Biomedical subjects
Publications and source records attributed to P Stead.
A novel synthetic strategy is described which may be used to prepare analogues of the antimalarial, fungal metabolite apicidin. Compared to the natural product, one analogue shows potent and selective activity in vitro against the parasite Trypanosoma brucei and low mammalian cell toxicity.
The biotransformation of the fungal protein synthesis inhibitor sordarin is reported. Nine taxonomically diverse organisms supported the isolation and identification of twelve modified products. The structural diversity of the biotransformation products observed and their value in supporting further chemistry is discussed.
We report the discovery of Eryloside F, a novel disaccharide of the steroidal carboxylic acid penasterol, isolated from an extract of the marine sponge Erylus formosus. The compound is a potent thrombin receptor antagonist, and furthermore inhibits human platelet aggregation in vitro.
We describe the isolation and characterisation of novel non-benzoquinone ansamycin metabolites related to geldanamycin from a culture of Streptomyces sp. S6699. The compounds possess potent inhibitory activity in a cell-based assay measuring inhibition of oncostatin M signalling in a reporter cell line utilising a secreted placental alkaline phosphatase (sPAP) readout. In this paper we report the isolation and structure elucidation of the compounds and describe some of their biological properties.
In cell-free Pseudomonas aeruginosa culture supernatants, we identified two compounds capable of activating an N-acylhomoserine lactone (AHL) biosensor. Mass spectrometry and NMR spectroscopy revealed that these compounds were not AHLs but the diketopiperazines (DKPs), cyclo(DeltaAla-L-Val) and cyclo(L-Pro-L-Tyr) respectively. These compounds were also found in cell-free supernatants from Proteus mirabilis, Citrobacter freundii and Enterobacter agglomerans [cyclo(DeltaAla-L-Val) only]. Although both DKPs were absent from Pseudomonas fluorescens and Pseudomonas alcaligenes, we isolated, from both pseudomonads, a third DKP, which was chemically characterized as cyclo(L-Phe-L-Pro). Dose-response curves using a LuxR-based AHL biosensor indicated that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) activate the biosensor in a concentration-dependent manner, albeit at much higher concentrations than the natural activator N-(3-oxohexanoyl)-L-homoserine lactone (3-oxo-C6-HSL). Competition studies showed that cyclo(DeltaAla-L-Val), cyclo(L-Pro-L-Tyr) and cyclo(L-Phe-L-Pro) antagonize the 3-oxo-C6-HSL-mediated induction of bioluminescence, suggesting that these DKPs may compete for the same LuxR-binding site. Similarly, DKPs were found to be capable of activating or antagonizing other LuxR-based quorum-sensing systems, such as the N-butanoylhomoserine lactone-dependent swarming motility of Serratia liquefaciens. Although the physiological role of these DKPs has yet to be established, their activity suggests the existence of cross talk among bacterial signalling systems.
We describe the discovery and properties of a prenylated p-terphenyl metabolite of the fungus Aspergillus candidus. The compound (1) possesses potent cytotoxic activity against a range of tumour and other hyper-proliferative cell lines. Cell cycle analysis shows that in mouse keratinocyte (BALB/MK) cells treated with 1, the cell cycle is arrested in early S phase, indicative of an antimetabolite effect. Furthermore, cellular cytotoxicity can be reversed by addition of exogenous pyrimidine but not purine nucleosides to the cell culture medium. It is therefore likely that compound 1 selectively inhibits pyrimidine biosynthesis, and it is this property which accounts for its potent cytotoxic properties.
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A range of Pseudomonas spp. and other Gram-negative bacteria were screened for induction of antimicrobial activity in response to the autoregulatory factor L-N-(3-oxohexanoyl)homoserine lactone. In one of these, P. aeruginosa ATCC 10145, the production of phenazine metabolites was shown to be inducible in a dose-dependent manner. The production of phenazine-1-carboxamide increased over 50-fold compared to control cultures when supplemented with 200 micrograms/ml of the autoregulator. In addition, the production of an unidentified polar antibacterial substance by this strain increased with autoregulator concentration.
The immune response to infection with M. tuberculosis depends on cytokine activation of effector cells. We therefore conducted a pilot study of recombinant human interleukin-2 (rhuIL-2) as an adjunct to multidrug therapy (MDT) to evaluate the safety of this approach and to determine whether IL-2 can enhance the cellular immune response in patients with pulmonary tuberculosis (TB). Patients included in this study presented with a wide range of extent and duration of infection, and were grouped into three categories for data analysis: (1) patients with newly diagnosed, acute-stage TB who were just beginning MDT; (2) patients who had received a minimum of 45 days MDT before the start of the study and who had responded to treatment; and (3) patients with multidrug-resistant (MDR) TB who had been on MDT for at least seven months without apparent beneficial clinical response. Twenty patients received 30 days of twice-daily intradermal injections of 12.5 micrograms of IL-2. Patients from all three groups showed improvement of clinical symptoms over the 30-day period of treatment with IL-2 and MDT. Results of direct smear for acid fast bacilli (AFB) demonstrated conversion to sputum-negative following IL-2 and MDT treatment in all newly diagnosed patients and in 5/7 MDR TB patients. (The size of the skin test response to purified protein derivative (PPD) of tuberculin increased during the 30-day IL-2 adjunctive therapy in newly diagnosed patients, but decreased or disappeared in the other two groups of treated patients.) Assays in vitro for phenotype distribution, natural killer (NK) cell activity, frequency of cells proliferating in response to exogenous IL-2, and antigen-induced blastogenesis demonstrated systemic responses to intradermally administered rhuIL-2. Levels of interferon-gamma (IFN-gamma) in plasma, peripheral blood mononuclear cell (PBMC) IFN-gamma mRNA and IFN-gamma mRNA in biopsy of site of skin test response to purified protein derivative (PPD) were highest in those patients with the most acute symptoms at the beginning of the study, and decreased during rhuIL-2 and MDT. IL-2 immunotherapy did not modify levels of mRNA expression for other cytokines. Patients receiving IL-2 did not experience clinical deterioration or significant side effects. These results suggest that IL-2 administration in combination with conventional MDT is safe and may potentiate the antimicrobial cellular immune response to TB.
