茶多酚单体L-EGCG对气相烟引起鼠肺细胞膜损伤的抑制作用

茶多酚(GTP)单体L-EGCG对香烟气相物质损伤鼠肺细胞膜的保护作用研究结果表明,L-EGCG能抑制香烟气相物质引发的脂质过氧化;用脂肪酸自旋标记物5-DOXYL和16-DOXYL标记鼠肺细胞膜,发现预先加入L-EGCG可抑制气相烟引起的膜浅层流动性改变,并与L-EGCG的浓度呈量效关系。在0.001到0.1mg/mL浓度范围内,L-EGCG本身对膜的浅层没有影响,而能使膜深层的流动性略有增大。由试验推测,L-EGCG的保护作用很可能是由于清除了气相烟中的自由基或脂质过氧化产生的脂类自由基。     

茶多酚单体L-EGCG对气相烟引起鼠肺细胞膜损伤的抑制作用

茶多酚(GTP)单体L-EGCG对香烟气相物质损伤鼠肺细胞膜的保护作用研究结果表明,L-EGCG能抑制香烟气相物质引发的脂质过氧化;用脂肪酸自旋标记物5-DOXYL和16-DOXYL标记鼠肺细胞膜,发现预先加入L-EGCG可抑制气相烟引起的膜浅层流动性改变,并与L-EGCG的浓度呈量效关系。在0.001到0.1mg/mL浓度范围内,L-EGCG本身对膜的浅层没有影响,而能使膜深层的流动性略有增大。由试验推测,L-EGCG的保护作用很可能是由于清除了气相烟中的自由基或脂质过氧化产生的脂类自由基。     

外源脂肪酸对莱氏衣原体膜流动性的影响——电子自旋共振与荧光偏振研究

生物膜类脂的物理性质直接影响膜的生理功能,膜的流动动性是反映膜脂物理状态的一个重要特征.本文采用电子自旋共振波谱及荧光偏振技术研究油酸,硬脂酸以及油酸和棕榈酸的混合物渗入莱氏衣原体膜后对膜流动性的影响.结果表明,上述外源脂肪酸均能增加膜的流动性,其中以油酸渗入膜后最为显著.油酸中双键的作用不仅仅局限于双键所在碳原子附近,而且能使整个膜脂双层各个层次上流动性都有增加.对于用荧光偏振和自旋标记顺磁共振二种技术所获得结果的异同也进行了初步讨论.     

银杏叶提取物对自由基所致脑神经细胞损伤的保护作用研究

从细胞分子水平,采用大脑皮层神经细胞分离技术,自旋标记技术,酶学方法及细胞染色技术探讨自由基对神经细胞的损伤及ＥＧｂ保护作用。结果显示,用自由基攻击的细胞其自旋标记膜的序参数Ｓ,旋转相关时间τｃ及膜蛋白巯基的Ｓ／Ｗ比对照组高（Ｐ＜０．０５）,提示：受自由基攻击的细胞膜,其流动性比正常减低,且蛋白质构象发生了改变。加ＥＧｂ保护后,可减少自由基引起的膜流动性及蛋白质构象改变,起到保护神经细胞的作用。经·ＯＨ攻击的神经细胞,其ＬＤＨ活性及死细胞比率都比对照组高,而ＥＧｂ对此有保护作用。本文结果说明一定浓度ＥＧｂ可保护大脑皮层神经细胞免受自由基的损伤。     

山莨菪碱对人红细胞膜蛋白及膜脂运动的影响

本文采用自旋标记顺磁共振波谱技术,研究了山茛菪碱对人红细胞膜蛋白和膜脂运动的影响.结果表明:用马来酰亚胺标记的人红细胞膜,加入山茛菪碱后,其顺磁共振波谱中强、弱固定化作用谱的峰值比增大,膜蛋白的运动受到限制.山茛菪碱对红细胞膜脂的作用部位主要在极性头部,并影响膜脂的流动性.本文还对山茛菪碱与红细胞膜作用的可能机制进行了讨论.     

