三隔镰刀菌大米培养物产生二苯甲醇的研究

<正> T-2毒素是镰刀菌属三隔镰刀菌Fusarium tricinctum(Corda)Sacc.的一种次生代谢产物,隶属单端孢霉烯簇毒素。发霉大米中毒症及豆荚中毒症等均与单端孢霉烯簇毒素有关。张树荣等从真菌培养物M-20中提取T-2毒素成功,我们对其     

镰刀菌毒素的研究 Ⅰ.T-2毒素产生菌株的筛选

<正> 镰刀菌属(Fusarium)是危害性最大的真菌之一,不仅侵染田间作物和库贮谷物,造成经济损失,而且产生毒素,引起人畜镰刀菌毒素中毒。黄曲霉毒素的致癌作用被证实后,更引起世界各国对镰刀菌毒素及其中毒现象的重视(孟昭赫等,1979)。镰刀菌产生的毒素主要是单端孢霉烯族化合物,其中 T-2毒素的毒性最强(Ronald,1981),它能引起兔子皮肤反应和抑制植物种子发芽等生物毒性效应(Burme-istes,1970;Ueno,1971)。国内尚未见人工培养产生 T-2毒素的报告。为给农业、食品卫生和环境保护等方面研究提供科学依据,本文报告 T-2毒素产生菌株的筛选。     

封闭环境气载镰孢菌及其T-2毒素发生规律的研究进展

本文阐述了普遍存在的镰孢菌可增强封闭式环境中群体对疾病的易感性,并通过其次生代谢产物(如T-2毒素)对人和动物造成严重危害及预防和控制气载镰孢菌及T-2毒素危害的相关问题,包括气载真菌和毒素采集、毒素痕量富集技术、不同环境中的镰孢菌变化规律与产毒基因研究现状。提出研究封闭式环境中气载镰孢菌及T-2毒素发生规律研究的必要性。     

利用Fusarium poae制备T-2毒素的培养条件和提取方法

比较2种不同优化方法对分离纯化梨孢镰孢菌(Fusarium poae)产生的T-2毒素的效果,并得到高纯度的T-2毒素,解决国内T-2毒素产业化问题.在优化Fusarium poae产毒培养条件的基础上,对Burmeister的提取方法和Gregory培养基进一步优化以最大限度地提高T-2毒素的产率,从而得到高纯度并且...     

自然环境中T-2毒素降解菌的筛选与鉴定

[目的]在自然环境中筛选和鉴定能够降解T-2毒素的菌株.[方法]取对虾养殖池水样、养殖池沉泥样品和对虾混合饲料中分离到的镰孢菌,于GYM产毒培养基中培养14 d后引入自然条件下气载细菌,继续培养至28 d,采用LC-MS/MS检测其中T-2毒素含量.然后结合稀释涂布、平板划线、革兰氏染色、镜检,从镰孢菌产毒培养液中毒素含量明显降低的菌悬液中筛选出T-2毒素降解菌,用16S rRNA分析方法对其进行系统发育分析及菌种鉴定,并验证两菌株的降毒能力与不同基质中二者的联合降毒能力.[结果]以从对虾养殖环境中分离到的5株镰孢菌作为试验菌种,在其产毒培养过程中分离到2株T-2毒素降解菌,16S rRNA鉴定结果分别为弯曲假单胞菌和尼泊尔葡萄球菌,对T-2毒素的降解率分别为90.9％和85.5％,但两者降毒能力并无显著差异(P＞0.05);其联合作用也有较好降毒效果,与两菌株单独作用无显著差异(P＞0.05),不同基质对其联合降毒作用影响不大(P＞0.05).[结论]新的T-2毒素降解菌的发现为进一步探明T-2毒素降解基因和开发T-2毒素生物降解酶提供了研究基础.     

