AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs) in in vitro models both of the non-inflamed and inflamed intestinal epithelium. METHODS: A repor...AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs) in in vitro models both of the non-inflamed and inflamed intestinal epithelium. METHODS: A reporter gene system in HT-29 cells was used to measure levels of NF-KB activation after challenge with bifidobacteria or after bacterial pre-treatment following LPS challenge. IL-8 protein and pro-inflammatory gene expression was investigated using normal HT-29 cells. RESULTS: None of the bifidobacteria tested induced activation of nuclear factor κB (NF-κB) indicating that bifidobacteria themselves do not induce inflammatory events in IECs. However, six out of eight bifidobacteria tested inhibited lipopolysaccharide- (LPS-) induced NF-κB activation in a dose- and strain-dependent manner. In contrast, NF-κB activation in response to challenge with tumor necrosis factor-α (TNF-α) was affected by none of the tested bifidobacteria, indicating that the inhibitory effect of bifidobacteria is specific for LPS-induced inflammation in IECs. As shown with two of the six inhibitionpositive bifidobacteria, LPS-induced inhibition of NF-κB activation was accompanied by a dose-dependent decrease of interleukin 8 (IL-8) secretion and by lower mRNA levels for IL-8, TNF-α, cyclooxygenase 2 (Cox-2), and intercellular adhesion molecule 1(ICAM-1). CONCLUSION: Some strains of bifidobacteria are effective in inhibiting LPS-induced inflammation and thus might be appropriate candidates for probiotic intervention in chronic intestinal inflammation.展开更多
RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequenc...RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0~60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.展开更多
Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous vir...Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.展开更多
A novel Eucommia antifungal peptide, named EAFP3, was isolated from the bark of Eucommia ul- moides by NaC1 extract, gel filtration and reverse phase high performance liquid chromatography. The molecular mass of EAFP3...A novel Eucommia antifungal peptide, named EAFP3, was isolated from the bark of Eucommia ul- moides by NaC1 extract, gel filtration and reverse phase high performance liquid chromatography. The molecular mass of EAFP3 is 4157.3 Da, and its partial amino acid sequence is -. LYQQLIAGITLNK.-. EAFP3 exerts an inhibitory activity against Candida albicans in vitro and the drug concentration required for 50% growth inhibition (IC50) is 31.25μg/mL.展开更多
Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleoti...Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide frag-ments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2-CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results: DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion: The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.展开更多
To investigate into the mechanisms underlying the irreversible sterility induced by gossypol, we studied the relationship between its inhibitory action on acrosomal enzymes and its antifertility effect.As shown by our...To investigate into the mechanisms underlying the irreversible sterility induced by gossypol, we studied the relationship between its inhibitory action on acrosomal enzymes and its antifertility effect.As shown by our result, after exposure to gossypol (l.25-60 μg/ml) for 15 min. in vitro,the sperms' ability to penetrate bovine cervical mucus and the fertility rate were significantly reduced. Also, following administration of gossypol (12.5 mg/kg/day) for six weeks, the rate of fertilization in vitro by hamster sperm was significantly decreased. In the gossypol-treated group, extracts of testis sperm delayed dispersion of cumulus cells, suggesting inhibition of hyaluronidase and other acrosomal enzymes. Furthermore, the acrosin and arylsulfatase activities were shown to be markedly inhibited. Thus, a parallelism was displayed between the reduction of fertility and the decreasc in acrosin and arylsulfatase activities in epididymis sperms.Besides, the inhibition was reversible and was dosage-and durationdependent. In conclusion, the assay of acrosin activity might serve as a useful tool for monitoring the irreversible sterility induced by gossypol,展开更多
