiology Research Laboratory, Department of Zoology, Molecular Biology & Genetics, Presidency University, College Street, Kolkata: 700 073, India, between Aug, 2010 and March 2011. Methodology: The wild strain of Rhizopus oryzae PR7 MTCC 9642 was subjected to classical mutagenesis by suspending 5 hyphal discs (0.5 cm) in 10ml of N-methyl-N'-nitro- N-nitrosoguanidine (MNNG) solutions of various concentrations (125-1000μg). The in situ cellulolytic activity of the colonies of the mutant strains on the plates were measured by using alcoholic iodine solution and the highest enzyme producing mutant was selected. The mutant strain was later cultivated in presence of various domestic wastes at various pH, temperature, time. The morphological alteration was also checked by staining with fluorescent dye. Results: Out of 50 mutants, strain A7 was selected that showed about 33% increase in endoglucanase synthesis utilizing orange bagasse as sole carbon source in a shake flask screen. The strain was found to have the same pH and temperature optima, but could achieve highest level of enzyme production earlier than that by its wild counterpart. Being a dimorphic fungus, the wild type strain of Rhizopus oryzae, showed a transformation to yeast like pelleted form, whereas the mutant strain A 7 showed persistent filamentous structure indicating the achievement of a structural stability in presence of environmental stress. Conclusion: The present mutant strain could ferment orange bagasse and showed an increased production of endoglucanase with minimized time consumption with greater mycelial stability against various environmental stresses. These achievements will definitely add economy in industrial production of endoglucanse at a nominal cost.
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Objective To investigate the effects of sodium butyrate (SB) on fetal gastric epithelial cells (GECs) in vitro damaged by N-methyl-N’-nitro-N-nitrosoguanidine (MNNG). Methods The fetal gastric epithelial cells were isolated, cultured and identified. Then the cells of the second generation were cocultured with 10 -5 mol/L, 10 -6 mol/L, 10 -7 mol/L SB for 4 h, then added with 10 -5 mol/L MNNG culturing for another 24 h. After the treated cells were finally cultured in normal culture medium for 40 d, the unscheduled DNA synthesis (UDS), lipid peroxidation (LPO) and ras p21 were detected. The cells were only cultured with 10 -5 mol/L MNNG served as negative control and without MNNG or SB as normal control. Results The level of UDS, the contents of lipid peroxidation products and the concentrations of ras p21 in GECs treated with 10 -5 mol/L and 10 -6 mol/L SB decreased obviously, and had significant differences with that of GECs treated without SB. Conclusion SB can effectively prevent the damage of GECs induced from MNNG.
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Objective To examine the chemo-preventive effect of cyclooxygenase-2(COX-2) inhibitor (celecoxib) in an animal model of stomach carcinogenesis. Methods Eighty-six male Wistar rats were divided into six groups. The rats were given water alone (group A, n=5), N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) (group B, n=16), 3 mg/kg of indomethacin daily (group C, n=16), 5 mg/kg of celecoxib daily (group D, n=17), 10 mg/kg of celecoxib daily (group E, n=16) or 20 mg/kg of celecoxib daily (group F, n=16). The animals in group B to F were given 10% sodium chloride (in the initial 6 weeks) and drinking water containing MNNG (100 ?g/ml) to induce gastric adenocacinoma. All animals received treatment for 40 weeks, and were sacrificed after death or at week 48. Gastric tumor was evaluated histologically. Results Among 86 rats, 26 rats died, and 60 rats completed the experiment. The incidences of gastric cancer were found 0 (0%) in group A, 12 (75.0%) in group B, 11 (68.8%) in group C, 12 (70.6%) in group D, 3(18.8%) in group E, and 5(31.3%) in group F. There were significant differences in tumor incidence (P=0.002), multiplicity (P=0.001) and volume (P=0.009) among different groups. When compared with group B, the group E had the greatest reduction in tumor incidence (P=0.004), tumor multiplicity ( P=0.006) and mean tumor volume (P=0.02). Treatment with indomethacin had no significant effect on tumor development. Conclusion While treatment with indomethacin had no significant effect on tumor development, treatment with celecoxib reduced gastric cancer incidence and growth in rats.
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Objective Using a model of H.pylori infected Mongolian gerbil , we observed the effect of H.pylori and N methyl N’ nitro N nitrosoguanidine (MNNG) on gastric mucosa, in an attempt to clarify the potential role of vitamin C in the prevention of gastric carcinoma. Methods A total of 160 Mongolian gerbils , eight week old, were randomly divided into five groups(each 32 animals): Group A, infected with H.pylori ; Group B, infected with H.pylori followed by MNNG administration; Group C, received MNNG without H.pylori infection; Group D, infected with H pylori followed by administration of MNNG and vitamin C; Group E as control. Eight animals from each group were killed at 12, 24, 36, 48 weeks, and histopathological changes in their stomachs were examined for chronic gastritis, intestinal metaplasia, atypical hyperplasia and adenoma. Results The incidences of intestinal metaplasia and dysplasia in groups A and B were significantly higher than those in the other groups( P
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AIM: To find out common transcription factor binding sites in the promoter regions of the encoding genes of the co-expressive proteins induced by N-methyl-N'-nitro-N- nitrosoguanidine (MNNG). METHODS: Using phylogenetic footprinting and TRANSFAC position weight matrix (PWM) searching program to predict the common transcription factor binding sites among the promoter regions of the genes encoding the co-expressive proteins. The predictive results were validated with electrophoresis mobility shift assay (EMSA). RESULTS: Eleven common transcription factor binding sites were predicted in the promoters of the co-expressive proteins, among them, besides the activator protein 1(AP1) which was previously identified to be activated in MNNG pretreated cells in this laboratory, the nuclear factor Y (NFY) and GATA binding factor (GATA) consensus oligonucleotides binding activity were found being increased in the nuclear extract of cells pre-treated with MNNG as demonstrated by EMSA. CONCLUSION: Phylogenetic footprinting can effectively decrease the false positive rate in predicting transcription factor binding sites. It is possible that NFY and GATA transcription factor binding sites are involved in the co-regulation of the MNNG induced co- expressive proteins. [
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A colistin producing strain Paenibacillus polymyxa AS1.541 was treated by N-methyl-N-nitro-N-nitrosoguanidine(NTG) for increasing yields of the antibiotic colistin.High-yield strains were obtained by selection of deregulated mutant which grow on media containing colistin,a self second metabolite,and ethionine,an analogue of methionine.Some of these mutants have higher yield of colistin than that of the parent strain.
