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Biomedical subjects

A Tait

Publications and source records attributed to A Tait.

At least 19 recordsLinked to original sources
PubMed1999

A high level of mixed Trypanosoma brucei infections in tsetse flies detected by three hypervariable minisatellites.

The issue of whether genetic exchange occurs at a significant frequency in natural populations of Trypanosoma brucei is controversial and one of the arguments against a high frequency has been the apparent lack of host infections with mixtures of trypanosome genotypes. Three minisatellite markers (MS42, CRAM, 292) within the coding regions of three genes have been identified and PCR based methods developed for detecting variation at these loci using crude lysates of infected blood as templates. Initial PCR analysis, using primers flanking the repeats, of DNA from two cloned stocks of the parasite has shown that two DNA fragments of different size were amplified from each stock. Analysis of the inheritance of these fragments into the F1 progeny of crosses demonstrated that the different size fragments were alleles that segregated in a Mendelian manner. The alleles at each of the three loci segregated independently consistent with their localisation on three different chromosomes. Analysis of a series of cloned isolates from tsetse flies showed that these loci were highly variable giving heterozygosities of 94% and the identification of 12 distinct alleles in a sample of 17 cloned isolates. In order to determine whether isolates are heterogeneous in terms of trypanosome genotype, the allelic variation at these three loci was examined in uncloned samples from tsetse flies isolated in Kiboko, Kenya and Lugala, Uganda. A significant proportion of the isolates (36% in Lugala and 47% in Kiboko) contained more than two alleles at one or more of the loci thus demonstrating that a high proportion of tsetse flies were infected with more than one genotype of trypanosomes. This was established, unequivocally, for two isolates by generating a series of cloned trypanosome lines from each and determining the genotype of each clone; one isolate (927) contained seven different genotypes with a high proportion of the possible combinations of alleles at each locus. These results indicate the possibility of frequent genetic exchange in the field, they imply that a significant proportion of mammalian hosts must contain mixtures of different trypanosome genotypes and they demonstrate the advantages of using minisatellite markers for the analysis of the population structure of T. brucei.

Alleles↗
PubMed1999

Evidence for localisation of a Theileria parasite AT hook DNA-binding protein to the nucleus of immortalised bovine host cells.

Immortalisation of bovine leukocytes by the macroschizont stage of the tick transmitted protozoan parasite, Theileria annulata, results in the clonal expansion of infected cells and dissemination throughout the bovine host. The parasite-encoded factors which induce this unique transformation event have not been defined to date. In this study, a gene family (TashAT) has been characterised that encodes polypeptides with homology to known DNA-binding proteins. Expression of TashAT genes occurs at the intracellular macroschizont stage of the parasite life cycle and during differentiation to the merozoite, negative regulation of TashAT genes is detected early relative to other macroschizont genes. Interestingly, the early reduction in TashAT expression coincides with the initial decrease in host cell proliferation. One member of the family, TashAT2, was characterised in detail and the predicted polypeptide sequence was found to harbor three AT hook DNA-binding domains. Antisera generated against two distinct regions of TashAT2 both located the antigen to the host cell nucleus and, combined with protein translation inhibition and immunoprecipitation studies, provide evidence that this polypeptide could be transported from the parasite to this location. Further evidence for this postulation was provided by transfection studies which demonstrated that TashAT2 does encode the structural information required for localisation to the nucleus of a mammalian cell. Thus, TashAT2 is a potential candidate for a parasite-encoded factor that modulates host cell gene expression and may be involved in the control of host cell proliferation.

Amino Acid Sequence↗
PubMed1999

A novel gene encoding a ras-like GTP-binding protein from Trypanosoma brucei: an evolutionary ancestor of the ras and rap genes of higher eukaryotes?

The ras superfamily of GTP binding proteins encompasses a wide range of family members, related by conserved amino-acid motifs, and act as molecular binary switches that play key roles in cellular processes. Gene duplication and divergence has been postulated as the mechanism by which such family members have evolved their specific functions. We have cloned and sequenced a ras-like gene, tbrlp, from the primitive eukaryote Trypanosoma brucei. The gene encodes a protein of 227 amino acids and contains the six conserved subdomains that designate it as a ras/rap subfamily member. However, the presence of key diagnostic residues characteristic of both the ras and rap families of GTP confuse the familial classification of this gene. Phylogenetic analysis of the GTP binding domain places its origins at the divergence point of the ras/rap families and suggests that tbrlp is an ancestral gene to the ras/rap genes of higher eukaryotes.

Amino Acid Sequence↗
PubMed1998

The molecular karyotype of the megabase chromosomes of Trypanosoma brucei and the assignment of chromosome markers.

