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C C Curtain

Publications and source records attributed to C C Curtain.

At least 19 recordsLinked to original sources
PubMed1992

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).

Amino Acid Sequence↗
PubMed1992

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.

Amino Acid Sequence↗
PubMed1989

Fatty-acid spin probe interactions with erythrocyte ghosts and liposomes prepared from erythrocyte ghosts.

A model for the binding of 5-nitroxide stearate, I(12.3), to human erythrocyte ghosts was developed by comparing spin probe interactions with ghosts and liposomes prepared from ghosts. At low probe/lipid (P/L less than 1/2500), I(12.3) binds to a similar class of high-affinity, noninteracting sites in both ghosts and liposomes, indicating that lipid moieties are responsible for probe uptake. Saturation occurs in both systems with increasing P/L, and, at higher loading (e.g., P/L = 1/360 for ghosts and liposomes), the probe inserts itself at initially dilute sites to form a class of low-affinity sites consisting of clusters of variable size. At still higher P/L ranges (greater than 1/100), much increased probe uptake was observed in ghosts than in liposomes, which was attributed to another class of low-affinity sites, representing nonspecific interactions of I(12.3) with membrane proteins. The nature of the spectral components and ultrafiltration experiments with ghosts labeled at high P/L indicate that both 'dilute' and 'clustered' I(12.3) are due to membrane-incorporated probe.

Binding Sites↗
PubMed1988

Thermotropic lipid phase separation in the human immunodeficiency virus.

The presence of thermodependent lipid domains in the envelope of the human immunodeficiency virus (HIV) was studied. HIV was propagated in Hut-78 cells and purified by differential-gradient centrifugation. Since the virus was highly infectious in cell culture and Western blots of detergent-inactivated HIV showed envelope proteins when exposed to sera containing anti-HIV antibodies, this viral preparation was not deficient in 'spike' or 'knob' particles. Electron spin resonance (ESR) studies of intact HIV labeled with 5-nitroxide stearate (5-NS) indicated that a temperature-dependent lipid phase separation occurs with a high onset at approx. 42 degrees C and a low onset at approx. 15 degrees C. Cooling below 42 degrees C induces 5-NS clustering. Similar phase separations with high onsets at approx. 37-38 degrees C were previously identified in 5-NS labeled human erythrocytes (cholesterol/phospholipid (C/P) molar ratio = 0.90) and cholesterol-loaded (C/P = 0.85-0.98) rat liver plasma membranes. These were attributed to a temperature-sensitive redistribution of endogenous lipid components such that 5-NS is excluded from cholesterol-rich domains and tends to reside in cholesterol-poor domains at low temperatures. Since HIV has a lipid envelope with a similarly high C/P of 0.88 (Aloia et al. (1988) Proc. Natl. Acad. Sci. USA 85, 900-904), cholesterol-rich and cholesterol-poor domains also probably exist in HIV at physiologic temperatures. The reduced stability and infectivity of HIV noted on heating above 42 degrees C may be due, in part, to the abolition of these thermodependent domains.

Acquired Immunodeficiency Syndrome↗
PubMed1988

Lipid composition and fluidity of the human immunodeficiency virus.

Lipid analyses of the human immunodeficiency virus (HIV) propagated in Hut 78 cells indicated a low total lipid/protein ratio, a high cholesterol/phospholipid molar ratio, and major phospholipids consisting of phosphatidylcholine, phosphatidylethanolamine, sphingomyelin, and phosphatidylserine; comparable lipid profiles were noted for human erythrocytes and other RNA viruses. Electron spin resonance (ESR) studies of HIV labeled with 5-nitroxide stearate (N-oxy-4',4'-dimethyloxazolidine derivative of ketostearate) showed a low "fluidity" at 37 degrees C, similar to other enveloped RNA viruses and erythrocytes and probably due to the high cholesterol/phospholipid ratio. Ethanol (50%) completely disrupts the envelope, contributing to the rapid inactivation of HIV by ethanol. Contrarily, heating to 57 degrees C causes much less fluidization, and this heating may play a role in the slower viral inactivation at high temperatures. Should a critical minimum ordering in the HIV envelope be necessary for viral stability and infectivity, manipulating the lipid composition or fluidizing the HIV membrane, or both, may provide an untried therapeutic approach.

Cholesterol↗
PubMed1987

Estimation of spin probe clustering in biological membranes.

