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J D Barry

Publications and source records attributed to J D Barry.

At least 55 records · Page 3Linked to original sources
PubMed1993

A major surface antigen of procyclic stage Trypanosoma congolense.

Five monoclonal antibodies (mAb) were raised that bound to the surface of procyclic stage Trypanosoma congolense with high intensity in immunofluorescence. Immunoblot analysis of trypanosome lysates using 3 of these mAb revealed a diffuse SDS-PAGE band of 36-40 kDa. The purified antigen did not react with Coomassie Blue or silver stains, but did stain blue with Stains-all, indicating acidity. For the one mAb tested, the epitope was periodate-sensitive and therefore probably glycan. Although this antigen shares properties with procyclin/PARP, which forms a surface coat on procyclic Trypanosoma brucei, a search in T. congolense for homologues of a procyclin/PARP gene revealed only non-coding sequence of partial similarity. Using a differential screen, a procyclic stage T. congolense cDNA clone was isolated that encoded a putative 256-amino acid protein containing 2 peptides chemically sequenced independently by Beecroft et al. [36]. The protein, termed glutamate and alanine-rich protein (GARP), has potential hydrophobic leader and tail sequences (the latter with potential for replacement by a glycosyl phosphoinositol anchor) and no potential N-linked glycosylation sites. It has no significant sequence homology with known proteins. Antibodies against a translational fusion of GARP bound specifically in Western blots to a band very similar to that detected by the mAb and also to the purified antigen. Immunogold electron microscopy revealed a dense packing of the antigen on the cell surface. It appears that procyclic T. brucei and T. congolense have major surface proteins with structural analogy, but with no sequence homology.

Amino Acid Sequence↗
PubMed1992

Molecular analysis of mutations in the Caenorhabditis elegans collagen gene dpy-7.

Collagens are a family of proteins contributing to the body structure of eukaryotes. They are encoded by a large and diverse gene family in the nematode Caenorhabditis elegans but by only a few genes in vertebrates. We have studied mutant alleles of the C. elegans dpy-7 gene, one of a large group of genes whose mutant phenotype is altered body form and several of which have previously been shown to encode cuticular collagens. We made use of the C. elegans physical map to screen specifically for collagen genes in the region of the X chromosome to which dpy-7 maps. This yielded a wild-type collagen gene clone which we showed, by micro-injection, could repair the dpy-7 mutant phenotype in transgenic animals. We cloned the homologous sequence from four dpy-7 mutant strains and by sequence analysis identified a single mutation in each case. All four mutations result in the substitution of a glycine with a larger residue in the conserved Gly-X-Y collagen domains. Similar substitutions in vertebrate collagens cause the heritable brittle bone disorder osteogenesis imperfecta. Whereas the human mutations are dominant, the dpy-7 mutations are recessive, and this may reflect different levels of complexity of collagenous macromolecular structures in the two organisms.

Alleles↗
PubMed1992

A developmentally regulated cysteine proteinase gene of Leishmania mexicana.

We have isolated a gene encoding a previously unreported class of trypanosomatid cysteine proteinase (CP) from the protozoan parasite Leishmania mexicana. The single-copy gene (lmcpa) [corrected]. has several unusual features that distinguish it from CP genes cloned from the related species Trypanosoma brucei and Trypanosoma cruzi. These include a shorter C-terminal extension of only 10 amino acids and a three-amino-acid insertion, GlyValMet, close to the predicted N-terminus of the mature protein. Northern blot analysis showed that the gene is expressed in all life-cycle stages but at higher levels in the amastigote stage in the mammal and in stationary phase promastigote cultures which contain the infective metacyclic form of the parasite. A precursor protein of 38 kDa was detected in amastigotes and stationary phase promastigotes with antisera specific to the LmCPa pro-region, but was barely detectable in early log-phase promastigotes. Anti-central domain antisera recognized the 38 kDa precursor and 24 and 27 kDa proteins. The major CPs of L. mexicana amastigotes, previously designated types A, B and C, were not detected with the antisera, suggesting that the gene codes for a previously uncharacterized CP in L. mexicana. The 24 kDa protein detected by the antiserum has no activity towards gelatin but apparently hydrolyses the peptide substrate BzPheValArgAMC. The relative levels of the 24 and 27 kDa proteins vary between the different life-cycle stages. The results indicate that expression of this CP is regulated at both the RNA and protein level.

Amino Acid Sequence↗
PubMed1992

Detection of antibody to immunodeficiency viruses by dot immunobinding assay.

