Myelin-Associated Calpain II

Anti-chicken muscle calpain (calcium-activated neutral protease) antibody (ACAb) was found to be absorbed by purified human brain myelin when titrated by enzyme-linked immunosorbent assay, suggesting the close association of the protease with myelin. To confirm this, calcium-dependent protease was extracted from myelin membrane and purified on a phenyl Sepharose CL 4B column. It was activated by calcium ion in the millimolar range, and therefore was determined to be calpain II. This enzyme fraction was electrophoresed and immunostained with ACAb, resulting in staining as a single band with apparent molecular weight of 80K. This protease degraded exogenous myelin-associated glycoprotein. From the present results, it is suggested that calpain is bound to myelin membrane and involved in the turnover of myelin proteins.     

Inhibition of Calpains by Calmidazolium and Calpastatin

Abstract

Several prolinal derivatives were synthesized and examined for their inhibitory activity on post-proline cleaving enzymes from Flavobacterium meningosepticum and bovine brain and their possible properties as nootropic agents. Almost all the compounds tested inhibited the activity of both enzymes at low IC₅₀ values of the order of nM, but a specificity difference was observed with alkylacyl-prolinal derivatives which strongly inhibited only the bacterial enzyme. Prolyl-prolinal derivatives were the most effective inhibitors for both enzymes. In the passive avoidance test using amnesic rats experimentally induced with scopolamine, the prolinal derivatives that have potent inhibitory activity toward post-proline cleaving enzymes showed also strong anti-amnesic activities at doses of 10 ~ 1000μg/kg, i.p. Some of the compounds showed a bell-shape dose dependency. These results suggest that the post-proline cleaving enzymes play an important role in the regulation of learning and memory consolidation in the brain and inhibitors of these enzymes are suggested as possible candidates for nootropic agents, particularly for an anti-amnesic drug.     

Effects of detergents on Ca2+-activated neural proteinase activity (calpain) in neural and non-neural tissue: A comparative study

Calcium activated neutral proteinase (mcalpain) activity was determined in brain and other tissue of rat. More than 60% of the brain mcalpain activity was present in the particulate fraction while only 30% was in cytosol. In contrast, particulate fractions of liver, kidney, muscle, and heart contained about 8–12% of tissue mcalpain activity while 88% was present in cytosol. Removal of the endogenous inhibitor calpastatin increased the tissue mcalpain activity severalfold. Triton X-100 and deoxycholate (DOC) stimulated the neural calpain activity by ten-fold while activity in non-neural tissue was unaffected. Incubation with other detergents, e.g. Triton N-57 and thioglucopyranoside, stimulated brain calpain activity five-fold while Brij-35 did not have any effect. Sodiumdodecylsulphate (SDS), on the other hand, inhibited the enzyme activity. Brain contained the lowest calpain activity compared to non-neural tissue. The calpain activity in muscle, kidney and heart was three-fold greater than liver. Immunoblot identification of the enzyme revealed that calpain was predominantly in the particulate fraction and less in cytosol of brain while it was present mainly in cytosol and less in the pellet fractions of non-neural tissue.     

Effects of Cadmium on Antioxidant Enzyme Activities in Sugar Cane

Sugar cane (Saccharum officinarum L. cv. Copersucar SP80-3280) seedlings were grown in nutrient solution with varying concentrations (0, 2 and 5 mM) of cadmium chloride for 96 h. Leaves were analysed for catalase (CAT), glutathione reductase (GR) and superoxide dismutase (SOD) activities. Although a clear effect of CdCl₂ on plant growth was observed, the activity of SOD was not altered significantly. However, the CAT activity decreased as the concentration of CdCl₂ increased. GR exhibits a significant increase in activity at 2 and 5 mM CdCl₂. CAT and SOD isoenzymes were further characterised by analysis in non-denaturing PAGE. Activity staining for SOD revealed up to seven isoenzymes in untreated control and 2 mM CdCl₂ treated plants, corresponding to Cu/Zn-SOD isoenzymes. At 5 mM CdCl₂, only six Cu/Zn-SOD isoenzymes were observed. No Fe-SOD and Mn-SOD isoenzymes were detected. For CAT, one band of activity was observed.     

