Effects of Spontaneous Bilayer Curvature on Influenza Virus–mediated Fusion Pores

Cells expressing the hemagglutinin protein of influenza virus were fused to planar bilayer membranes containing the fluorescent lipid probes octadecylrhodamine (R18) or indocarbocyanine (DiI) to investigate whether spontaneous curvature of each monolayer of a target membrane affects the growth of fusion pores. R18 and DiI lowered the transition temperatures for formation of an inverted hexagonal phase, indicating that these probes facilitate the formation of negative curvature structures. The probes are known to translocate from one monolayer of a bilayer membrane to the other in a voltage-dependent manner. The spontaneous curvature of the cis monolayer (facing the cells) or the trans monolayer could therefore be made more negative through control of the polarity of voltage across the planar membrane. Electrical admittance measurements showed that the open times of flickering fusion pores were shorter when probes were in trans monolayers and longer when in cis monolayers compared with times when probe was symmetrically distributed. Open times were the same for probe symmetrically distributed as when probes were not present. Thus, open times were a function of the asymmetry of the spontaneous curvature between the trans and cis monolayers. Enriching the cis monolayer with a negative curvature probe reduced the probability that a small pore would fully enlarge, whereas enriching the trans monolayer promoted enlargement. Lysophosphatidylcholine has positive spontaneous curvature and does not translocate. When lysophosphatidylcholine was placed in trans leaflets of planar membranes, closing of fusion pores was rare. The effects of the negative and positive spontaneous curvature probes do not support the hypothesis that a flickering pore closes from an open state within a hemifusion diaphragm (essentially a “flat” structure). Rather, such effects support the hypothesis that the membrane surrounding the open pore forms a three-dimensional hourglass shape from which the pore flickers shut.     

Mouse alcohol dehydrogenase 4: kinetic mechanism,substrate specificity and simulation of effects of ethanol on retinoid metabolism

Mouse ADH4 (purified, recombinant) has a low catalytic efficiency for ethanol and acetaldehyde, but very high activity with longer chain alcohols and aldehydes, at pH 7.3 and temperature 37°C. The observed turnover numbers and catalytic efficiencies for the oxidation of all-trans-retinol and the reduction of all-trans-retinal and 9-cis-retinal are low relative to other substrates; 9-cis-retinal is more reactive than all-trans-retinal. The reduction of all-trans- or 9-cis-retinals coupled to the oxidation of ethanol by NAD⁺ is as efficient as the reduction with NADH. However, the Michaelis constant for ethanol is about 100 mM, which indicates that the activity would be lower at physiologically relevant concentrations of ethanol. Simulations of the oxidation of retinol to retinoic acid with mouse ADH4 and human aldehyde dehydrogenase (ALDH1), using rate constants estimated for all steps in the mechanism, suggest that ethanol (50 mM) would modestly decrease production of retinoic acid. However, if the K_m for ethanol were smaller, as for human ADH4, the rate of retinol oxidation and formation of retinoic acid would be significantly decreased during metabolism of 50 mM ethanol. These studies begin to describe quantitatively the roles of enzymes involved in the metabolism of alcohols and carbonyl compounds.     

The effect of trans-10, cis-12 conjugated linoleic acid on gene expression profiles related to lipid metabolism in human intestinal-like Caco-2 cells

We conducted an in-depth investigation of the effects of conjugated linoleic acid (CLA) on the expression of key metabolic genes and genes of known importance in intestinal lipid metabolism using the Caco-2 cell model. Cells were treated with 80 μmol/L of linoleic acid (control), trans-10, cis-12 CLA or cis-9, trans-11 CLA. RNA was isolated from the cells, labelled and hybridized to the Affymetrix U133 2.0 Plus arrays (n = 3). Data and functional analysis were preformed using Bioconductor. Gene ontology analysis (GO) revealed a significant enrichment (P < 0.0001) for the GO term lipid metabolism with genes up-regulated by trans-10, cis-12 CLA. Trans-10, cis-12 CLA, but not cis-9, trans-11 CLA, altered the expression of a number of genes involved in lipid transport, fatty acid metabolism, lipolysis, β-oxidation, steroid metabolism, cholesterol biosynthesis, membrane lipid metabolism, gluconeogenesis and the citrate cycle. These observations warrant further investigation to understand their potential role in the metabolic syndrome.     

