分别采用邻苯三酚-鲁米诺化学发光体系、硫酸亚铁-鲁米诺-过氧化氢化学发光体系和过氧化氢-鲁米诺化学发光体系测定白腊、妃子笑、糯米糍和怀枝4个品种的荔枝皮原花青素(procyanidins of litchi pericarp,LPPC)体外清除超氧阴离子自由...分别采用邻苯三酚-鲁米诺化学发光体系、硫酸亚铁-鲁米诺-过氧化氢化学发光体系和过氧化氢-鲁米诺化学发光体系测定白腊、妃子笑、糯米糍和怀枝4个品种的荔枝皮原花青素(procyanidins of litchi pericarp,LPPC)体外清除超氧阴离子自由基、羟自由基和过氧化氢的能力。同时运用硫酸铜-邻菲啰啉-抗坏血酸-过氧化氢-DNA体系测定不同LPPC对诱导体系DNA损伤的保护作用。实验结果表明:白腊LPPC具有较好的体外清除超氧阴离子自由基和过氧化氢的能力;妃子笑呈现出较显著的羟自由基清除能力和保护DNA损伤的效果。不同品种的LPPC在不同化学发光体系中的抗氧化活性呈现较大差异。展开更多
研究了化学发光法测定山楂果肉原花青素(procyanidins of Hawthorn Sarcocarp简称HSPC)的体外抗氧化活性及其对DNA损伤的保护作用。运用邻苯三酚-鲁米诺化学发光体系测定了HSPC对超氧阴离子的清除作用,硫酸铜-邻菲啰啉-抗坏血酸-双氧水...研究了化学发光法测定山楂果肉原花青素(procyanidins of Hawthorn Sarcocarp简称HSPC)的体外抗氧化活性及其对DNA损伤的保护作用。运用邻苯三酚-鲁米诺化学发光体系测定了HSPC对超氧阴离子的清除作用,硫酸铜-邻菲啰啉-抗坏血酸-双氧水体系测定了HSPC对羟基自由基的清除作用,双氧水-鲁米诺体系测定了HSPC对体外双氧水的清除作用,采用硫酸铜-邻菲啰啉-抗坏血酸-双氧水-脱氧核糖核酸测定了HSPC对体外DNA损伤的保护作用。结果表明:当HSPC浓度为1mg/mL时,对超氧阴离子抑制率达87.7%,其半数抑制率浓度IC50值为130.8μg/mL;100μg/mL时对羟基自由基抑制率达91.7%,IC50值为61.73μg/mL;8μg/mL时对双氧水抑制率达91.6%,IC50值为0.19μg/mL;40μg/mL时对体外DNA损伤抑制率达80.7%,IC50值为22.16μg/mL。表明HSPC具有很好的体外清除活性氧和保护DNA损伤的活性。展开更多
采用四种化学发光体系研究艾蒿黄酮(flavonoids of Artemisia argyi Lé vl.et Vant,简称FAA)的体外抗氧化活性,及对DNA氧化损伤的保护作用。结果表明,艾蒿黄酮能有效地清除O2-·、·OH、H2O2、减轻或消除·OH对DNA的...采用四种化学发光体系研究艾蒿黄酮(flavonoids of Artemisia argyi Lé vl.et Vant,简称FAA)的体外抗氧化活性,及对DNA氧化损伤的保护作用。结果表明,艾蒿黄酮能有效地清除O2-·、·OH、H2O2、减轻或消除·OH对DNA的氧化损伤。艾蒿黄酮50%抑制浓度(IC50)分别为151.17、15.34、0.23、15.46mg/L。实验同时还比较了艾蒿黄酮和VC的抗氧化作用,结果表明,艾蒿黄酮抗氧化效应远远高于VC的。艾蒿黄酮是有效的、多功能的天然抗氧化剂和自由基清除剂,是值得进一步开发利用的天然产物。展开更多
AIM： To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus （DHBV） infection. METHODS： DuIFN-γ cDNA was cloned and expressed in COS-γ...AIM： To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus （DHBV） infection. METHODS： DuIFN-γ cDNA was cloned and expressed in COS-γ cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies, Ducks were vaccinated with DHBpreS/S DNA alone or coimmunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells （PBMCs） from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay （EUSA）. DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS： DuIFN-γ expressed by COS-γ was able to protect duck fibroblasts against vesicular stomatitis virus （VSV） infection in a dose-dependent fashion, and anti DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups.展开更多
Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-...Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.展开更多
Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune res...Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.展开更多
Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。...Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H2O2(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。展开更多
