Biomedical subjects

M A Ferguson

Publications and source records attributed to M A Ferguson.

177 records · Page 10Linked to original sources

PubMed1987

Direct measurement of inositol in bovine myelin basic protein.

Myelin basic protein has been isolated from bovine central-nervous-system myelin by four methods, none of which exposes the protein to acid. After purification the inositol content of both hydrolysed and unhydrolysed protein was quantified by g.c.-m.s. Basic protein prepared by all methods contained less than 4 mol % of inositol. It is concluded, contrary to a previous proposal, that covalent binding to phosphoinositides does not represent a general mechanism for attachment of this cytoplasmically-oriented protein to its membrane.

Animals↗

PubMed1987

Identification of phosphorylated 3-deoxy-manno-octulosonic acid as a component of Haemophilus influenzae lipopolysaccharide.

A phosphorylated 3-deoxy-manno-octulosonic acid (KDO) was released from the lipopolysaccharide (LPS) of the deep rough mutant (Rb+169) of Haemophilus influenzae by acid hydrolysis. Both phosphorylated and dephosphorylated KDO, produced by treatment with alkaline phosphatase, were identified by gas chromatography-mass spectrometry after trimethylsilylation. This technique provides a rapid and reliable method for the identification of phosphorylated KDO in LPS.

Alkaline Phosphatase↗

PubMed1986

Purification and characterization of a novel glycan-phosphatidylinositol-specific phospholipase C from Trypanosoma brucei.

A novel membrane-bound glycan-phosphatidylinositol-specific phospholipase C, which catalyzes the conversion of membrane form variant surface glycoproteins to soluble variant surface glycoproteins, with the release of sn-1,2-dimyristylglycerol, has been isolated from Trypanosoma brucei. The activity was solubilized from trypanosome membrane fractions in non-ionic detergent and purified by anion exchange chromatography on DEAE-cellulose followed by chromatography on phosphatidylinositol-Sepharose. The enzyme constitutes about 0.1% of the total cellular protein and has an apparent molecular weight of 39,800. The enzyme shows a head group specificity for molecules containing carbohydrate covalently linked to glycan-phosphatidylinositol, but can also act on the monoacyl derivative of membrane form variant surface glycoprotein. It shows no specific ion requirements but is stimulated by thiol-reducing agents and inhibited by ions that thiols chelate.

Animals↗

PubMed1986

Trypanosoma brucei brucei variant surface glycoprotein contains non-N-acetylated glucosamine.

The C-terminal amino acid of the variant surface glycoprotein of the parasitic protozoan Trypanosoma brucei brucei is glycosylated and the oligosaccharide has been shown to contain glucosamine. By acid hydrolysis, HNO2 deamination and 1H-n.m.r. studies we have demonstrated that the amino group of this glucosamine is not N-acetylated and is most probably unmodified.

Acetylation↗

PubMed1986

Biosynthesis of Trypanosoma brucei variant surface glycoproteins. N-glycosylation and addition of a phosphatidylinositol membrane anchor.

The variant surface glycoproteins (VSGs) of Trypanosoma brucei are synthesized with a hydrophobic COOH-terminal peptide that is cleaved and replaced by a glycophospholipid, which anchors VSG to the surface membrane. The kinetics of VSG processing were studied by metabolic labeling with [35S]methionine and [3H]myristic acid. The COOH-terminal oligosaccharide-containing structure remaining after phospholipase removal of dimyristyl glycerol from membrane-form VSG could be detected serologically within 1 min of polypeptide synthesis in two T. brucei variants studied. Addition of the oligosaccharide-containing structure was resistant to tunicamycin. VSGs synthesized in the presence of tunicamycin displayed lower apparent molecular weights, consistent with the complete inhibition of N-glycosylation at one (variant 117), two (variant 221), or at least three (variant 118) internal asparagine sites. In most experiments, N-glycosylation appeared to occur during or immediately after polypeptide synthesis but in a few cases N-glycosylation was delayed or incomplete. In all cases, addition of the COOH-terminal oligosaccharide-containing structure occurred normally. In dual-labeling studies, cycloheximide caused rapid inhibition of both [35S]methionine and [3H]myristic acid incorporation, suggesting that myristic acid addition also occurs immediately after polypeptide synthesis. Our data suggest that the complex ethanolamine-glycosyl-dimyristylphosphatidylinositol structure of membrane-form VSG is added en bloc within 1 min of completion of the polypeptide.

