蛋白质羰基化与衰老

不同来源的活性羰基类物质(reactive carbonyl species,RCS),主要是一些不饱和醛,如:4-羟基壬烯醛(4-hydroxy-trans-2-nonenal,HNE)、丙烯醛(aerolein,ACR)、丙二醛(malondialde-hyde,MDA)等,在生物体系中扮演中非常重要的作用,在氧化剂和自由基攻击生物体系而引起的细胞毒性过程中,RCS便是直接的致病因子[1].     

蛋白质氧化羰基化和肌肽的保护作用

目的 探讨肌肤的抗氧化、抗衰老作用的途径和机制。方法用H2O2诱导小鼠新鲜脑组织使其产生氧化碳基化蛋白，用DNPH法和定磷法检测碳基化蛋白的含量和氧化磷酸化水平。结果 在加入肌肤的保护组，碳基化蛋白含量低于损伤组，氧化磷酸化水平高于损伤组，各组差异明显。结论 肌肤对蛋白质氧化碳基化有明显的保护作用。     

分枝杆菌中原核类泛素蛋白化靶蛋白的功能分析

分枝杆菌中存在一种类似真核生物泛素的小分子蛋白,称为原核类泛素蛋白（prokaryotic ubiquitinlike protein,Pup）,蛋白被Pup共价标记后,或被蛋白酶体降解或参与修饰后的调节作用,可影响细菌活动的各个环节.结核分枝杆菌中目前发现可被Pup标记的共有58种蛋白;耻垢分枝杆菌被标记了41种蛋白,其中38种都可以在结核分枝杆菌中找到同源蛋白.这些标记后的靶蛋白涉及了至少6种机制和功能,包括分枝杆菌的毒力、信号通路、脂类的合成、细胞壁和（或）膜的合成、中间代谢,等等.其中大部分蛋白被标记后经由蛋白酶体降解,也有被Pup标记的蛋白参与信号传导和调控作用;通过对这些靶蛋白的比较分析,能够对结核分枝杆菌的耐药和致病机制提供进一步的解释,同时这些Pup标记的蛋白也可以作为潜在的药物靶点,对其的分析研究可为结核病的治疗和控制提供理论基础.     

蛋白质羰基化与慢病伴发的骨骼肌萎缩

慢性心力衰竭、慢性阻塞性肺疾病( COPD)、癌症等老年慢性病患者晚期均可能伴发恶病质,其特征是体重减轻及骨骼肌功能障碍.无论这些老年患者基础的心肺功能如何,其骨骼肌功能障碍和肌肉受损的程度,可以预测患者的死亡率〔1〕.维持足够的肌肉数量和保持肌肉良好的性能,有助于维持老年患者的日常活动并且提高其生存率.尽管造成老年慢...     

过表达parkin基因对蛋白酶体抑制剂致SH-SY5Y细胞损伤的影响

目的探讨过表达parkin基因能否抵抗蛋白酶体抑制剂lactacystin对SH-SY5Y细胞的特异性损伤。方法不同浓度的lactacystin(1、5、10、15、20μmol/L)分别作用于人多巴胺能SH-SY5Y细胞和人胶质瘤U251细胞24 h,用二甲基噻唑二苯基四唑溴盐(MTT)法检测细胞活力。选取10μmol/L和20μmol/L lactacystin处理SH-SY5Y细胞,Western印迹法检测细胞内多泛素化蛋白的含量。瞬时转染parkin至SH-SY5Y细胞,并以MTT法检测10μmol/L和20μmol/L lactacystin作用下的细胞活力,Western印迹法检测细胞内多泛素化蛋白的含量,以及用免疫荧光法检测泛素阳性包涵体的形成。结果lactacystin呈剂量依赖性地损伤多巴胺能SH-SY5Y细胞(细胞活力分别为104%、82%、72%、60%、50%),而对胶质瘤U251细胞则无毒性作用。parkin过表达不能缓解lactacystin的毒性作用,也没有增加细胞内多泛素化蛋白生成,但却促进泛素阳性包涵体的形成(增加5%)。结论蛋白酶体功能障碍有可能在多巴胺能神经元的选择性死亡中起关键作用,而Parkin蛋白在路易小体的形成过程中可能起重要作用。     

