Tolerance and cross-tolerance in chronic alcoholics: reduced membrane binding of ethanol and other drugs.

Membrane binding of ethanol, anesthetics, and hydrophobic molecules in brain synaptosomes and liver mitochondria from rats is conspicuously reduced after long-term consumption of ethanol. The membranes are resistant to structural disordering by both ethanol and halothane. Tolerance, cross-tolerance, and dependence in chronic alcoholics may in part result from membrane alterations that inhibit the binding of ethanol and other drugs.

A function of lung surfactant protein SP-B.

The primary function of lung surfactant is to form monolayers at the alveolar interface capable of lowering the normal surface tension to near zero. To accomplish this process, the surfactant must be capable of maintaining a coherent, tightly packed monolayer that avoids collapse during expiration. The positively charged amino-terminal peptide SP-B1-25 of lung surfactant-specific protein SP-B increases the collapse pressure of an important component of lung surfactant, palmitic acid (PA), to nearly 70 millinewtons per meter. This alteration of the PA isotherms removes the driving force for "squeeze-out" of the fatty acids from the primarily dipalmitoylphosphatidylcholine monolayers of lung surfactant. An uncharged mutant of SP-B1-25 induced little change in the isotherms, suggesting that a specific charge interaction between the cationic peptide and the anionic lipid is responsible for the stabilization. The effect of SP-B1-25 on fatty acid isotherms is remarkably similar to that of simple poly-cations, suggesting that such polymers might be useful as components of replacement surfactants for the treatment of respiratory distress syndrome.

Phase and morphology changes in lipid monolayers induced by SP-B protein and its amino-terminal peptide.

Both human lung surfactant protein, SP-B, and its amino-terminal peptide, SP-B1-25, inhibit the formation of condensed phases in monolayers of palmitic acid, resulting in a new fluid phase. This fluid phase forms a network, separating condensed-phase domains at coexistence. The network persists to high surface pressures, altering the nucleation, growth, and morphology of monolayer collapse structures, leading to lower surface tensions on compression and more reversible respreading on expansion. The network is stabilized by the low line tension between the fluid phase and the condensed phase as confirmed by the formation of "stripe" phases.

Compaction of DNA in an anionic micelle environment followed by assembly into phosphatidylcholine liposomes.

A difficult problem concerning the interaction of DNA with amphiphiles of opposite charge above their critical micelle concentration is the propensity for aggregation of the condensed DNA complexes. In this study, this problem was addressed by attenuating amphiphile charge density within a cholate micelle environment. The amphiphile consisted of a cationic peptide, acetyl-CWKKKPKK-amide, conjugated to dilaurylphos-phatidylethanolamine. In the presence of cholate, multiple equivalents of cationic charge were required to bring about the completion of DNA condensation. At the end point of condensation, stable, soluble DNA-micelle complexes were formed, which by dynamic light scattering exhibited apparent hydro-dynamic diameters between 30 and 60 nm. Aggregation, as measured by static light scattering at 90 degrees and by turbidity, was not observed until further additions of peptide-lipid conjugate were made beyond the end point of DNA condensation. Liposome complexes containing the non-aggregated, compacted DNA were formed by adding dioleoylphosphatidylcholine followed by removing the cholate by dialysis. The resulting complexes were distributed within a narrow density range, the DNA was quantitatively assembled into the liposomes, and liposomes without DNA were not detected. Small particles were formed with a mean hydrodynamic diameter of 77 nm. The liposomal DNA showed complete retention of its supercoiled form and no detectable sensitivity to DNase (25 U/10 microg DNA, 1.5 h, 37 degrees C). The use of an anionic, dialyzable amphiphile to attenuate charge inter-actions between DNA and cationic amphiphiles is a useful technology for the quantitative assembly of compacted DNA into conventional liposomes, with complete protection against nuclease activity.

Development of an effective gene delivery system: a study of complexes composed of a peptide-based amphiphilic DNA compaction agent and phospholipid.

