The chromosomal ~-lactamase of Pseudomonas aeruginosa SAlconst (a derepressed laboratory strain) was isolated and purified. Two peaks of activity were observed on gel permeation chromatography (one major peak mol. wt. 45 kD and one minor peak of 54 kD). Preparations from 12 clinical derepressed strains showed identical results. Chromosomal ~-lactamase production in both normal and derepressed P aeruginosa strains was induced both by iron restricted growth conditions and by penicillin G. The majority of the enzyme (80-90%) was found in the periplasm and cytoplasm but a significant amount (2-20%) was associated with the outer membrane (OM). The growth conditions did not affect the distribution of the enzyme between subcellular fractions although higher activity was found in the cells grown under iron limitation and/ or in the presence of ~-lactams. The penicillanate sulphone inhibitor, tazobactam, displayed irreversible kinetics whilst cloxacillin, cefotaxime, ampicillin and penicillin G were all competitive inhibitors of the enzyme. Similar results were obtained for the Enterobacter cloacae P99 [)-lactamase, but tazobactam displayed a non-classical kinetic pattern for the Staphylococcus aureus PC 1 ~-lactamase. The residues involved in f3-lactam hydrolysis by the P aeruginosa SAlconst enzyme were detennined by affinity labelling with tazobactam. A tryptic digestion fragment of the inhibited enzyme contained the amino acids D, T, S, E, P, G, A, C, V, M, I, Y, F, H, K, R. This suggests the involvement of the conserved SVSK, DAE and KTG motifs found in all penicillin sensitive proteins. A model of the 3-D structure of the active site of the P aeruginosa SAlconst chromosomal f3-!actamase was constructed from the published amino acid sequence of P aeruginosa chromosomal ~-lactamase and the a-carbon coordinates of the S. aureus PCI ~-lactamase by homology modelling and energy minimisation. The crystal structure of tazobactam was determined and energy minimised. Computer graphics docking identified Ser 72 as a possible residue involved in a secondary attack on the C5 position of tazobactam after initial ~-lactam hydrolysis by serine 70. The enhanced activity of tazobactam over sulbactam might be explained by the triazole substituent which might participate in favourable hydrogen bonding between N3 and active site residues. Keywords: Pseudomonas aeruginosa SAyonst, chromosomal ~-lactamase, tazobactam, molecular modelling, growth conditions, [)-lactam antibiotics, f3-lactamase inhibitors.

A transplantable colon adenocarcinoma of the mouse (MAC16) was utilized as a model of human cancer cachexia. The MAC16 tumour produced extensive weight loss in the host at small tumour burdens and without a reduction in either food or fluid intake. The weight loss was characterised by a decrease in both carcass fat and muscle mass which were directly proportional to the weight of the tumour. The weight loss has been correlated with the production of circulatory catabolic factors by the tumour, which degrade host muscle and adipose tissue in vitro. These factors were further characterised and have been shown to be distinct and separable by gel exclusion chromatography. The proteolytic factors (molecular weight > 150k daltons) were distinguishable from the lipolytic factors which appeared related with molecular weights of approximately 3.0, 1.5 and 0.7k daltons. Lipolytic factors of the same molecular weights were identified in other tumour models and in the body fluids of tumour-bearing animals and cancer patients. These factors were not present in healthy individuals or in patients with other weight-losing conditions. Various temperatures studied reversed the weight loss seen in the cachexia induced by the MAC16 adenocarcinoma in vivo. The effects of these treatments could be linked in vitro to the inhibition of the catabolic factors produced by the tumour. These results suggest that these factors may be responsible for the cachexia the tumour confers on its host. These factors may be useful in the understanding and therapy of cancer cachexia.

No description available.

No description available.

