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Biomedical subjects

S M Kelly

Publications and source records attributed to S M Kelly.

At least 109 records · Page 6Linked to original sources
PubMed1994

Development of recombinant Salmonellae expressing hybrid hepatitis B virus core particles as candidate oral vaccines.

This paper provides a review on the development of hepatitis core antigen as a vaccine carrier moiety and the use of recombinant Salmonella vaccine strains expressing hybrid HBcAg particles as live oral vaccines. Salmonella spp. can be attenuated by defined genetic means so that they become avirulent, yet preserve invasiveness after oral uptake. Oral immunization of mice with such avirulent candidate Salmonella typhimurium vaccine strains elicited serum antibody responses against a limited number of bacterial antigens. A highly immunogenic viral nucleocapsid antigen, hepatitis B virus core antigen (HBcAg) that can be expressed in prokaryotes was used as a carrier moiety for B-cell epitopes. Insertion sites with an enhanced immunogenicity for the carried epitopes were defined using HBV envelope protein virus neutralizing epitopes. An internal insertion site in HBcAg was found that drastically enhanced the immunogenicity of the foreign (pre-S1) epitope while reducing the immunogenicity of the carrier protein. Internally fused HBc/pre-S hybrid particles were expressed in Salmonella typhimurium and S. typhi vaccine strains. A single oral immunization of mice with such live recombinant S. typhimurium strains elicited a high titred serum anti-pre-S1 IgG response. Similarly, circumsporozoite repeat epitopes of three different malaria parasites were expressed as HBcAg/CS hybrids in recombinant S. spp. and were found to be highly immunogenic.

Administration, Oral↗
PubMed1994

Recombinant Salmonella vectors in vaccine development.

A diversity of means are available for the attenuation of Salmonella which can be used to immunize animals and humans orally to elicit mucosal, humoral and cellular immune responses. Avirulent Salmonellae can be genetically engineered to express foreign antigens and the recombinant avirulent Salmonellae are capable of stable, high-level expression of the foreign antigen in the orally immunized animal or human host. The resulting vaccines are safe, efficacious, and are easy and economical to use.

Administration, Oral↗
PubMed1993

Dissociation of the tetrameric phosphoglycerate mutase from yeast by a mutation in the subunit contact region.

Phosphoglycerate mutases from different sources exhibit a variety of quaternary structures (tetramer, dimer and monomer). To perturb the tetrameric structure of yeast phosphoglycerate mutase we have prepared a mutant enzyme in which Lys-168 in the subunit-contact region has been replaced by proline. The K168P mutant enzyme undergoes dissociation to dimers at low concentrations; thus on lowering the concentration from 200 micrograms/ml to 5 micrograms/ml the proportion of tetramer falls from 85% to 53%. The tetrameric structure of the wild-type enzyme remains intact over this range of concentrations. The mutant enzyme has similar kinetic properties to the wild-type enzyme, with kcat. being reduced by 26%. Far-u.v. c.d. studies show that there has been a small loss of helical structure in the mutant. Compared with wild-type enzyme, the K168P mutant enzyme is slightly less stable towards proteolysis by trypsin, but significantly less stable towards denaturation by guanidinium chloride, with the midpoint concentration of guanidinium chloride some 50% lower. After denaturation, the mutant enzyme could regain activity and quaternary structure when the guanidinium chloride concentration was lowered to 0.05 M. The properties of the mutant enzyme are discussed in terms of other dimeric phosphoglycerate and bisphosphoglycerate mutases which contain proline at position 168.

Circular Dichroism↗
PubMed1993

Characterization of the type I dehydroquinase from Salmonella typhi.

The type I dehydroquinase from the human pathogen Salmonella typhi was overexpressed in an Escherichia coli host and purified to homogeneity. The S. typhi enzyme was characterized in terms of its kinetic parameters, important active-site residues, thermal stability and c.d. and fluorescence properties. In all important respects, the enzyme from S. typhi behaves in a very similar fashion to the well-characterized enzyme from E. coli, including the remarkable conformational stabilization observed on reduction of the substrate/product mixture by NaBH4. This gives confidence that the information from X-ray studies on the S. typhi enzyme [Boys, Fawcett, Sawyer, Moore, Charles, Hawkins, Deka, Kleanthous and Coggins (1992) J. Mol. Biol. 227, 352-355] can be applied to other type I dehydroquinases. Studies of the quenching of fluorescence of the S. typhi enzyme by succinimide show that NaBH4 reduction of the substrate/product imine complex involves a dramatic decrease in the flexibility of the enzyme, with only very minor changes in the overall secondary and tertiary structure.