We recently described a basic technology to efficiently combine compacted DNA with phospholipids and hydrophobic peptides, to produce homogenous complexes that are completely resistant to nuclease. We have developed this technology further to form gene delivery complexes that transfect cells effectively in vitro. In addition to plasmid DNA, the complexes contained two basic components: (i) a DNA compacting peptide (-CGKKKFKLKH), either conjugated to lipid or extended to contain (WLPLPWGW-) and (ii) either phosphatidylethanolamine or phosphatidylcholine. Complexes containing a 5.5-fold charge equivalence (peptide charge/DNA charge) of WLPLPWGWCGKKKFKLKH and 5 nmol dimyristoleoylphosphatidylethanolamine/microg DNA produced the highest luciferase gene expression, exceeding 1 x 10(9) relative light units/s/mg protein (>3 microg luciferase per mg protein). These complexes transfected OVCAR-3, COS-7 and HeLa cells at either similar or superior levels when compared to polyethylenimine or lipofectamine complexes. With green fluorescent protein reporter gene, >50% of HeLa cells were positive 30 h after addition of these complexes. Furthermore, these optimal complexes were the least sensitive to pre-treatment of cells with chloroquine, indicating efficient endosomal escape. Our results indicated that self-assembling complexes of plasmid DNA, amphiphilic peptide and phosphatidylethanolamine are highly effective non-viral gene delivery systems.

Subzero temperature study of the inner mitochondrial membrane and related phospholipid membrane systems with the fluorescent probe, trans-parinaric acid.

The fluorescence intensity of trans-parinaric acid as a function of the temperature indicates a phase transition in bovine heart mitochondrial inner membranes below 0 degrees C. The comparison of the dye fluorescence intensity in intact inner mitochondrial membranes and in vesicles from extracted phospho lipids of mitochondria revealed a similar intensity increase with decreasing temperature. A synthetic phospholipid system of dioleoyl phosphatidylcholine was investigated because of its low phase transition temperature and showed a very definite intensity change at -25 degrees C. trans-Parinaric acid in membrane systems probes an environment of intermediate polarity; this was found from the excitation and emission spectra and from fluorescence decay.

Synthesis and degradation of cyclic nucleotides in the pituitary gland, ovary and testis of rats treated with D-Trp6-luteinizing hormone-releasing hormone.

The effect of administration of D-Trp6-Luteinizing Hormone-Releasing Hormone (LH-RH) on synthesis and degradation of cyclic nucleotides was studied in the rat. There were no significant changes in the rate of synthesis and degradation of cyclic AMP in the ovary, testis and pituitary gland of D-Trp6-LH-RH-treated rats as compared to controls. On the other hand, the levels of cyclic GMP and activity of guanylate cyclase were significantly higher in the ovary and testis as well as in the pituitary gland of animals which received the analog. The rate of hydrolysis of cyclic GMP was unchanged by the administration of D-Trp6-LH-RH. Interestingly, the cyclic CMP phosphodiesterase seemed to be activated in animals treated with D-Trp6-LH-RH.

Inhibition of mixtures of surfactant lipids and synthetic sequences of surfactant proteins SP-B and SP-C.

The respiratory distress syndrome of premature infants is caused by both surfactant deficiency and surfactant inhibition by capillary-alveolar leakage of serum factors. Dispersions of a standard surfactant lipid mixture, with and without various synthetic peptides, modeled on human surfactant proteins SP-B (residues 1-25, 49-66, 1-78) and SP-C (residues 1-10), were evaluated for inhibition by serum and by plasma constituents using a pulsating bubble surfactometer. Inhibition was derived from the changes in surface properties of these mixtures after addition of human serum or plasma constituents. Modified bovine surfactant (TA) containing native SP-B and SP-C was used as a control. In the absence of serum inhibitors, mixtures with synthetic peptides gave results similar to surfactant TA. However, inhibition was more evident in the dispersions with synthetic peptides when compared with surfactant TA. The peptide/phospholipid mixture with the entire sequence of SP-B and the first 10 residues of SP-C were more resistant to inhibition than mixtures with synthetic peptides containing fewer domains. Addition of calcium reduced the inhibitory effects of serum both in mixtures containing synthetic peptides and in surfactant TA. Therefore, synthetic SP-B and SP-C peptides in surfactant lipids, in cooperation with calcium, permit resistance to inhibition by several plasma constituents that probably inactivate surfactant by a variety of different mechanisms.

Membrane interactions of the synthetic N-terminal peptide of HIV-1 gp41 and its structural analogs.

