BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin...BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.展开更多
Strategies for labeling proteins with fluorophores are always important for biotechnology. Here we take a model protein(bovine serum albumin) and a typical fluorophore(rhodamine B) to demonstrate a direct labeling met...Strategies for labeling proteins with fluorophores are always important for biotechnology. Here we take a model protein(bovine serum albumin) and a typical fluorophore(rhodamine B) to demonstrate a direct labeling method just by physical adsorption. In combination with size exclusion chromatography and the Scartchard equation, we have developed a facile analysis method for calculating the binding constant and binding sites.The molecular docking method has been used to study the binding site in amino acid level.展开更多
A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination...A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination. When expressed in monkey kidney cell line COS-M6, this gene product (S309 protein) formed surface antigen (HBsAg) particles and secreted from the cells. It was stable within the cells and in the culture medium and could be immunoprecipitated with antisera directed against plasma-derived HBsAg or synthetic preS1 polypeptide. Isopycnic CsCl gradient centrifugation showed that the density of S309 protein particles (1.25 g/ml) was slightly higher than that of S protein particles. The S309 protein was readily secretable from hepatoma cell lines, and the amount secreted was comparable to that of the S protein. By contrast, only about 10% of the S309 protein was secreted from COS-M6 cells, and its appearance in culture medium was delayed. The efficiency of the secretion of the S309 protein can be improved when it is coexpressed with the S protein.展开更多
This study was designed to investigate the contribution of miRNA-122-binding site polymorphism at the IL-1A gene and its multiplicative interactions with hepatitis B virus （HBV） mutations in the risk of hepatocellul...This study was designed to investigate the contribution of miRNA-122-binding site polymorphism at the IL-1A gene and its multiplicative interactions with hepatitis B virus （HBV） mutations in the risk of hepatocellular carcinoma （HCC）. A total of 1021 healthy controls, 302 HBV surface antigen （HBsAg） seroclearance subjects, and 2011 HBsAg-positive subjects （including 1021 HCC patients） were enrolled in this study. Quantitative PCR was used to genotype rs3783553. HBV mutations were determined by direct sequencing. Multivariate logistic regression analyses were performed to test the associations of rs3783553, mutations, and their interactions with the risk of HCC. No significant association was found between rs3783553 and the risk of HCC among healthy controls, HBsAg seroclearance subjects, HBsAg-positive subjects without HCC, and all controls. Additionally, rs3783553 was not significantly associated with chronic HBV infection, liver cirrhosis, HBV e antigen seroconversion, abnormal alanine aminotransferase, and high viral load （ 〉 10^4 copies/ml）. However, the TTCA insertion allele of rs3783553 was significantly associated with an increased frequency of HBV C7A mutation compared with homozygous TTCA deletion carriers [（del/ins ＋ ins/ins） vs. del/del, adjusted odds ratio （OR）= 1.48, 95% confidence interval （CI）= 1.09-2.02, P = 0.013]. Multiplicative interaction of rs3783553 with HBV preS deletion significantly reduced the risk of HCC in males, with an adjusted OR of 0.64 （95% CI = 0.42-0.98; P = 0.041） after age and HBV genotype were adjusted. Although rs3783553 did not significantly affect genetic susceptibility to HBV-related HCC, its variant allele may predispose the host to selecting HBV C7A mutation during evolution and significantly reduce the risk of HCC caused by HBV preS deletion. This study provides an insight into the complex host-virus interaction in HBV-induced hepatocarcinogenesis and is helpful in determining HBsAg-positive subjects who are likely to develop HCC.展开更多
Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by cas...Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.展开更多
