银与光所产生的协同效应的微生物灭活机理及其家电产品中的应用

研究发现在使用紫外线(UV-A, 395 nm)进行照射时, 银溶液对微生物的灭活作用得到增强, 特别是对真核微生物的灭活作用得到显著增强。为解明这种银与光所产生的协同效应的微生物灭活机理, 使用电子自旋共振仪(Electron spin resonance, ESR)对溶液进行检测, 并采用扫描电子显微镜(SEM)以及测定线粒体酶活性等方法, 从微生物形态学及生理学特性方面对真核微生物细胞进行分析, 推测出了其作用机理。分析认为, 在光照下氧化银(Ag2O)被激活并与水分子发生反应产生羟基自由基(·OH)。羟基自由基破坏真核微生物的细胞壁, 失活其细胞内线粒体酶活性, 从而引起真核微生物细胞死灭。在实验中, 作为原核微生物的代表使用金黄色葡萄球菌(Staphylococcus aureus), 作为真核微生物的代表使用了白色念珠菌(Candida albicans)和须癣毛癣菌(Trichophyton Mentagrophytes), 并对各种类进行了检测对比。本文还阐述了把这项微生物增殖抑制技术具体应用于洗衣机的具体结果, 并进行了讨论。     

荆皮癣湿酊诱导红色毛癣菌凋亡的机制研究北大核心

【目的】本研究旨在探讨复方中药荆皮癣湿酊(Jingpixian tincture,JPXT)对红色毛癣菌(Trichophyton rubrum)的凋亡诱导作用,以阐明其可能的抗真菌作用机制。【方法】采用细胞计数试剂盒-8(cell counting kit-8,CCK-8)评价荆皮癣湿酊对红色毛癣菌生长活力的影响;流式细胞仪检测红色毛癣菌细胞内活性氧(reactive oxygen species,ROS)水平和线粒体膜电位(mitochondrial membrane potential,MMP)变化;Annexin V-FITC/PI染色荧光显微镜观察红色毛癣菌细胞磷脂酰丝氨酸(phosphatidylserine,PS)外翻情况;流式细胞术检测红色毛癣菌细胞凋亡率;FITC-VAD-FMK染色观察红色毛癣菌偏半胱天冬酶(metacaspase)活性;紫外分光光度计测定红色毛癣菌细胞色素C氧化酶的活性。【结果】荆皮癣湿酊处理后的红色毛癣菌细胞活力与MMP水平均有所降低,ROS水平显著升高,PS外翻与凋亡率明显增加,偏半胱天冬酶活性显著升高,细胞色素C氧化酶活性降低。【结论】荆皮癣湿酊可通过诱导菌体凋亡的方式发挥对红色毛癣菌的抗菌作用。     

基于叶酸修饰的S-TiO2的PDT体外灭活HL60细胞实验研究

文章主要研究叶酸修饰硫掺杂二氧化钛(FA-S-TiO_2)纳米颗粒光催化灭活作用并初步探讨FA-S-TiO_2增强了TiO_2光催化灭活效率的作用机理。利用固相反应的方法制备了S-TiO_2,并利用表面修饰的方法制备了FA-S-TiO_2。通过紫外-可见光吸收光谱(UV-Vis),荧光光谱,傅里叶红外光谱(FTIR),透射电镜(TEM)等手段对样品进行了表征。利用Cell Counting Kit-8(CCK-8)法分别检测TiO_2、S-TiO_2、FA-S-TiO_2对HL60细胞的灭活效应,并采用活性氧检测等方法进行了分析研究。细胞实验结果表明,在暗室条件下,随着叶酸修饰S-TiO_2的比例增加,细胞的存活率随之减少;在光照条件下,细胞的存活率明显下降。其中FA-S-TiO_2样品1对HL60细胞的灭活效率最高,达到72%。其相应的活性氧含量也是最高。同时,通过荧光光谱推测FA-S-TiO_2提高HL60细胞对该纳米颗粒摄取效率,进而增强PDT灭活效果。     

