Kiyotaka Machida

Farnesol-induced growth inhibition in Saccharomyces cerevisiae by a cell cycle mechanism.

The growth of budding yeast, Saccharomyces cerevisiae, was inhibited in medium containing 25 microM farnesol (FOH). The FOH-treated cells were still viable, and were characterized by a transition from budded to unbudded phase as well as a significant loss of intracellular diacylglycerol (DAG). FOH-induced growth inhibition could be effectively prevented by the coaddition of a membrane-permeable DAG analogue which can activate yeast protein kinase C (PKC). However, yeast cell growth was not initiated upon addition of the PKC activator when the cells had been pretreated with FOH for 20 min. The failure in cell growth recovery was believed to be due to a signalling-mediated cell cycle arrest in FOH-pretreated cells. Differential display analysis demonstrated that the expression of cell cycle genes encoding DNA ligase (CDC9) and histone acetyltransferase (HAT2) was strongly repressed in FOH-treated cells. Repression of the expression of these genes was effectively cancelled when cells were grown in medium supplemented with DAG. The authors propose an interference with a phosphatidylinositol-type signalling which is involved in cell cycle progression as a cause of FOH-induced growth inhibition in yeast cells.

FARNESOL-INDUCED GENERATION OF REACTIVE OXYGEN SPECIES DEPENDENT ON MITOCHONDRIAL TRANSMEMBRANE POTENTIAL HYPERPOLARIZATION MEDIATED BY F0F1-ATPASE IN YEAST

An isoprenoid farnesol (FOH) inhibited cellular oxygen consumption and induced mitochondrial generation of reactive oxygen species (ROS) in cells of Saccharomyces cerevisiae in correlation with hyperpolarization of the mitochondrial transmembrane potential (mtΔΨ). The FOH‐induced events were coordinately abolished with the F1‐ATPase inhibitor sodium azide as well as the F0F1‐ATPase inhibitor oligomycin, suggesting the dependence of ROS generation on mtΔΨ hyperpolarization mediated by the proton pumping function of F0F1‐ATPase as a result of ATP hydrolysis. The role of F1‐ATPase activity in mtΔΨ hyperpolarization was supported by the intracellular depletion of ATP in FOH‐treated cells and its protection with sodium azide. An indirect mechanism was suggested to exist in the regulation of F0F1‐ATPase by FOH to accelerate its ATP‐hydrolyzing activity.

Depletion of glutathione as a cause of the promotive effects of polygodial, a sesquiterpene on the production of reactive oxygen species in Saccharomyces cerevisiae.

The pungent sesquiterpenoid unsaturated dialdehyde, polygodial, exhibited a strong yeastcidal activity against the cells of Saccharomyces cerevisiae, in which production of reactive oxygen species (ROS) at a significant level could be detected with a fluorescent probe. The production of ROS in polygodial-treated cells was further confirmed by its elimination and the accompanying protection against yeastcidal effects in the presence of antioxidants such as L-ascorbate and alpha-tocopherol (alpha-TOH). Polygodial could accelerate ROS production only in cells of the wild-type grande strain but not in those of the respiratory-deficient petite mutant (rho0), indicating the role of the mitochondrial electron transport chain in the production of ROS. Unlike the case with antimycin A which accelerates ROS production by directly targeting the mitochondrial electron flow, polygodial caused depletion of cytoplasmic and mitochondrial glutathione which functions in estiminating ROS inevitably generated during aerobic growth. Polygodial-mediated depletion of intracellular glutathione was possibly dependent on a direct interaction between its enal moiety and the sulfhydryl group of the cysteine in glutathione by a Michael-type reaction.

Synthesis of uridine 5′-monophosphate glucose as an inhibitor of UDP-glucose pyrophosphorylase

Abstract Uridine 5′-monophosphate α- d -glucose (UMPG) was evaluated as a novel and potent inhibitor of the enzymatic reaction involved in sugar nucleotide metabolism. UMPG was synthesized by chemical coupling of 2,3,4,6-tetra- O -acetyl-α- d -glucopyranosyl bromide with uridine 5′-monophosphate (UMP) to give uridine 5′-monophosphate 2″,3″,4″,6″-tetra- O -acetyl-α- d -glucose (UMPTAG), followed by deacetylation of UMPTAG with sodium methoxide. In addition to UMPG, UMPTAG showed potent inhibitory activity toward yeast UDPG pyrophosphorylase (UDPG synthetase). UMPG and UMPTAG were competitive with UDPG in the pyrophosphorolytic reaction, with inhibition constants ( K i ) of 4.8 and 20.7 μM, respectively, but non-competitive with inorganic pyrophosphate. UMPG and UMPTAG also inhibited the enzyme non-competitively in the reverse reaction to synthesize UDPG from UTP and glucose 1-phosphate (G1P). The acetyl group of UMPTAG was thought to enhance its hydrophobic interaction, possibly with an active site region of the enzyme functional for binding with UDPG.

Functional interaction between membranous surface and colloidal inside of yeast cells as reflected by an isopreniod-promoted mitochondrial generation of reactive oxygen species

Publisher Summary This chapter explores the mechanism of isopreniod farnesol (FOH)-promoted generation of reactive oxygen species (ROS) in terms of signal transduction between membranous surface and colloidal inside of the yeast cells. Farnesol (FOH)-treated cells are characterized with 5–8 fold increase in the level of ROS generation at the initial 30 minute incubation while none of ROS generating response is observed against other isoprenoid compounds like geraniol, geranylgeraniol, and squalene. Dependence of FOH-induced growth inhibition on such an oxidative stress is confirmed by its protection with the coexistence of antioxidant such as a-tocopherol (α-TOH), probucol and AT-acetylcysteine (NAC). FOH can accelerate ROS generation only in cells of wild-type strain but not in those of the respiration-deficient petite mutant, representing the role of mitochondrial electron transport chain as its origin. Among the respiratory inhibitors, ROS generation can be effectively canceled with myxothiazol which inhibits oxidation of ubiquinol to ubisemiquinone radical by the Rieske iron–sulfur center of complex III but not with antimyicin A, an inhibitor of electron transport functional for further oxidation of ubisemiquinone radical to ubiquinone in Q-cycle of complex III. Cellular oxygen consumption is inhibited immediately upon extracellular addition of FOH whereas FOH and its possible metabolites fail to directly inhibit any of oxidase activities detected with isolated mitochondrial preparation. These findings suggest that FOH does not directly interact but indirectly interacted with mitochondrial electron transport chain viaa mechanism of signal transduction.