几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因分析

尖孢镰刀菌在与寄主的相互作用中分泌几个特定的富含半胱氨酸的小分子量蛋白进入木质部中启动致病力,被称为SIX(secreted in xylem)蛋白,为明确其在不同寄主中的作用,本研究比较分析了几种尖孢镰刀菌专化型中SIX1、SIX4、SIX6、SIX8同源基因序列。根据已完成的尖孢镰刀菌古巴专化型1号(Foc1)与4号生理小种(Foc4)全基因组测序序列信息及相关SIX基因序列设计引物,应用PCR方法扩增分析56株尖孢镰刀菌古巴专化型与18株其它专化型及非致病型尖孢镰刀菌菌与其它种或属共21株菌株中的SIX1、SIX4、SIX6、SIX8基因。结果表明:设计的SIX1、SIX4、SIX6、SIX8基因引物均不能从非致病性尖孢镰刀菌与其它镰刀属种或其它属的菌株DNA中扩增出目的条带;SIX1基因的2个引物均能从供试的Foc菌株DNA中扩增出目的条带,同时可从部分其它专化型菌株DNA中扩增出目的条带;SIX4基因引物仅能从供试的尖孢镰刀菌番茄专化型与部分甘蓝专化型菌株DNA中扩增出目的条带;SIX6引物仅能从供试Foc1、Foc2、Foc4菌株DNA中扩增出目的条带;SIX8基因引物能从所有供试的致病尖孢镰刀菌中DNA扩增出目的条带。研究发现的SIX6基因序列提供了快速鉴定尖孢镰刀菌古巴专化型的检测方法,同时为深入研究尖孢镰刀菌各个专化型中SIX基因的功能奠定基础。     

镰刀菌毒素

镰刀菌毒素黄秀琴,吴晶琼（华东师范大学生物系,上海２０００６２）镰孢霉（Ｆｕｓａｒｉｕｍ）也称镰刀菌,它所产生的镰刀菌毒素,不仅能使植物致病,而且还会污染食品和饲料,引起人及动物的中毒甚至死亡,但也有使有害昆虫致死,在生物防治上显示出具有极为重要的作...     

红外-核磁共振光谱-免疫亲和柱法解析梨孢镰孢菌代谢产物成分

利用红外光谱,核磁共振光谱结合免疫亲和柱的方法解析梨孢镰孢菌代谢产物成分,为真菌代谢产物的分析提供新的信息.将F.Poae菌株在GYM培养基上25℃条件下培养12 h后转至8℃培养12h,交替进行4周,将其代谢产物分离纯化、结晶,80℃干燥后用红外光谱议分析产物结构,然后利用免疫亲和柱特异性,比较产物经T-2免疫亲和柱纯化前后的1H核磁谱图.由红外谱图可判断目标组分存在与单端孢霉烯族毒素相同的特征官能团,初步判定产物为单端孢霉烯族毒素.通过1H核磁谱图比较T-2免疫亲和柱纯化前后物质结构一致.梨孢镰孢菌代谢产物成分为T-2毒素.红外-核磁共振光谱结合免疫亲和柱的方法解析梨孢镰孢菌代谢产物的方法在国内外尚未见报道.     

尖孢镰刀菌致病机理和化感作用研究进展

尖孢镰刀菌引起的枯萎病在生产中的防控相当困难。通过总结国内外相关文献,综述近年来有关尖孢镰刀菌致病机理和化感作用的研究进展。尖孢镰刀菌通过分泌毒素和细胞壁降解酶共同致病,谱系特异性区域的存在是其致病性强和宿主范围广的主要原因;在尖孢镰刀菌各专化型中已分离出大量致病相关基因;其他植物和拮抗微生物(木霉菌、丛枝菌根真菌、非致病性尖孢镰刀菌以及植物生长促生菌)可以分泌化感物质,作用于宿主植物和尖孢镰刀菌,直接抑制尖孢镰刀菌的生长或激活宿主植物的防御反应。未来有关尖孢镰刀菌致病机理研究应该在基因组测序基础上构建精细的遗传图谱;对化感作用的研究应当深入探讨分子机理,利用高通量测序等技术在转录组或蛋白组水平上明确宿主植物抗枯萎病相关基因,同时利用分子标记辅助育种来筛选新的抗枯萎病品种。     

小麦赤霉病镰孢菌毒素的生物合成及其分子调控

禾谷镰刀菌是小麦赤霉病的主要致病菌,其真菌次生代谢产生的单端孢霉烯类B型毒素,如雪腐镰刀菌烯醇(nivalenol,NIV)、脱氧雪腐镰刀菌烯醇(deoxynivalenol,DON)和其它乙酰化衍生物等污染小麦籽粒后对人畜健康构成威胁。综述了近年来国内外对小麦赤霉病镰孢菌单端孢霉烯类B型毒素生物合成的主要途径及分子调控研究进展,对毒素合成过程中的重要调控基因如TRI5、TRI7和TRI13在农业中的应用进行了阐述。     

