Ferroxidase Ii Activity and Serum Cholesterol

Ferroxidase II (Fox II) was developed in serum by acid incubation for 24h. The resulting activity showed a strong positive correlation with the serum cholesterol concentration in normal subjects and patients with hyperlipidaemia. The potentiating effect of cholesterol on developed Fox II has been confirmed by the in-vitro addition of cholesterol to serum. There was no significant correlation between developed Fox II and caeruloplasmin (ferroxidase I) or between cholesterol and caeruloplasmin.     

Ferroxidase Ii Activity and Serum Cholesterol

Ferroxidase II (Fox II) was developed in serum by acid incubation for 24h. The resulting activity showed a strong positive correlation with the serum cholesterol concentration in normal subjects and patients with hyperlipidaemia. The potentiating effect of cholesterol on developed Fox II has been confirmed by the in-vitro addition of cholesterol to serum. There was no significant correlation between developed Fox II and caeruloplasmin (ferroxidase I) or between cholesterol and caeruloplasmin.     

Continuous Monitoring of in Vztro Oxidation of Human Low Density Lipoprotein

The kinetics of the oxidation of human low densit) lipoprotein (LDL) can be measured continuously by monitoring the change of the 234 nm diene absorption. The time-course shows three consecutive phases, a lag-phase during which the diene absorption increases only weakly. a propagation phase with a rapid increase of the diene absorption and finally a decomposition phase. The increase of the dienes is highly correlated with the increase of MDA or lipid hydroperoxides. The duration of the lag-phase is determined by the endogenous antioxidants contained in LDL (vitamin E. carotenoids. retinylstearate). Water-soluble antioxidants (ascorbic acid. urate) added in micromolar concentrations prolong the lag-phase in a concentration-dependent manner. The determination of the lag-phase is a convenient and objective procedure for determining the susceptibility of LDL from different donors towards oxidation as well as effects of pro-and antioxidants.     

椰子种皮油提取物对低密度脂蛋白氧化的抑制作用

本文研究了椰子种皮油提取物对低密度脂蛋白氧化的抑制作用,以水溶性VE(Trolox)作为对照,测定椰子种皮油提取物的总酚含量,总抗氧化能力,检测提取物对低密度脂蛋白的氧化易感性,硫代巴比妥酸反应产物(TBARS)值及铜络合能力的影响。结果表明,椰子种皮油提取物的总酚含量为68.6 mg/g,总抗氧化能力也随着浓度增大而加强,说明该提取物具有较好的抗氧化活性。浓度为0.5 mg/mL的提取物能将低密度脂蛋白的延滞时间延长了两倍,丙二醛(MDA)含量也明显下降,表明椰子种皮油提取物对低密度脂蛋白的氧化具有一定的抑制作用。     

Lipoprotein Oxidation and Plasma Vitamin E in Nondiabetic Normotensive Obese Patients

Objective: To correlate the susceptibility of low‐(LDL) and very‐low‐density lipoprotein to oxidation in vitro and the concentrations of serum antibodies against malondialdehyde‐modified LDL and plasma vitamin E with the anthropometric and laboratory characteristics of obesity. Research Methods and Procedures: A total of 75 nondiabetic, normotensive obese patients were assigned to one of four groups according to their body mass index (BMI): moderately obese (30 ≤ BMI ≤ 34.9 kg/m², n = 11), severely obese (35 ≤ BMI ≤ 39.9 kg/m², n = 20), morbidly obese (40 ≤ BMI ≤ 50 kg/m², n = 29), and very severely obese (BMI > 50 kg/m², n = 15). Results: The oxidation lag time for LDL from patients with a BMI ≥35 kg/m² was shorter than that for LDL from non‐obese controls (n = 13), whereas very‐low‐density lipoprotein oxidation lag times were not significantly different. The serum antibodies against modified LDL were similar in all groups, whereas the plasma vitamin E concentrations of obese patients were decreased (p ≤ 0.01). There was a negative correlation between LDL oxidation lag time and BMI (r = ?0.35, p = 0.0008), and between plasma vitamin E and BMI (r = ?0.53, p < 0.0001) and waist‐to‐hip ratio (r = ?0.40, p = 0.0003). Discussion: The LDL of nondiabetic, normotensive obese patients is more readily oxidized, and plasma vitamin E concentrations are low. These are both risk factors for coronary heart disease.     