We present the molecular karyotype of the megabase chromosomes of Trypanosoma brucei stock TREU927/4 (927). We have identified 11 diploid chromosomes ranging in size from 1 to 5.2 Mb approximately and pairs of homologues differ in size by up to 15%. A total of 401 cDNA probes were hybridised to T. brucei stock 927 chromosomes and 168 chromosome-specific markers were defined. Most of these markers were hybridised to the separated chromosomal DNA of two other cloned field isolates and four F1 progeny clones from a laboratory cross. The chromosomes vary in size by up to two and a half times between stocks and the DNA content of the 11 pairs of homologues varies by up to 33% in different stocks. Stock 927 contains the smallest chromosomes and the least nuclear genomic DNA. Nevertheless, all 11 syntenic groups of cDNA probes are maintained in all stocks. In the F1 hybrids only we have identified one extra PFG band to which none of our probes hybridise. We have shown that probes thought to be specific for the bloodstream-form variant surface glycoprotein expression sites hybridise to different chromosomes in different stocks and may hybridise to either one or both of a homologous pair of chromosomes. We have also determined the chromosomal location of the ribosomal RNA gene arrays.

Animals↗
PubMed1998

Characterisation of a polo-like protein kinase gene homologue from an evolutionary divergent eukaryote, Trypanosoma brucei.

The polo-like protein kinase gene family (PLKs) encodes proteins which are involved in the control of exit from mitosis in higher eukaryotes. We have cloned and analysed a polo-like kinase, tbplk, from an evolutionary divergent eukaryote, Trypanosoma brucei. The gene encodes a 767 amino acid protein of predicted size 86.8 kDa with 50.4% identity to mammalian PLKs over the protein kinase catalytic domain and it possesses a conserved motif, the 'polo-box', which is found in all PLKs. Phylogenetic analysis demonstrates that this gene is clearly a member of the PLK family, although it has some distinctive features such as a large C-terminal insertion when compared with mammalian PLKs. The gene is single copy and expressed in both bloodstream and procyclic stage trypanosomes. Sequencing of tbplk from a number of trypanosome isolates reveals a length polymorphism in a run of asparagine residues within the coding region. The presence of PLKs in a wide range of organisms, including such a primitive organism as T. brucei, suggests that PLKs may have a key role in the function of the cell cycle.

Amino Acid Sequence↗
PubMed1998

Identification of a Theileria annulata antigen expressed in multiple stages of the parasite life cycle.

In order to identify sporozoite surface molecules which may be involved in invasion and could act as potential vaccine candidates, a number of Mabs were raised in mice against T. annulata sporozoites. These were assayed for their ability to block sporozoite invasion of bovine peripheral blood mononuclear (PBM) cells in vitro. One of these, Mab 4B11, was found to neutralize sporozoite invasion to a high degree and to recognize a group of sporozoite antigens on Western blots. A T. annulata lambdagt11 genomic expression library was screened with Mab 4B11 and a positive clone containing a 900-bp insert (KP8) analysed further. Data from Southern and Northern blotting indicated that the gene containing the KP8 sequence, termed sporozoite and macroschizont gene 2 (spm2), was expressed both in T. annulata sporozoites and in later parasite life-cycle stages, macroschizont-infected leucocytes and piroplasms. The KP8 sequence was expressed in E. coli as a fusion protein with glutathione-S-transferase (GST) using the vector pGEX1lambdaT. Bovine antiserum raised against GST-KP8 recognised a single high molecular weight molecule on Western blots corresponding to one of the antigens recognised by Mab 4B11, expressed in sporozoites, macroschizont-infected leucocytes, and piroplasms. While our evidence suggests that the spm2 molecule alone is not responsible for sporozoite neutralization, it is a multistage antigen likely to function both in T. annulata sporozoites and in subsequent parasite life-cycle stages.

Amino Acid Sequence↗
PubMed1998

Vision and driving: selective effect of optical blur on different driving tasks.

A test of static (high-contrast) visual acuity is the most prevalent vision test used to screen driver license applicants worldwide, even though research has largely failed to provide convincing empirical evidence for its role in traffic accidents. The purpose of this research was to evaluate the effect of visual acuity degradation on different components of the driving task. Participants were 24 young licensed drivers (15 men, 9 women) with normal vision. Driving performance was measured while participants wore modified swimmer's goggles to which blurring lenses were affixed in amounts necessary to produce binocular visual acuity levels of 20/20, 20/40 (the prevalent acuity standard for driving), 20/100, and 20/200. Driving performance was measured using the closed-road method of Wood and Troutbeck (1994). Acuity degradation produced significant decrements in road sign recognition and road hazard avoidance as well as significant increments in total driving time. Participants' abilities to estimate whether clearances between pairs of traffic cones were sufficiently wide to permit safe passage of the vehicle and to slalom through a series of traffic cones were relatively unaffected by acuity degradation. Thus the latter tasks appear to be mediated by aspects of central and peripheral vision that are relatively unaffected by optical blur. Potential applications of this type of research include the development of procedures for defining empirically justifiable vision standards for driver licensure.