Effects of azathioprine and its metabolites on repair mechanisms of the intestinal epithelium in vitro

Erosions and ulcerations of the intestinal epithelium are hallmarks of inflammatory bowel diseases (IBD). Intestinal epithelial cell migration (restitution) and proliferation are pivotal mechanisms for healing of epithelial defects after mucosal injury. In addition, the rate of apoptosis of epithelial cells may modulate intestinal wound healing. The purine antagonists azathioprine (AZA) and 6-mercaptopurine (6-MP) are widely used drugs in the treatment of IBD. In the present study, the hitherto unknown effects of AZA as well as its metabolites 6-MP and 6-thioguanine (6-TG) on repair mechanisms and apoptosis of intestinal epithelia were analysed. Intestinal epithelial cell lines (human Caco-2, T-84 and HT-29 cells, rat IEC-6 cells) were incubated with AZA, 6-MP or 6-TG for 24 h (final concentrations 0.1-10 microM). Migration of Caco-2 and IEC-6 cells was analysed by in vitro restitution assays. Caco-2 and IEC-6 cell proliferation was evaluated by measurement of [3H]thymidine incorporation into DNA. Apoptosis of Caco-2, T-84, HT-29 and IEC-6 cells was assessed by histone ELISA, 4'6'diamidino-2'phenylindole-dihydrochloride staining as well as flow cytometric analysis of Annexin V/propidium iodide (PI)-stained cells. Cell cycle progression was evaluated by PI staining and flow cytometry. Epithelial restitution was not significantly affected by any of the substances tested. However, proliferation of intestinal epithelial cells was inhibited in a dose-dependent manner (maximal effect 92%) by AZA, 6-MP as well as 6-TG. In HT-29 cells, purine antagonist-effected inhibition of cell proliferation was explained by a cell cycle arrest in the G2 phase. In contrast, AZA, 6-MP and 6-TG induced no cell cycle arrest in Caco-2, T-84 and IEC-6 cells. AZA, 6-MP as well as 6-TG induced apoptosis in the non-transformed IEC-6 cell line but not in human Caco-2, T-84 and HT-29 cells. In summary, AZA and its metabolites exert no significant effect on intestinal epithelial restitution. However, they profoundly inhibit intestinal epithelial cell growth via various mechanisms: they cause a G2 cell cycle arrest in HT-29 cells, induce apoptosis in IEC-6 cells and dose-dependently inhibit intestinal epithelial proliferation.     

Sister chromatid exchanges induced by cyclophosphamide in V79 cells cultured in diffusion chambers in mice

Chinese hamster cells V79 were cultured in diffusion chambers (DC) and implanted into mice. An exponential growth was observed from the 2nd to 4th day after implantation. The maximum growth was reached on the 6th day. After that, cell growth and viable cell counts decreased. Three days after implantation of DC with V79 cells, the hosts received 6 hourly injections of 0.2 ml of 5-bromodeoxyuridine (BUdR) solution at concentrations of 0.125 to 1.0 x 10(-2) M. DC were removed for chromosome and sister-chromatid exchanges (SCE) analyses 24 h after the first BUdR injection. The frequency of metaphases with differentially stained chromatids, with aberrations, and the number of SCE per cell increased with BUdR dose. The frequency of metaphases with differentially stained chromatids was also positively correlated with the duration of BUdR exposure or the number of hourly injections of BUdR-solution. The effects of cyclophosphamide (CY) in V79 cells in DC in mice were studied. Injections of CY at 2.5, 5, 10 and 15 microgram per gram of body weight to the hosts caused an increase in the number of SCE per cell in a linear manner. The results from this study indicate that V79 cells cultured in DC in mice may provide a potential test system for mutagenicity.     

