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A W Thomas

Publications and source records attributed to A W Thomas.

At least 91 records · Page 5Linked to original sources
PubMed1996

Nonradioactive characterization of low-level heteroplasmic mitochondrial DNA mutations by SSCP-PCR enrichment.

Mitochondrial DNA (mtDNA) mutations have been implicated in an increasing number of human diseases. Many of these mutations are heteroplasmic and are only present at low levels in readily accessible human tissue such as blood. The technique of single-stranded conformational polymorphism (SSCP) allows the detection of mtDNA variants from peripheral blood, but characterization of these variants by automated sequencing is hampered by the low level of heteroplasmy. We have therefore developed a technique for the enrichment of mtDNA mutations that allows reliable sequence data to be obtained even if the variant mtDNA represents only 1% of the total mtDNA. The procedure involves the excision, purification and subsequent PCR amplification of selected DNA fragments from SSCP gels. The techniques can be applied to other heterogeneous mutations such as mosaic mutations in skin biopsies or somatic oncogene mutations in tumor tissue.

DNA Mutational Analysis↗
PubMed1996

Geophysical variables and behavior: LXXX. Periodicities and energetic characteristics of a strobe-light luminosity during a geomagnetic storm.

A videotape of a "strobe-light" luminosity that occurred in northern Ontario for several minutes during the evening of 6 October, 1994, at the time of a geomagnetic storm, was analyzed frame by frame. Brightness of the flashes decreased over the time of the observation. The interflicker intervals displayed phase-shifted periodicities that would be compatible with a rotating electromagnetic dipole and with the observations of the witnesses. The time of the occurrence and the dynamics of the luminosity were consistent with the predictions of the tectonic strain hypothesis.

Electromagnetic Fields↗
PubMed1995

Animal research.

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Animal Experimentation↗
PubMed1995

The nucleotide sequence of a transposable haloalkanoic acid dehalogenase regulatory gene (dehRI) from Pseudomonas putida strain PP3 and its relationship with sigma 54-dependent activators.

The mobile genetic element, DEH found in Pseudomonas putida PP3 carries a 2-haloalkanoic acid dehalogenase structural gene, dehI, and its associated regulatory gene, dehRI. The nucleotide sequence of dehRI was determined. The gene had an open reading frame putatively encoding for a 64 kDa protein containing 571 amino acid residues. The protein was similar to previously published sequences of several other sigma 54-dependent activator proteins. Amino acid sequence analysis showed that the deduced DehRI protein clustered with the NifA nitrogenase regulatory activator family, and was most closely related, with 47.7% similarity, to a 'NifA-like' deduced partial sequence from a plasmid-encoded ORF in Pseudomonas sp. strain NS671, associated with L-amino acid production. The domain structure of DehRI was analysed by alignment with other NifA-like and NtrC-like sequences and showed a highly conserved central region of approximately 230 amino acids, and a potential DNA-binding domain. No homology was detected between the deduced DehRI and other sigma 54-dependent activator sequences at the N-terminus, a result which was consistent with that region being the domain which recognised inducer.

Amino Acid Sequence↗
PubMed1995

Maternally inherited diabetes mellitus: the role of mitochondrial DNA defects.

Several studies have shown a consistent maternal effect in the transmission of Type 2 diabetes (NIDDM). The mitochondrial encephalomyopathies are a group of diseases characterized by maternal inheritance and a variety of mitochondrial DNA defects. Diabetes is a feature of some of these disorders and therefore the hypothesis arose that mitochondrial DNA mutations might play a role in patients with diabetes but no other features of neurological disease. Recent studies have confirmed that a specific point mutation in the gene encoding the mitochondrial tRNA for leucine segregates with diabetes and nerve deafness in families from the UK, Holland, France and Japan. Mitochondrial gene deletions have also been reported. Affected subjects present with progressive insulin deficiency and may fall into the broad classifications of either Type 1 (IDDM) or Type 2 diabetes (NIDDM). Future studies are aimed at searching for other mitochondrial gene defects in diabetes and attempting to explain the mechanism of hyperglycaemia by the development of phenotypic expression systems. Although an exciting development in the genetics of diabetes, currently described mitochondrial mutations do not fully explain the maternal effect in the transmission of Type 2 diabetes.

DNA, Mitochondrial↗
PubMed1994

Conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, in field isolates and distinct species of Plasmodium.

