A mulfistep conversion system composed of phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） was used to synthesize methylcatechols and semialdehydes from o- and m-cresol for the firs...A mulfistep conversion system composed of phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） was used to synthesize methylcatechols and semialdehydes from o- and m-cresol for the first time. Docking studies displayed by PyMOL predicted that cresols and methylcatechols could be theoretically transformed by this multistep conversion system~ High performance liquid chromatography mass spectrometry （HPLC-MS） analysis also indicated that the products formed from multistep conversion were the corresponding 3-methylcatechol, 4-methylcatechol, 2- hydroxy-3-methyl-6-oxohexa-2,4-dienoic acid （2- hydroxy-3-methyl-ODA） and 2-hydroxy-5-methyl-6-oxo- hexa-2,4-dienoic acid （2-hydroxy-5-methyl-ODA）. The optimal cell concentrations of the recombinant E. coli strain BL21 （DE3） expressing phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） and pH for the multistep conversion of o- and m-cresol were 4.0 （g-L-1 cell dry weight） and pH 8.0, respectively. For the first step conversion, the formation rate of 3- methylcatechol （0.29μmol·L-1·min-1·mg-1cell dry weight） from o-cresol was similarly with that ofmethylca- techols （0.28 μmol·L-1·min-1·mg-1 cell dry weight） from m-cresol by strain PHrND. For the second step conversion, strain BphCLA_4 showed higher formation rate （0.83 μmol·L-1·min-1·mg-1 cell dry weight） for 2-hydroxy-3-methyl- ODA and 2-hydroxy-5-methyl-ODA from m-cresol, which was 1.1-fold higher than that for 2-hydroxy-3-methyl- ODA （0.77 μmol·L-1·min-1·mg-1. mglcell dry weight） from ocresol. The present study suggested the potential application of the multistep conversion system for the production of chemical synthons and high-value products.展开更多
Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. ...Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. Characterization of the crude C12O showed that the maximum activity was obtained at 40–70°C and pH 7.8–8.8. Metal ions had different influences on the activity of crude C12O. It was suggested that strain QYY possessed an inducible and ferric-dependent C12O. Kinetic studies showed that the value of V max and K m was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C12O was achieved by a HiTrap Q Sepharose column chromatography.展开更多
芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛...芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。展开更多
AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of...AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of 95 patients who received surgical resection from August 2008 to January 2010. All patients were randomly assigned to adjuvant treatment with or without celecoxib groups after surgery. We performed standard immunohistochemistry to assess the expression levels of IDO1/COX2 and evaluated the correlation of IDO1/COX2 with clinicopathological factors and overall survival(OS) outcomes.RESULTS The expression of nuclear IDO1 was significantly correlated with body mass index(P < 0.001), and IDO1 expression displayed no association with sex, age, tumor differentiation, T stage, N stage, carcinoembryonic antigen, cancer antigen 19-9, CD3+ and CD8+ tumor infiltrating lymphocytes, and COX2. In univariate analysis, we found that nuclear IDO1(P = 0.039), nuclear/cytoplasmic IDO1 [hazard ratio(HR) = 2.044, 95% confidence interval(CI): 0.871-4.798, P = 0.039], nuclear IDO1/COX2(HR = 3.048, 95%CI: 0.868-10.7, P = 0.0049) and cytoplasmic IDO1/COX2(HR = 2.109, 95%CI: 0.976-4.558, P = 0.022) all yielded significantly poor OS outcomes. Nuclear IDO1(P = 0.041), nuclear/cytoplasmic IDO1(HR = 3.023, 95%CI: 0.585-15.61, P = 0.041) and cytoplasmic IDO1/COX2(HR = 2.740, 95%CI: 0.764-9.831, P = 0.038) have significantly poor OS outcomes for the CRC celecoxib subgroup. In our multivariate Cox model, high coexpression of cytoplasmic IDO1/COX2 was found to be an independent predictor of poor outcome in CRC(HR = 2.218, 95%CI: 1.011-4.48, P = 0.047) and celecoxib subgroup patients(HR = 3.210, 95%CI: 1.074-9.590, P = 0.037).CONCLUSION Our results showed that cytoplasmic IDO1/COX2 coexpression could be used as an independent poor predictor for OS in CRC.展开更多
Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fet...Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs), and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC func- tions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflam- matory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regu- lated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.展开更多
Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxyge...Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase(IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma(HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immuno-suppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.展开更多
AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRN...AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRNA and FasL mRNA in KC pretreated with IFN-γwere studied with real-time polymerase chain reaction(PCR).The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography.Allogeneic T-cell response was used to confirm the inhibition of KC in vitro.The proliferation of lymphocytes was detected using[ 3 H]thymidine incorporation.Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS:Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ,and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6,which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody.KC expressing IDO could induce allogeneic T-cell apoptosisCONCLUSION:In addition to Fas/FasL pathway,IDO may be another mechanism for KC to induce immune tolerance.展开更多
基金The authors gratefully acknowledge the financial supports from the National Natural Science Foundation of China （Grant Nos. 51078054, 51108120, and 51178139） and the National Creative Research Group from the National Natural Science Foundation of China （No. 51121062）.