Genotoxicity testing of extracts of a Swedish moist oral snuff

The present study was designed to investigate the potential genotoxicity of aqueous and methylene chloride extracts of Swedish moist oral snuff. The test systems were selected to provide optimal data for the prediction of carcinogenicity in rodents and included assays for the induction of mutation in bacteria, sister-chromatid exchanges (SCE) in human lymphocytes, of chromosome aberrations and gene mutations in V79 Chinese hamster cells and of micronuclei in mouse bone marrow cells. In addition, the methylene chloride extract was tested for the induction of sex-linked recessive lethal mutations in Drosophila melanogaster. The aqueous extract of 'Snus' induced SCE in human lymphocytes and chromosome aberrations in V79 cells, the latter effect being observed both with and without metabolic activation. No induction of point mutations was detected with the Ames test or in V79 cells and the micronucleus test in mice was negative. It was demonstrated that the induction of chromosome aberrations without metabolic activation may be due to a high salt concentration, indicating that the clastogenic agent(s) in this extract required metabolic activation. The methylene chloride extract showed genotoxicity in the Ames test, the SCE test and the chromosome aberration test, whereas no induction of gene mutations in V79 cells was observed. Once again, the results suggested that metabolism is required for genotoxicity. The methylene chloride extract did not cause induction of micronuclei in mice or of sex-linked recessive lethal mutations in Drosophila melanogaster. These combined data on genotoxicity were analyzed using various models for the prediction of carcinogenicity. In a sequential testing model, the probabilities that the aqueous and methylene chloride extracts of 'Snus' are carcinogenic due to a genotoxic mechanism were both predicted to be low. Using carcinogenicity prediction by battery selection (CPBS), the probabilities of the methylene chloride and aqueous extracts being correctly identified as non-carcinogens are 71 and 77%, respectively. Up to date, the CPBS approach has been validated primarily for individual compounds, so some caution should at present be exercised in interpreting the results using this method. Based on these results, the carcinogenic potential of Swedish 'Snus' should be considered to be low, a conclusion in agreement with the low incidence of oral cancer in Sweden compared to other countries.     

Action spectra for killing and mutation of Chinese hamster cells exposed to mid- and near-ultraviolet monochromatic light

We have examined the response of Chinese hamster V79 cells to monochromatic light of selected wavelengths in the mid- to near-UV region, using cell survival and induction of mutants resistant to 6-thioguanine (6-TG) or ouabain (OUA) as end points. As the wavelength increased from 313 to 405 nm, the induction of mutants resistant to 6-TG and to OUA decreased to a greater degree than did cell survival. Cells resistant to OUA were induced with considerably lesser efficiency at wavelengths of 313 and 334 nm than cells resistant to 6-TG. No mutants resistant to either 6-TG or OUA were induced by 405-nm light, and no mutants resistant to OUA were induced by 365-nm light. Thus, cell killing and mutation induction have different action spectra, and furthermore, action spectra for mutation induction at the HGPRT and Na+/K+-ATPase loci are different from each other. These observations imply important differences in the cellular mechanisms, and/or lesions, for cell inactivation, induction of 6-TG and OUA resistance for V79 cells exposed to near-UV monochromatic light.     

绿茶抗氧化剂成分抑制突变作用的初步研究

绿茶水溶性提取物及茶叶中抗氧化剂成份具有明显的抑制AFB_1及Bap诱导的鼠伤寒沙门氏菌回复突变作用。这种抗氧化剂成份还可以抑制AFB_1和Bap诱导的V79细胞基因正向突变,以及AFB_1诱导的V79细胞SCE和染色体畸变。本实验结果提示,绿茶中抗氧化剂成份可能对AFB_1及Bap的致癌性具有抑制作用。本文就茶叶抗氧化剂抑制突变的可能机制进行了初步讨论。     

Mutagenic effects of TPA-induced clastogenic factor in Chinese hamster cells

Previous work in our laboratory has shown that the clastogenic and SCE-inducing effects of 12-O-tetradecanoylphorbol 12-acetate (TPA) are mediated by secondary products formed by the cell in response to the tumor promoter. A low-molecular-weight clastogenic factor (CF) was isolated from the medium of TPA-treated human leukocytes and caused chromosome aberrations and sister-chromatid exchanges (SCE) in fresh cultures not exposed to TPA itself. In the present study, we show that Chinese hamster fibroblasts (V79 cells) also produce CF when exposed to TPA. CF from V79 cells induced SCE not only in hamster cells, but also in human lymphocytes. Vice versa, CF from human leukocyte cultures induced SCE in hamster cells. It also increased the frequency of 6-thioguanine-resistant mutants in this cell system. All cyto- and geno-toxic effects of TPA-induced CF were prevented if the cells were treated with superoxide dismutase before exposure. The lipophilic CF seems to be derived from arachidonic acid of cell membranes released as a consequence of oxidative damage and subsequently degraded to genotoxic aldehydes in an autoxidative process. CF is formed only under culture conditions with low antioxidant content in culture media and sera. This may explain the discordant results obtained by different laboratories with regard to the genotoxic effects of TPA.     