羟基喜树碱诱导肝癌细胞凋亡前后线粒体疏水蛋白质组的定量分析

抗癌剂羟基喜树碱可以通过线粒体途径诱导肝癌细胞凋亡.应用定量蛋白质组学技术分析羟 基喜树碱诱导肝癌细胞凋亡前后的线粒体疏水蛋白质差异表达,探讨癌细胞凋亡机制及羟基 喜树碱的抗癌机理.分离提取羟基喜树碱诱导肝癌细胞凋亡前后的线粒体,并采用顺序抽提法提取疏水蛋白质;用含稳定同位素亲和标签的c-ICAT试剂标记蛋白,利用基于多维色谱线性离子阱/静电场轨道阱质谱联用技术的鸟枪(shotgun)法策略分析鉴定了在肝癌细胞凋亡前后的线粒体中表达量差异有显著统计学意义(P<0.05)的疏水蛋白144种,其中, 12种蛋白的表达量在凋亡细胞中下调,而表达量在羟基喜树碱诱导细胞凋亡后上调10倍以上的蛋白43种.这些蛋白主要与细胞分裂增殖、分化凋亡、能量代谢、核酸代谢以及信号转导相关.该研究结果为在亚细胞定量蛋白质组水平上深入探讨羟基喜树碱的作用机理提供了新的实验依据,亦为研究肿瘤细胞凋亡机制提供了新的思路.     

Angeli盐对二氢硫辛酰胺脱氢酶的可逆性灭活作用(英文)

二氢硫辛酰胺脱氢酶(dihydrolipoamide dehydrogenase,DLDH)是线粒体3个α-酮酸脱氢酶复合物(丙酮酸脱氢酶复合物、α-酮戊二酸脱氢酶复合物、支链氨基酸脱氢酶复合物)的关键成分,属于吡啶依赖性二硫化物氧化还原酶类,对活性氮自由基(reactive nitrogen species,RNS)和活性氧自由基(reactive oxygenspecies,ROS)造成的氧化修饰非常敏感。本研究探索由Angeli盐所产生的RNS对DLDH的修饰作用及机制。将大鼠脑线粒体分离,与不同浓度的Angeli盐作用,应用分光光度计、蓝色胶、基于二维电泳的蛋白质组学等手段,测定DLDH酶活性。结果显示,Angeli盐呈浓度依赖性方式灭活DLDH,过氧亚硝酸盐在同样条件下对DLDH酶活性无抑制作用,说明Angeli盐对DLDH的作用可能是非随机的。由于Angeli盐在生理pH条件下可分解为硝基阴离子(nitroxyl anion,HNO)和一氧化氮(nitric oxide,NO),故进一步分析了Angeli盐对DLDH的灭活作用是否由HNO引起,结果证实确实如此。最后,二维电泳Western blot结果显示,Angeli盐对DLDH的灭活伴随着DLDH蛋白质的S-亚硝基硫醇形成,提示S-亚硝基硫醇化可能是导致DLDH酶失活的原因。综上,本研究为研究Angeli盐灭活DLDH的机制提供了新证据。     

核壳结构CdS-TiO_2纳米颗粒体外光催化灭活白血病HL60细胞实验研究

本文研究了核壳结构CdS-TiO_2纳米颗粒光对HL60细胞光催化灭活作用,并初步探讨了CdS量子点增强TiO_2光催化灭活效率的作用机理。实验利用超声法制备了核壳结构CdS-TiO_2纳米颗粒,使用CCK-8法检测了其对白血病HL60细胞的光催化灭活效果,并采用活性氧分析和荧光发光光谱等分析手段对CdS量子点增强TiO_2光催化灭活效率的作用机理进行了分析。细胞实验结果表明,在暗室条件下,随着CdS包裹的TiO_2外壳的增厚,细胞的暗室存活率从28.5%逐渐上升至80%;在可见光照下,细胞的存活率显著下降。CdS-TiO_2纳米颗粒对HL60细胞的光催化灭活率均超过60%,其中CdS-TiO_2样品0.6对HL60细胞的光催化灭活效率最高,达到95%。根据荧光光谱和活性氧含量分析的结果推测,这可能是由于核壳结构的CdS内核与TiO_2外壳之间产生了有效的电子转移,抑制了TiO_2的空穴电子的复合,增强了TiO_2外壳的光催化活性,最终增加了TiO_2对HL60细胞的PDT灭活效果。     

皮肤癣菌体外蛋白水解酶活性测定

目的观察皮肤癣菌的体外蛋白水解酶活性;比较分离自不同感染部位的红色毛癣菌的体外蛋白水解酶活性。方法实验菌株包括来自不同感染部位的红色毛癣菌22株、须癣毛癣菌3株、犬小孢子菌5株,进行体外培养,并利用9-羟基乙酚噻唑标识的酪蛋白和酶标仪检测真菌细胞外蛋白水解酶的活性。结果须癣毛癣菌的体外蛋白水解酶活性高于红色毛癣菌和犬小孢子菌（P〈0.05）,而红色毛癣菌和犬小孢子菌之间无差异（P〉0.05）。红色毛癣菌的细胞外蛋白水解酶活性在分离自浅部感染部位的菌株之间无差异（P〉0.05）,但高于引起毛癣菌肉芽肿的菌株（P〈0.05）。结论不同的皮肤癣菌体外蛋白水解酶活性可能不同;分离自不同感染部位的同一菌种的体外蛋白水解酶活性也有可能不同。     