Polymer Partitioning and Ion Selectivity Suggest Asymmetrical Shape for the Membrane Pore Formed by Epsilon Toxin

Using poly-(ethylene glycol)s of different molecular weights, we probe the channels formed in planar lipid bilayers by epsilon toxin secreted by the anaerobic bacterium Clostridium perfringens. We find that the pore is highly asymmetric. The cutoff size of polymers entering the pore through its opening from the cis side, the side of toxin addition, is ∼500 Da, whereas the cutoff size for the polymers entering from the trans side is ∼2300 Da. Comparing these characteristic molecular weights with those reported earlier for OmpF porin and the α-Hemolysin channel, we estimate the radii of cis and trans openings as 0.4 nm and 1.0 nm, respectively. The simplest geometry corresponding to these findings is that of a truncated cone. The asymmetry of the pore is also confirmed by measurements of the reversal potential at oppositely directed salt gradients. The moderate anionic selectivity of the channel is salted-out more efficiently when the salt concentration is higher at the trans side of the pore.     

13C-NMR and spectrophotometric studies of alcohol-lipid interactions

The interactions of butanol and mixtures of butanol and ethanol with dipalmitoylphosphatidyl choline (DPPC) liposomes have been investigated by both spectrophotometric measurements and Fourier transform 13C nuclear magnetic resonance spectroscopy. The spectrophotometric experiments indicate that butanol exhibits the same effects on the thermotropic properties of DPPC as the other short chain alcohols, methanol, ethanol and propanol, which have been shown to be characteristic of the alcohol induced transition of the lipid to the interdigitated state. An additive effect of butanol and ethanol on the induction of the interdigitated phase in DPPC was also observed. A decrease in line width and increase in T1 of the choline methyl signal were observed in the 13C-NMR experiments conducted at 32 degrees C when butanol was added to DPPC in increasing amounts suggesting an increase of disorder in the head group region of the lipid. Addition of ethanol to the NMR sample containing butanol produced hysteresis in the heating and cooling curves characteristic of the interdigitated state. In the interdigitated state, the choline methyl signal exhibited a T1 value equal to that when the lipid is in the fluid state. The increase of mobility in the head group region in the interdigitated gel state relative to the bilayer gel can be rationalized by the increase in surface area in that site when the lipid interdigitates.     

Novel Escherichia coli hybrids with enhanced butanol tolerance

Hybrids between Escherichia coli and Lactobacillus brevis were generated via protoplast fusion. Growth kinetics of five hybrid strains and E. coli were used to evaluate the butanol tolerance of the novel strains under different conditions. The hybrid strains tolerated up to 2% (v/v) butanol compared to the 1% (v/v) maximum for E. coli. The growth inhibitory effects of butanol were also significantly less in several of the hybrids compared to E. coli. These results demonstrate the potential use of protoplast fusion to generate butanol-tolerant strains.     

Comparative study of the effect of aliphatic alcohols on energy-dependent accumulation of Ca2+ in intracellular membranes of smooth muscle cells

The effects of ethanol and other aliphatic alcohols on energy-dependent Ca2+ transport in endoplasmic reticulum and mitochondria were studied in digitonin-treated myometrium cells. The Ca2+ uptake in mitochondria increased (on 15-20%) with increasing methanol, ethanol and propanol concentrations in medium, whereas further rise of concentration inhibited this process. Treatments of myometrial cells with short-chain alcohols caused an inhibition of calcium uptake in endoplasmic reticulum. Butanol inhibited both calcium uptake in mitochondria and endoplasmic reticulum. Ca2+ accumulation in intracellular pools is inhibited by aliphatic alcohols in the following order of potency: butanol > propanol > ethanol > methanol. It is concluded that modifying effect of aliphatic alcohols on energy dependent calcium accumulation in intracellular membrane structures is defined as on origin of Ca(2+)-transporting system and (or) properties of these membrane structures so on properties of alcohols.     