Resveratrol Has No Effect on Lipoprotein Profile and Does Not Prevent Peroxidation of Serum Lipids in Normal Rats

Trans-resveratrol, one of the antioxidants found in red wine, has been the subject of controversial reports regarding its protective role against cardiovascular diseases. In this study we synthesized trans-resveratrol and injected it to rats (20 and 40 mg/kg body weight, once a day for 21 days, i.p.) to determine its effect on the serum lipid profile. Synthetic trans-resveratrol was an effective antioxidant in vitro against hydroxyl radical (I₅₀ = 33 μM). Resveratrol treatment, however, did not have any effect on either the lipid profile or on Cu⁺²-dependent formation of thiobarbi-turic-acid-reactive substances (TBARS) from protein-associated lipids. Since the amount of resveratrol used in these experiments was orders of magnitude higher than the amounts found in wine, these results suggest that if resveratrol has any effect against coronary heart diseases, it is not related to its antioxidant role on lipids or to changes in lipoprotein profile.     

Resveratrol Has No Effect on Lipoprotein Profile and Does Not Prevent Peroxidation of Serum Lipids in Normal Rats

Trans-resveratrol, one of the antioxidants found in red wine, has been the subject of controversial reports regarding its protective role against cardiovascular diseases. In this study we synthesized trans-resveratrol and injected it to rats (20 and 40 mg/kg body weight, once a day for 21 days, i.p.) to determine its effect on the serum lipid profile. Synthetic trans-resveratrol was an effective antioxidant in vitro against hydroxyl radical (I₅₀ = 33 μM). Resveratrol treatment, however, did not have any effect on either the lipid profile or on Cu⁺²-dependent formation of thiobarbi-turic-acid-reactive substances (TBARS) from protein-associated lipids. Since the amount of resveratrol used in these experiments was orders of magnitude higher than the amounts found in wine, these results suggest that if resveratrol has any effect against coronary heart diseases, it is not related to its antioxidant role on lipids or to changes in lipoprotein profile.     

Investigation of Lipid Peroxidation in Human Low Density Lipoprotein

Human plasma low density lipoprotein (LDL) exposed to oxygen saturated buffer becomes depleted of alpha-tocopherol within 3 to 6 hours. Thereafter, lipid peroxidation commences as evidenced by the loss of 18:2 (67nmol/mg LDL) and 20:4 (12nmol/mg LDL) and the concomitant formation of 4-hydroxy-nonenal (0.28 nmol/mg LDL) and fluorescent compounds. The major fluorophor in apo B of oxidized LDL has an excitation maximum at 355 nm and an emission maximum at 430 nm. A fluorophor with the same spectral properties is produced in apo B, if LDL is incubated with 4-hydroxynonenal, whereas malonal-dehyde gives a fluorophor with excitation and emission maxima at 400/470nm. Three-dimensional fluorescence spcetroscopy proved to be an useful tool in analysing the complex fluorescence of apo B.     

Analysis of Lipoprotein Diene Formation in Human Serum Exposed to Copper

The susceptibility of low density lipoprotein (LDL) to oxidative modification can be determined by analyzing the lag phase for initiation of diene formation in isolated LDL exposed to Cu^2+. However, the applicability of this assay for clinical studies is limited by the requirement of a preparative ultracentrifugation of LDL and that the influence of water soluble antioxidants and other lipoproteins is not accounted for. The present paper describes a modification of this assay allowing determination of lag phase for lipoprotein diene formation in serum. The formation of dienes in serum exposed to Cu²⁺ begins following the consumption of serum α-tocopherol, correlates to the formation of thiobarbituric acid reactive substances (r = 0.987, n = 8), is inhibited by the addition of ascorbic acid and is absent in lipoprotein-deficient serum. It is also accompanied by an increased mobility of serum lipoproteins on agarose gel electrophoresis and with an ability of serum to displace isolated copper-oxidized LDL from binding sites mediating degradation in mouse peritoneal macrophages. The coefficient of variance of the analysis is below 3%. It is concluded that this technique allows analysis of lipoprotein oxidation susceptibility in serum samples and may prove to be useful in clinical analysis of the lipoprotein oxidation susceptibility.     

Gender Differences in Lipoprotein Lipase Activity after Acute Exercise

Objective: To determine whether gender differences exist in lipoprotein lipase (LPL) activity in response to exercise and/or insulin. Exercise and insulin are known modulators of LPL activity in men, but this is less clear in women. LPL activity may predict propensity for obesity; therefore, understanding its modulators is of considerable importance. Research Methods and Procedures: Gender differences in skeletal muscle and adipose tissue LPL activity were determined after a single bout of exercise followed by a hyperinsulinemic/euglycemic clamp and compared with an identical rest day in healthy lean men (n = 10) and women (n = 10). Muscle and adipose tissue biopsies were obtained pre‐ (post‐exercise vs. rest) and post‐clamp. Results: Basal levels of muscle and adipose tissue LPL activity were not different between men and women. There was, however, a significant gender by day interaction for muscle LPL activity (p = 0.023) and adipose tissue LPL activity (p = 0.013). In muscle, this was because of a significant increase in LPL activity on the exercise vs. rest day in men (p < 0.001) but not women. Adipose tissue LPL activity also increased significantly in men on the exercise day relative to rest day (p = 0.04) but decreased in women (p = 0.10). The hyperinulinemic/euglycemic clamp had no independent effect on tissue LPL activity, in either gender, after rest or exercise. Discussion: In the 3 to 4 hours after exercise, muscle and adipose tissue LPL activity increased significantly in men, whereas LPL activity remained unchanged in women.     