Circular Dichroism↗
PubMed1993

An interaction of beta-amyloid with aluminium in vitro.

We have used circular dichroism spectroscopy to confirm that, in a membrane-mimicking solvent, A beta P(1-40) adopts a partially helical conformation and we have demonstrated the loss of this structure in the presence of physiologically relevant concentrations of aluminium. This is the first evidence of a direct biochemical interaction between aluminium and beta-amyloid and may have important implications for the pathogenesis of Alzheimer's disease.

Aluminum↗
PubMed1993

Substitution of His-181 by alanine in yeast phosphoglycerate mutase leads to cofactor-induced dissociation of the tetrameric structure.

The structure and stability of a mutated yeast phosphoglycerate mutase in which His-181 has been replaced by alanine have been studied. The secondary, tertiary and quaternary structures of the mutant enzyme in the absence of ligands are essentially identical to those of the wild-type enzyme as revealed by c.d., fluorescence and cross-linking studies. The mutant enzyme is slightly less stable than the wild-type enzyme towards denaturation by guanidium chloride (GdnHCl). On addition of cofactor 2,3-bisphosphoglycerate, the wild-type enzyme shows increased stability towards GdnHCl. However, addition of cofactor causes dramatic changes in the structure of the mutant enzyme, leading to dissociation of the tetrameric form to dimeric and monomeric species.

2,3-Diphosphoglycerate↗
PubMed1993

On the role of the C-terminus of alpha-calcitonin-gene-related peptide (alpha CGRP). The structure of des-phenylalaninamide37-alpha CGRP and its interaction with the CGRP receptor.

alpha-Calcitonin-gene-related peptide (alpha CGRP) lacking its C-terminal phenylalaninamide residue was found not to bind to its receptor as did full-length (amidated) alpha CGRP. Investigation of the structure of these peptides by c.d. and n.m.r. revealed no significant difference, so it seemed that the effect of deleting the C-terminal phenylalaninamide on the biological activity of alpha CGRP was not by disruption of the peptide's structure. Thus the C-terminal phenylalaninamide is an important factor in this ligand-receptor interaction, and the group itself may interact directly with the receptor.

Adenylyl Cyclases↗
PubMed1993

The unfolding and attempted refolding of the bacterial chaperone protein groEL (cpn60).

The unfolding of the bacterial chaperone protein groEL (cpn60) in solutions of guanidinium chloride (GdnHCl) has been studied. From the results of CD, fluorescence and light scattering, it is clear that major structural transitions in the protein occur over the range 1.0-1.5 M GdnHCl. The ATPase activity of the protein is lost at lower concentrations (0.75 M). After denaturation in concentrations of GdnHCl above 1.5 M, removal of the denaturing agent by dialysis results in very nearly complete regain of secondary structure (as judged by CD), but not the regain of correct tertiary or quaternary structure, or ATPase activity. The product was shown to be very sensitive to proteolysis by thermolysin, unlike the native protein, and not to show enhanced binding of ANS, a characteristic property of the 'molten globule' state of proteins. The results are discussed in relation to current information concerning the assembly of the groEL protein.

Adenosine Triphosphatases↗
PubMed1993

Helicobacter pylori increases gastric antral juxtamucosal pH.

In order to examine the effect of Helicobacter pylori colonization on the gastric mucus microclimate, antral juxtamucosal pH was measured in 47 patients attending as out patients for upper gastrointestinal endoscopy. The mean pH in 28 patients negative for H. pylori was 6.40 +/- 0.24 compared to 6.88 +/- 0.16 in 19 patients who were positive (P < 0.0001). In six of seven patients who agreed to a second study, H. pylori was eradicated and the mean pH fell from 6.81 +/- 0.17 to 6.08 +/- 0.16 (P < 0.001). The pH remained high in the one patient who remained positive (6.8 and 7.0). This study provides the first in vivo evidence that H. pylori can increase the antral juxtamucosal pH and suggests that ammonia production by the organism is capable of altering gastric mucus microclimate to impair the normal negative feedback controlling gastrin release. This observation may explain the coexistence of relative hypergastrinemia and H. pylori colonization in duodenal ulcer patients.

Female↗
PubMed1993

Unfolding and refolding of the NAD(+)-dependent isocitrate dehydrogenase from yeast.