Animals↗

PubMed1985

Glycosyl-sn-1,2-dimyristylphosphatidylinositol is covalently linked to Trypanosoma brucei variant surface glycoprotein.

The COOH terminus of the externally disposed variant surface glycoprotein (VSG) of the eukaryotic pathogenic protozoan Trypanosoma brucei strain 427 variant MITat 1.4 (117) is covalently linked to a novel phosphatidylinositol-containing glycolipid. This conclusion is supported by analysis of the products of nitrous acid deamination or Staphylococcus aureus phosphatidylinositol-specific phospholipase C treatment of purified membrane-form VSG. Lysis of trypanosomes is accompanied by release of soluble VSG, catalyzed by activation of an endogenous phospholipase C. The only apparent difference between membrane-form VSG and soluble VSG is the removal of sn-1,2-dimyristylglycerol. The COOH-terminal glycopeptide derived by Pronase digestion of soluble VSG was characterized by chemical modification and digestion with alkaline phosphatase. The results are consistent with the single non-N-acetylated glucosamine residue being the reducing terminus of the oligosaccharide and in a glycosidic linkage to a myo-inositol monophosphate that is probably myo-inositol 1,2-cyclic monophosphate. A partial structure for the VSG COOH-terminal moiety is presented. This structure represents a new type of eukaryotic post-translational protein modification and membrane anchor. We discuss the relevance of this structure to observations that have been made with other eukaryotic membrane proteins.

Animals↗

PubMed1985

Cysteine eliminates the feeder cell requirement for cultivation of Trypanosoma brucei bloodstream forms in vitro.

In all previous studies, bloodstream forms of Trypanosoma brucei could be grown in vitro only when supported by a feeder layer of mammalian fibroblasts. We have axenically cultivated bloodstream T. brucei by adding L-cysteine at regular intervals and appropriate concentrations. The optimum cysteine concentration depends on cell density and is close to physiological serum levels. At concentrations greater than 24 mg/liter (2 X 10(-4) M), cysteine was acutely toxic to trypanosome concentrations of 3 X 10(7)/ml. Toxicity was prevented by addition of pyruvate or catalase, which neutralize H2O2 produced by cysteine autoxidation. In uptake studies using [35S]cysteine and [35S]cystine, T. brucei efficiently incorporated only cysteine. The Km for cysteine uptake was 4 X 10(-4) M. Cystine supported axenic growth if low concentrations of 2-mercaptoethanol were added at regular intervals.

Animals↗

PubMed1985

Comparative compositions of cell surface glycoconjugates isolated from Trypanosoma cruzi epimastigotes.

Cell surface glycoconjugates of epimastigotes of Trypanosoma cruzi have been isolated and analyzed to give their amino acid and carbohydrate compositions. Those which have been investigated are a complex of three closely associated glycoproteins, GP24, GP31, GP37, and a lipopeptidophosphoglycan. The GP24-GP31-GP37 complex has an unusual amino acid composition with very low levels of hydrophobic amino acids, it contains 56% (w/w) carbohydrate, with mannose, galactose and glucosamine (presumably N-acetyl) being present in approximately equal quantities. The lipopeptidophosphoglycan also has low levels of hydrophobic amino acids and contains equal levels of mannose and galactose together with lesser amounts of (N-acetyl) glucosamine. The glycoconjugates are contrasted and compared with two other previously characterised cell surface glycoproteins (GP25 and GP72) from T. cruzi.

Amino Acids↗

PubMed1985

Acylation of a Plasmodium falciparum merozoite surface antigen via sn-1,2-diacyl glycerol.

The 195-kDa merozoite protein synthesized in schizonts of Plasmodium falciparum (Holder, A. A., and Freeman, R. R. (1982) J. Exp. Med. 156, 1528-1538) contains ester-linked fatty acid. Enzymatic treatment of the purified acylated protein established that the lipid is present as sn-1,2-diacyl glycerol, most probably linked to a phosphodiester at the 3-position of glycerol. The phosphodiglyceride is not directly esterified to an amino acid residue on the polypeptide backbone. The 195-kDa protein is processed to three fragments (83, 42, and 19 kDa) on the surface of free merozoites (Holder, A. A., and Freeman, R. R. (1984) J. Exp. Med. 160, 624-629), of which only the 42-kDa polypeptide is acylated.

Animals↗

PubMed1985

Trypanosoma brucei variant surface glycoprotein has a sn-1,2-dimyristyl glycerol membrane anchor at its COOH terminus.

