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Biomedical subjects

L M Thomas

Publications and source records attributed to L M Thomas.

At least 19 recordsLinked to original sources
PubMed1998

Development of a PCR test based on a gene region associated with the pathogenicity of Pasteurella multocida serotype B:2, the causal agent of haemorrhagic septicaemia in Asia.

We have developed a PCR assay to detect Pasteurella multocida serotype B:2, the causal agent of Haemorrhagic Septicaemia (HS) in Asia. Nucleotide sequence determination of a 16S rRNA-23S rRNA PCR product unique to B:2 strains was shown to share amino acid sequence homology with a bacteriophage Mu protein. Primers designed from this sequence when tested against a panel of isolates recovered from a wide geographical area and representing a large range of bacterial genera and species, were found to specifically amplify DNA from P. multocida, serotype B:2. Southern hybridisation confirmed the presence of this sequence in only the B:2 serotype of P. multocida, suggesting an association between bacterial virulence and the presence of bacteriophage genes in the bacterial genome. The results of this study demonstrate the potential application of PCR to the diagnosis of HS in cattle and buffalo in Asia. Application of PCR to support diagnosis of HS will greatly improve accuracy, laboratory response time, and will facilitate rational deployment of resources for controlling this disease.

Animals↗
PubMed1998

The specificity of antibody in chickens immunised to reduce intestinal colonisation with Campylobacter jejuni.

Poultry consumption has been identified as a major risk factor for human infection with Campylobacter jejuni in developed countries. C. jejuni is present in the gastrointestinal tract of broiler chickens at the time of slaughter, and faecal contamination of carcases during processing results in significant campylobacter loads on carcases. One approach to reducing the level of carcase contamination with C. jejuni is to control campylobacter infection in broiler chickens. To this end, the study described here investigated the specificity of antibody in serum and intestinal secretions of chickens that had been immunised with campylobacter antigens and then challenged with viable bacteria. The immunodominant antigens in the serum of birds that showed a 2-log reduction in caecal colonisation with C. jejuni included flagellin protein (61-63 Kd) and three additional antigens of 67, 73.5 and 77.5 Kd. Only flagellin and the 67 Kd antigen were recognised by IgG antibody in gastrointestinal secretions of the same birds. Antibody from chickens immunised with purified native flagellin protein recognised flagellin protein and the 67 Kd antigen in Western blots probed with serum, but only the flagellin proteins (61-63 Kd) in Westerns probed with gastrointestinal secretions. Analysis of the specificity of the response to flagellin protein using recombinant clones that expressed regions of the flagellin gene suggests that epitopes in each region of the flagellin protein were immunogenic. Of the immunodominant antigens, only flagellin appeared to be surface-exposed on viable C. jejuni, although conformational epitopes of flagellin appeared to be sensitive to the method of antigen purification. The results of this study suggest that flagellin and possibly the 67 Kd antigen may be valuable for immunological control of intestinal infection with C. jejuni in chickens, but that further work is required to purify these as vaccine candidates by using methods that preserve conformational epitopes.

Animals↗
PubMed1996

Marked enrichment of the alkenylacyl subclass of plasma ethanolamine glycerophospholipid with eicosapentaenoic acid in human subjects consuming a fish oil concentrate.