Objective: To purify the natural antikeratin autoantibody (AK auto-Ab) and observe its effects on the prolif eration of the cultured keratinocytes. Methods: Natural AK auto-Ab was purified by using keratin affinity co...Objective: To purify the natural antikeratin autoantibody (AK auto-Ab) and observe its effects on the prolif eration of the cultured keratinocytes. Methods: Natural AK auto-Ab was purified by using keratin affinity column, and then the titre and specificity of the Abs were studied by ELISA, immunoperoxidase staining and immuno-electronicmicroscope. The effect of the purified Abs on the cultured keratino-cytes was assayed by 3H-TdR incorporation. Results: Natural AK auto-Ab was obtained. The binding activity of IgG AK auto-Ab in purified Ah remained similar to that in pooled human sera. and the specificity of the obtained antibody is strong. The purified antibody could decrease the Il-TdR incorporation of the cultured keratinocytes in a dose-dependent manner. Conclusion: The method of punning AK auto-Ab is simple, practicable and reliable. Natural AK auto-Ab, existing in normal human individuals, has inhibitory etiect on the proliferation of the cultured keratinocytes.展开更多
Ancylostoma anticoagulant peptide 5 (AcAP5) is a strong inhibitor of human coagulation factor Xa (FXa). The N-terminal residues (N40) of AcAP5 contains a domain that could combine with FXa. In order to determine...Ancylostoma anticoagulant peptide 5 (AcAP5) is a strong inhibitor of human coagulation factor Xa (FXa). The N-terminal residues (N40) of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 (DE3). Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside (IPTG), and the interest protein was identified by SDS-PAGE and purified using one-step nickel (Ni) affinity chromatography. Under the optimal expres- sion condition (0.05 mM IPTG for 6 h at 37 ℃), the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.展开更多
A series of urea-based compounds containing purine moieties were designed and synthesized as novel non-ATP competitive CHK1 inhibitors. The biological evaluation showed that several target compounds exhibited more pot...A series of urea-based compounds containing purine moieties were designed and synthesized as novel non-ATP competitive CHK1 inhibitors. The biological evaluation showed that several target compounds exhibited more potent inhibitory effects against CHK1 than the lead compound. In addition, one particular compound displayed synergistic effects with gemcitabine against HT29 cells.展开更多
Glucocorticoid(GC) plays an important role in anti-inflammatory,anti-allergic effects and immunosuppression,and has become a widely used drug in clinical departments.However,GC also produces a number of serious side e...Glucocorticoid(GC) plays an important role in anti-inflammatory,anti-allergic effects and immunosuppression,and has become a widely used drug in clinical departments.However,GC also produces a number of serious side effects at the same time.After GC acting on human body,the syndrome change has some regular pattern and it can be treated on the basis of syndrome differentiation and stage to aim at further improving therapeutic efficacy.The Chinese medicine can reduce the side effects of GC when treating the primary disease,thus plays a role in Synergism and Detoxification.展开更多
文摘AIM: Different strains of bifidobacteria were analysed for their effects on HT-29 intestinal epithelial cells (IECs) in in vitro models both of the non-inflamed and inflamed intestinal epithelium. METHODS: A reporter gene system in HT-29 cells was used to measure levels of NF-KB activation after challenge with bifidobacteria or after bacterial pre-treatment following LPS challenge. IL-8 protein and pro-inflammatory gene expression was investigated using normal HT-29 cells. RESULTS: None of the bifidobacteria tested induced activation of nuclear factor κB (NF-κB) indicating that bifidobacteria themselves do not induce inflammatory events in IECs. However, six out of eight bifidobacteria tested inhibited lipopolysaccharide- (LPS-) induced NF-κB activation in a dose- and strain-dependent manner. In contrast, NF-κB activation in response to challenge with tumor necrosis factor-α (TNF-α) was affected by none of the tested bifidobacteria, indicating that the inhibitory effect of bifidobacteria is specific for LPS-induced inflammation in IECs. As shown with two of the six inhibitionpositive bifidobacteria, LPS-induced inhibition of NF-κB activation was accompanied by a dose-dependent decrease of interleukin 8 (IL-8) secretion and by lower mRNA levels for IL-8, TNF-α, cyclooxygenase 2 (Cox-2), and intercellular adhesion molecule 1(ICAM-1). CONCLUSION: Some strains of bifidobacteria are effective in inhibiting LPS-induced inflammation and thus might be appropriate candidates for probiotic intervention in chronic intestinal inflammation.
基金supported by the Grant No.2003AA208215 from the National High Technology Programs of Chinathe Grant No.30270311 from the National Natural Science Foundation of China.
