Modulation of antioxidant and detoxification responses induced by lipoic acid in the Pacific white shrimp Litopenaeus vannamei (Boone, 1931) subjected to hypoxia and re-oxygenation

The effect of lipoic acid supplementation and moderate hypoxia (3?mg/L), followed by re-oxygenation, was analyzed in terms of antioxidant and oxidative damage responses in juvenile shrimp Litopenaeus vannamei. Lipoic acid (LA)-enriched rations (D1: 76.4?±?6.4; D2: 196.4?±?70.2; and D3: 397.2?±?79.97?mg LA/kg) were offered to shrimps. A control group without LA adding was also run. After 45?days, LA-enriched ration increased the activity of the detoxifying enzyme glutathione-S-transferase in gills. Total antioxidant capacity against peroxy radicals was augmented in gills and hepatopancreas at doses D2 and D3. Doses D1 and D2 of LA reduced the levels of oxidative damage (lipid peroxidation) in gills and hepatopancreas. The results showed that certain LA doses (particularly D2) improved not only antioxidant responses but also weight gain. It can be concluded that LA triggered antioxidant and detoxification protection in L. vannamei, allowing the shrimp to cope with environmental stressful factors.     

Association of GSTT1 null,XPD 751 CC and XPC 939 CC genotypes with increased levels of genomic damage among hospital pathologists

Context: Hospital workers are at risk for genotoxic damage following occupationally exposure to xenobiotics. Pathologists are exposed to chemicals during their use in health care environments, particularly throughout inhalation of airborne agents, absorption through skin or contact with the patient’s body fluids.

Objective: We evaluated the level of genomic damage in a sample of 61 hospital pathologists (occupationally exposed to antineoplastic drugs and sterilizing agents) and 60 control subjects.

Materials and methods: Lymphocytes were analyzed by SCEs and CAs assays and genotyped for GSTT1, GSTM1, CYP1A1 Ile/Val, XPD (A751C) and XPC (A939C) gene polymorphisms.

Results: Pathologists showed significantly higher frequencies of SCEs and CAs with respect to control subjects. GSTT1 null genotype was found to be associated with higher SCEs and CAs frequencies, whereas XPD 751?CC and XPC 939?CC genotypes only with a higher level of SCEs.

Discussion and conclusions: The SCEs and CAs results are consistent with other published data, placing hospital workers as a category at risk for genotoxic damage caused by chronic exposure to xenobiotics. The higher levels of cytogenetic damage observed among GSTT1 null, XPD 751 and XPC 939?CC homozygote subjects confirm the importance of the genetic polymorphisms analysis associated to genotoxicological studies.     

Specific motifs of the V-ATPase a2-subunit isoform interact with catalytic and regulatory domains of ARNO

We have previously shown that the V-ATPase a2-subunit isoform interacts specifically, and in an intra-endosomal acidification-dependent manner, with the Arf-GEF ARNO. In the present study, we examined the molecular mechanism of this interaction using synthetic peptides and purified recombinant proteins in protein-association assays. In these experiments, we revealed the involvement of multiple sites on the N-terminus of the V-ATPase a2-subunit (a2N) in the association with ARNO. While six a2N-derived peptides interact with wild-type ARNO, only two of them (named a2N-01 and a2N-03) bind to its catalytic Sec7-domain. However, of these, only the a2N-01 peptide (MGSLFRSESMCLAQLFL) showed specificity towards the Sec7-domain compared to other domains of the ARNO protein. Surface plasmon resonance kinetic analysis revealed a very strong binding affinity between this a2N-01 peptide and the Sec7-domain of ARNO, with dissociation constant K_D = 3.44 × 10⁻⁷ M, similar to the K_D = 3.13 × 10⁻⁷ M binding affinity between wild-type a2N and the full-length ARNO protein. In further pull-down experiments, we also revealed the involvement of multiple sites on ARNO itself in the association with a2N. However, while its catalytic Sec7-domain has the strongest interaction, the PH-, and PB-domains show much weaker binding to a2N. Interestingly, an interaction of the a2N to a peptide corresponding to ARNO's PB-domain was abolished by phosphorylation of ARNO residue Ser₃₉₂. The 3D-structures of the non-phosphorylated and phosphorylated peptides were resolved by NMR spectroscopy, and we have identified rearrangements resulting from Ser₃₉₂ phosphorylation. Homology modeling suggests that these alterations may modulate the access of the a2N to its interaction pocket on ARNO that is formed by the Sec7 and PB-domains. Overall, our data indicate that the interaction between the a2-subunit of V-ATPase and ARNO is a complex process involving various binding sites on both proteins. Importantly, the binding affinity between the a2-subunit and ARNO is in the same range as those previously reported for the intramolecular association of subunits within V-ATPase complex itself, indicating an important cell biological role for the interaction between the V-ATPase and small GTPase regulatory proteins.     

