Cumene peroxide and Fe2+-ascorbate-induced lipid peroxidation and effect of phosphoglucose isomerase

Malondialdehyde (MDA) is one of cytotoxic aldehydes produced in cells as a result of lipid peroxidation and further MDA metabolism in cytoplasm is not known. In our experiments the liver fraction 10,000 g containing phosphoglucose isomerase and enzymes of the glyoxalase system was used and obtained experimental data shows that in this fraction there is an aggregate of reactions taking place both in membranes (lipid peroxidation) and outside membranes. MDA accumulation is relatively slow because MDA is a substrate of aldehyde isomerase (MDA ↔ methylglyoxal). The well known enzyme phosphoglucose isomerase acts as an aldehyde isomerase (Michaelis constant for this enzyme Km = 133 ± 8 μM). MDA conversion to methylglyoxal and further to neutral product D-lactate (with GSH as a cofactor) occurs in cytoplasm and D-lactate should be regarded as the end product of two different parametabolic reactions: lipid peroxidation or protein glycation.     

Aldehyde adducts generated during lipid peroxidation modification of proteins

Abstract

Various lines of evidence indicate that an important part in the pathogenesis of atherosclerosis is the modification of the plasma low-density lipoproteins (LDLs). A large number of pro-inflammatory and pro-atherogenic properties have been ascribed to the oxidatively modified LDLs and their components. There is considerable evidence to support the role of lipid peroxidation products, reactive aldehydes in particular, originating from the oxidized LDL as important signaling molecules in the context of the atherosclerotic lesion. These aldehydes generated during the peroxidation of LDL exhibit a facile reactivity with proteins, generating a variety of intra- and intermolecular covalent adducts on the apolipoprotein B-100 particle in LDL. Characterization of the aldehyde adducts generated in the protein is therefore critical in understanding the nature of the oxidized LDL. However, the majority of adducts generated during the oxidative modification of LDL have not yet been chemically characterized. In this review, the current status of aldehyde adducts quantitatively analyzed in the Cu²⁺-oxidized LDL is reviewed.     

Chilling Tolerance of Potato Plants Transformed with a Yeast-Derived Invertase Gene under the Control of the B33 Patatin Promoter

Tolerance to chilling was compared under in vitro conditions in potato plants (Solanum tuberosum L., cv. Désirée) transformed with a yeast-derived invertase gene under the control of the B33 class 1 tuber-specific promoter (the B33-inv plants) and potato plants transformed only with a reporter gene (the control plants). The expression of the inserted yeast invertase gene was proved by following the acid and alkaline invertase activities and sugar contents in the leaves under the optimum temperature (22°C). The total activities of acid and alkaline invertases in the B33-inv plants exceeded those in the control plants by the factors of 2–3 and 1.3, respectively. In the B33-inv plants, the activity of acid invertase twice exceeded that of the alkaline invertase, whereas the difference equaled 12% in the control plants. The contents of sucrose and glucose increased in the B33-inv plants by 21 and 13%, respectively, as compared to the control. Chilling at +3 and –1°C for 1, 3, and 6 h did not affect the rate of lipid peroxidation, as measured by the content of malonic dialdehyde (MDA) in the leaves of the genotypes under study. Only the longer exposures (24 h at +3 and –1°C and 7 days at +5°C) produced a significant decline in the MDA content in the B33-invplants, as compared to the control. Following short freezing (20 min at –9°C), the content of MDA increased by 50% in the leaves of the control plants, while in the B33-inv plants, cold-treated and control plants did not differ in the MDA content. The authors presume that the potato plants transformed with the yeast invertase gene acquire a higher tolerance to low temperatures as compared to the control plants, apparently due to the changes in sugar ratio produced by the foreign invertase.     

