氧化修饰低密度脂蛋白对巨噬细胞一氧化氮合酶基因表达的影响

氧化修饰LDL(OX-LDL)可抑制脂多糖(LPS)诱导的巨噬细胞NO释放, 而正常(N-LDL)和乙酰化LDL(AC-LDL)则没有抑制作用.OX-LDL对NO释放的抑制作用随LDL修饰程度的升高而增强,且具有浓度和时间效应.狭缝杂交结果显示OX-LDL处理可使LPS诱导的巨噬细胞NOS mRNA含量下降,提示OX-LDL对NO释放的抑制作用可能发生在转录水平.     

染料木素体外抑制人低密度脂蛋白氧化修饰作用

为探讨染料木素对人低密度脂蛋白(LDL)氧化修饰的影响,采用铜离子(10 umol/L)体外氧化LDL的方法,观察大豆异黄酮主要成分染料木素(genistein)对LDL氧化过程中脂质过氧化产物丙二醛(MDA)含量和维生素E(VitE)水平的影响。结果:10 umol/LCuSO4与100 mg/L LDL共同孵育18 h,MDA含量明显升高,VitE含量明显降低,染料木素(0.25、1.25、2.5、12.5、25、50、125、250 umol/L)能显著降低MDA含量,升高VitE含量(P<0.01,P<0.05,P<0.02),且呈剂量依赖性。提示一定浓度范围的染料木素体外有抗LDL氧化修饰作用。     

活性氧信号传导作用的研究进展

活性氧的信号传导作用已经为大量研究结果所证实,氧化还原修饰靶分子是其信号传导的主要机制.活性氧的信号传导作用几乎与所有已知的信号传导途径相关,蛋白酪氨酸激酶、蛋白激酶C、分裂刺激因子激活的蛋白激酶、转录因子NF-κB、AP-1及Ca^2＋、环鸟酸苷等信号分子都参与活性氧的信号传导作用.但是,有关活性氧信号传导作用还有许多问题有待阐明.     

氧化修饰高密度脂蛋白的研究进展

血浆高密度脂蛋白(HDL)与低密度脂蛋白(LDL)一样可以在体内外发生氧化修饰,引起其理化性质发生一系列的改变,如多不饱和脂肪酸过氧化,卵磷脂水解,蛋白质发生聚合或分解等.活体内HDL可能在动脉壁巨噬细胞、内皮细胞及中性粒细胞、单核细胞的作用下发生氧化修饰.氧化修饰HDL可能通过清道夫受体途径代谢.氧化修饰HDL产生多种致动脉粥样硬化作用.维生素E、C的摄入可能有助于防止脂蛋白的氧化.     

巴郎山刺叶高山栎叶片δ13C对海拔高度的响应

在卧龙自然保护区,按海拔梯度选择了三个刺叶高山栎分布地点(2600m、3050m和3500m),对各研究地点刺叶高山栎进行了叶片d¹³C、光合、CO2扩散导度、养分以及形态等参数的测量,以期揭示刺叶高山栎叶片d¹³C随着海拔的响应规律及原因。结果表明：首先,随着海拔的升高,目标树种叶片d¹³C随之增加,且海拔每升高1000m,d¹³C增加2.0‰;其次,随着海拔升高,大气压强降低,因而导致的叶片扩散导度(包括气孔导度(gs)和叶肉细胞导度(gm))的降低是叶片d¹³C随着海拔升高而升高的主要原因;同时,随着海拔升高而增加的叶片厚度(LMA)也在一定程度上促进了这种响应趋势;最后,叶片N含量随着海拔升高而更多被分配给非光合系统的趋势促进了羧化效率及光合速率的降低,进而对刺叶高山栎叶片d¹³C海拔响应起到了一定的抑制作用,但程度不大;此外,光合氮利用效率(PNUE)在反映植物叶片的氮含量与其吸收和固定大气CO2能力之间的关系方面及对d¹³C的指示作用方面较单位面积氮含量(Narea)和单位重量氮含量(Nmass)更鲜明和准确。     

稀土La3+跨PC12细胞膜行为研究

使用AR-CM-M1C阳离子测定系统,发展Fura-2荧光测定技术,将其应用于测定细胞内游离稀土离子La³⁺,并以此研究了La³⁺跨PC12细胞（大鼠嗜铬细胞瘤细胞）膜的行为.结果表明：在模拟细胞内离子组分,pH=7.05的溶液中,测得La³⁺-Fura-2的表观解离常数为3.27×10^-11 mol·L^-1.对于PC12细胞,静息条件下La³⁺不能跨越细胞膜进入胞内.与钙离子通道相关的KCl和去甲肾上腺素均不能刺激稀土La³⁺过膜.用哇巴因（ouabain）使胞内Na⁺超载后,La³⁺可过膜进入细胞内,且过膜量与胞外La³⁺浓度和胞内Na⁺超载程度有一定的浓度依赖关系,提示La³⁺可以经由Na⁺／La³⁺交换机制过膜而进入细胞内.     