迷迭香酸对羟自由基所致小鼠肝线粒体损伤的保护作用

探索迷迭香酸对羟自由基致小鼠肝脏线粒体氧化损伤的保护作用。采用羟自由基(.OH),诱导小鼠肝线粒体损伤后,通过测定线粒体肿胀度、膜流动性、丙二醛(MDA)含量及琥珀酸脱氢酶(SDH)活性等指标以确定迷迭香酸对小鼠肝线粒体羟自由基损伤的保护作用。结果迷迭香酸剂量依赖地抑制线粒体肿胀,提高膜流动性,降低MDA的生成,增强SDH活性,差异显著。本实验证明迷迭香酸可以抑制.OH所致的线粒体损伤。     

对羟基苯甲酸和间苯三酚对棉花幼苗根系线粒体功能和根系生长的影响

以中国大面积种植的早熟棉中棉所50(CCRI-50)为材料,设置水培试验,研究不同浓度(0.8、4.0、20.0 mmol·L^-1)的对羟基苯甲酸和间苯三酚对棉花苗期根系线粒体活性氧产生、抗氧化酶活性变化及线粒体特性的影响.结果表明: 对羟基苯甲酸和间苯三酚抑制了棉花根系生长,降低了根系线粒体超氧化物歧化酶(SOD)、过氧化氢酶(CAT)、过氧化物酶(POD)和线粒体膜H⁺-ATPase活性,增加了O₂^-·产生速率和H₂O₂含量;对羟基苯甲酸和间苯三酚处理还增大了线粒体细胞通透性转换孔(MPTP)开放程度,降低了线粒体膜流动性和细胞色素Cyt c/a 值.0.8 mmol·L^<sup>-1的对羟基苯甲酸和间苯三酚处理间线粒体功能差异相对较小,4.0和20.0 mmol·L^-1时,对羟基苯甲酸处理对根系生长和线粒体功能的抑制作用高于间苯三酚处理.总之,对羟基苯甲酸和间苯三酚均抑制了棉苗根系生长和线粒体功能,且浓度越高,抑制作用越强.对羟基苯甲酸和间苯三酚处理间存在差异,浓度高于4.0 mmol·L^-1时,对羟基苯甲酸比间苯三酚具有更强的抑制作用.     

羟基自由基对兔脑微粒体膜脂及膜蛋白的损伤

本文研究了过氧化氢与亚铁离子体系产生的羟基自由基对兔脑微粒体脂质过氧化作用及对膜上(Na~++K~+)-ATP酶活性的影响.结果表明,羟基自由基导致兔脑微粒体脂质过氧化,增加丙二醛的含量.羟基自由基还使微粒体膜巯基数下降,(Na~++K~+)-ATP酶活力受到抑制.阿魏酸钠对抑制微粒体脂质过氧化及对膜巯基和(Na~++K~+)-ATP酶均有保护作用.自旋捕集实验结果进一步证明药物对羟基自由基的猝灭作用.     

Different inactivation behaviors of MS-2 phage and Escherichia coli in TiO2 photocatalytic disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO(2) photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (.OH), which is generated on the surface of UV-illuminated TiO(2), plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H(2)O(2) and O(2).(-), etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe(2+), were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O(2).(-) and H(2)O(2), according to the present results.     

Visible light inactivation of bacteria and fungi by modified titanium dioxide.

Visible light induced photocatalytic inactivation of bacteria (Escherichia coli, Staphylococcus aureus, Enterococcus faecalis) and fungi (Candida albicans, Aspergillus niger) was tested. Carbon-doped titanium dioxide and TiO2 modified with platinum(IV) chloride complexes were used as suspension or immobilised at the surface of plastic plates. A biocidal effect was observed under visible light irradiation in the case of E. coli in the presence of both photocatalysts. The platinum(IV) modified titania exhibited a higher inactivation effect, also in the absence of light. The mechanism of visible light induced photoinactivation is briefly discussed. The observed detrimental effect of photocatalysts on various microorganism groups decreases in the order: E. coli > S. aureus approximately E. faecalis>C. albicans approximately A. niger. This sequence results most probably from differences in cell wall or cell membrane structures in these microorganisms and is not related to the ability of catalase production.     

