Switching the mode of metabolism in the yeast Saccharomyces cerevisiae

The biochemistry of most metabolic pathways is conserved from bacteria to humans, although the control mechanisms are adapted to the needs of each cell type. Oxygen depletion commonly controls the switch from respiration to fermentation. However, Saccharomyces cerevisiae also controls that switch in response to the external glucose level. We have generated an S. cerevisiae strain in which glucose uptake is dependent on a chimeric hexose transporter mediating reduced sugar uptake. This strain shows a fully respiratory metabolism also at high glucose levels as seen for aerobic organisms, and switches to fermentation only when oxygen is lacking. These observations illustrate that manipulating a single step can alter the mode of metabolism. The novel yeast strain is an excellent tool to study the mechanisms underlying glucose-induced signal transduction. © 2004 European Molecular Biology Organization.

The mode of action of a lipid mobilising factor in cancer cachexia

Cachexia is characterised by a progressive weight loss due to depletion of both skeletal muscle and adipose tissue. The loss of adipose tissue is due to the production of a tumour-derived lipid mobilising factor (LMF), which has been shown to directly induce lipolysis in isolated epididymal murine white adipocytes. The administration of LMF to a non-tumour bearing mice produced a rapid weight loss, with a specific reduction in carcass lipid with also some redistribution of lipid with the accumulation of lipid in the liver. There was also up-regulation of uncoupling protein-1 and -2 mRNA and protein expression in brown adipose tissue, suggesting that an adaptive process occurs due to increased energy mobilisation. There was also up-regulation of UCP-2 in the livers of LMF treated mice, suggesting a protective mechanism to the build up of lipid in the livers, which would produce free radical by-products. LMF was also shown to stimulate cyclic AMP production in CHO-K1 cells transfected with human -3 adrenergic receptors and inhibited by the -β3 antagonist SR59230A. LMF binding was also inhibited by SR59230A in isolated receptors. This suggests that LMF mediates its effects through a β3 adrenergic receptor. There were also changes in glucose and fatty acid uptake in LMF treated mice, which suggests metabolic changes are occurring. The study suggests that a tumour derived lipolytic factor acts through the 3 adrenoceptor producing effects on lipid mobilisation, energy expenditure and glucose metabolism.

Studies on the mode of action and beta-lactamase inhibitory properties of novel oxapenem compounds

Four novel oxapenem compounds were evaluated for their ß-lactamase inhibitory and antibacterial properties. Two (AM-112 and AM-113) displayed intrinsic antibacterial activity with MICs of between 2 to 16µg/ml and 0.5-2µg/ml against Escherichia coli and methicillin-sensitive and -resistant Staphylococcus aureus, respectively. The isomers of these compounds, AM-115 and AM-114 did not display significant antibacterial activity. Combination of the oxapenems with ceftazidime afforded protection against ß-lactamase-producing strains, including hyperproducers of class C enzymes and extended-spectrum ß-lactamase enzymes. A fixed 4µg/ml concentration of AM-112 protected a panel of eight cephalosporins against hydrolysis by class A and class C ß-lactamase producers. In vivo studies confirmed the protective effect of AM-112 for ceftazidime against ß-lactamase producing S. aureus, Enterobacter cloacae and E. coli strains in a murine intraperitoneal infection model. Each of the oxapenems inhibited class A, class C and class D ß-lactamases isolated from whole cells and purified by isoelectric focusing. AM-114 and AM-115 were as effective as clavulanic acid against class A enzymes. AM-112 and AM-113 were less potent against these enzymes. Class C and class D enzymes proved very susceptible to inhibition by the oxapenems. Molecular modelling of the oxapenems in the active site of the class A. TEM-1 and class C P99 enzymes identified a number of potential sites of interaction. The modelling suggested that Ser-130 in TEM-1 and Tyr-150 in P99 were likely candidates for cross-linking of the inhibitor, leading to inhibition of the enzyme. Morphology studies indicated that sub-inhibitory concentrations of the oxapenems caused the formation of round-shaped cells in E. coli DC0, indicating inhibition of penicillin-binding protein 2 (PBP2). The PBP affinity profile of AM-112 was examined in isolated cell membranes of E. coli DC0, S. aureus NCTC 6571, Enterococcus faecalis SFZ and E. faecalis ATCC 29213, in competition with a radiolabelled penicillin. PBP2 was identified as the primary target for AM-112 in E. coli DC0. Studies on S. aureus NCTC 6571 failed to identify a binding target. AM-112 bound to all the PBPs of both E. faecalis strains, and a concentration of 10µg/ml inhibited all the PBPs except PBP3.