胡杨质膜的纯化及其H＋-ATPase活性的研究

用Dextran T-500, PEG 3350两相分配法分离并纯化了悬浮培养的胡杨细胞质膜.不同聚合物浓度(5.5%、5.7%、5.9%、6.1%、6.3%、6.5%)和KCl浓度(0、5、10、15 mmol/L)对分离效果影响的研究结果表明, 采用聚合物浓度为5.9%和无盐存在的两相分配体系可获得纯度较高的胡杨细胞质膜.纯化的质膜H^＋-ATPase的活力提高8倍,且酶定向程度较高,这为进一步研究胡杨细胞质膜特性及获得高纯度H^＋-ATPase提供了良好基础.     

Ca2+参与水霉菌丝细胞壁修饰的证据

以细胞壁崩溃酶-Driselflse短时间处理水霉(Saprozegma ferax)菌丝,pH5．0时可使原生质从菌丝亚顶端喷出,pH6．0～8．0时则不导致该现象发生;适当浓度EGTA的存在,可提高pH5．0时酶解引起的原生质喷出频率、使pH6．0～8．0时生长菌丝的顶端原生质也喷出、并且喷出多发生在菌丝最顶端;外加CaCl₂．不抑制菌丝顶端原生质的喷出,排除了Ca²⁺抑制酶活性的可能。随后的跟踪观察显示,长时间以缺Ca²⁺培养介质培养菌丝,同样能够导致菌丝顶端原生质喷出。上述研究结果表明,培养介质中Ca²⁺和H⁺对菌丝完整性的维持起调节作用,细胞壁上的Ca²⁺可能参与了水霉菌丝细胞壁物理特性的修饰。     

大鼠海马CA1区锥体细胞上一种Ca2+依赖性KATP通道

K_ATP通道在细胞的新陈代谢与膜兴奋性的耦联中起重要作用.采用膜片钳的内面向外式记录方法,在成年大鼠海马CA1区锥体细胞上记录到一种被胞浆侧ATP和甲糖宁（tolbutamide,一种K_ATP通道阻断剂）抑制的Ca²⁺依赖性钾离子通道.在细胞膜内外的K⁺浓度均为140 mmol/L时,通道的电导为(204±21) pS,翻转电位为(3.57±1.13) mV,通道无整流性.通道开放概率及ATP对通道的抑制作用均呈现电压依赖性.该K_ATP通道与以往报道的“经典”K_ATP通道有显著不同,其活动受膜电位、胞内Ca²⁺和ATP三重调节,表明这是一种新型的K_ATP通道.上述结果表明在海马神经元上至少有两种性质不同的K_ATP通道,提示神经元可能通过不同性质的K_ATP通道感受细胞内的代谢状态,进而调节细胞膜的兴奋性.     

关于吖啶橙测定溶酶体H＋－ATPase转运H＋的研究

揭示了吖啶橙的吸收光谱和荧光光谱对其浓度依赖性上的区域性特征,分析了测定溶酶体H^＋转运时合理选用吖啶橙浓度及溶酶体用量的重要性、机理和原则,探讨了其与溶酶体的温育时间和K^＋／H^＋交换对测定H^＋转运的明显影响.     

A fibre cocktail of fenugreek, guar gum and wheat bran reduces oxidative modification of LDL induced by an atherogenic diet in rats

