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Biomedical subjects

S J Ward

Publications and source records attributed to S J Ward.

At least 37 records · Page 2Linked to original sources
PubMed1999

Characterization of Helicobacter pylori PldA, a phospholipase with a role in colonization of the gastric mucosa.

BACKGROUND & AIMS: Phospholipase activity may play a role in the pathogenicity of Helicobacter pylori. Furthermore, some drugs that are effective against H. pylori infection are phospholipase inhibitors. Scrutiny of the H. pylori 26695 genome sequence revealed the presence of a putative protein with homology to Esherichia coli outer membrane phospholipase A (PldA). The aim of this study was to investigate the role of this putative PldA in the pathogenicity of H. pylori. METHODS: An isogenic pldA mutant was constructed and analyzed for in vitro phospholipase A(2) and hemolytic activity. Adherence of the mutant to human gastric adenocarcinoma cells and the ability to colonize mice were also investigated. RESULTS: The pldA mutant showed a marked reduction in phospholipase A(2) and hemolytic activity compared with the wild-type strain. The mutant was unable to colonize mice at 2 and 8 weeks, but it did induce a significant immune response. In contrast, the ability of the mutant to adhere to human gastric adenocarcinoma cells was unaffected. CONCLUSIONS: The results suggest a role for PldA in colonization of the gastric mucosa and possibly tissue damage after colonization.

Animals↗
PubMed1999

Retinoids and mammalian development.

All vertebrate embryos require retinoic acid (RA) for fulfilment of the developmental program encoded in the genome. In mammals, maternal homeostatic mechanisms minimize variation of retinoid levels reaching the embryo. Retinol is transported as a complex with retinol-binding protein (RBP): transplacental transfer of retinol and its uptake by the embryonic tissues involves binding to an RBP receptor at the cell surface. Embryonic tissues in which this receptor is present also contain the retinol-binding protein CRBP I and the enzymes involved in RA synthesis; the same tissues are particularly vulnerable to vitamin A deficiency. In the nucleus, the RA signal is transduced by binding to a heterodimeric pair of retinoid receptors (RAR/RXR). In general, the receptors show functional plasticity, disruption of one RAR or RXR gene having minor or no effects on embryogenesis. However, genetic studies indicate that RXR alpha is essential for normal development of the heart and eye. Excess RA causes abnormalities of many systems; altered susceptibility to RA excess in mice lacking RAR gamma or RXR alpha suggests that the teratogenic signal is transduced through different receptors compared with physiological RA function in the same tissue.

Animals↗
PubMed1999

Local and systemic neutralizing antibody responses induced by intranasal immunization with the nontoxic binding domain of toxin A from Clostridium difficile.

Fourteen of the 38 C-terminal repeats from Clostridium difficile toxin A (14CDTA) were cloned and expressed either with an N-terminal polyhistidine tag (14CDTA-HIS) or fused to the nontoxic binding domain from tetanus toxin (14CDTA-TETC). The recombinant proteins were successfully purified by bovine thyroglobulin affinity chromatography. Both C. difficile toxin A fusion proteins bound to known toxin A ligands present on the surface of rabbit erythrocytes. Intranasal immunization of BALB/c mice with three separate 10-microg doses of 14CDTA-HIS or -TETC generated significant levels of anti-toxin A serum antibodies compared to control animals. The coadministration of the mucosal adjuvant heat labile toxin (LT) from Escherichia coli (1 microg) significantly increased the anti-toxin A response in the serum and at the mucosal surface. Importantly, the local and systemic antibodies generated neutralized toxin A cytotoxicity. Impressive systemic and mucosal anti-toxin A responses were also seen following coadministration of 14CDTA-TETC with LTR72, an LT derivative with reduced toxicity which shows potential as a mucosal adjuvant for humans.

Administration, Intranasal↗
PubMed1999

Immunogenicity of a Salmonella typhimurium aroA aroD vaccine expressing a nontoxic domain of Clostridium difficile toxin A.