锌_7-与镉_7-金属硫蛋白对羟自由基损伤大鼠红细胞膜保护作用的比较

用电子自旋共振自旋标记物氮氧自由基硬脂酸和马来酰亚胺标记大鼠红细胞膜脂和膜蛋白,测定膜脂流动性和膜蛋白构象改变,以硫代巴比妥酸法测定脂质过氧化产物丙二醛含量．结果表明,锌7-与镉7-金属硫蛋白对羟自由基引起的膜脂流动性减低、脂质过氧化反应增强双膜蛋白构象改变有明显抑制作用,而且,前者的作用明显强于后者．     

巨噬细胞细胞内吞过程中膜受体流动性的测量——两种标记受体方法的比较

本文首次实现了细胞内吞过程中膜受体流动性的测量。实验选择巨噬细胞膜和伴刀豆凝集素A(ConA),分别用Con A-Biotin Avi-din-FITC(ABC法)和Con A-FITC(直接法)两种方法标记巨噬细胞膜Con A受体,比较了这两种方法标记的巨噬细胞Con A受体的荧光强度;利用FRAP(Fluorescence RecoveryAfter Rhotobleaching)技术,分别用两种标记方法测量了巨噬细胞Con A受体的流动性。结果显示Con A-Biotin Avidin-FITC标记的巨噬细胞受体的平均荧光强度比用Con A-FITC标记的平均荧光强度高大约3倍;直接标记法应用于细胞内吞过程中受体流动性的测量在方法学上存在着很大的缺陷,ABC标记法适合于测量细胞内吞过程中膜表面受体的流动性的变化,且灵敏度高、误差小;ABC方法标记受体的测量结果显示,Con A刺激后巨噬细胞膜表面Con A受体的扩散系数和荧光恢复率与静息状态相比呈下降趋势。     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

用马来酰亚胺自旋标记研究库存血红细胞膜蛋白质构象

用两种马来酰亚胺自旋标记物—马来酰亚胺Ⅰ和马来酰亚胺Ⅴ研究了红细胞膜蛋白质构象及巯基结合位点性质在ACD-B方库存血保存期间的动态变化。结果发现,在35天的血液保存期间,马来酰亚胺Ⅰ所标记红细胞膜的S/w值很快下降到一低水平,而马来酰亚胺Ⅴ所标记红细胞膜的旋转相关时间则呈现迅速下降后缓慢升高的双相性变化。作者结合膜蛋白构象及其周围微观环境进行了讨论。     

羟基自由基对兔脑微粒体膜脂及膜蛋白的损伤

本文研究了过氧化氢与亚铁离子体系产生的羟基自由基对兔脑微粒体脂质过氧化作用及对膜上(Na~++K~+)-ATP酶活性的影响.结果表明,羟基自由基导致兔脑微粒体脂质过氧化,增加丙二醛的含量.羟基自由基还使微粒体膜巯基数下降,(Na~++K~+)-ATP酶活力受到抑制.阿魏酸钠对抑制微粒体脂质过氧化及对膜巯基和(Na~++K~+)-ATP酶均有保护作用.自旋捕集实验结果进一步证明药物对羟基自由基的猝灭作用.     

Membrane reception of thyroid hormones]

Comparative and competitive analyses of thyroxine (T4) and triiodothyronine (T3) binding to highly purified rat liver, brain and lung cell plasma membranes were carried out. The dependence of hormone binding on the time, temperature and concentration was studied. The effects of trypsin and partial delipidation on the binding parameters of thyroid hormones were investigated. Two thyroid hormone-binding sites were detected in cell plasma membranes of all tissues under study. The maximal binding of T4 to rat liver membranes and the maximal binding of T3 to rat brain membranes was observed in all experiments, the affinity for T3 being higher than that for T4. An important role of both protein and lipid components of plasma membranes in the membrane reception of thyroid hormones is proposed.     

Differential effects of Ca2+-calmodulin on adenylate cyclase activity cyclase activity in mouse and rat pancreatic islets.

