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Biomedical subjects

J T Buckley

Publications and source records attributed to J T Buckley.

At least 91 records · Page 5Linked to original sources
PubMed1983

Purification and reconstitution in lipid bilayer membranes of an outer membrane, pore-forming protein of Aeromonas salmonicida.

We have purified a major outer membrane protein from Aeromonas salmonicida. This 42-kilodalton protein shared several physical characteristics with enterobacterial porins in that it was noncovalently associated with the peptidoglycan, it was released from the peptidoglycan in the presence of 0.1 M NaCl and sodium dodecyl sulfate, and its mobility on sodium dodecyl sulfate-polyacrylamide gels was dependent on the solubilization temperature before electrophoresis. When added to the aqueous solution bathing a planar bilayer membrane it caused the conductance of the membrane to increase by several orders of magnitude. At lower protein concentrations, single channels with an average conductance of 1.6 nS in 1 M KCl were incorporated into the membrane in a stepwise fashion. Evidence that the protein formed a large, relatively nonselective, water-filled channel was obtained by performing single-channel experiments at different NaCl concentrations and in a variety of different salts. Current through the channel was a linear function of the applied voltage, and no evidence of voltage gating was observed. In addition, we obtained evidence for a 43-kilodalton channel-forming protein in the outer membrane of A. hydrophila with a similar single-channel conductance as the 42-kilodalton protein in 1 M NaCl.

Aeromonas↗
PubMed1982

Substrate specificity of bacterial glycerophospholipid:cholesterol acyltransferase.

The substrate specificity of a bacterial analogue of the plasma enzyme lecithin:cholesterol acyltransferase (LCAT) has been examined with small unilamellar liposomes and Triton mixed micelles. In contrast to LCAT, the microbial enzyme is capable of using all of the naturally occurring phospholipids as acyl donors. In general reaction rate depends more on the length or degree of unsaturation of the acyl chains than on the nature of the phospholipid head group. Among a series of disaturated phosphatidylcholines in liposomes, dilauroylphosphatidylcholine is the preferred acyl donor. Like LCAT, the enzyme will catalyze acyl transfer by using other alcohols in addition to cholesterol. Of saturated straight chain primary alcohols 1-decanol is the preferred acyl acceptor. Cholesterol, however, is a far better acceptor than any non-sterol alcohol tested. Other steroids with equatorial hydroxyls at position C-3 and trans-fused A:B rings will also act as acceptors whereas those steroids with axial hydroxyls at C-3 or cis-fused rings are inhibitors of acyl transfer. The ability of steroids to act as acyl acceptors may be due to the nature of their interaction with the phospholipid acyl donor.

Acyltransferases↗
PubMed1982

Membrane glycoprotein receptor and hole-forming properties of a cytolytic protein toxin.

Aerolysin, a cytolytic bacterial exotoxin, was radioiodinated by using the Iodogen reagent. Binding of the labeled toxin to rat erythrocytes was inhibited by the native protein and by anti-aerolysin antibody. Toxin, once bound, was not removed by the addition of a large excess of free aerolysin. Binding of the radioactive toxin to erythrocytes of different species paralleled the hemolytic specificity of the unlabeled toxin. Pretreatment of the rat erythrocytes with trypsin, which removed a major membrane glycoprotein, resulted in a dramatic decrease in binding, whereas chymotrypsin treatment had no effect. Binding was inhibited by a glycoprotein fraction isolated from these cells but not by a total rat erythrocyte glycolipid preparation. Aerolysin caused the formation of holes in erythrocytes which were sized by measuring the release of labeled molecular weight markers. Glucagon (molecular weight 3550) and smaller molecules entrapped in human or rat erythrocytes were released by treatment with aerolysin, whereas methoxyinulin (molecular weight 5500) and larger molecules were not. Aerolysin also caused the release of glucose from large unilamellar lipid vesicles. The results indicate that a specific glycoprotein receptor facilitates the interaction of aerolysin with erythrocyte membranes. Binding is followed by the formation of discrete holes or pores, and this results in cell rupture by a colloid-osmotic process.

Aeromonas↗
PubMed1982

Purification and partial characterization of a bacterial phospholipid: cholesterol acyltransferase.