The C-terminal repeat domain of Clostridium difficile toxin A harbors toxin-neutralizing epitopes and is considered to be a candidate component of a vaccine against C. difficile-associated disease (CDAD). Fourteen of the 38 C-terminal toxin A repeats (14CDTA) were cloned into pTECH-1 in frame with the immunogenic fragment C of tetanus toxin (TETC) to generate plasmid p56TETC. Expression of the TETC-14CDTA fusion protein was driven from the anaerobically inducible nirB promoter within attenuated Salmonella typhimurium BRD509 (aroA aroD). The TETC-14CDTA fusion protein was purified and shown to bind to known toxin A receptors found on the surface of rabbit erythrocytes. Intranasal (i.n.) and intragastric (i.g.) immunization with 10(7) and 10(10) CFU, respectively, of BRD509(p56TETC) generated significant (P < 0.05) anti-toxin A serum responses after a single dose. Antibody titers were elevated following a boosting dose with either live vaccine or a subcutaneous injection of 0.5 microgram of purified 14CDTA protein. Importantly, serum from mice immunized with BRD509(p56TETC) neutralized toxin A cytotoxicity. Both i.n. and i.g. immunizations also generated toxin A-specific immunoglobulin A on the pulmonary and intestinal mucosa, respectively. Intranasal vaccination induced consistently higher serum and mucosal anti-toxin A antibody responses. Significant anti-tetanus toxoid serum and mucosal antibodies were also generated by both immunization routes. The availability of live attenuated Salmonella typhi for human use may allow the development of a multivalent mucosal vaccine against CDAD, tetanus, and typhoid.

Administration, Intranasal↗
PubMed1999

Induction for senior house officers. Part I: The hospital programme.

The study was set up to examine how an induction programme can be designed to meet the needs and expectations of senior house officers (SHOs) beginning a new post in a hospital. A total of 63 SHOs, in five hospitals in the Anglia region, participated in standardised structured interviews up to 3 months after starting their post, by sorting a list of 38 possible induction topics in order of priority. Trainees' early induction needs were found to centre on day-one service activities, i.e., information that enables them to undertake their service work efficiently and effectively, is particularly valued and required early. Information related to clinical education and training is also valued, but many trainees prefer it to be provided after they have been in post for a week or two. Much induction information can also be effectively conveyed in writing, with little or no need for additional formal presentation. We conclude that first-day induction programmes which strive to satisfy the range of trainee needs in one session fail to take account of the different priorities that trainees attach to induction information. Trainees do not want all information at once. Information of prime concern to clinical tutors, namely education, training and generic skills, is generally favoured after a week or two. To ensure that trainees' hospital induction needs are met in an efficient and effective way, programmes must be flexible and timely, and supported by comprehensive written information.

Education, Medical, Graduate↗
PubMed1999

Induction for senior house officers. Part II: The departmental programme.

This study was designed to examine the content and usefulness of departmental induction programmes to senior house officers (SHOs) and to explore perceptions of the usefulness of a range of topics to these trainees. A total of 64 SHOs, in five hospitals in the Anglia region, participated in semi-structured interviews up to 3 months after starting their post. The interviews investigated the content of any induction received and also examined what the trainees would have liked to have received. Almost half (29) of the SHOs also completed a questionnaire which examined the perceived usefulness of various induction topics for a trainee starting a new post. The results showed that, although a departmental induction programme is considered important and highly valued, a substantial minority of SHOs had not received one. Of this minority, 75% would have welcomed an induction. Where an induction had taken place, the focus was primarily upon timetables, tours and meeting people, however, a quarter of the questionnaire sample had not received a service timetable, a third had not met any consultants, two-thirds had received no introduction to clinical management, and two-thirds had received no information about how their consultants manage their patients. Thus, although many trainees receive an induction, important information may not be covered. A staged approach to departmental induction is recommended, using a short, high-quality, and comprehensive induction programme. Flexible and timely programmes, supported by comprehensive written information, will contribute to meeting the needs of trainees in an efficient and effective way.

Education, Medical, Graduate↗
PubMed1998

Prenatal bias in sex ratios in a marsupial, Antechinus agilis.

Biased sex ratios of young of birds and mammals clearly occur and may have an adaptive significance, but we rarely know the stage at which a bias is generated, or the mechanism. If a bias is generated prior to birth, studies of marsupials may be insightful because gestation is short and neonates are relatively undifferentiated. This study investigated whether biased sex ratios in Antechinus agilis are generated in the brief period between birth and the attachment of young to the mother's teats. When all young born, or just pouch young, or supernumerary young were considered, litters were strongly biased towards females (0.32 males), and there was no significant difference across the groups, so a bias is generated before birth in this species. Evidence from counts of corpora lutea suggests that embryo loss during gestation cannot account fully for the level of bias observed. Therefore, prefertilization mechanisms must contribute to the generation of sex-biased litters in this marsupial.

Animals↗
PubMed1998

Three-dimensional quantitative structure-activity relationship study of the cannabimimetic (aminoalkyl)indoles using comparative molecular field analysis.

