兔肝细胞膜高密度脂蛋白受体酶联免疫检测法的研究

用辣根过氧化物酶标记羊抗人apoAI-IgG,建立了检测兔肝细胞膜HDL受体的酶联免疫吸附检测法.测定时, HDL结合量按抗体-配体-抗配体抗体酶交联物反应制作的标准曲线确定;膜蛋白非特异吸附则用与酶交联的抗体来源相同的同种动物血浆HDL平行抑制试验消除.实验测得正常家兔肝细胞膜HDL受体K_d值为7.17±1.18 mg/L,B_max值为(622.5±146.1)mg/g(n=7).     

Specificity of Auxin-binding Sites on Maize Coleoptile Membranes as Possible Receptor Sites for Auxin Action

Dissociation coefficients of auxin-binding sites on maize (Zea mays L.) coleoptile membranes were measured, for 48 auxins and related ring compounds, by competitive displacement of ¹⁴C-naphthaleneacetic acid from the binding sites. The sites bind with high affinity several ring compounds with acidic side chains 2 to 4 carbons long, and much more weakly bind neutral ring compounds and phenols related to these active acids, most phenoxyalkylcarboxylic acids, and arylcarboxylic acids except benzoic acid, which scarcely binds, and triiodobenzoic acids, which bind strongly. Specificity of the binding is narrowed in the presence of a low molecular weight “supernatant factor” that occurs in maize and other tissues. Activity of many of the analogs as auxin agonists or antagonists in the cell elongation response was determined with maize coleoptiles. These activities on the whole roughly parallel the affinities of the binding sites for the same compounds, especially affinities measured in the presence of supernatant factor, but there are some quantitative discrepancies, especially among phenoxyalkylcarboxylic acids. In view of several factors that can cause receptor affinity and biological activity values to diverge quantitatively among analogs, the findings appear to support the presumption that the auxin-binding sites may be receptors for auxin action.     

人血浆HDL对动脉粥样硬化家兔肝细胞膜LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化(As)模型家兔通过静脉注射人血浆HDL制剂,观察HDL对As家兔肝细胞膜LDL受体活性的影响.结果发现,摄取高胆固醇饲料的As家兔,其肝细胞膜LDL受体K_d值虽无明显变化但B_max值显著减小（P＜0.01,与正常对照组比较);注射HDL制剂后,As家兔肝细胞膜LDL受体K_d值仍无明显改变,但B_max值却显著回升（P＜0.01,与高脂组比较).表明人血浆HDL具有增加As家兔肝细胞膜LDL受体活性的作用.     

动物细胞膜的分离纯化及其在病毒受体研究中的应用

细胞膜是动物细胞与胞外环境之间的屏障。病毒只有与细胞膜上的病毒受体特异性结合 ,才能进入细胞 ,进而启动其增殖周期。因此 ,病毒受体是病毒学研究的重要组成部分。分离纯化病毒受体所在的细胞膜作为病毒受体研究的实验材料 ,已经在许多病毒的研究中得到应用 ,并取得了很好的效果。现就动物细胞膜的分离纯化及其在病毒受体研究中的应用作一综述。     

人血浆HDL对动脉粥样硬化家兔肝细胞膜 LDL受体活性影响的研究

高胆固醇饲料喂养造成的动脉粥样硬化（Ａｓ） 模型家兔通过静脉注射人血浆ＨＤＬ 制剂, 观察ＨＤＬ 对Ａｓ家兔肝细胞膜ＬＤＬ受体活性的影响． 结果发现, 摄取高胆固醇饲料的Ａｓ 家兔, 其肝细胞膜ＬＤＬ 受体 Ｋｄ 值虽无明显变化但Ｂｍａｘ 值显著减小（ Ｐ＜ ０－０１ , 与正常对照组比较） ; 注射ＨＤＬ 制剂后, Ａｓ 家兔肝细胞膜ＬＤＬ受体Ｋｄ 值仍无明显改变, 但Ｂｍａｘ 值却显著回升（ Ｐ＜ ０－０１ , 与高脂组比较） ． 表明人血浆ＨＤＬ 具有增加Ａｓ 家兔肝细胞膜ＬＤＬ 受体活性的作用．     

Electromagnetic Enhancement Effect Caused by Aggregation on SERS-Active Gold Nanoparticles