Inactivation of Escherichia coli by photochemical reaction of ferrioxalate at slightly acidic and near-neutral pHs

Fenton chemistry, which is known to play an effective role in degrading toxic chemicals, is difficult to apply to disinfection in water treatment, since its reaction is effective only at the acidic pH of 3. The presence of oxalate ions and UV-visible light, which is known as a photoferrioxalate system, allows the Fe(III) to be dissolved at slightly acidic and near-neutral pHs and maintains the catalytic reaction of iron. This study indicates that the main oxidizing species in the photoferrioxalate system responsible for microorganism inactivation is OH radical. Escherichia coli was used as an indicator microorganism. The CT value (OH radical concentration x contact time; used to indicate the effect of the combination of the concentration of the disinfectant and the contact time on inactivation) for a 2-log inactivation of E. coli was approximately 1.5 x 10(-5) mg/liter/min, which is approximately 2,700 times lower than that of ozone as estimated by the delayed Chick-Watson model. Since the light emitted by the black light blue lamp is similar to sunlight in the specific wavelength range of 300 to 420 nm, the photoferrioxalate system, which can have a dual function, treating water for both organic pollutants and microorganisms simultaneously, shows promise for the treatment of water or wastewater in remote or rural sites. However, the photoferrioxalate disinfection system is slower in inactivating microorganisms than conventional disinfectants are.     

Photoinactivation of cellular catalase by ultraviolet radiation

Summary

Photoinactivation of catalase is found to be similar in solution and in human normal skin fibroblasts exposed to ultraviolet B, ultraviolet A and near visible light, and the kinetics of such photoinactivation obey first order processes. The action spectrum, measured for the first time in cells, suggests that catalase photoinactivation in solution and in cells proceeds via similar routes. In both systems, no protective effect was observed with diethyldithiocarbamate, a superoxide dismutase inhibitor, with desferrioxamine, an iron chelator which impedes the production of hydroxyl radical via the Fenton reaction, and with vitamin E which scavenges peroxyl radical to protect against membrane peroxidative process. While the absence of protection by these inhibitors may be anticipated for the photoinactivation of catalase in solution, the lack of effect in cells suggests that reactive oxygen species produced by endogenous photosensitization are not responsible for the enzyme inactivation. Moreover, the already established protective effect of ethanol in solution is also observed in cells, supporting the view that photoinactivation in solution and in cells is due to the same primary events.     

Photochemical studies and ultraviolet sensitization of Escherichia coli thymidylate kinase by various halogenated substrate analogs.

The effect of 5-iodo-2'-deoxyuridine monophosphate (IdUMP), various 5-halogenated-5'-azido-2', 5' -dideoxyuridine derivatives, 2'-deoxy-6-azauridine (AzdUrd), and its halogenated analogs on the ultraviolet sensitization of Escherichia coli thymidylate kinase has been investigated. Only those compounds iodinated in position 5 enhance the rate of ultraviolet inactivation of this enzyme. However, 5'-azido nucleosides with iodo, bromo, chloro, or fluoro substituents in position 5 neither protect nor sensitize thymidylate kinase to ultraviolet inactivation. Thymidine 5'-monophosphate partially protects the enzyme against ultraviolet inactivation either in the presence or absence of ultraviolet-sensitizing iodinated analogs. Magnesium ion does not enhance the ultraviolet inactivation of thymidylate kinase by 5-iodinated nucleoside analogs. The kinatic data support an active site-directed enhancement of the enzyme to ultraviolet inactivation by 5-iodo-2'-deoxyuridine monophosphate, since the concentration of IdUMP required to attain 50% maximal enhancement is 0.24 mM which is in good agreement with its Ki of 0.18 mM. When either [125I]IdUMP or [2-14C]IdUMP was irradiated with the enzyme, both radioactivities were associated with the enzyme, however only with the 14C analog was the amount bound at half-saturation essentially equal to the amount required to inactivate the enzyme by 50%. These data support the hypothesis that the active entity in the enhancement by IdUMP of thymidylate kinase inactivation during ultraviolet irradiation is the uridylate free radical which is formed photochemically from IdUMP. Photochemical studies of 6-azauracil (AzUra), 2'-deoxy-6-azauridine, and 5-iodo-2'-deoxy-6-azauridine (IAzdUrd) were performed. Photolysis of IAzdUrd in the presence of a hydrogen donor yields AzdUrd which upon further photolysis yields the photohydrate. The photohydrate of AzdUrd when incubated in the dark at pH 5.2 is 90% converted back to AzdUrd, whereas the photohydrate of AzUra is only partially (20%) converted to AzUra. The rate of deiodination of IAzdUrd is 2.1-fold greater than that of IdUMP. Although the Ki of IdUMP and IAzdUrd is similar, the increased photosensitivity of the aza analog accounts for the much greater enhancement of ultraviolet inactivation of thymidylate kinase. The ability of a compound to enhance the ultraviolet inactivation of deoxythymidylate kinase is correlated with the potential of the compound to produce a free radical rather than a photohydrate when the enzyme-substrate analog complex is irradiated.     