The subcellular localization of the beta-galactoside-binding protein, or lectin, from rat lung was investigated by the specific binding of anti-lectin immunoglobulin G to subcellular fractions. We used both adult and immature (12-day-old) rats; the immature rat lungs have an 8-10-fold greater concentration than adult rat lungs [Powell & Whitney (1980) Biochem. J. 188, 1-8]. In both groups of animals we observed greater specific binding of anti-lectin immunoglobulin G to intracellular membrane (mitochondrial and microsomal fractions) than to plasma membranes. Pre-incubation of membrane fractions with lactose resulted in a marked diminution of anti-lectin immunoglobulin G binding. In the adult rat lung most (approx. 80%) of the lectin activity was membrane-associated. In the immature rat lung only approx. 30% of the lectin activity was membrane associated and most of the beta-galactoside-binding protein appeared to be a soluble cytoplasmic component. The rat lung beta-galactoside-binding protein appeared to have a broad but predominantly intracellular location, being associated with membranes through one of its galactoside-binding sites.     

A bromoacetylated analogue of cyanopindolol: an irreversible antagonist at rat beta-adrenoceptors

A high affinity, chemically reactive cyanopindolol derivative. N8-bromoacetyl-N1-3'-(2-cyano-4-indolyloxy)-2'-hydroxypropyl-[Z]-1 ,8-diamino-p-menthane (Br-CYP) was synthesized and its interaction with beta-adrenoceptors characterized. Studies with rat heart, lung, brain, and red blood cell membranes indicated that the compound displaced 3H-dihydroalprenolol (3H-DHA) from beta-adrenoceptors with IC50 values in the nanomolar range. The concentration of functional beta-adrenoceptors in membranes was markedly reduced when membranes were preincubated with Br-CYP and then extensively washed prior to assay. (+/-)Alprenolol and (-)isoproterenol, but not (+)isoproterenol, when included in the preincubation prevented this reduction in binding sites by Br-CYP. Br-CYP was active in vivo when injected intraperitoneally into rats. A dose of 10 micrograms/kg reduced the concentration of binding sites in membranes from heart by 30%, lung by 36%, and RBC by 70%, but did not affect sites on brain membranes 16 hours after injection. Higher doses blocked virtually all the 3H-DHA binding sites in the peripheral organs studied. Br-CYP reduced the concentration of beta-adrenoceptors in membranes from these same tissues (but not brain tissue) as long as two weeks after injection with recovery of binding occurring more rapidly in heart tissue than lung and red blood cells. These results suggest that Br-CYP may be a useful compound for in vivo studies of the biochemistry and pharmacology of beta-adrenergic systems.     

Cigarette smoke extract increases albumin flux across pulmonary endothelium in vitro

Cigarette smoking causes lung inflammation, and a characteristic of inflammation is an increase in vascular permeability. To determine if cigarette smoke could alter endothelial permeability, we studied flux of radiolabeled albumin across monolayers of porcine pulmonary artery endothelium grown in culture on microporous membranes. Extracts (in either dimethylsulfoxide or phosphate-buffered saline) of cigarette smoke in a range estimate of concentrations simulating cigarette smoke exposure to the lungs in vivo caused a dose-dependent increase in albumin flux that was dependent on extracellular divalent cations and associated with polymerization of cellular actin. The effect was reversible, independent of the surface of endothelial cells exposed (either luminal or abluminal), and due primarily to components of the vapor phase of smoke. The effects occurred without evidence of cell damage, but subtle morphological changes were produced by exposure to the smoke extracts. These findings suggest that cigarette smoke can alter permeability of the lung endothelium through effects on cytoskeletal elements.     

High affinity thyroxine binding to purified rat liver plasma membranes.

Two sets of high-affinity thyroxine binding sites (K_D 0.39 ± 0.06 nM and 23 ± 5 nM) were detected on purified rat liver plasma membranes. Thyroxine is bound with high stereospecificity regarding iodine substituents and alanine side chain modifications of the molecule. Thyroxine binding is inhibited by -SH blocking agents and proteases. The highest affinity thyroxine binding site is also affected by phospholipase A and is distinct from triiodothyronine binding sites present in the membrane preparations; arguments are given for its plasmalemma origin.     