We report a morphology-correlated surface-enhanced Raman scattering (SERS) from molecules on the surface of individual clusters of gold nanoparticles of two types and compare the signal from clusters of two, three, four, and five nanoparticles with the signal from single particles. Cluster geometry and particle morphology are determined from transmission electron microscopy for both clusters of 78- to 133-nm nanospheres and clusters of ~250-nm-etched cylindrical particles with crevices and sharp edges, formed in templates. Scattering from molecules on etched cylinders, but not spheres, is sufficiently strong to allow spectra to be collected from single particles illuminated at 632.8 nm. SERS intensities from clusters of cylinders are found to scale linearly with particle number, whereas, for nanospheres, the scaling is non-linear. The linear scaling of SERS from cylinders is a reflection of the high enhancement provided by the sharp features of the individual particles; whereas, the non-linear scaling of SERS from clusters of spheres is found to be consistent with the near-field enhancement from inter-particle coupling simulated for clusters of spheres arranged in representative-observed geometries.     

Uncoating of Poliovirus by Isolated Plasma Membranes

Poliovirus type I LSc strain labeled with (14)C-uridine was adsorbed onto isolated plasma membranes and incubated with them. When membranes from Hep-2 or Vero cells were used, 22% of the label was converted to a trichloroacetic acid-soluble form, when trypsin or ribonuclease was added, the fraction rendered soluble was increased, and when the two enzymes were added in sequence, 85% or more of the label became trichloroacetic acid-soluble. This labilization of poliovirus could be reproduced when butanol-solubilized proteins from membranes were substituted for the whole plasma membranes, but it did not occur with membranes from polio-virus-resistant calf kidney or BHK-21 cells.     

Evidence for the Existence of Imidazoline-Specific Binding Sites in Synaptosomal Plasma Membranes of the Bovine Brainstem

Abstract: Nonadrenergic imidazoline-specific binding sites were characterized pharmacologically in crude cerebral membrane preparations, but little is known about their subcellular localization in neurons. As in the brain-stem these sites are involved in cardiovascular regulation and peripherally imidazolines modulate neurotransmitter release, we tried to determine a possible (pre)synaptic localization in brainstem. We found a specific enrichment in (entire) synaptosome, purified synaptosomal plasma membrane (37 fmol/mg), and mitochondrial (83 fmol/mg) fractions as compared with other membrane fractions (3–8 fmol/mg). Synaptosomes appeared to be free of postsynaptic structures, and purified synaptosomal plasma membranes were devoid of mitochondrial material, as determined by electron microscopy and by comparison with the distribution of marker enzymes such as monoamine oxidase. These results show for the first time that these extramitochondrial imidazoline-specific sites are neuronal and are located on presynaptic terminals. We found high affinities for unlabeled p -iodoclonidine (subnanomolar), clonidine (0.2 n M ), and efaroxan (11 n M ), but idazoxan did not compete significantly for the p -[¹²⁵I]iodoclonidine binding in these membranes. Therefore, these sites can be classified as I₁ imidazoline receptors. In summary, we describe for the first time that high-affinity I₁ receptors of the bovine brainstem are located on (pre)synaptic membranes.     

Activation of the Liver X Receptor Stimulates Trans-intestinal Excretion of Plasma Cholesterol

