Study of Mechanism of Proinsulin Gene Mutations in Diabetes Mellitus / 天津医药

Objective To construct several human proinsulin mutants plasmid related to diabetes and to express in INS-1 (Insulin secreting beta cell derived line) cell. Methods Human mild proinsulin gene was used as template , and site-directed mutagenesis PCR was employed to generate four human proinsulin plasmid mutants. Each mutant plasmid was sequenced then transfected with empty plasmid and mild plasmid into INS-1 cell by liposome 2000. Insulin value in each cell solution was determined by radioimmunoassay. Results Proinsulin mutants plasmid were confirmed by sequencing. In-sulin values in culture solution of H-C(B19)G、H-L(B11)P、H-R(S6)C mutants are less than those in wild type and H-F (B25)L(P＜0.05). Comparison of insulin values between H-C(B19)G、H-L(B11)P、H-R(S6)C groups were not statistically significant(P>0.05), and all these three groups showed no significant differences with empty plasmid group statistically (P>0.05).Insulin value of H-F(B25)L was of no significant differences statistically with empty plasmid(P>0.05). Conclu-sion Four human proinsulin mutants plasmid were constructed and expressed successfully in INS-1 cell, and different mu-tants plasmid result in diabetes through different mechanism.

Effect of mutations in the 5'untranslated region pyrimidine-rich tract on the virulence variation of coxsackievirus B1 / 中国地方病学杂志

Objective To evaluate the infectivity and virulence variation caused by mutations in the 5' untranslated region(5'UTR)pyrimidine-rich tract of coxsackievirus B1(CVB1)genome.Methods Five pyrimidines in the 5'UTR pyrimidine-rich tract(nt563-nt573)of CVB1 genome were substituted with purines by site-directed mutagenesis.The mutant,CVB1/m563-573,was purified by plaque assay,and subjected to infectivity and virulence assessments by means of cytopathic effect(CPE),plaque forming,one-step growth curve,and 50% lethal dose(LD50)assays.Results Sequencing data revealed that the sequence of pyrimidine-rich tract in the 5'UTR of CVB1/m563-573 mutant was exactly identical to our design(C565A,U567C,U568A,U570A,and U572G).CPE assay showed that the infectivity of CVB1/m563-573 was weaker than that of its prototype CVB1/wt(A490=0.710±0.074,0.812±0.092)though no significant difference could be observed(t=-2.204,P>0.05).Plaque forming assay showed that the plaque quantities of CVB1/m563-573 were(6.40±1.52)×103,(11.60±2.19)×103 pfu/L and the plaque diameters of CVB1/m563-573 were(2.00±0.35),(2.47±0.41)mm at 46 and 58 hours pestinfection,respectively.The plaque quantities of CVB1/wt were(8.40±2.51)×103,(11.80±1.92)×103 pfu/L and the plaque diameters of CVB1/wt were(1.80±0.27),(2.85±0.44)mm,respectively.There was no significant difference between the plaque quantities and sizes of CVB1/m563-573 and CVB1/wt(t=8.000,0.985,10.000,9.000,all P>0.05).One-step growth curve demonstrated that the numbers(lg)of CVB1/m563-573 progenies at time-points of 3,5,7 h postinfection were 2.10±0.09,4.28±0.03,7.44±0 and that of CVB1/wt progenies were 2.80±0.02,4.77±0.02,8.55±0.01,respectively.The replication of CVB1/m563-573 was significantly slower than that of CVB1/wt at all three time-points(t=-13.151,-24.319,-47.714,all P<0.01).The LD50 of CVB1/m563-573(3.10×109 pfu/L)and CVB1/wt(1.26×107 pfu/L)indicated that the virulence of CVB1/m563-573 was significantly weakened compared to that of CVB1/wt.Conclusions The infectivity and virulence of CVB1 are weakened by substitution of pyrimidines with purines in the pyrimidine-rich tract of CVB1 5'UTR.Site-directed mutagenesis in the pyrimidine-rich tract may be a strategy for developing attenuated CVB vaccine.

Construction of recombinant hING4 gene and its inhibitory effect on the growth of lung cancer NCI-H460 cells / 肿瘤

Objective: To construct a recombinant human inhibitor of growth 4 (hING4) gene and observe its growth inhibition effect on lung adenocarcinoma NCI H460 cells. Methods: The GFP-labeled recombinant adenovirus vector hING4 (Ad-hING4) was constructed using pcDNA3. 0-mING4 plasmid as a template and site-specific mutagenesis technique. RT-PCR and fluorescence microscope were used to detect the expression of hING4 in NCI H460 cells. Bax and Bcl-2 protein expression were detected by Western blotting. Colony formation assay and flow cytometry were used to observe the effect of hING4 on the proliferation and apoptosis of H460 cells. Results: DNA sequence analysis and PCR indicated that Ad-hING4 were successfully constructed. Ad-hING4 was expressed in lung cancer H460 cells and had obvious cytopathic effect (CPE). Western blotting suggested that hING4 gene caused up-regulation of Bax expression and down-regulation of Bcl-2 expression. Colony formation assay and flow cytometry showed that hING4 inhibited the proliferation of H460 cells and induced apoptosis of lung cancer cells. Conclusion: Ad-hING4 gene was successfully constructed. It inhibited the growth of lung cancer H460 cells in vitro.

Homo-dimerization of RyR1 C-terminus via charged residues in random coils or in an alpha-helix

To investigate the mechanism by which the C-terminus (4,938-5,037) of the ryanodine receptor 1 (RyR1) homo-tetramerizes, forming a functional Ca2+ -release channel, the structural requirements for the tetramerization were studied using site-directed mutagenesis. Alanine-substitutions at five charged residues, E4976, H5003, D5026, E5033 and D5034, significantly decreased the formation of homo-dimers (reduced by > 50%). Interaction between the C-terminus and cytoplasmic loop I (4,821-4,835) required two positively charged residues, H4832 and K4835. Based on the predicted protein secondary structures, all seven charged residues are located in random coils. Paired alanine-substitutions at six negatively charged residues (E4942A/D4953A, D4945A/E4952A and E4948A/ E4955A) of the alpha-helix (4,940-4,956) in the C-terminus increased homo-dimerization. Therefore, the homo-tetramerization of RyR1 may be mediated by intra- and/or inter-monomer electrostatic interactions among the C-terminal charged residues in random coils or in an alpha-helix.