Tomihiko Higuti

Phytochemical flavones isolated from Scutellaria barbata and antibacterial activity against methicillin-resistant Staphylococcus aureus

A crude extract prepared from Scutellaria barbata D. Don (Lamiaceae) was analyzed in the effort to discover antibacterial compounds against high-level strains of methicillin-resistant Staphylococcus aureus (MRSA). Apigenin and luteolin were isolated from the plant as active constituents against the bacteria. These flavonoid congeners were selectively toxic to S. aureus, including the MRSA and methicillin-sensitive S. aureus strains.

Alkyl gallates, intensifiers of beta-lactam susceptibility in methicillin-resistant Staphylococcus aureus.

ABSTRACT We found that ethyl gallate purified from a dried pod of tara (Caesalpinia spinosa) intensified β-lactam susceptibility in methicillin-resistant and methicillin-sensitive strains of Staphylococcus aureus (MRSA and MSSA strains, respectively). This compound and several known alkyl gallates were tested with MRSA and MSSA strains to gain new insights into their structural functions in relation to antimicrobial and β-lactam susceptibility-intensifying activities. The maximum activity of alkyl gallates against MRSA and MSSA strains occurred at 1-nonyl and 1-decyl gallate, with an MIC at which 90% of the isolates tested were inhibited of 15.6 μg/ml. At concentrations lower than the MIC, alkyl gallates synergistically elevated the susceptibility of MRSA and MSSA strains to β-lactam antibiotics. Such a synergistic activity of the alkyl gallates appears to be specific for β-lactam antibiotics, because no significant changes were observed in the MICs of other classes of antibiotics examined in this study. The length of the alkyl chain was also associated with the modifying activity of the alkyl gallates, and the optimum length was C5 to C6. The present work clearly demonstrates that the length of the alkyl chain has a key role in the elevation of susceptibility to β-lactam antibiotics.

Coumarins and γ-pyrone derivatives from Prangos pabularia: antibacterial activity and inhibition of cytokine release

The n-hexane and ethyl acetate extracts of the stems and the ethyl acetate extracts of roots from Prangos pabularia afforded an gamma-pyrone derivative and furanocoumarin derivatives with three glucose and gamma-pyrone (pabularin A, B and C), along with 26 previously known compounds (18 coumarins, six terpenoids and two glycosides). Their structures were established on the basis of spectroscopic studies. Of these, 16 coumarin derivatives isolated from P. pabularia were tested for antibacterial activity and inhibition of cytokine release.

Sesquiterpenoids from the fruits of Ferula kuhistanica and antibacterial activity of the constituents of F. kuhistanica

Ethyl acetate extracts of the air-dried fruits of Ferula kuhistanica afforded three daucane esters: kuhistanicaol H, I and J, together with nine other known compounds. Their structures were established on the basis of spectroscopic evidence. Isolated compounds in this paper and previously reported compounds from the roots and stems of F. kuhistanica were tested for antibacterial activity. Some of them were selectively toxic against Gram-positive bacteria, including methicillin-sensitive and methicillin-resistant Staphylococcus aureus (MSSA and MRSA).

Cell-surface H+-ATP synthase as a potential molecular target for anti-obesity drugs

Here we show that the cell‐surface expression of the alpha subunit of H+‐ATP synthase is markedly increased during adipocyte differentiation. Treatment of differentiated adipocytes with small molecule inhibitors of H+‐ATP synthase or antibodies against alpha and beta subunits of H+‐ATP synthase leads to a decrease in cytosolic lipid droplet accumulation. Apolipoprotein A‐I, which has been shown to bind to the ectopic β‐chain of H+‐ATP synthase and inhibit the activity of cell‐surface H+‐ATP synthase, also was found to inhibit cytosolic lipid accumulation. These results suggest that the cell‐surface H+‐ATP synthase has a previously unsuspected role in lipid metabolism in adipocytes.

Rhodamine 6G, inhibitor of both H+-ejections from mitochondria energized with ATP and with respiratory substrates.

Rhodamine 6G inhibited ATP hydrolysis by oligomycin-sensitive ATPase, purified from rat liver mitochondria, in good accord with the dose-response curve for its inhibition of energy transduction of ATP synthesis in mitochondria, but it did not inhibit ATP hydrolysis by purified F1. Rhodamine 6G also inhibited both H+-ejections from mitochondria energized with respiratory substrates and with ATP. The present findings show that the inhibitory effect of rhodamine 6G on energy transduction is not due to a modification of the transport system for adenine nucleotides, Pi, and respiratory substrates, and that the inhibition sites of rhodamine 6G are on components related with H+-ejection by redox components and also on F0.