Effect of 6-Phosphogluconate on Phosphoglucose Isomerase in Rat Brain In Vitro and In Vivo

The activity of phosphoglucose isomerase, its kinetic properties, and the effect of 6-phosphogluconate on its activity in the forward (glucose 6-phosphate----fructose 6-phosphate) and the reverse (fructose 6-phosphate----glucose 6-phosphate) reactions were determined in adult rat brain in vitro. The activity of phosphoglucose isomerase (in nmol/min/mg of whole brain protein) was 1,865 +/- 20 in the forward reaction and 1,756 +/- 32 in the reverse reaction at pH 7.5. It was 1,992 +/- 28 and 2,620 +/- 46, respectively, at pH 8.5. The apparent Km and Vmax of phosphoglucose isomerase were 0.593 +/- 0.031 mM and 2,291 +/- 61 nmol/min/mg of protein, respectively, for glucose 6-phosphate and 0.095 +/- 0.013 mM and 2,035 +/- 98 nmol/min/mg of protein, respectively, for fructose 6-phosphate. The activity of phosphoglucose isomerase was inhibited intensely and competitively by 6-phosphogluconate, with an apparent Ki of 0.048 +/- 0.005 mM for glucose 6-phosphate and 0.042 +/- 0.004 mM for fructose 6-phosphate as the substrate. With glucose 6-phosphate as the substrate, at concentrations from 0.05 to 0.5 mM, the activity of the enzyme was inhibited completely in the presence of 0.5-2.0 mM 6-phosphogluconate. With 0.05-0.2 mM fructose 6-phosphate as the substrate, it was inhibited greater than or equal to 85% at the same concentrations of the inhibitor. No significant changes were observed in the values of Km, Vmax, and Ki for phosphoglucose isomerase in the brain of 6-aminonicotinamide-treated rats.(ABSTRACT TRUNCATED AT 250 WORDS)     

Mechanisms of oxidative modification of low density lipoproteins under conditions of oxidative and carbonyl stress

Low-molecular-weight aldehydes (glyoxal, methylglyoxal, 3-deoxyglucosone) generated on autooxidation of glucose under conditions of carbonyl stress react much more actively with amino groups of L-lysine and epsilon-amino groups of lysine residues of apoprotein B-100 in human blood plasma low density lipoproteins (LDL) than their structural analogs (malonic dialdehyde (MDA), 4-hydroxynonenal) resulting on free radical oxidation of lipids under conditions of oxidative stress. Glyoxal-modified LDL aggregate in the incubation medium with a significantly higher rate than LDL modified by MDA, and MDA-modified LDL are markedly more poorly absorbed by cultured human macrophages and significantly more slowly eliminated from the rat bloodstream upon intravenous injection. Studies on kinetics of free radical oxidation of rat liver membrane phospholipids have shown that ubiquinol Q(10) is the most active lipid-soluble natural antioxidant, and suppression of ubiquinol Q(10) biosynthesis by beta-hydroxy-beta-methylglutaryl coenzyme A reductase inhibitors (statins) is accompanied by intensification of lipid peroxidation in rat liver biomembranes and in LDL of human blood plasma. Injection of ubiquinone Q(10) protects the human blood plasma LDL against oxidation and prevents oxidative stress-induced damages to rat myocardium. A unified molecular mechanism of atherogenic action of carbonyl-modified LDL in disorders of lipid and carbohydrate metabolism is discussed.     

Neuroprotective role of hydralazine in rat spinal cord injury‐attenuation of acrolein‐mediated damage

Acrolein, an α,β‐unsaturated aldehyde and a reactive product of lipid peroxidation, has been suggested as a key factor in neural post‐traumatic secondary injury in spinal cord injury (SCI), mainly based on in vitro and ex vivo evidence. Here, we demonstrate an increase of acrolein up to 300%; the elevation lasted at least 2 weeks in a rat SCI model. More importantly, hydralazine, a known acrolein scavenger can provide neuroprotection when applied systemically. Besides effectively reducing acrolein, hydralazine treatment also resulted in significant amelioration of tissue damage, motor deficits, and neuropathic pain. This effect was further supported by demonstrating the ability of hydralazine to reach spinal cord tissue at a therapeutic level following intraperitoneal application. This suggests that hydralazine is an effective neuroprotective agent not only in vitro, but in a live animal model of SCI as well. Finally, the role of acrolein in SCI was further validated by the fact that acrolein injection into the spinal cord caused significant SCI‐like tissue damage and motor deficits. Taken together, available evidence strongly suggests a critical causal role of acrolein in the pathogenesis of spinal cord trauma. Since acrolein has been linked to a variety of illness and conditions, we believe that acrolein‐scavenging measures have the potential to be expanded significantly ensuring a broad impact on human health.

