Biomedical subjects

Nobuo Kato

Publications and source records attributed to Nobuo Kato.

At least 55 records · Page 3Linked to original sources

PubMed2004

Identification of (+)-phyllocladene, (--)-sandaracopimaradiene, and (+)-kaurene as new fungal metabolites from fusicoccin-producing Phomopsis amygdali F6.

A chemical analysis of the diterpene hydrocarbons produced by fusicoccin-producing fungus Phomopsis amygdali F6 identified five phyllocladene-related tri- and tetracyclic diterpene hydrocarbons. The presence of (+)-phyllocladene, (--)-sandaracopimaradiene, (+)-isopimara-8,15-diene, and (+)-pimara-8(14),15-diene in the fungus was demonstrated by GC-MS, 1H-NMR, and [alpha]D measurements. (+)-Kaurene was also identified by GC-MS and chiral capillary GC. The possible biosynthetic relationship of these metabolites is discussed.

Ascomycota↗

PubMed2004

Fusicocca-3(16),10(14)-diene, and beta- and delta-araneosenes, new fusicoccin biosynthesis-related diterpene hydrocarbons from Phomopsis amygdali.

Further isolation and examination of fusicoccane hydrocarbons biosynthetically related to fusicoccin from Phomopsis amygdali allowed us to identify new fungal diterpene hydrocarbons of fusicoccadiene and araneosene. These were assigned as (+)-fusicocca-3(16),10(14)-diene, and (+)-beta- and (+)-delta-araneosenes. These findings led to the experimental clarification of the structures of the biosynthetic hydrocarbon intermediates presumed earlier.

Ascomycota↗

PubMed2004

Erinacol (cyatha-3,12-dien-14beta-ol) and 11-O-acetylcyathin A3, new cyathane metabolites from an erinacine Q-producing Hericium erinaceum.

In our search for new cyathane metabolites related to the biosynthesis of erinacine Q in Hericium erinaceum, we isolated a novel cyatha-3,12-dien-14beta-ol named erinacol together with known 11-O-acetylcyathatriol (the erinacine Q aglycon) and new metabolite 11-O-acetylcyathin A(3) from the mycelial extract. The structure of each compound was determined by spectral methods. Possible biosynthetic relationships of these metabolites are discussed from their structural features.

Basidiomycota↗

PubMed2004

The pro-peptide of Streptomyces mobaraensis transglutaminase functions in cis and in trans to mediate efficient secretion of active enzyme from methylotrophic yeasts.

Transglutaminase (TGase) from the actinomycete Streptomyces mobaraensis is a useful enzyme in the food industry, and development of an efficient production system for it would be desirable. Herein we report secretion of TGase in an enzymatically active form by methylotrophic yeasts as expression hosts. Secretory production of active TGase required a pro-peptide from TGase. When an artificial Kex2-endopeptidase recognition site was placed between the pro-peptide and mature TGase, secretion and in vitro maturation of TGase depended on Kex2-dependent cleavage. Unexpectedly, coexpression of unlinked pro-peptide with mature TGase yielded efficient secretion of the active enzyme. These results indicate that the pro-peptide from TGase functions not only in an intramolecular but also in an intermolecular manner. Site-directed mutagenesis of putative N-glycosylation sites increased the productivity of the active TGase further. A recombinant Candida boidinii strain was found to secrete active TGase up to 1.83 U/ml (about 90 mg/l) after 119 h of cultivation.

Candida↗

PubMed2004

Three new thermotolerant methylotrophic yeasts, Candida krabiensis sp. nov., Candida sithepensis sp. nov., and Pichia siamensis sp. nov., isolated in Thailand.

Five strains of thermotolerant methylotrophic yeasts isolated in Thailand were found to represent three new species in the genera Pichia and Candida, based on phylogenetic analysis of D1/D2 domain of 26S rDNA, in addition to the morphological, physiological, biochemical and chemotaxonomic characterization. Three strains, FS96 and FS101 from flowers and M02 from tree flux, were characterized by ubiquinone Q7, multilateral budding, and the formation of hat-shaped ascospores that are liberated at maturation. These strains showed identical nucleotide sequences in the D1/D2 domain and formed a cluster with Candida thermophila, "Pichia salicis" and Pichia angusta. They differed by 1.9% of nucleotide substitutions from Candida thermophila, the nearest species. They were considered to represent a single new species and are described as Pichia siamensis sp. nov. Two strains, N051 and S023, isolated from soil did not produce ascospores, proliferated by multilateral budding, did not demonstrate urease or DBB color reaction, and lacked sexual stages. These characteristics correspond to the genus Candida. Strains N051 and S023 differed by 2.8% and 1.9% of nucleotide substitutions in the D1/D2 domain from the nearest species, Candida nemodendra and Candida ovalis, respectively, and are considered to represent respective new species. N051 and S023 are described as Candida krabiensis sp. nov. and Candida sithepensis sp. nov., respectively.

Candida↗

PubMed2003

Small-conductance Ca2+-dependent K+ channels are the target of spike-induced Ca2+ release in a feedback regulation of pyramidal cell excitability.

