Objective To analyze the relationship between tumor necrosis factor-alpha (TNFα) gene promoter -308 polymorphism and myasthenia gravis (MG) in Chinese and analyze secretion of TNFα in peripheral blood mononuclear ce...Objective To analyze the relationship between tumor necrosis factor-alpha (TNFα) gene promoter -308 polymorphism and myasthenia gravis (MG) in Chinese and analyze secretion of TNFα in peripheral blood mononuclear cells (PBMC) in MG patients. Methods A biallelic polymorphism at position -308 in the promoter of TNFα gene was screened by PCR amplification and NcoI recognition site. One hundred and twenty-three MG cases and 115 healthy controls were included in this study. MG patients were classified to different groups according to clinical type, age at onset, and sex respectively. PBMC were isolated from 20 patients and 20 healthy controls, and then cultured in the presence or absence of phytohemag- glutinin (PHA) and acetycholine receptors (AchR). The supernatants were harvested after incubation and stored until TNFα was assayed by enzyme-linked immunosorbent assay. Results The frequency of TNFα-308 allele 2 (A) was found significantly increase in MG patients and showed a trend especially in late onset (≥ 40 years) and male patients (P < 0.05). The allele A had no relationship with thymic pathogenesis in MG patients. But frequency of allele A was significantly higher in general type than in ocular type (P < 0.05). MG patients had a higher inducible level of TNFα by PHA and AchR, and could be down regulated after treatment. Conclusion Polymorphism in TNFα gene promoter -308 is associated with onset of MG. The microsatellite allele TNFα2 confer risk for the development of MG in Chinese patients. MG patients have a higher inducible level of TNFα.展开更多
Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fr...Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fragment discloses several motifs similar to the conservative domains of prokaryotic promoters, including -10 box, -35 box, parallel SD fragment essential to transcription initiation, and the translation initiation site ATG. Southern blotting of PA7 indicates that the PA7 fragment comes from P. pseudoalcaligenes genome and has probably one copy. The PA7 fragment is subcloned by PCR, and the result shows that the 5’-flanking fragment from 889 to 1120 bp has promoter activity, which can be enhanced by the 0.7Kb fragment at 5’ end. The fragments of pPA7 and pPA7-2 are transferred into pseudomonas pseudoaligenes by electroporation, and the significant higher kanamycin resistance of transformants than that of control indicates that the PA7 fragment has the promoter activity in P. pseudoaligene.展开更多
Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during initial substrate preorganization s...Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during initial substrate preorganization step, oxidosqualene cyclases （OSCs） are roughly segregated into protosteryl cation group that mainly catalyzes tetracyclic products and dammarenyl cation group which mostly generates pentacyclic products. However, in contrast to well-studied cascade of ring-forming reactions, little is known about the mechanism underlying the initial sub- strate folding process. Previously, we have identified a cucurbitadienol synthase （Bi） and its null allele bi （C393Y） from cucumber. By integration of homology modeling, residue coevolution and site-directed mutagenesis, we discover that four covarying amino acids including C393 constitute a dynamic domain that may be involved in substrate folding process for Bi. We also reveal a group of co-conserved residues that closely associated with the segregation of plant OSCs. These residues may act col- laboratively in choice of specific substrate folding inter- mediate for OSCs. Thus, engineer plant OSCs from into five-ringed producer. our findings open a door to four-ringed skeleton catalysts展开更多
文摘Objective To analyze the relationship between tumor necrosis factor-alpha (TNFα) gene promoter -308 polymorphism and myasthenia gravis (MG) in Chinese and analyze secretion of TNFα in peripheral blood mononuclear cells (PBMC) in MG patients. Methods A biallelic polymorphism at position -308 in the promoter of TNFα gene was screened by PCR amplification and NcoI recognition site. One hundred and twenty-three MG cases and 115 healthy controls were included in this study. MG patients were classified to different groups according to clinical type, age at onset, and sex respectively. PBMC were isolated from 20 patients and 20 healthy controls, and then cultured in the presence or absence of phytohemag- glutinin (PHA) and acetycholine receptors (AchR). The supernatants were harvested after incubation and stored until TNFα was assayed by enzyme-linked immunosorbent assay. Results The frequency of TNFα-308 allele 2 (A) was found significantly increase in MG patients and showed a trend especially in late onset (≥ 40 years) and male patients (P < 0.05). The allele A had no relationship with thymic pathogenesis in MG patients. But frequency of allele A was significantly higher in general type than in ocular type (P < 0.05). MG patients had a higher inducible level of TNFα by PHA and AchR, and could be down regulated after treatment. Conclusion Polymorphism in TNFα gene promoter -308 is associated with onset of MG. The microsatellite allele TNFα2 confer risk for the development of MG in Chinese patients. MG patients have a higher inducible level of TNFα.
文摘Promoter-probe vector pSUPV4 is used to clone the promoter of Pseudomonas pseudoalcaligenes directly in E. coli, and the recombinant pPA7, which has the highest kanamycin resistance, is obtained. Sequencing the PA7 fragment discloses several motifs similar to the conservative domains of prokaryotic promoters, including -10 box, -35 box, parallel SD fragment essential to transcription initiation, and the translation initiation site ATG. Southern blotting of PA7 indicates that the PA7 fragment comes from P. pseudoalcaligenes genome and has probably one copy. The PA7 fragment is subcloned by PCR, and the result shows that the 5’-flanking fragment from 889 to 1120 bp has promoter activity, which can be enhanced by the 0.7Kb fragment at 5’ end. The fragments of pPA7 and pPA7-2 are transferred into pseudomonas pseudoaligenes by electroporation, and the significant higher kanamycin resistance of transformants than that of control indicates that the PA7 fragment has the promoter activity in P. pseudoaligene.
基金supported by funding from the National Natural Science Foundation of China (31401886)the Agricultural Science and Technology Innovation Program
文摘Sterols and triterpenes are structurally diverse bioactive molecules generated through cyclization of linear 2,3-oxidosqualene. Based on carbocationic intermediates generated during initial substrate preorganization step, oxidosqualene cyclases （OSCs） are roughly segregated into protosteryl cation group that mainly catalyzes tetracyclic products and dammarenyl cation group which mostly generates pentacyclic products. However, in contrast to well-studied cascade of ring-forming reactions, little is known about the mechanism underlying the initial sub- strate folding process. Previously, we have identified a cucurbitadienol synthase （Bi） and its null allele bi （C393Y） from cucumber. By integration of homology modeling, residue coevolution and site-directed mutagenesis, we discover that four covarying amino acids including C393 constitute a dynamic domain that may be involved in substrate folding process for Bi. We also reveal a group of co-conserved residues that closely associated with the segregation of plant OSCs. These residues may act col- laboratively in choice of specific substrate folding inter- mediate for OSCs. Thus, engineer plant OSCs from into five-ringed producer. our findings open a door to four-ringed skeleton catalysts
