Biomedical subjects

G A Cross

Publications and source records attributed to G A Cross.

At least 55 records · Page 3Linked to original sources

PubMed1993

Inhibition of glycosylphosphatidylinositol biosynthesis in Leishmania mexicana by mannosamine.

In several Leishmania species, glycosylated inositol phospholipids exist as free lipids, as membrane protein anchors, and as the membrane-binding moieties of the lipophosphoglycans. Both the glycolipid-anchored cell surface metalloproteinase, gp63, and the lipophosphoglycans have been proposed to be involved in cell invasion. Moreover, the lipophosphoglycans have been implicated in the survival of Leishmania in the parasitophorous vacuole of the host macrophage. In this report we show that mannosamine effectively inhibits the biosynthesis of both free glycosylated inositol phospholipids and the lipophosphoglycans of Leishmania mexicana. [3H]Mannosamine is incorporated into glycosylated inositol phospholipids, but not significantly into lipophosphoglycans when added as a radiochemical tracer at a subinhibitory concentration. The reversible inhibitory effect of mannosamine may be useful for studying precursor/product relationships during the biosynthesis of free glycosylated inositol phospholipids, glycolipid anchors, and the lipophosphoglycans. The implications of these data for the mode of action of mannosamine are discussed.

Animals↗

PubMed1993

Autonomously replicating single-copy episomes in Trypanosoma brucei show unusual stability.

We have obtained several autonomously replicating plasmids in procyclic Trypanosoma brucei. Two of these have been analyzed in detail. Both exist as monomeric single-copy episomes, which nevertheless demonstrate significant stability. These episomes transform procyclics at frequencies that are at least 60-fold higher than those achieved earlier with non-replicating molecules. A modified version of one of these episomes is able to replicate autonomously in bloodstream form T. brucei but it is unstable in this developmental stage of the organism. These episomes will prove useful as shuttle vectors, in functional complementation studies, in the construction of promoter-traps for the isolation of transcriptional promoters in T. brucei, and importantly, as models for the analysis of DNA replication in this ancient eukaryotic lineage.

Animals↗

PubMed1993

Molecular species analysis of phospholipids from Trypanosoma brucei bloodstream and procyclic forms.

We present a quantitative description of the molecular species composition of the major phospholipid classes in bloodstream and procyclic forms of Trypanosoma brucei. Phospholipid classes were resolved by 2-dimensional thin-layer chromatography. Diradylglycerols were released from individual phospholipid classes by phospholipases C, converted into benzoate derivatives and separated into diacyl, alkylacyl and alk-1-enylacyl subclasses. Individual molecular species were quantitated and identified by HPLC and the assignments were confirmed by mass spectrometry. Comparison of the diacyl species of PC, PE and PI in bloodstream trypanosomes showed major differences in the relative amounts of individual molecular species between the different classes but not striking changes in the degree of saturation or overall chain length. In contrast, in procyclic trypanosomes the relative amounts of diacyl molecular species with polyunsaturated fatty acyl chains decreased in the order of PC > PE >> PI. Also, the alkylacyl and alk-1-enylacyl subclasses of PC and PE in bloodstream trypanosomes comprised a single molecular species, 18:0 18:2. Such exclusivity was not observed in procyclic trypanosomes among the same phospholipid subclasses, although 18:0 18:2 was the predominant species. Almost all the PI of bloodstream forms contained one 18:0 acyl species, which is consistent with the composition of the PI used for glycosylphosphatidylinositol synthesis.

Acetylglucosamine↗

PubMed1993

A possible role for the 3'-untranslated region in developmental regulation in Trypanosoma brucei.

A series of Trypanosoma brucei transfection vectors was constructed in which transcription of the luciferase gene was driven by the procyclic acidic repetitive protein (procyclin) promoter. The untranslated regions surrounding the luciferase gene were derived from the actin, fructose bisphosphate aldolase, or PARP loci. Trans-splicing of the resulting transcripts occurred as expected, but the site of 3' polyadenylation was upstream of the position anticipated. The nature of the 3'-untranslated region was crucial to the level of expression in bloodstream forms.

Animals↗

PubMed1993

Rapid isolation of DNA from trypanosomatid protozoa using a simple 'mini-prep' procedure.