The unfolding of the NAD(+)-dependent isocitrate dehydrogenase from yeast in guanidinium chloride (GdnHCl) has been monitored by changes in c.d. and fluorescence. Major structural changes occur over the range of GdnHCl concentrations from 0.5 to 1.5 M, although loss of catalytic activity is complete at 0.3 M. After incubation in GdnHCl, activity can be regained on dilution; however, the extent of this regain is dependent on the initial concentration of GdnHCl and is very small at a concentration of 2 M or above. Under these conditions there is only limited regain of the secondary and tertiary structure of the enzyme. Considerably more structure and activity can be regained if the concentration of GdnHCl is lowered by dialysis. The implications of these results for the folding and assembly of the enzyme are discussed.

Circular Dichroism↗
PubMed1993

Live oral avirulent Salmonella vaccines.

Infection of animals and humans with Salmonella is a consequence of oral consumption of food or fluids contaminated with Salmonella. Once in the intestine, Salmonella usually attach to, invade, and proliferate in enterocytes or the cells of the gut associated lymphoid tissue (GALT). The latter route of infection can lead to disease or to an asymptomatic carrier state or stimulate the induction of mucosal, systemic and cellular immune responses. Infection of animals with virulent invasive Salmonella can result in suppression of the immune responses which in turn can facilitate the establishment of a carrier state. It is possible to attenuate Salmonella by introducing mutations that (i) confer auxotrophy, (ii) interfere with sugar metabolism and LPS biosynthesis or (iii) affect some global means of regulating genes needed for the full display of virulence. Oral immunization of animals such as mice and chickens with avirulent Salmonella strains usually is not associated with suppression but rather with stimulation of mucosal, systemic and cellular immune responses. Vaccination by injection of killed vaccines or bacterins does not lead to the induction of either mucosal or cellular immune responses, and humoral immunity may be relatively short lived. Thus, killed vaccines are inferior to orally administered live avirulent Salmonella vaccines which induce a long-lasting protective immunity. In this manuscript we discuss desirable attributes of a safe, efficacious live attenuated Salmonella vaccine, describe attenuated Salmonella mutants so far isolated and their properties and present information on the evaluation of a live attenuated Salmonella oral vaccine for poultry.(ABSTRACT TRUNCATED AT 250 WORDS)

Administration, Oral↗
PubMed1992

The secondary structure of influenza A M2 transmembrane domain. A circular dichroism study.

Using circular dichroism, this study investigated the secondary structure of the influenza A M2 transmembrane domain. When reconstituted into 1,2-dioleoyl-sn-glycero-3-phosphocholine liposomes, the M2 transmembrane domain was found to adopt a predominantly alpha-helical secondary structure which was unaffected by both temperature and the addition of 1-aminoadamantane hydrochloride. Reconstitution into 1,2-dioleoyl-sn-glycero-3-phosphoglycerol liposomes resulted in a marked decrease in helical content.

Amantadine↗
PubMed1992

Transglutaminase catalyses the modification of glutamine side chains in the C-terminal region of bovine beta-lactoglobulin.

The transglutaminase-catalysed incorporation of primary amines (putrescine and monodansylcadaverine) into bovine beta-lactoglobulin has been studied. In the presence of 1 mM-dithiothreitol between 1 and 2 mol of amine can be incorporated per mol of beta-lactoglobulin subunit. There is very little incorporation of amines in the absence of reducing agent. By isolating and sequencing the modified peptides, the sites of modification have been identified as Gln-159 (preferred) and Gln-155. C.d. has been used to study the structure of beta-lactoglobulin over a range of pH values and in the presence or absence of dithiothreitol. The results are discussed in terms of the X-ray-crystallographically determined structure of beta-lactoglobulin.

Amino Acid Sequence↗
PubMed1992

A comparison of the enzymological and biophysical properties of two distinct classes of dehydroquinase enzymes.