     

The Metabolism of Low Molecular Weight Hydrocarbon Gases in Man

The metabolism of ethane and pentane in man is demonstrated to occur from the uptake of an enriched atmosphere of these gases in a rebreathe spirometer circuit. Dithiocarb, an inhibitor of alkane metabolism, reduced uptake and increased the respiratory excretion of these gases. This effect was least marked for the slowly metabolised ethane. Therefore the endogenous production of ethane as measured by respiratory excretion is less affected. However pentane is rapidly metabolised and this limits the use of simple respiratory excretion of pentane as a measure of in vivo lipid peroxidation.     

Aldehyde oxidase and alcohol dehydrogenase genetics in the mouse. New alleles for the Aox-2 and Adh-3 loci

The genetic variability of one of the liver isozymes of aldehyde oxidase (AOX-B₂ or AOX-2) and the stomach isozyme of alcohol dehydrogenase (ADH-C₂) has been examined among strains of mice. Evidence is presented for a fourth allele of Aox-2 and a third allele of Adh-3 . The hybrid allozyme pattern for mouse liver AOX was consistent with a dimeric subunit structure for this enzyme.     

Acrolein involvement in sensory and behavioral hypersensitivity following spinal cord injury in the rat

Growing evidence suggests that oxidative stress, as associated with spinal cord injury (SCI), may play a critical role in both neuroinflammation and neuropathic pain conditions. The production of the endogenous aldehyde acrolein, following lipid peroxidation during the inflammatory response, may contribute to peripheral sensitization and hyperreflexia following SCI via the TRPA1‐dependent mechanism. Here, we report that there are enhanced levels of acrolein and increased neuronal sensitivity to the aldehyde for at least 14 days after SCI. Concurrent with injury‐induced increases in acrolein concentration is an increased expression of TRPA1 in the lumbar (L3–L6) sensory ganglia. As proof of the potential pronociceptive role for acrolein, intrathecal injections of acrolein revealed enhanced sensitivity to both tactile and thermal stimuli for up to 10 days, supporting the compound's pro‐nociceptive functionality. Treatment of SCI animals with the acrolein scavenger hydralazine produced moderate improvement in tactile responses as well as robust changes in thermal sensitivity for up to 49 days. Taken together, these data suggest that acrolein directly modulates SCI‐associated pain behavior, making it a novel therapeutic target for preclinical and clinical SCI as an analgesic.

     

泛素化在调控铁死亡中的作用

铁死亡是一种由脂质过氧化驱动的铁依赖性的新的细胞死亡方式,越来越多的证据表明,铁死亡与各种病理状态有关,如神经退行性疾病、糖尿病肾病、癌症等,脂质过氧化驱动的铁死亡可能促进或抑制这些疾病的发生发展,细胞中抗氧化系统通过抑制脂质过氧化在抵抗铁死亡过程中发挥着重要作用。铁死亡的关键通路有以SLC7A11-GPX4为关键分子的氨基酸代谢通路、以铁蛋白或转铁蛋白为主的铁代谢通路,以及脂质代谢通路。铁死亡的发生受到细胞内蛋白质的调节,这些蛋白质会发生各种翻译后修饰,包括泛素化修饰。泛素-蛋白酶体系统（ubiquitin-proteasome system,UPS）是细胞内主要降解系统之一,通过酶促级联反应催化泛素分子标记待降解蛋白,随后由蛋白酶体识别并降解目标蛋白质。UPS根据其降解底物的不同在调节铁死亡的反应中发挥双重作用。UPS通过促进铁死亡关键分子（如SLC7A11、GPX4、GSH）以及抗氧化系统成分（如NRF2）的泛素化降解从而促进铁死亡,也可以通过促进脂质代谢通路中相关分子（如ACSL4、ALOX15）的泛素化降解从而抑制铁死亡。本综述介绍泛素化修饰在调控铁死亡进程中作用的最新研究进展,总结了已发表的关于E3泛素连接酶和去泛素酶调控铁死亡的研究,归纳了泛素连接酶、去泛素酶调控铁死亡的作用靶点,有助于确定人类疾病中新的预后指标,为这些疾病提供潜在的治疗策略。     