High sensitivity of Chinese hamster epithelial liver cells to toxic analogues of purines

The resistance of Chinese hamster epithelial liver cells (CHEL) and Chinese hamster fibroblasts (V79) towards toxic purine analogues has been determined. The liver cells are more sensitive than fibroblasts to 6-thioguanine (6-TG), 8-azaguanine (8-AZ) and 2,6-diaminopurine (DAP). The hypoxanthine-guanine (HGPRT) and adenine phosphoribosyl transferase (APRT) activities of extracts of CHEL cells were lower than those of corresponding extracts of V79. The level of 5'-nucleotidase was about 5-fold higher in the epithelial cells. It appears that HGPRT and APRT activities of extracts of liver epithelial cells are masked or reduced by 5'-nucleotidase activity and other inhibitors. The significance of these findings is discussed.     

Gamma-tocopherol is less effective than alpha-tocopherol in preventing oxidant-induced sister chromatid exchanges in Chinese hamster V79 cells.

Although alpha-tocopherol (alpha-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, gamma-tocopherol (gamma-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of gamma-TOC and a high alpha-TOC:gamma-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the gamma-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 microM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of gamma-TOC (319.8 +/- 66.2 ng/10(6) cells) into V79 cells compared to alpha-TOC (66.9 +/- 6.4 ng/10(6) cells). Gamma-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, alpha-TOC acetate was partially protective against both genotoxins, whereas alpha-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of gamma-TOC into V79 cells, alpha-TOC but not gamma-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.     

Enhancing effects of cytosine arabinoside on ethyl methanesulfonate-induced 6-thioguanine resistance mutations in Chinese hamster V79 cells

We studied the synergistic enhancement effects of two chemicals which are different in their mechanism of action on DNA in cells. The test chemicals used were ethyl methanesulfonate (EMS) as an alkylating agent and cytosine arabinoside (Ara-C) as an analogue of cytidine. For determination of mutagenesis we measured the induction of resistance to 6-thioguanine (6-TG) in Chinese hamster V79 cells. EMS had a strong mutagenic effect on V79 cells, but for Ara-C the results were less clear. In this study, Ara-C had no detectable effect in inducing mutation up to a concentration of 5 X 10(-4) M. The mutation frequency of combined treatment with EMS and Ara-C was significantly higher than that obtained with EMS alone. These results indicate that Ara-C had an enhancing effect on mutations induced by EMS.     

γ-tocopherol is less effective than α-tocopherol in preventing oxidant-induced sister chromatid exchanges in Chinese hamster V79 cells

Although α-tocopherol (α-TOC) is the most biologically active form of vitamin E and is found at high levels in plasma, γ-tocopherol (γ-TOC) has also been found to be a powerful antioxidant in vitro and constitutes up to 70% of the dietary intake of TOC. Low plasma levels of γ-TOC and a high α-TOC:γ-TOC ratio may be associated with coronary heart disease, suggesting that there may be a positive protective role for the γ-form of TOC. In this study the ability of different forms of vitamin E to protect against sister chromatid exchanges (SCE) induced by either hydrogen peroxide or menadione was investigated. Chinese hamster V79 cells were pre-treated with 10 μM TOC for 24 h, and then challenged with a genotoxin. After a 24 h pre-treatment, there was a greater incorporation of γ-TOC (319.8 ± 66.2 ng/10⁶ cells) into V79 cells compared to α-TOC (66.9 ± 6.4 ng/10⁶ cells). γ-TOC did not protect the cells against SCE induced by either hydrogen peroxide or menadione, α-TOC acetate was partially protective against both genotoxins, whereas α-TOC completely abolished the oxidant induced SCE. These results demonstrate that, despite a greater incorporation of γ-TOC into V79 cells, α-TOC but not γ-TOC was more effective at inhibiting oxidatively-induced SCE in V79 cells.