Biomedical subjects
Publications and source records attributed to T Higuti.
Four quinic acid gallates were isolated from the dried pods of Tara [Caesalpinia spinosa (Molina) Kuntze]. These compounds intensified the susceptibility of methicillin-resistant Staphylococcus aureus (MRSA) to oxacillin. 3,4,5-Tri-O-galloylquinic acid methyl ester (2) was the most effective compound of them.
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.
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).
The effects of alkane derivatives, 1-alcohols (ROH), aliphatic amine hydrochlorides (RNH(2).HCl) and sodium aliphatic carboxylates (ROONa), on the proton pumping activity of bacteriorhodopsin (bR) in a purple membrane have been examined. Photocurrents in bR in the purple membrane adsorbed onto polyester thin film were recorded before and after exposure to these test substances. The peak photocurrent in bR was reversibly suppressed by each substance. From the dose-response curve, the concentrations required to reduce the peak capacitive current by 50% were determined for each homolog and then the standard free energies per CH(2) for the adsorption of the alkane derivatives to the site of action were estimated: -3.13 kJ mol(-1) for ROH, -3.05 kJ mol(-1) for RNH(3)(+), and -2.95 kJ mol(-1) for ROO(-). The proton pumping activity of bR was mainly suppressed by the hydrophobic interaction with the additive. The relative potencies of the functional groups of the alkane derivatives were almost comparable between 1-octanol (C(8)OH) and octylamine hydrochloride (C(8)NH(3)(+)) and about 10 times less effective for sodium octanoate (C(8)OO(-)) than for others. The addition of C(8)OH or C(8)OO(-) changed the absorption spectra of bR with a maximum at 560 nm to the spectra of the intermediate state with a maximum at 480 nm, while the C(8)NH(3)(+) decreased the intensity of the 560 nm band only with no blue-shift by the 480 nm band. We conclude that the action of the alkane derivatives is nonspecific and directed to all organized purple membrane structures and that the binding sites of the ROH and ROO(-) are different from that of RNH(3)(+).
A careful investigation of the subterranean part of Vitex rotundifolia has shown that this plant contains five novel lignans having a 1-phenylnaphthalene-type skeleton together with four known lignans. These structures were elucidated on the basis of spectroscopic data. Furthermore, some of the isolated compounds showed antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA).
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.
Little is known about the relationship between the stoichiometry of polypeptides of multisubunit enzyme complexes and the absolute amount of each transcript of the complexes in mammalian tissues. Here we showed that the absolute amounts of the transcripts of most subunits of rat H+-ATP synthase examined greatly differed in the different tissues, showing the following hierarchy of tissue-specificity: heart > kidney > brain approximately liver. However, surprisingly, there was no difference in the expression pattern of these in terms of the molar ratio of each transcript, indicating a nearly similar stoichiometric expression pattern irrespective of tissue or age of the rat. Therefore, the present finding clearly indicates that most of the transcripts of the 16 subunits of rat H+-ATP synthase were concertedly and synchronously expressed, having a constant expression pattern of the transcripts, irrespective of tissue or age of the rats. This is the first report of the absolute amounts of the transcripts of this multisubunit enzyme.
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 (beta-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.
This study was conducted to find constituents of an annual herb, Xanthium sibiricum Patr er Widd, with effective antibacterial activity against methicillin-resistant Staphylococcus aureus (MRSA). By monitoring antibacterial activity against MRSA strains, it was shown that a sesquiterpene lactone, identified as [3aR-(3a alpha, 7 beta,8a beta)]-3,3a,4,7,8,8a-hexahydro-7- methyl-3-methylene-6-(3-oxo-1-butenyl)-2H-cyclohepta[b]furan-2-one, or xanthatin, isolated from leaves of the herb, had outstandingly potent activity against S. aureus species, including MRSA; its activity against MRSA and MSSA strains was similar. Other bacteria, e.g. Staphylococcus epidermidis, Klebsiella pneumoniae, Bacillus cereus, Pseudomonas aeruginosa and Salmonella typhi, were also susceptible at the concentrations tested but the compound had no inhibitory effect on some other bacteria, including Escherichia coli. The results show that xanthatin has outstandingly potent activity against strains of S. aureus but that the activity of the compound is highly species-specific.
Examination of the EtOH extract of the fruiting bodies of Terminalia chebula RETZ. led to the isolation of two potent antimicrobial substances against even methicillin-resistant strains of Staphylococcus aureus. On the basis of spectroscopic evidence, the two isolates have been identified as gallic acid and its ethyl ester.
The oligomycin sensitivity conferring protein (OSCP) of Saccharomyces cerevisiae is a component of the stalk FA sector of the mitochondrial F1Fo-ATP synthase complex (mtATPase). The subunits comprising this sector have been implicated in the coupling of proton flux through the membrane Fo sector to ATP synthesis on the catalytic F1 sector. We tested whether the yeast subunit encoded by the ATP5 gene could be functionally replaced by a mammalian homologue, rat OSCP. The yeast and rat OSCP proteins have 58% homology in terms of conserved amino acids. A vector-borne rat cDNA encoding the OSCP precursor was introduced into a yeast strain lacking endogenous OSCP as a result of its disrupted ATP5 gene. The resultant yeast cells showed essentially a wildtype growth rate at 18, 28 and 35 degrees C, on nonfermentable substrates. Expression of mammalian FA subunit homologues in yeast should provide a productive new approach to generating information on the structure and function of mtATPase.
