Action of Cystine in the Cytotoxic Response of Escherichia Coli Cells Exposed to Hydrogen Peroxide

Cystine markedly enhanced the cytotoxic response of Escherichia coli cells to concentrations of hydrogen peroxide resulting in mode one killing, but displayed little effect in mode two killed cells. The effect of cystine was concentration-dependent over a range of 5-50 μM and did not further increase at higher levels. Cystine had similar effects in other bacterial systems.

In order to sensitize the cells to the oxidative injury, the amino acid must be present during exposure to the oxidant since no enhancement of the cytotoxic response can be observed in cystine pre-loaded cells. In addition, no further enhancement of cytotoxicity could be detected when cystine was added before and left during challenge with the oxidant. The enhancing effect of cystine on oxidative injury of E. coli cells appears to be directly mediated by the amino acid and in fact cysteic acid, the most likely oxidation product, had no effect on the killing of bacterial cells elicited by hydrogen peroxide. Other disulfide compounds such as oxidized glutathione, cystamine and dithionitrobenzoic acid only slightly increased the susceptibility of bacteria to the oxidant. The effect of the disulfides was not concentration-dependent over a range of 200-800 μM and was statistically significant only for cystamine.

Taken together, these results indicate that cystine markedly increases the cytotoxic response of bacteria to hydrogen peroxide and suggest that the amino acid might impair the cellular defence machinery against hydrogen peroxide. This effect may involve a thiol-disulfide exchange reaction at the cell membrane level.     

过氧化氢在水杨酸诱导的蚕豆气孔关闭中的作用

许多植物病原菌可通过气孔进入叶片组织,因此减小气孔开度有利于提高植物的抗性。我们通过表皮条分析和激光扫描共聚显微镜得到的证据表明在保卫细胞中过氧化氢可能是水杨酸信号的中间环节。SA可以浓度依赖的方式诱导气孔关闭（图1A）,H2O2也有类似的作用（图1B）。100μmol/L的水杨酸诱导的气孔关闭作用可明显地被20U/ml的过氧化氢酶或10μmol/L的Vc逆转,但CAT和Vc单独处理时诱导气孔开放的作用很微弱。单细胞中基于荧光探针DCFH的时间进程实验表明直接外加（图版I）或显微注射100μmol/L的SA均可诱导保卫细胞中H2O2产生,但以显微注射双蒸水作为对照时对DCFH荧光无影响（图版II）。这些结果暗示了植物被病原菌感染时可能通过产生H2O2导致气孔关闭而阻止病原菌继续通过气孔侵入。     

黄芪总黄酮对DNA损伤防护作用的研究

用DNA解旋荧光检测法(FADU)研究了黄芪总黄酮(TFA)对γ射线和H₂O₂所致V79细胞DNA链断裂的防护作用. 结果表明TFA对这两种损伤因子所致的DNA损伤均有不同程度的防护作用, 当TFA浓度达到0.4g/L和0.6g/L时, 分别对H₂O₂和γ射线所致的损伤有保护作用(P<0.05), 而浓度增至0.8g/L和1.2g/L时, 分别对两种因素所致的DNA链断裂损伤有非常显著的防护效果(P<0.01), 对H₂O₂的防护效果优于对γ射线.     

Free Radicals and the Ischemia-Evoked Extracellular Accumulation of Amino Acids in Rat Cerebral Cortex

The effects of free radical generating systems on basal and ischemia/reperfusion-evoked release of amino acids into cortical superfusates was examined in the rat using the cortical cup technique. Xanthine oxidase plus xanthine significantly enhanced GABA levels 358 fold over controls during 20 min of four vessel occlusion. Glutamate and phosphoethanolamine release following reperfusion were also elevated. Prostaglandin synthase plus arachidonic acid significantly enhanced the ischemia-evoked release of all amino acids (aspartate 360 fold; glutamate 433 fold; glycine 6 fold; GABA 689 fold; phosphoethanolamine 69 fold) and increased the pre-ischemic levels of glutamate, glycine and phosphoethanolamine. Administration of H₂O₂ plus ferrous sulfate significantly elevated both pre-ischemic amino acid release and ischemia-evoked release. A role for free radical generating systems in the development of ischemic injury is supported by the ability of superoxide dismutase plus catalase to reduce ischemia-evoked amino acid efflux into cortical superfusates. Thus, the species of free radical produced, as well as the amount generated, may alter the pattern of amino acid release under both ischemic and non-ischemic conditions.     

Role of Riboflavin and Certain Amino Acids in the Excystment of Schizopyrenus russelli*

SYNOPSIS. A simple medium, consisting of riboflavin and a mixture of L-glutamic acid, L-tryptophan, L-isoleucine, L-serine, and L-proline has been shown to induce rapid and mass scale excystment in Schizopyrenus russelli. Whereas percentage excystment was found to depend on the concentration of riboflavin and amino acids, more than 80% of cysts were found to excyst within 4 hr, when adequate amounts of these were supplied. Several individual amino acids, particularly L-glutamic acid, L-tryptophan, and L-proline, also supported considerable excystment, but riboflavin was always indispensable.     