Comparison of the Effect of Calpain Inhibitors on Two Extralysosomal Proteinases: The Multicatalytic Proteinase Complex and m-Calpain

Abstract: The potencies of three peptide aldehyde inhibitors of calpain (calpain inhibitors 1 and 2 and calpeptin) as inhibitors of four catalytic activities of the multicatalytic proteinase complex (MPC) were compared with their potencies as inhibitors of m-calpain. The chymotrypsinlike activity (cleavage after hydrophobic amino acids) and the caseinolytic activity (degradation of β-casein) of MPC were strongly inhibited by calpain inhibitors 1 and 2 (IC₅₀ values in the low micromolar range). Cleavage by MPC after acidic amino acids (peptidylglutamyl-peptide bond hydrolyzing activity) and basic amino acids (trypsinlike activity) was inhibited less effectively, declining moderately with increasing concentrations of calpain inhibitors 1 and 2. Calpeptin only weakly inhibited the four MPC activities, yet was the most potent inhibitor of m-calpain. These results indicate that caution must be exercised when calpain inhibitors 1 and 2 are used to infer calpain function. Calpeptin may be a better choice for such studies, although its effect on other cysteine or serine proteinases remains to be determined.     

Antioxidant Activities of a Low Etherified Pectin from the Seagrass Zostera marina

In experiments on male white mice we initiated the induction of peroxidation of lipids by lead acetate, tetrachloromethane, sodium nitrite, or SOVOL (a mixture of polychlorinated biphenyls). Then the animals received intragastral injections of pectin isolated from the seagrass Zostera marina, in the form of a 1% gel, at 100 mg/kg for 2 weeks. It has been found that the low etherified pectin normalizes the level of malonic dialdehyde and the activity of glutathione reductase and glutathione peroxidase in the liver.     

亚硒酸钠对小麦幼苗中谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及谷胱甘肽含量的影响

用1.0 mg·L-1的亚硒酸钠根施小麦幼苗,测定亚硒酸钠对谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性以及还原性谷胱甘肽含量的结果表明,外源亚硒酸钠对麦苗地上部的谷胱甘肽过氧化物酶和谷胱甘肽转硫酶活性均有诱导作用,使麦苗体内的谷胱甘肽含量水平增加.     

Activities of Antioxidant Systems During Germination of Chenopodium rubrum Seeds

The activities of superoxide-dismutase (SOD), catalase (CAT) and peroxidase (POD), and concentrations of glutathione and ascorbate have been studied during the first stages of germination in Chenopodium rubrum L. seeds. The highest CAT and SOD activity was found prior to radicle protrusion, while POD activity was maximal at the time of radicle protrusion and seedling development, new POD isozymes simultaneously appearing. The concentrations of total, reduced and oxidized glutathione showed similar changes during germination, the highest values being detected at the time of radicle protrusion. Ascorbic acid was present in the seeds in a detectable concentration only at the time preceding radicle protrusion, while its oxidized form dehydroascorbic acid was detected during the whole germination period studied. Gibberellic acid (GA₃, 160 M) had no effect on germination percentage, but in presence of GA₃, SOD and CAT activity notably increased prior to radicle protrusion, and oxidized glutathione concentration decreased in further germination.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25-2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

A Simultaneous Visualization of the Antioxidant Enzymes Glutathione Peroxidase and Catalase on Polyacrylamide Gels