文摘RNA interference (RNAi) is triggered by the presence of a double-stranded RNA (dsRNA), and results in the silencing of homologous gene expression through the specific degradation of an mRNA containing the same sequence. dsRNAmediated RNAi can be used in a wide variety of eucaryotes to induce the sequence-specific inhibition of gene expression.Synthetic 21-23 nucleotide (nt) small interfering RNA (siRNA) with 2 nt 3' overhangs was recently found to mediate efficient sequence-specific mRNA degradation in mammalian cells. Here, we studied the effects of synthetic siRNA duplexes targeted to SARS coronavirus structural proteins E, M, and N in a cell culture system. Among total 26 siRNA duplexes, we obtained 3 siRNA duplexes which could sequence-specifically reduce target genes expression over 80% at the concentration of 60 nM in Vero E6 cells. The downregulation effect was in correlation with the concentrations of the siRNA duplexes in a range of 0~60 nM. Our results also showed that many inactive siRNA duplexes may be brought to life simply by unpairing the 5' end of the antisense strands. Results suggest that siRNA is capable of inhibiting SARS coronavirus genes expression and thus may be a new therapeutic strategy for treatment of SARS.
基金Inserm, France Université Louis Pasteur, France+3 种基金the European Union (Virgil Network of Excellence)the DeutscheForschungsgemeinschaft (Ba1417/11-1), Germanythe ANRchair of excellence program and ANRS, FranceInserm "PosteVert" research fellowship in the framework of Inserm EuropeanAssociated Laboratory Inserm U748-Department of Medicine Ⅱ,University of Freiburg, Germany
文摘Hepatitis C virus (HCV) is a major cause of hepatitis world-wide. The majority of infected individuals develop chronic hepatitis which can then progress to liver cirrhosis and hepatocellular carcinoma. Spontaneous viral clearance occurs in about 20%-30% of acutely infected individuals and results in resolution of infection without sequaelae. Both viral and host factors appear to play an important role for resolution of acute infection. A large body of evidence suggests that a strong, multispecific and long-lasting cellular immune response appears to be important for control of viral infection in acute hepatitis C. Due too the lack of convenient neutralization assays, the impact of neutralizing responses for control of viral infection had been less defined. In recent years, the development of robust tissue culture model systems for HCV entry and infection has finally allowed study of antibody-mediated neutralization and to gain further insights into viral targets of host neutralizing responses. In addition, detailed analysis of antibody-mediated neutralization in individual patients as well as cohorts with well defined viral isolates has enabled the study of neutralizing responses in the course of HCV infection and characterization of the impact of neutralizing antibodiesfor control of viral infection. This review will summarize recent progress in the understanding of the molecular mechanisms of antibody-mediated neutralization and its impact for HCV pathogenesis.
基金the National Natural Science Foundation of China(30660146)the Ministry of Sciences of Technology Program of China(2003CCA02600)
文摘A novel Eucommia antifungal peptide, named EAFP3, was isolated from the bark of Eucommia ul- moides by NaC1 extract, gel filtration and reverse phase high performance liquid chromatography. The molecular mass of EAFP3 is 4157.3 Da, and its partial amino acid sequence is -. LYQQLIAGITLNK.-. EAFP3 exerts an inhibitory activity against Candida albicans in vitro and the drug concentration required for 50% growth inhibition (IC50) is 31.25μg/mL.
基金the Main Project of Ministry of Education of China (No. 207150)
文摘Objective: To construct the small interfering RNA (siRNA) expression vector of carcino-embryonic antigen (CEA) and inhibit the expression of CEA in EC9706 cells by RNA interference. Methods: Two pairs of oligonucleotide sequences were designed and synthesized according to the encoding sequence of mRNA of CEA. The annealed oligonucleotide frag-ments were cloned into pRNAT-U6.2 expression vector and identified by sequencing. The recombinant plasmid pRNAT-U6.2-CEA was transfected into EC9706 cells. The expression of CEA in the stable transfected cells was assayed by real time PCR and Western blot. Results: DNA sequencing showed that the oligonucleotide fragments were correctly inserted into pRNAT-U6.2 vector, and CEA expression in the transfected cells was down-regulated significantly by pRNAT-U6.2-CEA at both the mRNA and protein levels. Conclusion: The siRNA expression vector of CEA is successfully constructed and inhibits CEA expression in EC9706 cells. This facilitates further studies of the function of CEA at the molecular level.