Nuclear factor kB（NF-kB） is a DNA-binding transcription factor. Characterizing its genomic binding sites is crucial for understanding its gene regulatory function and mechanism in cells. This study characterized the...Nuclear factor kB（NF-kB） is a DNA-binding transcription factor. Characterizing its genomic binding sites is crucial for understanding its gene regulatory function and mechanism in cells. This study characterized the binding sites of NF-kB ReIA/p65 in the tumor neurosis factor-a（TNFa） stimulated HeLa cells by a precise chromatin immunoprecipitation-sequencing（ChIP-seq）. The results revealed that NF-kB binds nontraditional motifs（nt-motifs） containing conserved GGAA quadruplet. Moreover, nt-motifs mainly distribute in the peaks nearby centromeres that contain a larger number of repetitive elements such as satellite, simple repeats and short interspersed nuclear elements（SINEs）. This intracellular binding pattern was then confirmed by the in vitro detection, indicating that NF-kB dimers can bind the nontraditional kB（nt-KB） sites with low affinity. However, this binding hardly activates transcription.This study thus deduced that NF-kB binding nt-motifs may realize functions other than gene regulation as NF-kB binding traditional motifs（t-motifs）. To testify the deduction, many ChIP-seq data of other cell lines were then analyzed. The results indicate that NF-kB binding nt-motifs is also widely present in other cells. The ChIP-seq data analysis also revealed that nt-motifs more widely distribute in the peaks with low-fold enrichment. Importantly, it was also found that NF-kB binding nt-motifs is mainly present in the resting cells, whereas NF-kB binding t-motifs is mainly present in the stimulated cells. Aston？ishingly, no known function was enriched by the gene annotation of nt-motif peaks. Based on these results, this study proposed that the nt-KB sites that extensively distribute in larger numbers of repeat elements function as a nuclear reservoir of NF-kB. The nuclear NF-kB proteins stored at nt-KB sites in the resting cells may be recruited to the t-KB sites for regulating its target genes upon stimulation.展开更多
基金Supported by Recurrent Grants from Institut National de la Santéet de la Recherche Médicale to the INSERM U1048.
文摘BACKGROUND Despite overt insulin resistance,adipocytes of genetically obese Zucker rats accumulate the excess of calorie intake in the form of lipids.AIM To investigate whether factors can replace or reinforce insulin lipogenic action by exploring glucose uptake activation by hydrogen peroxide,since it is produced by monoamine oxidase(MAO)and semicarbazide-sensitive amine oxidase(SSAO)in adipocytes.METHODS 3H-2-deoxyglucose uptake(2-DG)was determined in adipocytes from obese and lean rats in response to insulin or MAO and SSAO substrates such as tyramine and benzylamine.14C-tyramine oxidation and binding of imidazolinic radioligands[3H-Idazoxan,3H-(2-benzofuranyl)-2-imidazoline]were studied in adipocytes,the liver,and muscle.The influence of in vivo administration of tyramine+vanadium on glucose handling was assessed in lean and obese rats.RESULTS 2-DG uptake and lipogenesis stimulation by insulin were dampened in adipocytes from obese rats,when compared to their lean littermates.Tyramine and benzylamine activation of hexose uptake was vanadate-dependent and was also limited,while MAO was increased and SSAO decreased.These changes were adipocyte-specific and accompanied by a greater number of imidazoline I2 binding sites in the obese rat,when compared to the lean.In vitro,tyramine precluded the binding to I2 sites,while in vivo,its administration together with vanadium lowered fasting plasma levels of glucose and triacylglycerols in obese CONCLUSION The adipocytes from obese Zucker rats exhibit increased MAO activity and imidazoline binding site number.However,probably as a consequence of SSAO down-regulation,the glucose transport stimulation by tyramine is decreased as much as that of insulin in these insulin-resistant adipocytes.The adipocyte amine oxidases deserve more studies with respect to their putative contribution to the management of glucose and lipid handling.
基金Supported by the National Natural Science Foundation of China(Nos.21171086 and 81160213)Inner Mongolia Autonomous Region science and Technology Department(No.211-202077)+2 种基金Inner Mongolia Grassland Talent(No.108-108038)Natural Science Foundation of Inner Mongolia Autonomous Region of China(No.2013MS1121)Inner Mongolia Agricultural University(Nos.109-108040,211-109003 and 211-206038)
文摘Strategies for labeling proteins with fluorophores are always important for biotechnology. Here we take a model protein(bovine serum albumin) and a typical fluorophore(rhodamine B) to demonstrate a direct labeling method just by physical adsorption. In combination with size exclusion chromatography and the Scartchard equation, we have developed a facile analysis method for calculating the binding constant and binding sites.The molecular docking method has been used to study the binding site in amino acid level.
