彗星系统定量检测柠檬醛损伤黄曲霉DNA的研究

理化因素致细胞DNA损伤,彗星测试提供了一个直观的方法。采用新型SCGE图象分析系统(IMI10),将细胞显微分光光度分析与显微成像及图象分析结合,直接检测柠檬醛致黄曲霉核DNA损伤,与国际流行的SCGE图象分析系统相比,具有分析速度快、便于分析,同时具有中英文可切换界面和多格式输出打印特点。该系统使彗星试验的检测时间缩短2/3,并提高了准确性,可实现对活细胞多种结构参数、细胞内分子与膜的变化状况同时进行长时间连续的动态瞬间监测,具有广阔应用前景。     

显微多道分光光度法研究受柠檬醛损伤后黄曲霉细胞形态变化

采用从山苍籽油分离出的柠檬醛作为抗菌药物,产毒和无毒的黄曲霉细胞（Aspergillus flavus cell, AFC）作为药物作用的靶,以显微多道分光光度法和显微图像分析法对被该醛所损伤的AFC进行图像捕获,测定受损伤后细胞内吸收光谱、截面积、周长、长轴长及短轴长所发生的变化。发现在410nm和665nm处产毒AFC存在特征吸收峰,受该醛损伤后产毒和无毒AFC的吸收光谱波峰均发生迁移,且峰面积增大;截面积等4类形态参数的数值随柠檬醛浓度升高而减少;为质膜物理化学及细胞内生理生化指标变化提供了理论依据。表明柠檬醛不仅破坏质膜的选择性通透性,而且使细胞质膜结构改变并进入细胞,与靶分子及靶细胞器作用而引发一系列新的生理生化现象的出现。实现对活态细胞受药物作用后形态及生物大分子动态变化的快速、实时、在位的测定,对抗菌药物作用于细胞并使其发生形态结构及靶分子的变化提供了必要的物理参数,在药物抗菌理论及方法研究上具有重要意义。     

柠檬醛顺反异构体对黄曲霉超微结构及膜功能的影响

研究柠檬醛顺反异构体（香叶醛和橙花醛）抗菌性是阐明该醛抗菌机理核心所在.用酶转化法合成香叶醛和橙花醛后,用液态或气态的异构单体分别对黄曲霉孢子及菌丝体进行毒化.采用透射电镜、多维显微及激光拉曼散射技术对毒化的黄曲霉孢子及菌丝体进行显微结构观察和膜相关参数的测定.结果表明,无论是液体还是气体毒化方式,柠檬醛顺反异构体单独存在时均有抗黄曲霉作用;二者混合物的抗菌总活性与单体相比表现出一定程度协同性;二个异构单体的抗菌作用不仅表现为破坏黄曲霉超微结构,而且还反映在损伤其细胞膜体积调节功能及变形能力.     

柠檬醛胁迫环境下黄曲霉线粒体的畸变

通过对黄曲霉细胞受柠檬醛损伤后线粒体形态畸变的透射电镜观察,发现柠檬醛所产生胁迫环境影响线粒体DNA复制系统,产生增生变异的巨型线粒体而与之应答。丙二醛法测定黄曲霉细胞内自由基,结果表明药物进入细胞后还通过诱发自由基使线粒体损伤,致使氧化还原系统及细胞能量代谢途径受到影响。     