An iterative spectral subtraction technique has been developed which accurately estimates the proportion of 'dilute' and 'clustered' I(12, 3) (i.e., 5-nitroxide stearate) in human erythrocyte ghosts at 37 degrees C, even if subtractant spectra free from probe-probe interactions cannot be measured due to technical limitations. Gordon et al. ((1985) J. Membrane Biol. 84, 81-95) earlier showed that I(12, 3) occupies a class of high-affinity sites in ghosts at probe/total lipid ratios (P/L) less than 1/2250. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. Although this model allows determination of the dilute/clustered probe ratio, it requires subtraction of experimental spectra with a 'magnetically dilute' spectrum obtained using P/L less than 1/4600. The new methodology accurately profiles the % probe clustering in human erythrocyte ghosts over the entire P/L range, even if the lowest P/L for the subtractant spectrum contains substantial probe-probe interactions (i.e., P/L of 1/604 or 1/303). Application of either the subtraction technique in Gordon et al. (1985) or the iterative subtraction protocol described here should allow determination of probe clustering in a wide range of I(12, 3)-labeled biological membranes.

Cyclic N-Oxides↗
PubMed1987

Electron spin resonance spectroscopy in the study of lymphoid cell receptors.

ESR spectroscopy is a sensitive method which can be used to monitor a wide range of changes in and near the plasma membranes of lymphoid cells during such events as ligand-induced receptor patching and capping, activation and endocytosis and phagocytosis. The advantages of the technique are rapidity, sensitivity, small sample size (10(6)-10(7) cells), and nondestructive nature. Spin labels that are attached to a range of intrinsic and extrinsic molecules give information about the fluidity and polarity of the environment in which they are located. Because of the occurrence of interaction when probes are sequestered in restricted regions of the membrane, ESR spectroscopy is also a valuable technique for measuring the formation of domains in the cell membrane.

Cell Membrane↗
PubMed1987

ESR spectroscopy in the study of antigen processing--uptake of spin-labelled antigens by macrophages.

Macrophages were briefly pulsed with a spin-labelled synthetic polypeptide, poly(L-tyrosine:L-glutamic acid) poly DL-alanine:poly L-lysine (n-TGAL) in the presence and absence of anti-TGAL-antibody, and the electron spin resonance (ESR) spectra of the cell suspension compared with the spectrum of free n-TGAL in solution. Spectral analysis indicated two cell-associated n-TGAL pools, one composed of freely rotating label held in an aqueous environment, susceptible to protease digestion and ascorbate reduction, and a second highly concentrated pool, sequestered intracellularly, and held within a highly ordered, polar microenvironment. The ESR analyses were completed within minutes of antigen pulsing, employed very small numbers of live cells, and did not damage the cells being tested. The utility of the technique in screening fatty acid-antigen conjugates for macrophage uptake was demonstrated.

Animals↗
PubMed1985

Spin probe clustering in human erythrocyte ghosts.

A model has been developed for 5-nitroxide stearate, I(12,3), distribution in human erythrocyte ghosts which accurately predicts ESR spectral alterations observed with increased probe/total lipid (P/L) at 37 degrees C. This spin probe occupies a class of high-affinity, noninteracting sites at low loading. Saturation occurs with increasing probe concentration, and, at higher loading, the probe inserts itself at initially dilute sites to form membrane-bound clusters of variable size. No 'low' probe remains at high P/L where all I(12,3) clusters in a 'concentrated' phase. This model allows determination of the dilute/clustered probe ratio, and shows that I(12,3) segregates in erythrocytes at what might otherwise be considered low P/L (e.g., 1/359). These findings validate the earlier use of empirical parameters to estimate probe sequestration in biological membranes.

Cyclic N-Oxides↗
PubMed1985

A spin label study of the ionic strength dependent conformational change in the human Ia molecule.

Purified human Ia molecules were labelled with maleimide or isothiocyanate spin labels or by reacting "TEMPAMINE" spin label with the neuraminic acid of their carbohydrate residues. It was found that increasing the ionic strength from 0.05 to 0.75 markedly increased the dipolar interaction between the maleimide-attached labels, but no effect was found of ionic strength or motion on dipolar interaction with the other two labels. The effect of increasing ionic strength could be blocked by the prior addition of Ia-specific antibody, but could not be reversed by the addition of antibody after ionic strength was increased. These findings complement an earlier finding that increasing ionic strength over the range 0.05-0.75 has an inhibitory effect on the combination of Ia with its antibody. Because the maleimide spin labels attach predominantly to SH groups it is suggested that increasing ionic strength causes conformational changes in the immunoglobulin loop region which alter the accessibility of the Ia antibody-binding site.

Antibody Specificity↗
PubMed1979

Lymphocyte surface modulation and glycosphingolipids.