基金This research was supported in part by U.S.Public Health Service Grants CA-07175 and CA-22443 from the National Institutes of Health
文摘A human hepatitis B virus (HBV) gene, which encodes the major surface antigen protein(S protein) carrying the hepatocyte receptor-binding site, was constructed with site-directed mutagenesis and in vitro recombination. When expressed in monkey kidney cell line COS-M6, this gene product (S309 protein) formed surface antigen (HBsAg) particles and secreted from the cells. It was stable within the cells and in the culture medium and could be immunoprecipitated with antisera directed against plasma-derived HBsAg or synthetic preS1 polypeptide. Isopycnic CsCl gradient centrifugation showed that the density of S309 protein particles (1.25 g/ml) was slightly higher than that of S protein particles. The S309 protein was readily secretable from hepatoma cell lines, and the amount secreted was comparable to that of the S protein. By contrast, only about 10% of the S309 protein was secreted from COS-M6 cells, and its appearance in culture medium was delayed. The efficiency of the secretion of the S309 protein can be improved when it is coexpressed with the S protein.
文摘This study was designed to investigate the contribution of miRNA-122-binding site polymorphism at the IL-1A gene and its multiplicative interactions with hepatitis B virus （HBV） mutations in the risk of hepatocellular carcinoma （HCC）. A total of 1021 healthy controls, 302 HBV surface antigen （HBsAg） seroclearance subjects, and 2011 HBsAg-positive subjects （including 1021 HCC patients） were enrolled in this study. Quantitative PCR was used to genotype rs3783553. HBV mutations were determined by direct sequencing. Multivariate logistic regression analyses were performed to test the associations of rs3783553, mutations, and their interactions with the risk of HCC. No significant association was found between rs3783553 and the risk of HCC among healthy controls, HBsAg seroclearance subjects, HBsAg-positive subjects without HCC, and all controls. Additionally, rs3783553 was not significantly associated with chronic HBV infection, liver cirrhosis, HBV e antigen seroconversion, abnormal alanine aminotransferase, and high viral load （ 〉 10^4 copies/ml）. However, the TTCA insertion allele of rs3783553 was significantly associated with an increased frequency of HBV C7A mutation compared with homozygous TTCA deletion carriers [（del/ins ＋ ins/ins） vs. del/del, adjusted odds ratio （OR）= 1.48, 95% confidence interval （CI）= 1.09-2.02, P = 0.013]. Multiplicative interaction of rs3783553 with HBV preS deletion significantly reduced the risk of HCC in males, with an adjusted OR of 0.64 （95% CI = 0.42-0.98; P = 0.041） after age and HBV genotype were adjusted. Although rs3783553 did not significantly affect genetic susceptibility to HBV-related HCC, its variant allele may predispose the host to selecting HBV C7A mutation during evolution and significantly reduce the risk of HCC caused by HBV preS deletion. This study provides an insight into the complex host-virus interaction in HBV-induced hepatocarcinogenesis and is helpful in determining HBsAg-positive subjects who are likely to develop HCC.
基金We thank for the funding support from the National Natural Science Foundation of China(No.81260550)Key Laboratory Construction Project of Traditional Chinese Medicine for Prevention and Treatment of Five Sense Organ Diseases in Hunan Province(No.2017TP1018)Key Subject Construction Project of Traditional Chinese Medicine Ophthalmology of the State Administration of Traditional Chinese Medicine(No.ZK1801YK015).