The GTP-Insensitive Component of High-Affinity [3H]8-Hydroxy-2-(Di-n-Propylamino)tetralin Binding in the Rat Hippocampus Corresponds to an Oxidized State of the 5-Hydroxytryptamine1A Receptor

Previous studies on central 5-hydroxytryptamine1A (5-HT1A) receptors have consistently shown the existence of a GTP-insensitive component of agonist binding, i.e., binding of [3H]8-hydroxy-2-(di-n-propylamino)tetralin ([3H]8-OH-DPAT) that persists in the presence of 0.1 mM GTP or guanylylimidodiphosphate (GppNHp). The molecular basis for this apparent heterogeneity was investigated pharmacologically and biochemically in the present study. The GppNHp-insensitive component of [3H]8-OH-DPAT binding increased spontaneously by exposure of rat hippocampal membranes or their 3-[3-(cholamidopropyl)dimethylammonio]-1-propane sulfonate-soluble extracts to air; it was reduced by preincubation of solubilized 5-HT1A binding sites in the presence of dithiothreitol and, in contrast, reversibly increased by preincubation in the presence of various oxidizing reagents like sodium tetrathionate or hydrogen peroxide. In addition, exposure of hippocampal soluble extracts to short-cross-linking reagents specific for thiols produced an irreversible increase in the proportion of GppNHp-insensitive over total [3H]8-OH-DPAT binding. The pharmacological properties of this GppNHp-insensitive component of [3H]8-OH-DPAT binding were similar to those of 5-HT1A sites in the absence of nucleotide. Sucrose gradient sedimentation of solubilized 5-HT1A binding sites treated by dithiothreitol or sodium tetrathionate showed that oxidation prevented the dissociation by GTP of the complex formed by the 5-HT1A receptor binding subunit (R[5-HT1A]) and a guanine nucleotide-binding protein (G protein). Moreover, the oxidation of -SH groups by sodium tetrathionate did not prevent the inactivation of [3H]8-OH-DPAT specific binding by N-ethylmaleimide, in contrast to that expected from an interaction of both reagents with the same -SH groups on the R[5-HT1A]-G protein complex. These data suggest that the appearance of GTP-insensitive [3H]8-OH-DPAT specific binding occurs as a result of the (spontaneous) oxidation of essential -SH groups (different from those preferentially inactivated by N-ethylmaleimide) on the R[5-HT1A]-G protein complex.     

Binding sites of a novel neuropeptide pituitary-adenylate-cyclase-activating polypeptide in the rat brain and lung

Pituitary-adenylate-cyclase-activating polypeptide (PACAP) is a novel 38-amino-acid neuropeptide isolated from ovine hypothalamic tissues based on its activity of stimulating adenylate cyclase of rat pituitary cells. Binding sites for PACAP were studied in rat tissue membranes using a 27-amino-acid N-terminal derivative of PACAP [PACAP(1-27)] labelled with 125I. Particularly high specific binding sites of 125I-PACAP(1-27) were noted in the hypothalamus, brain stem, cerebellum and lung. Specific binding sites are also present in the pituitary gland, but at a lower concentration, and mainly in the anterior lobe. Very low concentration of 125I-PACAP(1-27)-binding sites were found in the colon, aorta and kidney membranes and no binding sites were detected in the pancreas and testis. Maximal binding of 125I-PACAP(1-27) was observed at pH 7.4. Interaction of 125I-PACAP(1-27) with its binding site was rapid, specific and saturable as well as time, pH and temperature dependent. PACAP(1-27) is more potent than PACAP in displacing the binding of 125I-PACAP(1-27) with brain membranes [concentration that inhibits 50% of the binding (IC50) = 7.45 +/- 1.52 nM and 11.45 +/- 3.65 nM, respectively; mean +/- SEM, n = 4] and lung membranes (IC50 = 4.41 +/- 0.87 nM and 10.68 +/- 3.09 nM, respectively). Vasoactive intestinal peptide displaced the binding of 125I-PACAP(1-27) in lung membrane (IC50 = 16.88 +/- 5.14 nM) but not in brain membranes. The equilibrium binding of 125I-PACAP(1-27) at 4 degrees C was characterized by a single class of binding site for the brain membrane with a dissociation constant (Kd) of 2.46 +/- 0.53 nM and a maximal binding capacity (Bmax) of 8.44 +/- 3.13 pmol/mg protein, but there were two classes of binding site for lung membranes with Kd of 1.02 +/- 0.51 nM and 5.19 +/- 0.99 nM, and Bmax of 2.84 +/- 0.72 pmol/mg protein and 9.13 +/- 1.89 pmol/mg protein, respectively. These findings suggest that subtypes of PACAP-binding sites exist and PACAP may have a physiological role in the hypothalamus/pituitary axis as well as in other regions of the brain and lung.     

