To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas re...To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.展开更多
Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2...Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2β-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes,was sequenced and analyzed. Using polymerase chain reaction (PCR),the tissue distribution and expression time pattern of Ap2β-NY were determined. Results:Ap2β-NY was identified and has been deposited in the GenBank (AY341427).The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis.PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis,spleen,thymus,prostate,ovary,blood leukocyte and brain,but not in the heart,placenta,lung,liver,skeletal muscle,kidney and pancreas.In addition,Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome,which implied that,in the testis,Ap2β-NY was restrictively ex- pressed in germ cells.Conclusion:Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.展开更多
Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta s...Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3’RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.展开更多
Objective To explore the association between the three polymorphisms [ C825T, C1429T and G（-350）A] of the gene encoding the G protein beta 3 subunit （GNB3） and hypertension by performing a case-control study in th...Objective To explore the association between the three polymorphisms [ C825T, C1429T and G（-350）A] of the gene encoding the G protein beta 3 subunit （GNB3） and hypertension by performing a case-control study in the northern Han Chinese population. Methods We recnaited 731 hypertensive patients and 673 control subjects （the calculated power value was 〉 0.8）. Genotyping was performed to identify C825T, C1429T and G（-350）A polymorphisms using the TaqMan assay. Comparisons of allelic and genotypic frequencies between cases and controls were made by using the chi-square test. Logistic regression analyses were performed to investigate the relationships between the three polymorphisms of GNB3 gene under different genetic models （additive, dominant and recessive models）. Results The genotype dis- tribution and allele frequencies of C825T, C1429T and G（-350）A polymorphisms did not differ significantly between hypertensive patients and control subjects, either when the full sample was assessed, or when the sample was stratified by gender. No significant association was observed between C825T, C 1429T and G（-350）A polymorphisms and the risk of essential hypertension in any genetic model. Linkage dis- equilibrium was only detected between C825T and C 1429T polymorphisms. Haplotype analyses observed that none of the three estimated haplotypes significantly increased the risk of hypertension. Conclusions Our study suggested that the GNB3 gene polymorphisms [C825T, C 1429T and G（-350）A] were not significantly associated with essential hypertension in northern Han Chinese population.展开更多
Background: Sickle cell disease and sickle cell trait are common erythrocyte disorders that are most often caused by a point mutation (rs334, designated HbS) in the hemoglobin beta gene (HBB);however of this fact, the...Background: Sickle cell disease and sickle cell trait are common erythrocyte disorders that are most often caused by a point mutation (rs334, designated HbS) in the hemoglobin beta gene (HBB);however of this fact, there is extreme variability in occurrence and clinical presentation of sickle cell disease which may be explained by some other genetic changes associated with the gene. In the present study we examined the association between HBB gene polymorphism rs33949930 T>C in the occurrence of sickle cell disease in Saudi Arabia population. Materials and Methods: A case control study of 100 sickle cell disease patients and 100 healthy controls from Tabuk, Saudi Arabia. HBB gene rs33949930 T>C polymorphism was analyzed using Allele specific polymerase chain reaction technique. Results: It was observed that the genotype percentages TT, TC and CC among the patients with sickle cell disease were 63.0%, 35.0% and 2.0% and healthy controls were 68.0%, 27.0% and 5.0% respectively. Allele frequency for T allele was observed to be fT = 0.20 and fT = 0.19, where as for C allele was fC = 0.80 and fC = 0.81 among cases and controls respectively (p = 0.29). Compared to the TT genotype, the odds ratio of 1.4 (95% CI 0.76 - 2.57), risk ratio of 1.2 (95% CI 0.86 - 1.65) and risk difference of 8.4 (-6.66 - 23.38) for heterozygous