长链非编码RNA在脂质代谢中的作用

脂质代谢是人体三大代谢之一，在激素等信号分子的调控下，脂质代谢处于稳定平衡的状态。当稳态被打破，血液中甘油三酯（triglyceride，TG）、胆固醇等水平发生变化，最终引起动脉粥样硬化（atherosclerosis，AS）、肥胖等脂质代谢疾病。长链非编码RNA（long noncoding RNA，lncRNA）是一组不具备蛋白质编码能力，长度大于200个核苷酸的RNA，近来研究发现，lncRNA与机体代谢、炎症和免疫系统以及血管功能的调控密切相关。大量文献表明，lncRNA参与脂质代谢调控，因而有望成为一些脂质代谢疾病的潜在治疗靶点。     

大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达

本文用穿梭质粒载体pSE-3,进行了大肠杆菌葡萄糖异构酶基因在变铅青链霉菌中的克隆与表达。把含有1.6kb葡萄糖异构酶基因的pX1200(4.3kb)质粒与pGEM-3(2.9kb)质粒分别用EcoRI酶解,T4DNA连接酶连接,转化E.Coli HB101(Xy1~-,Neo(?)),得到的重组质粒被命名为pX1203(7.2kb);将pX1203与穿梭质粒载体pSE-3分别用HindⅢ酶解,T_4DNA连接酶连接,转化E.Coli HB101,在含有新霉素的木糖培养基上筛选到转化重组体,其重组质粒被命名为pSE×100(10.6kb);把pSE×100转化变铅青链霉菌TK54的原生质体,在硫链丝菌肽(50μg/ml)和新霉素(50μg/ml)的平皿上得到了重组体。经质粒提取,酶切分析,再转化和葡萄糖异构酶活性测定,结果表明,大肠杆菌的葡萄糖异构酶基因确实已在链霉菌中克隆和表达。     

Measurement of Aldehydes in Low Density Lipoprotein by High Performance Liquid Chromatography

A modified procedure is presented for the HPLC determination of nanomolar concentrations of n-alkanals, hydroxyalkenals, malondialdehyde and furfural in biological fluid. The modifications allow aldehyde profile analysis of small samples of fresh, human, low density lipoprotein (LDL), enabling more detailed studies of LDL fatty acid peroxidation. Aldehydes are reacted with 1,3-cyclohexanedione to produce fluorescent derivatives which are separated by gradient, reversed phase, high performance liquid chromatography (HPLC). Analysis time has been reduced by shortening the sample preparation. Sensitivity has been increased by miniaturization of the derivatisation procedure, reducing required sample size. Recoveries of added aldehydes have been improved. In addition, the method presented allows determination of three further aldehydes, not measured previously by CHD methods: malondialdehyde, formaldehyde and furfural. Recovery and variability data and concentrations of aldehydes found in human LDL are given. The capacity of the method for further development, to enable determination of other aldehydes such as the trans, 2-alkenals, is also demonstrated.     

Critical role of acrolein in secondary injury following ex vivo spinal cord trauma

The pathophysiology of spinal cord injury (SCI) is characterized by the initial, primary injury followed by secondary injury processes in which oxidative stress is a critical component. Secondary injury processes not only exacerbate pathology at the site of primary injury, but also result in spreading of injuries to the adjacent, otherwise healthy tissue. The lipid peroxidation byproduct acrolein has been implicated as one potential mediator of secondary injury. To further and rigorously elucidate the role of acrolein in secondary injury, a unique ex vivo model is utilized to isolate the detrimental effects of mechanical injury from toxins such as acrolein that are produced endogenously following SCI. We demonstrate that (i) acrolein-Lys adducts are capable of diffusing from compressed tissue to adjacent, otherwise uninjured tissue; (ii) secondary injury by itself produces significant membrane damage and increased superoxide production; and (iii) these injuries are significantly attenuated by the acrolein scavenger hydralazine. Furthermore, hydralazine treatment results in significantly less membrane damage 2 h following compression injury, but not immediately after. These findings support our hypothesis that, following SCI, acrolein is increased to pathologic concentrations, contributes significantly to secondary injury, and thus represents a novel target for scavenging to promote improved recovery.     