The dot immunobinding assay (DIA), a modified enzyme immunoassay (EIA), has been demonstrated to be a highly sensitive and specific assay for the detection of antibody to a number of viruses. Different laboratory procedures are available for detecting antibody to the immunodeficiency viruses; however, these procedures require a certain amount of sophisticated equipment and trained personnel. Further, commercial kits for detecting antibody to human immunodeficiency virus, as now available, are not easy to use in the nonlaboratory setting. The DIA, as described herein, may be formatted to test up to 30 serum samples and is designed to be used in the absence of laboratory equipment. To determine the effectiveness of the DIA as a test kit for the detection of HIV and human T-cell leukemia virus type I (HTLV-I) antibodies, the kit was compared with commercial EIA and Western blot (WB; immunoblot) kits. Testing approximately 1,000 human serum samples for HIV antibody by DIA and EIA revealed a total agreement of 98.1%, a specificity of 99.0%, and a sensitivity of 95.9%. For 804 serum samples tested (200 were tested independently in two laboratories), eight results were discrepant: four DIA negatives which were EIA borderline positive and four DIA positives which were EIA negative. Testing the eight discrepant sera by immunofluorescence assay and WB resulted in their being either negative or indeterminate. The four DIA positives were indeterminate by WB. Close agreement was obtained when the remaining sera were compared by DIA, EIA, and WB. Of interest was finding that the DIA results compared favorably with those obtained by WB. Twenty-six suspect HTLV-I-positive serum samples tested by DIA also gave results comparable to those obtained by EIA and WB.

Blotting, Western↗
PubMed1991

tRNAs of Trypanosoma brucei. Unusual gene organization and mitochondrial importation.

In Trypanosoma brucei the U snRNA B gene (J. C. Mottram, K. L. Perry, P. M. Lizardi, R. Lührmann, N. Agabian, and R. G. Nelson (1989) Mol. Cell. Biol. 9, 1212-1223) is very tightly linked with other small RNA genes coding for tRNA(ACGArg), tRNA(CUULys), and a approximately 275-nucleotide RNA (RNA X) of unknown function. A similar genomic organization is found at the U6 snRNA locus, where the U6 gene is linked to tRNA(CGUThr) and tRNA(GUATyr) genes. The tRNA(Lys) and tRNA(Arg) genes are members of a multigene family, whereas the tRNA(Thr) and tRNA(Tyr) genes are single copy. Two additional tRNA(CUULys) genes and one tRNA(UUULys) gene were also isolated and sequenced and, together with a sequence previously published (D. A. Campbell (1989) Nucleic Acids Res. 17, 9479), appear to represent the entire gene family. Probes for tRNA(Lys), tRNA(Arg), tRNA(Thr), and tRNA(Tyr) were found to hybridize with mitochondrial and cytoplasmic tRNAs but not with mitochondrial DNA. This supports the hypothesis that mitochondrial tRNAs may be nuclear-encoded and imported from the cytosol into the mitochondrion.

Animals↗
PubMed1991

Trypanosoma brucei rhodesiense: characterisation of stocks from Zambia, Kenya, and Uganda using repetitive DNA probes.

We have previously described a system for characterising the relationships between trypanosome stocks of the T.brucei group based on Southern blotting with repetitive DNA probes followed by cluster analysis of resultant banding patterns (G. Hide et al. Molec. Bioch. Parasitol. 39, 213-226, 1990). In this study, we extend this analysis to examine the relationships between trypanosome stocks isolated from major sleeping sickness foci in Zambia, Kenya, and Uganda. We show that the trypanosome strains responsible for disease in Zambia are quite distinct from those sampled from the Kenya/Uganda foci. Furthermore, the human serum resistant stocks isolated from the Kenya/Uganda foci which were isolated from man (or from animals) were found to form a tight group in the cluster analysis, while stocks isolated from nonhuman sources in the same area or stocks from elsewhere were found in separate groups. Thus, the human infective trypanosome strains found in these foci may have common origins and have, perhaps, arisen by clonal selection from a common source.

Animals↗
PubMed1991

Expression site-associated genes transcribed independently of variant surface glycoprotein genes in Trypanosoma brucei.