This paper compares the biophysical and mechanistic properties of a typical type I dehydroquinase (DHQase), from the biosynthetic shikimate pathway of Escherichia coli, and a typical type II DHQase, from the quinate pathway of Aspergillus nidulans. C.d. shows that the two proteins have different secondary-structure compositions; the type I enzyme contains approx. 50% alpha-helix while the type II enzyme contains approx. 75% alpha-helix. The stability of the two types of DHQase was compared by denaturant-induced unfolding, as monitored by c.d., and by differential scanning calorimetry. The type II enzyme unfolds at concentrations of denaturant 4-fold greater than the type I and through a series of discrete transitions, while the type I enzyme unfolds in a single transition. These differences in conformational stability were also evident from the calorimetric experiments which show that type I DHQase unfolds as a single co-operative dimer at 57 degrees C whereas the type II enzyme unfolds above 82 degrees C and through a series of transitions suggesting higher orders of structure than that seen for the type I enzyme. Sedimentation and Mr analysis of both proteins by analytical ultracentrifugation is consistent with the unfolding data. The type I DHQase exists predominantly as a dimer with Mr = 46,000 +/- 2000 (a weighted average affected by the presence of monomer) and has a sedimentation coefficient s0(20,w) = 4.12 (+/- 0.08) S whereas the type II enzyme is a dodecamer, weight-average Mr = 190,000 +/- 10,000 and has a sedimentation coefficient, s0(20,w) = 9.96 (+/- 0.21) S. Although both enzymes have reactive histidine residues in the active site and can be inactivated by diethyl pyrocarbonate, the possibility that these structurally dissimilar enzymes catalyse the same dehydration reaction by the same catalytic mechanism is deemed unlikely by three criteria: (1) they have very different pH/log kcat. profiles and pH optima; (2) imine intermediates, which are known to play a central role in the mechanism of type I enzymes, could not be detected (by borohydride reduction) in the type II enzyme; (3) unlike Schiff's base-forming type I enzymes, there are no conserved lysine residues in type II amino acid sequences.

Bacterial Proteins↗
PubMed1992

Paternal cyclophosphamide treatment causes postimplantation loss via inner cell mass-specific cell death.

Treatment of the father with the anticancer alkylating agent cyclophosphamide has negative effects on embryonic development in the rat. Four-week treatment of male rats with a low dose of cyclophosphamide causes a dramatic, dose-dependent increase in postimplantation death of the progeny. Several recent studies have indicated that the paternal genome is required for the development of the extraembryonic tissues. Thus, the purpose of this study was to determine which tissues of the implanting embryo were affected by paternal exposure to cyclophosphamide. Male Sprague-Dawley rats were given cyclophosphamide (6 mg/kg/day) or saline by gavage and bred to untreated female rats after 4 weeks of treatment. Pregnant female rats were killed on day 7 of gestation, and implantation sites were dissected from the uterus, fixed, embedded in Epon for semithin serial sectioning, and stained for subsequent light microscopy. Strikingly, many of the implantation sites of affected embryos sired by treated males displayed an apparently normal trophectoderm enclosing a region of dying cells, containing dark-stained pyknotic nuclei. Very few or no inner cell mass-derived embryonic cells were present in these implantation sites. Therefore, there is a selective death of inner cell mass-derived cells in day 7 implantation sites obtained from the progeny of cyclophosphamide-treated males. The results of this study suggest that treatment of the male with cyclophosphamide can affect paternal genes specifically required for development of the inner cell mass cells of the embryo, without an apparent effect on those genes required for normal trophectoderm.

Animals↗
PubMed1992

Reactivation of denatured citrate synthase.

1. The imported mitochondrial enzyme citrate synthase can be partially (less than or equal to 45%) reactivated after denaturation in guanidinium chloride, if the concentration of the denaturing agent is lowered by dialysis, rather than by dilution, when essentially no reactivation is observed. 2. The presence of a reducing agent (dithiothreitol) is necessary for regain of activity. 3. Optimum regain of activity occurs at enzyme concentrations of about 10-20 micrograms/ml; at higher concentrations there is significant formation of aggregates.

Animals↗
PubMed1992

Unfolding and refolding of hen egg-white riboflavin binding protein.

The unfolding and refolding of riboflavin-binding protein (RfBP) from hen egg-white induced by addition of guanidinium chloride (GdnHCl), and its subsequent removal by dialysis have been studied by c.d. and fluorescence for both the native and reduced protein. The reduction of its nine disulphide bonds causes a reduction in the secondary structure (alpha-helix plus beta-sheet) from 63% to 33% of the amino acid residues. Unfolding of the native protein occurred in two phases; the first involving a substantial loss of tertiary structure, followed by a second phase involving loss of secondary structure at higher GdnHCl concentrations. By contrast this biphasic behaviour was not discernible in the reduced protein. The loss of ability to bind riboflavin occurred after the first phase of unfolding. Comparison of unfolding of the holoprotein and apoprotein suggested that riboflavin has only a small stabilizing effect on the unfolding process. After removal of GdnHCl, the holoprotein, apoprotein and reduced protein assumed their original conformation. The significance of the results in relation to various models for protein folding is discussed.

Animals↗