Antioxidant Activity of Quercetin against Metmyoglobin-induced Oxidation of Fish Oil-bile Salt Emulsion

The antioxidative effect of quercetin was examined in metmyoglobin-induced oxidation of a fish oil-bile salt emulsion (average diameter of particles; 2.0 μm) to evaluate its effectiveness during the digestion of highly oxidizabile oils. The activity of quercetin increased with the lowering of the initial peroxide value (PV) of the oil and its effectiveness was superior to that of α-tocopherol. A synergistic antioxidant effect was observed upon the addition of quercetin and α-tocopherol irrespective of the initial PV of the oils, and quercetin was consumed faster than α-tocopherol. The loss of quercetin was larger than that of α-tocopherol when cumene hydroperoxide and metmyoglobin were mixed in a trimyristin-bile salt emulsion. In an ultrafiltration experiment on emulsified oil with a membrane filter of 100 nm pore size, the recovery of quercetin in the filtrate was higher than that of α-tocopherol. These data suggest that quercetin was an antioxidant in the digestion of fish oil. The effectiveness seems to come from its distribution in the emulsified oil, different from that of α-tocopherol, and its ability to scavenge radicals generated from the reaction of lipid hydroperoxides with metmyoglobin.     

Oxidative Stress and Heat Shock Protein Induction in Human Cells

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis.

Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.     

Oxidative Stress and Heat Shock Protein Induction in Human Cells

Agents which induce heat shock protein synthesis in cultured monolayers of Hela cells such as hyperthermia, ethanol and sodium arsenite can also cause increases in the levels of lipid peroxidation as determined by the formation of TBA-products. The heat induced increases may be diminished by addition to the medium of mannitol or EGTA. These compounds are known to depress heat shock protein synthesis.

Following hyperthermia there is also a decrease in protein synthesis. In vitro studies indicate possible damage to ribosomes, and since the heat induced loss of protein synthetic capacity can be increased by superoxide dismutase inhibitors, and prevented by mannitol, such effects may be linked to the increases observed in lipid peroxidation. It is suggested that a connection exists between lipid peroxidation and heat shock protein gene activation.     

Oxidative Modification of Low-Density Lipoprotein by the Human Hepatoma Cell Line HepG2

The human hepatoblastoma cell line HepG2 is a liver model commonly used for lipid metabolism studies. Numerous cell types have been found to oxidize low-density lipoprotein (LDL) but, to our knowledge, the effects of HepG2 cells on LDL have not been investigated. We found that LDL is modified by HepG2 cells through a peroxidative mechanism, as judged by an increase in TBARS content (which was prevented in the presence of the antioxidants vitamin E, 2, 6-di-tert-butyl-cresol and probucol), increased degradation by J774 macrophages, decreased internalization by MRC5 fibroblasts, and aggregation of apo B. Aspirin and allopurinol, which inhibit cyclooxygenase and xanthine-oxidase activities, respectively, had no effect on HepG2-induced LDL modification, and neither did catalase, which dismutates hydrogen peroxide; or mannitol, which scavenges hydroxyl radicals. In contrast, superoxide dismutase, a superoxide anion scavenger, and glutamate and threonine, which alter cellular cystine uptake, prevented LDL modifications, as did the removal of cysteine/cystine from the culture medium. Oxidation of LDL by HepG2 cells might thus involve superoxide anion production and/or thiol metabolism.     

NMR Analysis of Low-Density Lipoprotein Oxidatively-Modified in vztro

Human plasma low density lipoprotein has been oxidized at different stages in vitro and analysed by ¹H. ¹³C, and ³¹P NMR spectroscopy and by biochemical methods. Information was obtained on: a) structure mobilities of lipids and on lipid-protein interactions: b) conjugated and oxo-dienes; c) polyunsaturated/ monounsaturated fatty acid chains; d) lysophosphatidylcholine production. The results show that the NMR approach is particularly useful for the assessment of structural modification in oxidized LDL.     

Sequential Oxidative Damage, and Changes in Iron-Binding and Iron-Oxidising Plasma Antioxidants During Cardiopulmonary Bypass Surgery

Cardiopulmonary bypass patients undergoing heart valve replacement surgery appear to be under oxidative stress, when compared with normal healthy controls, by showing increased levels of protein and lipid damage. During bypass surgery two further episodes of oxidative stress occur. The first is seen when patients are placed on extracorporeal blood circulation and oxygenation which results in a rise in lipid peroxides and thiobarbituric acid-reactive substances. The second phase of oxidative stress occurs during reperfusion of the myocardium following removal of the aortic cross clamp. Coincident with evidence of increased oxidative damage to lipids during these latter phases of oxidative stress were decreases in plasma iron-binding and iron-oxidising antioxidant activities.     

