Condensed complexes of cholesterol and phospholipids.
Mixtures of dihydrocholesterol and phospholipids form immiscible liquids in monolayer membranes at the air-water interface under specified conditions of temperature and 2-dimensional pressure. In recent work it has been discovered that a number of these mixtures exhibit two upper miscibility critical points. Pairs of upper critical points can be accounted for by a theoretical model that implies the cooperative formation of molecular complexes of dihydrocholesterol and phospholipid molecules. These complexes are calculated to be present in the membranes both above and below the critical points. Below the critical points the complexes form a separate phase, whereas above the critical points the complexes are completely miscible with the other lipid components. The cooperativity of complex formation prompts the use of the terminology condensed complex. (+info)
Influence of exogenous thiols on inorganic mercury-induced injury in renal proximal and distal tubular cells from normal and uninephrectomized rats.
Inorganic mercury (Hg(2+)) induced time- and concentration-dependent cellular injury in freshly isolated proximal tubular (PT) and distal tubular (DT) cells from normal (control) rats or uninephrectomized (NPX) rats. PT cells from NPX rats were more susceptible than PT cells from control rats, and DT cells were slightly more susceptible than PT cells to cellular injury induced by Hg(2+) (not bound to a thiol). Preloading cells with glutathione increased Hg(2+)-induced cellular injury in PT cells from control rats. However, coincubation of PT or DT cells from control or NPX rats with Hg(2+) and glutathione (1:4) provided significant protection relative to incubations with Hg(2+) alone. No support was obtained for a role for gamma-glutamyltransferase in glutathione-dependent protection. However, the organic anion carrier does appear to play a role in accumulation and toxicity of mercuric conjugates of cysteine in PT cells from control, but not NPX, rats. Coincubation with Hg(2+) and cysteine (1:4) had little effect on, or slightly enhanced, Hg(2+)-induced cellular injury at low concentrations of Hg(2+) in all cells studied. Coincubation with Hg(2+) and albumin (1:4) markedly protected PT and DT cells from control and NPX rats at all concentrations except the highest concentration of Hg(2+) in DT cells from NPX rats. 2,3-Dimercapto-1-propanesulfonic acid protected cells both when preloaded or added simultaneously with Hg(2+). Thus, renal cells from NPX rats are more susceptible to Hg(2+)-induced injury, PT and DT cells respond differently to exposure to Hg(2+), and thiols can significantly modulate the toxic response to Hg(2+). (+info)
Electric field effect on cholesterol-phospholipid complexes.
Monolayer mixtures of dihydrocholesterol and phospholipids at the air-water interface are used to model membranes containing cholesterol and phospholipids. Specific, stoichiometric interactions between cholesterol and some but not all phospholipids have been proposed to lead to the formation of condensed complexes. It is reported here that an externally applied electric field of the appropriate sign can destabilize these complexes, resulting in their dissociation. This is demonstrated through the application of an electric field gradient that leads to phase separations in otherwise homogeneous monolayers. This is observed only when the monolayer composition is close to the stoichiometry of the complex. The electric field effect is analyzed with the same mean field thermodynamic model as that used previously to account for pairs of upper miscibility critical points in these mixtures. The concentrations of dihydrocholesterol, phospholipid, and complex vary strongly and sometimes discontinuously in the monolayer membrane in the field gradient. The model is an approximation to a two-dimensional liquid in which molecules freely exchange between free and complexed form so that the chemical potentials are constant throughout the membrane. The calculations are illustrated for a complex of about 15 molecules, composed of 5 cholesterol molecules and 10 phospholipid molecules. (+info)
Selective destabilization of acidic phospholipid bilayers performed by the hepatitis B virus fusion peptide.