老年代谢综合征患者冠状动脉狭窄程度与蛋白酶体20S的相关性

目的探讨老年代谢综合征(MS)患者冠状动脉狭窄程度与蛋白酶体20S的相关性。方法选择行冠状动脉造影术的老年MS患者204例,根据造影结果分冠心病组103例和非冠心病组101例。评估各支冠状动脉狭窄病变程度,检测蛋白酶体20S水平、活性及TNF-α。结果与非冠心病组比较,冠心病组蛋白酶体20S水平、活性及TNF-α明显升高(P<0.01)。冠心病患者Gensini积分及TNF-α与蛋白酶体20S(r=0.484,r=0.352,P<0.01)和活性(r=0.417,r=0.396,P<0.01)呈显著正相关。回归分析显示,Gensini积分和TNF-α是蛋白酶体20S水平及活性的主要影响因素。结论老年MS患者冠状动脉狭窄程度与蛋白酶体20S水平及活性密切相关。     

蛋白质Z、蛋白质Z依赖性蛋白酶抑制物与卒中

蛋白质Z(proteinz,PZ)是一种维生素K依赖性蛋白,作为蛋白质Z依赖性蛋白酶抑制物(protein Z-dependent protease inhibitor,ZPI)的辅助因子,在磷脂和钙离子存在的条件下,能抑制凝血因子Ⅹa,使ZPI活性提高近1 000倍,从而在m栓形成过程中发挥作用.ZPI可单独抑制凝血因子Ⅺa.ZPI的活性也在抑制凝血因子Ⅹa和Ⅺa的过程中被消耗.文章对Pz、ZPI的生物特性及与卒中的关联做了综述.     

衰老机制的研究进展及应用

衰老机制与抗衰老研究一直是生命科学研究的热门话题，人体衰老的原因是多方面的，衰老的机制也极为复杂。本文将综述衰老机制的几大重点学说：自由基一线粒体衰老学说、褪黑素一松果腺衰老学说、羰基毒化衰老学说、端粒学说、基因衰老学说等的研究进展及应用。     

精液蛋白质质谱的研究进展

蛋白质组学是一种复杂生命作用机理的阐述工具,其中蛋白质质谱技术可以揭示参与生命活动蛋白质的类别和性质。蛋白质组学应用于精液蛋白质质谱的研究,可以发现不育等疾病的特异性蛋白质标志物,探索其发病机制并得出治疗途径,对生育调控和不育的治疗均有重要意义。该文就精液蛋白质质谱的研究进展进行综述。     

抗心律失常肽的研究进展

1980年Aonuma等从牛的心室肌中分离、提取出一种活性多肽,具有强大的抗心律失常及抗血小板血栓形成作用,命名为抗心律失常肽(Antiar-rhythmic peptid,AAP),动物实验结果表明其作用类似奎尼丁,现综述如下。 一、AAP的结构特点及体内分布 1.AAP的结构特点:AAP由47个氨基酸组成,其神经末端为组氨酸,分子量约5500～10000,在体内较稳定,半衰期约10分钟,抗心律失常作用在离体心肌细胞上及药物诱发动物心律失常模型上证实,作用类似肾上腺素,不能被β-受体阻滞剂所阻断,能对抗低钾及高钙诱发的培养心肌细胞的     

Effect of aging and late onset dietary restriction on antioxidant enzymes and proteasome activities,and protein carbonylation of rat skeletal muscle and tendon

