Ca2+-independent,protein-mediated fusion of chromaffin granule ghosts with liposomes

We have investigated the interaction between isolated membrane vesicles from chromaffin granules and large unilamellar phospholipid vesicles (liposomes). Mixing of membrane lipids has been monitored continuously, utilizing the fluorescence resonance energy transfer assay described by Struck et al. ((1982) Biochemistry 20, 4093–4099). To demonstrate coalescence of the internal vesicle volumes the transfer of colloidal gold from the liposomes to the interior of the granule membrane vesicles has been examined. Efficient fusion of the liposomes with the granule membranes was observed. Significant fusion occurred in the absence of Ca²⁺, although the extent of interaction was enhanced in its presence. The sensitivity of the interaction to pretreatment of the granule membranes with trypsin showed the fusion reaction to be a protein-mediated process.     

Evidence for a New Member of the Myosin I Family from Mammalian Brain

Myosin I is an actin-based motor responsible for powering a wide variety of motile activities in amebae and slime molds and has been found previously in vertebrates as the lateral bridges within intestinal epithelial cell microvilli. Although neurons exhibit extensive cellular and intracellular motility, including the production of ameboid-like growth cones during development, the proteins responsible for the motor in these processes are unknown. Here, we report the isolation of a partially purified protein fraction from bovine brain that is enriched for a 150-kDa protein; immunochemical and biochemical analyses suggest that this protein possesses a number of functional properties that have been ascribed to myosin I from various sources. These properties include an elevated K(+)-EDTA ATPase, a modest actin-activated Mg(2+)-ATPase, the ability to bind calmodulin, and a ready association with phospholipid vesicles made from phosphatidylserine, but not from phosphatidylcholine. The combination of these properties, together with a molecular mass of 150 kDa (most myosin I molecules found to date have molecular masses in the range 110-130 kDa) yet recognition by an anti-myosin I antibody, suggests the presence of a new member of the myosin I family within mammalian brain.     

Engineering Escherichia coli membrane phospholipid head distribution improves tolerance and production of biorenewables

Economically competitive microbial production of biorenewable fuels and chemicals is often impeded by toxicity of the product to the microbe. Membrane damage is often identified as a major mechanism of this toxicity. Prior efforts to strengthen the microbial membrane by changing the phospholipid distribution have largely focused on the fatty acid tails. Herein, a novel strategy of phospholipid head engineering is demonstrated in Escherichia coli. Specifically, increasing the expression of phosphatidylserine synthase (+pssA) was found to significantly increase both the tolerance and production of octanoic acid, a representative membrane-damaging solvent. Tolerance of other industrially-relevant inhibitors, such as furfural, acetate, toluene, ethanol and low pH was also increased. In addition to the increase in the relative abundance of the phosphoethanolamine (PE) head group in the +pssA strain, there were also changes in the fatty acid tail composition, resulting in an increase in average length, percent unsaturation and decreased abundance of cyclic rings. This +pssA strain had significant changes in: membrane integrity, surface potential, electrochemical potential and hydrophobicity; sensitivity to intracellular acidification; and distribution of the phospholipid tails, including an increase in average length and percent unsaturation and decreased abundance of cyclic rings. Molecular dynamics simulations demonstrated that the +PE membrane had increased resistance to penetration of ethanol into the hydrophobic core and also the membrane thickness. Further hybrid models in which only the head group distribution or fatty acid tail distribution was altered showed that the increase in PE content is responsible for the increase in bilayer thickness, but the increased hydrophobic core thickness is due to altered distribution of both the head groups and fatty acid tails. This work demonstrates the importance of consideration of the membrane head groups, as well as a modeling approach, in membrane engineering efforts.     

The synthesis and properties of a functional fluorescent cholesterol analog

The synthesis of a new fluorescent cholesterol analog is described. The analog contains a cholesterol nucleus attached via a hydrophilic spacer to N-4-nitrobenzo-2-oxa-1,3-diazole. Since the cholesterol moiety is not perturbed this molecule probably interacts with lipid bilayers in much the same way as cholesterol itself does. The compound can be readily incorporated into small unilamellar vesicles by sonicating a mixture of it with egg yolk phosphatidylcholine in a buffer. Furthermore, the analog can be incorporated into preformed membranes either by exchange from vesicles containing the analog or by uptake from sonicated micelles of the analog. Thus this analog shows potential as a useful tool for studying the interactions of cholesterol with cell membranes.     