Bongkrekic acid and atractyloside inhibits chloride channels from mitochondrial membranes of rat heart

The aim of this work was to characterize the effect of bongkrekic acid (BKA), atractyloside (ATR) and carboxyatractyloside (CAT) on single channel properties of chloride channels from mitochondria. Mitochondrial membranes isolated from a rat heart muscle were incorporated into a bilayer lipid membrane (BLM) and single chloride channel currents were measured in 250/50 mM KCl cis/trans solutions. BKA (1-100 μM), ATR and CAT (5-100 μM) inhibited the chloride channels in dose-dependent manner. The inhibitory effect of the BKA, ATR and CAT was pronounced from the trans side of a BLM and it increased with time and at negative voltages (trans-cis). These compounds did not influence the single channel amplitude, but decreased open dwell time of channels. The inhibitory effect of BKA, ATR and CAT on the mitochondrial chloride channel may help to explain some of their cellular and/or subcellular effects.     

The Diphtheria Toxin Channel-forming T Domain Translocates Its Own NH2-Terminal Region Across Planar Bilayers

The T domain of diphtheria toxin, which extends from residue 202 to 378, causes the translocation of the catalytic A fragment (residues 1–201) across endosomal membranes and also forms ion-conducting channels in planar phospholipid bilayers. The carboxy terminal 57-amino acid segment (322–378) in the T domain is all that is required to form these channels, but its ability to do so is greatly augmented by the portion of the T domain upstream from this. In this work, we show that in association with channel formation by the T domain, its NH₂ terminus, as well as some or all of the adjacent hydrophilic 63 amino acid segment, cross the lipid bilayer. The phenomenon that enabled us to demonstrate that the NH₂-terminal region of the T domain was translocated across the membrane was the rapid closure of channels at cis negative voltages when the T domain contained a histidine tag at its NH₂ terminus. The inhibition of this effect by trans nickel, and by trans streptavidin when the histidine tag sequence was biotinylated, clearly established that the histidine tag was present on the trans side of the membrane. Furthermore, the inhibition of rapid channel closure by trans trypsin, combined with mutagenesis to localize the trypsin site, indicated that some portion of the 63 amino acid NH₂-terminal segment of the T domain was also translocated to the trans side of the membrane. If the NH₂ terminus was forced to remain on the cis side, by streptavidin binding to the biotinylated histidine tag sequence, channel formation was severely disrupted. Thus, normal channel formation by the T domain requires that its NH₂ terminus be translocated across the membrane from the cis to the trans side, even though the NH₂ terminus is >100 residues removed from the channel-forming part of the molecule.     

Gating of a voltage-dependent channel (colicin E1) in planar lipid bilayers: the role of protein translocation

Summary The voltage-dependent channel formed in planar lipid bilayers by colicin E1, or its channel-forming C-terminal fragments, is susceptible to destruction by the nonspecific protease pepsin under well-defined conditions. In particular, pepsin acts only from thecis side (the side to which colicin has been added) and only upon channels in the closed state. Channels in the open state are refractory to destruction bycis pepsin, and neither open nor closed channels are destroyed bytrans pepsin. Colicin E1 channels are normally turned on bycis positive voltages and turned off bycis negative voltages. For large (>80 mV) positive voltages, however, channels inactivate subsequent to opening. Associated with the inactivated state, some channels become capable of being turned on bycis negative voltages and turned off bycis positive voltages, as if the channel-forming region of the molecule has been translocated across the membrane. Consistent with this interpretation is the ability now oftrans pepsin to destroy these reversed channels when they are closed, but not when they are open, whereascis pepsin has no effect on them in either the open or closed state. Our results indicate that voltage gating of the E1 channel involves translocation of parts of the protein across the membrane, exposing different domains to thecis andtrans solutions in the different channel states.     