Background LDL (low-density lipoprotein) oxidation is a key trigger factor for the development of atherosclerosis. Relatively few studies exist on the impact of dietary fibre on LDL oxidation. This study was undertaken to evaluate the influence of a novel fibre mix of fenugreek seed powder, guar gum and wheat bran (Fibernat) on LDL oxidation induced by an atherogenic diet. Method Male Wistar albino rats were administered one of the following diets: (1) a control diet that was fibre-free (Group I); (2) an atherogenic diet containing 1.5% cholesterol and 0.1% cholic acid (Group II) or (3) an atherogenic diet supplemented with Fibernat (Group III). Peroxidative changes in low-density lipoprotein (LDL) and the oxidative susceptibility of LDL and the LDL + VLDL (very low-density lipoprotein) fraction were determined. As a corollary to the oxidative modification theory, the titer of autoantibodies to oxidised LDL (oxLDL) was determined at various time points of the study. In addition, plasma homocysteine (tHcy) and lipoprotein (Lp (a)), apolipoprotein (apoB), cholesterol, triglyceride, phospholipid and α-tocopherol content of LDL were determined. Results A decrease in malonaldehyde (MDA) content (p < 0.05) and relative electrophoretic mobility (REM) of LDL was observed in the group III rats as compared to the group II rats. An increase in lag time to oxidation (p < 0.01) and decrease in maximum oxidation (p < 0.01) and oxidation rate (p < 0.01) were observed in the LDL + VLDL fraction of group III rats. In group II rats, formation of autoantibodies to oxLDL occurred at an earlier time point and at levels greater than in the group III rats. Fibernat, had a sparing effect on LDL α-tocopherol, which was about 51% higher in the group III rats than in the group II rats; apo B content of LDL was reduced by 37.6% in group III rats. LDL of group III rats displayed a decrease in free and ester cholesterol (p < 0.01) as compared to that of group II. A decrease in plasma homocysteine (p < 0.01) and an increase in GSH (p < 0.05) were also observed in group III rats when compared with that of group II. Conclusion Fibernat administration appears to combat oxidative stress resulting in a trend to lower oxidative modification of LDL. In addition, the cholesterol and apo B content of LDL were reduced significantly with a sparing effect on LDL α-tocopherol. This novel fibre preparation could be an effective diet therapy and therefore needs further investigation.     

Increased oxidazability of plasma low density lipoprotein from patients with coronary artery disease

Oxidative modification of lipoproteins may play a crucial role in the pathogenesis of atherosclerosis. This study was designed to examine whether increased lipid peroxides and/or oxidative susceptibility of plasma lipoproteins occur in patients with coronary artery disease. The levels of lipid peroxides, estimated as thiobarbituric acid-reactive substances (TBARS), were significantly greater in the plasma and very low density lipoprotein (VLDL) of symptomatic patients with coronary artery disease than in those of healthy persons, but the TBARS levels of low density lipoprotein (LDL) and high density lipoprotein (HDL) showed insignificant difference between patients and normals. To evaluate the oxidative susceptibility of lipoproteins, we employed in vitro Cu²⁺ oxidation of lipoproteins monitored by changes in fluorescenece, TBARS level, trinitrobenzene sulfonic acid (TNBS) reactivity, apolipoprotein immunoreactivity and agarose gel electrophoretic mobility. While VLDL and LDL of normal controls were oxidazed at 5–10 μM Cu²⁺, pooled VLDL and LDL of patients with coronary artery disease were oxidized at 1–2.5 μM Cu²⁺, i.e., at relatively lowver oxidative stress. At 5 μM Cu²⁺, VLDL and LDL of patients with coronary artery disease still showed at faster oxidation rate, judged by the rate of fluorescence increase, higher TBARS level, less TNBS reactivity, greater change in apo B immunoreactivity and higher electrophoretic mobility than those of normal controls. However, the difference on the oxidizability of HDL was insignificant for patients vs. normals. In conclusion, we have shown that plasm VLDL and LDL of patients with coronary artery disease are more susceptible to in vitro oxidative modification than those of health persons. The data suggest that enhanced oxidizability of plasma lipoproteins may be important factor influencing the development of coronary artery disease.     

Enzymatic modification of low density lipoprotein by purified lipoxygenase plus phospholipase A2 mimics cell-mediated oxidative modification

Low density lipoprotein (LDL) can be oxidatively modified by cultured endothelial cells or by cupric ions, resulting in increased macrophage uptake of the lipoprotein. This process could be relevant to the formation of macrophage-derived foam cells in the early atherosclerotic lesion. The mechanism of endothelial cell modification of LDL is unknown. In the present work we show that incubation of LDL with purified soybean lipoxygenase, in the presence of pure phospholipase A2, can mimic endothelial cell-induced oxidative modification. Typically, incubation with lipoxygenase plus phospholipase A2 caused: 1) generation of about 15 nmol of thiobarbituric acid-reactive substances per mg of LDL protein; 2) a 4- to 7-fold increase in the rate of subsequent macrophage degradation of the LDL; 3) a 10-fold decrease in recognition by fibroblasts; 4) a marked increase in electrophoretic mobility in agarose gels; and, 5) disappearance of intact apoprotein B on SDS polyacrylamide gels. Degradation of the enzymatically modified LDL by macrophages was competitively inhibited by endothelial cell-modified LDL and by polyinosinic acid, but only partially suppressed by acetylated LDL. The lipoxygenase plus phospholipase A2-induced modification of LDL is not necessarily identical to endothelial cell modification, but it is a useful model for studying the mechanism of oxidative modification of LDL. This work also represents the first example of oxidative modification of LDL by specific enzymes leading to enhanced recognition by macrophages.     