Alteration in human platelet fatty acid levels with the consumption of fish oils containing eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3) have been well documented, but changes in the fatty acid composition of plasma plasmalogenic phospholipid under similar circumstances have not been delineated. In the present study, subjects consumed the fish oil concentrate (MaxEPA) for 6 wk followed immediately by a 6-wk recovery period with no fish oil ingestion. Plasma total choline glycerophospholipid (GPC) and ethanolamine glycerophospholipid (GPE) subclasses isolated from blood samples obtained at 0, 3, 6, 9 and 12 wk of the experimental period were analyzed for fatty acid composition via thin-layer and gas-liquid chromatographic techniques. Consumption of fish oil for 3 or 6 wk significantly elevated the content of n-3 fatty acids while concomitantly decreasing n-6 fatty acid levels in plasma total GPC and in diacyl and alkenylacyl (plasmalogen) GPE. Alkenylacyl GPE exhibited the greatest alteration of both n-3 and n-6 fatty acid levels. Following 6 wk of supplementation with fish oil, EPA rose by 24.6 mol% in alkenylacyl GPE compared to increases of 6.7 and 7.1 mol% in diacyl GPE and total GPC, respectively. The increase in EPA (from 5.0 to 29.6 mol%) in plasma alkenylacyl GPE represents amongst the highest enrichment of EPA in any lipid yet reported in human subjects. DHA also rose by 8.0, 4.8, and 3.1 mol% in alkenylacyl GPE, diacyl GPE, and total GPC, respectively. Alkenylacyl GPE exhibited the greatest mol% decline (by 18.7 mol%) in arachidonic acid (AA, 20:4n-6) following 6 wk of fish oil supplementation. The corresponding decreases of AA in diacyl GPE and total GPC were 8.7 and 1.8 mol%, respectively. Following the 6 wk recovery period, n-3 and n-6 fatty acid levels had returned to pre-supplementation values. The marked enrichment of alkenylacyl GPE in n-3 fatty acids, especially EPA, may be of significance with respect to a unique role for this plasma phospholipid subclass in attenuating certain lipoprotein-mediated cardiovascular effects as observed with fish/fish oil consumption.

Acylation↗
PubMed1994

A new class of substituted 1,2,4-triazolo-1,3,4-thiadiazepines.

A series of 3-aryloxymethyl-(5-nitro-2-furyl)-6-phenyl-1,2,4- triazolo[3,4-b][1,3,4]thiadiazepine compounds have been synthesized recently by a new route. Reported here are the structures of two such compounds with para-substituted aryloxymethyl groups: one has a chloro group, 3-(4-chlorophenyl-oxymethyl)-8-(5-nitro-2-furyl)-6-phenyl-1,2,4- triazolo[3,4-b][1,3,4]thiadiazepine, C22H14C1N5O4S, TD1, and the other a methyl group, 3-(p-tolyloxy-methyl)-8-(5-nitro-2-furyl)-6-phenyl-1,2,4- triazolo-[3,4-b][1,3,4]thiadiazepine, C23H17N5O4S, TD7. The nitrofuryl and phenyl groups on the thiadiazepine ring are each found to adopt a similar conformation in the two structures, whereas the aryloxymethyl substituents on the triazole rings are conformationally different from each other. Each thiadiazepine ring adopts a boat conformation with the S atom at the apex. The interplanar angle between the triazole ring and the thiadiazepine ring is 30 degrees for both compounds. The conformation of the aryloxy-methyl group is dependent on the intermolecular interactions that arise as a result of the polarity of the para substituent. The Cl group in TD1 is involved in a C-Cl...O non-bonded interaction with a Cl...O distance of 3.100 (3) A and a C-Cl...O angle of 138.4 (1) degree. TD7 has a stacking interaction involving the nitrofuryl groups.

Crystallography, X-Ray↗
PubMed1994

Crystal structure of cyclo(Pro-Gly)3:Li complex: a model for ion transport by cyclo(Pro-Gly)3.

The crystal structure of cyclo(Pro-Gly)3 (PG3) complex with LiSCN (C22H30N7O6SLi) has been solved by x-ray diffraction. The crystals belong to the space group R3 in the hexagonal setting with unit cell parameters of a = 12.581(1), c = 29.705(3) A, V = 4072.0 A3, Z = 6, M(r) = 527.53, Dc = 1.23 g/cm3. The crystal structure was solved by direct methods using the program SHELXS-86 and refined to an R value of 5.3% for 1645 reflections (I > 2 sigma I). There are two conformers in the crystal structure. One conformer has three carbonyls on one side and three on the other side of the peptide plane. The other conformer has all six of the carbonyls on the same side of the peptide plane. Both of these conformers bind independently to a Li ion. Based on the conformers of the Li complex and other reported ion complexes formed by PG3, we propose a model for the transport of ions across the lipid membrane. The features of the model are as follows: (1) PG3 forms a hexameric stack in a lipid bilayer when complexing and transporting metal ions. (2) It undergoes a conformational flipping in order pass the ion along the channel. The energy required for the conformational change involved in the flipping of the PG3 molecule may be provided by the applied potential during ion transport.