Although several methods for isolating genomic DNA from trypanosomatid protozoa exist, all are time-consuming and cumbersome. Faster, simpler and efficient protocols for preparation of DNA from these protozoa are needed to ease the screening of mutants and transfectants. We describe the use of a bacterial lysis method to isolate chromosomal DNA from a wide range of trypanosomatids. The method is based on the finding reported by He et al., who noticed that phenol/chloroform treatment of Escherichia coli cells in the presence of LiCl and Triton X-100 solubilizes plasmid DNA, while precipitating unwanted chromosomal DNA and denatured cellular proteins. In applying this lysis method to the isolation of episomal DNA from transfected trypanosomatids, we found that, unlike bacterial genomic DNA, chromosomal DNA of trypanosomatids was soluble in the phenol/chloroform/Triton/LiCl mixture. This observation prompted us to use the bacterial lysis method as a routine protocol for extraction of DNA from trypanosomatids.

Animals↗

PubMed1993

Crithidia fasciculata contains a transcribed leishmanial surface proteinase (gp63) gene homologue.

We have cloned and analysed a gene from the insect parasite, Crithidia fasciculata, with homology to the gp63 metalloendoproteinase gene of Leishmania. The Crithidia gene homologue is arranged as a multicopy family comprised of approximately 7 genes. The mature transcript is 4.0 kb. The predicted amino acid sequence has significant homology with Leishmania gp63s, contains a zinc-binding motif and a potential site for addition of a glycosylphosphatidylinositol anchor. Demonstration of a gp63 homologue in C. fasciculata, a monogenetic parasite, suggests that the molecule may play a role in parasite survival within the insect gut.

Amino Acid Sequence↗

PubMed1993

Deletion of an immunodominant Trypanosoma cruzi surface glycoprotein disrupts flagellum-cell adhesion.

Null mutants of the Trypanosoma cruzi insect stage-specific glycoprotein GP72 were created by targeted gene replacement. Targeting plasmids were constructed in which the neomycin phosphotransferase and hygromycin phosphotransferase genes were flanked by GP72 sequences. These plasmids were sequentially transfected into T. cruzi epimastigotes by electroporation. Southern blot analyzes indicated that precise replacement of the two genes had occurred. No aberrant rearrangements occurred at the GP72 locus and no GP72 gene sequences had been translocated elsewhere in the genome. Western blots confirmed that GP72 is not expressed in these null mutants. The morphology of the mutants is dramatically different from wild-type. In both mutant and wild-type parasites, the flagellum emerges from the flagellar pocket. In the null mutant the normal attachment of the flagellum to the cell membrane of the parasite is lost.

Amino Acid Sequence↗

PubMed1993

The surface trans-sialidase family of Trypanosoma cruzi.

Trypanosomes cannot synthesize sialic acids. Infectious stages of the life cycle of the human pathogen Trypanosoma cruzi express a cell-surface glycolipid-anchored trans-sialidase, which can transfer sialic acid between glyco-conjugates. Sialic acid is transferred from host cell-surface and serum sialylglycoproteins to trypanosome cell-surface glycoconjugates. The transfer reaction is specific for donors with terminal alpha-2,3-linked sialic acid, and terminal beta-1,4-linked galactose is the preferred acceptor. In the absence of an acceptor, the enzyme acts as a hydrolase, but cleavage is less efficient than transfer. Trans-sialidase activity is attributable to a few members of a large family of T. cruzi surface glycoproteins, many of which are simultaneously expressed. The functions of the trans-sialidase surface glycoprotein family are unknown but may be important for adhesion, invasion, virulence, or pathogenicity. A trans-sialidase is also expressed in the procyclic forms of Trypanosoma brucei.

Amino Acid Sequence↗

PubMed1993

Gene deletion suggests a role for Trypanosoma cruzi surface glycoprotein GP72 in the insect and mammalian stages of the life cycle.