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Objective To establish a malignant transformed human fetal osteoblastic human cells from the immortalized cell line hFOB1.19 in order to explore the molecular mechanism in tumorigenesis of osteosarcoma. Methods hFOB1.19 cells were treated sequentially by an initiated factor, N-methyl-N’-nitro-N-nitrosoguanidine (MNNG) and a promoter, 12-O-tetradecanoyl phorbol-13-acetate (TPA). The features of malignancy of transformed cells were identified by cell morphology, DNA content analysis, colony forming frequency on soft agar and tumorigenetic test in nude mice. Results Continuous passaging after the treatments resulted in the formation of a few paramorph foci and exhibited in an extensively random orientation. The poly-ploid of DNA was 81.08% in experiment group, much higher than that in control group (55.03%). Compared with that of negative control cell, colony formation efficiency of transformed cells in semisolid agar showed a a significant increase. The transformed cells formed tumors subcutaneously in the nude mice, which were verified to be poorly differentiated osteosarcoma histopathological examination. Conclusion Mocking the process of malignant transformation of human cells, we establish malignant transformed immortalized human fetal osteoblastic human cells (hFOB1.19) model.
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AIM: To understand whether endoplasmic reticulum stress (ER-stress) is involved in DNA-damaging agent/carcinogen induced cell responses. METHODS: Three DNA-damaging agents/carcinogens different in the mode of action, ie, alkylating agent N-methyl-N’-nitro-N-nitrosoguanidine (MNNG), bulky adduct forming agent benzo[a] pyrene-7, 8-dihydrodiol-9, 10-epoxide (BPDE) and cross-linking agent mitomycin C (MMC) were selected. SDS-PAGE and immunoblotting were used to examine the protein levels of GRP78/BiP, GDADD153/CHOP and activation state of endoplasmic reticulum located caspase-12 in FL cells before and after MNNG, BPDE or MMC exposure. RESULTS: Immunoblotting showed that the protein level of endoplasmic reticulum specific proteins GRP78/BiP and GADD153/CHOP were significantly increased and endoplasmic reticulum located caspase-12 was activated in low concentration of MNNG (0.25 and 1 ?mol/L) and BPDE (5 and 50 nmol/L)-treated cells. MMC at all of the three concentration used (5, 50 and 500 ?mol/L) decreased the expression of GRP78/BiP, while it has no effects on CHOP and caspase-12. CONCLUSIONS: Both low concentration MNNG and BPDE could trigger the ER-stress in the exposed cells, while MMC could induce the down-regulation of the GRP78/BiP protein, which plays an important mediating role in the induction of ER-stress and may thus change the responsiveness against ER-stress inducers. It is suggested that ER-stress might partially mediate the cellular responses excited by exposure to some DNA-damaging agents/carcinogens.
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The addition of the carcinogen, N-methyl N’-nitro N-nitrosoguanidine, to a cell-free system consisting of purified polysome and ‘pH 5 enzyme’ fraction resulted in a marked inhibition of incorporation of (14C)-leucine into polypeptides. The extent of inhibition was remarkably high if the cell-free system contained limiting amount of ‘pH 5 enzyme’ fraction. Under this condition, the rate of inhibition was dependent on the concentration of carcinogen. Some component present in the ‘pH 5 enzyme’ fraction was inferred to be the susceptible factor, since the inhibition at low concentration of carcinogen could be reversed by increasing the amount of this fraction in the polysomal system. It was ascertained that tRNA was the primary target of carcinogenic action. Evidence suggested that functions attributed to tRNA such as aminoacylation and ribosomal transfer were both affected in a characteristic way by the action of the carcinogenic N-nitroso compound.
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A model system which simulated the conditions of the human stomach was used in the experiments. Precursors NaNO2 and MNG formed MNNG and resulted in the positive mutagenic response to Salmonella typhimurium TA 100. When the concentration of the precursors was 50 mM NaNO2 and 100 mM MNG, the induced average number of revertant per plate was 4327 showed an approximate twenty fold spontaneous revertants. With diluting of the precursors, the mutagenic response was lower, showing a dose-response relationship between the mutagenic activity and the precursors concentration. It was found that, when precursor concentration was lower than 22.2 mM NaNO2 and 44.4 mM MNG, the formation of MNNG was blocked completely by the Chinese Kiwi fruit juice and mutation was inhibited. In the same concentration without the Chinese Kiwi fruit juice, the induced revertant was an approximate 13 fold of the spontaneous revertant. The Chinese Kiwi fruit could not block the formation of the MNNG when the precursors concentration was higher than 33.3 mM NaNO2, but it inhibited the mutagenic activity partly. Compared with the Chinese Kiwi fruit juice, the effect of the ascorbic acid solution in the same concentration was much less.It was demonstrated with TLC that MNNG was formed in the model system.