Many studies have shown that lifelong dietary restriction (DR) can retard aging processes. Very few reports, however, are found that examined the effect of late onset DR on biochemical parameters in aging animals [Goto, S., Takahashi, R., Araki, S., Nakamoto, H., 2002b. Dietary restriction initiated in late adulthood can reverse age-related alterations of protein and protein metabolism. Ann. NY Acad. Sci. 959, 50–56]. We studied the effect of every-other-day feeding, initiated at the age of 26.5 months and continued for 3.5 months, on antioxidant enzymes, protein carbonyls, and proteasomes of the gastrocnemius muscle and tendon in rats. Age-related increase in the activity and content of Cu, Zn-SOD and the content of Mn-SOD was attenuated by the DR in both tissues. The same was true for glutathione peroxidase and catalase activities. Significant increase with age in protein reactive carbonyl derivatives (RCD) in the tendon was noted that was partially reversed by the DR. No significant change of RCD, however, was observed in the skeletal muscle. The age-related and DR-induced changes of the RCD in the tendon appeared to be associated with proteasome activity that decreases with age and increases by the DR. It is suggested that the late onset DR can have beneficial effects on the locomotive functions by reducing age-associated potentially detrimental oxidative protein damage in the tendon.     

Increased rate of lipid peroxidation and protein carbonylation in experimental diabetic pregnancy

Aims/hypothesis. Maternal Type I (insulin-dependent) diabetes mellitus is associated with an increased risk for fetal malformations and spontaneous abortions. Although the pathogenic mechanism is not fully understood, reactive oxygen species have been shown to contribute to the pathogenesis in experimental studies. By measuring 8-iso-PGF₂α and protein carbonyls, radical oxygen damage to lipids and proteins can be estimated. The aim of this study was to investigate the status of lipid peroxidation and protein carbonylation in mothers and fetuses in experimental diabetic pregnancy. Methods. Non-pregnant and pregnant rats with and without streptozotocin-induced diabetes were studied after 4 weeks of diabetes or at gestational day 19, respectively. Gross morphology of the offspring was studied and 24 h urine, plasma, amniotic fluid, maternal and fetal livers were collected. Concentrations of 8-iso-PGF₂α, 15-keto-DH-PGF₂α and other oxidative stress variables were measured. Results. Malformation and resorption rates were increased in diabetic litters, whereas fetal weights were decreased in the control rats. There were no statistically significant differences in maternal plasma concentrations of 8-iso-PGF₂α, but plasma protein carbonyl content was increased in the diabetic groups. Pregnancy increased 24 h urinary excretion of 8-iso-PGF₂α in diabetic rats but not in the control rats. There was no difference in the amniotic fluid concentration of 8-iso-PGF₂α between the normal and the diabetic group. However, in the diabetic group there was a correlation between the uterine horn concentration of 8-iso-PGF₂α and the percentage of resorptions. Conclusions/interpretation. In diabetic pregnancy, both diabetes and pregnancy are promoting oxygen radical damage. Fetal oxidative stress markers do not clearly reflect fetal morphology. [Diabetologia (2001) 44: 766–774] Received: 11 December 2000 and in revised form: 18 January 2001     

Modulation of glutathione peroxidase activity by age-dependent carbonylation in glomeruli of diabetic mice

Aims

Low levels of reactive oxygen species and resulting oxidative protein modifications may play a beneficial role in cellular function under stress conditions. Here we studied the influence of age-dependent protein carbonylation on expression and activity of the anti-oxidative selenoenzyme glutathione peroxidase (GPx) in insulin-deficient Ins2^Akita mice and type 2 diabetic obese db/db mice in context of diabetic nephropathy.

糖尿病视网膜组织中自由基防御机能变化的实验研究

观察了糖尿病大鼠在不同病程时视网膜组织中超氧化物歧化酶（ＳＯＤ）、过氧化氢酶（ＣＡＴ）和谷胱甘肽过氧化物酶（ＧＳＨ－Ｐｘ）的活性及脂质过氧化物（ＬＰＯ）水平的变化。结果显示：在各实验时点，糖尿病大鼠视网膜组织中ＳＯＤ和ＣＡＴ活性明显低于对照组，ＬＰＯ水平显著升高，ＳＯＤ／ＬＰＯ和ＣＡＴ／ＬＰＯ明显降低，且随病程延长改变更明显。提示糖尿病大鼠视网膜组织中自由基防御机能明显降低，这种变化可能在糖尿病视网膜病变的发生发展中起重要作用。     