Recent studies have indicated that direct intestinal secretion of plasma cholesterol significantly contributes to fecal neutral sterol loss in mice. The physiological relevance of this novel route, which represents a part of the reverse cholesterol transport pathway, has not been directly established in vivo as yet. We have developed a method to quantify the fractional and absolute contributions of several cholesterol fluxes to total fecal neutral sterol loss in vivo in mice, by assessing the kinetics of orally and intravenously administered stable isotopically labeled cholesterol combined with an isotopic approach to assess the fate of de novo synthesized cholesterol. Our results show that trans-intestinal cholesterol excretion significantly contributes to removal of blood-derived free cholesterol in C57Bl6/J mice (33% of 231 μmol/kg/day) and that pharmacological activation of LXR with T0901317 strongly stimulates this pathway (63% of 706 μmol/kg/day). Trans-intestinal cholesterol excretion is impaired in mice lacking Abcg5 (−4%), suggesting that the cholesterol transporting Abcg5/Abcg8 heterodimer is involved in this pathway. Our data demonstrate that intestinal excretion represents a quantitatively important route for fecal removal of neutral sterols independent of biliary secretion in mice. This pathway is sensitive to pharmacological activation of the LXR system. These data support the concept that the intestine substantially contributes to reverse cholesterol transport.Reverse cholesterol transport (RCT)³ is defined as the flux of excess cholesterol from peripheral tissues toward the liver followed by biliary secretion and subsequent disposal via the feces (1). Accumulation of cholesterol in macrophages in the vessel wall is considered a primary event in the development of atherosclerosis and, therefore, removal of excess cholesterol from these cells is of crucial importance for prevention and/or treatment of atherosclerotic cardiovascular diseases. It is generally accepted that HDL is the obligate transport vehicle in RCT and that plasma HDL levels reflect the capacity to accommodate this flux. In line herewith, HDL-raising therapies are currently considered as a promising strategy for prevention and treatment of atherosclerotic cardiovascular diseases (2). In the “classical” scenario, the liver has a central role in RCT (3). Biliary secretion of free cholesterol, facilitated by the heterodimeric ABC-transporter ABCG5/ABCG8 (4), and hepatic conversion of cholesterol into bile acids followed by fecal excretion are referred to as the main routes for quantitatively important elimination of cholesterol from the body. Fecal excretion of sterols is stimulated upon whole body activation of the liver X receptor (LXR, NR1H2/3), a member of the nuclear receptor family for which oxysterols have been identified as natural ligands (5). LXR regulates expression of several genes involved in RCT and activation of LXR by synthetic agonists leads to elevated plasma HDL-cholesterol levels, increased hepatobiliary cholesterol secretion, reduced fractional intestinal cholesterol absorption and increased fecal sterol loss (6). LXR is thus considered an attractive target for therapeutic strategies aimed at stimulation of RCT, which, however, will require approaches to circumvent potential detrimental consequences of LXR activation such as induction of lipogenesis.Recent studies indicate that the classical concept of RCT may require reconsideration. Studies in apoA-I-deficient mice revealed that the magnitude of the centripetal cholesterol flux from the periphery to the liver is not related to the concentration of HDL-cholesterol or apoA-I in plasma (7). Furthermore, Abca1^−/− mice that completely lack plasma HDL show unaffected rates of hepatobiliary cholesterol secretion and fecal sterol loss (8). Additionally, mice lacking both Abcg5 and Abcg8 do not show a reduction in fecal neutral sterol excretion to the extent expected on the basis of their strongly reduced hepatobiliary cholesterol secretion (9). Recent studies by Plösch et al. (6) have revealed that increased fecal neutral sterol loss upon general LXR activation cannot be attributed to the increased hepatobiliary cholesterol secretion only, suggesting a major contribution of the intestine in excretion of cholesterol. This potential role of the intestine in cholesterol removal from the body has been corroborated by Kruit et al. (10), who showed that fecal sterol loss is not affected in Mdr2^−/− (Abcb4^−/−) mice that have a dramatic reduction in biliary cholesterol secretion (11). Moreover, intravenously administered [³H]cholesterol could be recovered in the neutral sterol fraction of the feces in these mice and fecal excretion of neutral sterols was stimulated upon treatment with an LXR agonist (10). However, the exact quantitative contribution of the direct intestinal pathway under physiological conditions has not directly been determined so far. Very recently, intestinal perfusion studies in mice revealed that, in the presence of mixed micelles as cholesterol acceptors in the intestinal lumen, murine enterocytes indeed have a high capacity to secrete cholesterol via a specific process that is most active in the proximal part of the small intestine (12). In addition, it was shown that direct trans-intestinal cholesterol excretion (TICE) could be stimulated by a high fat diet. The existence of a non-biliary route for fecal neutral sterol excretion is further supported by very recent studies by Brown et al. (13) in mice with targeted deletion of hepatic ACAT2.The present study provides insight into the relative and absolute contributions of several cholesterol fluxes relevant to total fecal sterol loss in mice, making use of a panel of stable isotope tracers. Our results show that TICE is a major route for removal of blood-derived free cholesterol and that pharmacological LXR activation strongly stimulates this arm of the reverse cholesterol transport pathway.     