Cis-monolignols in Fagus grandifolia and their possible involvement in lignification

Lignification in all plant species is assumed to occur exclusively via the dehydrogenative polymerization of the trans (E) monolignols, p-coumaryl, coniferyl and sinapyl alcohols. This assumption may have to be revised somewhat due to the presence of both E(trans) and Z (cis) isomers of p-hydroxy substituted cinnamic acids in grasses, and cis-coniferyl and cis-sinapyl alcohols in beech bark (Fagus grandifolia). This suggests that lignification of these tissues may use either cis- or trans-monolignols. By means of H₂O₂/peroxidase induction, we have prepared synthetic dehydrogenative polymerized (DHP) lignin from both cis- and trans-coniferyl alcohols. Under the tests employed, the degradation products from both DHPs were identical suggesting that either cis- or trans-monolignols are suitable substrates for this enzyme and, therefore, lignification. An alternative hypothesis is that the cis-monolignols accumulate in beech bark because they are not suitable substrates. Therefore, it would follow that the enzymes involved in lignification in vivo are highly specific.     

Isolation and characterization of butanol-tolerant <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

Objectives

A new solvent-tolerant species, Staphylococcus aureus, was isolated and characterized during the screening of butanol-tolerant microorganisms.

Results

Three isolates of S. aureus were obtained as contaminants during improvement of butanol tolerance of E. coli K12. Their cell dry weights were 135 % that of K12 in the absence of butanol stress. S. aureus had a growth advantage over K12 when cultured with various concentrations of butanol. It can tolerate up to 3 % (v/v) butanol, while most solventogenic bacteria can tolerate only 2 % (v/v) butanol. The addition of 10–20 g glucose/l enhanced its butanol tolerance. The relative cell biomass of the S. aureus was 71–306 % that of E. coli under 5.5–10 % (v/v) ethanol stress, indicating ethanol resistance.

Conclusions

This is the first study to observe butanol-tolerant S. aureus. As this organism can be genetically manipulated, it could have a wide array of applications.

     

Organization of SNAREs within the Golgi Stack

Antero- and retrograde cargo transport through the Golgi requires a series of membrane fusion events. Fusion occurs at the cis- and trans-side and along the rims of the Golgi stack. Four functional SNARE complexes have been identified mediating lipid bilayer merger in the Golgi. Their function is tightly controlled by a series of reactions involving vesicle tethering and SM proteins. This network of protein interactions spatially and temporally determines the specificity of transport vesicle targeting and fusion within the Golgi.At steady state, the Golgi maintains its structural and functional organization despite a massive lipid and protein flow. A balanced anterograde and retrograde membrane flow are required to constantly recycle the transport machinery and cargo containers (vesicles). In the absence of efficient recycling, directional net cargo transport would cease and the Golgi would collapse. Thus, transport vesicles constantly leave and enter at both sides of the Golgi stack and bud and fuse along the rims of the cisternae. To maintain the compartmental identity, vesicle fusion occurs in a specific and orchestrated manner. These fusion events are mediated by a cascade of reactions centered around the membrane fusion proteins SNAREs (SNAP receptors) (Söllner et al. 1993b).     

Inhibition of forskolin-stimulated cardiac adenylate cyclase activity by short-chain alcohols

The diterpene forskolin stimulated rat cardiac adenylate cyclase activity at least 20-fold and potentiated the effect of NaF. The stimulatory effect of forskolin was reduced in the presence of Gpp(NH)p. Ethanol markedly reduced the stimulation of adenylate cyclase by forskolin while potentiating NaF and Gpp(NH)p stimulation. The inhibitory effect of ethanol on forskolin stimulation appeared to be of a mixed type with both a competitive and a non-competitive component. Three other short-chain linear alcohols (methanol, propanol, butanol) also inhibited forskolin-stimulation, this effect being proportional to the number of carbon atoms.     

