Search PubMed⌕ Search

Biomedical subjects

A Mellors

Publications and source records attributed to A Mellors.

At least 55 records · Page 3Linked to original sources
PubMed1986

The phospholipase A1 of Trypanosoma brucei does not release myristate from the variant surface glycoprotein.

[3H]Myristoyl-labeled variant surface glycoprotein (VSG) has been isolated from Trypanosoma brucei by reverse phase high performance liquid chromatography and used as substrate for the conversion by trypanosomal enzymes of membrane-form VSG to soluble VSG. Conversion is detected by the release of myristoyl-containing lipids. The major lipolytic enzyme of T. brucei, phospholipase A1, is effective for the hydrolysis of myristoyl esters of p-nitrophenol, in a colorimetric assay. However, the phospholipase is unable to cleave the myristoyl ester linkage of VSG. The phospholipase can be separated from the myristoyl-releasing activity of trypanosome homogenate by centrifugation, affinity chromatography, and anion-exchange chromatography. Elution profiles on anion-exchange high performance liquid chromatography also indicate that the phospholipase is inactive against VSG. A small amount of myristoyl-releasing activity associated with the purified phospholipase is probably due to contamination with a phosphodiesterase which releases myristoyl-containing diglyceride from VSG.

Animals↗
PubMed1985

A tumor promoter enhances the phosphorylation of polyphosphoinositides while decreasing phosphatidylinositol labelling in lymphocytes.

12-O-Tetradecanoyl-phorbol-13-acetate, a comitogen for lymphocytes, suppresses the concanavalin A-induced accumulation of 3 2P-phosphatidyl-inositol, in mouse spleen lymphocytes incubated with 3 2P-orthophosphate. The comitogenic tumor promoter does not affect the rate of de novo synthesis of phosphatidylinositol, as measured by [3H]-glycerol incorporation. Mitogen stimulates the incorporation of [3 2P]-phosphate into phosphatidylinositol, phosphatidylinositol-4-phosphate and phosphatidylinositol-4,5-bisphosphate, but the tumor promoter suppresses only the increased labelling of the phosphatidylinositol, while it enhances phosphorylation of the polyphosphoinositides.

Animals↗
PubMed1985

Co-mitogenic tumor promoters suppress the phosphatidylinositol response in lymphocytes during early mitogenesis.

The tumor-promoting agents 12-O-tetradecanoyl-phorbol-13-acetate (TPA) and phorbol-12,13-dibenzoate inhibited the increased accumulation of [32P]phosphatidylinositol (PI) induced in mouse spleen lymphocytes by mitogenic lectins in the presence of [32P]orthophosphate. Similar inhibition of [32P]PI levels by TPA was seen in human tonsil T-lymphocytes stimulated with phytohemagglutinin. Only co-mitogenic phorbol esters prevented the [32P]PI accumulation during early mitogenesis. No increased 32P-labelling due to mitogen or decreases due to TPA was observed when cells were equilibrated with [32P]orthophosphate for 24 h prior to stimulation with mitogen, from which it is concluded that the total concentrations of phosphatidylcholine (PC) and PI are unaffected by mitogen or co-mitogen. The [32P]PI elevation but not the [32P]PC elevation was proportional to T-cell mitogenic potency for the lectins concanavalin A, divalent succinyl concanavalin A and phytohemagglutinin, and was prevented in each case by 5 X 10(-8) M TPA. Escherichia coli lipopolysaccharide did not give increased 32P incorporation into PI or PC, and TPA had no effect on 32P labelled phospholipid levels in the presence of this B-cell mitogen. The results indicate that the phosphatidylinositol response is not an invariable correlate of T-cell mitogenesis by polyclonal mitogens.

Animals↗
PubMed1985

Physical aspects of the inhibition of enzymes by hydrocarbons: the inhibition of alpha-chymotrypsin by chlorinated aromatics and alkanes.

The inhibition of alpha-chymotrypsin by a series of chlorinated hydrocarbons, including polychlorinated biphenyls, has been studied. The solubility of the hydrocarbons was determined by autocorrelation analysis of light scattering. Kinetic analysis indicated that inhibition of the enzyme occurs when 1-2 molecules of inhibitor bind per molecule of enzyme. Chlorinated aromatics including polychlorinated biphenyls were more potent than monochloroalkanes in inhibition of the enzyme. Considerable inhibition was seen when some compounds were present as micelles. Molar volume correlations suggest that chlorinated hydrocarbons exert effects on soluble enzymes similar to their effects on membrane-bound enzymes, and that a membrane lipid phase is not essential for this type of inhibition.

Chymotrypsin↗
PubMed1984

Isolation of phospholipase A1 from Trypanosoma brucei.