A simple and sensitive method for the simultaneous visualization of glutathione peroxidase and catalase on polyacrylamide gels is described. The procedure included: (I) running samples on a 7. 5% polyacryla-mide gel, (2) soaking the gel in a certain concentration of reduced glutathione (0.25–2.0 mM). (3) soaking the gel in GSH plus H_zO_z or cumene hydroperoxide, (4) finally staining with a 1% ferric chloride I% potassium ferricyanide solution. The best concentration of glutathione for simultaneous visualization of glutathione peroxidase and catalase was 0.25rnM; I.5mM glutathione was the best concentration for visualization of glutathione peroxidase alone. The method is sensitive enough to detect catalase and glutathione peroxidase in mouse liver homogenates and also it is specific for glutathione peroxidase since other peroxidases such as lactoperoxidase, horseradish peroxidase and glutathione S-transferase cannot be visualized. Using this method, it was found that unlike catalase. glutathione peroxidase is heat resistant (68°C. 1min), but sensitive to 10mM sodium iodoacetate.     

低氧胁迫对黄瓜幼苗根系无氧呼吸酶和抗氧化酶活性的影响

对两个抗低氧胁迫能力不同的黄瓜品种进行营养液水培,研究了低氧胁迫下植株根系中无氧呼吸酶和抗氧化酶活性的变化。结果表明,低氧胁迫下,黄瓜植株生长受到抑制,鲜重和干重显著降低,根系中蛋白质含量降低,而根系中乳酸脱氢酶（LDH）、丙酮酸脱羧酶（PDC）、乙醇脱氢酶（ADH）、超氧化物歧化酶（SOD）、过氧化物酶（POD）和过氧化氢酶（CAT）活性在低氧胁迫下显著提高,且提高的幅度与品种抗低氧胁迫能力的强弱有关,与“中农8号”相比,抗低氧性胁迫能力较强的“绿霸春四号”根系内LDH活性增幅较小,而ADH、PDC、SOD、POD和CAT活性增幅较大。说明较高的ADH、PDC、SOD、POD、CAT活性和较低的LDH活性有利于增强幼苗植株抗低氧胁迫的能力。     

NaCl 胁迫对甘草生长及抗氧化酶活性的影响

目的:通过分析不同浓度NaCl胁迫下甘草生长和抗氧化酶活性的变化探讨甘草对盐分胁迫的适应性机理;方法:采用不同浓度的NaCl溶液(配成Hoagland液)处理盆栽一年生甘草,分别于35d、70d和105d取样,测定甘草株高、地上部分鲜、干重、根鲜、干重及甘草叶片SOD、POD、CAT活性,分析各生长指标与抗氧化酶活性的相关性;结果:NaCl胁迫70d和105d,0.6%和0.9%处理组的株高、地上部分鲜、干重及根鲜、干重均显著低于CK,SOD、POD及CAT活性均显著高于CK,经相关性分析得知,SOD、POD及CAT活性与各生长指标均负相关,其中POD活性与各生长指标极显著负相关;结论:甘草对NaCl胁迫的响应有胁迫时间和浓度的依赖性,在遭遇胁迫时,通过改变自身的生长和提高抗氧化酶活性,来提高机体的抗盐能力。     

Lipid Peroxidation and Antioxidant Enzyme Activities in Vascular and Alzheimer Dementias

It has been reported that oxidative stress may play a role in the pathogenesis of dementia of the Alzheimer type (AD) and the cerebral ischemia which causes vascular dementia (VD). We measured malondialdehyde (MDA) levels and superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GPx) and glutathione reductase (GR) activities in blood samples from patients with AD and VD and in healthy non-demented controls (CTR) which similar ages to the patients, in order to evaluate the degree of oxidative stress in patients with AD and VD. A sample of 150 subjects consisting of 50 patients with AD; 50 patients with VD and 50 CTR, aged from 65 to 85 years on, was analyzed. Most of the changes observed were in SOD activity and MDA levels. Catalase activity were least affected. Significant differences were observed in SOD and GR activity between males and females in CRT and in patients with AD, but not in VD. We have found a decrease in antioxidant enzymes activities (SOD, CAT, GPx and GR) in patients with AD and VD and significant differences were observed between CRT and AD patients for ages from 65 to 74, 75 to 84 and from 85 years to 94 years in SOD activity and MDA levels (P < 0.001). MDA levels increase with age in VD, AD and CTR. No significant variation with respect to sex were detected, but significant variations in MDA levels were detected between CRT and patients with VD and AD (P < 0.001). We conclude that oxidative stress plays an important role in the brain damage for both AD and VD, being observed higher levels of oxidative stress for AD that for VD.     