Adult↗
PubMed1997

Trypanosoma brucei: lack of cross-resistance to melarsoprol in vitro by cymelarsan-resistant parasites.

We have examined cross-resistance between trypanocidal drugs using a well-characterised drug-sensitive line, 247, and its cymelarsan-resistant derivative, 247melCyR. The cymelarsan-resistant line was cross-resistant to trimelarsen and melarsen oxide, and partially cross-resistant to two diamidines, pentamidine and berenil (diminazene aceturate). It was cross-resistant to lipid-soluble melarsoprol in vivo but to only a trivial degree in two in vitro assays. The potential role of adenosine transport in arsenical-induced killing of parasites was investigated. Adenosine, adenine, and the diamidines, but not inosine, were able to inhibit killing of drug-sensitive STIB 247 trypanosomes by cymelarsan and melarsen oxide in a concentration-dependent manner. These results are consistent with the view that these arsenical compounds enter trypanosomes via an adenosine-specific transporter. Melarsoprol-induced killing of trypanosomes was unaffected, however, by either purine and to only a slight degree by the diamidines. These data suggest that melarsoprol can enter trypanosomes by a route other than through an adenosine transporter and that there may be two mechanisms contributing to arsenical resistance in this drug-resistant line of trypanosomes.

Adenine↗
PubMed1997

Ostertagia circumcincta: isolation of a partial cDNA encoding an unusual member of the mitochondrial processing peptidase subfamily of M16 metallopeptidases.

A reverse-transcriptase polymerase chain reaction (PCR) procedure was used to isolate an Ostertagia circumcincta partial cDNA encoding a protein with general primary sequence features characteristic of members of the mitochondrial processing peptidase (MPP) subfamily of M16 metallopeptidases. The structural relationships of the predicted protein (Oc MPPX) with MPP subfamily proteins from other species (including the model free-living nematode Caenorhabditis elegans) were examined, and Northern analysis confirmed the expression of the Oc mppx gene in adult nematodes.

Amino Acid Sequence↗
PubMed1997

A computer-interfaced vertometer system for contact lenses.

BACKGROUND: The conventional vertometer (lensometer) is difficult to use for accurate measurement of contact lenses whose back vertex power (BVP) varies across the optic zone. BVPs of multifocal rigid and soft contact lenses have previously been measured using a conventional vertometer incorporating the Scheiner principle, which makes use of two light paths equidistant from the center of the lens. METHODS: We have developed a computer-based vertometer system based on the Scheiner principle which can be used to produce a profile of BVPs across rigid and soft contact lenses. A computer interface to the vertometer allows rapid acquisition of readings and software-based ray tracing derives in-air dioptric power readings across the optic zone at specific ray heights. RESULTS: Results are presented for soft and rigid, and spherical and aspheric surface contact lenses. CONCLUSIONS: The system improves the measurement resolution of a standard vertometer and shows an acceptable level of precision and accuracy for most applications.

Contact Lenses↗
PubMed1996

Conservation of neutralizing determinants between the sporozoite surface antigens of Theileria annulata and Theileria parva.

The sporozoite surface antigens SPAG-1 of Theileria annulata and p67 of Theileria parva are postulated to contain determinants necessary for host cell invasion and/or recognition and are both being considered as candidates for inclusion in subunit vaccines. Preliminary data suggest that these are related molecules. In this paper we describe the investigation of the relationship between these sporozoite antigens further by analysis of the immunological cross-reactivity using Mabs and sera raised against each antigen. The cross-reactions were examined by carrying out Western blots, IFA tests, and in vitro sporozoite neutralization assays. In addition, sequence comparison data which clearly establish that these surface antigens are encoded by related genes are presented. The regions of SPAG-1 identified as containing cross-reactive epitopes recognized by p67 antiserum correlated to regions of high predicted homology between p67 and SPAG-1, which are located at their respective N- and C-termini. Furthermore, p67 and SPAG-1 were found to contain cross-reactive determinants responsible for neutralization of sporozoite infectivity in vitro, and at least some of these were located in the C-termini of both molecules. The relevance of these findings to the possible roles for these molecules in host cell invasion is discussed.

