The structure of synaptosomal plasma membrane

Synaptosomal plasma membrane (SPM), like other plasma membranes, is composed of lipids and proteins and appears to conform to the "fluid mosaic" model proposed by Singer and Nicolson. SPM components show a marked asymmetry with respect to their inside-outside distribution, with the majority of proteins being external. The lateral distribution of proteins is also somewhat asymmetric; this asymmetry is maintained by the immobilization of species at the junction.     

Do salicylates and ascorbate increase the outer membrane permeability to hydrophobic antibiotics in Pseudomonas aeruginosa?

Acetylsalicylate and ascorbate have earlier been shown to increase the outer membrane (OM) permeability of Pseudomonas aeruginosa to a hydrophobic probe compound, nitrocefin. In order to elucidate whether these drugs increase the OM permeability to a wider set of hydrophobic compounds, the OM permeability to three other hydrophobic probes (rifampin, fusidic acid and sodium deoxycholate) was studied in the presence of salicylates or ascorbate. A high concentration (300 micrograms/ml, equal to 1.7 mM) of L-ascorbate decreased the minimum inhibitory concentration (MIC) of rifampin against P. aeruginosa by a factor of approximately 3. As a sharp contrast, the reference compound, polymyxin B nonapeptide (PMBN) which has a strong OM permeability-increasing action, decreased the MIC by a factor of approximately 100, at a concentration as low as 3 micrograms/ml (equal to 3 microM). If the assays were performed in a low ionic strength medium (L broth diluted 1/5 with water) instead of L broth, ascorbate was somewhat more effective. The MIC of fusidic acid was even less influenced by ascorbate. Additionally, ascorbate did not potentiate the bacteriolytic action of sodium deoxycholate, whereas the control compound hexametaphosphate had a marked effect. Furthermore, salicylate and acetylsalicylate sensitised, in all conditions tested, P. aeruginosa to none of the three probes. The results suggest that ascorbate and salicylates lack any significant OM permeability-increasing action.     

Polyene antibiotics. IX. An improved method for the preparation of methyl esters of polyene antibiotics.

An improved general method for the preparation of methyl esters of polyene antibiotics is discussed. Using this method methyl esters of pimaricin, nystatin, eurocidin, hamycin, hamycins A and B, aureofungin, partricins A and B, candimycin, candicidin, and amphotericin B have been prepared and their physical properties are reported. The biological activities of hamycins A and B and their methyl esters are also described.     

Detergent extraction from rat synaptosomal plasma membranes reveals difference in mu and delta opioid receptor binding

Rat synaptosomal plasma membranes were extracted with a detergent (CHAPS, a zwitterionic derivative of cholic acid). mu and delta opioid receptor binding and adenylate cyclase activities were tested in the intact membranes and in the supernatants from detergent treated membranes. The 6000 X g/8 min. supernatant contained mu receptor binding equal to 33% of the mu receptor binding measured in the untreated membranes. When the detergent treated membranes were sedimented at (50,000 X g/10 min.), 23% of the mu receptor binding was recovered in the supernatant. After a 100,000 X g/30 min. centrifugation the supernatant contained 10% of the mu receptor binding when compared to untreated membranes. Of the delta receptor binding found in intact membranes, 10% or less was recovered in the 3 supernatants described above. Furthermore, the mu and delta receptor binding were distributed differently among particles in the supernatants. This indicates differences in the chemical properties of the mu and delta opioid receptors. Adenylate cyclase assays showed that the G/F site of this enzyme complex was inactivated in the supernatants from detergent treated membranes parallel to the delta receptor binding decrease. However, the catalytic part of adenylate cyclase was present in the supernatants and seemed resistant to the detergent.     

Phospholipid bilayer permeability of beta-lactam antibiotics

Liposomes containing penicillinase or cephalosporinase were prepared from the phospholipids of Escherichia coli. After free beta-lactamase was inactivated by clavulanic acid or penicillanic acid sulfone followed by separation of inactivated enzyme and inhibitor from liposomes by gel filtration, the permeability of these liposomes to ampicillin, cefazolin and cephaloridine was estimated by measuring the hydrolysis of these antibiotics by the entrapped enzymes. The permeability parameter C (minute-1 microM lipid-1) of ampicillin, cefazolin and cephaloridine was calculated to be 2.35 X 10(-4), 0.33 X 10(-4) and 0.52 X 10(-4), respectively. The lipid bilayer permeability of these antibiotics was also measured by using the liposomes containing these antibiotics. About half of the initially entrapped ampicillin was released from the liposomes within 80 minutes, while no significant release of cefazolin and cephaloridine could be detected during the same period. These results clearly indicates that the lipid bilayer membrane is more permeable to ampicillin than cefazolin and cephaloridine, and they are consistent with the observations of Sawai et al., who showed that ampicillin was a more effective antibacterial drug than cefazolin and cephaloridine against the porin-deficient mutants.     