We have explored the biological function of a surface glycoprotein (GP72) of Trypanosoma cruzi by studying a null mutant parasite, generated by targeted gene deletion. GP72 deletion affected parasite morphology in several stages of the life cycle. Insect midgut (epimastigote) forms had a detached flagellum (apomastigote) in the null mutant. The abnormal flagellar phenotype persisted during development of the infective (metacyclic) forms but there was no impairment in the acquisition of complement resistance, sialidase expression or cell infectivity. The GP72 null mutant could efficiently infect and proliferate in mouse macrophages and non-phagocytic L6E9 cells. The mammalian stages of the life cycle also showed major morphological abnormalities. During early subcultures in L6E9 cells, few extracellular fully flagellated forms, expressing markers characteristic of trypomastigotes, were seen. The extracellular population consisted almost exclusively of rounded forms with short flagella (micromastigote), which expressed an amastigote-specific surface marker and no sialidase. The propagation of the parasite was not affected, despite the apparent lack of the trypomastigote forms, which are thought to be primarily responsible for cell invasion. After some subcultures, the extracellular population changed to about equal numbers of micromastigotes and a range of flagellated forms that still did not include true trypomastigotes. Instead, the kinetoplast remained close to the nucleus and the flagellum emerged from the middle of the cell (mesomastigote). Half of the flagellum adhered to the cell body and the remainder was free at the anterior end. In Triatoma infestans, the survival of the mutant was dramatically reduced, suggesting that either GP72 itself, or the altered properties of the flagellum, were critical for establishment in the insect vector.

Animals↗

PubMed1992

High-efficiency clonal growth of bloodstream- and insect-form Trypanosoma brucei on agarose plates.

This report describes a method for growing both bloodstream- and procyclic-form Trypanosoma brucei as colonies on agarose plates. Procyclic colonies, which took 2 weeks to develop, grew with approximately 17% plating efficiency on SDM-79/0.65% agarose supplemented with 20% (vol/vol) conditioned medium. Bloodstream forms were adapted to in vitro growth in liquid HMI-9 medium and then spread on HMI-9/0.65% agarose plates, where they grew to visible colonies in 3-5 days. Plating efficiencies were from 3 to 80%, depending upon the trypanosome variant and experiment. Colonies were proven to be the result of growth from a single cell and contained approximately 10(6) cells at maturity. Colonies were transferred to filters and probed for multicopy and single-copy genes. Potential uses of this method in conjunction with classical and reverse genetic approaches to studying trypanosomes are discussed.

Animals↗

PubMed1992

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. In vitro biosynthesis of intermediates in the construction of the GPI anchor of the major procyclic surface glycoprotein.

The African trypanosome, Trypanosoma brucei, expresses two abundant stage-specific glycosylphosphatidylinositol (GPI)-anchored glycoproteins, the procyclic acidic repetitive protein (PARP or procyclin) in the procyclic form, and the variant surface glycoprotein (VSG) in the mammalian bloodstream form. The GPI anchor of VSG can be readily cleaved by phosphatidylinositol (PI)-specific phospholipase C (PI-PLC), whereas that of PARP cannot, due to the presence of a fatty acid esterified to the inositol. In the bloodstream form trypanosome, a number of GPIs which are structurally related to the VSG GPI anchor have been identified. In addition, several structurally homologous GPIs have been described, both in vivo and in vitro, that contain acyl-inositol. In vivo the procyclic stage trypanosome synthesizes a GPI that is structurally homologous to the PARP GPI anchor, i.e. contains acyl-inositol. No PI-PLC-sensitive GPIs have been detected in the procyclic form. Using a membrane preparation from procyclic trypanosomes which is capable of synthesizing GPI lipids upon the addition of nucleotide sugars we find that intermediate glycolipids are predominantly of the acyl-inositol type, and the mature ethanolamine-phosphate-containing precursors are exclusively acylated. We suggest that the differences between the bloodstream and procyclic form GPI biosynthetic intermediates can be accounted for by the developmental regulation of an inositol acylhydrolase, which is active only in the bloodstream form, and a glyceride fatty acid remodeling system, which is only partially functional in the procyclic form.

Aging↗

PubMed1992

Galactose-containing glycosylphosphatidylinositols in Trypanosoma brucei.

Many eukaryotic surface glycoproteins, including the variant surface glycoproteins (VSGs) of Trypanosoma brucei, are synthesized with a carboxyl-terminal hydrophobic peptide extension that is cleaved and replaced by a complex glycosylphosphatidylinositol (GPI) membrane anchor within 1-5 min of the completion of polypeptide synthesis. We have reported the purification and partial characterization of candidate precursor glycolipids (P2 and P3) from T. brucei. P2 and P3 contain ethanolamine-phosphate-Man alpha 1-2Man alpha 1-6Man alpha 1-GlcN linked glycosidically to an inositol residue, as do all the GPI anchors that have been structurally characterized. The anchors on mature VSGs contain a heterogenously branched galactose structure attached alpha 1-3 to the mannose residue adjacent to the glucosamine. We report the identification of free GPIs that appear to be similarly galactosylated. These glycolipids contain diacylglycerol and alpha-galactosidase-sensitive glycan structures which are indistinguishable from the glycans derived from galactosylated VSG GPI anchors. We discuss the relevance of these galactosylated GPIs to the biosynthesis of VSG GPI anchors.