Amino Acid Sequence↗
PubMed1996

Characterisation of cloned lines of Trypanosoma brucei expressing stable resistance to MelCy and suramin.

Two cloned drug-sensitive stocks of Trypanosoma brucei (STIB 247 and STIB 386) were each used to generate cloned lines expressing resistance to the melaminophenyl arsenical drug cymelarsan (247MelCyR and 386MelCyR) and to suramin (247SurR and 386SurR). The drug-resistance phenotypes were stable after passaging in mice in the absence of drug pressure and three of the lines were transmitted through tsetse flies with no alteration of drug-resistance. There was no evidence of cross-resistance between melCy and suramin in vivo. Twenty-four hour growth inhibition assays were conducted on bloodstream and procyclic forms in axenic in vitro cultures. Suramin-resistance was expressed in bloodstream forms but not in the procyclic stage, while the melCy-resistant lines expressed melCy-resistance in both stages. No cross-resistance between melCy and suramin was observed. Cross-resistance between melCy and another arsenical drug, melB (melarsoprol), was observed in vivo, but to only a very limited extent in vitro. We propose that this difference between the in vivo and in vitro results for melB may indicate that an alteration in a surface adenosine transporter responsible for reduced melCy uptake was bypassed by melB over 24 hours in vitro.

Animals↗
PubMed1996

The isolation and characterization of genomic and cDNA clones coding for a cdc2-related kinase (ThCRK2) from the bovine protozoan parasite Theileria.

The tick-transmitted protozoan parasites Theileria annulata and Theileria parva are important intracellular pathogens of domestic cattle in tropical and subtropical regions. Proliferative phases take place within both lymphocytes and erythrocytes. The lymphocyte is stimulated to enter the cell cycle by the parasite and the multinucleate parasite can establish a state in which karyokinesis and cytokinesis occur in phase with the host cell. The link between parasite nuclear division and cytokinesis is altered during the formation of merozoites (a non-dividing, invasive, extracellular stage). These features imply a high degree of control over parasite nuclear division and cytokinesis. Two different approaches have been used to identify clones from both species which are extremely highly conserved homologues. These encode a cdc2-related kinase which is > 60% identical to eukaryotic cyclin-dependent kinases of the p34cdc2/p32CDK2 subfamily. There is typical conservation of kinase domains, implying an in vivo protein kinase activity for the polypeptide. The PSTAIRE region, implicated in cyclin binding, is well conserved suggesting that ThCRK2 will bind cyclin molecules closely related to the eukaryotic A/B-type cyclins. However, there is divergence in certain key motifs potentially associated with binding of molecules that regulate the activity of the kinase. Expression patterns of RNA and protein indicate that ThCRK2 is likely to function in all dividing stages of the parasite and, taken together, the results point to a central role in the regulation of nuclear division.

Amino Acid Sequence↗
PubMed1995

Differential transcription and expression of ornithine decarboxylase in embryos of replicated mouse lines divergently selected for lean body mass.

Embryos of replicated mouse lines divergently selected for high or low lean body mass have been shown to differ approximately twofold in the activity of ornithine decarboxylase during embryogenesis and that this difference has been shown to be associated with a restriction enzyme polymorphism in the structural gene [Gray and Tait (1993) Genet. Res. 62, 31-37]. In the present paper we report that the differences in enzyme activity are due to changes in transcription and show that the enzyme produced has the same specific activity in all selected lines, precluding any significant structural change in the enzyme being associated with the differences in the structural gene.

Animals↗
PubMed1995

Trypanosoma brucei: inheritance of kinetoplast DNA maxicircles in a genetic cross and their segregation during vegetative growth.

The inheritance of maxicircle DNA was determined using a polymorphic EcoRI restriction site in the maxicircle variable region. In 11 hybrid progeny from a genetic cross of two stocks of Trypanosoma brucei, 7 progeny apparently inherited maxicircles uniparentally from either parent, in agreement with the results from previous studies, but in 4 progeny inheritance was biparental. Three subclones from 2 of these 4 progeny were made, and in these the maxicircles of only one parental type were detected. These data are considered in terms of a simple model whereby half the maxicircle genomes are inherited from each parent into progeny at meiosis with subsequent stochastic segregation at each mitotic division. This model generates a quantitative prediction as to the period of time required for fixation of inheritance to a uniparental pattern which provides a reasonable fit to the experimental data. These results provide an explanation as to why previous studies have shown that maxicircles are (apparently) inherited uniparentally: the kinetoplast is a unitary organelle inherited faithfully at cell division, but the maxicircles that it contains are best considered as a population that divides stochastically. Consideration of the model also explains why maxicircle populations are homogeneous.

Animals↗