Inhibition of Yersinia pestis DNA adenine methyltransferase in vitro by a stibonic acid compound: identification of a potential novel class of antimicrobial agents.

BACKGROUND AND PURPOSE: Multiple antibiotic resistant strains of plague are emerging, driving a need for the development of novel antibiotics effective against Yersinia pestis. DNA adenine methylation regulates numerous fundamental processes in bacteria and alteration of DNA adenine methlytransferase (Dam) expression is attenuating for several pathogens, including Y. pestis. The lack of a functionally similar enzyme in humans makes Dam a suitable target for development of novel therapeutics for plague. EXPERIMENTAL APPROACH: Compounds were evaluated for their ability to inhibit Dam activity in a high-throughput screening assay. DNA was isolated from Yersinia grown in the presence of lead compounds and restricted to determine the effect of inhibitors on DNA methylation. Transcriptional analysis was undertaken to determine the effect of an active inhibitor on virulence-associated phenotypes. KEY RESULTS: We have identified a series of aryl stibonic acids which inhibit Dam in vitro. The most active, 4-stibonobenzenesulfonic acid, exhibited a competitive mode of inhibition with respect to DNA and a K(i) of 6.46 nM. One compound was found to inhibit DNA methylation in cultured Y. pestis. The effects of this inhibition on the physiology of the cell were widespread, and included altered expression of known virulence traits, including iron acquisition and Type III secretion. CONCLUSIONS AND IMPLICATIONS: We have identified a novel class of potent Dam inhibitors. Treatment of bacterial cell cultures with these inhibitors resulted in a decrease in DNA methylation. Expression of virulence factors was affected, suggesting these inhibitors may attenuate bacterial infectivity and function as antibiotics.

An evaluation of dirlotapide to reduce body weight of client-owned dogs in two placebo-controlled clinical studies in Europe.

The clinical efficacy for weight loss and safety of dirlotapide in dogs were evaluated in two multi-centre studies with parallel designs. Overweight, adult dogs (n = 245) of various breeds were randomized to treatment with dirlotapide or placebo in a 2:1 ratio. Dirlotapide was administered orally once daily to dogs at an initial dose of 0.05 mg/kg/day commencing on day 0 and doubled after 14 days. Every 28 days, dogs were examined, weighed, body condition scores (BCS) were recorded, and dose was adjusted to meet weight loss targets. Each study comprised three consecutive phases: weight-loss (up to day 196); weight-stabilization (84 days); and post-treatment (28 days). pre-treatment feeding and exercise regimens were continued during treatment. Dirlotapide-treated dogs showed mean weight loss of 15.9% (study A) and 14.0% (study B) by the end of weight loss phase (up to day 196). Percentage weekly weight losses for dirlotapide were significantly greater than for placebo (P < or = 0.0002). Emesis and diarrhoea were experienced in both treatments but were more frequent with dirlotapide; resolution was spontaneous. BCS improved for 75.7-82.5% of dogs on dirlotapide treatment compared with 15.4-41.4% for placebo. Mean dirlotapide dosage at end of weight-loss phase was 0.38 (study A) and 0.29 (study B) mg/kg initial body weight/day. Dirlotapide was found to be clinically safe and effective in the reduction of body weight in overweight dogs.

Culicoides antigen extract stimulates equine blood mononuclear (BMN) cell proliferation and the release of eosinophil adherence-inducing factor(s).

Intradermal injection of a Culicoides antigen extract (CAgX) induces T lymphocyte and eosinophil accumulation in the skin of horses with sweet itch. Blood mononuclear (BMN) cells from normal ponies proliferate when stimulated by mitogen (phytohaemagglutinin, PHA) or antigen (tetanus toxoid, TT) and, as shown here, release soluble factor(s) that induce eosinophil adherence. CAgX also caused concentration dependent proliferation of BMN cells from sweet itch and normal ponies [stimulation index: 29 (13) and 17 (7) for BMN cells from sweet itch and normal ponies, respectively during the active phase of disease; 4 microg protein ml(-1)CAgX; 168 h]. A heat labile factor(s) which caused eosinophil adherence was also released [sweet itch ponies: 6.0 (1.6) per cent adherence versus 1.3 (0.4) per cent; normal ponies: 6.6 (0.5) per cent adherence versus 0.9 (0.1) per cent for supernatants from CAgX (4 microg protein ml(-1); 48 hours) stimulated versus unstimulated BMN cells, respectively]. These results suggest that soluble proteins released from T lymphocytes could affect eosinophil function in the lesional skin of sweet itch horses.

