Mechanism of Inactivation of Bacteriophage J1 by Glutathione

Mechanism of inactivation of a double-stranded DNA phage, phage Jl of Lactobacillus casei, by reduced form of glutathione (GSH) was studied.

Air (oxygen) bubbling, oxidizing agents and transition metal ions enhanced the rate of inactivation of the phage by GSH. Partial oxidation of GSH resulted in a more rapid rate of inactivation. In contrast, nitrogen bubbling, reducing agents, chelating agents and radical scavengers prevented the inactivation. Fully oxidized GSH had no phagocidal effect. These results indicate that the inactivating effect of GSH requires the presence of molecular oxygen and is caused by free radical involved in the mechanism of GSH oxidation.

The target of GSH in the phage particle was not the tail protein but DNA. GSH reacted with phage DNA and caused single-strand scissions in the DNA, as exhibited by alkaline sucrose gradient centrifugation; thus inactivating phage.     

Some Dinophycean Red Tide Plankton Species Generate a Superoxide Scavenging Substance

Recent studies indicate that some raphidophycean red tide flagellates produce substances able to scavenge superoxide, whereas there have been no reports on superoxide scavenger production by dinophycean red tide flagellates. In this study, we examined the superoxide-scavenging activity of aqueous extracts from dinophycean red tide flagellates, Gymnodinium spp., Scrippsiella trochoidea, and Karenia sp., by a luminol analog L-012-dependent chemiluminescence (CL) method and an electron spin resonance (ESR)-spin trapping method, and compared the activity to that of raphidophycean red tide flagellates, Chattonella spp., Heterosigma akashiwo, and Fibrocapsa japonica. In the experiment applying the L-012-dependent CL method, only the aqueous extracts from raphidophycean red tide flagellates showed superoxide-scavenging activity. On the other hand, applying the ESR-spin trapping method, we found that the aqueous extracts from dinophycean red tide flagellates also showed superoxide-scavenging activity. This is the first report on the production of a superoxide-scavenger by dinophycean red tide flagellates.     

In vitro scavenging activity for reactive oxygen species by N-substituted indole-2-carboxylic acid esters.

The hydroxyl radical (HO*)- and superoxide anion radical (O* (2))-scavenging activity, as well as the singlet oxygen ((1)O(2))-quenching property of N-substituted indole-2-carboxylic acid esters (INDs) were investigated by deoxyribose degradation assay, a chemiluminescence method and the electron spin resonance (ESR) spin-trapping technique. This novel group of compounds was developed as a search for cyclooxygenase-2 (COX-2)-selective enzyme inhibitors. The results obtained demonstrated that of the 16 compounds examined, five inhibited light emission from the superoxide anion radical (O* (2))-DMSO system by at least 60% at a concentration of 1 mmol/L, nine prevented the degradation of deoxyribose induced by the Fenton reaction system (range 3-78%) or scavenged hydroxyl radicals (HO*) directly (range 8-93%) and 14 showed the (1)O(2)-quenching effect (range 10-74%). These results indicate that majority of the indole esters tested possess the ability to scavenge O(-) (2) and HO radicals and to quench (1)O(2) directly, and consequently may be considered effective antioxidative agents.     

Mechanism of Inactivation of Bacteriophage J 1 by Dithiothreitol,Cysteine, Mercaptoethanol,or Thioglycollate

The mechanism of inactivation of a double-stranded DNA phage, phage J1 of Lactobacilluscasei, by reducing agents containing thiol group(s) other than glutathione was studied mainly with dithiothreitol (DTT).

Air bubbling, oxidizing agents, and transition metal ions enhanced the rate of phage inactivation by DTT. Partial oxidation of DTT resulted in a more rapid rate of phage inactivation. In contrast, nitrogen bubbling, reducing agents including high concentrations of DTT itself, chelating agents, and radical scavengers prevented phage inactivation. Fully oxidized DTT had no phagocidal effect. These results indicate that the inactivating effect of DTT requires the presence of molecular oxygen and is indirectly caused by free radicals involved in the mechanism of DTT oxidation. The target attacked by DTT in phage particle was not protein but DNA; DTT reacted with DNA to produce single-strand scissions in DNA, which were the cause of inactivation of phage.