Animals↗

PubMed1992

Sequence homology and absence of mRNA defines a possible pseudogene member of the Trypanosoma cruzi gp85/sialidase multigene family.

A genomic clone, pTt21, containing DNA apparently transcribed specifically in Trypanosoma cruzi trypomastigotes, was obtained by differentially screening a genomic library with trypomastigote and epimastigote cDNA. This 3444-bp clone contained open reading frames at each end, separated by a 1.8-kb non-coding region. The translated polypeptide from the 3' open reading frame (ORF2) of 1037 bp had 25-30% identity with 5 recently published T. cruzi gp85/sialidase sequences, and 20-25% identity with bacterial sialidases. Rabbit antiserum raised against an Escherichia coli fusion protein derived from the 5' open reading frame (ORF1) identified a surface antigen of 160 kDa, specifically expressed in trypomastigotes. A probe containing the first 211 bp from ORF1 was used to obtain a complete copy (c1821) of a gene that was closely related to ORF1, and encoded another member of the gp85/sialidase family. c1821 encodes a protein of 897 amino acids, but assignment of the N-terminus of the polypeptide was not possible. The 5'-most start codon is an unfavourable context to act as a translation initiator, it does not align with the initiator methionines of other gp85/sialidase sequences, nor is it followed by a signal peptide sequence characteristically found in other gp85/sialidase sequences. Although homology with the 5' ends of other gp85/sialidase sequences decays towards the 5' end of c1821, alignment of c1821 with 4 other gp85/sialidases indicated that the coding sequence should extend upstream at least 160 amino acids. In this region of c1821 there are multiple stop codons in each frame. The presence of the stop codons, the alignment data and our inability to amplify reverse transcribed mRNA using four internal primers, suggest that c1821 may not be present as a mature mRNA and is a pseudogene. Comparison of the apparently non-repetitive 3' coding domain of c1821 with the corresponding repetitive domains of two other members of the gp85/sialidase family revealed a high degree of similarity in nucleotide but not in amino acid sequence, and c1821 may thus represent an evolutionary intermediate between sub-families of the gp85/sialidase superfamily.

Amino Acid Sequence↗

PubMed1991

Stable transformation of Leptomonas seymouri by circular extrachromosomal elements.

To define the cis-acting sequences necessary for gene expression and DNA replication in trypanosomatids, we have developed a selectable vector that can be grown in Escherichia coli and maintained stably in the insect trypanosomatid Leptomonas seymouri. The vector is relatively small (6 kilobase pairs) and contains a portion of the L. seymouri alpha-tubulin gene positioned in-frame with a truncated neomycin phosphotransferase gene that confers resistance to the aminoglycoside G418. This construct is maintained in cells as a high-copy-number circular extrachromosomal element containing several head-to-tail copies of the transforming plasmid. In L. seymouri, alpha-tubulin-neomycin phosphotransferase fusion RNAs are polyadenylylated and possess a trans-spliced mini-exon. Additional DNA sequences can be inserted into the vector, propagated, and expressed in transformed cells.

Animals↗

PubMed1991

Developmental variation of glycosylphosphatidylinositol membrane anchors in Trypanosoma brucei. Identification of a candidate biosynthetic precursor of the glycosylphosphatidylinositol anchor of the major procyclic stage surface glycoprotein.

The major surface antigen of the mammalian bloodstream form of Trypanosoma brucei, the variant surface glycoprotein (VSG), is attached to the cell membrane by a glycosylphosphatidylinositol (GPI) anchor. The VSG anchor is susceptible to phosphatidylinositol-specific phospholipase C (PI-PLC). Candidate precursor glycolipids, P2 and P3, which are PI-PLC-sensitive and -resistant respectively, have been characterized in the bloodstream stage. In the insect midgut stage, the major surface glycoprotein, procyclic acidic repetitive glycoprotein, is also GPI-anchored but is resistant to PI-PLC. To determine how the structure of the GPI anchor is altered at different life stages, we characterized candidate GPI molecules in procyclic T. brucei. The structure of a major procyclic GPI, PP1, is ethanolamine-PO4-Man alpha 1-2Man alpha 1-6 Man alpha 1-GlcN-acylinositol, linked to lysophosphatidic acid. The inositol can be labeled with [3H]palmitic acid, and the glyceride with [3H]stearic acid. We have also found that all detectable ethanolamine-containing GPIs from procyclic cells contain acylinositol and are resistant to cleavage by PI-PLC. This suggests that the procyclic acidic repetitive glycoprotein GPI anchor structure differs from that of the VSG by virtue of the structures of the GPIs available for transfer.