文摘A mulfistep conversion system composed of phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） was used to synthesize methylcatechols and semialdehydes from o- and m-cresol for the first time. Docking studies displayed by PyMOL predicted that cresols and methylcatechols could be theoretically transformed by this multistep conversion system~ High performance liquid chromatography mass spectrometry （HPLC-MS） analysis also indicated that the products formed from multistep conversion were the corresponding 3-methylcatechol, 4-methylcatechol, 2- hydroxy-3-methyl-6-oxohexa-2,4-dienoic acid （2- hydroxy-3-methyl-ODA） and 2-hydroxy-5-methyl-6-oxo- hexa-2,4-dienoic acid （2-hydroxy-5-methyl-ODA）. The optimal cell concentrations of the recombinant E. coli strain BL21 （DE3） expressing phenol hydroxylase （PHrND） and 2,3-dihydroxy-biphenyl 1,2-dioxygenase （BphCLA_4） and pH for the multistep conversion of o- and m-cresol were 4.0 （g-L-1 cell dry weight） and pH 8.0, respectively. For the first step conversion, the formation rate of 3- methylcatechol （0.29μmol·L-1·min-1·mg-1cell dry weight） from o-cresol was similarly with that ofmethylca- techols （0.28 μmol·L-1·min-1·mg-1 cell dry weight） from m-cresol by strain PHrND. For the second step conversion, strain BphCLA_4 showed higher formation rate （0.83 μmol·L-1·min-1·mg-1 cell dry weight） for 2-hydroxy-3-methyl- ODA and 2-hydroxy-5-methyl-ODA from m-cresol, which was 1.1-fold higher than that for 2-hydroxy-3-methyl- ODA （0.77 μmol·L-1·min-1·mg-1. mglcell dry weight） from ocresol. The present study suggested the potential application of the multistep conversion system for the production of chemical synthons and high-value products.
基金Supported by the National Natural Science Foundation of China (Grant No. 50608011)the 39th Postdoctoral Funds of China (Grant No. 20060390983)
文摘Sphingomonas xenophaga QYY, capable of growing significantly on more than ten kinds of aromatic compounds as sole carbon source, was used to study characterization of catechol 1,2-dioxygenase (C12O) in cell extracts. Characterization of the crude C12O showed that the maximum activity was obtained at 40–70°C and pH 7.8–8.8. Metal ions had different influences on the activity of crude C12O. It was suggested that strain QYY possessed an inducible and ferric-dependent C12O. Kinetic studies showed that the value of V max and K m was 0.25 μmol catechol/L/mg protein/min and 52.85 μmol/L, respectively. In addition, the partial purification of C12O was achieved by a HiTrap Q Sepharose column chromatography.
文摘芝麻是八大类食物过敏原之一,快速准确识别芝麻过敏原对预防其过敏有重要意义。核酸适配体可以高效识别靶标过敏原,在过敏原检测中有良好的应用前景。为了获得芝麻主要过敏原Ses i 2的特异性核酸适体,本研究以Ses i 2为靶标,通过磁珠筛选法(磁珠-SELEX)开展10轮筛选,经由高通量测序获得6条候补序列(S1~S6),并进行家族性、同源性分析及二级结构预测。结果表明,6条候选核酸适体的重复率可达46.38%,其自由能在-9.02到-2.47 kcal·moL^(-1)之间,根据自由能能量稳定原则,S1和S5吉布斯自由能最低最稳定,分别为-6.70和-9.02 kcal·moL^(-1)。利用ELISA试验进行亲和力测试,结果表明核酸适体S1和S2的亲和能力较强,S1:KD=67.02 nmol·L^(-1),R2=0.925 8,S2:KD=97.65 nmol·L^(-1),R2=0.795 1。核酸适体S1与过敏原Ses i 2的结合力和其他过敏原蛋白相比有显著差异,可视为具有特异性。本研究最终获得一条兼具良好亲和力和特异性的核酸适体S1,为芝麻过敏原快速检测提供了技术支撑。
基金Supported by the National Natural Science Foundation of China,No.81502459
文摘AIM To evaluate indoleamine-2,3-dioxygenase 1/cyclooxygenase 2(IDO1/COX2) expression as an independent prognostic biomarker for colorectal cancer(CRC) patients.METHODS We retrospectively studied the medical records of 95 patients who received surgical resection from August 2008 to January 2010. All patients were randomly assigned to adjuvant treatment with or without celecoxib groups after surgery. We performed standard immunohistochemistry to assess the expression levels of IDO1/COX2 and evaluated the correlation of IDO1/COX2 with clinicopathological factors and overall survival(OS) outcomes.RESULTS The expression of nuclear IDO1 was significantly correlated with body mass index(P < 0.001), and IDO1 expression displayed no association with sex, age, tumor differentiation, T stage, N stage, carcinoembryonic antigen, cancer antigen 19-9, CD3+ and CD8+ tumor infiltrating lymphocytes, and COX2. In univariate analysis, we found that nuclear IDO1(P = 0.039), nuclear/cytoplasmic IDO1 [hazard ratio(HR) = 2.044, 95% confidence interval(CI): 0.871-4.798, P = 0.039], nuclear IDO1/COX2(HR = 3.048, 95%CI: 0.868-10.7, P = 0.0049) and cytoplasmic IDO1/COX2(HR = 2.109, 95%CI: 0.976-4.558, P = 0.022) all yielded significantly poor OS outcomes. Nuclear IDO1(P = 0.041), nuclear/cytoplasmic IDO1(HR = 3.023, 95%CI: 0.585-15.61, P = 0.041) and cytoplasmic IDO1/COX2(HR = 2.740, 95%CI: 0.764-9.831, P = 0.038) have significantly poor OS outcomes for the CRC celecoxib subgroup. In our multivariate Cox model, high coexpression of cytoplasmic IDO1/COX2 was found to be an independent predictor of poor outcome in CRC(HR = 2.218, 95%CI: 1.011-4.48, P = 0.047) and celecoxib subgroup patients(HR = 3.210, 95%CI: 1.074-9.590, P = 0.037).CONCLUSION Our results showed that cytoplasmic IDO1/COX2 coexpression could be used as an independent poor predictor for OS in CRC.