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.     

Protective role of arzanol against lipid peroxidation in biological systems

This study examines the protective effect of arzanol, a pyrone–phloroglucinol etherodimer from Helichrysum italicum subsp. microphyllum, against the oxidative modification of lipid components induced by Cu²⁺ ions in human low density lipoprotein (LDL) and by tert-butyl hydroperoxide (TBH) in cell membranes. LDL pre-treatment with arzanol significantly preserved lipoproteins from oxidative damage at 2 h of oxidation, and showed a remarkable protective effect on the reduction of polyunsaturated fatty acids and cholesterol levels, inhibiting the increase of oxidative products (conjugated dienes fatty acids hydroperoxides, 7β-hydroxycholesterol, and 7-ketocholesterol). Arzanol, at non-cytotoxic concentrations, exerted a noteworthy protection on TBH-induced oxidative damage in a line of fibroblasts derived from monkey kidney (Vero cells) and in human intestinal epithelial cells (Caco-2), decreasing, in both cell lines, the formation of oxidative products (hydroperoxides and 7-ketocholesterol) from the degradation of unsaturated fatty acids and cholesterol. The cellular uptake and transepithelial transport of the compound were also investigated in Caco-2 cell monolayers. Arzanol appeared to accumulate in Caco-2 epithelial cells. This phenol was able to pass through the intestinal Caco-2 monolayers, the apparent permeability coefficients (P_app) in the apical-to-basolateral and basolateral-to-apical direction at 2 h were 1.93 ± 0.36 × 10⁻⁵ and 2.20 ± 0.004 × 10⁻⁵ cm/s, respectively, suggesting a passive diffusion pathway. The results of the work qualify arzanol as a potent natural antioxidant with a protective effect against lipid oxidation in biological systems.     

Purification and Immunological Properties of NAD+-linked Malate Dehydrogenase Isozymes from Euglena gracilis

We investigated the hypolipidemic effects of young persimmon fruit (YP) on apolipoprotein E-deficient C57BL/6.KOR-ApoE^shl mice. These mice exhibited higher plasma cholesterols, except for high-density lipoprotein (HDL), and lower plasma HDL cholesterol than C57BL/6.Cr mice that had the same genetic background as the C57BL/6.KOR-ApoE^shl mice. Male C57BL/6.KOR-ApoE^shl mice (n=5) were fed a diet supplemented with dry YP, Hachiya-kaki, at a concentration of 5% (w/w) for 10 weeks. YP treatment significantly lowered plasma chylomicron, very low-density lipoprotein (VLDL) and low-density lipoprotein (LDL) cholesterols, and triglyceride, and this response was accompanied by an elevation of fecal bile acid excretion. In the liver, sterol regulatory element binding protein-2 gene expression was significantly higher in mice fed YP, while the mRNA and protein levels of the LDL receptor did not change. These results indicate that acceleration of fecal bile acid excretion is a major mechanism of the hypolipidemic effect induced by YP in C57BL/6.KOR-ApoE^shl mice.     

Copper- and magnesium protoporphyrin complexes inhibit oxidative modification of LDL induced by hemin,transition metal ions and tyrosyl radicals

The oxidative modification of LDL may play an important role in the early events of atherogenesis. Thus the identification of antioxidative compounds may be of therapeutic and prophylactic importance regarding cardiovascular disease. Copper-chlorophyllin (Cu-CHL), a Cu²⁺-protoporphyrin IX complex, has been reported to inhibit lipid oxidation in biological membranes and liposomes. Hemin (Fe³⁺-protoporphyrin IX) has been shown to bind to LDL thereby inducing lipid peroxidation. As Cu-CHL has a similar structure as hemin, one may assume that Cu-CHL may compete with the hemin action on LDL. Therefore, in the present study Cu-CHL and the related compound magnesium-chlorophyllin (Mg-CHL) were examined in their ability to inhibit LDL oxidation initiated by hemin and other LDL oxidizing systems. LDL oxidation by hemin in presence of H₂O₂ was strongly inhibited by both CHLs. Both chlorophyllins were also capable of effectively inhibiting LDL oxidation initiated by transition metal ions (Cu²⁺), human umbilical vein endothelial cells (HUVEC) and tyrosyl radicals generated by myeloperoxidase (MPO) in presence of H₂O₂ and tyrosine. Cu- and Mg-CHL showed radical scavenging ability as demonstrated by the diphenylpicrylhydracylradical (DPPH)-radical assay and estimation of phenoxyl radical generated diphenyl (dityrosine) formation. As assessed by ultracentrifugation the chlorophyllins were found to bind to LDL (and HDL) in serum. The present study shows that copper chlorophyllin (Cu-CHL) and its magnesium analog could act as potent antagonists of atherogenic LDL modification induced by various oxidative stimuli. As inhibitory effects of the CHLs were found at concentrations as low as 1 μmol/l, which can be achieved in humans, the results may be physiologically/therapeutically relevant.     