文摘Objective To observe the effects of total flavonoids of chrysanthemum and medicated serum on the expression of related proteins in the lacrimal tissue and dry-eye cell models of male rabbits with dry eye caused by castration.Methods(1)150 male Japanese rabbits were randomly divided into five groups,with 30 rabbits in each group:normal control group(group A),sham group(group B),model group(group C),androgen control group(group D)and total flavonoids of chrysanthemum treatment group(group E).The androgen deficiency dry-eye model was established by bilateral castration in groups C,D and E.Normal saline was administered to groups A,B and C by gavage;androgen(testosterone propionate)was injected into muscle in group D;and group E was given total flavonoids of chrysanthemum by gavage.All white rabbits were tested the Schirmer I test(SIT)and tear break-up time(BUT).After euthanasia,tear gland tissue was harvested so that we could observe pathological changes in the expression of related inflammatory factors in the lacrimal gland tissue.The expression of interleukin-1β(IL-1β),tumor necrosis factor-α(TNF-α)and transforming growth factor-β1(TGF-β1)was detected in the lacrimal gland tissue by immunohistochemistry.Reverse transcription PCR was used to quantitatively detect expression of TGF-β1 mRNA.(2)Male Wistar rat lacrimal epithelial cells were used to establish a model of eye stem cell apoptosis caused by androgen levels.The blank control group was set up without androgen culture,the control group with androgen culture,and the total flavonoids of chrysanthemum group without androgen.The MTT method was used to determine the optimal intervention dosage of drug-containing plasma.Western blot and QPCR were used to detect the expression of AR mRNA,NF-κB phosphorylated protein and TGF-β1 in lacrimal epithelial cells,and the androgen-like effect of total flavonoids of chrysanthemum was observed.Results(1)Immunohistochemistry showed that groups A,B,D and E had significantly lower expression of IL-1βand TNF-αthan group C(P<0.05);among these,group E had slightly higher expression than group D(P>0.05).RT-PCR results showed that the relative expression of TGF-β1 mRNA in groups A,B,D and E was significantly higher than in group C(P<0.05),and the relative expression of TGF-β1 mRNA in groups D and E was higher than that in groups A and B(P<0.05).(2)Using the MTT method,the final concentration of interfering cells was calculated to be 13.2%.The expression of AR protein,NF-κB and TGF-β1 in the chrysanthemum flavonoid plasma intervention and testosterone propionate intervention groups was enhanced,and there were significant differences relative to the blank group(P<0.01).The expression level of NF-κB in the total flavonoid containing plasma intervention group was lower than that in the testosterone propionate intervention group(P<0.01).Conclusions The total flavonoids of chrysanthemum can inhibit IL-1βand TNF-αexpression in the lacrimal gland tissue of castrated male rabbits with dry eye to increase synthesis of TGF-β1 mRNA and TGF-β1,thereby inhibiting the inflammatory response.The medicated plasma with total flavonoids of chrysanthemum promotes expression of AR mRNA,upregulating expression of NF-κB,further promoting upregulation of TGF-β1 protein expression in lacrimal epithelial cells,inhibiting inflammation by regulating related proteins,and ultimately alleviating the symptoms of dry eye.
基金supported by the National Natural Science Foundation of China(Nos.61571119 and 81502853)the Natural Science Foundation of Jiangsu Province(BK20151026)
文摘Nuclear factor kB（NF-kB） is a DNA-binding transcription factor. Characterizing its genomic binding sites is crucial for understanding its gene regulatory function and mechanism in cells. This study characterized the binding sites of NF-kB ReIA/p65 in the tumor neurosis factor-a（TNFa） stimulated HeLa cells by a precise chromatin immunoprecipitation-sequencing（ChIP-seq）. The results revealed that NF-kB binds nontraditional motifs（nt-motifs） containing conserved GGAA quadruplet. Moreover, nt-motifs mainly distribute in the peaks nearby centromeres that contain a larger number of repetitive elements such as satellite, simple repeats and short interspersed nuclear elements（SINEs）. This intracellular binding pattern was then confirmed by the in vitro detection, indicating that NF-kB dimers can bind the nontraditional kB（nt-KB） sites with low affinity. However, this binding hardly activates transcription.This study thus deduced that NF-kB binding nt-motifs may realize functions other than gene regulation as NF-kB binding traditional motifs（t-motifs）. To testify the deduction, many ChIP-seq data of other cell lines were then analyzed. The results indicate that NF-kB binding nt-motifs is also widely present in other cells. The ChIP-seq data analysis also revealed that nt-motifs more widely distribute in the peaks with low-fold enrichment. Importantly, it was also found that NF-kB binding nt-motifs is mainly present in the resting cells, whereas NF-kB binding t-motifs is mainly present in the stimulated cells. Aston？ishingly, no known function was enriched by the gene annotation of nt-motif peaks. Based on these results, this study proposed that the nt-KB sites that extensively distribute in larger numbers of repeat elements function as a nuclear reservoir of NF-kB. The nuclear NF-kB proteins stored at nt-KB sites in the resting cells may be recruited to the t-KB sites for regulating its target genes upon stimulation.