A glycerophospholipid:cholesterol acyltransferase has been purified to near homogeneity from cell-free culture supernatants of Aeromonas salmonicida. The characteristics of the enzyme distinguish it from bacterial phospholipases; however, it shares several properties with the lecithin:cholesterol acyltransferase of mammalian plasma. Thus, the enzyme inhibits 2-positional specificity as an acyltransferase and it will act as a phospholipase A2 in the absence of cholesterol. Furthermore, it has no divalent cation requirement and it is stimulated both by albumin and by human apolipoprotein A-I. Unlike the mammalian acyltransferase, however, the bacterial enzyme is not specific for phosphatidylcholine and in addition it can use human erythrocyte membranes as substrates. Similar to Naja naja phospholipase A2, it acts asymmetrically on intact erythrocytes.

Acyltransferases↗
PubMed1982

Incorporation and turnover of essential fatty acids in phospholipids and neutral lipids of rainbow trout.

1. The turnover of rainbow trout (Salmo gairdneri) lipid was monitored in fish receiving essential fatty acids (EFA) replete and EFA deficient diets using [1-14C]-linolenic acid (C18:3 omega 3). 2. Forty percent of administered radioactivity was recovered in total lipid after 7 days. 3. Eighty-five percent of incorporated label was recovered in C18:3 omega 3 derivatives, primarily docosa-hexaenoic acid (22:6 omega 3). 4. Over 10 weeks, 14C activity in neutral lipid (NL) remained constant whereas that in phospholipid (PL) declined due to loss of label from shorter chain omega 3 fatty acids. The mass and specific activity of longer chain omega 3 fatty acids remained constant. 5. There was an accumulation of 14C activity and C22:6 omega 3 in PE at the expense of PC. 6. The pattern of incorporation of 32P inorganic phosphate into molecular classes of phosphatidylcholine (PC) of EFA deficient fish indicated that the rate of turnover of docosahexaenoyl PC was not diminished by EFA deficiency. 7. It is hypothesized that retention of C22:6 omega 3 in EFA deficiency is due to the presence of an efficient recycling system during membrane turnover.

Animals↗
PubMed1982

Chronic exposure of coho salmon to sublethal concentrations of copper.--I. Effect on growth, on accumulation and distribution of copper, and on copper tolerance.

1. Coho salmon, exposed to sublethal levels of aqueous copper (1/4 and 1/2 LC50), lost appetite and ceased growing or showed decreased rates of growth. 2. Recovery of appetite and growth rate was faster in fish exposed to 1/4 of the LC50 than in those exposed to 1/2 of the LC50. 3. Copper levels were elevated in liver gill and kidney of exposed fish with the liver tissue accumulating a much larger amount of the metal than any other tissue. 4. The concentration of liver copper became constant at about the time that growth rate recovered. 5. The exposed fish exhibited much higher resistance to elevated aqueous copper levels than did the controls. 6. The results suggest that coho salmon may become acclimated to higher levels of copper and that acclimated fish are more tolerant to copper than control animals.

Animals↗
PubMed1982

Chronic exposure of coho salmon to sublethal concentrations of copper--II. Distribution of copper between high- and low-molecular-weight proteins in liver cytosol and the possible role of metallothionein in detoxification.

1. Liver cytosol samples from juvenile coho salmon exposed over a 14-week period to 70 micrograms/l (1/4 LC50) or 140 micrograms/l (1/2 LC50), were separated into high-molecular-weight and low-molecular-weight fractions by column chromatography on Sephadex G-75, and compared to similar samples obtained from control fish. 2. The levels of copper in the low-molecular-weight function of fish exposed to 70 micrograms/l were not significantly increased over control values for the first 6 weeks but then increased rapidly, while those of fish exposed to 140 micrograms/l increased substantially in the first 2-4 weeks of exposure, then levelled off. 3. In the exposed fish the levels of copper in the high-molecular-weight fraction were not significantly elevated above control levels over the first 8-10 weeks but then increased significantly. 4. The low-molecular-weight fractions of exposed fish were shown to contain increasing levels of metallothionein. The metallothionein was isolated on DEAE-cellulose and characterized by amino acid analyses.

Animals↗
PubMed1981

Purification and disposition of a surface protein associated with virulence of Aeromonas salmonicida.

Virulent strains of Aeromonas salmonicida observed by electron microscopy were characterized by an outer layer exhibiting a tetragonal repeat pattern. Attenuated strains had a 2.5 X 10(3)- to 5 X 10(3)-fold reduction in virulence and lost the outer layer, autoaggregating properties, and a 49-kilodalton protein (A protein) simultaneously. The A protein is the major protein component of outer membrane fractions of virulent strains. A variety of radiolabeling studies showed that this protein was surface localized and that it provided an effective barrier against iodination of other outer membrane proteins with either lactoperoxidase or diazoiodosulfanilic acid; A protein was not labeled with lactoperoxidase but was specifically labeled with diazoidosulfanilic acid. The A protein was purified by selective extraction with detergent and guanidine hydrochloride, and its amino acid composition was determined. The properties of A protein are compared with those of other bacterial surface layer proteins.