The proteasome and its role in nuclear protein maintenance

The cellular proteome is in a dynamic state of synthesis and degradation. Degradation of extracellular proteins is mainly mediated non-specifically by the lysosomes or due to released proteases, while the proteolysis of intracellular including nuclear proteins is catalyzed by the ubiquitin-proteasome pathway. Furthermore, the proteasomal system is largely responsible for the removal of unfolded and oxidatively damaged proteins. Taking into account the role of ubiquitin and proteasome system in protein metabolism, studies of its spatial organization within the cell are of great importance. For the understanding of cellular, including nuclear, protein maintenance the distribution of the proteasomes in both the nucleus and the cytosol and their response upon oxidative stress is of great interest. Although, the functional diversity of the cells is ensured by the three dimensional organization of the nucleus, nuclear proteins are also prone to oxidation and have to be removed from the cellular environment by the nuclear proteasome. Interestingly, nuclear proteins are partly degraded within the nucleus, whereas some are exported from the nucleus to the cytosol. Proteasomes are transported unidirectionally from the cytoplasm to the nucleus with a possible countervail during mitosis. This review is focused largely on the specifics of cellular proteasome distribution and on nuclear protein maintenance under physiological and oxidative stress conditions.     

Progress in the understanding of the protein C anticoagulant pathway

A natural anticoagulant pathway denoted the protein C system provides specific and efficient control of blood coagulation. Protein C is the key component of the system and circulates in the blood as a zymogen to an anticoagulant serine protease. Activation of protein C is achieved on the surface of endothelial cells by thrombin bound to the membrane protein thrombomodulin. The endothelial protein C receptor stimulates the activation of protein C on the endothelium. Activated protein C (APC) modulates blood coagulation by cleaving a limited number of peptide bonds in factor VIIIa (FVIIIa) and factor Va (FVa), cofactors in the activation of factor X and prothrombin, respectively. Vitamin K-dependent protein S stimulates the APC-mediated regulation of coagulation. Not only is protein S involved in the degradation of FVIIIa, but so is FV, which in recent years has been found to be a Janus-faced protein with both procoagulant and anticoagulant potentials. A number of genetic defects affecting the anticoagulant function of the protein C system, eg, APC resistance (Arg506Gln or FV Leiden) and deficiencies of protein C and protein S constitute major risk factors of venous thrombosis. The protein C system also has anti-inflammatory and antiapoptotic potentials, the molecular mechanisms of which are beginning to be unraveled. APC has emerged in recent years as a useful therapeutic compound in the treatment of severe septic shock. The beneficial effect of APC is believed be due to both its anticoagulant and its anti-inflammatory properties.     

Accelerated protein evolution analysis reveals genes and pathways associated with the evolution of mammalian longevity

The genetic basis of the large species differences in longevity and aging remains a mystery. Thanks to recent large-scale genome sequencing efforts, the genomes of multiple species have been sequenced and can be used for cross-species comparisons to study species divergence in longevity. By analyzing proteins under accelerated evolution in several mammalian lineages where maximum lifespan increased, we identified genes and processes that are candidate targets of selection when longevity evolves. We identified several proteins with longevity-specific selection patterns, including COL3A1 that has previously been related to aging and proteins related to DNA damage repair and response such as DDB1 and CAPNS1. Moreover, we found that processes such as lipid metabolism and cholesterol catabolism show such patterns of selection and suggest a link between the evolution of lipid metabolism, cholesterol catabolism, and the evolution of longevity. Lastly, we found evidence that the proteasome–ubiquitin system is under selection specific to lineages where longevity increased and suggest that its selection had a role in the evolution of longevity. These results provide evidence that natural selection acts on species when longevity evolves, give insights into adaptive genetic changes associated with the evolution of longevity in mammals, and provide evidence that at least some repair systems are selected for when longevity increases.     

褪黑素及其代谢物抗氧化作用研究进展

褪黑素是强有力的抗氧化剂,其抗氧化作用包括直接清除自由基,间接刺激抗氧化酶活性及基因表达,刺激谷胱甘肽合成,减少线粒体电子传递链电子渗漏,减少细胞因子产生,限制炎性反应扩散.另外,褪黑素和其他扰氧化剂有协同效应,褪黑素家族级联式自由基清除反应也是其抗氧化作用的重要特点.最近的研究表明,植物中也含有褪黑素,在一些中药中,褪黑素的含量超过1000ng/g干重,这些褪黑素水平含量高的中药,传统上被用于治疗与自由基损伤相关的疾病.     