genotype of HBB rs33949930 T>C was observed in relation to sickle cell disease. In addition, some difference in the laboratory values was observed among sickle cell disease patients with the different variants of HBB gene rs33949930 T>C polymorphism, especially the carriers of heterozygous TC genotype;however, the difference doesn’t reach to statically significant number. Conclusion: Present study suggested that there was not any significant association between HBB gene rs33949930 T>C polymorphism and occurrence of sickle cell disease. However, the heterozygous TC genotype of the polymorphism showed some higher ratios among cases as compared to healthy control group.展开更多
目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA des...目的:探讨抑制素β亚基A反义RNA1(INHBA-AS1)对宫颈癌HeLa细胞EMT和鸟氨酸代谢途径的影响及其机制。方法:体外常规培养HeLa细胞,实验分为10组:对照组、阴性对照(NC)组、sh-INHBA-AS1组、PluriSIn 1[硬脂酰辅酶A去饱和酶(stearyl CoA desaturase,SCD)抑制剂]组、NC+PluriSIn 1组、sh-INHBA-AS1+PluriSIn 1组、10058-F4(c-Myc抑制剂)组、NC+10058-F4组、sh-INHBA-AS1+10058-F4组、sh-INHBA-AS1+OE-c-Myc组。平板克隆实验检测各组细胞的增殖能力,FCM检测各组细胞的凋亡情况,Transwell小室实验检测各组细胞的侵袭、迁移能力,qPCR法检测各组细胞中INHBA-AS1、c-Myc、SCD和EMT相关基因(N-cadherin、TGF-β、ZEB1)mRNA的表达,WB法检测各组细胞中c-Myc、SCD、EMT相关(N-cadherin、TGF-β、ZEB1)、S-腺苷-甲硫氨酸脱羧酶(SAMDC)和亚精胺/精胺N1-乙酰转移酶(SSAT)蛋白的表达,ELISA检测各组细胞上清液中鸟氨酸脱羧酶(ODC)的含量。结果:敲减INHBA-AS1表达使HeLa细胞的增殖、侵袭和迁移能力显著降低(均P<0.05)而细胞凋亡率显著升高(P<0.05),q PCR、WB法检测结果显示,敲减INHBA-AS1均可显著抑制HeLa细胞中c-Myc、SCD、N-cadherin、TGF-β、ZEB1和SAMDC的表达(均P<0.05),而促进SSAT的表达(P<0.05),并降低HeLa细胞上清液中ODC的含量(P<0.05)。与c-Myc抑制剂和SCD抑制剂单独处理相比,其联合敲减INHBA-AS1后上述作用更加显著(均P<0.05);与sh-INHBA-AS1组相比,进一步过表达c-Myc后HeLa细胞的增殖能力显著升高(P<0.05)、SCD和N-cadherin蛋白表达水平显著升高(P<0.05)、细胞上清液中ODC含量显著升高(P<0.05)。结论:INHBA-AS1可通过c-Myc调控SCD的表达,从而影响HeLa细胞鸟氨酸代谢和EMT进程,进而促进HeLa细胞的增殖、侵袭和迁移能力。展开更多
文摘To demonstrate the ability of the Nativis signal transduction technology (Butters et al. 2014) to modulate the expression of algae mRNA and protein, we tested if we can alter specific enzyme levels in Chlamydomonas reinhardtii. We inhibited the synthesis of the enzyme tryptophan synthase beta subunit (MAA7) by applying the signal derived from a published siRNA (Zhao et al. 2009). With lower levels of MAA7, Chlamydomonas reinhardtii can grow in the presence of the prodrug 5-Fluoroindole (5-FI), because less 5-Fluoroin-dole can be converted to the toxic 5-Fluoro-L-tryptophan (5-FT). We find a 24% (±5%) increase of growth with the signal versus no signal. To see if that effect was due to the reduction of the amount of mRNA encoding MAA7, we used Real-Time Quantitative PCR (RT-QPCR) to measure the levels of MAA7 mRNA. To normalize the MAA7 mRNA level, we compared them to the levels of a mRNA that is not affected by the signal (G protein beta subunit-like polypeptide, Cblp). Two conditions increase the effectiveness of the signal. One can either treat the cell cultures during the logarithmic growth phase (starting the cultures at density of 0.104 OD at 750 nm). Or one can treat the cultures at a later stage of the logarithmic growth, but treating them for a longer time (8.7% versus 3.5% of the culture time). Under these conditions we found around a 50% decrease in the mRNA levels for MAA7. Treating the cultures at the earlier growth phase or at a later growth phase is less effective, with only a 20% effect.
文摘Aim:To identify a novel isoform of adaptin 2 beta subunit (named Ap2β-NY) and to investigate its relationship with testicular development and spermatogenesis.Methods:Using a human testis cDNA microarray,a clone (Ap2β-NY), which was strongly expressed in adult testes but weakly expressed in embryo testes,was sequenced and analyzed. Using polymerase chain reaction (PCR),the tissue distribution and expression time pattern of Ap2β-NY were determined. Results:Ap2β-NY was identified and has been deposited in the GenBank (AY341427).The expression level of Ap2β-NY in the adult testis was about 3-fold higher than that in the embryo testis.PCR analysis using multi-tissue cDNA indicated that Ap2β-NY was highly expressed in the testis,spleen,thymus,prostate,ovary,blood leukocyte and brain,but not in the heart,placenta,lung,liver,skeletal muscle,kidney and pancreas.In addition,Ap2β-NY was variably expressed in the testes of patients with spermatogenesis-disturbance and spermatogenesis-arrest but not expressed in those of Sertoli-cell-only syndrome,which implied that,in the testis,Ap2β-NY was restrictively ex- pressed in germ cells.Conclusion:Ap2β-NY is an isoform of Ap2β and may be involved in regulating the process of spermatogenesis and testis development.