Effects of phenazepam and aliphatic alcohols on epileptic complex created in the rat brain cortex

Effects of phenazepam and aliphatic alcohols (methanol, ethanol, butanol, and propanol) on associated epileptic complex created in the rat brain cortex by applications of strychnine were studied. Phenazepam considerably suppressed the epileptic foci and their complexes in a dose-dependent manner. A comparison of anticonvulsive effects in a series of aliphatic alcohols methanol-ethanol-propanol-butanol showed that all these substances are potent anticonvulsants. Propanol and butanol reduce epileptic seizures most intensively, but, at the same time, they are most toxic. Application of labelled⁴C-ethanol showed that anticonvulsive effects strongly correlate with changes in the ethanol concentration in the blood.Neirofiziologiya/Neurophysiology, Vol. 26, No. 6, pp. 443–448, November–December, 1994.     

Interaction of alcohols with proteins

The kinetics of denaturation of egg albumin have been determined for methanol, ethanol, propanol, and butanol. The reactions are first order in respect to protein but between 11th and 18th order for the alcohols. The denaturation reaction is characterized by a large temperature coefficient with little or no dependence on pH. There is a marked change of pH when proteins are denatured. A series of eight proteins has been studied. There is surprisingly little difference in susceptibility to alcohol denaturation between the various proteins. Methanol, ethanol, propanol, and butanol are strongly bound to egg albumin—butanol being the most strongly bound. The binding of alcohol is probably accompanied by protein dehydration. The polyhydric alcohols' behavior is much different. These alcohols do not denature proteins and the protein is hydrated. Sucrose produces the greatest degree of hydration.     

Productive hemifusion intermediates in fast vesicle fusion driven by neuronal SNAREs

An in vitro fusion assay uses fluorescence microscopy of labeled lipids to monitor single v-SNARE vesicle docking and fusion events on a planar lipid bilayer containing t-SNAREs. For vesicles and bilayer comprising phosphatidylcholine (POPC, 84-85% by mol) and phosphatidylserine (DOPS, 15% by mol), previous work demonstrated prompt, full fusion (τ_fus = 25 ms). Substitution of 20-60% phosphatidylethanolamine (DOPE) for phosphatidylcholine in the v-SNARE vesicle with either 0 or 20% DOPE included in the t-SNARE bilayer gives rise to hemifusion events. Labeled lipids diffuse into the planar bilayer as two temporally distinct waves, presumably hemifusion of the outer leaflet followed by inner leaflet (core) fusion. The fusion kinetics with DOPE is markedly heterogeneous. Some vesicle/docking site pairs exhibit prompt, full fusion while others exhibit hemifusion. Hemifusion events are roughly half productive (leading to subsequent core fusion within 20 s) and half dead-end. In qualitative accord with expectations from studies of protein-free vesicle-vesicle fusion, the hemifusion rate k_hemi is 15-20 times faster than the core fusion rate k_core, and the fraction of hemifusion events increases with increasing percentage of DOPE. This suggests similar underlying molecular pathways for protein-free and neuronal SNARE-driven fusion. Removal of phosphatidylserine from the v-SNARE vesicle has no effect on docking or fusion.     

Ethanol Reduces Norepinephrine-Stimulated Melatonin Synthesis in Rat Pinealocytes

In this study, the in vitro effects of ethanol on norepinephrine-stimulated cyclic AMP (cAMP), N-acetyltransferase (NAT), and melatonin (MT) production were examined in dispersed rat pinealocytes. Cellular cAMP content was determined 15 min after treatment; whereas NAT activity and MT release in the medium were determined 4.5 h after treatment. It was found that ethanol less than or equal to 200 mM had no effect on norepinephrine-stimulated cAMP response, whereas 25 mM ethanol resulted in a significant inhibition of norepinephrine-stimulated NAT and MT levels. Furthermore, ethanol was equally effective in inhibiting the dibutyryl cAMP-stimulated NAT and MT levels. The inhibitory action of ethanol was not due to a direct effect or a delay in the onset of NAT activity. When alcohols with different chain lengths were used, it was found that their inhibitory potencies were related to their chain lengths with butanol greater than propanol greater than ethanol greater than methanol. Taken together, these findings indicate that (1) ethanol has an inhibitory action on norepinephrine-stimulated MT synthesis, (2) one site of ethanol action is distal to cAMP elevation, and (3) the inhibitory effect of ethanol on pineal MT synthesis appears to be secondary to its hydrophobic action.     