Aeromonas↗
PubMed1981

Loss of virulence during culture of Aeromonas salmonicida at high temperature.

The effect of growth temperature on the loss of virulence of the fish pathogen Aeromonas salmonicida was investigated. Three virulent strains were grown in Trypticase soy broth at temperatures ranging from 22 to 30 degrees C. Growth at a higher-than-optimal temperature (26 to 27 degrees C for the three strains studied) resulted in the selection of spontaneous attenuated derivatives in the initial bacterial population. For example, virulent bacteria represented less than 10% of the population of a culture grown at 30 degrees C, and attenuated derivatives were easily isolated by streaking the culture on solid medium and picking single colonies. Virulent strains autoaggregated during growth and possessed a cell wall layer (A-layer) external to the outer membrane, as previously described. Attenuated strains did not autoaggregate and did not possess the A-layer. The A-layer apparently shielded bacteriophage receptors and a mannose-specific yeast agglutinin located in the outer membrane. Thus, virulent strains exhibited impaired adsorption of phages, whereas attenuated strains were phage sensitive. Furthermore, attenuated strains agglutinated yeast cells but virulent strains did not. The attenuated strains had higher maximum growth temperatures than their virulent parent strains, and this accounts for their selection at high temperatures. It is proposed that the A-layer contributes significantly to the physical properties of the A. salmonicida cell envelope and that these physical properties of the A. salmonicida cell envelope and that these physical change upon loss of the A-layer to permit growth at a higher-than-usual temperature.

Adsorption↗
PubMed1981

Purification and some properties of the hemolytic toxin aerolysin.

Aerolysin, the hemolytic toxin produced by Aeromonas hydrophila, has been purified by a combination of salt fractionation, gel filtration, and ion-exchange and hydroxyapatite chromatography. The resulting protein has a molecular weight of 51 500 and appears homogeneous by polyacrylamide gel electrophoresis in sodium dodecyl sulphate. It is free of detectable protease and phospholipase activities. The purified protein can be separated into two active components with pIs of 5.39 and 5.46 by isoelectric focusing. Both components are found in the original culture supernatant indicating that the multiplicity is not due to proteolysis during isolation. Purified aerolysin is unstable even at 25 degrees C and its hemolytic action is inhibited by certain reducing agents including ferrous iron and cysteine. It appears to be the only toxin hemolytic to human cells that is produced by A. hydrophila under the conditions described.

Aeromonas↗
PubMed1980

Distribution and mobility of omega 3 fatty acids in rainbow trout fed varying levels and types of dietary lipid.

The availability of essential fatty acids in fish neutral lipid to tissue phospholipids was determined under conditions of adequate and inadequate essential fatty acid intake as well as during fasting. Juvenile rainbow trout were fed a semi-purified diet containing varying levels of cod liver oil, with or without supplementary olein. Fatty acid analysis indicated that in all treatments the neutral lipid pool was not turned over during feeding but was enhanced by exogenous or endogenously synthesized fatty acids. Fish that received diets devoid of essential fatty acids maintained virtually all of the docosahexenoic acid originally present in each lipid pool. Fish fed diets containing essential fatty acids deposited them in proportion to the dietary levels. After a 4-week fast, no change was noted in the relative levels of fatty acids in neutral lipid indicating that all fatty acids in neutral lipid were catabolized equally--including essential fatty acids. During fasting there was a selective retention of docosahexenoic and linoleic acids in the phospholipid pool.

Animals↗
PubMed1980

Identification and characterization of outer membrane fragments released by Aeromonas sp.

Aeromonas hydrophila and Aeromonas salmonicida were shown to release "blebs" or fragments averaging 20-30 nm in diameter. The protein composition of the fragments was very similar to that of the corresponding outer membrane by sodium dodecyl sulphate--polyacrylamide gel electrophoresis and the fragments were shown to contain phospholipid and lipopolysaccharide. The results therefore indicate that the blebs are derived from the outer membrane of these organisms. Fragments isolated directly by differential ultracentrifugation were indistinguishable from fragments isolated by salt fractionation and gel filtration in chemical composition, protein composition, and density. However fragments isolated directly contained much less glycerophospholipid-cholesterol acyltransferase than those isolated by salt fractionation. The potential usefulness of membrane fragments to the bacteria and as a tool in the study of outer membrane structure and function is discussed.