文摘Indoleamine 2, 3-dioxygenase (IDO) is a rate-limiting enzyme for the tryptophan catabolism. In human and murine cells, IDO inhibits antigen-specific T cell proliferation in vitro and suppresses T cell responses to fetal alloantigens during murine pregnancy. In mice, IDO expression is an inducible feature of specific subsets of dendritic cells (DCs), and is important for T cell regulatory properties. However, the effect of IDO and tryptophan deprivation on DC func- tions remains unknown. We report here that when tryptophan utilization was prevented by a pharmacological inhibitor of IDO, 1-methyl tryptophan (1MT), DC activation induced by pathogenic stimulus lipopolysaccharide (LPS) or inflam- matory cytokine TNF-α was inhibited both phenotypically and functionally. Such an effect was less remarkable when DC was stimulated by a physiological stimulus, CD40 ligand. Tryptophan deprivation during DC activation also regu- lated the expression of CCR5 and CXCR4, as well as DC responsiveness to chemokines. These results suggest that tryptophan usage in the microenvironment is essential for DC maturation, and may also play a role in the regulation of DC migratory behaviors.
文摘Tumor cells induce an immunosuppressive microen-vironment which leads towards tumor immune escape. Understanding the intricacy of immunomodulation by tumor cells is essential for immunotherapy. Indoleamine 2,3-dioxygenase(IDO) is an immunosuppressive enzyme which mediates tumor immune escape in various cancers including hepatocellular carcinoma(HCC). IDO up-regulation in HCC may lead to recruitment of regulatory T-cells into tumor microenvironment and therefore inhibit local immune responses and promote metastasis. HCC associated fibroblasts stimulate natural killer cells dysfunction through prostaglandin E2 and subsequently IDO promotes favorable condition for tumor metastasis. IDO up-regulation induces immuno-suppression and may enhance the risk of hepatitis C virus and hepatitis B virus induced HCC. Therefore, IDO inhibitors as adjuvant therapeutic agents may have clinical implications in HCC. This review proposes future prospects of IDO not only as a therapeutic target but also as a prognostic marker for HCC.
基金Supported by Natural Science Foundation of Fujian Province,No.2007J0073Young Talents Innovation Foundation of Fujian Province,No.2006F3033
文摘AIM:To explore the possibility and mechanism of inhibiting allogeneic T-cell responses by Kupffer cells (KC)pretreated with interferon-γ(IFN-γ)in vitro. METHODS:The expressions of indoleamine 2,3-dioxygenase(IDO)mRNA and FasL mRNA in KC pretreated with IFN-γwere studied with real-time polymerase chain reaction(PCR).The catabolism of tryptophan by IDO from KC was analyzed by high performance liquid chromatography.Allogeneic T-cell response was used to confirm the inhibition of KC in vitro.The proliferation of lymphocytes was detected using[ 3 H]thymidine incorporation.Cell cycle and lymphocyte apoptosis were evaluated by flow cytometric assay. RESULTS:Real-time PCR revealed IDO mRNA and FasL mRNA expressions in KC pretreated with IFN-γ,and IDO catabolic effect was confirmed by a decrease in tryptophan and increase in kynurenine concentration. KC expressing IDO and FasL in BABL/c mice acquired the ability to suppress the proliferation of T-cells from C57BL/6,which could be blocked by addition of 1-methyl-tryptophan and anti-FasL antibody.KC expressing IDO could induce allogeneic T-cell apoptosisCONCLUSION:In addition to Fas/FasL pathway,IDO may be another mechanism for KC to induce immune tolerance.