A peptide corresponding to the N-terminal region of the S protein of hepatitis B virus (Met-Glu-Asn-Ile-Thr-Ser-Gly-Phe-Leu-Gly-Pro-Leu-Leu-Val-Leu-Gln) has been previously demonstrated to perform aggregation and destabilization of acidic liposome bilayers and to adopt a highly stable beta-sheet conformation in the presence of phospholipids. The changes in the lipid moiety produced by this peptide have been followed by fluorescence depolarization and electron microscopy. The later was employed to determine the size and shape of the peptide-vesicle complexes, showing the presence of highly aggregated and fused structures only when negatively charged liposomes were employed. 1,6-Diphenyl-1,3,5-hexatriene depolarization measurements showed that the interaction of the peptide with both negatively charged and zwitterionic liposomes was accompanied by a substantial reduction of the transition amplitude without affecting the temperature of the gel-to-liquid crystalline phase transition. These data are indicative of the peptide insertion inside the bilayer of both types of liposomes affecting the acyl chain order, though only the interaction with acidic phospholipids leads to aggregation and fusion. This preferential destabilization of the peptide towards negatively charged phospholipids can be ascribed to the electrostatic interactions between the peptide and the polar head groups, as monitored by 1-(4-(trimethylammoniumphenyl)-6-phenyl-1,3, 5-hexatriene fluorescence depolarization analysis. (+info)
Trimeric ring-like structure of ArsA ATPase.
ArsA protein is the soluble subunit of the Ars anion pump in the Escherichia coli membrane which extrudes arsenite or antimonite from the cytoplasm. The molecular weight of the subunit is 63 kDa. In the cell it hydrolyzes ATP, and the energy released is used by the membrane-bound subunit ArsB to transport the substrates across the membrane. We have obtained two-dimensional crystals of ArsA in the presence of arsenite on negatively-charged lipid monolayer composed of DMPS and DOPC. These crystals have been studied using electron microscopy of negatively-stained specimens followed by image processing. The projection map obtained at 2.4 nm resolution reveals a ring-like structure with threefold symmetry. Many molecular assemblies with the same ring-shape and dimensions were also seen dispersed on electron microscopy grids, prepared directly from purified ArsA protein solution. Size-exclusion chromatography of the protein sample with arsenite present revealed that the majority of the protein particles in solution have a molecular weight of about 180 kDa. Based on these experiments, we conclude that in solution the ArsA ATPase with substrate bound is mainly in a trimeric form. (+info)
Condensed complexes, rafts, and the chemical activity of cholesterol in membranes.
Epifluorescence microscopy studies of mixtures of phospholipids and cholesterol at the air-water interface often exhibit coexisting liquid phases. The properties of these liquids point to the formation of "condensed complexes" between cholesterol and certain phospholipids, such as sphingomyelin. It is found that monolayers that form complexes can incorporate a low concentration of a ganglioside G(M1). This glycolipid is visualized by using a fluorescently labeled B subunit of cholera toxin. Three coexisting liquid phases are found by using this probe together with a fluorescent phospholipid probe. The three liquid phases are identified as a phospholipid-rich phase, a cholesterol-rich phase, and a condensed complex-rich phase. The cholera toxin B labeled ganglioside G(M1) is found exclusively in the condensed complex-rich phase. Condensed complexes are likely present in animal cell membranes, where they should facilitate the formation of specialized domains such as rafts. Condensed complexes also have a major effect in determining the chemical activity of cholesterol. It is suggested that this chemical activity plays an essential role in the regulation of cholesterol biosynthesis. Gradients in the chemical activity of cholesterol should likewise govern the rates and direction of intracellular intermembrane cholesterol transport. (+info)
Effects of 2,3-dimercapto-1-propanesulfonic acid (DMPS) on tissue and urine mercury levels following prolonged methylmercury exposure in rats.