Phospholipase A1 from Trypanosoma brucei brucei has been purified 380-fold by column chromatography on phosphatidylcholine-Sepharose affinity columns followed by DEAE-cellulose anion-exchange chromatography and Sephacryl S-200 molecular exclusion chromatography. Octyl-Agarose hydrophobic column chromatography can be substituted for the PC-Sepharose column. The molecular weight of trypanosomal PLase A1 was found to be 26,000 by SDS-polyacrylamide gel electrophoresis.

Animals↗
PubMed1983

Mitogen effects on lipid metabolism during lymphocyte activation.

Mitogenic and non-mitogenic lectins have been compared for their abilities to stimulate acetate and choline incorporation into mouse spleen lymphocyte lipids. The concns required for maximal acetate incorporation correspond to those required for maximal blastogenesis. The non-mitogenic lectins tested had no effect on acetate incorporation. Concanavalin A also stimulated acetate incorporation into splenic lymphocyte lipids of athymic mice which do not undergo T-cell blastogenesis. 4,4'-Diaminodiphenylsulfone (dapsone) at 150 micrograms/ml inhibited the incorporation of choline into mouse spleen lymphocyte phosphatidylcholine by 40%, but had no significant effect on concanavalin A induced mitogenesis when the inhibitor was present during the first few hours of transformation. Enhanced turnover of phospholipids appears to be a parallel but non-essential event in the early stages of mitogenesis.

Acetates↗
PubMed1983

Proteolysis of sialoglycoprotein by Pasteurella haemolytica cytotoxic culture supernatant.

Proteolytic enzyme activity releasing sialo glycopeptides from 3H-labeled human erythrocyte ghosts was detected in cytotoxic (leukotoxic) culture supernatants from 9 of 12 Pasteurella haemolytica serotypes. Microcrystalline cellulose thin-layer chromatograms of radioactive water-soluble products showed the following two radioactive peaks: a high-mobility minor peak (Rf, 0.54 to 0.74), identified as sialic acid, and a low-mobility major peak (Rf, 0.18 to 0.21), partially characterized as a trichloroacetic acid-soluble, sialic acid-rich fragment with a molecular weight of greater than 3,500, not extractable by chloroform. The sialic acid content of this fragment after treatment with Clostridium perfringens neuraminidase was estimated to be 7.2 X 10(-2) mumol mg-1. The presence of neuraminidase as a separate activity in some culture supernatants was confirmed. It is considered to be responsible for the observed release of free sialic acid. Preliminary studies with the crude enzyme showed that it has a broad pH optimum around pH 7.0 and that activity is not affected by inhibitors of trypsin, chymotrypsin, thermolysin, thio and serine enzymes, nor by an inhibitor of neuraminidase, 2,3-dehydro-2-deoxy-N-acetylneuraminic acid. Activity was, however, inhibited by o-phenanthroline at a high concentration after prolonged treatment. The enzyme hydrolyzed glycophorin at a rate four times higher than the rate for casein. Free glycophorin inhibited the enzyme-induced release of radioactive products from 3H-labeled ghosts. It is speculated that the novel enzyme is a neutral protease, probably metal-dependent, with specificity for sialoglycopeptides. The possible relationship of this protease to the previously reported host species-specific leukotoxicity of P. haemolytica and its potential role in virulence is discussed.

Caseins↗
PubMed1981

A lysosomal enzyme involved in diphosphatidylglycerol degradation.

A soluble lysosomal phosphodiesterase in rat liver that hydrolyzes monoacylglycerophosphorylglycerophosphorylglycerol (AGPGPGase) was shown to be distinct from a lysosomal acid phosphodiesterase IV (PDase IV) which catalyzes the hydrolysis of bis(p-nitrophenyl) phosphate. The criteria used to distinguish lysosomal AGPGPGase from PDase IV were: separation on ion exchange celluloses, dissimilar inhibition patterns and different rates of inactivation on concentration. The lysosomal PDase IV activity was competitively inhibited by inorganic phosphate with a Ki value of 0.33 mM phosphate and was inhibited by a number of organophosphoryl compounds including AGPGPG, phosphatidylcholine, phosphatidylinositol, ATP and 4-methylumbelliferylpyrophosphate.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1981

The phospholipases of pathogenic and non-pathogenic Trypanosoma species.

Four species of trypanosome were examined for phospholipase activities using 1-[3H]palmitoyl-2-acyl-sn-glycero-3-phosphocholine and 1-acyl-2[14C]linoleoyl-sn-glycero-3-phosphocholine as substrates. The major activity in each species is a phospholipase A1 (EC 3.1.1.32) which does not require calcium. The most effective of the detergents tested for activation of the enzyme from each species, and the Ph optima, are as follows: Trypanosoma brucei, 0.125% Triton X-100 at pH 6.0-8.5; T. congolense, 0.5 mM linoleate at pH 6.0; T. theileri, 0.1% Triton X-100 at pH 6.75; T. lewisi, 0.2 mM sodium dodecyl sulfate at pH 5.2. The specific activity of the enzyme from a pathogenic species, T. brucei, is very high (145 nmol/min/mg/protein) and could contribute to the tissue damage characteristically caused by this parasite. The level in T. lewisi, a non-pathogenic species, is relatively low (1 nmol/min/mg). The levels in T. theileri (31 nmol/min/mg) and T. congolense (10 nmol/min/mg are intermediate. These results are compatible with the hypothesis that phospholipases contribute to the pathogenicity of trypanosomes.