Specific binding of crotoxin to brain synaptosomes and synaptosomal membranes

Crotoxin, the presynaptic neurotoxin from Crotalus durissus terrificus, was iodinated and used to demonstrate high affinity, specific binding to guinea-pig (Cavia porcellus) brain synaptosomes and synaptosomal membrane fragments. 125I-crotoxin binding to the membrane fragments displays two binding plateaus, (Kd1 = 4 nM and Kd2 = 87 nM, Bmax1 = 2 and Bmax2 = 4 pmoles/mg membrane protein), but binding to whole synaptosomes revealed only one plateau (Kd = 2 nM and Bmax = 5 pmoles/mg membrane protein). Rosenthal analyses of Scatchard plots yielded similar binding constants in the presence or absence of 0.025% Triton X-100. In addition to equilibrium analyses, kinetic analyses of 125I-crotoxin binding to synaptosomal membrane fragments gave a Kd-value of 3 nM. The Kd value was not significantly changed by the exclusion of added calcium, but the binding site number was lowered. Crotoxin binding was inhibited by the acidic subunit of crotoxin and several presynaptic neurotoxins, which were classified according to their inhibitory properties as, strong (acidic subunit of crotoxin, Mojave toxin, concolor toxin, taipoxin and pseudexin), moderate (ammodytoxin A and textilotoxin), weak (notexin and scutoxin A), very weak (notechis II-5) and non-inhibitory (basic subunit of crotoxin, beta-bungarotoxin, Crotalus atrox and porcine pancreatic phospholipases A2, dendrotoxin, and notechis III-4). Purified acidic subunit of crotoxin, the most potent competitor of crotoxin binding, was somewhat more competitive than intact crotoxin and the other strong inhibitors on a molar basis. Strong, moderate and weak inhibitor groups each differed from the preceding group by requiring about a ten fold increase in concentration to effect a 50% inhibition of crotoxin binding. The weak group was therefore at least two-orders of magnitude less effective than the strong inhibition shown by the acidic subunit of crotoxin. Treatment of synaptosomal membranes with protease K lowered 125I-crotoxin binding, whereas treatment with trypsin did not. Iodinated, phospholipase A2 from C. atrox venom showed no specific binding to whole synaptosomes. Our results demonstrate the presence and describe some of the properties of high affinity, specific binding sites in brain tissue for crotoxin and related presynaptic neurotoxins.     

Comparison of several procedures for the preparation of synaptosomal plasma membrane vesicles

Synaptosomal plasma membrane vesicles (SPMV) were isolated from fresh bovine cerebral cortex by several well-known procedures, and the best procedure was selected by enzymatic and morphological standards. The SPMV isolated by the modified procedure of Smith and Loh showed the highest purity. The specific activities of Na, K-ATPase, acetylcholinesterase and 5′-nucleotidase were about 5.6-fold, 2.5-fold and 3.3-fold, respetively, enriched in the plasma membrane fraction as compared to those of the crude homogenate. Electron microscopic examination also showed that the membranes were in vesicular form.     

Solubilization of dopamine-D2 receptors from synaptosomal membranes of the bovine caudate nucleus.

Dopamine D2-receptors were solubilized from synaptosomal membranes of the bovine caudate nucleus using different detergents. They were labelled with [3H]-spiperone and assayed by polyethylene glycol precipitation. CHAPS was found to be the best solubilizing agent among all detergents used. Optimal conditions for solubilization were: 0.25% CHAPS, 3.5 mg ml-1 protein, 25 min, 4 degrees C and the yield of D2-receptors was 18.6%. Addition of some sulphobetain detergents increased the extent of solubilization, 125 mM NaCl and 0.25 M sucrose decreased it, while SH-group protecting agents (2 mM dithiothreitol and 6 mM beta-mercaptoethanol), as well as MEGA-9 and MEGA-12 were almost ineffective. -log IC50 values for solubilized dopamine D2-receptors are in linear correlation with the corresponding values for membrane-bound receptors (r = 0.962, slope factor 0.96) and Kd value of solubilized receptors was 3.61 +/- 0.94 nM, while that of membrane-bound receptors was 1.25 +/- 0.10 nM. Specific binding of [3H]-spiperone to the solubilized receptors resolved by linear sucrose density gradient centrifugation shows two maxima, one in the first several fractions from the bottom and the other with an apparent S value of 7.3.     