The present study describes the implementation of comparative molecular field analysis (CoMFA) to develop two 3D-QSAR (quantitative structure-activity relationship) models (CoMFA models 1 and 2) of the cannabimimetic (aminoalkyl)indoles (AAIs) for CB1 cannabinoid receptor binding affinity, based on pKi values measured using radioligand binding assays that displace two different agonist ligands, [3H]CP-55940 and [3H]WIN-55212-2. Both models exhibited a strong correlation between the calculated steric-electrostatic fields and the observed biological activity for the respective training set compounds. In light of the basicity of the morpholine nitrogen in the AAIs, separate CoMFA models were built for the AAIs as unprotonated and protonated species. Comparison of the statistical parameters resulting from these CoMFA models failed to provide unequivocal evidence as to whether the AAIs are protonated or neutral as receptor-bound species. Although the training sets of CoMFA model 1 and CoMFA model 2 differed with respect to composition and to the choice of displacement radioligand in each biological assay, their CoMFA StDevCoeff contour plots reveal similarities in terms of identifying those regions around the AAIs that are important for CB1 cannabinoid receptor binding such as the sterically favored region around the C3 aroyl group and the sterically forbidden region around the indole ring. When the experimental pKi values for the training set compounds to displace the AAI radioligand [3H]WIN-55212-2 were plotted against the pKi values as predicted for the same compounds to displace the cannabinoid radioligand [3H]CP-55940, the correlation was moderately strong (r = 0.73). However, the degree of correlation may have been lowered by the structural differences in the compounds comprising the training sets for CoMFA model 1 and CoMFA model 2. Taken together, the results of this study suggest that the binding site region within the CB1 cannabinoid receptor can accommodate a wide range of structurally diverse cannabimimetic analogues including the AAIs.

Animals↗
PubMed1998

Effects of dopamine antagonists and accumbens dopamine depletions on time-constrained progressive-ratio performance.

Four experiments were conducted to determine the effects of dopamine (DA) antagonists and DA depletions on progressive-ratio responding for food reinforcement. On this schedule, ratio requirement increased by one response after each reinforcer was obtained, and rats were tested in 30-min sessions. Response rates and highest ratio completed were reduced in a dose-related manner by systemic injections of the D1 antagonist SCH 23390, and also by the D2 antagonists haloperidol and raclopride. Drug-treated rats also showed reductions in time to complete the last ratio, demonstrating that they had stopped responding before the end of the session. DA depletions produced by injections of 6-OHDA directly into the nucleus accumbens substantially decreased both the number of responses and the highest ratio completed. The deficits in response number and highest ratio completed induced by DA depletions persisted through the first 3 weeks of postsurgical testing, with some recovery by the fourth week. However, the deficits resulting from dopamine depletions were largely a manifestation of a decrease in response rate; although time to complete the last ratio was significantly reduced by dopamine depletions in the first few days of testing, rats recovered on this measure by the fifth day after surgery. Although previous work has shown that performance on several schedules (e.g., continuous, low value ratios, variable interval) is relatively unaffected by accumbens DA depletions, the present data demonstrate that such depletions do produce a substantial and persistent impairment of progressive ratio response output. Rats with accumbens DA depletions appear to have deficits in maintaining the high work output necessary for responding at large ratio values. The relative sparing of responding on some simple schedules, together with the present progressive ratio results, suggest that rats with accumbens DA depletions remain directed toward the acquisition and consumption of food, but they show deficits in work output for food.

Animals↗
PubMed1997

A flagellar-specific ATPase (FliI) is necessary for flagellar export in Helicobacter pylori.

Although flagellar motility is essential for the colonisation of the stomach by Helicobacter pylori, little is known about the regulation of flagellar biosynthesis in this organism. We have identified a gene in H. pylori, designated fliI, whose deduced amino acid sequence revealed extensive homology with the FliI/LcrB/InvC family of proteins which energise the export of flagellar and other virulence factors in several bacterial species. An isogenic mutant of fliI was non-motile and synthesised reduced amounts of flagellin and hook protein subunits. The majority (> 99%) of mutant cells were completely aflagellate. These results suggest that FliI is a novel ATPase involved in flagellar export in H. pylori.

Adenosine Triphosphatases↗
PubMed1997

Differential expression of transcripts encoding retinoid binding proteins and retinoic acid receptors during placentation of the mouse.