The extent of structural conservation of the Plasmodium falciparum sporozoite surface protein gene, STARP, recently characterized in the T9/96 clone, has been analyzed using the polymerase chain reaction. Results from Ivory Coast and Thai clones, field isolates originating from Brazil and Kenya and laboratory-maintained strains strongly suggest that this gene has a highly conserved structure throughout this species. This structure includes a complex repetitive central domain consisting of a mosaic region followed by tandem 45-amino acid-encoding (Rp45) and 10-amino acid-encoding (Rp10) repeat regions. Limited size variation in this domain appeared to result from highly localized duplication events in the Rp45 and Rp10 regions. No size variation was observed in the 5' and 3' coding non-repetitive regions, but minor size polymorphism was found in the single intron at the 5' end of the gene. No evidence was found of distinct families of polymorphic types, as has been observed with the blood-stage MSA-1, MSA-2 and S-antigens. The sequence of the STARP homologue in the phylogenetically close chimpanzee parasite, Plasmodium reichenowi, has also been elucidated and reveals high sequence conservation, although interesting differences were detected in the composition of the Rp10 region, known in P. falciparum to contain B- and T-cell epitopes. Finally, DNA hybridization reveals the presence in rodent malaria species of sequences containing homology to the STARP non-repetitive (though not the repetitive) regions, which would suggest that a similar, conserved gene may exist in these species.

Amino Acid Sequence↗
PubMed1994

Differential localization of full-length and processed forms of PF83/AMA-1 an apical membrane antigen of Plasmodium falciparum merozoites.

A well conserved 83-kDa apical membrane antigen of Plasmodium falciparum, PF83/AMA-1, is the analogue of PK66/AMA-1, a 66-kDa P. knowlesi protective merozoite protein. PK66/AMA-1 is expressed in late-stage schizonts; is localized within the merozoite apex; and is processed to a 44/42-kDa doublet at, or around, the time of schizont rupture. The processed forms can associate with the merozoite surface. We were interested to further analyze the timing of synthesis and processing, and subcellular localization of PF83/AMA-1, a malaria vaccine candidate, using monoclonal antibodies (mAbs) developed against PF83/AMA-1. Using [35S]methionine metabolically labeled asexual blood stage parasites, in combination with indirect single and dual immunofluorescence, we have determined that, in similar fashion to PK66/AMA-1, protein expression of PF83/AMA-1 is restricted to late-stage schizonts with greater than 8 nuclei. PF83/AMA-1 is post-synthetically processed rapidly by cleavage of an N-terminal peptide to a 66-kDa molecule. Both the 83- and the 66-kDa molecules are initially localized at the merozoite apex. In P. falciparum (7G8 strain and CVD-1 clone) the full-length 83-kDa molecule remains apically restricted following merozoite release. However, the processed 66-kDa form can become circumferentially associated with the merozoite surface at or around the time of schizont rupture and merozoite release. After merozoite invasion a processed form of PF83/AMA-1 is present in early ring stage parasites. Comparative analysis of a rhoptry associated protein RAP-1, shows a co-ordinated and compartmentalized release of rhoptry components.

Amino Acid Sequence↗
PubMed1994

Aspects of immunity for the AMA-1 family of molecules in humans and non-human primates malarias.

The apical membrane antigen (AMA-1) family of malaria merozoite proteins is characterised by a high degree of inter-species conservation. Evidence that the protein (PK66/AMA-1) from the simian parasite Plasmodium knowlesi was protective in rhesus monkeys suggested that the 83kDa P. falciparum equivalent (PF83/AMA-1) should be investigated for protective effects in humans. Here we briefly review pertinent comparative data, and describe the use of an eukaryotic full length recombinant PF83/AMA-1 molecule to develop a sensitive ELISA for the determination of serological responses in endemic populations. The assay has revealed surprisingly high levels of humoral response to this quantitatively minor antigen. We also show that PK66/AMA-1 inhibitory mAb's are active against merozoites subsequent to release from schizont-infected red cells, further implicating AMA-1 molecules in red cell invasion.

Animals↗
PubMed1994

Chimpanzees and supporting models in the study of malaria pre-erythrocytic stages.

Chimpanzees are being used in the study of immune response to Plasmodium falciparum malaria pre-erythrocytic stages (MPES). Responses induced by immunisation with recombinant/synthetic antigens and by irradiated sporozoites are being evaluated in a model system that is phylogenetically close to humans and that is amenable to limited manipulation not possible in humans. The value of chimpanzees for the in-depth study of immunological mechanisms at work in MPES-induced protection are discussed. A total number of 7 chimpanzees have been used to evaluate the immune response to recombinant antigens, and 5 have been challenged with large numbers of sporozoites, followed by surgical liver-wedge resection, in order to generate infected liver tissue for histological and immunological studies. As a complementary model, SCID mice carrying live, transplanted human and primate hepatocytes have been inoculated with sporozoites and infection of transplanted cells has been monitored by histological and immunological methods. In ongoing experiments chimpanzees are being immunised with MPES-derived lipopeptides that have been shown to overcome MHC restriction in mice, and with irradiated sporozoites.

Animals↗
PubMed1994

Geophysical variables and behavior: LXXVII. Increased geomagnetic activity and decreased pleasantness of spontaneous narratives for percipients but not agents.