Structural and functional studies assessed the membrane actions of the N terminus of HIV-1 glycoprotein 41000 (gp41). Earlier site-directed mutagenesis has shown that key amino acid changes in this gp41 domain inhibit viral infection and syncytia formation. Here, a synthetic peptide corresponding to the N terminus of gp41 (FP; 23 residues, 519-541), and also FP analogs (FP520V/E with Val-->Glu at residue 520; FP527L/R with Leu-->Arg at 527; FP529F/Y with Phe-->Tyr at 529; and FPCLP1 with FP truncated at 525) incorporating these modifications were prepared. When added to human erythrocytes at physiologic pH, the lytic and aggregating activities of the FP analogs were much reduced over those with the wild-type FP. With resealed human erythrocyte ghosts, the lipid-mixing activities of the FP analogs were also substantially depressed over that with the wild-type FP. Combined with results from earlier studies, theoretical calculations using hydrophobic moment plot analysis and physical experiments using circular dichroism and Fourier transform infrared spectroscopy indicate that the diminished lysis and fusion noted for FP analogs may be due to altered peptide-membrane lipid interactions. These data confirm that the N-terminal gp41 domain plays critical roles in the cytolysis and fusion underlying HIV-cell infection.

Cytochrome c-cytochrome oxidase interaction at subzero temperatures.

Cytochrome oxidase forms two distinctive compounds with oxygen at --105 and --90 degrees C, one appears to be oxycytochrome oxidase (Compound A) and the other peroxycytochrome oxidase (Compound B). The functional role of compound B in the oxidation of cytochrome c has been examined in a variety of mitochondrial preparations. The rate and the extent of the reaction have been found to be dependent upon the presence of a fluid phase in the vicinity of the site of the reaction of cytochrome c and cytochrome oxidase. The kinetics of cytochrome c oxidation and of the slowly reacting component of cytochrome oxidase are found to be linked to one another even in cytochrome c depleted preparations, but under appropriate conditions, especially low temperatures, the oxidation of cytochrome c precedes that of this component of cytochrome oxidase. Based upon the identification of the slowly reacting components of cytochrome oxidase with cytochrome c, various mechanisms are considered which allow cytochrome c to be oxidized without the intervention of cytochrome a at very low temperatures, and tunneling seems an appropriate mechanism.

In vivo modification of plant membrane phospholipid composition.

Tomato seedlings treated with ethanolamine showed altered phospholipid composition. The changes included altered acyl chain composition as well as changes in the relative amounts of the phospholipid classes. Specifically, there was an increase in phosphatidylethanolamine and phosphatidylserine with a concomitant decrease in phosphatidylcholine and no overall increase in phospholipids. Treatment with ethanolamine increased the relative amount of C18 acyl chains (especially 18 : 2) in phosphatidylethanolamine and phosphatidylcholine at the expense of 16 : 0 and 16 : 1. Acyl composition of other phospholipid classes were unchanged. Labeled ethanolamine was incorporated mostly into phosphatidylethanolamine and phosphatidylcholine. Ethanolamine-stimulated incorporation of labeled oleate was entirely into acyl chains and appeared only as 18 : 1 and 18 : 2. There was greater incorporation, but less conversion of 18 : 1 to 18 : 2 with choline. Stearate was incorporated but desaturated.

Structure-function relationships of bovine pulmonary surfactant proteins: SP-B and SP-C.

Pulmonary surfactant contains at least three unique proteins: SP-A, SP-B and SP-C. SP-B and SP-C from bovine surfactant are markedly hydrophobic and have molecular masses between 3 and 26 kDa. We identify surfactant proteins under nonreducing conditions on polyacrylamide gels with approximate molecular mass of 5, 14, 26 kDa (SP-5, 14, 26) when organic solvent-soluble material is eluted from a Sephadex LH-20 size exclusion column followed by separation on a high-performance reverse-phase chromatography system. These bands correspond to monomeric SP-C, oligomeric SP-C and oligomeric SP-B, respectively. Computer analysis (Eisenberg-hydrophobic moment) of sequences for these proteins suggests that SP-B contains surface-seeking amphiphilic segments. In contrast, SP-C resembles a more hydrophobic transmembrane anchoring peptide. Dispersions containing dipalmitoylphosphatidylcholine, phosphatidylglycerol, palmitic acid and multimeric SP-B and SP-C duplicate the surface activity of natural surfactant when assayed in a pulsating bubble surfactometer. We speculate that oligomers of SP-B and monomers and oligomers of SP-C may act cooperatively in affecting surfactant function. An important function of SP-B and SP-C may be to affect the ordering of surfactant lipids so that rates of transport of surfactant lipids to the hypophase surface in the alveoli are enhanced.