Endothelin binding sites in porcine aortic and rat lung membranes

High-affinity binding sites for endothelin were identified on porcine aortic and rat lung membranes. Interaction of 125I-labelled endothelin with its binding site was specific, saturable, time- and temperature-dependent but dissociation of receptor-bound ligand was minimal. Maximal binding was observed at pH 7.0 in porcine aorta and at pH 3.1 in the rat lung. Treatment of membranes with trypsin destroyed the binding site in both tissues. Porcine endothelin showed a higher affinity for receptors in both tissues compared to rat endothelin. Vasoactive peptides and Ca2+ channel antagonists did not interact with this site suggesting high specificity of binding. Analysis of saturation binding showed that the number of binding sites was 1250 +/- 104 and 1650 +/- 170 fmol/mg protein and the affinity of binding sites was 0.47 +/- 0.15 and 0.16 +/- 0.07 nM in the aorta and the lungs respectively (n = 5). Presence of protease inhibitors did not alter binding suggesting that the label was stable under the incubation conditions. This was further confirmed by HPLC. Removal of the endothelium from the aorta did not change the binding characteristics of this tissue. Ca2+ and Mg2+ ions caused an increase in binding by increasing the affinity. Binding was completely abolished in the presence of Triton and dithiothreitol. The binding sites identified in this study may be responsible for the actions of endothelin in the aorta and the lung.     

Characterization of methadone receptor subtypes present in human brain and lung tissues

In addition to their use in pain control, opioids can function as regulators of tumor cell growth. We have found that the therapeutic opioid, methadone, significantly inhibits the in vitro and in vivo growth of human lung cancer cells, and this effect appears to be mediated by specific, high affinity, non-conventional opioid binding sites. The present study indicates the existence of multiple subtypes of binding sites mediating the peripheral and central nervous system actions of this drug. Pharmacological and biochemical characterizations of the methadone binding sites expressed in human brain and normal lung tissues indicate that these sites are distinct from each other and from other opioid receptor types present on human and rat brain membranes, as well as those expressed in human lung cancer cells. The identification of distinct methadone receptor types in the different tissues could lead to the development of more selective and less toxic drugs targeted toward the tumor Cells.     

Binding of laminin to brain gangliosides and inhibition of laminin-neuron interaction by the gangliosides

Binding of laminin to glycolipids of neuronal membranes was studied with a thin-layer chromatography overlay assay. The major brain ganglioside GD1A was the main binding component, when chromatograms containing the same molar amount of the different brain gangliosides and the brain sulfatide were incubated with laminin at physiological ionic strength. The possible role of laminin binding to brain gangliosides in laminin-neuron interactions was studied with adhesion assays. It was found that binding of rat brain neurons to laminin is blocked by 10-40 microM brain gangliosides but not by sulfatide. The inhibition by the gangliosides is suggested to be due to competition with the cell surface interaction sites of laminin and not to binding of the gangliosides to the cells. Our findings support the idea that the adhesive and neurite-promoting effect of laminin is dependent on its interaction with gangliosides at the neuronal cell surfaces.