Biological Transport, Active↗
PubMed1994

Structural characteristics of diproline: a new crystal form of tBoc-Pro-Pro-OH.

The structure of a new crystalline form of tBoc-Pro-Pro-OH (C15 H24 N2 O5) has been determined. The crystals were monoclinic, P2(1), a = 14.667(5), b = 16.600(4), c = 15.502(3) A, beta = 117.84(2) degrees, V = 3337.2 A3 and Z = 8, Dc = 1.24 g/cm3. There are four molecules in the asymmetric unit, each displaying polyproline-type structure but differing in the proline pucker. All four molecules display a twist conformation in the first proline ring, with molecules A, B and C being beta gamma T (P approximately 183 degrees, tau approximately 33 for A and B, tau approximately 18 for C) and molecule D between gamma beta T and gamma E (P = 10 degrees, tau approximately 38). The second residue of all four molecules has an envelope conformation. Molecules A and B display an alpha E conformation (P approximately 126 degrees, tau approximately 25) and molecules C and D display a beta E conformation (P approximately 168 degrees, tau approximately 37). The molecules are hydrogen-bonded (O...OH), forming helical channels along the alpha-axis.

Amino Acid Sequence↗
PubMed1993

A comparison of ELISA, FAST-ELISA and gel diffusion tests for detecting antibody to equine infectious anaemia virus.

Sera of sixteen horses with clinical signs of EIA from six different outbreaks and sera of 100 uninfected horses were used to validate an ELISA for EIA diagnosis. The antigen used was a recombinant protein derived from the amino-terminal portion of the transmembrane envelope protein of EIA (gp45). Reactivity between positive and negative sera could be clearly distinguished. Comparison with the traditional agar gel immunodiffusion test (commonly called the Coggins test) showed that the ELISA was superior in sensitivity. Comparison of this ELISA with the FAST-ELISA system showed that the latter was less sensitive. Although the FAST-ELISA was much faster to perform, it could not be recommended as a diagnostic test in its present form, because the margin between reactivity by a positive serum and a negative serum was not high.

Animals↗
PubMed1993

Structural characteristics of human salivary statherin: a model for boundary lubrication at the enamel surface.

A three-dimensional structural model for salivary statherin in aqueous phase has been developed using structure prediction, circular dichroism, molecular modeling, and mechanics. The relevant structural features of statherin are N-terminal helix segment connected to a long poly-L-proline type II segment, which is followed by a short extended structure. Using this model, the hydroxyapatite binding ability of statherin has been explained. The hydroxyapatite binding region is comprised of the N-terminal acidic residues (Asp-pSer-pSer-Glu-Glu) and Glu-26, which are clustered together in space. Partial conformational unfolding and oriented aggregation of several statherin molecules at the enamel surface provides an amphipathic film that is responsible for the boundary lubrication exhibited by statherin.

Actinomyces viscosus↗
PubMed1992

A soluble recombinant fusion protein of the transmembrane envelope protein of equine infectious anaemia virus for ELISA.

The use of the bacterial expression vector, pGex, to produce an abundant, soluble fusion protein of gp45 from equine infectious anaemia virus is described. Purification of the recombinant protein was achieved by one step affinity chromatography on immobilized glutathione using competitive elution so no harsh conditions were required. This provides a readily available antigen that is defined, plentiful and cheap. Yields of 3.5 mg of purified soluble protein/litre of bacterial culture were obtained. This antigen was found to be suitable for ELISA. Background reactivity to either the glutathione-S-transferase (GST) fusion partner by immune sera or the EIA-GST fusion protein by normal sera were negligible.

Amino Acid Sequence↗
PubMed1991

Recombinant fusion proteins of protein A and protein G with glutathione S-transferase as reporter molecules.