Full length synthetic surfactant proteins, SP-B and SP-C, reduce surfactant inactivation by serum.

The failure of some infants with respiratory distress syndrome to respond to therapy with surfactant may be explained in part by inactivation of surfactant caused by leakage of plasma constituents into air spaces. Surfactant-associated proteins (SP-A, SP-B and SP-C) reduce the susceptibility of surfactants to inactivation in vitro. To study this phenomenon further, we used full length synthetic proteins, SP-B [1-78] and SP-C [1-31], mixed with surfactant lipids in different ratios and different concentrations. Equilibrium and minimum surface tensions of these mixtures, with or without serum and calcium, were measured using a pulsating surfactometer. Mixtures containing both SP-B and SP-C had optimal minimum and equilibrium surface tensions of < 5 and < 28 mN/m, respectively. Mixtures with SP-B had optimal minimum surface tensions, but equilibrium surface tensions averaged 35 mN/m. Mixtures with SP-C had high minimal (19 mN/m) and high equilibrium surface tensions (35 mN/m). When serum was added to these mixtures, the least inactivation was found with mixtures containing 3% protein at 1:1 ratio of SP-B/SP-C with 2 mM calcium chloride. These data indicate that SP-B and SP-C, particularly in the presence of calcium, reduce surfactant inactivation that may be caused by plasma constituents. The results lead to the hypothesis that charge interactions among ions, lipids, surfactant proteins, and serum inactivators are a major element in pathophysiological surfactant inactivation.

The amino-terminal peptide of HIV-1 glycoprotein 41 fuses human erythrocytes.

The ability of synthetic peptides based on the amino-terminus of HIV-1 glycoprotein 41,000 (gp41) to fuse human erythrocytes was investigated. Previous site-directed mutagenesis studies have shown an important role for the N-terminal gp41 domain in HIV-fusion, in which replacement of hydrophobic amino acids with polar residues inhibits viral infection and syncytia formation. Here, a synthetic peptide (FP; 23 amino acid residues 519-541) corresponding to the N-terminus of HIV-1 gp41, and also a FP analog (FP526L/R) with Arg replacing Leu-526, were prepared with solid phase techniques. The lipid mixing and leakage of resealed ghosts triggered by these peptides were examined with fluorescence quenching techniques. Peptide-induced aggregation of human erythrocytes was studied using Coulter counter sizing and scanning electron microscopy (SEM). Using resealed erythrocyte ghosts at physiologic pH, FP induces rapid lipid mixing between red cell membranes at doses previously shown to hemolyze intact cells. FP also causes leakage from resealed ghosts, and promotes the formation of multicelled aggregates with whole erythrocytes. Contrarily, similar FP526L/R concentrations did not induce red cell lysis, lipid mixing, leakage or aggregation. Since the fusogenic potency of FP and FP526L/R parallels earlier gp41 mutagenesis studies showing that substitution of Arg for Leu-526 blocks fusion activity, these data suggest that the N-terminal gp41 domain in intact HIV participates in fusion.

The amino-terminal peptide of HIV-1 glycoprotein 41 lyses human erythrocytes and CD4+ lymphocytes.