Characterisation of lymphocyte subpopulations in the skin and circulation of horses with sweet itch (Culicoides hypersensitivity).

Circulating lymphocyte numbers are elevated in horses with the allergic skin disease sweet itch and skin lesions are typified by an infiltrate of eosinophils and mononuclear cells, the latter of which have not been fully characterised. The aim of the present study was to characterise the lymphocyte subpopulations in the circulation and skin of ponies with sweet itch by flow cytometry and a newly developed modified alkaline phosphatase immunohistochemical technique. Sweet itch ponies were found to have significantly greater numbers of circulating CD5+ and CD4+ T-lymphocytes than normal animals. Increased numbers of CD3+ T-lymphocytes, most of which were CD4+, and eosinophils were present in the skin of these animals following intradermal injection of a Culicoides antigen extract (97 +/- 21 vs. 449 +/- 49 CD3+ T-lymphocytes/mm2 in deep dermis of vehicle vs. antigen injected sites; 83 +/- 8% CD4+ T-lymphocytes at antigen injected site). T-lymphocytes, which are thought to be important in the pathogenesis of human allergic skin disease, may therefore contribute to the development of sweet itch lesions via the release of cytokines which can cause eosinophil accumulation and activation. An understanding of the pathology of this disease may lead to a more rational approach to therapy.

Inhibition of antigen-induced cutaneous responses of ponies with insect hypersensitivity by the histamine-1 receptor antagonist chlorpheniramine.

A whole-body extract of Culicoides impunctatus induced a biphasic increase in oedema formation in ponies with insect hypersensitivity, with maxima after one and eight hours. The Culicoides antigen did not induce similar responses in ponies with no previous history of the disease. In insect-hypersensitive ponies the local administration of chlorpheniramine (12 micrograms) completely inhibited oedema formation in response to histamine (0.04 microgram) and to Culicoides antigen (0.5 microgram) at one hour, and the response to Culicoides antigen at eight hours was inhibited by 63 per cent. Chlorpheniramine also partially inhibited the accumulation of eosinophils and neutrophils induced by Culicoides antigen after two hours.

Equine peripheral blood mononuclear cells proliferate in response to tetanus toxoid antigen.

It has been reported that equine peripheral blood mononuclear cells (PBMNs) do not proliferate in response to tetanus toxoid (TT) (Frayne and Stokes 1995, Research in Veterinary Science 59, 79-81). Here we demonstrate that lymphocyte proliferation responses to TT, which are characteristic of a recall antigen, may be achieved under certain culture conditions. Given that TT vaccination is routinely applied to many horses, TT is a suitable antigen for the investigation of cellular immune responses by peripheral blood mononuclear cells in the horse.

Thermal-insensitive moiré interferometry.

An optical arrangement has been devised that will permit the measurement of one component of the relative displacement, and therefore the corresponding strain, in a surface at nonambient temperatures by use of coherent optics. The method is based on the technique of moiré interferometry with illumination at grazing incidence. A demonstration of principle is described, with the beams passing through the flame of a blowtorch.

Grating speckle method for in-plane displacement measurement.

A high sensitivity technique combining lensless speckle photography and a grid technique is proposed and demonstrated. A high frequency grid is applied to the specimen surface and contact specklegrams are made in white light before and after loading, in the double exposure mode. Subsequent optical processing yields the various components of in-plane displacement. The sensitivity can be chosen equivalent to the grid frequency or multiples thereof. The method is simple and gives low-noise fringe patterns with high contrast. It is suitable for application in the mechanical laboratory and field environments, because vibration isolation precautions are not necessary.

Fabrication of holographic gratings using a moving point source.

In the manufacture of holographic gratings using coherent light there is an associated problem of optical noise, the conventional remedies for which involve various restrictions. In this work a moving point source is adopted and analysis indicates that if the path is a circle several of these restrictions are removed. The scheme is implemented and the results are highly satisfactory with high-efficiency gratings being produced at frequencies up to 2400 lines/mm, in sizes up to 65 mm square. Significant operational advantages also accrue in relation to alignment procedures and light utilization.