褪黑素对邻苯二甲酸(2-乙基己基)酯致雄性小鼠生殖细胞DNA损伤的拮抗作用

目的探讨邻苯二甲酸(2-乙基己基)酯(DEHP)致小鼠睾丸细胞DNA损伤及褪黑素(MT)对此损伤的拮抗作用。方法将40只CL57BL/6J雄性小鼠随机分为4组,包括对照组、MT组、DEHP组和MT+DEHP联合组。MT采用腹腔注射(剂量为15 mg·kg-1),DEHP灌胃染毒(染毒剂量为1000 mg·kg-1),每天染毒1次,连续30 d。检测睾丸组织中谷胱甘肽过氧化物酶(GSH-Px)、超氧化物歧化酶(SOD)活力和丙二醛(MDA)、8-羟基脱氧鸟嘌呤(8-OHd G)含量。单细胞凝胶电泳(彗星实验)检测睾丸细胞DNA损伤,慧星图像软件测定慧星尾长、慧尾DNA百分含量、尾矩及Olive尾矩。结果与对照组比较,DEHP组小鼠睾丸组织GSH-Px和SOD活力降低,MDA和8-OHd G含量增加,睾丸细胞彗星尾长、彗尾DNA百分含量、尾矩、Olive尾矩均显著增加,差异均有统计学意义(P<0.05);与DEHP组比较,MT+DEHP联合组小鼠睾丸组织GSH-Px和SOD活力升高,MDA和8-OHd G含量降低,睾丸细胞DNA损伤程度减轻,差异均有统计学意义(P<0.05)。结论 DEHP造成小鼠睾丸明显的氧化应激,并引起睾丸细胞DNA的损伤;MT可拮抗因DEHP染毒导致的睾丸氧化损伤。     

溴氰菊酯对真鲷肝胰脏组织及细胞DNA的损伤

为研究溴氰菊酯亚急性染毒对真鲷肝胰脏的毒性作用,将真鲷分为5组进行不同剂量半静置染毒.染毒25 d后取真鲷肝胰脏进行组织切片显微观察,并采用彗星试验技术对其肝胰脏细胞进行DNA损伤分析.结果表明: 在0.025、0.125、0.250和0.375 μg·L^-1暴露浓度下,真鲷肝胰脏组织出现了不同程度的淤血、细胞核浓缩、细胞坏死等病理性损伤,且染毒浓度越高组织细胞损伤越显著.与空白对照组相比,各染毒浓度组肝胰脏细胞DNA均有不同程度损伤,彗星拖尾率、彗尾DNA相对含量、Olive距等指标均与对照有显著性差异.一元回归分析表明,染毒浓度与拖尾率、彗星尾长等参数呈极显著正相关; 染毒浓度与各指标均呈线性关系,回归方程拟合度(R)极高,范围为0.909～0.996.表明溴氰菊酯对真鲷肝胰脏组织和细胞DNA均可产生不同程度损伤,且损伤程度与染毒浓度之间具有高度线性相关关系.     

紫外辐射诱导烟草原生质体中DNA损伤的彗星电泳检测

以烟草原生质体为材料,采用彗星电泳检测用0.5W·m^-2紫外线以不同时间（0、5、10、30、60和120s）诱导的烟草原生质体中DNA的损伤。结果表明,在0～10s的时间内代表DNA损伤程度的尾矩、Olive尾矩等参数与紫外线照射时间具有良好的时间依赖关系。本文建立的烟草原生质体体系采用彗星电泳技术,可以快速而灵敏地检测紫外线对植物细胞的损伤程度。     

对二甲苯对皱纹盘鲍肝胰腺细胞DNA的损伤

为研究对二甲苯对皱纹盘鲍肝胰腺的毒性作用,设置4个浓度(0.5、1.0、1.5和2.0 mg·L^-1)和对照组,开展为期21 d的对二甲苯对皱纹盘鲍的亚慢性毒性试验,采用彗星试验技术进行皱纹盘鲍肝胰腺细胞DNA损伤分析,采用CASP分析软件对拖尾率、彗星尾长、彗尾DNA相对含量、Olive矩等损伤指标进行统计。结果表明: 与对照组相比,各染毒组皱纹盘鲍肝胰腺细胞DNA均受到损伤,且损伤程度存在显著性差异。随着染毒浓度的增加,肝胰腺细胞DNA受损程度加重,高浓度甚至可以引发细胞凋亡,呈现一定的剂量-损伤效应。中浓度对二甲苯短时间暴露即可对皱纹盘鲍肝胰腺细胞造成DNA损伤,随着暴露时间延长,细胞DNA受损程度加重,呈现一定的时间-损伤效应。但长时间暴露细胞DNA各损伤指标有所减小,这可能与细胞自身的DNA修复机制和生物体解毒系统的代谢机制有关。研究表明,对二甲苯可对皱纹盘鲍肝胰腺细胞产生氧化损伤,导致DNA断裂,高浓度的对二甲苯长时间暴露可导致其细胞凋亡。     