Integration Efficiency in DNA-Induced Transformation of PNEUMOCOCCUS. II. Genetic Studies of Mutant Integrating All the Markers with a High Efficiency

Transformation of the pneumococcus mutant 401 by DNA's bearing the standard reference marker and several other markers belonging to two unlinked loci has shown that differences in the integration efficiencies of these markers were considerably reduced in this strain compared to the wild-type strain Cl(3). The sensitivities of mutant 401 to ultraviolet light and to X-ray irradiation are the same as those of Cl(3). However, in 401 all the markers tested are more resistant to inactivation as shown by transformation of 401 and Cl(3) by ultraviolet-irradiated DNA. The increase in resistance is greater for low efficiency (LE) markers than for high efficiency (HE) markers.-The decreased discrimination between LE and HE markers in strain 401 is not due to a mechanism related to modification of markers in the transforming DNA by the recipient cells, nor are the proteins inducing competence of the cells responsible for the differences in the integration efficiencies of various markers.-Genetic studies of the fate of recombinants as well as the measure of the amount of DNA taken up have shown that all the markers are integrated in strain 401 by the same recombination process, that specific to high efficiency markers.     

Inactivation of Escherichia coli by Photochemical Reaction of Ferrioxalate at Slightly Acidic and Near-Neutral pHs

Fenton chemistry, which is known to play an effective role in degrading toxic chemicals, is difficult to apply to disinfection in water treatment, since its reaction is effective only at the acidic pH of 3. The presence of oxalate ions and UV-visible light, which is known as a photoferrioxalate system, allows the Fe(III) to be dissolved at slightly acidic and near-neutral pHs and maintains the catalytic reaction of iron. This study indicates that the main oxidizing species in the photoferrioxalate system responsible for microorganism inactivation is OH radical. Escherichia coli was used as an indicator microorganism. The CT value (OH radical concentration × contact time; used to indicate the effect of the combination of the concentration of the disinfectant and the contact time on inactivation) for a 2-log inactivation of E. coli was approximately 1.5 × 10⁻⁵ mg/liter/min, which is approximately 2,700 times lower than that of ozone as estimated by the delayed Chick-Watson model. Since the light emitted by the black light blue lamp is similar to sunlight in the specific wavelength range of 300 to 420 nm, the photoferrioxalate system, which can have a dual function, treating water for both organic pollutants and microorganisms simultaneously, shows promise for the treatment of water or wastewater in remote or rural sites. However, the photoferrioxalate disinfection system is slower in inactivating microorganisms than conventional disinfectants are.     

Different Inactivation Behaviors of MS-2 Phage and Escherichia coli in TiO2 Photocatalytic Disinfection

Despite a wealth of experimental evidence concerning the efficacy of the biocidal action associated with the TiO₂ photocatalytic reaction, our understanding of the photochemical mechanism of this particular biocidal action remains largely unclear. It is generally accepted that the hydroxyl radical (·OH), which is generated on the surface of UV-illuminated TiO₂, plays the main role. However, our understanding of the exact mode of action of the hydroxyl radical in killing microorganisms is far from complete, and some studies report that other reactive oxygen species (ROS) (H₂O₂ and O₂·⁻, etc.) also play significant roles. In particular, whether hydroxyl radicals remain bound to the surface or diffuse into the solution bulk is under active debate. In order to examine the exact mode of action of ROS in inactivating the microorganism, we tested and compared the levels of photocatalytic inactivation of MS-2 phage and Escherichia coli as representative species of viruses and bacteria, respectively. To compare photocatalytic microbial inactivation with the photocatalytic chemical degradation reaction, para-chlorobenzoic acid, which rapidly reacts with a hydroxyl radical with a diffusion-limited rate, was used as a probe compound. Two different hydroxyl radical scavengers, tert-butanol and methanol, and an activator of the bulk phase hydroxyl radical generation, Fe²⁺, were used to investigate their effects on the photocatalytic mode of action of the hydroxyl radical in inactivating the microorganism. The results show that the biocidal modes of action of ROS are very different depending on the specific microorganism involved, although the reason for this is not clear. It seems that MS-2 phage is inactivated mainly by the free hydroxyl radical in the solution bulk but that E. coli is inactivated by both the free and the surface-bound hydroxyl radicals. E. coli might also be inactivated by other ROS, such as O₂·⁻ and H₂O₂, according to the present results.