N-(3-Oxohexanoyl)-L-homoserine lactone (HSL) (I) is the autoregulator controlling carbapenem antibiotic biosynthesis in Erwinia carotovora ATCC 39048. The chemical synthesis and biological evaluation of analogues of HSL are described. These include alterations of chirality, side-chain modifications, ring size and ring hetero atom. A number of compounds are reported which are capable of restoring the phenotype to a HSL negative mutant but at higher concentrations than HSL. A-factor, the autoregulator of streptomycin biosynthesis in Streptomyces griseus, was not active as an inducer of carbapenem biosynthesis in E. carotovora.
Erwinia carotovora A.T.C.C. 39048 produces the antibiotic 1-carbapen-2-em-3-carboxylic acid. A number of mutants with a carbapenem-non-producing phenotype were selected as part of an investigation into the molecular and genetic basis of carbapenem biosynthesis. Cross-feeding studies revealed that the mutants fell into two discrete groups. Group 1 mutants were found to secrete a diffusible low-molecular-mass compound which restored carbapenem production in group 2 mutants. This compound was isolated from the spent culture supernatant of a group 1 mutant using solvent extraction, hydrophobic-interaction chromatography and silica-gel chromatography, and finally purified by reverse-phase semipreparative h.p.l.c. M.s. and n.m.r. spectroscopy revealed that the compound was N-(3-oxohexanoyl)homoserine lactone. Both D- and L-isomers were synthesized, and subsequent analysis by c.d. established that the natural product has the L-configuration. Although carbapenem production was restored by both isomers, dose-response curves indicated that the L-isomer has greater activity, with an induction threshold of about 0.5 micrograms/ml. N-(3-Oxohexanoyl)-L-homoserine lactone is, therefore, an autoregulator of carbapenem biosynthesis rather than a biosynthetic intermediate. This compound is already known for its role in autoinduction of bioluminescence in the marine bacterium Vibrio fischeri. It is also structurally-related to the A- and I-factors which are known to regulate production of antibiotics in some Streptomyces species. Its association in this work with the regulation of carbapenem biosynthesis implies a broader role for autoregulator-controlled gene expression in prokaryotes.
Micro-organisms have evolved complex and diverse mechanisms to sense environmental changes. Activation of a sensory mechanism typically leads to alterations in gene expression facilitating an adaptive response. This may take several forms, but many are mediated by response-regulator proteins. The luxR-encoded protein (LuxR) has previously been characterised as a member of the response-regulator superfamily and is known to respond to the small diffusible autoinducer signal molecule N-(beta-ketocaproyl) homoserine lactone (KHL). Observed previously in only a few marine bacteria, we now report that KHL is in fact produced by a diverse group of terrestrial bacteria. In one of these (Erwinia carotovora), we show that it acts as a molecular control signal for the expression of genes controlling carbapenem antibiotic biosynthesis. This represents the first substantive evidence to support the previous postulate that the lux autoinducer, KHL, is widely involved in bacterial signalling.
OBJECTIVE: To determine the impact of the introduction of a plastic shield-shaped device (Needleguard, Biosafe, Auckland, New Zealand) and education program designed to allow safer recapping, on recorded rates of needlestick injury. DESIGN: A before-after trial with a two-year duration of follow-up. SETTING: Tertiary referral hospital. PARTICIPANTS: Nursing and other hospital personnel. RESULTS: Prospectively collected baseline data, together with the results of an anonymous questionnaire of 25% of the hospital nursing staff, defined a reported needlestick injury rate of 6.9 per hundred full-time nursing staff per year. In the pre-intervention period, there were 6.7 needlestick injuries per 100 nursing staff members per year reported. This increased to 15.4 (p less than .0001) needlestick injuries per 100 nursing staff members per year after the intervention. An anonymous survey undertaken at both time periods suggests that the apparent increase in officially reported needlestick injuries is due to an increase in the willingness of nurses to now report previously unreported needlestick injuries. CONCLUSIONS: The impact of the safety device and education program was the more accurate reporting of needlestick injuries; many nursing staff continued to resheath needles contrary to hospital policy. Many staff simply did not use the newly designed safety device. Approaches to improving compliance with such safety devices are considered.
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SHARP is a hospital-based, self-help program for treating alcoholic veterans, which involves them in assuming major roles in governing the program and in helping one another. Because follow-up support is essential to maintaining sobriety, SHARP created on active social support system in the community. The evidence of SHARP's success is shown by results of a follow-up study that surveyed former patients' levels of drinking and life functioning.