Vitamin E Protects Against Oxidative Damage Caused by Formaldehyde in the Liver and Plasma of Rats

Abstract Background: During myocardial ischemia, accumulation of end products from anaerobic glycolysis (hydrogen ions (H⁺), lactate) can cause cellular injury, consequently affecting organ function. The cells' ability to buffer H⁺ (buffering capacity (BC)) plays an important role in ischemic tolerance. Age related differences in myocardial lactate and H⁺ accumulation (one hour of ischemia) as well as differences in BC, myoglobin (Mb) and histidine (His) contents in the left (LV) and right (RV) ventricles were assessed in neonatal compared to adult pigs. The BC of the septum was also compared. Methods and Results: Neonatal RV and LV had lactate accumulations of 43% and 63% and significantly greater H⁺ (p < 0.004) compared to the adult. In the neonate LV, BC was 17% significantly poorer (p = 0.0001), had 33% lower Mb (p = 0.0002) and 15% lower His content (p = 0.0004) when compared to the adult. In the RV, despite similar BC between the neonate and adult, myoglobin content was 36% (p = 0.0004) lower in the neonate. The neonate septum had a BC that was 11% lower than that of the adult. With maturation, the adult LV had a BC that was 10% greater (p < 0.01) than the RV while the septum mirrored that of the LV. Conclusions: During maturation to adulthood, the BC of the septum begins to closely resemble the LV. Neonatal hearts have a potentially greater vulnerability to acid-base disturbances during ischemia in both ventricles when compared to hearts of adults. This is due to lower levels of myoglobin and histidine in the young, which could render them more susceptible to injury during ischemia.Condensed Abstract During myocardial ischemia, H⁺ and lactate accumulation may pose deleterious effects on the heart. The ability to buffer H⁺ (buffering capacity, BC) affects ischemic tolerance. Although lactate accumulation during 1 h of global ischemia was similar between ventricles of neonatal and adult swine, H⁺ accumulation was greater and BC, Mb and His content were lower. With maturation, LV BC was higher than the RV while septum developmentally resembled the LV. Thus, hearts of neonates may be at a greater risk of ischemic injury compared to hearts of adults. (Mol Cell Biochem xxx: 1–7, 2005)     

Opioid-Inhibited Adenylyl Cyclase in Rat Brain Membranes: Lack of Correlation with High-Affinity Opioid Receptor Binding Sites

Opioid agonists bind to GTP-binding (G-protein)-coupled receptors to inhibit adenylyl cyclase. To explore the relationship between opioid receptor binding sites and opioid-inhibited adenylyl cyclase, membranes from rat striatum were incubated with agents that block opioid receptor binding. These agents included irreversible opioid agonists (oxymorphone-p-nitrophenylhydrazone), irreversible antagonists [naloxonazine, beta-funaltrexamine, and beta-chlornaltrexamine (beta-CNA)], and phospholipase A2. After preincubation with these agents, the same membranes were assayed for high-affinity opioid receptor binding [3H-labeled D-alanine-4-N-methylphenylalanine-5-glycine-ol-enkephalin (mu), 3H-labeled 2-D-serine-5-L-leucine-6-L-threonine enkephalin (delta), and [3H]ethylketocylazocine (EKC) sites] and opioid-inhibited adenylyl cyclase. Although most agents produced persistent blockade in binding of ligands to high-affinity mu, delta, and EKC sites, no change in opioid-inhibited adenylyl cyclase was detected. In most treated membranes, both the IC50 and the maximal inhibition of adenylyl cyclase by opioid agonists were identical to values in untreated membranes. Only beta-CNA blocked opioid-inhibited adenylyl cyclase by decreasing maximal inhibition and increasing the IC50 of opioid agonists. This effect of beta-CNA was not due to nonspecific interactions with G(i), Gs, or the catalytic unit of adenylyl cyclase, as neither guanylylimidodiphosphate-inhibited, NaF-stimulated, nor forskolin-stimulated activity was altered by beta-CNA pretreatment. Phospholipase A2 decreased opioid-inhibited adenylyl cyclase only when the enzyme was incubated with brain membranes in the presence of NaCl and GTP. These results confirm that the receptors that inhibit adenylyl cyclase in brain do not correspond to the high-affinity mu, delta, or EKC sites identified in brain by traditional binding studies.     