Functional studies assessed the cytolytic activity of the amino terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of the Human Immunodeficiency Virus Type-1 (HIV-1). Synthetically prepared FP-I efficiently hemolyzed human red blood cells at 37 degrees C, with 40% lysis at 32 microM. Kinetic studies indicated that FP-I induced maximal hemolysis in 30 min, probably through tight binding of the peptide with the red cell membrane. The Phe-Leu-Gly-Phe-Leu-Gly (residues 526-531) motif in FP-I apparently plays a critical role in lysis of red cells, since no hemolytic activity was observed for an amino-acid-substituted FP-I in which the unique Phe-Leu-Gly-Phe-Leu-Gly was converted to Ala-Leu-Gly-Ala-Leu-Gly. As neither smaller constituent peptides (e.g., residues 519-524 and residues 526-536) nor a N-terminal flanking peptide (e.g., residues 512-523) induced red cell hemolysis, the entire 23-residue (519-541) sequence of FP-I may be required for hemolytic activity. FP-I was also cytolytic with CD4(+)-bearing Hut-78 cells, with 40% lysis at approx. 150 microM. These results are consistent with an earlier hypothesis that the N-terminal peptide of gp41 may partially contribute to the in vivo cytopathic actions of HIV-1 infection (Gallaher, W.R. (1987) Cell 50, 327-328).

The amino-terminal peptide of HIV-1 glycoprotein 41 interacts with human erythrocyte membranes: peptide conformation, orientation and aggregation.

Structural studies assessed interactions between the amino-terminal peptide (FP-I; 23 residues 519-541) of the glycoprotein 41,000 (gp41) of Human Immunodeficiency Virus Type-1 (HIV-1) and human erythrocyte membranes and simulated membrane environments. Peptide binding was examined at sub-hemolytic (approx. less than 5 microM) and hemolytic (greater than or equal to 5 microM) doses (Mobley et al. (1992) Biochem. Biophys. Acta 1139, 251-256), using circular dichroism (CD) and Fourier-transform infrared (FTIR) measurements with FP-I, and electron spin resonance (ESR) studies employing FP-I spin-labeled at either the amino-terminal alanine (FP-II; residue 519) or methionine (FP-III; position 537). In the sub-lytic regime, FP-I binds to both erythrocyte lipids and dispersions of SDS with high alpha-helicity. Further, ESR spectra of FP-II labeled erythrocyte ghosts indicated peptide binding to both lipid and protein. In ghost lipids, FP-II was monomeric and exhibited low polarity and rapid, anisotropic motion about its long molecular axis (i.e., alpha-helical axis), with restricted motion away from this axis. The spin-label at the amino-terminal residue (Ala-519) is insensitive to the aqueous broadening agent chromium oxalate and buried within the hydrophobic core of the membrane; the angle that the alpha-helix (residues 519-536) makes to the normal of the bilayer plane is either 0 degree or 40 degrees. Contrarily, ESR spectra of ghost lipids labeled with sub-lytic doses of FP-III indicated high mobility and polarity for the reporter group (Met-537) at the aqueous-membrane interface, as well as extreme sensitivity to chromium oxalate. At lytic FP-I doses, CD and FTIR showed both alpha-helix and beta-structure for peptide in ghost lipids or detergent, while ESR spectra of high-loaded FP-II in ghost membranes indicated peptide aggregates. Membrane aggregates of FP-I may be involved in hemolysis, and models are suggested for N-terminal gp41 peptide participation in HIV-induced fusion and cytolysis.

Dicynthaurin: an antimicrobial peptide from hemocytes of the solitary tunicate, Halocynthia aurantium.

We isolated a novel antimicrobial peptide, dicynthaurin, from hemocytes of a tunicate, Halocynthia aurantium. The native peptide had a mass of approximately 6.2 kDa and was composed of two 30-residue monomers without sequence homology to any previously identified peptides (ILQKAVLDCLKAAGSSLSKAAITAIYNKIT). Most cynthaurin molecules were C-terminally amidated and were linked covalently by a single cystine disulfide bond. When performed in membrane-mimetic environments, circular dichroism studies of dicynthaurin revealed largely alpha-helical conformations. Dicynthaurin's broad-spectrum activity encompassed Gram-positive (Micrococcus luteus, Staphylococcus aureus, Listeria monocytogenes) and Gram-negative bacteria (Escherichia coli, Pseudomonas aeruginosa), but not Candida albicans, a fungus. Although dicynthaurin was purified from a marine invertebrate, its antimicrobial activity was optimal at NaCl concentrations below 100 mM. This suggests that the antimicrobial actions of this molecule may take place intracellularly (e.g., within a phagosome) rather than extracellularly.

Circular minidefensins and posttranslational generation of molecular diversity.

