Yuki Sakai

A respiratory-driven and an artificially driven ATP synthesis in mutants of Vibrio parahaemolyticus lacking H+-translocating ATPase

Mutants of Vibrio parahaemolyticus lacking the H+-translocating ATPase were isolated to evaluate both the role of this enzyme and the possibility of the involvement of other cation-translocating ATPase in the energy transduction in this organism. Dicyclohexylcarbodiimide-sensitive ATPase activity which represents the H+-translocating ATPase was not detected either in the membrane vesicles or in the cytosol of the mutants. Three major subunits, alpha, beta and gamma, of the H+-translocating ATPase were missing in the membranes of the mutants. Although ATP was synthesized in wild type cells when an artificial H+ gradient was imposed, little ATP was synthesized in the mutants. However, we observed a large ATP synthesis driven by the respiration not only in the wild type but also in the mutants. The respiratory-driven ATP synthesis in wild type was inhibited by an H+ conductor, carbonylcyanide m-chlorophenylhydrazone, by about 50%. On the other hand, the ATP synthesis in the mutants was not affected by the H+ conductor. Since this organism possesses a respiratory Na+ pump, Na+-coupled ATP synthesis might take place. In fact, we observed some ATP synthesis driven by an artificially imposed Na+ gradient both in the wild type and the mutant.

Na+ / adenosine co-transport in Vibrio parahaemolyticus

Adenosine transport in Vibrio parahaemolyticus was studied. Na+ greatly stimulated adenosine uptake. Addition of adenosine to a cell suspension under anaerobic conditions elicited Na+ uptake, and the Na+ uptake was inhibited by monensin, an Na+ ionophore. Imposition of an electrochemical potential of Na+ or a membrane potential in energy-depleted cells elicited adenosine uptake. Therefore, adenosine transport in this organism was concluded to proceed by an Na+/adenosine co-transport mechanism. The Na+/adenosine co-transport system was induced when cells were grown in the presence of adenosine, and repressed by glucose. Although Na+ uptake elicited by adenosine was reduced by glucose, it was enhanced by methyl alpha-glucoside, which reduced the intracellular ATP level. Thus, the effects of glucose and the glucoside on the Na+/adenosine co-transport system did not seem to be due to inducer exclusion, but to be related to the intracellular ATP level.

Indene Compounds Synthetically Derived from Vitamin D Have Selective Antibacterial Action on Helicobacter pylori

Helicobacter pylori infects the human stomach and is closely linked with the development of gastric cancer. When detected, this pathogen can be eradicated from the human stomach using wide-spectrum antibiotics. However, year by year, H. pylori strains resistant to the antibacterial action of antibiotics have been increasing. The development of new antibacterial substances effective against drug-resistant H. pylori is urgently required. Our group has recently identified extremely selective bactericidal effects against H. pylori in (1R,3aR,7aR)-1-[(1R)-1,5-dimethylhexyl]octahydro-7a-methyl-4H-inden-4-one (VDP1) (otherwise known as Grundmanns ketone), an indene compound derived from the decomposition of vitamin D3 and proposed the antibacterial mechanism whereby VDP1 induces the bacteriolysis by interacting at least with PtdEtn (dimyristoyl-phosphatidylethanolamine [di-14:0 PtdEtn]) retaining two 14:0 fatty acids of the membrane lipid constituents. In this study, we synthesized new indene compounds ((1R,3aR,7aR)-1-((2R,E)-5,6-dimethylhept-3-en-2-yl)-7a-methyloctahydro-4H-inden-4-one [VD2-1], (1R,3aR,7aR)-1-((S)-1-hydroxypropan-2-yl)-7a-methyloctahydro-1H-inden-4-ol [VD2-2], and (1R,3aR,7aR)-7a-methyl-1-((R)-6-methylheptan-2-yl)octahydro-1H-inden-4-ol [VD3-1]) using either vitamin D2 or vitamin D3 as materials. VD2-1 and VD3-1 selectively disrupted the di-14:0 PtdEtn vesicles without destructing the vesicles of PtdEtn (dipalmitoyl-phosphatidylethanolamine) retaining two 16:0 fatty acids. In contrast, VD2-2, an indene compound lacking an alkyl group, had no influence on the structural stability of both PtdEtn vesicles. In addition, VD2-1 and VD3-1 exerted extremely selective bactericidal action against H. pylori without affecting the viability of commonplace bacteria. Meanwhile, VD2-2 almost forfeited the bactericidal effects on H. pylori. These results suggest that the alkyl group of the indene compounds has a crucial conformation to interact with di-14:0 PtdEtn of H. pylori membrane lipid constituents whereby the bacteriolysis is ultimately induced.

Selective Monoprotection of Symmetrical Diols in a Flow Reactor

Desymmetrization reactions provide a powerful approach for the construction of complex molecules. Various methods have been developed for the selective monoprotection of symmetrical diols; however, their application to large-scale operations is limited. In this study, the monotetrahydropyranylation of symmetrical diols in a flow reactor has been developed, whereby the length of the flow reactor tube and the amount of acid were optimized. A higher selectivity for the monoprotected derivative was observed when the reaction was performed in a flow reactor compared with that observed in a conventional batch experiment. The efficient flow method developed herein can be applied to large-scale synthesis by numbering up the flow reactor without affecting the selectivity and yield. Since monoprotection can be achieved without using a large excess of diol, our developed flow method is effective when expensive diol must be used.

Concise Synthesis of Hydroxy β-Methyl Fatty Acid Ethyl Esters

Hydroxy β-methyl fatty acid ethyl esters bearing different carbon chain lengths and varying hydroxyl group positions were successfully synthesized from symmetric diols. These fatty acid derivatives are useful intermediates of chemical probes for metabolic analyses of fatty acid.