微量液基稀释法测定中药活性成分的体外抗曲霉菌活性*

通过测定中药活性成分肉桂醛和柠檬醛对常见深部条件致病性真菌黄曲霉、烟曲霉的抗菌活性,为建立中药抗曲霉菌药敏试验标准提供参考依据。参照美国国家临床试验标准化委员会(National Committee for Clinical Laboratory Standards,NCCLS)提出的标准,用微量液基稀释法分别测定肉桂醛和柠檬醛对黄曲霉、烟曲霉的抗菌活性。肉桂醛对黄曲霉、烟曲霉最低抑菌浓度(Minimal inhibitory concentration,MIC)分别为：0.100μg/mL,0.050μg/mL,柠檬醛对黄曲霉、烟曲霉的MIC分别为：2．600μg/mL、0．650μg/mL。中药活性成分肉桂醛和柠檬醛具有高效抗曲霉作用。该研究可为制定中药抗曲霉作用评价标准提供参考依据。     

超高灵敏度荧光显微成像技术对光敏剂细胞内分布的初步研究

目的：探讨应用基于ICCD的超高灵敏度荧光显微成像系统研究光敏剂细胞内分布的可行性。方法：传代培养内皮细胞、食管癌细胞和肺癌细胞,将不同浓度血卟啉单甲醚(HMME)与细胞共同孵育不同时间。采用荧光显微镜及ICCD组成的荧光显微成像系统采集不同浓度及不同孵育时间条件下HMME的荧光图像,并采用计算机图像处理技术进行图像增强、滤波后计算其细胞浆与细胞核的平均荧光强度比值。同时应用激光共聚焦显微镜图像采集进行对比。结果：HMME浓度为5μg／ml时,荧光显微镜采集到HMME的荧光图像;HMME浓度升高到160μg／ml,激光共聚焦显微镜获得HMME的荧光图像。两组图像的特点都为胞浆中荧光强度较高,细胞核区荧光较弱;细胞浆与细胞核的比值约为2～3：1。结论：荧光显微镜和ICCD采集细胞内光敏剂的荧光图像灵敏度高,方法可靠、实用。HMME较多分布在细胞质中,细胞核吸收较少。     

Effects of citral on Aspergillus flavus spores by quasi-elastic light scattering and multiplex microanalysis techniques

Litsea cubeba (Lour.) Pers. is a kind of medicinal plantin China. The first report about the antibacterial and anti-phlogistic function of Litsea cubeba (Lour.) Pers. and itsoil appeared in the Zhong Yao Da Ci Dian [1]. Since 1980s,many studies showed that Litsea cubeba oil had wideantibacterial and antifungal activity [2–4]. The antibioticfunctions of Litsea cubeba oil are attributable mainly tocitral [5–7], which amounts to 60%–80% of the essentialoil [8]. Pattnaik [9] reported that…     

柠檬醛抗黄曲霉作用的分子机理

以多组分山苍子[Litsea cubeba(Lour)Per]香精油作为复合中药模型。以该香精油中主要抗菌成分柠檬醛为中药靶部位,以能分泌致癌毒素的黄曲霉单细胞作为药物作用对象,吸收当今医学影像领域先进的科学技术,采用多学科交叉策略,将多维显微、瑞利光散射(Rayleigh scattering)、电镜与生化分析4项技术构筑平台, 从细胞、亚细胞和生物大分子三个水平,研究柠檬醛作用于黄曲霉的动静态过程,阐明模拟的中药方剂靶部位对细胞整体的作用规律.发现该醛不仅能改变黄曲霉细胞膜的形态结构、物理学特性及其生物学功能(如对物质吸收的选择通透性,细胞体积调节机制等),而且使细胞膜产生脂质过氧化损伤;进入细胞后,既作用于细胞器(如线粒体、细胞核等),使其产生损伤及区域性分布;又通过干扰细胞内大分子拥挤状态,导致细胞内生物大分子构象的改变、高含量类大分子缔合反应不可逆增强以及因生化反应区域效应丧失而产生的新陈代谢紊乱,揭示该醛能使黄曲霉孢子失去萌发力、菌丝体生长被抑制及产生孢子的能力,在于黄曲霉细胞膜、细胞器及大分子失去了正常结构、功能及相关的调节机制.在实现对柠檬醛抗黄曲霉机理阐明的过程中,在研究思路和方法上进行全新的探索.     

