Biomedical subjects
J Davies
Publications and source records attributed to J Davies.
In situ observation of streptavidin-biotin binding on an immunoassay well surface using an atomic force microscope.
Polystyrene microtitre wells are commonly used as supports for the enzyme-linked immunosorbent assay (ELISA) method of biomolecular detection, which is employed in the routine diagnosis of a variety of medical conditions. We have used an atomic force microscope (AFM) to directly monitor specific molecular interactions between individual streptavidin and biotin molecules on such wells. This was achieved by functionalising an AFM probe with biotin and monitoring the adhesive forces between the probe and a streptavidin coated immunoassay well. The results demonstrate that the AFM may be employed as an analytical tool to study the interactions between biomolecules involved in immunoassay systems.
Inhibition of the self-cleavage reaction of the human hepatitis delta virus ribozyme by antibiotics.
Human hepatitis delta virus (HDV) poses a health threat in populations where chronic hepatitis B is endemic. It is a single-stranded RNA virus of 1700 nucleotides and both genomic and antigenomic sequences contain ribozymes which are important for viral replication. Using ribozyme constructs we show that several classes of antibiotics inhibit the self-cleavage reaction of the HDV ribozyme. Antibiotics of the aminoglycoside, peptide and tetracycline classes all inhibit HDV cleavage in vitro at micromolar concentrations. Neomycin (an aminoglycoside) inhibits HDV self-cleavage with a Ki value of 28 (+/- 10) microM. Neomycin inhibition can be reversed by increasing magnesium ion concentration in a competitive manner. Lead acetate cleaves positions G76, A42 and G28, which surround the ribozyme cleavage site. Both Mg2+ and neomycin prevent lead cleavage. Footprinting experiments using base-specific chemical probes revealed enhanced modifications of a set of bases by neomycin, overlapping with the above mentioned lead cleavages. These observations may indicate that neomycin directly displaces divalent metal ions essential for catalysis.
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Synovial tissue responses following treatment of rheumatoid arthritis with the humanized monoclonal antibody CAMPATH-1H.
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Antiviral properties of extract of Opuntia streptacantha.
An extract of the cactus plant Opuntia streptacantha inhibited intracellular virus replication and inactivated extracellular virus. Inhibition of virus replication also occurred following pre-infection treatment--a favourable finding in terms of in-vivo limitation of virus disease. There was inhibition of both DNA and RNA virus replication, for example, herpes simplex virus, equine herpes virus, pseudorabies virus, influenza virus, respiratory syncytial virus and human immunodeficiency virus, with normal protein synthesis in uninfected cells at extract concentrations which were 15-fold in excess of 50% viral inhibitory concentrations (1 mg/ml). The active inhibitory component(s) of the extract appeared to be protein in nature and resided mainly in the wall of the plant rather than in the cuticle or inner sap. The extract was non-toxic on oral administration to mice, horses and human patients; the non-toxicity of intravenous administration of 70 mg to a mouse representing at least fifty tissue culture 50% viral inhibitory dosages encourages clinical trial of this extract in virus disease of human and veterinary species.
The discrimination of IgM and IgG type antibodies and Fab' and F(ab)2 antibody fragments on an industrial substrate using scanning force microscopy.
We have previously employed scanning force microscopy (SFM) to study antibody-antigen molecular interactions on microtiter wells used for enzyme linked immunosorbant assays (ELISA). Here we demonstrate the ability of SFM to image and discriminate different types of antibody and antibody fragments bound to an ELISA well surface. The samples studied include a type IgG antibody with a proportion of bound IgM and two-dimensional films of whole IgG antibody, and Fab' and F(ab)2 antibody fragments. Molecular resolution is achieved in each case despite the size of substrate features exceeding most of the molecular dimensions observed. Analysis of the data shows that the SFM overestimates molecular dimensions by an approximately constant amount, which is proposed to principally result from the effects of a finite probe size and not from deformation of the molecular species due to the imaging forces employed.
An outbreak of herpes rugbiorum managed by vaccination of players and sociosexual contacts.
An outbreak of herpes rugbiorum involved nine players including the scrum half and the full back. The infection was characterized by significant constitutional upset with decreased levels of general fitness and match performance for 1-4 months following the outbreak; one player had herpetic lesions on his right eyelid and corneum. Every infected player, 15 non-infected players and five sociosexual contacts received two vaccinations with intracellular subunit vaccine NFU. Ac. HSV-1 (S-MRC5). None of the players or contacts developed cutaneous herpetic recurrence during a follow-up period of 3 years; the player with ocular disease had one recurrence at 30 months following the original episode. These findings encourage consideration of prophylactic or post-exposure vaccination of participants in rugby or other contact sports with this or other appropriate herpes simplex vaccine.
Affinity improvement of single antibody VH domains: residues in all three hypervariable regions affect antigen binding.