The membrane form of Trypanosoma brucei variant surface glycoprotein (mfVSG) is acylated with ester-linked tetradecanoic (myristic) acid (Ferguson, M. A. J., and Cross, G. A. M. (1984) J. Biol. Chem. 259, 3011-3015). Comparative analysis of Pronase peptides from mfVSG and soluble VSG localizes the site of mfVSG acylation to a COOH-terminal oligosaccharide structure. Chemical and enzymatic treatment of the acylated Pronase mfVSG fragment revealed that the myristic acid is present as a diglyceride (sn-1,2-dimyristin) that is probably linked to the COOH-terminal oligosaccharide via a phosphodiester bond between the sn-3-glycerol hydroxyl and a sugar hydroxyl group. The endogenous membrane-associated enzyme, which quantitatively cleaves myristic acid from mfVSG to produce soluble VSG, releases diglyceride, as would be expected of a phospholipase C.

Acylation↗

PubMed1984

Myristylation of the membrane form of a Trypanosoma brucei variant surface glycoprotein.

A variant surface glycoprotein (VSG) of the parasitic protozoan Trypanosoma brucei was purified following the direct solubilization of trypanosomes in a boiling detergent solution. This material behaved as the amphiphilic membrane form of the glycoprotein previously described (Cardoso de Almeida, M. L., and Turner, M. J. (1983) Nature (Lond.) 302, 349-352). Analysis of this material showed that it contained ester-linked tetradecanoic (myristic) acid. After biosynthetic labeling of trypanosomes with [3H]myristic acid, it was shown that the release of the VSG coat from the parasite membrane, as the soluble form of the VSG, occurred concomitantly with the loss of myristic acid from the glycoprotein. The results suggest that VSG is attached to the parasite membrane via a covalently linked myristic acid-containing lipid moiety.

Animals↗

PubMed1983

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.

Acetylglucosamine↗

PubMed1982

The detection of phosphonolipids in the protozoan Trypanosoma cruzi.

2-Aminoethylphosphonate was detected in the acid hydrolysates of the phosphonolipids and the lipopeptidophosphoglycan of Trypanosoma cruzi, the causative agent of Chagas' disease. This finding represents the first evidence of phosphonolipids in a zooflagellate. By comparison, no phosphonolipids were detected in Trypanosama brucei, indicating that phosphonolipids are not a ubiquitous feature of the Order Kinetoplastidia.

Animals↗

PubMed1981

Cell surface antigens of Trypanosoma cruzi: use of monoclonal antibodies to identify and isolate an epimastigote specific glycoprotein.

A monoclonal antibody was produced by cell fusion from mice immunized with Trypanosoma cruzi epimastigotes. The antibody was epimastigote specific but not strain specific; the antibody bound to Y, Peru and Tulahuén epimastigotes but did not bind to Y amastigotes or trypomastigotes. The antigen recognised by the monoclonal antibody was a 72 000 molecular weight cell surface glycoprotein which represented only 0.04% of the whole cell protein. Analysis of the glycoprotein purified by antibody affinity chromatography revealed a carbohydrate content of 52% by weight which was composed of glucosamine, mannose, galactose, glucose and three different pentoses: fucose, xylose and ribose. Immunisation with the purified glycoprotein did not protect mice from a lethal infection of T. cruzi.

Animals↗

PubMedDate not supplied

Transient-evoked otoacoustic emissions in a representative population sample aged 18 to 25 years.

Transient-evoked otoacoustic emissions (TEOAEs) were obtained from 688 ears of a group of 345 young adults aged 18 to 25 years, using the Otodynamics ILO88 in the standard, non-linear mode. Normative data for TEOAEs obtained from 186 otologically normal (ON) ears are presented. In 5 ON ears, there was no recordable response, despite hearing threshold levels better than 20 dB. The main factors affecting the TEOAE level were (1) gender, where females had larger responses on average than males; (2) tympanometric measures, where ears with entirely normal tympanometric measures had larger responses than those with minor tympanometric abnormalities; (3) click stimulus intensity measured in the ear canal, which correlated positively with TEOAE level; and (4) hearing threshold level at 0.5 kHz, which correlated negatively with amplitude. There was also a small effect of social noise exposure in the 2-kHz region of the TEOAE, where the response was lower in those subjects exposed to significant social noise.

Acoustic Impedance Tests↗