Central thermoregulatory effects of lactate in the toad Bufo paracnemis

Hypoxia induces a regulated decrease in body temperature (Tb; anapyrexia) in organisms ranging from protozoans to mammals, but very little is known about the mechanisms involved. Several candidates have been suggested to mediate hypoxia-induced anapyrexia, among them lactate, which is a classical companion of hypoxic stress in vertebrates. The present study was designed to assess the central thermoregulatory effects of lactate in Bujo paracnemis. Toads equipped with a temperature probe were tested over a thermal gradient (10–40°C). Lactate injected systemically (4.0 mmol kg⁻¹) caused a significant reduction of Tb from 24.6±2.1 to 17.4±3.9°C. To assess the role of central thermoregulatory mechanisms, a lower dose (0.4 mmol kg⁻¹) of lactate was injected into the fourth cerebral ventricle or systemically. Intracerebroventricular injection of lactate caused a similar decrease in Tb, whereas systemic injection caused no change. The data indicate that lactate may play a role in hypoxia-induced anapyrexia in central rather than peripheral sites.     

CBFβ-SMMHC Affects Genome-wide Polycomb Repressive Complex 1 Activity in Acute Myeloid Leukemia

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Interaction between the δ-endotoxin produced by Bacillus thuringiensis ssp. entomocidus and liposomes

The δ-endotoxin produced by Bacillus thuringiensis ssp. entomocidus induced the release of encapsulated [¹⁴C]sucrose from reverse-phase vesicles composed of phosphatidylcholine and cholesterol. No such release was detected when the phospholipid component of the vesicles was either phosphatidylethanolamine, phosphatidylglycerol, or sphingomyelin. The toxin-induced release was competitively inhibited by negatively charged organic ions while positively charged organic ions, apart from choline chloride, had no such effect. The existence of a polar head group in the phospholipid as well as intermolecular hydrogen bonding at the membrane surface, was found to be of major importance in the toxin-liposome interaction.     

Effect of the sodium/potassium ratio on glyceraldehyde-3-phosphate dehydrogenase interaction with red cell vesicles

Binding of glyceraldehyde 3-phosphate to glyceraldehyde-3-phosphate dehydrogenase, the membrane protein known as Band 6, causes shifts in the ³¹P nuclear magnetic resonance spectrum of the substrate (Fossel, E.T. and Solomon, A.K. (1977) Biochim. Biophys. Acta 464, 82–92). We have studied the resonance shifts produced by varying the sodium/potassium ratio, at constant ionic strength, in order to examine the relationship between the cation transport system and glyceraldehyde-3-phosphate dehydrogenase. Alteration of the potassium concentration at the extracellular face of the vesicle affects the conformation of glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face, thus showing that a conformation change induced by a change in extracellular potassium can be transmitted across the membrane. Alterations of the sodium concentration at the cytoplasmic face also affect the enzyme conformation, whereas sodium changes at the extracellular face are without effect. In contrast, there is no sidedness difference in the effect of potassium concentrations. The half-values for these effects are like those for activation of the red cell (Na⁺ + K⁺)-ATPase. We have also produced ionic concentration gradients across the vesicle similar to those Glynn and Lew ((1970) J. Physiol. London 207, 393–402) found to be effective in running the cation pump backwards to produce adenosine triphosphate in the human red cell. The sodium/potassium concentration dependence of this process in red cells is mimicked by ³¹P resonance shifts in the (glyceraldehyde 3-phosphate/glyceraldehyde-3-phosphate dehydrogenase/inside out vesicle) system. These experiments provide strong support for the existence of a functional linkage between the membrane (Na⁺ + K⁺)-ATPase and the glyceraldehyde-3-phosphate dehydrogenase at the cytoplasmic face.