Animals↗

PubMed1991

A glycosylphosphatidylinositol protein anchor from procyclic stage Trypanosoma brucei: lipid structure and biosynthesis.

Cells of the insect (procyclic) stage of the life cycle of the African trypanosome, Trypanosoma brucei, express an abundant stage-specific glycosylated phosphatidylinositol (GPI) anchored glycoprotein, the procyclic acidic repetitive protein (PARP). The anchor is insensitive to the action of bacterial phosphatidylinositol-specific phospholipase C (PI-PLC), suggesting that it contains an acyl-inositol. We have recently described the structure of a PI-PLC resistant glycosylphosphatidylinositol, PP1, which is specific to the procyclic stage, and have presented preliminary evidence that the phosphatidylinositol portion of the protein-linked GPI on PARP has a similar structure. In this paper we show, by metabolic labelling with [3H]fatty acids, that the PARP anchor contains palmitate esterified to inositol, and stearate at sn-1, in a monoacylglycerol moiety, a structure identical to PP1. Using pulse-chase labelling, we show that both fatty acids are incorporated into the GPI anchor from a large pool of metabolic precursors, rather than directly from acyl-CoA. We also demonstrate that the addition of the GPI anchor moiety to PARP is dependent on de novo protein synthesis, excluding the possibility that incorporation of fatty acids into PARP can occur by a remodelling of pre-existing GPI anchors. Finally we show that the phosphatidylinositol (PI) species that are utilized for GPI biosynthesis are a subpopulation of the cellular PI molecular species. We propose that these observations may be of general validity since several other eukaryotic membrane proteins (e.g. human erythrocyte acetylcholine esterase and decay accelerating factor) have been reported to contain palmitoylated inositol residues.

Acylation↗

PubMed1991

An mRNA-dependent in vitro translation system from Trypanosoma brucei.

We have demonstrated efficient protein synthesis in a cell-free system from the bloodstream form of Trypanosoma brucei. This system was able to translate endogenous mRNA, added mRNA, or (apparently at much lower efficiency) three synthetic RNA transcripts lacking 5' mini-exon and 3' poly(A) sequences. Translation was resistant to chloramphenicol and greater than 95% inhibited by low concentrations of anisomycin and puromycin, but only partially inhibited by cycloheximide. Variant surface glycoprotein synthesized from endogenous mRNA was sensitive to endoglycosidase H, indicating the co-translational glycosylation potential of the system. Two proteins translated ab initio from the corresponding in vitro-transcribed RNAs showed no evidence of signal sequence cleavage or glycosylation. Efficient processing occurred when the same RNAs were translated in a rabbit reticulocyte lysate supplemented with canine pancreatic microsomes but not with trypanosome microsomes.

Animals↗

PubMed1991

An 85-kilodalton surface antigen gene family of Trypanosoma cruzi encodes polypeptides homologous to bacterial neuraminidases.

We have determined the sequence of a cDNA (Tt34c1) encoding a Trypanosoma cruzi trypomastigote stage-specific 85-kDa surface glycoprotein (gp85). Within the peptide sequence of Tt34c1 are two 8-amino acid motifs, Ser-X-Asp-X-Gly-X-Thr-Trp, that are characteristic of bacterial neuraminidases. Analysis of the Tt34c1 sequence predicts the presence of an amino-terminal signal sequence and a hydrophobic carboxy-terminus that is probably replaced by a glycosyl phosphatidylinositol membrane anchor. Gp85 is encoded by an extensive multigene family that is distributed throughout the genome and can be divided into subsets on the basis of oligonucleotide hybridisation patterns. By sequencing products of polymerase chain reaction (PCR) amplification of the 5' end of trypomastigote gp85 mRNA we show that multiple copies of the gene family are transcribed simultaneously in a trypanosome population. Comparison of the sequence of the PCR clones and another gp85 cDNA showed a highly conserved region 5' of the first methionine extending 180 nt into the coding sequence. Insertions and point mutations were observable outside these homologous sequences demonstrating the variant nature of the gp85 mRNAs.

Amino Acid Sequence↗