Involvement of Reactive Oxygen Species in Plant Mitochondrial Response to Low-Temperature Stress

The review presents data for the generation of the reactive oxygen species (ROS) in mitochondria during low-temperature stress. The relation of lipid peroxidation processes in membranes to the induction of low-temperature stress is demonstrated. The data for the involvement of the mitochondrial uncoupling proteins and the processes of uncoupling oxidation and phosphorylation in the regulation of mitochondrial ROS formation during low-temperature stress are considered.     

Involvement of free radicals and oxidative stress in NAFLD/NASH

Abstract

Non-alcoholic fatty liver disease (NAFLD) is now the most common liver disease affecting high proportion of the population worldwide. NAFLD encompasses a large spectrum of conditions ranging from fatty liver to non-alcoholic steatohepatitis (NASH), which can progress to cirrhosis and cancer. NAFLD is considered as a multifactorial disease in relation to the pathogenic mechanisms. Oxidative stress has been implicated in the pathogenesis of NAFLD and NASH and the involvement of reactive oxygen species (ROS) has been suggested. Many studies show the association between the levels of lipid oxidation products and disease state. However, often neither oxidative stress nor ROS has been characterized, despite oxidative stress is mediated by multiple active species by different mechanisms and the same lipid oxidation products are produced by different active species. Further, the effects of various antioxidants have been assessed in human and animal studies, but the effects of drugs are determined by the type of active species, suggesting the importance of characterizing the active species involved. This review article is focused on the role of free radicals and free radical-mediated lipid peroxidation in the pathogenesis of NAFLD and NASH, taking characteristic features of free radical-mediated oxidation into consideration. The detailed analysis of lipid oxidation products shows the involvement of free radicals in the pathogenesis of NAFLD and NASH. Potential beneficial effects of antioxidants such as vitamin E are discussed.     

Role of lipid peroxidation in iron-induced cellular calcium overload

Calcium overload is the common pathway leading to cell injury. The role of iron-induced lipid peroxidation in the modification of Ehrlich carcinoma cells calcium homeostasis has been studied. There is a lack of correlation between that modification and the value of lipid peroxidation. The stability characteristics of low-mol-weight iron complexes affect lipid peroxidation and, to a lesser extent, cellular calcium uptake. Lipid peroxidation appears not as a triggering factor of cellular calcium homeostasis modification, but as a concomitant phenomenon.     

Tamoxifen,an anticancer drug,is an activator of human aldehyde dehydrogenase 1A1

The modulation of aldehyde dehydrogenase (ALDH) activity has been suggested as a promising option for the prevention or treatment of many diseases. To date, only few activating compounds of ALDHs have been described. In this regard, N‐(1,3‐benzodioxol‐5‐ylmethyl)?2,6‐dichlorobenzamide has been used to protect the heart against ischemia/reperfusion damage. In the search for new modulating ALDH molecules, the binding capability of different compounds to the active site of human aldehyde dehydrogenase class 1A1 (ALDH1A1) was analyzed by molecular docking, and their ability to modulate the activity of the enzyme was tested. Surprisingly, tamoxifen, an estrogen receptor antagonist used for breast cancer treatment, increased the activity and decreased the K_m for NAD⁺ by about twofold in ALDH1A1. No drug effect on human ALDH2 or ALDH3A1 was attained, showing that tamoxifen was specific for ALDH1A1. Protection against thermal denaturation and competition with daidzin suggested that tamoxifen binds to the aldehyde site of ALDH1A1, resembling the interaction of N‐(1,3‐benzodioxol‐5‐ylmethyl)?2,6‐dichlorobenzamide with ALDH2. Further kinetic analysis indicated that tamoxifen activation may be related to an increase in the K_d for NADH, favoring a more rapid release of the coenzyme, which is the rate‐limiting step of the reaction for this isozyme. Therefore, tamoxifen might improve the antioxidant response, which is compromised in some diseases. Proteins 2015; 83:105–116. © 2014 Wiley Periodicals, Inc.