We purified two new minidefensins (RTD-2 and RTD-3) from the bone marrow of rhesus monkeys. Both were circular octadecapeptides that contained three intramolecular disulfide bonds and were homologous to RTD-1, a circular (theta) defensin previously described by Tang et al. (Science, 286, 498-502, 1999). However, whereas the 18 residues of RTD-1 represent spliced nonapeptide fragments derived from two different demidefensin precursors, RTD-2 and -3 comprise tandem nonapeptide repeats derived from only one of the RTD-1 precursors. Thus, circular minidefensins are products of a novel posttranslational system that generates effector molecule diversity without commensurate genome expansion. A system wherein two demidefensin genes can produce three circular minidefensins might allow n such genes to produce (n/2)(n+1) peptides.

Conformation and molecular topography of the N-terminal segment of surfactant protein B in structure-promoting environments.

Although the effects of surfactant protein B (SP-B) on lipid surface activity in vitro and in vivo are well known, the relationship between molecular structure and function is still not fully understood. To further characterize protein structure-activity correlations, we have used physical techniques to study conformation, orientation, and molecular topography of N-terminal SP-B peptides in lipids and structure-promoting environments. Fourier transform infrared (FTIR) and CD measurements of SP-B1-25 (residues 1-25) in methanol, SDS micelles, egg yolk lecithin (EYL) liposomes, and surfactant lipids indicate the peptide has a dominant helical content, with minor turn and disordered components. Polarized FTIR studies of SP-B1-25 indicate the long molecular axis lies at an oblique angle to the surface of lipid films. Truncated peptides were similarly examined to assign more accurately the discrete conformations within the SP-B1-25 sequence. Residues Cys-8-Gly-25 are largely alpha-helix in methanol, whereas the N-terminal segment Phe-1-Cys-8 had turn and helical propensities. Addition of SP-B1-25 spin-labeled at the N-terminal Phe (i.e., SP-B1-25) to SDS, EYL, or surfactant lipids yielded electron spin resonance spectra that reflect peptide bound to lipids, but retaining considerable mobility. The absence of characteristic radical broadening indicates that SP-B1-25 is minimally aggregated when it interacts with these lipids. Further, the high polarity of SP-B1-25 argues that the reporter on Phe-1 resides in the headgroup of the lipid dispersions. The blue-shift in the endogenous fluorescence of Trp-9 near the N-terminus of SP-B1-25 suggests that this residue also lies near the lipid headgroup. A summary model based on the above physical experiments is presented for SP-B1-25 interacting with lipids.

Methionine oxidation within the cerebroside-sulfate activator protein (CSAct or Saposin B).

The cerebroside-sulfate activator protein (CSAct or Saposin B) is a small water-soluble glycoprotein that plays an essential role in the metabolism of certain glycosphingolipids, especially sulfatide. Deficiency of CSAct in humans leads to sulfatide accumulation and neurodegenerative disease. CSAct activity can be measured in vitro by assay of its ability to activate sulfatide-sulfate hydrolysis by arylsulfatase A. CSAct has seven methionine residues and a mass of 8,845 Da when deglycosylated. Mildly oxidized, deglycosylated CSAct (+16 Da), separated from nonoxidized CSAct by reversed-phase high-performance liquid chromatography (RP-HPLC), showed significant modulation of the in vitro activity. Because oxidation partially protected against CNBr cleavage and could largely be reversed by treatment with dithiothreitol, it was concluded that the major modification was conversion of a single methionine to its sulfoxide. High-resolution RP-HPLC separated mildly oxidized CSAct into seven or more different components with shorter retention times than nonoxidized CSAct. Mass spectrometry showed these components to have identical mass (+16 Da). The shorter retention times are consistent with increased polarity accompanying oxidation of surface-exposed methionyl side chains, in general accordance with the existing molecular model. A mass-spectrometric CNBr mapping protocol allowed identification of five of the seven possible methionine-sulfoxide CSAct oxoforms. The most dramatic suppression of activity occurred upon oxidation of Met61 (26% of control) with other residues in the Q60MMMHMQ66 motif falling in the 30-50% activity range. Under conditions of oxidative stress, accumulation of minimally oxidized CSAct protein in vivo could perturb metabolism of sulfatide and other glycosphingolipids. This, in turn, could contribute to the onset and progression of neurodegenerative disease, especially in situations where the catabolism of these materials is marginal.