BACKGROUND: Through antibody engineering, immunoglobulins can be tailored for their particular application. In this respect, small recognition units are desired for the targeting of antigens in obstructed locations like solid tumors. OBJECTIVES: To design efficient, minimum size recognition units, heavy chain variable regions (VH) had previously been modified for the use as antigen specific, single domain antibody fragments. To develop a rational approach to improve affinity, antigen binding is investigated here by analysing the effect of randomisations of CDR1 and 2 residues in VH domains specific for hapten and protein ligands. STUDY DESIGN: Randomised repertoires were displayed on phage and affinity selected to improve and analyse antigen binding. Affinities of newly selected VH domains were determined in their soluble format to assess the role of modified residues in binding. RESULTS: In four of five randomisation experiments, a new VH with an improved antigen affinity compared to the primary VH was selected. Dissociation constants decreased from 160 nM to 25 nM or 47 nM (CDR1 or CDR2 randomisation of an anti-Ox VH), from 300 nM to 31 nM (CDR2 randomisation of an anti-NIP VH) and from 3.1 microM to 1.6 microM (CDR2 randomisation of an anti-lysozyme VH). CONCLUSIONS: Thus the affinity of VH domains can be improved after site specific, secondary randomisations in CDR1 and CDR2, phage display and antigen selection. As differences in the CDR3 sequences had formed the only difference between the primary VH domains used in this study, the effect of CDR1 and CDR2 mutations of affinity is consistent with a participation of all three CDRs in antigen binding by single VH domains.
Bleomycin resistance in Staphylococcus aureus clinical isolates.
Among clinical isolates of Staphylococcus aureus tested for resistance to the antibiotic bleomycin, 197 were found to be resistant; most of them were also resistant to tobramycin and contained plasmids. DNA dot-blot hybridization analysis of the bleomycin resistant isolates with an 171 bp probe derived from the plasmid pUB110 indicated that 43 strains (22%) carried pUB110-like bleomycin resistance DNA sequences. Analysis of bacterial cell lysates derived from the bleomycin resistant isolates indicated that many contained bleomycin-binding properties (BBP), that prevent DNA damage by the antibiotic. Of 13 strains that were analysed by DNA gel electrophoresis and Southern blot DNA hybridization, six were found to carry pUB110-like bleomycin resistance DNA sequences. These studies indicate that there may be more than one genetic determinant for bleomycin resistance in S. aureus whose DNA is protected.
Single antibody domains as small recognition units: design and in vitro antigen selection of camelized, human VH domains with improved protein stability.
Folding stabilities of camelized human antibody VH domains were studied through the determination of their melting points in thermodenaturation experiments. The melting point of a VH domain originating from a synthetic library of human VHs, which had been optimized for the use as small recognition units through the mimicking of camelid antibody heavy chains occurring naturally without light chain, was 56.6 degrees C compared with 71.2 degrees C of the original human VH. Its stability was improved (melting point 61.6 degrees C) through three mutations to mimic camelid VHs even further: Va137 was replaced by phenylalanine and two cysteines were introduced at position 33 and 100b. The resulting VH folded properly and formed a second intradomain disulphide between the extra cysteines. The new mutations were then built constitutively into a phage-display VH library, from which antigen-specific VHs were selected. Two were analysed for stability with melting points of 72.6 and 75.3 degrees C. Thus secondary camelization enabled the isolation of VHs with improved folding stabilities exceeding even that of the original human VH. This indicates an effect on folding stability for some mutations specific in the light chain lacking camelid heavy chains.
Susceptibility of Actinomyces israelii to antibiotics, sodium hypochlorite and calcium hydroxide.
Actinomyces israelii has been repeatedly implicated as a cause of failure of endodontic therapy. This study investigated the antimicrobial effect of antibiotics as well as intracanal medicaments, sodium hypochlorite solution and calcium hydroxide, on this important pathogen. Growth of A. israelii was inhibited by low concentrations of antibiotics, yet high concentrations were not bactericidal for A. israelii over 1 week. When A. israelii was exposed for 2-6 weeks at concentrations equivalent to clinical serum levels, the antibiotics were lethal. The results reveal a species-specific antibiotic tolerance for A. israelii. Both sodium hypochlorite solution and calcium hydroxide were found to be highly effective in killing A. israelii.
The effect of insulin treatment and of islet transplantation on the resistance artery function in the STZ-induced diabetic rat.