Expression site-associated genes (ESAGs) of Trypanosoma brucei are found upstream of variant surface glycoprotein (VSG) genes in bloodstream expression sites. There are at least 6 different ESAGs in each of these expression sites, and each ESAG is repetitive in the genome. ESAGs are believed to reside only in VSG expression sites and to be co-transcribed with the VSG gene from a common alpha-amanitin-insensitive promoter. Our results show that this is not always true. The transcriptionally active 1.22 metacyclic expression site contains no ESAGs, but ESAGs are highly transcribed in these cells. The level of transcription indicates that more than one copy of each of these genes is active. Furthermore, some of these genes are transcribed, to produce steady state RNA, in procyclic culture cells which do not express the VSG gene: there is differential expression of ESAGs between the bloodstream and procyclic phases of the trypanosome life cycle. Thus ESAGs can be transcribed outwith an active VSG gene expression site and in the absence of expression of the VSG.

Animals↗
PubMed1991

The dynamics of antigenic variation and growth of African trypanosomes.

Antigenic variation in African trypanosomes, which is a simple strategy for survival in the immune host, is rendered complex by its magnitude. For protection from nonspecific immunity and escape from specific immunity, each trypanosome is covered by a replaceable surface coat composed of the variant surface glycoprotein (VSG), which specifies the variable antigen type (VAT) of the trypanosome. Antigenic variation is the process by which the trypanosome switches from one coat to another. Here, David Barry and Michael Turner consider this phenomenon within the context of the course of trypanosome infection.

Journal Article↗
PubMed1990

Duplicative activation mechanisms of two trypanosome telomeric VSG genes with structurally simple 5' flanks.

In the mammalian bloodstream, African trypanosomes express variant surface glycoprotein (VSG) genes from a family of long and complex telomeric expression sites. VSG switching generally occurs by the duplication of different VSG genes into these sites by gene conversion involving a series of 70 base pair (70bp) repeats in the 5' flank. In contrast, when VSG is first synthesised by trypanosomes in the tsetse fly at the metacyclic stage, a separate set of telomeric expression sites is activated. These latter telomeres appear not to act as recipients in gene conversion. We have found that the structure of two such expression sites is simple, with very short 70bp repeat regions and very little other sequence in common with bloodstream expression sites. However, the two telomeres readily act as donors in VSG gene conversion in the bloodstream and we show for one a consistent association of the conversion 5' end point with the short 70bp repeat region. These findings help explain why a very predictable set of VSGs is expressed in the tsetse fly and have implications for VSG gene conversion mechanisms.

Animals↗
PubMed1990

The identification of Trypanosoma brucei subspecies using repetitive DNA sequences.

We describe the use of repetitive DNA probes to characterise the relationships between different stocks of African trypanosomes representing the subspecies of Trypanosoma brucei. Probes derived from the ribosomal RNA genes (coding region and nontranscribed spacer) and another repetitive DNA sequence were used to characterise trypanosome stocks by Southern blotting. Numerical taxonomy methods applied to the resulting restriction enzyme patterns were used to derive a dendrogram depicting the relationships between the stocks examined. We show that three groups of West African human infective stocks can be distinguished: firstly, a group containing exclusively T. b. gambiense; secondly, a group which is indistinguishable from animal isolates in West Africa; and thirdly, a single stock which is indistinguishable from East African T. b. rhodesiense. In addition, we observe that T. b. rhodesiense stocks from East Africa are indistinguishable from animal isolates from the same area. Finally, we show that a group of T. b. rhodesiense stocks, isolated from a 1978 sleeping sickness outbreak in Zambia, are probably derived from a single parasite strain, and that this strain is distinct from T. b. rhodesiense parasites from Kenya and Uganda.

Animals↗
PubMed1990

Distinct, developmental stage-specific activation mechanisms of trypanosome VSG genes.

The metacyclic form of African trypanosomes is the first to express genes for the Variant Surface Glycoprotein (VSG) and it uses an unusually predictable subset of the VSG gene repertoire. We have developed a model system for the analysis of metacyclic VSG (M-VSG) gene expression and have used this to demonstrate that, for two M-VSG genes, different modes of expression operate in the insect and mammalian phases of the life-cycle. In metacyclic-derived clones, these genes are expressed in situ, whereas they are routinely activated by duplication in bloodstream trypanosomes. The expression loci for both M-VSG genes studied are structurally simple and we present a model, based on this, for the maintenance of a separate M-VSG repertoire and expression system.

Animals↗
PubMed1989

A cysteine proteinase cDNA from Trypanosoma brucei predicts an enzyme with an unusual C-terminal extension.