即时浸酸显著降低滞育家蚕卵的还原型与氧化型谷胱甘肽比值

即时浸酸在阻止家蚕Bombyx mori卵滞育发动的同时, 显著提高了家蚕卵H₂O₂含量。还原型谷胱甘肽（reduced glutathione, GSH）与氧化型谷胱甘肽（oxidized glutathione, GSSG）的比值是一种氧化胁迫状态的动态指标。为了调查即时浸酸是否造成滞育家蚕卵氧化胁迫, 本研究利用分光光度法分别测定了滞育家蚕卵和5 min即时浸酸滞育家蚕卵中GSH和GSSG含量以及谷胱甘肽转移酶（glutathione-S-transferase, GST）活性。结果表明: 处理后24 h, 即时浸酸处理家蚕卵的总谷胱甘肽（GSH+2GSSG）含量、 GSH含量、 GSSG含量、 GSH/GSSG比值和GST活性分别相当于同期滞育家蚕卵的204%, 78%, 550%, 14%和97%。据此推测, 即时浸酸在阻止滞育发动的同时, 可能通过促进GSH氧化为GSSG, 而显著降低了GSH/GSSG比值, 使家蚕卵处于过氧化状态。     

中国北方人群谷胱甘肽转硫酶P1基因多态性及其与胃癌遗传易感性的关系

为了分析中国北方人群谷胱甘肽转硫酶P1基因(glutathione-S-transferase P1, GSTP1)多态性分布, 同时探讨GSTP1基因多态性及其与幽门螺杆菌(H. pylori)既往感染联合作用对胃癌发病风险的影响, 采用多聚酶链反应-限制性片段长度多态性(PCR-RFLP)技术检测1,612例外周血DNA GSTP1的多态性; 采用ELISA方法检测血清H. pylori IgG。结果显示, (1) 中国北方人群GSTP1基因Val等位基因分布频率为22%, 胃癌高、低发区GSTP1 Val等位基因分布频率有显著性差异(0.23/0.20); (2) 以Ile/Ile基因型为参照组与其他两种基因型比较进行胃癌的风险分析, 结果显示携带Val/Val基因型的个体患胃癌的危险性最大, 其OR为5.588 (3.256 ~ 9.591); 携带Val等位基因的个体患胃癌危险性是非携带Val等位基因个体的1.587倍; (3) 以H. pylori IgG(-)并携带GSTP1基因纯合野生型(Ile/ Ile)的个体为参照, H. pylori IgG(+)并携带纯合多态基因型(Val/Val)的个体患胃癌的风险最高, OR为17.571(6.207 ~ 49.742)。说明GSTP1 Val等位基因的分布存在人群及地区差异。携带GSTP1 Val等位基因的个体胃癌发病风险增高。GSTP1 Val等位基因纯合型与H. pylori感染对于胃癌的发生具有交互作用。     

转GST和CAT1基因水稻根系抗氧化系统对干旱高温胁迫的响应(英文)