Association of Polymorphic Markers of the Antioxidant Enzyme Genes with Diabetic Polyneuropathy in Type 1 Diabetes Mellitus

The allele and genotype frequency distributions of polymorphic markers of genes coding for antioxidant enzymes were compared for type 1 diabetes mellitus patients with or without diabetic polyneuropathy (DPN). The groups (total 180 patients) had nonoverlapping (polar) phenotypes. Group DPN+ included 86 patients with DPN and diabetic record no more than 5 years. Control group DPN– included patients without DPN and diabetic record of at least 10 years. Comparative analysis with Fisher's exact test revealed a significant difference in allele and genotype frequency distributions of the (–262) polymorphic marker of the CAT gene. Polymorphic markers C1167T of the CAT gene, Pro/Leu of the GPX1 gene, 0/+ of the GSTT1 gene, and 0/+ of the GSTM1 gene showed no significant difference in allele or genotype frequency distribution. On this evidence, these markers were not associated with DPN in the sample examined.     

PFOS对再生东亚三角涡虫抗氧化酶活性的影响

为了探究全氟辛烷磺酸(Perfluorooctane sulfonate,PFOS)对再生涡虫抗氧化酶活性的影响,以再生中的涡虫作为实验材料,采用酶活性的测定的方法来探究PFOS对再生中东亚三角涡虫中抗氧化酶活性的影响。结果表明,PFOS刺激涡虫产生应激反应。在再生早期,PFOS胁迫会造成涡虫的氧化损伤和脂质的过氧化,并表现出随着PFOS浓度的升高,涡虫氧化损伤程度加剧的趋势;再生5 d时,机体产生抗氧化的反应来消除体内的氧自由基;在再生的后期,由于涡虫对药物的耐受作用,涡虫的抗氧化反应不显著。     

铅胁迫对3种藓类植物抗氧化酶活性的影响

采用水培实验研究了重金属Pb对3种藓类植物尖叶拟船叶藓(Dolichomitriopsis diversiformis)、湿地匍灯藓(Plagiomnium acutum)和匍枝青藓(Brachythecium procumbens)的抗氧化酶(SOD、POD和CAT)活性的影响.结果表明,(1)在1~200 mg·L-1的铅胁迫浓度范围内,Pb对3种藓类植物的SOD活性均具有显著的促进作用,对Pb胁迫的种间敏感性差异为尖叶拟船叶藓>湿地匍灯藓>匍枝青藓;(2)POD活性对Pb胁迫的反应趋势有一定差别,尖叶拟船叶藓和匍枝青藓的POD活性随Pb浓度的增加先升后降,湿地匍灯藓的POD活性变化较为平缓,种间敏感性差异为匍枝青藓>尖叶拟船叶藓>湿地匍灯藓;(3)在100 mg·L-1时Pb对湿地匍灯藓CAT活性表现为显著促进作用外,其余在低浓度下显示无显著影响,在高浓度下表现为抑制效应,种间敏感性差异为尖叶拟船叶藓>匍枝青藓>湿地匍灯藓.3种藓类植物的抗氧化酶体系中对Pb胁迫的抵抗起关键作用的是SOD,可作为藓类植物受Pb胁迫的敏感生理指标.3种植物中对Pb胁迫的反应较为敏感的是尖叶拟船叶藓.     