A cDNA for a Trypanosoma brucei cysteine proteinase has been cloned and sequenced. The deduced protein can be divided into four domains, based on homologies with other cysteine proteinases: the pre-, pro- and central regions show considerable homology to the cathepsin L class of mammalian enzymes, whilst the long C-terminal extension distinguishes the trypanosome enzyme from all mammalian cysteine proteinases reported. This 108 amino acid extension, which includes 9 contiguous prolines near the junction with the central domain, appears likely to be processed in part to produce the mature enzyme, and may be involved in targeting the protein within the cell. The trypanosome genome contains more than 20 copies of the cysteine proteinase gene arranged in a long tandem array.

Amino Acid Sequence↗
PubMed1989

High frequency of antigenic variation in Trypanosoma brucei rhodesiense infections.

Rates at which Trypanosoma brucei rhodesiense trypanosomes switch from expression of one variable antigen type (VAT) to that of another have been determined in cloned populations that have been recently tsetse-fly transmitted. Switching rates have been determined between several, specific pairs of VATs in each population. High rates of switching were observed in 2 cloned trypanosome lines, each derived from a separate cyclical transmission of the same parental stock and each expressing a different major VAT. Five estimates of the switching rate between one particular pair of VATs were consistently high (approximately 1 x 10(-3) switches/cell/generation). These high switching rates were similar both in bloodstream populations of mice and in populations confined to subcutaneously implanted growth chambers in mice, thus indicating that the interaction of the bloodstream population with other trypanosome populations in the lymphatics or extravascular sites in systemic infections did not influence the estimates of the rate of switching. Fourteen estimates were made of VAT-specific switching rates in bloodstream infections involving 8 combinations from among 6 VATs. Switching rate estimates were VAT-specific and showed considerable variation between different combinations of VATs--from 1.9 x 10(-6) to 6.9 x 10(-3) switches/cell/generation. The rates of switching to different metacyclic-VATs were, however, very similar. Summation of between 3 and 5 VAT-specific switching rate values in each of 4 experiments conducted in bloodstream infections has provided minimum estimates of the overall rate of antigenic variation: 2.0-9.3 x 10(-3) switches/cell/generation. These values are between 20 and 66,000-fold higher than previously published estimates.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Morphology of the uvula in obstructive sleep apnea.

Alterations in pharyngeal structure and function are considered fundamental in the pathogenesis of obstructive sleep apnea (OSA). However, little is known about morphologic features of the pharynx in patients with OSA. We therefore studied the tissue composition of the uvula (midsagittal section) in patients with OSA, using a quantitative, morphometric point-counting technique. Uvula tissue was obtained by uvulopalatopharyngoplasty (UPPP) in 33 patients (mean number of apneas per hour of sleep = 32.7 +/- 5.2) and by autopsy in 22 normal subjects not known to have OSA. All statistical comparisons were controlled for differences caused by age and body mass index. Patients with OSA had a significantly greater percentage of muscle in the uvula (18.1 +/- 1.9% versus 9.3 +/- 2.1%, p = 0.02) than did normal subjects. A significant difference in fat content was also found (9.5 +/- 1.4% in patients versus 4.0 +/- 1.0% in normal subjects, p less than 0.02). These differences between patients with OSA and control subjects could not be accounted for by anthropometric or sex differences. The percentage of uvula fat tissue was significantly related to the frequency of apneas and hypopneas in sleep (r = 0.43, p less than 0.01). Uvula morphology in 6 nonapneic snorers undergoing UPPP was similar to that of patients with OSA. We conclude that the uvula in patients with OSA contains more muscle and fat than the uvula in control subjects, possibly contributing to pharyngeal narrowing in OSA.

Female↗
PubMed1988

Loss of variable antigen during transformation of Trypanosoma brucei rhodesiense from bloodstream to procyclic forms in the tsetse fly.

A pleomorphic line of Trypanosoma brucei rhodesiense expressing a single variable antigen was used to quantify the rate of loss of the surface coat from bloodstream forms transforming to procyclics in the tsetse fly, Glossina morsitans, and in in vitro culture. Loss of variable antigen occurred at similar rates in the crop and anterior portion of the midgut of tsetse flies and in in vitro culture, but in the posterior portion of the fly midgut it occurred 2-3 times faster. The posterior portion of the midgut is the most important site for transformation of bloodstream-form trypanosomes to procyclics, and the dynamics of at least one component of this process are therefore not accurately paralleled in vitro.

Animals↗