Previous studies in man have shown that following dosing with L--3,4-dihydroxyphenylalanine (L-DOPA) and cotrimoxazole, plasma biopterins were raised. By analogy with dihydropteridine reductase deficient children in whom plasma biopterins are greatly elevated and the observations that these preparations were dihydropteridine reductase inhibitors, it was assumed that these raised plasma levels were due to increased efflux from tissues which resulted in tissue depletion of biopterins. In some human disease states such as senile dementia of the Alzheimer type lowered plasma biopterins were observed; by analogy with tetrahydrobiopterin synthesis deficient children these reduced plasma biopterins were attributed to lowered tetrahydrobiopterin synthesis and concomitant low tissue biopterin levels. Because of ethical considerations it was not possible to measure directly the tissue biopterins changes in either case. The Wistar rat was used as a model for human tetrahydrobiopterin metabolism, since tissues not normally accessible for study in humans, such as the brain and liver, could be examined for their effects on tetrahydrobiopterin metabolism after administration of the various agents. Plasma total biopterins in normal conditions were found to be much higher than in healthy humans. The elevation of plasma total biopterins concentration following the administration of dihydropteridine reductase inhibitors to humans, such as L-DOPA and cotrimoxazole was not observed in the rat. However, the administration of inhibitors of de novo tetrahydrobiopterin biosynthesis, such as diaminohydroxypyrimidine (DAHP) and bromocriptine was shown to decrease plasma biopterins concentration. In general, hepatic biopterins were decreased after administration of both dihydropteridine reductase inhibitors and de novo biosynthesis inhibitors. Drugs which are direct (bromocriptine) or indirect (L-DOPA and Sinemet Plus) agonists at dopamine receptors were investigated and were shown to decrease hepatic total biopterins concentration, but had no effect on brain biopterins. Bromocriptine was demonstrated as a potent inhibitor of de novo tetrahydrobiopterin biosynthesis in vivo and in vitro. Cotrimoxazole decreased brain tetrahydrobiopterin concentration. DAHP was effective in causing hyperphenylalaninaemia due to tetrahydrobiopterin deficiency in the rat. p-hydroxyphenylacetate was shown to be an effective inhibitor of dihydropteridine reductase in vivo. Phenylacetate administration had no observable effect on tetrahydrobiopterin metabolism, but did cause tyrosinaemia. It is proposed that scopolamine reduces tetrahydrobiopterin turnover. Lead and aluminium exposure caused deranged tetrahydrobiopterin metabolism. Aluminium, but not lead decreased brain choline acetyltransferase activity. Phenylalanine loading in normal human subjects was followed by an elevation in plasma biopterins which was not observed after tyrosine loading. Plasma N : B ratios correlated well with VEP latencies after tyrosine loading, but not after phenylalanine loading in healthy subjects. The use of derived pterin measurements as an indicator of tetrahydrobiopterin turnover or tetrahydrofolate status is discussed in the text.

The hepatotoxicity of the industrial solvent and investigational anti-tumour agent N-methylformamide (NMF, HOCNHCH3) and several structural analogues was assessed in mice. NMF and its ethyl analogue (NEF) were equipotent hepatotoxins causing extensive centrilobular necrosis and damage to the gall bladder. Pretreatment of mice with SKF525A did not influence the toxicity of these N-alkylformamides. Replacement of the formyl hydrogen of NMF with deuterium or methyl significantly reduced its hepatotoxicity. An in vitro model for the study of the toxicity and metabolism of N-alkylformamides was developed using isolated mouse hepatocytes. The cytotoxicity of NMF in vitro was concentration-dependent with maximal toxicity being achieved at concentrations of 5mM or above. The cytotoxic potential of related amides correlated well with their in vivo hepatotoxic potential. Pretreatment of mice with buthionine sulphoximine (BSO), which depleted hepatocytic levels of glutathione to 15% of control values, exacerbated the cytotoxicity of NMF towards the hepatocytes. NMF (1mM or above), incubated with isolated mouse hepatocytes, depleted intracellular glutathione levels to 26% of control values within 4h. Depletion of glutathione was quantitatively matched by the formation of a carbamoylating metabolite. Metabolism was dependent on the concentration of NMF and was drastically reduced in incubations of hepatocytes isolated from mice pretreated with BSO. The carbamoylating metabolite, S-(N-methylcarbamoyl)-glutathione (SMG), was identified in vitro using FAB-MS. The generation of SMG was subject to a large primary H/D kinetic isotope effect when the formyl hydrogen was replaced with deuterium. Likewise, glutathione depletion and metabolite formation were reduced or abolished by the deuteration or methylation of the formyl moiety of NMF. NEF, like NMF, depleted hepatocytic glutathione levels and was metabolised to a carbamoylating metabolite. Radioactivity derived from 14C-NMF and 14C-NEF, labelled in the alkyl moieties, was found to be irreversibly associated with microsomal protein on incubation in vitro. Binding was dependent on the presence of NADPH and was mostly abolished in the presence of reduced glutathione. SKF525A failed to influence the binding.

Influences on general practitioner prescribing of drugs continue to be of interest and importance as cost containment becomes central to Government health policy. This thesis employs a plurality of research methods including quantitative and qualitative survey techniques for example, questionnaires, interviews and prescription analyses to investigate some of the factors which may influence GP prescribing such as information sources, hospital consultants and in particular the community pharmacist. When the use and influence of drug information sources by GPs was examined, the community pharmacist was given a relatively low rating as a source but a high rating, similar to that of the consultant, for helpfulness. Influences are needed to improve prescribing and reduce the incidence of iatrogenic disease for the benefit of the patient. The education and expertise of pharmacists and their familiarity with local prescribing habits places them in a unique position to meet the needs of local GPs. As 96.5% of the public always or nearly always take their prescriptions to the same pharmacy, patient medication records, now kept by 77.5% of pharmacies, provide a valuable check on the appropriateness and safety of patients' medication. The barriers to the pharmacist's greater involvement were shown to be suspicion by GPs of pharmacists' motivation, isolation of many community pharmacists, difficulties in leaving the pharmacy for domiciliary visits, residential home care and GP practice meetings. These barriers must be lowered if the pharmacist is to have a greater influence and involvement. It was concluded that changes are necessary in pharmaceutical education, staff training, organisation and remuneration. Some changes in the targeting of remuneration to the pharmaceutical care services provided and registration of patients with pharmacies would contribute greatly to these aims.

No description available.

No description available.

No description available.