The mode of action of bradykinin

The action of bradykinin on transepithelial transfer of sodium and water in isolated rat jejunum and on smooth muscle contraction of rat terminal ileum has been investigated. (1) Bradykinin was shown to stimulate transfer at low control transfer, inhibit transfer at high control transfer and have no effect at intermediate transfer in rat jejunal sacs. Stimulation of transfer occurred only when bradykinin was in the serosal solutiun while inhibition of transfer occurred whether bradykinin was in the aerosal or mucosal solution. Bradykinin-induced stimulation of transfer was not affected by adrenalectomy, nephrectomy, combined adrenalectomy-nephrectomy,  nor maintenance on 1% saline drinking solution or low sodium diet pretreatment. Meclofenamic acid abolished the bradykinin-induced inhibition of water transfer while prostaglandins A1, E1 aud F2α all potentiated this action. Theophylline inhibited water transfer and potentiated the bradykinin-induced inhibition of water transfer. Cyclic AMP and dibutyryl cyclic AMP both inhibited water transfer and the bradykinin-induced inhibition of water transfer was potentiated by the latter. ( 2 ) Bradykinin-induced contractions of rat terminal ileum were little affected by hyoscine while those of acetylcholine were abolished. Anoxia reduced markedly responses tv bradykinin while those of acetylcholine were little affected . Theophylline reduced the responses of rat terminal ileum to bradykinin significantly more than those to acetylcholine. Aspirin and indomethacin reduced markedly the responses to bradykinin while not affecting those to acetylcholine and PGT2. Meslofenamic acid at a concentration of 3.4 µM blocked bradykinin-induced contractions but had no effect on those to acctylcholine, PGE2 or PGF2 and at a concentration of 17. 0 µM drastically reduced bradykinin responses but also reduced those to acetylcholine, PGE2 and PGF2α• Flufenamic acid drastically reduced responses to bradykinin while not affecting those to acetylcholine and PGE2 and slightly affecting those to PGF2α. Polyphloretin phosphate reduced responses to bradykinin, PGF2α and PGE2 but not acetylcholine . Diphloretin phosphate reduced responses to bradykinin, PGF2 and PGE2 in a dose dependent manner but not those to acetylcholine. SC 19220 , in a dose dependent manner, inhibited responses to bradykinin and PGE2 but not to acetylcholine and PGF2. 7 oxa - 13 -prostynoic acid non specifically reduced responses to acetylcholine, bradykinin and PGE2. Bradykinin, in the presence of SQ 20881 , increased the release of prostaglandin-like activity from rat terminal ileum and this was reduced or abolished in the presence of indomethacin, aspirin, meclofenamic acid or flufenamio acid. The extract of PG-like activity did not appear as PGE, PGA or PGFon TLC, but included a substance with similar mobility as 15-Keto-prosta-glandin E2.

Studies on the mode of cytotoxicity of imidazotetraziones

The irnidazotetrazinones are a novel group of anti tumour agents which have demonstrated good activity against a range of murine tumours and human xenografts. They possess a structure activity relationship similar to the anti tumour triazenes, with the chloroethyl (mitozolomide) and methyl (temozolomide) analogues being active antitumour agents, whilst the ethyl (CCRG 82019) and higher homologues are inactive. This thesiS attempts to elucidate the biological mechanisms responsible for the strict structure-activity relationship observed amongst the imidazotetrazinones. Mitozolomide is the only agent chemically capable of cross-linking DNA , which has been suggested to be responsible fo r the cytotoxicity of this group of agents. Only mitozolomide and ternozolornide Exhibit a marked ditferential toxicity towards the 0 -alkylguanine-DNA alkyltransferase deficient GM892A (Mer-) cell line rather than the proficient Raji cell line (Mer+). The rate of uptake of imidazotetrazinones into cells is similar for all three agents in both cell lines, and does not explain the differing sensitivities to these agents. The effect of drug treatment on the incorporation of precursors into macromolecules, and their pool sizes, was examined. Temozolomide administration was found to alter de novo protein synthesis in both GM892A and Raji cells. Flow cytometric analysis revealed that temozolomide and CCRG 82019 block cells in late S/G2/M phase of the cell cycle , similar to that observed with mitozolomide. The extent of reaction of all three drugs with isolated macromolecules and cellular macromolecules was determined, and differences found, with cellular repair processes influencing the number of alkyl lesions remaining bound to macromolecules. The specific bases formed in calf thymus DNA after treatment with either temozolornide and CCRG 82019 was measured, and it was found that the types and relative amounts of lesions formed, differed, as well as the total level of alkylation. Whereas DNA extracted from imidazotetrazinone treated cells is not affected in its ability to support RNA polymerase activity, an effect is observed on the ability to extract DNA polymerase from drug treated cells. This may suggest that the alkylated DNA must be in intact chromatin for the lesion to manifest its effects. Temozolomide and methyl methanesulphonate do got appear to act with a synergistic mode of action. The 0 -position of guanine is suspected to be a critical site for the action of these types of drugs.

Do gender and year of study affect the ability of the theory of planned behaviour to predict binge-drinking intentions and episodes?

Background: The present study tested the utility of the theory of planned behaviour (TPB), augmented with anticipated regret, as a model to predict binge-drinking intentions and episodes among female and male undergraduates and undergraduates in different years of study. Method: Undergraduate students (N = 180, 54 males, 126 females, 60 per year of study) completed baseline measures of demographic variables, binge-drinking episodes (BDE), TPB constructs and anticipated regret. BDE were assessed one-week later. Results: The TPB accounted for 60% of the variance in female undergraduates' intentions and 54% of the variance in male undergraduates' intentions. The TPB accounted for 57% of the variance in intentions in first-year undergraduates, 63% of the variance in intentions in second-year undergraduates and 68% of the variance in intentions in final-year undergraduates. Follow-up BDE was predicted by intentions and baseline BDE for female undergraduates as well as second- and final-year undergraduates. Baseline BDE predicted male undergraduates’ follow-up BDE and first-year undergraduates’ follow-up BDE. Conclusion: Results show that while the TPB constructs predict undergraduates’ binge-drinking intentions, intentions only predict BDE in female undergraduates, second- and final-year undergraduates. Implications of these findings for interventions to reduce binge drinking are outlined.