文摘To investigate into the mechanisms underlying the irreversible sterility induced by gossypol, we studied the relationship between its inhibitory action on acrosomal enzymes and its antifertility effect.As shown by our result, after exposure to gossypol (l.25-60 μg/ml) for 15 min. in vitro,the sperms' ability to penetrate bovine cervical mucus and the fertility rate were significantly reduced. Also, following administration of gossypol (12.5 mg/kg/day) for six weeks, the rate of fertilization in vitro by hamster sperm was significantly decreased. In the gossypol-treated group, extracts of testis sperm delayed dispersion of cumulus cells, suggesting inhibition of hyaluronidase and other acrosomal enzymes. Furthermore, the acrosin and arylsulfatase activities were shown to be markedly inhibited. Thus, a parallelism was displayed between the reduction of fertility and the decreasc in acrosin and arylsulfatase activities in epididymis sperms.Besides, the inhibition was reversible and was dosage-and durationdependent. In conclusion, the assay of acrosin activity might serve as a useful tool for monitoring the irreversible sterility induced by gossypol,
文摘Objective: To purify the natural antikeratin autoantibody (AK auto-Ab) and observe its effects on the prolif eration of the cultured keratinocytes. Methods: Natural AK auto-Ab was purified by using keratin affinity column, and then the titre and specificity of the Abs were studied by ELISA, immunoperoxidase staining and immuno-electronicmicroscope. The effect of the purified Abs on the cultured keratino-cytes was assayed by 3H-TdR incorporation. Results: Natural AK auto-Ab was obtained. The binding activity of IgG AK auto-Ab in purified Ah remained similar to that in pooled human sera. and the specificity of the obtained antibody is strong. The purified antibody could decrease the Il-TdR incorporation of the cultured keratinocytes in a dose-dependent manner. Conclusion: The method of punning AK auto-Ab is simple, practicable and reliable. Natural AK auto-Ab, existing in normal human individuals, has inhibitory etiect on the proliferation of the cultured keratinocytes.
基金National Technology Graveness Special Purpose Fund (Grant No. 2009zx09301-010)
文摘Ancylostoma anticoagulant peptide 5 (AcAP5) is a strong inhibitor of human coagulation factor Xa (FXa). The N-terminal residues (N40) of AcAP5 contains a domain that could combine with FXa. In order to determine whether N40 protein has FXa inhibitory effect, we cloned, expressed and purified the protein for activity evaluation. The DNA fragment coding N40 was amplified by PCR, cloned into pET-30a to construct recombinant plasmid pET30a-N40, and subsequently transformed into E. coli, BL21 (DE3). Expression of N40 was induced by isopropyl ~3-D-l-thiogalactopyranoside (IPTG), and the interest protein was identified by SDS-PAGE and purified using one-step nickel (Ni) affinity chromatography. Under the optimal expres- sion condition (0.05 mM IPTG for 6 h at 37 ℃), the purity of N40 reached 90%. We also evaluated the inhibition activity of N40 protein on FXa, finding the ICso was 4.58× 10 5 mol/L, This study suggests the N40 of AcAP5 could combine with FXa to inhibit FXa activity.
基金National Natural Science Foundation of China(Grant No.21302007)
文摘A series of urea-based compounds containing purine moieties were designed and synthesized as novel non-ATP competitive CHK1 inhibitors. The biological evaluation showed that several target compounds exhibited more potent inhibitory effects against CHK1 than the lead compound. In addition, one particular compound displayed synergistic effects with gemcitabine against HT29 cells.
基金Supported by Projects in the National Science & Technology Pillar Program in the Eleventh Five-year Plan Period (No. 2006BA104A10)State Traditional Chinese Medicine Department Research Foundation (No. 200707016)National Natural Science Foundation of China (No. 81072764)
文摘Glucocorticoid(GC) plays an important role in anti-inflammatory,anti-allergic effects and immunosuppression,and has become a widely used drug in clinical departments.However,GC also produces a number of serious side effects at the same time.After GC acting on human body,the syndrome change has some regular pattern and it can be treated on the basis of syndrome differentiation and stage to aim at further improving therapeutic efficacy.The Chinese medicine can reduce the side effects of GC when treating the primary disease,thus plays a role in Synergism and Detoxification.