Effect of Docosahexaenoic Acid and Ascorbate on Peroxidation of Retinal Membranes of ODS Rats

The ability of a range of dietary flavonoids to inhibit low-density lipoprotein (LDL) oxidation in vitro was tested using a number of different methods to assess oxidative damage to LDL. Overall quercetin was the most effective inhibitor of oxidative damage to LDL in vitro. On this basis, a diet enriched with onions and black tea was selected for a dietary intervention study that compared the effect on the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo in healthy human subjects of a high flavonoid diet compared with a low flavonoid diet. No significant difference was found in the Cu²⁺ ion-stimulated lag-time of LDL oxidation ex vivo between the high flavonoid and low flavonoid dietary treatments (48 ± 1.6 min compared to 49 ± 2.1 min).     

Catabolism of native and oxidized low density lipoproteins: in vivo insights from small animal positron emission tomography studies

Summary. The human organism is exposed to numerous processes that generate reactive oxygen species (ROS). ROS may directly or indirectly cause oxidative modification and damage of proteins. Protein oxidation is regarded as a crucial event in the pathogenesis of various diseases ranging from rheumatoid arthritis to Alzheimer’s disease and atherosclerosis. As a representative example, oxidation of low density lipoprotein (LDL) is regarded as a crucial event in atherogenesis. Data concerning the role of circulating oxidized LDL (oxLDL) in the development and outcome of diseases are scarce. One reason for this is the shortage of methods for direct assessment of the metabolic fate of circulating oxLDL in vivo. We present an improved methodology based on the radiolabelling of apoB-100 of native LDL (nLDL) and oxLDL, respectively, with the positron emitter fluorine-18 (¹⁸F) by conjugation with N-succinimidyl-4-[¹⁸F]fluorobenzoate ([¹⁸F]SFB). Radiolabelling of both nLDL and oxLDL using [¹⁸F]SFB causes neither additional oxidative structural modifications of LDL lipids and proteins nor alteration of their biological activity and functionality, respectively, in vitro. The method was further evaluated with respect to the radiopharmacological properties of both [¹⁸F]fluorobenzoylated nLDL and oxLDL by biodistribution studies in male Wistar rats. The metabolic fate of [¹⁸F]fluorobenzoylated nLDL and oxLDL in rats in vivo was further delineated by dynamic positron emission tomography (PET) using a dedicated small animal tomograph (spatial resolution of 2 mm). From this study we conclude that the use of [¹⁸F]FB-labelled LDL particles is an attractive alternative to, e.g., LDL iodination methods, and is of value to characterize and to discriminate the kinetics and the metabolic fate of nLDL and oxLDL in small animals in vivo.     

Calcium Antagonists Prevent Monocyte and Endothelial Cell-Induced Modification of Low Density Lipoproteins

Low density lipoprotein (LDL) incubated in the presence of the calcium antagonists verapamil, nifedipine and flunarizine were more resistant than control LDL to human monocyte- or endothelial cell-induced modification, as assessed by electrophoretic mobility in agarose gel, thiobarbituric acid reactive substance content, and degradation by J774 macrophages. The efficiency of the drugs was: flunarizine > nifedipine > veraparml. Moreover, a 24 h preculture with calcium antagonists significantly impaired the ability of cells to modify LDL in the absence of the drugs. All the studied drugs also inhibited copper-induced autooxidation of LDL. None of the studied calcium antagonists, at concentrations up to 10^-4 M, significantly reacted with free radicals as assessed by the l,1-diphenyl-2-picrylhydrazyl test. It is suggested that such a protective effect of calcium antagonists against LDL peroxidation could play a role in the previously reported antiatherogenic effect of these drugs.