柠檬醛损伤黄曲霉线粒体生化机理的研究

应用生物化学方法并结合扫描电镜,研究柠檬醛掺入黄曲霉细胞,并通过损伤线粒体(Mt),导致抑制其生长的机理。结果表明,在药物致敏浓度时,菌丝体经该醛作用后,胞内Mt呈不规则增多,氧化还原反应系统受到破坏,与对照组相比,柠檬醛组的琥珀酸脱氢酶(Succinate Dehydrogenase,SDH)、苹果酸脱氢酶(Malate Dehydrogenase,MDH)活性分别呈不可逆下降271%和242%,随着药物浓度的升高,SDH、MDH的活性直至消失;以琥珀酸、α酮戊二酸和丙酮酸为底物时,线粒体呼吸速率分别下降24.1%、14.3%和36.1%,提示柠檬醛能使菌丝体DNA、RNA、脂类和蛋白质等生物合成受到抑制,促进细胞死亡。     

柠檬醛对黄曲霉质膜损伤机制的初步研究

与正常生长的黄曲霉对照 ,通过测定经柠檬醛毒化的菌丝体对还原糖和蛋白质利用率、[Na+ ,K+ ] ATPase分解ATP活性、细胞电解质渗出率、并结合扫描电镜和快速显微多道分光光度法观察菌丝体细胞及孢子形态变化 ,结果表明经该醛MIC毒化后 ,菌丝体细胞及孢子表面疏松而粗糙 ;隘痕缩小并关闭 ;电导率增加 5 2 8% ;对还原糖和蛋白质的利用速率分别下降 6 1 5 %和 44 3 % ;孢子萌发率下降至 6 1 4% ;该醛能明显改变细胞质膜的分子结构 ,使其失去选择通透性而抑制菌丝体生长和孢子萌发率。     

Osmotically induced removal of water from fungal cells as determined by a spin probe technique

Effects of physical environment on plasma membrane semipermeability and osmotic induction of changes in aqueous cytoplasmic volume were studied in vegetative and spore cells of a plant pathogenic fungus, Fusarium sulphureum. A direct method, employing a spin probe molecule that partitioned between intracellular aqueous and hydrophobic phases, allowed measurement of reversible water movement out of macroconidial cells and chlamydospores exposed to solutions of high osmolarity. Equilibrium distribution of the spin probe between intracellular aqueous and lipid phases was more rapid than movement of water in and out of the cells. The extent of water removal was exponentially dependent on osmotic strength. Some cells became irreversibly permeable to divalent cations on treatment with sodium chloride above 1.5 osmolar but addition of sucrose to the suspension medium at equivalent osmolar concentrations caused water removal without adversely affecting the viability. Sucrose also protected the plasma membrane against damage during freeze-drying. Induction of plasma membrane damage by osmotic shock or freeze-drying permitted rapid permeation of nickel ions. Neither slow equilibration of intracellular components with divalent paramagnetic cations nor partial permeability of damaged plasma membranes to these ions was observed.     

Soil fungistasis: elevation of the exogenous carbon and nitrogen requirements for spore germination by fungistatic volatiles in soils.

Axenic, washed conidia of Fusarium solani f. sp. phaseoli, Aspergillus flavus, and Verticillium albo-atrum were placed on washed Difco purified agar discs along with an inorganic salt solution containing various levels of carbon and nitrogen substrates. These discs were exposed to volatiles from six soils (pH 5.1-8.6). Fusarium solani macroconidial germination was inhibited mostly by volatiles from soils of pH 5.1, 6.1, 7.0, and 7.5, but high levels of glucose and NH4Cl reversed this inhibition, raising germination to that of no-soil, no-carbon or nitrogen controls. Conidial germination of A. flavus was inhibited mainly by volatiles from high pH (7.0, 7.8, and 8.6) soils, and increased levels of glucose plus an amino acid mixture nullified this inhibition. Volatiles from soils of pH 5.1, 6.1, and 7.5 stimulated A. flavus conidial germination. Assays after the removal of CO2 from the air above soil of pH 5.1 demonstrated that volatiles inhibitory to A. flavus were produced by this soil. Assays indicated that a KOH-soluble compound was a fungistatic soil volatile to F. solani macroconidial germination. The nullification by carbon and nitrogen substrates of F. solani and A. flavus inhibition caused by soil volatiles parallels that for soil fungistasis. Conidial germination of V. albo-atrum was markedly stimulated by volatiles in all soils tested, and was not affected by removal of CO2. Inhibitory soil volatiles may increase the nutritional requirements for spore germination of certain fungi.     