This was true also for L-cysteine, 2-mercaptoethanol, and thioglycollate.

Possible mechanisms by which these thiols fail to inactivate phage at high thiol concentrations are also discussed.     

Mechanism of Copper-Catalyzed Oxidation of Glutathione

The mechanism of copper-catalyzed glutathione oxidation was investigated using oxygen consumption, thiol depletion, spectroscopy and hydroxyl radical detection. The mechanism of oxidation has kinetics which appear biphasic. During the first reaction phase a stoichiometric amount of oxygen is consumed (1 mole oxygen per 4 moles thiol) with minimal *OH production. In the second reaction phase, additional (excess) oxygen is consumed at an increased rate and with significant hydrogen peroxide and *OH production. The kinetic and spectroscopic data suggest that copper forms a catalytic complex with glutathione (1 mole copper per 2 moles glutathione). Our proposed reaction mechanism assumes two parallel processes (superoxide-dependent and peroxide-dependent) for the first reaction phase and superoxide-independent for the second phase. Our current results indicate that glutathione, usually considered as an antioxidant, can act as prooxidant at physiological conditions and therefore can participate in cellular radical damage.     

牡丹花水提液对氧自由基的清除作用

牡丹花水提液加入O 2和·OH的产生及检测系统中,能显著降低介导的氮兰四唑(NBT)的光化学还原和·OH作用下的水杨酸羟基化作用.这提示牡丹花水提液具有显著的清除氧自由基的活性,红色牡丹花水提液清除氧自由基的效能高于浅色花.     

Characterization of the superoxide anion radical scavenging activity by tetracycline antibiotics in aprotic media

The tetracycline family antibiotics are widely used as human and veterinary treatments. The drugs are effective as antibiotics and also show antimicrobial and non‐microbial action. However, the antioxidant properties of tetracyclines have not been characterized in aprotic media. To better understand their biological functions, the in vitro superoxide anion radical () scavenging activities of tetracycline, chlortetracycline, oxytetracycline, doxycycline and methacycline were characterized, along with a very efficient scavenger, tiron, in dimethyl sulphoxide (DMSO), using ultra‐weak chemiluminescence (CL). We found that tetracycline, chlortetracycline and doxycycline efficiently inhibited CL from the ‐generating system at concentration levels of 0.02–1.0 mmol/L. Methacycline and oxytetracycline were the scavengers at concentration levels of 0.01–0.1 mmol/L, whereas when their concentration was lowered the drugs were capable of generating , leading to CL enhancement. For all the data obtained in this study, the scavenging activity for the compounds tested decreased in the following order: tetracycline > doxycycline > chlortetracycline > tiron methacycline > oxytetracycline. These results indicate that the tetracycline drugs directly alter redox chemistry in aprotic media. Copyright © 2011 John Wiley & Sons, Ltd.     

Radical and Superoxide Scavenging Activities of Matairesinol and Oxidized Matairesinol

The radical and superoxide scavenging activities of oxidized matairesinols were examined. It could be assumed that the free benzylic position was important for higher radical scavenging activity. The different level of activity was observed between 7′-oxomatairesinol (Mat 2) and 7-oxomatairesinol (Mat 3). The activity of 8-hydroxymatairesinol was lower than that of matairesinol (Mat 1). The superoxide scavenging activity of the oxidized matairesinols was also demonstrated for the first time. It is assumed that the pKa value of phenol in the oxidized matairesinols affected this activity.     

Mechanism of the Inactivation of the Bacteriophage T1 in Aerosols

The mechanism of inactivation of bacteriophage T(1) in aerosols was studied by using (32)P-labeled phage. During inactivation, viability decreased in parallel with the adsorption of (32)P to the host, showing either that inactivated phage does not adsorb or that the deoxyribonucleic acid (DNA) has left the coat. A (32)P band at the density of free DNA was found when inactivated phage was analyzed in a CsCl gradient.     