The regions encoding the IgG-binding domains of protein A (PA) and protein G (PG) were cloned into the bacterial expression vector pGEX. Both proteins were expressed in Escherichia coli as fusion proteins with glutathione S-transferase (PA-GST and PG-GST) and were found to be soluble, abundant and easily purified in one step from the bacterial lysate by affinity chromatography on immobilized glutathione. Yields of 50 mg/litre of cultures were obtained. Both purified fusion proteins were shown to be functional in a variety of immunochemical procedures. In radial diffusion tests, PA-GST precipitated IgG from human, squirrel monkey, rabbit, dog, cat and pig but not mouse, sheep, goat, cow, horse or chicken. PG-GST formed precipitin bands with IgG from human, rabbit, mouse, pig, sheep, goat, cow and horse but not squirrel monkey, dog, cat and chicken IgG. The fusion proteins were shown to function as effective detection reagents in ELISA and Western blotting. Glutathione agarose beads with bound fusion protein were shown to be useful for immunoprecipitation.

Base Sequence↗
PubMed1991

Eicosanoid-dependent and -independent formation of individual [14C]stearoyl-labelled lysophospholipids in collagen-stimulated human platelets.

Low level collagen activation of platelets is mediated via the release of arachidonic acid (AA) from membrane phospholipids and the formation of thromboxane A2 (TxA2). To assess the specific phospholipids undergoing deacylation via phospholipase A2 thereby providing source(s) of releasable AA, we have measured the individual lysophospholipid formations in platelets prelabelled with [14C]stearic acid and incubated with a low level (2 micrograms/ml) or a high level (10 micrograms/ml) of collagen in the absence or presence of BW755C, a dual inhibitor of cyclooxygenase and lipoxygenase activities. Collagen activation resulted in the generation of [14C]stearoyl-labelled lysophosphatidylinositol (lysoPI), lysophosphatidylcholine (lysoPC), lysophosphatidylethanolamine (lysoPE) and lysophosphatidylserine. BW755C significantly inhibited these collagen-induced changes, suggesting that much of the lysophospholipid, and therefore AA release, was eicosanoid-mediated. At the lower level of collagen, considerable generation of [14C]lysoPE was maintained even in the presence of BW755C, suggesting an eicosanoid-independent degradation of phosphatidyl-ethanolamine. The TxA2-dependent release of AA was also investigated in U-46619-stimulated platelets. This TxA2 mimetic induced considerable formation of the 14C-labelled lysophospholipids, including lysoPI and lysoPC, but not lysoPE. These results suggest that an eicosanoid-independent degradation of phosphatidylethanolamine via phospholipase A2 at lower collagen levels may provide a source of the initial AA for conversion to TxA2 and the subsequent deacylation of phosphatidylinositol, phosphatidylcholine, and also phosphatidylserine.

4,5-Dihydro-1-(3-(trifluoromethyl)phenyl)-1H-pyraz↗
PubMed1991

BW755C or staurosporine inhibits collagen-stimulated phosphoinositide phosphorylation in platelets.

Stimulation of platelets by collagen results in increased formation of the polyphosphoinositides, phosphatidylinositol phosphate (PtdInsP) and phosphatidylinositol bisphosphate (PtdInsP2) through stimulation of phosphoinositide kinase activities. We investigated a possible regulatory role of endogenous thromboxane formation and protein kinase C (PKC) activation in the induction of phosphoinositide phosphorylation following collagen stimulation, as well as following stimulation by the thromboxane mimetic, U-46619. Human platelets were prelabeled with [3H]inositol and stimulated with collagen (2 micrograms/mL) or U-46619 (1 microM), in the absence or presence of either the cyclo-oxygenase/lipoxygenase inhibitor, BW755C, or staurosporine, a putative inhibitor or PKC. Collagen stimulation resulted in a time-dependent increase in [3H]inositol-labeled PtdInsP and PtdInsP2 which was completely inhibited in the presence of BW755C. Addition of U-46619 to BW755C-treated, collagen-stimulated platelets restored the increased polyphosphoinositide formation. Stimulation of platelets with U-46619 alone also resulted in increased formation of [3H]PtdInsP and [3H]PtdInsP2, but this was not affected by the presence of BW755C. These results suggest that the collagen-induced activation of phosphoinositide kinases was dependent upon thromboxane formation, but that U-46619-induced phosphoinositide formation was rather independent of further thromboxane production. Pretreatment of platelets with staurosporine, prior to agonist addition, completely blocked the collagen-stimulated rise in radiolabeled PtdInsP and the U-46619-induced PtdInsP and PtdInsP2 generations, suggesting that protein kinase, possibly PKC, may play a role in the activation of phosphoinositide kinases by these agonists.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1991