A total of 48 adults were tested in 24 separate pairs either during the morning (10:00 A.M.) or evening (10:00 P.M.). One member of each pair was instructed to record (write) impressions and descriptions of 8 different art pieces (5 min. each). At the same time the other member of the pair sat in a comfortable chair within an acoustic chamber in another room and wrote an ongoing narrative. The evaluation and activity dimensions of the words that composed the narratives were scored by the Whissell Dictionary of Affect in Language. The increased global geomagnetic activity two days before the experiments was significantly associated (rhos about 0.60) with the use of more unpleasant words for people who sat in the quiet chamber only. Implications for the hypothesis of affective concordance between emotionally bonded human beings and its role in episodes of potentially veridical telepathic experiences are discussed.

Adult↗
PubMed1994

High prevalence of natural antibodies against Plasmodium falciparum 83-kilodalton apical membrane antigen (PF83/AMA-1) as detected by capture-enzyme-linked immunosorbent assay using full-length baculovirus recombinant PF83/AMA-1.

The 83-kilodalton (kD) apical membrane antigen of Plasmodium falciparum (PF83/AMA-1) is a potential asexual blood stage vaccine component. This antigen has been expressed as a full-length, nonfusion, recombinant baculovirus protein (PF83-7G8-1) using the authentic predicted signal peptide for appropriate postsynthetic routing. When purified by a novel high-performance, ion exchange chromatography (HPIEC) method, PF83-7G8-1 induced polyclonal antibodies in rats that immunoprecipitated both 83- and 66-kD forms of PF83/AMA-1 from 35S-methionine metabolically labeled parasite extracts. Using HPIEC-purified PF83-7G8-1 in combination with a rat monoclonal antibody against the highly conserved carboxy-terminal (CT) region of PF83/AMA-1, we developed a CT-capture-enzyme-linked immunosorbent assay to measure naturally acquired responses against the entire PF83/AMA-1 molecule. Analysis of populations from villages in Guinea-Bissau and in an area of high malarial transmission in Senegal demonstrated a very high prevalence (94-100%) of naturally acquired serum IgG responses to PF83/AMA-1. Analysis of these natural responses showed that PF83/AMA-1 may be a well-recognized asexual parasite antigen. A statistically significant age-related change in antibody levels to PF83/AMA-1 was observed in Guinea-Bissau. No such correlation was observed in the Senegalese population, although an age-related antibody response was seen for total parasite antigen. No significant correlation was observed between PF83/AMA-1 responses and the parameters of parasite load and malaria-related fever.

Adolescent↗
PubMed1993

Ion-exchange-immunoaffinity purification of a recombinant baculovirus Plasmodium falciparum apical membrane antigen, PF83/AMA-1.

A two-step purification regime has been developed for a quantitatively minor, putatively transmembrane, M(r) 83,000, apical membrane blood stage vaccine candidate antigen of Plasmodium falciparum (PF83/AMA-1), that has been expressed as a full-length baculovirus recombinant protein, PF83-7G8-1. The first step utilizes a new approach to high-performance ion-exchange chromatography (HPIEC) in which elution conditions are not only defined by charge, but also by hydrophobicity. HPIEC fractionation involves successive sodium chloride gradient anion-exchange elutions (A and B), where a change in the non-ionic detergent polyoxyethylenealkylether C10E5 concentration between elutions A and B (from 0.01% to 0.1% (w/v) respectively), results in a fraction that comprises from 2% to 9% PF83-7G8-1. Subsequent column immunoaffinity purification of this fraction on Q-Sepharose CL 4B-28G2dc1 mAb yields a PF83-7G8-1 preparation that is 56% pure. Rat mAb 28G2dc1 recognizes a C-terminal region that is conserved and cross reactive within the AMA-1 family, thus permitting recombinant and native full-length AMA-1 molecules from other species to be purified for molecular analysis. Immunological and molecular characterisation of the vaccine-related characteristics of purified PF83/AMA-1 are now underway.

Animals↗
PubMed1992

Environmentally directed mutations in the dehalogenase system of Pseudomonas putida strain PP3.

Favourable mutations involving the two dehalogenases (DehI and DehII) of Pseudomonas putida PP3 and derivative strains containing the cloned gene for DehI (dehI) occurred in response to specific environmental conditions, namely: starvation conditions; the presence of dehalogenase substrates (halogenated alkanoic acids--HAAs) which were toxic to P. putida; and/or the presence of a potential growth substrate. Fluctuation tests showed that these mutations were environmentally directed by the presence of HAAs. The mutations were associated with complex DNA rearrangements involving the movement of dehI located on a transposon DEH. Some mutations resulted in switching off the expression of either one or both of the dehalogenases, events which were effective in protecting P. putida from toxic compounds in its growth environment. Other mutations partially restored P. putida's dehalogenating capability under conditions where toxic substrates were absent. Restoration of the capability to untilize HAAs was favoured when normal growth substrates were present in the environment.

Culture Media↗