The bacterial outer-membrane permeability of beta-lactam antibiotics.

Two penicillins and 5 cephalosporins were evaluated for their ability to pass through the outer-membranes of Proteus morganii, Citrobacter freundii and Escherichia coli. Cefazolin, ceftezole and cephaloridine showed high permeability through the outer-membranes of these Gram-negative bacteria. Benzylpenicillin and cephalothin, on the contrary, showed low permeability. The outer-membrane permeability of ampicillin and cephalexin varied from species to species. C. freundii was found to have the highest barrier against both the penicillins and the cephalosporins, and E. coli appeared to have a low barrier against the cephalosporins. The hydrophobic character of the beta-lactam antibiotics, which was estimated by a reversed-phase thin-layer chromatography was closely related to the outer-membrane permeability. In general, the more hydrophilic antibiotic showed the higher outer-membrane permeability. However, cephaloridine, the most lipophilic compound among the antibiotics tested, showed good permeability.     

几丁聚糖膜通透性的研究

几丁聚糖是由几丁质脱乙酰而来的一种线性高分子多糖,其β－1,4糖苷键在酸性条件下不稳定,易发生长链的水解,本文通过醋酸降解法制备了不同相对分子质量的几相聚,经测定降解前后产物的脱乙酰度未发生改变,将降解的几丁聚糖聚配制成3％的醋酸溶液,50度烘干后用碱中和制成几丁聚糖膜,膜透明,吸水后有弹性,强度好,将膜固定在自制的通透装置中研究了比较不同相对分子质量几个聚糖膜,膜透明,吸水后有弹性,强度好,将膜固定在自制的通透装置中研究比较了不同相对分子质量几丁聚糖膜对氯化钠,L－Tyr,葡萄糖以及牛血清白蛋白的通透性,结果表明膜对4种物质均有通透性,且通透性与相对分子质量相关,分子量越小,对4种物质的通透性越好。β     

Pharmacology of ionic channels in excitable membranes

Methods are now available for recording the behaviour of single ion channels in biological membranes. These include patch clamp, noise analysis and voltage-jump techniques. Their use is illustrated at the motor end-plate where it has been shown that the decay of end-plate currents is determined by the rate of closing of channels opened by acetylcholine. The effects of a variety of pharmacological agents can be explained in terms of changes in channel behaviour. Drugs may change the properties of the channel or its environment, block open channels or prolong the lifetime of acetylcholine in the synaptic cleft. Voltage-activated channels can also be characterised using patch clamp and noise analysis. A variety of drugs block sodium, potassium and calcium channels.     

Characterization of the increase in vascular permeability induced by vascular permeability factor in vivo.

1. Vascular permeability factor (VPF) is a protein secreted from a variety of human and rodent tumour and normal tissue cells. In addition to mediating angiogenesis and endothelial cell growth, VPF has been reported to be a potent mediator of increased microvascular permeability in vivo. In this study we have investigated these permeability changes in vivo using a quantitative model of local plasma leakage in rabbit skin. 2. Our results reveal that VPF is a potent mediator of plasma leakage which, in the rabbit, depends on a synergistic interaction with arteriolar vasodilators such as prostaglandin E2. The requirement for an exogenous vasodilator further suggest that VPF does not act to increase blood flow in this model. 3. We show that this response does not require the presence of circulating neutrophils and in this respect is similar to direct-action permeability increasing mediators such as histamine and bradykinin. Similarly, the time course of plasma leakage induced by VPF resembles that of direct-action mediators, where the greatest response occurs over the first 30 min. In contrast, the neutrophil-dependent plasma leakage induced by the active component of zymosan-activated plasma, C5ades arg, was maintained at a similar level over 2.5 h. 4. Further, using mediator antagonists and enzyme inhibitors we demonstrate that the mechanism of action of VPF is not via activation of histamine, kinin, or platelet-activating factor pathways.