Inhibition of phosphatidic acid production and lysophospholipid formation in collagen-stimulated human platelets by staurosporine, a protein kinase inhibitor.

To investigate a possible regulatory role of protein kinase C (PKC) on collagen-induced phospholipase activity, human platelets were prelabelled with either [3H] arachidonic acid or [14C]stearic acid and stimulated with collagen (2 micrograms/ml) in the presence or absence of the protein kinase inhibitor, staurosporine (1 microM). The collagen-induced release of [3H]arachidonic acid and formation of [14C]stearoyl-labelled lysophospholipids was inhibited by prior incubation with staurosporine, as was the formation of 3H-labelled thromboxane B2, thereby suggesting inhibition of the collagen-induced phospholipase A2 activity. The degradation of phosphatidylinositol (PI) and elevation of phosphatidic acid (PA) in platelets prelabelled with either radiotracer were also completely blocked by staurosporine pretreatment, indicating a suppression of collagen-stimulated phospholipase C activity. Suppressed phospholipase C activity may have been due to diminished thromboxane A2 formation since treatment with the dual cyclo-oxygenase/lipoxygenase inhibitor, BW755C, also resulted in an inhibition of the collagen-stimulated loss of 14C-labelled PI and rise in PA by 75-80%. Our results suggest that protein kinase, possible PKC, may be involved in the regulation of these phospholipases in collagen-stimulated human platelets.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1987

The formation of lysophosphatidylinositol phosphate in human platelet microsomes.

The formation of lysophosphatidylinositol phosphate (lysoPIP) from lysophosphatidylinositol (lysoPI) via kinase activity was studied in microsomal preparations from human platelets. For this purpose, [3H]lysoPI or [3H]phosphatidylinositol ([3H]PI) was prepared and incubated in the presence or absence of ATP, MgCl2 and Triton X-100, and the appearances of radioactivity in [3H]lysoPIP and [3H]phosphatidylinositol phosphate ([3H]PIP), respectively, were monitored using thin layer chromatography. Both lysoPI and PI phosphorylations were completely dependent upon the presence of ATP and MgCl2 in the incubation medium; Triton X-100 addition stimulated both reactions, with the stimulation of PI conversion being considerably greater than that for lysoPI can be converted to lysoPIP by phosphorylation in human platelet microsomes. The potential significance of this enzymatic reaction in stimulated cells is discussed in relation to the generation of inositol-1,4,5-trisphosphate, an important intracellular second messenger.

Blood Platelets↗
PubMed1987

Temporalis muscle-galea flap in craniofacial reconstruction.

With the advent of increasing technological and surgical sophistication in craniofacial surgery, reconstructive efforts are challenged to provide a reliable means of compartmentalization. When dural integrity is compromised in the face of nasopharyngeal or paranasal communication, the risk of ascending infection and potential life-threatening meningitis mandate cranial and facial compartments, separated by sufficient and healthy soft tissues. This paper describes a method of providing pedicled soft tissue coverage and support for the contents of the anterior cranial fossa using a temporalis muscle-galea rotation flap. The vascularized myofascial tissues, capable of carrying skin and bone grafts, are well suited to cover and protect large areas of the skull base. Several cases are described to show the advantages and disadvantages of the technique.

Adult↗