Superoxide,Hydrogen Peroxide and Hydroxyl Radical in D1/D2/cytochrome b-559 Photosystem II Reaction Center Complex

Spin-trapping electron spin resonance (ESR) was used to monitor the formation of superoxide and hydroxyl radicals in D1/D2/cytochrome b-559 Photosystem II reaction center (PS II RC) Complex. When the PS II RC complex was strongly illuminated, superoxide was detected in the presence of ubiquinone. SOD activity was detected in the PS II RC complex. A primary product of superoxide, hydrogen peroxide, resulted in the production of the most destructive reactive oxygen species, *OH, in illuminated PS II RC complex. The contributions of ubiquinone, SOD and H(2)O(2) to the photobleaching of pigments and protein photodamage in the PS II RC complex were further studied. Ubiquinone protected the PS II RC complex from photodamage and, interestingly, extrinsic SOD promoted this damage. All these results suggest that PS II RC is an active site for the generation of superoxide and its derivatives, and this process protects organisms during strong illumination, probably by inhibiting more harmful ROS, such as singlet oxygen.     

酶解骨胶原多肽的抗氧化特性研究

本文主要研究了酶解法制备的骨胶原多肽的总抗氧化能力、羟自由基清除作用和抑制超氧阴离子自由基的能力.通过与谷胱甘肽(GSH)的比较发现,溶液浓度在1130～150 mg/mL时,该骨胶原多肽的总抗氧化能力为GSH的71.92%.溶液浓度在2.5～20 mg/mL时,该多肽羟自由基清除作用为谷胱甘肽的1.36倍.溶液浓度为10～150 mg/mL时,多肽抑制超氧阴离子自由基的能力低于谷胱甘肽.     

The Mechanism of Inactivation of T4 Bacteriophage by Tritium Decay

Coliphage T4 was used as a model system to study the mechanism of biological inactivation produced by tritium decay. Experimentally, tritiated precursors were incorporated into phage DNA (thymidine-³H) or into phage protein (³H-amino acids). The ratio of killing efficiencies for decays originating in phage DNA to those originating in phage protein was 2.6. Inactivation by decays from labeled amino acids was assumed to occur exclusively from β-particle irradiation of phage DNA. If decays originating in DNA are due solely to irradiation of DNA, then the killing efficiencies reflect the energy transfer paths in phage DNA for decays originating in phage DNA and in the protein coat. The energy transfer paths were determined for the two cases with the help of a computer and found to be very nearly equal to the experimentally determined ratio (2.6). The killing efficiencies for decays originating in phage DNA were 0.12 and for decays originating in protein 0.046.     

Inactivation of Lactobacillus Bacteriophage PL-1 by Microwave Irradiation

The effect of microwave irradiation on the survival of bacteriophage PL-1, which is specific for Lactobacillus casei, was studied using a commercial 2,450 MHz microwave oven. The phages were inactivated by microwave irradiation according to almost first-order reaction kinetics. The rate of phage inactivation was not affected by the difference in the continuous or intermittent irradiation, nor by the concentrations of phages used, but was affected by the volume of phage suspensions, which prevented the loss of generated heat. Microwave irradiation of phage suspensions produced a number of ghost phages with empty heads, but fragmentation of the tail was hardly noticed. The breakage of phage genome DNA was primarily caused by the heat generated by microwave irradiation, whereas the phage DNA was not affected by the same temperature achieved by heat from outside. Thus we concluded that the phage-inactivating effect of microwave irradiation was mainly attributed to a thermal microwave effect, which was much stronger than a simple thermal exposure.     

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。     

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。     

A comparison of chemical systems for luminometric determination of antioxidant capacity towards individual reactive oxygen species.