Haemolytic Disease of the Newborn Caused by Anti-Lan Antibody

The first recorded example of anti-Lan associated with haemolytic disease of the newborn is reported. This emphasizes the importance of screening for atypical antibodies early in pregnancy, even though prophylactic use of anti-D immunoglobulin will eventually reduce the incidence of haemolytic disease due to anti-D antibody.     

胎膜早破导致胎膜绒毛膜羊膜炎的细菌培养及药敏结果分析

目的:分析胎膜早破(premature rupture of memberane,PROM)所致孕妇绒毛膜羊膜炎的细菌培养及药敏结果,探讨胎膜早破引起的绒毛膜羊膜炎主要致病菌的病原学特征,为围产期感染用药提供科学依据。方法:对2011年6月至2013年9月在我院分娩的597例破膜时间大于24小时的PROM孕妇的胎膜进行细菌培养及药敏试验。结果:597例破膜时间大于24小时的PROM孕妇的胎膜细菌培养中,86例培养阳性,阳性率为14.41%;培养阳性病例中革兰氏阴性杆菌占73.26%(63/86),革兰氏阳性菌球菌占34.88%(30/86),培养最多的菌种是大肠埃希菌,占60.47%(52/86)。革兰氏阴性杆菌对亚胺培南、哌拉西林/他唑巴坦、头孢哌酮/舒巴坦、庆大霉素、头孢西丁、左氧氟沙星敏感;革兰氏阳性球菌对亚胺培南、丁胺卡那、哌拉西林/他唑巴坦、替考拉宁、万古霉素、利奈唑胺、庆大霉素、头孢西丁、左氧氟沙星敏感。结论:革兰氏阴性杆菌是PROM相关的绒毛膜羊膜炎的主要致病菌,应根据病原学特征合理应用抗生素。     

A Rapid and Reproducible Hcmogenization Procedure for the Isolation of Plasma Membranes from Rat Liver

A simplified modification of the Neville procedure for the isolation of plasma membranes from rat liver is described in which cells are broken by low-shear homogenizetion with a Polytron homogenizer. Plasma membranes are recovered from the homogenates by differential and discontinuous sucrose gradient centrifugetions. The procedure provides plasma membrane fractions enriched 25-fold for AMPase, a marker enzyme for the plasma membrane of rat liver, with a combined contamination from endoplasmic reticulum, Golgi apparatus and mitochondria of less than 10% The procedure is uncomplicated, reproducible, and yields enzymatically active plasma membrane fractions of high purity.     

亚硒酸钠诱导的晶状体蛋白质聚合作用

不仅在体内,而且在体外亚硒酸钠可引起晶状体蛋白质聚合。将亚硒酸钠加到pH7.4的晶状体蛋白质溶液中,在37℃保温30min后观察到蛋白质溶液变混浊,随时间的延长混浊程度加重并有沉淀形成。经SDS聚丙烯酰胺凝胶电泳发现,加硒保温后形成的不溶性蛋白质中有大量的高分子聚合物。当加入二硫苏糖醇后混浊的蛋白质溶液变清,其中的高分子聚合物也基本消失,我们还发现;在亚硒酸钠使晶状体蛋白质变混浊的同时,蛋白质巯基减少,而蛋白质结合的硒量增加,且二者之间有较固定的比例关系,即蛋白质上每增加一个硒原子,蛋白质巯基就减少4.26个。当用二硫苏糖醇还原后,68％的硒从蛋白质中释放出来。这些结果表明,亚硒酸钠可引起大鼠晶状体水溶性蛋白质聚合,其可能方式如下:4PSH+SeO_3~-→PSSP+PS-Se-SP+H_2O+2OH~-这可能是亚硒酸钠诱发白内障的主要原因。     

Comparison of Insulin Binding Proteins In Plasma and Nuclear Membranes of Obese and Lean Mouse Liver