重组人骨形成蛋白-3羧基端肽在大肠杆菌中表达及其诱骨活性

以双顺反子表达载体,在大肠杆菌中经ＩＰＴＧ诱导表达了人骨形成蛋白－３羧基端肽段（ｈＢＭＰ－３Ｃ）,表达量占菌体总蛋白量的１８．５％．目的蛋白为２５ｋＤ、含ｈＢＭＰ－３Ｃ端２１５个氨基酸残基组成的肽段,包括ｈＢＭＰ－３成熟肽和一部分前肽．表达产物以包涵体的形式存在,用含ＴｒｉｔｏｎＸ－１００的洗涤液和５ｍｏｌ／Ｌ以下脲溶液连续洗涤,可获得较高纯度的重组人骨形成蛋白－３Ｃ端肽．经复性处理成可溶性蛋白,植入小鼠肌肉内,第１４ｄ组织切片显示有软骨细胞和软骨基质形成,第２１ｄ可见成骨细胞和骨基质形成．将ｒｈＢＭＰ－３Ｃ与脱矿去免疫原性异种骨粒复合后作小鼠肌肉植入试验,２１ｄ组织切片上可见硬质骨形成．结果表明：大肠杆菌表达的ｈＢＭＰ－３Ｃ经复性后具有诱骨活性,糖基化并非ＢＭＰ－３活性所必需．     

人脑及红细胞膜乙酰胆碱酯酶的芳基酰胺酶活性

采用联合亲和层析法从人小脑及红细胞膜中纯化了ＡＣｈＥ,纯化的人脑及红细胞ＡＣｈＥ在ＳＤＳ－ＰＡＧＥ上呈一主带,分子量约为６６０００。人脑ＡＣｈＥ制备酯酶与酰胺酶比活性分别为１２９９与１４３Ｕ／ｍｇ,人红细胞ＡＣｈＥ制备分别为４５８４与７４７Ｕ／ｍｇ。人脑及红细胞ＡＣｈＥ制备的酯酶与酰胺酶活性最适ｐＨ较接近,在ｐＨ７．５－８．０之间,酯酶活性底物ＡＴＣｈ对其芳基酰胺酶活性有抑制作用。ＩＣ_（５０）分别为１０．２×１０~（－３）及３×１０~（－３）ｍｏｌ／Ｌ。梭曼对其酯酶及酰胺酶活性均有明显抑制作用,说明二者均需活性中心丝氨酸参与。     

Enolase Activity and Isoenzyme Distribution in Human Brain Regions and Tumors

Abstract: The distribution of enolase (EC 4.2.1.11) activity and isoenzymes in various regions of human brain at different ages (from 23 weeks of gestation to 95 years) and in brain tumors has been determined. Total enolase activity increased in all regions with age. No significant differences were found in the relative proportions of αα-, αγ-, and γγ-enolase isoenzymes in the various brain regions, determined by agarose gel electrophoresis. Type αα-enolase was the predominant isoenzyme, and αγ-enolase represented a substantial proportion of the total enolase activity. Astrocytomas, anaplastic astrocytomas, glioblastomas, and meningiomas possessed lower enolase activity than normal brain. Among astrocytic tumors, total enolase activity correlated with malignancy. Astrocytomas possessed the lowest and glioblastomas the highest enolase activity. All tumors possessed a higher proportion of αα-enolase and a lower proportion of γγ-enolase than the normal human brain. Among astrocytic tumors, glioblastomas were the tumors with the highest proportion of αα-enolase and lowest proportion of γγ-enolase.     

人sTNFR1基因的克隆、融合表达与生物学活性

采用RT-PCR,从Hela细胞的mRNA中扩增人sTNFR1基因,构建含有目的片段的T载体克隆及原核表达载体pMAL-c2x重组质粒亚克隆,转化入大肠杆菌,测序证实其序列与基因数据库中sTNFR1基因一致。经异丙基-β-D半乳糖苷酶（IPTG）诱导表达,淀粉树脂亲和层析法纯化,得到融合蛋白sTNFR1-MBP。结果显示：经Western-blotting检测,sTNFR1-MBP具有免疫活性;MTT检测目的蛋白可有效地封闭TNFα对QSG7701的细胞毒性作用;流式细胞术检测目的蛋白对TNF-α诱导QSG7701凋亡有抑制作用;sTNFR1-MBP具有良好的生物活性,为今后的研究打下了基础。