衰老叶片和叶绿体中H_2O_2的累积与膜脂过氧化的关系

在自然衰老和ABA处理的叶片和叶绿体中活性氧H_2O_2均比对照明显增高。外加H_2O_2刺激水稻叶绿体膜脂过氧化作用。叶绿体的丙二醛含量随H_2O_2浓度、光照时间、光照强度及叶绿体完整性而变化。AsA、GSH、SOD、甘露醇和过氧化氢酶对外源H_2O_2引起的膜脂过氧化有缓解作用,Fe~(2+)有刺激作用。而H_2O_2对叶绿体过氧化损伤主要是转化为OH之故。     

Effects of Ionotropic Excitatory Amino Acid Receptor Antagonists on Glutamate Transport and Transport-Mediated Changes in Extracellular Excitatory Amino Acids in the Rat Striatum

Abstract: This study examined the effects of intrastriatal administration of ionotropic excitatory amino acid receptor antagonists on biochemical markers of excitatory amino acid transmission in the rat striatum. High-affinity glutamate uptake was measured ex vivo on striatal homogenates 15 min after the local administration of either 6,7-dinitroquinoxaline-2,3-dione (DNQX), a non-NMDA receptor antagonist, or dl -2-amino-5-phosphonopentanoic acid (AP5), a competitive NMDA antagonist, at various doses (10–500 pmol injected). DNQX induced a dose-dependent increase in glutamate uptake rate, related to an increase in the V _max of the transport process, whereas no significant change in glutamate uptake was detected after AP5 administration. Similar results were obtained from animals subjected to excitotoxic lesion of striatal neurons by kainate administration 15 days before the injection of DNQX or AP5. In a parallel series of experiments using in vivo microdialysis we showed that DNQX (10⁻⁵ M ) in the dialysis probe diminished by ∼30–40% the increases in the concentrations of glutamate and aspartate elicited by l - trans -pyrrolidine-2,4-dicarboxylic acid (1 m M ). These data suggest that presynaptic glutamate transmission in the rat striatum may undergo facilitatory autoregulatory processes involving ionotropic non-NMDA receptors and highlight the view that transporters for glutamate may be potent regulatory sites for glutamatergic transmission.     

拟南芥AtDAD1 超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H2O2抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H2O2有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

中国田鼠卵巢细胞gpt基因自发和亚砷酸钠诱发突变的分子分析

本文研究了亚砷酸钠对CHO-AS52细胞gpt基因的致突变作用。实验结果表明,亚砷酸钠能诱发该基因发生突变,且其突变频率随砷浓度的增加而增高。PCR分析指出,绝大多数亚砷酸钠诱发的CHO-AS52突变体的gpt基因完全缺失。在CHO-AS52细胞自发的、50μmol/L和100μmol/L亚砷酸钠诱发的突变体中,gpt基因完全缺失者所占比率分别为36.00％、54.72％及66.67％。对亚砷酸钠诱发的非缺失型gpt基因突变的PCR产物直接进行DNA序列分析表明,在9个突变细胞克隆中,有2个发生移码突变,其余7个突变细胞克隆的gpt基因结构未发现改变,碱基的改变可能发生在基因启动子区。     

The Tetraspanin CD9 Influences the Adhesion, Spreading, and Pericellular Fibronectin Matrix Assembly of Chinese Hamster Ovary Cells on Human Plasma Fibronectin

The role of CD9 in cell adhesion and spreading on adhesive proteins was investigated using a transfected Chinese hamster ovary (CHO) cell system. CD9 cell surface expression resulted in reduced adhesion and increased spreading on fibronectin (Fn). Whereas mock-transfected (mock CHO) and na?ve CHO cells assumed a typical fibroblast spindle shape morphology, CD9-transfected (CD9-CHO) cells were polygonal with many filipodial projections and exhibited a twofold greater surface area. The spread morphology of CD9-CHO cells, but not mock CHO cells, was inhibited by PB1 mAb blockade of alpha(5)beta(1), suggesting that the coexpression of alpha(5)beta(1) and CD9 influenced cell activity on Fn. The second extracellular loop of CD9 was implicated in regulation of adhesion since reduced CD9-CHO cell adhesion on Fn was reversed by either anti-CD9 antibody ligation to the second extracellular loop or with cells expressing a CD9 mutant lacking the second extracellular loop domain. Using cell adhesion assays and ELISA, we demonstrated CD9 binding to the HEP2/IIICS region of Fn. Finally, CD9 expression resulted in a twofold reduction in Fn-rich pericellular matrix assembly. Our observations show that CD9 dramatically influences CHO cell interactions with Fn and suggest that CD9 has an important role in modulating cell-extracellular matrix interactions.     