Glutathione metabolism in primate lenses: A phylogenetic study of glutathione synthesis and glutathione redox cycle enzyme activities

Lens wet weights, soluble protein, and activities of γ-glutiamylcysteine synthetase, glutathione synthetase, glutathione peroxidase, and glutathione reductase were determined in primate lenses. The primary sources of lenses were middle-aged adult animals. The Primates, from 23 genera, were categorized into six superfamilies: hominoids (five species), Old World monkeys (seven species), New World monkeys (five species), tarsiers (two species), lemurs (six species), and lorisids (three species). Significant differences between various groups or combinations of groups were noted for γ-glutamylcysteine synthetase, glutathione peroxidase, and glutathione reductase activities. Lenticular γ-glutamylcysteine synthetase activity was very low in the Old World simian lenses and highest in the prosimians. Glutathione peroxidase activity was extraordinarily high in lenses of Old World monkeys. Glutathione reductase activity was low in all the prosimians but tenfold higher in hominoid lenses with intermediate values in monkeys of both the Old World and New World. Glutathione synthetase activity was variable, and no clear pattern which might be useful for primate classification was noted. Lenticular activity ratios of glutathione synthetase:γ-glutamylcysteine synthetase were highest in the Old World simians and lowest in the prosimians. These data with emphasis upon Aotus and the tarsiers were examined with regard to phylogenetic relationships. © 1994 Wiley-Liss, Inc.     

NaCl胁迫对甘草生长及抗氧化酶活性的影响

目的：通过分析不同浓度NaCl胁迫下甘草生长和抗氧化酶活性的变化探讨甘草对盐分胁迫的适应性机理;方法：采用不同浓度的NaCl溶液（配成Hoagland液）处理盆栽一年生甘草,分别于35d、70d和105d取样,测定甘草株高、地上部分鲜、干重、根鲜、干重及甘草叶片SOD、POD、CAT活性,分析各生长指标与抗氧化酶活性的相关性;结果：NaCl胁迫70d和105d,0．6％和0．9％处理组的株高、地上部分鲜、干重及根鲜、干重均显著低于CK,SOD、POD及CAT活性均显著高于CK,经相关性分析得知,SOD、POD及CAT活性与各生长指标均负相关,其中POD活性与各生长指标极显著负相关;结论：甘草对NaCl胁迫的响应有胁迫时间和浓度的依赖性,在遭遇胁迫时,通过改变自身的生长和提高抗氧化酶活性,来提高机体的抗盐能力。     

Impact of Oestradiol and Progesterone on Antioxidant Activity in Normal Human Breast Epithelial Cells in Culture

The risk of developing breast cancer increases after long term use of oestrogen and progestagen, and carcinogenesis in the breast is partly due to oxidative damage to DNA bases. Therefore, we studied the effects of 17 β-oestradiol and progesterone on the antioxidative status and the vulnerability to oxidative stress exhibited by normal human breast epithelial cells in culture. After exposure to hydrogen peroxide, cells grown with oestradiol alone or with both oestradiol and progesterone showed significantly decreased viability compared to cells grown in medium without added hormones. There was, however, no difference in hydrogen peroxide degradation rate between controls and hormone treated cultures. When desferrioxamine was added, the viability increased and the hydrogen peroxide degradation rate decreased. The levels of several antioxidants were altered in cells grown in the presence of oestradiol and progesterone: the concentrations of glutathione reductase and catalase decreased significantly while the levels of glutathione peroxidase and reduced glutathione did not change. The alterations in enzyme activity and cell vulnerability were more pronounced in cultures treated with a combination of oestradiol and progesterone.

We conclude that the redox balance in the cultured normal human breast epithelial cells was altered by treatment with oestradiol and progesterone, and that this change led to the increased death of cells subsequently exposed to hydrogen peroxide. This effect may have implications for sex hormone dependent diseases of the breast.     

Activities of Antioxidant Enzymes during Senescence of Prunus Armeniaca Leaves

During the period of senescence of apricot leaves changes in photosynthetic pigment contents and in the activities of some antioxidant enzymes (superoxide dismutase, catalase, peroxidase and ascorbate peroxidase) were analysed. Significant changes in pigment contents were, in most cases, correlated with changes in activities of the antioxidant enzymes. Modifications in superoxide dismutase and catalase isoform patterns were also observed during the progression of senescence. Both enzyme activities and isoenzyme patterns proved to be genotype-dependent.