A tethered bilayer assembled on top of immobilized calmodulin to mimic cellular compartmentalization

Background

Biomimetic membrane models tethered on solid supports are important tools for membrane protein biochemistry and biotechnology. The supported membrane systems described up to now are composed of a lipid bilayer tethered or not to a surface separating two compartments: a ”trans” side, one to a few nanometer thick, located between the supporting surface and the membrane; and a “cis” side, above the synthetic membrane, exposed to the bulk medium. We describe here a novel biomimetic design composed of a tethered bilayer membrane that is assembled over a surface derivatized with a specific intracellular protein marker. This multilayered biomimetic assembly exhibits the fundamental characteristics of an authentic biological membrane in creating a continuous yet fluid phospholipidic barrier between two distinct compartments: a “cis” side corresponding to the extracellular milieu and a “trans” side marked by a key cytosolic signaling protein, calmodulin.

Methodology/Principal Findings

We established and validated the experimental conditions to construct a multilayered structure consisting in a planar tethered bilayer assembled over a surface derivatized with calmodulin. We demonstrated the following: (i) the grafted calmodulin molecules (in trans side) were fully functional in binding and activating a calmodulin-dependent enzyme, the adenylate cyclase from Bordetella pertussis; and (ii) the assembled bilayer formed a continuous, protein-impermeable boundary that fully separated the underlying calmodulin (trans side) from the above medium (cis side).

Conclusions

The simplicity and robustness of the tethered bilayer structure described here should facilitate the elaboration of biomimetic membrane models incorporating membrane embedded proteins and key cytoplasmic constituents. Such biomimetic structures will also be an attractive tool to study translocation across biological membranes of proteins or other macromolecules.     

Modulation of lipid droplet size and lipid droplet proteins by trans-10,cis-12 conjugated linoleic acid parallels improvements in hepatic steatosis in obese,insulin-resistant rats

The isomer-specific effects of conjugated linoleic acid (CLA) on hepatic steatosis were assessed in fa/fa Zucker rats, a model for insulin resistance and the metabolic syndrome. Eight weeks of feeding trans-10,cis-12 CLA significantly improved glucose tolerance without changing body weight or visceral adipose mass. The trans-10,cis-12 isomer was also associated with reduced liver lipid content, improved liver function and reduced inflammation; these effects were not observed in rats fed the cis-9,trans-11 CLA isomer. Reduced liver lipid content did not correlate with activation of AMP-activated protein kinase or suppressed activation of sterol-regulatory element binding protein-1, two key regulators of hepatic lipid metabolism. Interestingly, rats fed cis-9,trans-11 CLA had fewer cytoplasmic lipid droplets in hepatocytes compared to rats fed control diet, but these droplets were larger in size. Conversely, fa/fa rats fed the trans-10,cis-12 CLA isomer had greater numbers of hepatic lipid droplets that were smaller in size, resulting in overall lower total lipid within these droplets. Changes in lipid droplets were associated with lower hepatic levels of PERILIPIN-2 (formerly known as adipophilin) in rats fed trans-10,cis-12 CLA, whereas amounts of other members of the PERILIPIN family of lipid droplet proteins were unaffected by dietary CLA. However, CLA isomers differentially affected the subcellular localization of these proteins. Treatment of H4IIE rat hepatoma cells with CLA isomers neither prevented nor reversed, but rather induced cytoplasmic lipid droplet formation, suggesting that the anti-steatotic effects of trans-10,cis-12 CLA are likely indirect and potentially mediated via increased lipid utilization by peripheral tissues.