Abstract

Plasma membranes obtained from obese (ob/ob) and lean (+/+ or +/ob) mouse livers were chemically crosslinked to [¹²⁵I] -insulin and examined by electrophoresis and autoradiography. The pattern of crosslinked hormone was qualitatively similar in obese and lean plasma membranes. A major insulin binding protein of approximately M 120,000 was observed. Two additional bands were apparent, one which remained near the top of the gel and one about M 90,000. A minor band at approximately M 50,000 was also detected. For each of the insulin binding proteins a reduction in the amount of [¹²⁵I]-insulin bound was observed with obese plasma membranes as compared with lean. For all proteins the insulin binding was specific as determined by competition with unlabeled hormone. In addition to plasma membrane receptors, insulin has also been reported to bind to nuclear membranes. The autoradiographic patterns of gels of [¹²⁵]-insulin bound and crosslinked to nuclear membranes from obese and lean mouse livers indicated the presence of proteins of the same M as those described for plasma membranes. Nuclear membrane proteins bound less insulin than plasma membranes and, again, the obese was decreased relative to the lean. Contamination of the nuclear membrane fraction by plasma membranes was ruled out. Scatchard analyses of [¹²⁵]-insul in bound to plasma and nuclear membranes indicated that the decrease in hormone binding in the obese mouse is a result of a reduction in the absolute number of receptors. The findings presented in this study provide additional support for this conclusion by demonstrating that membranes from obese mice are comprised of the same set of apparently unaltered insulin binding proteins. Further, the presence of similar insulin binding proteins in both nuclear and plasma membranes suggests a physiological relationship between these structures with respect to hormone binding and/or in the mechanism of action of insulin.     

Interactions of Malondialdehyde and 4-Hydroxynonenal with Rat Liver Plasma Membranes and their Effect on Binding of Prostaglandin E2 by Specific Receptors

We investigated effect of aldehydic products of lipid peroxidation, malondialdehyde (MDA) and 4-hydrox-ynonenal (HNE) on prostaglandin (PG) E₂ receptors of liver plasma membranes. The modification of the membranes by MDA diminished PGE₂ binding, decreasing receptor affinity for PGE₂ and receptor density whereas HNE increased PGE₂ binding, enhanced receptor density but did not changed receptor affinity. ESR study showed the decrease of the whole membrane fluidity after modification by MDA whereas HNE lowered membrane fluidity only in the internal zone of lipid bilayer and increased it in the surface area. The possible effects of membrane changes caused by MDA and HNE on PGE₂ receptor parameters are discussed.     

Identification of Purine Binding Sites on Torpedo Acetylcholine Receptor

Abstract

Electrophysiological studies from this and other laboratories have suggested a direct action of ATP on nicotinic acetylcholine receptors (nAChR). To determine the site of binding of this purine derivative, we have covalently modified the nAChR from Torpedo marmorata electrocytes employing 2-[³H]-8-azido-ATP as a photoactivable affinity label. Covalently attached radioactivity was predominantly found in the β-polypeptide of the receptor. Based on the results of protection studies with several nAChR ligands whose target sites at the receptor are known, we conclude that the purine site(s) differ from those of acetylcholine and of physostigmine, galanthamine and related ligands, and those of local anesthetics.     

Differential Regulation of Kainate Receptor Trafficking by Phosphorylation of Distinct Sites on GluR6

Kainate receptors are widely expressed in the brain, and are present at pre- and postsynaptic sites where they play a prominent role in synaptic plasticity and the regulation of network activity. Within individual neurons, kainate receptors of different subunit compositions are targeted to various locations where they serve distinct functional roles. Despite this complex targeting, relatively little is known about the molecular mechanisms regulating kainate receptor subunit trafficking. Here we investigate the role of phosphorylation in the trafficking of the GluR6 kainate receptor subunit. We identify two specific residues on the GluR6 C terminus, Ser⁸⁴⁶ and Ser⁸⁶⁸, which are phosphorylated by protein kinase C (PKC) and dramatically regulate GluR6 surface expression. By using GluR6 containing phosphomimetic and nonphosphorylatable mutations for these sites expressed in heterologous cells or in neurons lacking endogenous GluR6, we show that phosphorylation of Ser⁸⁴⁶ or Ser⁸⁶⁸ regulates receptor trafficking through the biosynthetic pathway. Additionally, Ser⁸⁴⁶ phosphorylation dynamically regulates endocytosis of GluR6 at the plasma membrane. Our findings thus demonstrate that phosphorylation of PKC sites on GluR6 regulates surface expression of GluR6 at distinct intracellular trafficking pathways, providing potential molecular mechanisms for the PKC-dependent regulation of synaptic kainate receptor function observed during various forms of synaptic plasticity.