文摘分别采用邻苯三酚-鲁米诺化学发光体系、硫酸亚铁-鲁米诺-过氧化氢化学发光体系和过氧化氢-鲁米诺化学发光体系测定白腊、妃子笑、糯米糍和怀枝4个品种的荔枝皮原花青素(procyanidins of litchi pericarp,LPPC)体外清除超氧阴离子自由基、羟自由基和过氧化氢的能力。同时运用硫酸铜-邻菲啰啉-抗坏血酸-过氧化氢-DNA体系测定不同LPPC对诱导体系DNA损伤的保护作用。实验结果表明:白腊LPPC具有较好的体外清除超氧阴离子自由基和过氧化氢的能力;妃子笑呈现出较显著的羟自由基清除能力和保护DNA损伤的效果。不同品种的LPPC在不同化学发光体系中的抗氧化活性呈现较大差异。
文摘研究了化学发光法测定山楂果肉原花青素(procyanidins of Hawthorn Sarcocarp简称HSPC)的体外抗氧化活性及其对DNA损伤的保护作用。运用邻苯三酚-鲁米诺化学发光体系测定了HSPC对超氧阴离子的清除作用,硫酸铜-邻菲啰啉-抗坏血酸-双氧水体系测定了HSPC对羟基自由基的清除作用,双氧水-鲁米诺体系测定了HSPC对体外双氧水的清除作用,采用硫酸铜-邻菲啰啉-抗坏血酸-双氧水-脱氧核糖核酸测定了HSPC对体外DNA损伤的保护作用。结果表明:当HSPC浓度为1mg/mL时,对超氧阴离子抑制率达87.7%,其半数抑制率浓度IC50值为130.8μg/mL;100μg/mL时对羟基自由基抑制率达91.7%,IC50值为61.73μg/mL;8μg/mL时对双氧水抑制率达91.6%,IC50值为0.19μg/mL;40μg/mL时对体外DNA损伤抑制率达80.7%,IC50值为22.16μg/mL。表明HSPC具有很好的体外清除活性氧和保护DNA损伤的活性。
文摘采用四种化学发光体系研究艾蒿黄酮(flavonoids of Artemisia argyi Lé vl.et Vant,简称FAA)的体外抗氧化活性,及对DNA氧化损伤的保护作用。结果表明,艾蒿黄酮能有效地清除O2-·、·OH、H2O2、减轻或消除·OH对DNA的氧化损伤。艾蒿黄酮50%抑制浓度(IC50)分别为151.17、15.34、0.23、15.46mg/L。实验同时还比较了艾蒿黄酮和VC的抗氧化作用,结果表明,艾蒿黄酮抗氧化效应远远高于VC的。艾蒿黄酮是有效的、多功能的天然抗氧化剂和自由基清除剂,是值得进一步开发利用的天然产物。
基金Supported by the Major State Basic Research Development Program of China, 973 Program, No. G2002CB512803the National Natural Science Foundation of China, No. 30070693the Science and Technology Foundation of Shanghai, No. 02DJ14002
文摘AIM： To detect the effects of DNA vaccines in combination with duck IFN-γ gene on the protection of ducks against duck hepatitis B virus （DHBV） infection. METHODS： DuIFN-γ cDNA was cloned and expressed in COS-γ cells, and the antiviral activity of DuIFN-γ was detected and neutralized by specific antibodies, Ducks were vaccinated with DHBpreS/S DNA alone or coimmunized with plasmid expressing DuIFN-γ. DuIFN-γ mRNA in peripheral blood mononuclear cells （PBMCs） from immunized ducks was detected by semi-quantitative competitive RT-PCR. Anti-DHBpreS was titrated by enzyme-linked immunosorbent assay （EUSA）. DHBV DNA in sera and liver was detected by Southern blot hybridization, after ducks were challenged with high doses of DHBV. RESULTS： DuIFN-γ expressed by COS-γ was able to protect duck fibroblasts against vesicular stomatitis virus （VSV） infection in a dose-dependent fashion, and anti DuIFN-γ antibodies neutralized the antiviral effects. DuIFN-γ in the supernatant also inhibited the release of DHBV DNA from LMH-D2 cells. When ducks were co-immunized with DNA vaccine expressing DHBpreS/S and DuIFN-γ gene as an adjuvant, the level of DuIFN-γ mRNA in PBMCs was higher than that in ducks vaccinated with DHBpreS/S DNA alone. However, the titer of anti-DHBpreS elicited by DHBpreS/S DNA alone was higher than that co-immunized with DuIFN-γ gene and DHBpreS/S DNA. After being challenged with DHBV at high doses, the load of DHBV in sera dropped faster, and the amount of total DNA and cccDNA in the liver decreased more significantly in the group of ducks co-immunized with DuIFN-γ gene and DHBpreS/S DNA than in other groups.