1. This study was designed to investigate the influence of insulin treatment and islet transplantation on the smooth muscle contractility and endothelium-dependent and independent relaxation of resistance arteries in the chemically induced streptozotocin (STZ) diabetic rat after 6-8 weeks, and 12-14 weeks of diabetes, compared to non-diabetic age-matched controls. 2. The morphology, and contractile responses to high potassium physiological salt solution (KPSS), KPSS containing 10(-5) M noradrenaline (NAK), and concentration-response curves to noradrenaline (NA) of mesenteric resistance arteries were recorded, along with the endothelium-dependent relaxation responses to acetylcholine (ACh) and bradykinin (BK), and endothelium-independent relaxation to sodium nitroprusside (SNP). Concentration-response curves were then repeated in the presence of a nitric oxide synthase inhibitor, NG-nitro-L-arginine (L-NOARG). 3. Insulin-treated diabetic rats in the 12 week study demonstrated enhanced vascular contractility to KPSS, NAK and NA, compared to age-matched non-diabetic controls. 4. Incubation with L-NOARG resulted in both a significant increase in maximum contractile response, and sensitivity (pD2) to NA in the untreated diabetic group (6 weeks). A significant shift in sensitivity was also seen in the insulin-treated diabetic group. In the 12 week study, incubation with L-NOARG resulted in an increased maximum contractile response and sensitivity to NA in the insulin-treated diabetics. An increase in sensitivity was also observed in the untreated diabetic group. 5. Endothelium-dependent relaxation to ACh was significantly augmented in the untreated diabetics (6-weeks), compared to the control group. In the 12-week study, relaxation to both ACh and BK was not significantly different in any of the experimental groups when compared to the sham-operated non-diabetic controls. 6. Incubation with L-NOARG resulted in a significant attenuation of the maximum relaxation response to ACh and BK in all of the experimental groups, in the 6- and the 12-week study. 7. There was no significant difference in the maximum relaxation response or sensitivity to sodium nitroprusside between the diabetic groups and their age-matched controls in either the 6-week or the 12-week study. 8. The results of this study suggest an enhanced release of nitric oxide in the early stages of diabetes, which is more evident in the untreated diabetic rats than the insulin treated, and appears to normalize as the duration of diabetes progresses. This study also shows that the alteration in vascular reactivity of the resistance arteries can be restored to within normal limits by the transplantation of islets of Langerhans, and that islet transplantation is an effective strategy in the correction of the metabolic abnormalities associated with insulin-dependent diabetes.
A prospective study of self-esteem in the prediction of eating problems in adolescent schoolgirls: questionnaire findings.
A number of authors have emphasized the importance of self-esteem in the aetiology of the eating disorders anorexia nervosa and bulimia nervosa. Evidence for such theorizing, however, mainly derives from clinical observations on people being treated for eating disorders. This study is the first prospective study to investigate the role of self-esteem in aetiology prior to the onset of an eating disorder. Self-esteem was measured in 594 schoolgirls aged 11-12 using the Rosenberg Self-Esteem Scale (Rosenberg, 1965). Almost 400 of these girls were successfully followed up at age 15-16 and they completed a questionnaire examining eating and other psychological problems. Results showed that girls with low self-esteem at age 11-12 were at significantly greater risk of developing the more severe signs of eating disorders, as well as other psychological problems, by the age of 15-16. It is argued that more research is needed to replicate and extend these findings. The results also give weight to the case for examining the potential role of self-esteem enhancement in the prevention of eating disorders.
Apramycin resistance as a selective marker for gene transfer in mycobacteria.
We have explored the potential of using the apramycin resistance gene as a marker in mycobacterial gene transfer studies. Shuttle plasmids available for both electroporation and conjugation studies have been constructed, and we have successfully validated the use of the apramycin resistance gene as a component of cloning vectors for Mycobacterium smegmatis, M. bovis BCG, and M. tuberculosis.
Distribution of thiols in microorganisms: mycothiol is a major thiol in most actinomycetes.
Mycothiol [2-(N-acetylcysteinyl)amido-2-deoxy-alpha-D-glucopyranosyl- (1-->1)-myo-inositol] (MSH) has recently been identified as a major thiol in a number of actinomycetes (S. Sakuda, Z.-Y. Zhou, and Y. Yamada, Biosci. Biotech. Biochem. 58:1347-1348, 1994; H. S. C. Spies and D. J. Steenkamp, Eur. J. Biochem. 224:203-213, 1994; and G. L. Newton, C. A. Bewley, T. J. Dwyer, R. Horn, Y. Aharonowitz, G. Cohen, J. Davies, D. J. Faulkner, and R. C. Fahey, Eur. J. Biochem. 230:821-825, 1995). Since this novel thiol is more resistant than glutathione to heavy-metal ion-catalyzed oxidation, it seems likely to be the antioxidant thiol used by aerobic gram-positive bacteria that do not produce glutathione (GSH). In the present study we sought to define the spectrum of organisms that produce MSH. GSH was absent in all actinomycetes and some of the other gram-positive bacteria studied. Surprisingly, the streptococci and enterococci contained GSH, and some strains appeared to synthesize it rather than import it from the growth medium. MSH was found at significant levels in most actinomycetes examined. Among the actinobacteria four Micrococcus species produced MSH, but MSH was not found in representatives of the Arthrobacter, Agromyces, or Actinomyces genera. Of the nocardioforms examined, Nocardia, Rhodococcus, and Mycobacteria spp. all produced MSH. In addition to the established production of MSH by streptomycetes, we found that Micromonospora, Actinomadura, and Nocardiopsis spp. also synthesized MSH. Mycothiol production was not detected in Propionibacterium acnes or in representative species of the Listeria, Staphylococcus, Streptococcus, Enterococcus, Bacillus, and Clostridium genera. Examination of representatives of the cyanobacteria, purple bacteria, and spirochetes also gave negative results, as did tests of rat liver, bonito, Candida albicans, Neurospora crassa, and spinach leaves. The results, which indicate that MSH production is restricted to the actinomycetes, could have significant implications for the detection and treatment of infections with actinomycetes, especially those caused by mycobacteria.
Epidemiology of adult Still's disease.
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