拟南芥AtDAD1超量表达植株对H2O2抗性的研究

构建拟南芥AtDAD1超量表达载体,以农杆菌介导的方法转化拟南芥哥伦比亚生态型,比较AtDAD1超量表达植株和野生型植株表现型的差异,以及两者对H₂O₂抗性的不同。实验显示,AtDAD1转基因拟南芥生长较野生型拟南芥更为强壮,对高浓度H₂O₂有较强的耐受力。测定两者糖含量,发现AtDAD1转基因拟南芥叶片糖的含量明显高于野生型拟南芥叶片。以上结果表明,AtDAD1基因可能参与植物生长发育,并可能在拟南芥抵抗凋亡的过程中发挥重要的作用。     

The Effect of Quercetin on the Accumulation of Carbohydrates and Amino Acids in Tomato Fruits

We studied the effects of low concentrations of quercetin on the contents of sugars and amino acids in ripe tomato (Lycopersicon esculentumMill.) fruits. In treated plants, the content of glucose increased by 1.5–4.5 times, whereas the total content of amino acids decreased by 1.5 times. The glucogenic amino acids, glutamic and aspartic acid, decreased most substantially, viz. by 1.7 and 1.6 times, respectively. The mechanism of the quercetin-induced enhancement of gluconeogenesis and suppression of glycolysis, both resulting in the accumulation of glucose, are discussed.     

Role of Glial Cells for the Basal and Ca2+-Dependent K+-Evoked Release of Transmitter Amino Acids Investigated by Microdialysis

The role of glial cells for the inactivation and synthesis of precursors for amino acid transmitters was studied in the brains of anesthetized rats in vivo using the microdialysis technique. The dialysis probes were inserted stereotactically into each neostriatum. One neostriatum was treated with the gliotoxin fluorocitrate, whereas the contralateral side served as a control. The basal efflux of amino acids, reflecting the extracellular level, was measured as well as the efflux during depolarization with 100 mM K+ in the dialysis stream. The potassium-evoked efflux of transmitter amino acids was calcium dependent and thus considered to reflect release from the transmitter pool. gamma-Aminobutyric acid (GABA) and glutamate release from the treated side was higher than the control value during the first 2-3 h, a result indicating an important role of glial cells in the inactivation of released transmitter. After 6-7 h with fluorocitrate, the release of glutamate was lower than the control value, a result indicating an important role of glial cells in the synthesis of precursors for the releasable pool of glutamate. The role of glutamine for the production of transmitter glutamate and GABA in vivo was further investigated by inhibiting glutamine synthetase with intrastriatally administered methionine sulfoximine. The release of gluatamate into the dialysis probe decreased to 54% of the control value, whereas the release of GABA decreased to 22% of the control value, a result indicating that glutamine may be more important for transmitter GABA than for transmitter glutamate.     

Cultivation of Microorganisms in the Cultural Medium Made from Squid Internal Organs and Accumulation of Polyunsaturated Fatty Acids in the Cells

The disposal and more efficient utilization of marine wastes is becoming increasingly serious. A culture media for microorganisms has been prepared from squid internal organs that are rich in polyunsaturated fatty acids (PUFAs). Both freshwater and marine bacteria grew well in this medium and some bacteria accumulated PUFAs in their lipids, suggesting uptake of exogenous PUFAs. Higher PUFA accumulations were observed in Escherichia coli mutant cells defective either in unsaturated fatty acid biosynthesis or fatty acid degradation, or both, compared to those without these mutations. Therefore, PUFA accumulation in cells can be improved by genetic modification of fatty acid metabolism in the bacteria. Squid internal organs would be a good source of medium, not only for marine bacteria but also for freshwater bacteria, and that this process may be advantageous to make efficient use of the fishery wastes and to produce PUFA-containing microbial cells and lipids.     

The Action of Hydrogen Peroxide on the Formation of Thiobarbituric Acid-Reactive Material From Microsomes, Liposomes Or From Dna Damaged By Bleomycin Or Phenanthroline. Artefacts in the Thiobarbituric Acid Test