以携带谷胱甘肽转移酶(GST)和过氧化氢酶(CAT1)的转基因水稻和非转基因水稻(Oryza sativa L.) 品种'中花11'的根系为材料, 比较分析了二者在PEG 6000、38℃及PEG 6000和38℃复合胁迫下抗氧化系统特别是抗坏血酸-谷胱甘肽循环系统的变化.结果显示, 6% PEG处理时,转基因水稻的CAT、GST、超氧化物歧化酶(SOD)、谷胱甘肽还原酶(GR)和脱氢抗坏血酸还原酶(DHAR)的活性都显著高于非转基因水稻;38℃处理时,前者的CAT、GST、SOD和GR的活性则显著低于后者;6% PEG和38℃复合处理时,前者的CAT、GST、抗坏血酸过氧化物酶(APX)和DHAR的活性也都显著高于后者,但前者的SOD和GR活性则显著低于后者.6%PEG 诱导的转基因水稻根系的抗坏血酸氧还状态显著低于非转基因水稻,但二者的谷胱甘肽氧还状态无显著差异; 而6% PEG和38℃同时处理时,转基因水稻的谷胱甘肽氧还状态则显著高于非转基因水稻,但二者的抗坏血酸氧还状态差异不显著.研究发现,干旱和高温复合胁迫时,转基因水稻和非转基因水稻的抗氧化组分的变化均不等于这2种单一胁迫的叠加;GST和CAT1基因的转入对水稻抗氧化系统内源功能相关组分尤其是抗坏血酸-谷胱甘肽循环系统产生了一定的影响,两种水稻的根系可能利用不同的抗氧化组分调节机制对这些胁迫做出应答.     

The cytosolic subunit p67 of the NADPH-oxidase complex does not bind NADPH

The NADPH-oxidase of phagocytic cells is a multicomponent enzyme that generates superoxide. It comprises a membrane flavocytochrome b₅₅₈ and four cytosolic proteins; p67^phox, p47^phox, p40^phox and Rac. The NADPH-binding site of this complex was shown to be located on the flavocytochrome b₅₅₈. However, a number of studies have suggested the presence of another site on the p67^phox subunit which is the key activating component. Using several approaches like tryptophan quenching fluorescence measurement, inhibition by 2′,3′-dialdehyde NADPH, and free/bound NADPH concentration measurements, we demonstrate that no NADPH binds on p67^phox, thus definitively solving the controversy on the number and location of the NADPH-binding sites on this complex.     

Trypanothione: A unique bis-glutathionyl derivative in trypanosomatids

Background

Trypanosomatids are early-diverging eukaryotes devoid of the major disulfide reductases – glutathione reductase and thioredoxin reductase – that control thiol-redox homeostasis in most organisms. These protozoans have evolved a unique thiol-redox system centered on trypanothione, a bis-glutathionyl conjugate of spermidine. Notably, the trypanothione system is capable to sustain several cellular functions mediated by thiol-dependent (redox) processes.

Scope of review

This review provides a summary of some historical and evolutionary aspects related to the discovery and appearance of trypanothione in trypanosomatids. It also addresses trypanothione's biosynthesis, physicochemical properties and reactivity towards biologically-relevant oxidants as well as its participation as a cofactor for metal binding. In addition, the role of the second most abundant thiol of trypanosomatids, glutathione, is revisited in light of the putative glutathione-dependent activities identified in these organisms.

Major conclusions

Based on biochemical and genome data, the occurrence of a thiol-redox system that is strictly dependent on trypanothione appears to be a feature unique to the order Kinetoplastida. The properties of trypanothione, a dithiol, are the basis for its unique reactivity towards a wide diversity of oxidized and/or electrophilic moieties in proteins and low molecular weight compounds from endogenous or exogenous sources. Novel functions have emerged for trypanothione as a potential cofactor in iron metabolism.

General significance

The minimalist thiol-redox system, developed by trypanosomatids, is an example of metabolic fitness driven by the remarkable physicochemical properties of a glutathione derivative. From a pharmacological point of view, such specialization is the Achilles' heel of these ancient and deadly parasites. This article is part of a Special Issue entitled Cellular functions of glutathione.     

Do-it-yourself histidine-tagged bovine enterokinase: A handy member of the protein engineer's toolbox

Enterokinase, a two-chain duodenal serine protease, activates trypsinogen by removing its N-terminal propeptide. Due to a clean cut after the non-primed site recognition sequence, the enterokinase light chain is frequently employed in biotechnology to separate N-terminal affinity tags from target proteins with authentic N-termini. In order to obtain large quantities of this protease, we adapted an in vitro folding protocol for a pentahistidine-tagged triple mutant of the bovine enterokinase light chain. The purified, highly active enzyme successfully processed recombinant target proteins, while the pentahistidine-tag facilitated post-cleavage removal. Hence, we conclude that producing enterokinase in one's own laboratory is an efficient alternative to the commercial enzyme.