Possible Role of Mitochondrial Remodelling on Cellular Triacylglycerol Accumulation

Mitochondrial fusion and fission processes play a role in a variety of cell functions, including energy metabolism, cell differentiation and programmed cell death. Still, it is not clear how these processes contribute to the cell functions. Here, we investigated the role of mitochondrial remodelling on lipid metabolism in adipocytes. In 3T3-L1 pre-adipocytes, the morphology of mitochondria is organized as a continuous reticulum. Upon differentiation of adipocytes manifested by cellular triacylglycerol (TG) accumulation, mitochondrial morphology altered from filamentous to fragmented and/or punctate structures. When the mitochondrial fusion was induced in adipocytes by silencing of mitochondrial fission proteins including Fis1 and Drp1, the cellular TG content was decreased. In contrast, the silencing of mitochondrial fusion proteins including mitofusin 2 and Opa1 increased the cellular TG content followed by fragmentation of mitochondria. It also appears that polyphenolic phytochemicals, negative regulators of lipid accumulation, have mitochondrial fusion activity and that there is a good correlation between mitochondrial fusion activity and the cellular TG accumulation-reducing activity of the phytochemicals. These results suggest that cellular TG accumulation is regulated, at least in part, via mitochondrial fusion and fission processes.

Stoichiometry of subunit e in rat liver mitochondrial H+-ATP synthase and membrane topology of its putative Ca2+-dependent regulatory region

Previous studies have revealed that residues 34-65 of subunit e of mitochondrial H(+)-ATP synthase are homologous with the Ca(2+)-dependent tropomysin-binding region for troponin T and have suggested that subunit e could be involved in the Ca(2+)-dependent regulation of H(+)-ATP synthase activity. In this study, we determined the content of subunit e in H(+)-ATP synthase purified from rat liver mitochondria, and we also investigated the membrane topology of a putative Ca(2+)-dependent regulatory region of subunit e using an antibody against peptide corresponding to residues 34-65 of subunit e. Quantitative immunoblot analysis of subunit e in the purified H(+)-ATP synthase revealed that 1 mol of H(+)-ATP synthase contained 2 mol of subunit e. The ATPase activity of mitoplasts, in which the C-side of F(0) is present on the outer surface of the inner membrane, was significantly stimulated by the addition of the antibody, while the ATPase activity of submitochondrial particles and purified H(+)-ATP synthase was not stimulated. The antibody bound to mitoplasts but not to submitochondrial particles. These results suggest that the putative Ca(2+)-dependent regulatory region of subunit e is exposed on the surface of the C-side of F(0) and that subunit e is involved in the regulation of mitochondrial H(+)-ATP synthase activity probably via its putative Ca(2+)-dependent regulatory region.

Sidedness of inhibition of energy transduction in oxidative phosphorylation in rat liver mitochondria by ethidium bromide.

Ethidium bromide, a new type of inhibitor of energy transduction in oxidative phosphorylation, inhibited ATP synthesis in intact mitochondria but not in submitochondrial particles, the latter being inside-out relative to the membranes of intact mitochondria. Ethidium bromide incorporated inside the submitochondrial particles inhibited ATP synthesis in the particles. The decrease of the membrane potential by valinomycin (plus KCl) inhibited only slightly the energy-dependent binding of ethidium bromide to the mitochondria. The present results show clearly that ethidium bromide inhibited energy transduction in oxidative phosphorylation by acting on the outer side (C-side) of the inner mitochondrial membrane, perhaps by neutralizing negative charges created on the surface of the C-side, and that it had no inhibitory activity on the inner side (M-side) of the membrane. Th present results show also that the energy-dependent binding of ethidium is not due to electrophoretic transport down the membrane potential; ethidium may bind to negative charges on the surface of the C-side. The present study suggest that an anisotropic distribution of electric charge in the inner mitochondrial membrane is an intermediary high energy state of oxidatvie phosphorylation.

Gene Expression of Subunit c(P1), Subunit c(P2), and Oligomycin Sensitivity-conferring Protein May Play a Key Role in Biogenesis of H+-ATP Synthase in Various Rat Tissues

Mammalian H+-ATP synthase is a supramolecule composed of at least 14 subunits that have a constant stoichiometry. Nevertheless the coordinate regulation of the gene expressions of various subunits remains obscure. To clarify the coordinate transcriptional regulatory system of mammalian H+-ATP synthase, we determined the absolute amount of nine species of mRNAs for eight nuclear-encoded subunits of H+-ATP synthase in different tissues of 8-week-old rats by use of the synthetic mRNAs and 32P-labeled DNA probes for each mRNA. Our quantitative analyses of the transcripts of H+-ATP synthase revealed that nine species of the subunits in different tissues of 8-week-old rats were divisible into two groups: a high transcript gene (HTG) group (β-subunit, subunit b, subunit d, subunit e, and Factor 6) and a low transcript gene (LTG) group (subunit c(P1), subunit c(P2), IF1, and oligomycin sensitivity-conferring protein). The transcription step of LTG could constitute a bottleneck in the biogenesis of H+-ATP synthase. Thus, the transcriptional regulatory system of the LTG may play a key role in the biogenesis of mammalian H+-ATP synthase. The HTG were transcribed in a tissue-specific manner that corresponds with energy demand in the tissues. However, there was no tissue specificity in subunit c(P2). Furthermore, the tissue specificity of the transcript of IF1 differed substantially from that of HTG, suggesting that it could be crucial in the protection of mitochondrial membrane under abnormal conditions.