Analysis of damage due to moist heat treatment of spores of Bacillus subtilis

Aims:  To determine conditions for generation and recovery of Bacillus subtilis spore populations heavily damaged by moist heat treatment.
Methods and Results:  Bacillus subtilis spores were treated with moist heat and spore viability was assessed on different media. A rich medium and several minimal media gave similar spore recoveries after moist heat treatment, but lack of glucose in minimal media greatly decreased spore recovery. High NaCl levels also greatly decreased the recovery of moist heat-treated spores on minimal media, and addition of good osmoprotectants reversed this effect. Moist heat treatment did not decrease spore recovery on minimal media with high salt through DNA damage or by eliminating spore germination, but by affecting spore outgrowth.
Conclusions:  Conditions for generating B. subtilis spore populations with high levels of conditional moist heat damage have been determined. The major conditional damage appears to be in spore outgrowth, perhaps because of damage to one or more important metabolic enzymes.
Significance and Impact of the Study:  This work has provided new insight into the mechanism of B. subtilis spore killing by moist heat.     

Treatment with oxidizing agents damages the inner membrane of spores of Bacillus subtilis and sensitizes spores to subsequent stress

AIMS: To determine if treatment of Bacillus subtilis spores with a variety of oxidizing agents causes damage to the spore's inner membrane. METHODS AND RESULTS: Spores of B. subtilis were killed 80-99% with wet heat or a variety of oxidizing agents, including betadine, chlorine dioxide, cumene hydroperoxide, hydrogen peroxide, Oxone, ozone, sodium hypochlorite and t-butylhydroperoxide, and the agents neutralized and/or removed. Survivors of spores pretreated with oxidizing agents exhibited increased sensitivity to killing by a normally minimal lethal heat treatment, while spores pretreated with wet heat did not. In addition, spores treated with wet heat or the oxidizing agents, except sodium hypochlorite, were more sensitive to high NaCl in plating media than were untreated spores. The core region of spores treated with at least two oxidizing agents was also penetrated much more readily by methylamine than was the core of untreated spores, and spores treated with oxidizing agents but not wet heat germinated faster with dodecylamine than did untreated spores. Spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents. CONCLUSIONS: Treatment of spores with oxidizing agents has been suggested to cause damage to the spore's inner membrane, a membrane whose integrity is essential for spore viability. The sensitization of spores to killing by heat and to high salt after pretreatment with oxidizing agents is consistent with and supports this suggestion. Presumably mild pretreatment with oxidizing agents causes some damage to the spore's inner membrane. While this damage may not be lethal under normal conditions, the damaged inner membrane may be less able to maintain its integrity, when dormant spores are exposed to high temperature or when germinated spores are faced with osmotic stress. Triggering of spore germination by dodecylamine likely involves action by this agent on the spore's inner membrane allowing release of the spore core's depot of dipicolinic acid. Presumably dodecylamine more readily alters the permeability of a damaged inner membrane and thus more readily triggers germination of spores pretreated with oxidizing agents. Damage to the inner spore membrane by oxidizing agents is also consistent with the more rapid penetration of methylamine into the core of treated spores, as the inner membrane is likely the crucial permeability barrier to methylamine entry into the spore core. As spores of strains with very different levels of unsaturated fatty acids in their inner membrane exhibited essentially identical resistance to oxidizing agents, it is not through oxidation of unsaturated fatty acids that oxidizing agents kill and/or damage spores. Perhaps these agents work by causing oxidative damage to key proteins in the spore's inner membrane. SIGNIFICANCE AND IMPACT OF THE STUDY: The more rapid heat killing and germination with dodecylamine, the greater permeability of the spore core and the osmotic stress sensitivity in outgrowth of spores pretreated with oxidizing agents is consistent with such agents causing damage to the spore's inner membrane, even if this damage is not lethal under normal conditions. It may be possible to take advantage of this phenomenon to devise improved, less costly regimens for spore inactivation.