The aim of the study was to investigate the reactive oxygen species (ROS) production in the hypoxanthine-xanthinoxidase (HX-XO), hydrogen peroxide-ferrous sulphate (H2O2-FeSO4) and hydrogen peroxide (H2O2) systems by using various concentrations of ROS scavengers, such as superoxide dismutase (SOD), dimethylthiourea (DMTU) or catalase (CAT). Luminol (0.8 mmol/L) was dissolved in a borate buffer, pH 9.0, and was used as a luminophor in the chemiluminescence (CL) measurements. In the HX-XO system SOD, CAT and DMTU deepened the CL signal, whereas in the H2O2-FeSO4 system, only CAT and DMTU deepened the CL signal, and in the H2O2 system SOD and CAT increased and DMTU deepened the CL signal. Electron spin resonance (ESR) measurements were performed only in the H2O2-FeSO4 system. 5,5-dimethyl-pyrroline-N-oxide (DMPO) was used as a spin trap. According to typical ESR spectra, .OH was produced in this chemical system. It can be concluded that the chemical systems do not produce single reactive oxygen species but a mixture of them.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Thioctic Acid and Dihydrolipoic Acid are Novel Antioxidants Which Interact With Reactive Oxygen Species

Thioctic acid (TA) and its reduced form dihydrolipoic acid (DHLA) have recently gained somc recognition as useful biological antioxidants. In particular, the ability of DHLA to inhibit lipid peroxidation has been reported. In the present study, the effects of TA and DHLA on reactive oxygen species (ROS) generated in the aqueous phase have been investigated. Xanthine plus xanthine oxidase-generated superoxide radicals (O₂), detected by electron spin resonance spectroscopy (ESR) using DMPO as a spin trap. were eliminated by DHLA but not by TA. The sulhydryl content of DHLA, measured using Ellman's reagent decreased subsequent to the incubation with xanthine plus xanthine oxidase confirming the interaction between DHLA and O₂^-. An increase of hydrogen peroxide concentration accompanied the reaction between DHLA and O₂x, suggesting the reduction of O₂^- by DHLA. Competition of O₂^- with epinephrine allowed us to estimate a second order kinetic constant of the reaction between O₂^- and DHLA, which was found to be a 3.3 × 10⁵ M^-1 s^-1. On the other hand, the DMPO signal of hydroxyl radicals (HO ·) generated by Fenton's reagent were eliminated by both TA and DHLA. Inhibition of the Fenton reaction by TA was confirmed by a chemiluminescence measurement using luminol as a probe for HO ·. There was no electron transfer from Fe²⁺ to TA or from DHLA to Fe^{3 +} detected by measuring the Fe²⁺ -phenanthroline complex. DHLA did not potentiate the DMPO signal of HO · indicating no prooxidant activity of DHLA. These results suggest that both TA and DHLA possess antioxidant properties. In particular. DHLA is very effective as shown by its dual capability by eliminating both O₂^-; and HO ·.     

Thermal Inactivation of Aeromonas hydrophila As Affected by Sodium Chloride and Ascorbic Acid

The combined effects of sodium chloride (0, 1.0, 1.5, and 3.0%) and ascorbic acid (0, 1.0, and 2.0 mmol/liter) with mild heat (46°C) on the survival of Aeromonas hydrophila were evaluated. Because of the nonlinear nature of the survivor curves obtained, several equations yielding an R² (coefficient of multiple determination) of 1 were tested. The equation that most closely fit the curvature of the observed data set was a hyperbolic function. Equation coefficients were combined to obtain a so-called death value. This value (46.67% explained variance) was calculated by extracting the larger eigenvalue and the relative eigenvector from the correlation matrix of the coefficients. the effects of the experimental factors on the death value were described by a quadratic response surface model. Results revealed that the death value was not influenced by the presence of ascorbic acid. However, increased mortality resulted from the interaction between sodium chloride and ascorbic acid.     

百合皂苷的提取、纯化及其对自由基的清除作用

本文用正交实验法对百合总皂甙的提取工艺中温度、乙醇浓度、固液比例、回流时间和提取次数5个因素进行研究,优选出简便可靠、且适合工业化生产的百合总皂甙的提取工艺。其最佳提取工艺条件是:温度为60℃,乙醇浓度为70%,固液比例为1∶6,提取时间3h,提取次数3次。以溴邻苯三酚红(BPR)为显色剂,基于Co(II)H2O2体系反应产生的羟自由基(·OH)使溴邻苯三酚红(BPR)的颜色发生变化,采用756型紫外可见分光光度计测定其吸光度的变化值,研究百合皂苷在此体系中清除羟自由基的作用,对照实验表明:百合总皂苷提取物对羟自由基的清除作用比人参皂苷强。