Gender difference as regards myocardial protein oxidation in aged rats: male rats have increased oxidative protein damage

Objective The reasons for the difference in life expectancy between males and females are still unknown. Previous studies have provided compelling evidence for the presence of oxidized proteins, and lipids in advanced human atherosclerotic lesions. The gender factor responsible for such protein oxidation is unknown and controversial. Our aim was to reveal the difference between myocardial protein and lipid oxidation parameters of male and female aged rats. Methods We investigated the relation between myocardial protein carbonyl (PCO) and other protein oxidation parameters such as advanced oxidation protein products (AOPP), nitrotyrosine (NT), protein hydroperoxide (P-OOH), and protein thiol (P-SH). Our study also covered other oxidative stress parameters, such as total thiol (T-SH), non-protein thiol (Np-SH), 4-hydroxyalkenal (4-HAE), malondialdehyde (MDA), reduced glutathione (GSH), and the glutathione disulfide (GSSG). Results Among the studied parameters, myocardial PCO, AOPP, NT, Np-SH, GSH, Fe²⁺ levels and the redox index (RI) of male rats were significantly higher than in the female group. On the other hand, P-OOH, P-SH, T-SH, 4-HAE, and MDA levels were all found to be not different. Conclusions These data support the hypothesis that elevated levels of PCO, AOPP, and NT contribute to the extent of protein, but not lipid, oxidation in aged male rats. We are of the conviction that the increased myocardial Np-SH, GSH and RI levels that we have determined in aged male rats may be a protective factor in propagation of protein oxidation. Our findings support our conviction that protein and lipid oxidation, in the myocardial tissue of aged rats, have a controlling role in differing regulating mechanisms through gender differences.     

C-reactive protein impairs coronary arteriolar dilation to prostacyclin synthase activation: Role of peroxynitrite

Endothelium-derived vasodilators, i.e., nitric oxide (NO), prostacyclin (PGI₂) and prostaglandin E₂ (PGE₂), play important roles in maintaining cardiovascular homeostasis. C-reactive protein (CRP), a biomarker of inflammation and cardiovascular disease, has been shown to inhibit NO-mediated vasodilation. The goal of this study was to determine whether CRP also affects endothelial arachidonic acid (AA)-prostanoid pathways for vasomotor regulation. Porcine coronary arterioles were isolated and pressurized for vasomotor study, as well as for molecular and biochemical analysis. AA elicited endothelium-dependent vasodilation and PGI₂ release. PGI₂ synthase (PGI₂-S) inhibitor trans-2-phenyl cyclopropylamine blocked vasodilation to AA but not to serotonin (endothelium-dependent NO-mediated vasodilator). Intraluminal administration of a pathophysiological level of CRP (7 μg/mL, 60 min) attenuated vasodilations to serotonin and AA but not to nitroprusside, exogenous PGI₂, or hydrogen peroxide (endothelium-dependent PGE₂ activator). CRP also reduced basal NO production, caused tyrosine nitration of endothelial PGI₂-S, and inhibited AA-stimulated PGI₂ release from arterioles. Peroxynitrite scavenger urate failed to restore serotonin dilation, but preserved AA-stimulated PGI₂ release/dilation and prevented PGI₂-S nitration. NO synthase inhibitor L-NAME and superoxide scavenger TEMPOL also protected AA-induced vasodilation. Collectively, our results suggest that CRP stimulates superoxide production and the subsequent formation of peroxynitrite from basal released NO compromises PGI₂ synthesis, and thus endothelium-dependent PGI₂-mediated dilation, by inhibiting PGI₂-S activity through tyrosine nitration. By impairing PGI₂-S function, and thus PGI₂ release, CRP could promote endothelial dysfunction and participate in the development of coronary artery disease.