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Biomedical subjects

G A Cross

Publications and source records attributed to G A Cross.

At least 109 records · Page 6Linked to original sources
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

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↗
PubMed1985

Variations in the organization of repetitive DNA sequences in the genomes of Plasmodium falciparum clones.

Repetitive DNA in cultured Plasmodium falciparum was examined by restriction digestion and transfer hybridization, using cloned repetitive DNA probes. The arrangement of repetitive DNA was unstable: after 6 months culture of a cloned population, variations could be detected. Significant differences can be seen between clones derived from a single isolate, and are even more marked between isolates of diverse geographical origin. No cross-species conservation was seen for the sequences examined, and no relationship was observed between their representation in the P. falciparum genome and the potential for sexual differentiation.

Animals↗
PubMed1985

Coordinate transcription of variant surface glycoprotein genes and an expression site associated gene family in Trypanosoma brucei.

Trypanosoma brucei variant surface glycoprotein (VSG) genes are activated either by duplicative (DA) transposition of the gene to a pre-activated expression site or by nonduplicative (NDA) activation of a previously silent telomeric gene. We have obtained a recombinant clone spanning the 5' barren region of the expression linked copy of the duplicated VSG gene 117a. By DNA sequence and hybridization analyses we have identified a pleomorphic family of 14-25 non-VSG genes that lie upstream of both DA and NDA VSG expression sites. These expression site associated genes (ESAGs) encode 1.2 kb poly(A)+ mRNAs that are specifically transcribed from the active VSG expression telomere in mammalian bloodstream stages of T. brucei but, in common with VSG genes, are not transcribed in procyclic culture forms. cDNA and genomic sequences predict open reading frames that are conserved in the two ESAGs examined.

Animals↗
PubMed1984

Structure of the variant glycoproteins and surface coat of Trypanosoma brucei.

The pathogenic African trypanosomes have a unique mechanism for antigenic variation. Each cell is covered by a surface coat consisting of about seven million essentially identical glycoprotein molecules drawn from a large repertoire of variants, each encoded by an individual gene. Amino acid sequence variation extends throughout the molecule but reduces from the amino terminus to the carboxy terminus, where certain features, especially the grouping of cysteine residues, are quite conserved. The range of diversity within the thousand or so variant glycoprotein genes that exist in each cell is large. New variants may arise instantaneously by segmental gene conversion. Variant surface glycoproteins are synthesized with amino terminal signal sequences and hydrophobic carboxy terminal tails. The tails are extraordinarily conserved. After synthesis, they are replaced by a complex glycolipid structure in which myristic (dodecanoic) acid serves to anchor the polypeptide to the surface membrane. Enzymic cleavage of myristic acid releases variant glycoproteins from the surface coat.

Amino Acid Sequence↗
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↗
PubMed1984

Release and purification of Trypanosoma brucei variant surface glycoprotein.

Conditions affecting the solubilization of variant surface glycoprotein (VSG) from Trypanosoma brucei have been investigated. The results obtained form the basis for a convenient and efficient method for VSG purification. VSG release from the cell surface was temperature-dependent, following osmotic lysis at 0 degree C, and was inhibited by low concentrations of Zn2+ but not by tosyl-lysine chloromethyl-ketone (TLCK), phenylmethylsulfonylfluoride (PMSF), or iodoacetamide. These and other results eliminated the possibility that release was due to proteolytic cleavage of the C-terminal hydrophobic tail present on newly synthesized VSG. Bolton and Hunter reagent reacted with several components on living cells.

Animals↗
PubMed1984

Histidine-rich protein genes and their transcripts in Plasmodium falciparum and P. lophurae.

The presence of histidine-rich protein (HRP) related genes and gene products in Plasmodium falciparum was demonstrated using a synthetic pentahistidine-encoding oligonucleotide and a cloned HRP cDNA probe prepared from the avian parasite P. lophurae. In Northern blotting experiments, two knobby clones of P. falciparum were found to contain a 3500 nucleotide RNA species that hybridized with the oligonucleotide and HRP cDNA probes. As this component had the expected size for an mRNA encoding an 80-90 kDa protein and was absent from two knobless clones of P. falciparum, we concluded that it represented a 'knob protein' mRNA. Using the restriction enzyme EcoRI, three identical cross-hydribizing HRP gene fragments were found in the DNA of both knobby and knobless clones of P. falciparum. These fragments differed in size from those present in P. lophurae. These results suggest that the absence of knob protein mRNA in knobless clones is not due to loss of the corresponding gene(s).

Animals↗
PubMed1982

The effect of microwaves on nutrient value of foods.

Microwave cooking has gained considerable importance as an energy-saving, convenient, and time-saving cooking method. This article reviews the state of the art of microwave cooking and the existing publishing data on the effects of microwave cooking on nutritive values of moisture, protein, carbohydrate, lipid, minerals, and vitamins. Most reports indicated that microwave cooking resulted in higher moisture losses compared with conventional methods. Overall, the nutritional effects of microwaves on protein, lipid, and minerals appear minimal. There is no report on the effects of microwaves on carbohydrate fraction in foods. A large amount of data is available on the effects of microwaves on vitamins. It is concluded that there are only slight differences between microwave and conventional cooking on vitamin retention in foods. In conclusion, no significant nutritional differences exist between foods prepared by conventional and microwave methods. Any differences reported in the literature are minimal.

Animals↗
PubMed1981

Molecular cloning of foot and mouth disease virus genome and nucleotide sequences in the structural protein genes.

Foot and mouth disease virus (FMDV), of the family Picornaviridae, consists of a single-stranded RNA (approximately 8,000 nucleotides), the translation of which is initiated on the 3' side of a 150-nucleotide poly(C) tract and yields a single polyprotein which is processed by host cell proteases into four primary products (Fig. 1). One or more virus-specified proteases further cleave these into the final products, the capsid proteins (VP1-4) being derived from the precursor p88 (for review see ref. 5). There are seven serotypes of the virus and as it has been shown that the immunizing activity of FMDV particles is associated primarily with VP1 (refs 6, 7), it seems likely that antigenic variation in FMDV is a result of changes in the structure of this protein. To further our understanding of this variation and as a first step in the possible development of FMDV vaccines from genetically manipulated microorganisms, we report here the construction and analysis of recombinant plasmids containing cDNA copies of the RNA. Comparison of the deduced amino acid sequence with the known polypeptide sequences shows that the NH2-termini of VP2 and VP3 are conserved between the A and O serotypes whereas that of VP1 (the immunizing antigen) varies by as much as 42% between serotypes.

Amino Acid Sequence↗