Animals↗
PubMed1981

Lysophospholipase 1 in Trypanosoma brucei.

Protein fractions from Trypanosoma brucei brucei showed lysophospholipase 1 activity (E.E.3.1.1.5), against the substrate 1-acyl-sn-glycero-3-phosphocholine, and also phospholipase A1 activity (E.C.3.1.1.4) by hydrolysis of the 1-acyl bond of 1,2-diacyl-sn-glycero-3-phosphocholine. Both enzyme activities were eluted together and showed a 12-fold purification following Sephacryl S-200 column chromatography. A final 96-fold increase in activity was obtained by electrophoresis on nondenaturing polyacrylamide gels to yield a band containing both enzymic activities. Phospholipase A1 showed maximum activity between pH 6.0--8.5 and lysophospholipase 1 had a pH optimum of 8.5. Both activities were found mainly in the soluble fraction of disrupted trypanosomes and were similarly inhibited by N-ethylmaleimide and p-chloromercuribenzoic acid. Although Triton X-100 stimulated phospholipase A1 activity, it inhibited lysophospholipase 1 activity. The Km value for the lysophospholipase 1 was found to be 0.15 mM. It was not possible to resolve separate activities for lysophospholipase 1 and phospholipase A1 and the ratio of the two activities was approximately 1 : 10 for a variety of preparations and treatments. It is probable that a single enzyme displays both activities.

Chromatography↗
PubMed1980

Inhibition of acyltransferase in lymphocytes by concanavalin A.

The effects of concanavalin A and succinylated concanavalin A on the transformation of mouse splenic lymphocytes, and on early biochemical events in the transformation, were compared. 1. The transformation of lymphocytes is biphasic with respect to concanavalin A concentration with optimal activation at about 1 microgram/ml. Activation by succinyl concanavalin A is not biphasic over a range of lectin concentration of 1--16 microgram/ml. 2. In intact lymphocytes cultured for 4 h, the enzyme Acyl-CoA:1-acylglycero-3-phosphocholine O-acyltransferase (EC 2.3.1.23) was not activated by concanavalin A but was inhibited at all concentrations tested, and was about 60% inhibited at 16 micrograms concanavalin A per ml. Succinyl concanavalin A gave little or no inhibition at similar concentrations. 3. Lymphocytes become committed to divide while their acyltransferase activities are markedly inhibited by concanavalin A. 4. The inhibition of acyltransferase by concanavalin A can be lifted by displacing the lectin from the cells by alpha-methylmannoside. Lowered enzyme activity is not caused by cell agglutination or by direct cross-linking of lectin receptors. It is unlikely that the inhibition of acyltransferase is due to indirect cross-linking via the cytoskeleton since colchicine did not reverse the inhibition. 5. The inhibition of acyltransferase and the reduced stimulation of transformation by higher levels of concanavalin A appear to be due to hydrophobic interaction of the lectin with the plasma membrane, as shown by liposome aggregation studies.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1980

Accumulation of phospholipase A1 in tissue fluid of rabbits infected with Trypanosoma brucei.

Samples of tissue fluid were obtained from plastic cages implanted subcutaneously in rabbits. A phospholipase A1 similar to that found in Trypanosoma brucei appeared in the tissue fluid about seven days after infection with this parasite, increasing with parasite burden, and reaching levels of more than 5 nmol phosphatidylcholine hydrolysed per min per ml tissue fluid. The large amount of phospholipase A1 found free in the tissue fluid appeared to be of trypanosomal origin and was either secreted by living parasites or released from dying organisms. Phospholipase A1 was detectable in blood plasma from the infected rabbits, but at a level considerably lower than in the tissue fluid. An inhibitor of the phospholipase was present in the plasma after the first two week of infection which may be partly responsible for this lower level.

Animals↗
PubMed1980

Studies on the generation of biologically active substances by Trypanosoma theileri in vitro.

Trypanosoma theileri was grown in bovine primary spleen cell cultures. These organisms were tested for phospholipase activity which was found to be very low, for haemolytic activity which was very weak and for lymphocyte mitogenicity which was found to be absent. High concentrations of T theileri were found to activate bovine complement probably by both the alternate and classical pathways. There was no evidence that these organisms exerted any significant toxic effect in vitro or in vivo. This is compatible with the known low pathogenicity of this species.

Animals↗