文摘Single-strand breaks （SSBs） can occur in cells either directly, or indirectly following initiation of base excision repair （BER）. SSBs generally have blocked termini lacking the conventional 5＇-phosphate and 3＇-hydroxyl groups and require further processing prior to DNA synthesis and ligation. XRCC1 is devoid of any known enzymatic activity, but it can physically interact with other proteins involved in all stages of the overlapping SSB repair and BER pathways, including those that conduct the rate-limiting end-tailoring, and in many cases can stimulate their enzymatic activities. XRCC1^-/- mouse fibroblasts are most hypersensitive to agents that produce DNA lesions repaired by monofunctional glycosylase-initiated BER and that result in formation of indirect SSBs. A requirement for the deoxyribose phosphate lyase activity of DNA polymerase β （pol β） is specific to this pathway, whereas pol β is implicated in gap-filling during repair of many types of SSBs. Elevated levels of strand breaks, and diminished repair, have been demonstrated in MMS- treated XRCC1^-/-, and to a lesser extent in pol β^-/- cell lines, compared with wild-type cells. Thus a strong correlation is observed between cellular sensitivity to MMS and the ability of cells to repair MMS-induced damage. Exposure of wild-type and polβ^-/- cells to an inhibitor of PARP activity dramatically potentiates MMS-induced cytotoxicity. XRCC1^-/- cells are also sensitized by PARP inhibition demonstrating that PARP-mediated poly（ADP-ribosyl）ation plays a role in modulation of cytotoxicity beyond recruitment of XRCC 1 to sites of DNA damage.
基金supported by the Innovation Platform Open Fund of Hunan Provincial Education Department (11K010)a research fund from Hunan Provincial Science and Technology Development (2008TP4033-2)
文摘Human cytomegalovirus virions contain three major glycoprotein complexes (gC I, II, III), all of which are required for CMV infectivity. These complexes also represent major antigenic targets for anti-viral immune responses. The gC II complex consists of two glycoproteins, gM and gN. In the current study, DNA vaccines expressing the murine cytomegalovirus (MCMV) homologs of the gM and gN proteins were evaluated for protection against lethal MCMV infection in a mouse model. Humoral and cellular immune responses, spleen viral titers, and mice survival and body-weight changes were examined. The results showed that immunization with gM or gN DNA vaccine alone was not able to offer good protection, whereas co-immunization with both gM and gN induced an effective neutralizing antibody response and cellular immune response, and provided mice with complete protection against a lethal MCMV challenge. This study provides the first in vivo evidence that the gC II (gM-gN) complex may be able to serve as a protective subunit antigen for future HCMV vaccine development.
文摘Dps(DNAprotection during starvation)蛋白是原核生物中特有的一类具有铁离子结合和抗氧化损伤功能的重要蛋白。利用体外PCR扩增技术和体内同源重组方法,获得了耐辐射奇球菌(Deinococcus radiodurans)dps全基因(DRB0092)缺失突变株。对突变株和野生型分别进行不同浓度过氧化氢(H2O2)处理,结果表明:与野生型菌株R1相比,dps突变株在低浓度H2O2(≤10mmol/L)条件下存活率急剧下降,而高浓度(≥30mmol/L)下则完全致死。Native-PAGE活性染色结果显示,稳定生长期dps突变株体内两种过氧化氢酶(KatA和KatB)的活性较野生型R1分别上调2.3倍和2.6倍。通过质粒构建和大肠杆菌诱导表达,获得可溶性Dps蛋白。体外结合和DNA保护实验结果显示:Dps具有明显的DNA结合功能,并能保护质粒DNA免受羟自由基攻击。本研究证明,Dps蛋白在耐辐射奇球菌抗氧化体系中发挥重要作用,可能对该菌极端抗性机制有重要贡献。