Using the fluorescent antibody method, identical localization of ligand and glycosphingolipids was found in human peripheral blood B lymphocytes patched and capped with polyvalent anti-immunoglobulin. cAMP also showed the same pattern of localization. It is suggested that glycosphingolipids are hydrogen bonded to receptor-bearing membrane macromolecules and are borne with them into the ligand-induced patches and caps. Here the asymmetric carbon chains of the glycosphingolipids modify the local lipid environment, leading to activation of the adenyl cyclase in the membrane which produces the cAMP.

Antibodies, Anti-Idiotypic↗
PubMed1978

Changes in lipid ordering and state of aggregation in lymphocyte plasma membranes after exposure to mitogens.

An electron spin probe study was made of the effect of a number of mitogenic agents on the ordering and state of aggregation of the plasma membrane lipids of lymphocytes. These agents, which included phytohemagglutinin, Concanavalin A, the calcium ionophore A23187 and periodate, caused a 20% decrease in lipid ordering in the region of the bilayer probed by 5-nitroxide stearic acid. The corresponding methyl ester probe showed marked probe-probe interaction under the same conditions indicating an aggregation of lipids in the area probed by this label. Studies with mixed lipid vesicles and ganglioside-free cells indicate that these areas are rich in glycolipids capable of hydrogen bonding to the ester probe. The decrease in ordering and the increase in aggregation of the membrane lipids were correlated with the patching and capping of the ligand-receptor complexes. Furthermore, the disappearance of fluorescent ligand from the surface of treated cells corresponded with the return of the spectral parameters of the probes to control cell values. It was concluded that glycolipids might play an important role in ligand-induced cell surface changes either as bearers of receptor groups, as in the case of some gangliosides, or in association by hydrogen-bonding with receptor proteins.

Animals↗
PubMed1977

Proteins retained with hyaluronic acid during ultrafiltration of synovial fluid.

The identity of the proteins associated with hyaluronic acid after ultrafiltration of bovine synovial fluid was examined. With discontinuous filtration, alpha2 macro- and IgM globulins were retained in hyaluronic complex both from synovial fluid and from a similar mixture of hyaluronic acid and serum. With continuous filtration and stirring to prevent formation of gels, protein was retained with hyaluronic acid in similar proportions. This protein showed immunologic identity with serum albumin, but differed in amino acid composition. After comparison with results of gel filtration, it was concluded that the identity of the retained proteins can be determined by the concentration of hyaluronic acid without variation in other ionic conditions.

Albumins↗
PubMed1976

The binding of dehydroheliotridine to DNA and the effect of it and other compounds on repair synthesis in main and satellite band DNA.

This study was aimed at elucidating the mechanisms of the preferential depression of satellite DNA synthesis by dehydroheliotridine (DHH). DHH was found to induce repair synthesis to the same extent in both main and satellite band DNA in cultured sheep lymphocytes. This was also the case with acridine orange, nitrogen mustard (HN2) and ethyl methane-sulphonate (EMS). Using analytical equilibrium ultracentrifugation no difference was found between the extents of in vitro binding of DHH by main and satellite band DNA. From these results it was concluded that the depression of the synthesis of satellite DNA could not be explained by either its preferential binding of DHH or by less effective repair mechanisms. Radiolabelled DHH when added to synchronized cultures of ovine kidney cells was found to be preferentially bound to the satellite DNA (one DHH molecule to 6000 nucleotides) compared with the main band DNA (one to 10 000). When 5-bromodeoxyuridine (BUdR) was added to the cultures no DHH label was found in the heavy, semiconservatively replicated DNA band. From these findings it is suggested that attack may occur during mitosis where all of the satellite DNA may be undergoing synthesis at the same time, thus explaining the increased amount of DHH bound to the satellite.

Animals↗
PubMed1975

Decreased synthesis of hepatic satellite DNA in pyrrolizidine alkaloidosis of the sheep.

the proportion of heavy satellite in the DNA isolated from the livers of sheep suffering from experimental pyrrolizidine alkaloidosis is significantly lower (3.5%) than that found in the DNA from lovers of normal sheep (12%). Dehydroheliotridine, the major unbound, relatively stable metabolite of lasiocarpine and heliotrine, the alkaloids used in the study, was found to inhibit selectively the semiconservative replication of the satellite DNA in cultures of ovine kidney cells. It is suggested that the inhibition of the synthesis of satellite DNA may be related to an attack by the metabolite on the pericentromeric region where the majority of the satellite sequences are located.

Animals↗