Methylmercury, a potent neurotoxicant, accumulates in the brain as well as the kidney during chronic exposure. We evaluated the capacity of 2,3-dimercapto-1-propanesulfonic acid (DMPS), a tissue-permeable metal chelator, to reduce brain, kidney, and blood Hg levels and to promote Hg elimination in urine following exposure of F-344 rats to methylmercury hydroxide (MMH) (10 ppm) in drinking water for up to 9 weeks. Inorganic (Hg2+) and organic (CH3Hg+) mercury species in urine and tissues were assayed by cold vapor atomic fluorescence spectroscopy (CVAFS). Following MMH treatment for 9 weeks, Hg2+ and CH3Hg+ concentrations were 0.28 and 4.80 microg/g in the brain and 51.5 and 42.2 microg/g in the kidney, respectively. Twenty-four hours after ip administration of a single DMPS injection (100 mg/kg), kidney Hg2+ and CH3Hg+ declined 38% and 59%, whereas brain mercury levels were slightly increased, attributable entirely to the CH3Hg+ fraction. Concomitantly, Hg2+ and CH3Hg+ in urine increased by 7.2- and 28.3-fold, respectively, compared with prechelation values. A higher dose of DMPS (200 mg/kg) was no more effective than 100 mg/kg in promoting mercury excretion. In contrast, consecutive DMPS injections (100 mg/kg) given at 72-h intervals significantly decreased total mercury concentrations in kidney, brain, and blood. However, the decrease in brain and blood mercury content was restricted entirely to the CH3Hg+ fraction, consistent with the slow dealkylation rate of MMH in these tissues. Mass balance calculations showed that the total amount of mercury excreted in the urine following successive DMPS injections corresponds quantitatively to the total amount of mercury removed from the kidney, brain, and blood of MMH-exposed rats. These findings confirm the efficacy of consecutive DMPS treatments in decreasing mercury concentrations in target tissue and in reducing overall mercury body burden. They demonstrate further that the capacity of DMPS to deplete tissue Hg2+ is highly tissue-specific and reflects the relative capacity of the tissue for methylmercury dealkylation. In light of this observation, the inability of DMPS to reduce Hg2+ levels in brain or blood may explain the inefficacy of DMPS and similar chelating agents in the remediation of neurotoxicity associated with prolonged MMH exposure. (+info)
Quantitative evaluation of urinary porphyrins as a measure of kidney mercury content and mercury body burden during prolonged methylmercury exposure in rats.
Changes in urinary porphyrin excretion patterns (porphyrin profiles) during prolonged mercury exposure are attributable to mercury accumulation in the kidney and to consequent effects of Hg2+ on renal porphyrin metabolism. In the present study, we evaluated the quantitative relationship of urinary porphyrin concentrations to mobilizable renal mercury content, using the metal chelator 2,3-dimercapto-1-propanesulfonic acid (DMPS) to modulate kidney mercury levels. Rats exposed to methylmercury hydroxide (MMH) at 10 ppm in drinking water for 6 weeks were treated with up to 3 consecutive doses of DMPS (100mg/kg, ip) at 72-h intervals. Consistent with previous findings, the concentrations of pentacarboxyl- (5-) and copro- (4-) porphyrins and of an atypical porphyrin specific to mercury exposure, precoproporphyrin, were significantly elevated in urine of MMH-exposed rats, compared with that of rats exposed to distilled water (dH2O) for the same period. Consecutive DMPS treatments of MMH-exposed rats significantly decreased kidney concentrations of total, as well as Hg2+ and CH3Hg+ species, and promoted increased urinary mercury excretion. Concomitantly, DMPS treatment decreased both kidney and urinary porphyrin concentrations, consistent with depletion of renal mercury levels. Regression analyses demonstrated a high correlation (r approximately 0.9) between prechelation urinary porphyrins and postchelation urinary mercury levels and also between prechelation urinary porphyrins and prechelation kidney mercury concentrations. These findings demonstrate that urinary porphyrin concentrations relate quantitatively to DMPS-mobilizable mercury in the kidney and, therefore, serve as a biochemical measure of renal mercury content. (+info)