基金ThisprojectwassupportedbytheNationalNaturalScienceFoundationofChina (No 3 0 0 70 683 )andDoctorFoundationofNationalMinistryofEducation (No 2 0 0 0 45 )
文摘Background Nowadays it is now a focus topic in schistosomiasis research to find ideal vaccine candidates and new drug targets for developing anti-schistosomiasis vaccine. We cloned a new gene, casein kinase Ⅱ beta subunit, of Schistosoma japonicum (S. japonicum) and express it in Escherichia coli (E.coli).Methods The ESTs obtained in our laboratory were analyzed by homologous searching, and a new gene was recognized. The full-length cDNA of the new gene was obtained by joining the 3’RACE PCR fragment and the EST clone. To express the new gene, the cDNA was cloned into pGEX-4T-1 vector and then transformed into E.coli JM109. The recombinant protein was analyzed by SDS-PAGE and Western-blot. Results A 908 bp cDNA was isolated from S. japonicum and identified to be casein kinase Ⅱ beta subunit gene by sequence analysis. The open reading frame of the gene encodes a protein of 217 amino acids exhibiting 75.8%, 75.8%, 73.9%, 68.2%, 51.6% identity to the amino acids sequence of the corresponding genes of Homo sapiens (H. sapiens), Xenopus laevi (X. laevi), Drosophila melanogaster (D. melanogaster), Caenorhabditis elegan (C. elegan), and Schizosaccharomyces pombe (S. promber) respectively. The predicted molecular weight of the protein was 24.921 kDa. The new cDNA sequence had been submitted to GenBank, and its accession number is AY241391. This cDNA was subcloned into the pGEX-4T-1 vector and expressed in E.coli JM109.The recombinant protein could be recognized by the S. japonicum infected rabbit serum. Conclusion The full-length cDNA sequences encoding S. japonicum casein kinase Ⅱ beta subunit were firstly sequenced, cloned, and expressed in E.coli.
基金grants of the National High Technology Research and Development Program，grants of the National Eleventh Five-year Plan Program from the Ministry of Science and Technology of China，Beijing Natural Science Foundation
文摘Objective To explore the association between the three polymorphisms [ C825T, C1429T and G（-350）A] of the gene encoding the G protein beta 3 subunit （GNB3） and hypertension by performing a case-control study in the northern Han Chinese population. Methods We recnaited 731 hypertensive patients and 673 control subjects （the calculated power value was 〉 0.8）. Genotyping was performed to identify C825T, C1429T and G（-350）A polymorphisms using the TaqMan assay. Comparisons of allelic and genotypic frequencies between cases and controls were made by using the chi-square test. Logistic regression analyses were performed to investigate the relationships between the three polymorphisms of GNB3 gene under different genetic models （additive, dominant and recessive models）. Results The genotype dis- tribution and allele frequencies of C825T, C1429T and G（-350）A polymorphisms did not differ significantly between hypertensive patients and control subjects, either when the full sample was assessed, or when the sample was stratified by gender. No significant association was observed between C825T, C 1429T and G（-350）A polymorphisms and the risk of essential hypertension in any genetic model. Linkage dis- equilibrium was only detected between C825T and C 1429T polymorphisms. Haplotype analyses observed that none of the three estimated haplotypes significantly increased the risk of hypertension. Conclusions Our study suggested that the GNB3 gene polymorphisms [C825T, C 1429T and G（-350）A] were not significantly associated with essential hypertension in northern Han Chinese population.
文摘Background: Sickle cell disease and sickle cell trait are common erythrocyte disorders that are most often caused by a point mutation (rs334, designated HbS) in the hemoglobin beta gene (HBB);however of this fact, there is extreme variability in occurrence and clinical presentation of sickle cell disease which may be explained by some other genetic changes associated with the gene. In the present study we examined the association between HBB gene polymorphism rs33949930 T>C in the occurrence of sickle cell disease in Saudi Arabia population. Materials and Methods: A case control study of 100 sickle cell disease patients and 100 healthy controls from Tabuk, Saudi Arabia. HBB gene rs33949930 T>C polymorphism was analyzed using Allele specific polymerase chain reaction technique. Results: It was observed that the genotype percentages TT, TC and CC among the patients with sickle cell disease were 63.0%, 35.0% and 2.0% and healthy controls were 68.0%, 27.0% and 5.0% respectively. Allele frequency for T allele was observed to be fT = 0.20 and fT = 0.19, where as for C allele was fC = 0.80 and fC = 0.81 among cases and controls respectively (p = 0.29). Compared to the TT genotype, the odds ratio of 1.4 (95% CI 0.76 - 2.57), risk ratio of 1.2 (95% CI 0.86 - 1.65) and risk difference of 8.4 (-6.66 - 23.38) for heterozygous genotype of HBB rs33949930 T>C was observed in relation to sickle cell disease. In addition, some difference in the laboratory values was observed among sickle cell disease patients with the different variants of HBB gene rs33949930 T>C polymorphism, especially the carriers of heterozygous TC genotype;however, the difference doesn’t reach to statically significant number. Conclusion: Present study suggested that there was not any significant association between HBB gene rs33949930 T>C polymorphism and occurrence of sickle cell disease. However, the heterozygous TC genotype of the polymorphism showed some higher ratios among cases as compared to healthy control group.