Subunit c is normally present as an inner mitochondrial membrane component of the F0 section of the ATP synthase complex, but in the late infantile form of neuronal ceroid lipofuscinosis (NCL) it was also found in lysosomes in high concentrations. To explore the mechanism of storage of subunit c, the rates of degradation and synthesis of subunit c were measured in fibroblast cell types from controls and patients with the late infantile form of NCL. The radiolabel from subunit c decreased with time in control cells, whereas no apparent loss of radioactivity of subunit c was found in patients' cells. There were no significant differences between control cells and cells with disease in the degradation of cytochrome oxidase subunit IV, an inner membrane protein of mitochondria. A combination of pulse-chase and subcellular fractionation analysis showed that a delay of intramitochondrial loss from prelabeled subunit c was seen in all diseased cells tested. Lysosomal appearance of labeled subunit c could be detected after chase for more than 1 week and its radioactivities were variable among diseased cell types. The biosynthetic rate of subunit c was almost the same in both control and patient cells. Northern blotting analyses showed that mRNAs for P1 and P2 genes had no significant difference in lengths and amounts between control and patient cells. Results suggest a specific failure in the degradation of subunit c after its normal inclusion in mitochondria and its consequent accumulation in lysosomes. This is the first direct evidence to show a delay of subunit c degradation in the cells from the late infantile form of NCL.
Eukaryotic proteasomes from an evolutionarily conserved multi-gene family and are thought to have originated from a common ancestral gene and diverged into alpha-type and beta-type subgroups. To understand the molecular basis of the proteasome genes, we isolated and characterized two human proteasome genes econding the alpha-type HC3 and beta-type HC5 subunit. The functional genes for HC3 and HC5 are similar in being approximately 15 kb in length, but differ in having exon numbers of 9 and 6, respectively. Analyses of about 2.5 to 3.0 kb of the 5'-flanking regions of these two genes revealed the absence of TATA and CAAT promoter elements. However, two or three GC boxes were found. By analysis of the transcriptional regulatory activities in the 5'-flanking regions of the two genes, these GC boxes were found to function coordinately as promoters of the two genes. Interestingly, the HC3 gene possesses an additional silencer element in the 5'-upstream region near the first exon. This element is also able to repress the promoter activities of other genes, such as the HC5 and the type 1 glucose transporter genes, irrespective of whether it has a sense or antisense orientation, indicating that it acts as a general transcriptional silencer. The HC5 gene does not have this silence element, and its promoter activity is five to ten times that of HC3. These results show that the human proteasomal HC3 and HC5 genes differ not only in their genomic structures, such as their numbers of exons and their exon-intron organizations, but also in the mechanisms regulating their transcription, suggesting that they diverged at an early stage of evolution.
Two cDNAs encoding different signal peptides (61 and 66 amino acid residues) and the same mature protein have been cloned from a human cDNA library with a rat subunit c cDNA. The amino acid sequence of the mature human subunit c of mitochondrial H(+)-ATP synthase was completely identical with those of the mature bovine, sheep and rat subunit c.
Four cDNAs for the import precursors of oligomycin sensitivity conferring protein (OSCP), ATPase inhibitor protein (IF1) and subunit cs (encoded by P1 and P2 genes) of rat mitochondrial H(+)-ATP synthase have been cloned from a rat cDNA library. The import precursors and the mature polypeptides of rat OSCP, IF1, subunit c (P1) and subunit c (P2) consisted of 23/190, 25/82, 61/75 and 66/75 amino acids, respectively.
Subunit d of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high performance liquid chromatography. The partial amino acid sequence of the subunit was determined by automated Edman degradation of the peptide fragments. The nucleotide sequence of subunit d of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 581 nucleotides including a coding region for the import precursor of subunit d and noncoding regions on the 5'- and 3'- sides. The possible precursor of subunit d and its mature polypeptide deduced from the open reading frame consisted of 161 and 160 amino acid residues with molecular weights of 18,763 and 18,631, respectively. Subunit d is a hydrophilic protein with an isoelectric point of 6.19. The sequence of the rat subunit d is highly homologous with that of subunit d of bovine heart and slightly similar to that of the subunit d of the yeast mitochondria. However, it had no homology with the sequence of any of the subunits of bacterial or chloroplast H(+)-ATP synthase.
Subunit e of H(+)-ATP synthase from rat liver mitochondria was isolated from the purified enzyme by reverse-phase high-performance liquid chromatography. The amino acid sequence of the subunit was determined by automated Edman degradation of the whole protein and derived peptides. The nucleotide sequence of the import precursor of subunit e of rat liver H(+)-ATP synthase was determined from a recombinant cDNA clone isolated by screening a rat hepatoma cell line H4TG cDNA library with a probe DNA. The sequence was composed of 289 nucleotides including a coding region for the import precursor of subunit e and noncoding regions on the 5'- and 3'-sides. The possible import precursor of subunit e and its mature polypeptide deduced from the open reading frame consisted of 71 and 70 amino acid residues with molecular weights of 8254 and 8123, respectively. Subunit e is a basic hydrophilic protein with an isoelectric point of 9.78. The sequence of the rat subunit e is highly homologous with that of subunit e of bovine heart, but has no homology with any subunit of bacterial or chloroplast H(+)-ATP synthase. The function of subunit e is unknown. However, a homology search in the database of the National Biomedical Research Foundation revealed that residues 34-65 of subunit e are homologous with residues 90-117 of troponin T, and with residues 529-561 of h-caldesmon and residues 289-319 of l-caldesmon, which are the homologous sequences corresponding to the Ca(2+)-dependent tropomyosin-binding region of troponin T.