Incubation of rat-liver microsomes, previously azide-treated to inhibit catalase, with H₂O₂ caused a loss of cytochrome P-450 but not of cytochrome b₅. This loss of P-450 was not prevented by scavengers of hydroxyl radical, chain-breaking antioxidants or metal ion-chelating agents. Application of the thiobarbituric acid (TBA) assay to the reaction mixture suggested that H₂O₂ induces lipid peroxidation, but this was found to be due largely or completely to an effect of H²O₂ on the TBA assay. By contrast, addition of ascorbic acid and Fe(III) to the microsomes led to lipid peroxidation and P-450 degradation: both processes were inhibited by chelating agents and chain-breaking antioxidants, but not by hydroxyl radical scavengers. H₂O₂ inhibited ascorbate/Fe (III)-induced microsomal lipid peroxidation, but part of this effect was due to an action of H₂O₂ in the TBA test itself. H₂O₂ also decreased the colour measured after carrying out the TBA test upon authentic malondialdehyde, tetraethoxypropane, a DNA-Cu²⁺/o-phenanthroline system in the presence of a reducing agent, ox-brain phospholipid liposomes in the presence of Fe(III) and ascorbate, or a bleomycin-iron ion/DNA/ascorbate system. Caution must be used in interpreting the results of TBA tests upon systems containing H₂O₂.     

Irradiation of Cells with Ultraviolet-A (320-400 nm) in the Presence of Cell Culture Medium Elicits Biological Effects Due to Extracellular Generation of Hydrogen Peroxide

Biological effects of ultraviolet A (UVA) irradiation have been ascribed to the photochemical generation of singlet oxygen. Not all effects described in the literature, however, are explicable solely by the generation of singlet oxygen, but rather resemble effects elicited by hydrogen peroxide (H 2 O 2 ). Here, we show that when cells are kept in cell culture media during exposure to UVA, stress kinases, including ERK 1 and ERK 2 as well as Akt (protein kinase B), are activated, whereas there is no or only minor activation when cells are kept in phosphate-buffered saline during irradiation. Indeed, the exposure of cell culture media to UVA (30 J/cm 2 ) results in the generation of significant amounts of H 2 O 2 , with concentrations of about 100 &#119 M. H 2 O 2 concentrations are at least three-fold higher in HEPES-buffered culture media after UVA irradiation. From experiments with solutions of riboflavin, tryptophan or HEPES, as well as combinations thereof, it is concluded that riboflavin mediates the photooxidation of either tryptophan or HEPES, resulting in the generation of H 2 O 2 . Thus, if signaling effects of UVA radiation are to be investigated in cell culture systems, riboflavin and HEPES/tryptophan should be avoided during irradiation because of artificial H 2 O 2 generation. It should be taken into account, however, that in vivo tryptophan and riboflavin might play an important role in the generation of reactive oxygen species by UVA as both substances are abundant in living tissues.     

Functional Coupling of the NK1 Tachykinin Receptor to Phospholipase D in Chinese Hamster Ovary Cells and Astrocytoma Cells

Abstract: In [³H]myristic acid-prelabeled Chinese hamster ovary cells stably expressing the rat NK₁ tachykinin receptor, the selective NK₁ agonist [Pro⁹]substance P ([Pro⁹]SP) time and concentration dependently stimulated the formation of [³H]phosphatidylethanol in the presence of ethanol. This [Pro⁹]SP-induced activation of phospholipase D (PLD) was blocked by NK₁ receptor antagonists and poorly or not mimicked by NK₂ and NK₃ agonists, respectively. In confirmation of previous observations, [Pro⁹]SP also stimulated the hydrolysis of phosphoinositides, the release of arachidonic acid, and the formation of cyclic AMP (cAMP). All these [Pro⁹]SP-evoked responses could be mimicked by aluminum fluoride, but they remained unaffected in cells pretreated with pertussis toxin, suggesting that a G_i/G_o protein is not involved in these different signaling pathways. The activation of PLD by [Pro⁹]SP was sensitive to external calcium and required an active protein kinase C because the inhibition of this kinase (Ro 31-8220) or its down-regulation (long-term treatment with a phorbol ester) abolished the response. In contrast, a cAMP-dependent process was not involved in the activation of PLD because the [Pro⁹]SP-evoked response was neither affected by Rp-8-bromoadenosine 3′,5′-cyclic monophosphorothioate nor mimicked by cAMP-generating compounds (cholera toxin or forskolin) or by 8-bromo-cyclic AMP. A functional coupling of NK₁ receptors to PLD was also demonstrated in the human astrocytoma cell line U 373 MG stimulated by SP or [Pro⁹]SP. These results suggest that PLD activation could be an additional signaling pathway involved in the mechanism of action of SP in target cells expressing NK₁ receptors.     

On the Relative Content of G,C Bases in Codons of Amino Acids Corresponding to Class I and II Aminoacyl-tRNA Synthetases

We have analyzed the relative G,C content from protein coding regions of 530 organisms and found that the ratio of the G,C content of the codons of the amino acids correspondingto Class II and Class I aminoacyl-tRNA synthetases decreases in a statistically significant way from prokaryotes to animals.This can be interpreted assuming that an initial asymmetry between the G,C content of codons of Class I and II amino acids existed and has decreased in the course of evolution.