We report the distribution of transcripts from genes encoding the retinol binding protein (RBP), the cellular retinol binding proteins (CRBP I, II) and retinoic acid binding proteins (CRABP I, II), the retinaldehyde dehydrogenase type 2 (RALDH-2), the retinoic acid receptors (RARs), and the retinoid X receptors (RXRs) in mouse placental tissues from 6.5 to 19.5 days postcoitum (dpc). During early placentation, RBP and RALDH-2 gene expression are restricted to the endoderm of the visceral yolk sac and the outer uterine epithelium, respectively, whereas CRBP I transcripts are detected in the visceral yolk sac and in the presumptive chorioallantoic placenta. By 15.5 dpc, CRBP I expression is down-regulated in the yolk sac where CRBP II becomes strongly expressed. Expression of CRBP II is also detected in the trophoblastic giant cells. Throughout placentation, the expression patterns of the CRABP I and II genes partly overlap in the decidual tissue and the vacuolar zones of the decidua, suggesting a role for these binding proteins in sequestering free retinoic acid from maternal blood, thus regulating its availability to the embryo. RAR alpha is ubiquitously expressed in all placental tissues, except in trophoblastic giant cells, at all stages studied. During early placentation, RAR beta and RAR gamma are co-expressed in the decidua but differentially expressed in the chorionic region (RAR beta, 10.5 to 12.5 dpc) and the presumptive labyrinth (RAR gamma, 7.5 to 12.5 dpc). During the same stages, RXR alpha is strongly expressed in the presumptive placenta. RAR gamma remains weakly expressed in the labyrinth until 15.5 dpc, whereas RXR alpha exhibits a strong expression in this zone until birth, suggesting a role for these receptors in the development and function of the definitive placenta.

Animals↗
PubMed1997

The functional basis of tissue-specific retinoic acid signalling in embryos.

Retinoic acid (RA) is essential for normal embryonic development. In mammals it is sequestered from the maternal circulation in the form of retinol. In rodents, embryonic uptake relies upon the presence of retinol binding protein (RBP) in the yolk sac, probably involving an RBP receptor. The molecular activity of RA in the nucleus is well established, but less is known about cytoplasmic events including tissue-specific intraembryonic RA synthesis and intracellular transport of both retinol and RA. The cellular binding proteins for retinol and RA may play important roles in these processes.

Journal Article↗
PubMed1997

Identification of a mechanism to localize generation of retinoic acid in rat embryos.

Vitamin A (retinol) is essential for normal mammalian development. However, its biological activity depends upon its conversion to retinoic acid (RA), a local mediator of cellular proliferation and differentiation. Previous studies have shown that embryonic RA is found specifically in tissues known to depend upon vitamin A for normal development and that its production follows uptake of maternal retinol. The aim of this study was to identify the mechanism for tissue-specific generation of RA in developing rat embryos. Here we show immunohistochemical localization of the retinol binding protein receptor, cellular retinol binding protein, retinol dehydrogenase and retinal dehydrogenase in rat embryos (presomitic to the 25-30 somite pair stage). These proteins are proposed to be responsible for cellular uptake of retinol, its intracellular transport and its conversion to RA. Thus, they potentially constitute the entire metabolic pathway from vitamin A to RA. All four proteins were detected specifically in tissues that are known to depend upon vitamin A for normal development including the yolk sac, heart, gut, notochord, somites, sensory placodes and the limb. Furthermore, our previous studies have demonstrated that uptake of retinol into the yolk sac depends upon a retinol binding protein receptor. Here we provide evidence that this mechanism functions also in the heart. Colocalization of cellular retinol binding protein, retinol and retinal dehydrogenase with the retinol binding protein receptor in tissues dependent upon vitamin A for normal development suggests that coordinate functioning of these proteins is responsible for cellular uptake of circulating retinol and its metabolism to RA. This is the first evidence of a tissue-specific mechanism for generation of RA from its precursor retinol in the developing embryo.

Alcohol Oxidoreductases↗
PubMed1997

A retinol-binding protein receptor-mediated mechanism for uptake of vitamin A to postimplantation rat embryos.

The major form of dietary vitamin A in blood is retinol bound to retinol-binding protein (RBP), but it is not clear how maternal retinol reaches the embryo. We demonstrate here that uptake of radiolabeled retinol from holo-RBP to the visceral yolk sac of cultured rat embryos exhibits receptor-mediated characteristics (Km = 4.1 x 10(-7) M, Vmax = 3.0 x 10(-12) mol/sec per gram yolk sac protein) and also demonstrate the presence in the yolk sac endoderm of a protein previously described as an RBP receptor. Furthermore, transport of radiolabeled retinol from the yolk sac to the embryo proper can be markedly decreased by inhibition of visceral yolk sac RBP synthesis. Thus embryonic accumulation of retinol depends on a relay of retinol from maternal RBP to RBP synthesized in the yolk sac, via interaction with a visceral yolk sac RBP receptor.

Animals↗