Aeromonas↗
PubMed1979

Distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species.

The distribution of glycerophospholipid-cholesterol acyltransferase in selected bacterial species was examined. Enzyme activity was demonstrated in cell-free growth media from all members of the family Vibrionaceae which were tested except Plesiomonas shigelloides. In each case, enzyme was produced in exponential to early stationary phase and was excluded from Sepharose 6B, indicating a complex of high molecular weight. In a limited survey of other families, Stahylococcus aureus was the only organism outside the Vibrionaceae which was shown to produce the enzyme. In this case, however, the enzyme exhibited much less activity against erythrocyte membranes and appeared to have a lower molecular weight. The reasons for these differences and the importance of the acyltransferase as a biochemical identification tool are discussed.

Acyltransferases↗
PubMed1978

Presence of glycerophospholipid: cholesterol acyltransferase and phospholipase in culture supernatant of Aeromonas hydrophila.

Human erythrocyte membrane glycerophospholipids are deacylated by Aeromonas hydrophila 13-h culture supernatants, resulting in the production of cholesterol ester, free fatty acid, and water-soluble phosphates. This activity appears to be due to the actions of an acyltransferase (phosphatide:cholesterol acyltransferase, EC 2.3.1 group) and a phospholipase (phosphatide acyl-hydrolase). The enzyme activities are produced simultaneously in late exponential/early stationary phase, are precipitated together from the culture supernatant with 85% ammonium sulfate, and are eluted together near the void volume during gel filtration on Sepharose 6B. These results suggest that A. hydrophila produces a multienzyme complex with an unusual mode of action on membrane lipids. The complex is distinct from the hemolytic factor aerolysin, which is also produced by A. hydrophila.

Acyltransferases↗
PubMed1978

Coisolation of glycophorin A and polyphosphoinositides from human erythrocyte membranes.

The lipid composition of purified erythrocyte membrane glycophorin was measured. Diphosphoinositide, triphosphoinositide, and phosphatidylserine are the major phospholipids in glycophorin preparation. Nearly all of the radioactive diphosphoinositide and triphosphoinositide extracted from erythrocyte membranes by lithium dïodosalicylate are recoverd in purified glycophorin. There appeared to be no significant enrichment of other acidic membrane phospholipids in the protein. The results do not permit a firm conclusion as to whether the polyphosphoinositides are associated specifically with the membrane protein or whether fortuitous binding has occurred during purification.

Erythrocyte Membrane↗
PubMed1977

Properties of human erythrocyte phosphatidylinositol kinase and inhibition by adenosine, ADP and related compounds.

1. An improved assay for the enzyme phosphatidylinositol kinase is described. The kinase activity is increased more than 10-fold by the addition of mercaptoethanol and exogenous phosphatidylinositol in the presence of Triton X-100. The enzyme is solubilized by non-ionic detergents. 2. Phosphatidylinositol kinase is inhibited by physiological concentrations of ADP and by similar levels of adenosine. Cyclic AMP, AMP and theophylline inhibit at higher concentrations. Caffeine is much less effective than theophylline as an inhibitor. Guanine nucleotides do not appreciably inhibit the kinase. All the inhibitors tested seemed to compete with ATP. Theophylline also reduced the rate of polyphosphoinositide synthesis in intact cells. 3. Possible roles of polyphosphoinositides in energy charge maintenance and inversion and resealing are discussed.

Adenosine↗
PubMed1977

Effect of neomycin and ionophore A23189 on ATP levels and turnover of polyphosphoinositides in human erythrocytes.

The relationship of polyphosphoinositide metabolism to erythrocyte ATP levels was examined. Turnover of polyphosphoinositides was not closely dependent on ATP as it is reported to be in yeast. Neomycin increased 32P incorporation into diphosphoinositide and to a lesser extent into triphosphoinositide without affecting intracellular ATP. Treatment of intact cells with ionophore A23187 resulted in a decrease of at least 80% in polyphosphoinositide levels which followed a decrease in cellular ATP and an increase in phosphatidate. The results indicate that polyphosphoinositide turnover does not regulate energy charge in the erythrocyte. However some of the events which follow treatment of erythrocytes with metabolic inhibitors or calcium and ionophore may be related to the accompanying decrease in polyphosphoinositide levels.

Adenosine Triphosphate↗