Cooperative regulation of inosiol-1,4,5-trisphosphate receptors (IP(3)Rs) by Ca(2+) and IP(3) has been increasingly recognized, although its functional significance is not clear. The present experiments first confirmed that depolarization-induced Ca(2+) influx triggers an outward current in visual cortex pyramidal cells in normal medium, which was mediated by apamin-sensitive, small-conductance Ca(2+)-dependent K(+) channels (SK channels). With IP(3)-mobilizing neurotransmitters bath-applied, a delayed outward current was evoked in addition to the initial outward current and was mediated again by SK channels. Calcium turnover underlying this biphasic SK channel activation was investigated. By voltage-clamp recording, Ca(2+) influx through voltage-dependent Ca(2+) channels (VDCCs) was shown to be responsible for activating the initial SK current, whereas the IP(3)R blocker heparin abolished the delayed component. High-speed Ca(2+) imaging revealed that a biphasic Ca(2+) elevation indeed underlays this dual activation of SK channels. The first Ca(2+) elevation originated from VDCCs, whereas the delayed phase was attributed to calcium release from IP(3)Rs. Such enhanced SK currents, activated dually by incoming and released calcium, were shown to intensify spike-frequency adaptation. We propose that spike-induced calcium release from IP(3)Rs leads to SK channel activation, thereby fine tuning membrane excitability in central neurons.

Animals↗

PubMed2003

Semi-wet peptide/protein array using supramolecular hydrogel.

The protein microarray is a crucial biomaterial for the rapid and high-throughput assay of many biological events where proteins are involved. In contrast to the DNA microarray, it has not been sufficiently established because of protein instability under the conventional dry conditions. Here we report a novel semi-wet peptide/protein microarray using a supramolecular hydrogel composed of glycosylated amino acetate. The spontaneous gel-formation and amphiphilic properties of this supramolecular hydrogel have been applied to a new type of peptide/protein gel array that is compatible with enzyme assays. Aqueous cavities created in the gel matrix are a suitable semi-wet reaction medium for enzymes, whereas the hydrophobic domains of the fibre are useful as a unique site for monitoring the reaction. This array system overcomes several drawbacks of conventional protein chips, and thus can have potential applications in pharmaceutical research and diagnosis.

Equipment Design↗

PubMed2003

Age-dependent emergence of oscillatory signal flow between the primary and secondary visual cortices in rat brain slices.

Developmental changes in dynamics of signal propagation between the primary (Oc1) and secondary visual cortex (Oc2) were investigated by using optical recording methods with voltage-sensitive dyes. Propagating oscillatory optical responses were evoked by our previously reported procedure, and were recorded on stimulation to white matter of Oc1 in rat visual cortex slices. In immature slices, evoked signals spread out from the stimulation site by way of deep layers, but were restricted largely to Oc1. In mature slices, however, evoked signals spread upward from the stimulation site at first, and then spread out along layer II/III, finally to penetrate Oc2. More remarkably, after this initial signal was attenuated, oscillatory responses emerged and spread back from Oc2 to Oc1 by way of layer II/III, suggesting that the origin of backpropagating oscillation is located in Oc2. The initial forward component was dependent on both N-methyl-D-aspartate (NMDA) and non-NMDA receptors, and the subsequent backward components were dependent only on NMDA receptors. These results suggest that the extent of corticocoritcal signal propagation within the visual cortex grows wider horizontally during maturation, so that information interchange may become easier between the Oc1 and Oc2.

2-Amino-5-phosphonovalerate↗

PubMed2003

Modification of a ubiquitin-like protein Paz2 conducted micropexophagy through formation of a novel membrane structure.

Microautophagy is a versatile process in which vacuolar or lysosomal membranes directly sequester cytosolic targets for degradation. Recent genetic evidence suggested that microautophagy uses molecular machineries essential for macroautophagy, but the details of this process are still unknown. In this study, a ubiquitin-like protein Paz2 essential for micropexophagy in the yeast Pichia pastoris has been shown to receive modification through the function of Paz8 and Gsa7, yielding a modified form Paz2-I, similar to the ubiquitin-like lipidation of Aut7 that is essential for macroautophagy in Saccharomyces cerevisiae. We identified a novel membrane structure formed after the onset of micropexophagy, which we suggest is necessary for the sequestration of peroxisomes by the vacuole. Assembly of this newly formed membrane structure, which is followed by localization of Paz2 to it, was found to require a properly functioning Paz2-modification system. We herein show that Paz2 and its modification system conduct micropexophagy through formation of the membrane structure, which explains the convergence between micropexophagy and macroautophagy with regard to de novo membrane formation.

Amino Acid Sequence↗

PubMed2003

Age-dependent appearance of an insulo-parietal cortical signal propagation that elicits a synchronized population oscillation in the parietal cortex in rats.

We investigated postnatal development of a functional connectivity from the gustatory insular cortex to the parietal cortex, which is known to contain many more neurons responding to oral somesthetic stimulation than the insular cortex, in slices obtained from 9-35-day-old rats. Field potentials were evoked by stimulation to the insular cortex. In the mature cortex, insular stimulation elicited a solitary field potential in both the insular and parietal cortices and, as the simulation continued, the initial solitary potential came to be followed by a population oscillation of field potential in the parietal cortex, but not in the insular cortex. In the immature cortex, by contrast, insular stimulation failed to evoke both the initial solitary potential and the subsequent population oscillation in the parietal cortex. In the mature cortex, application of neither thapsigargin nor AP5 prevented elicitation of the initial solitary potential in the parietal cortex, but either of them abolished the parietal oscillation. In immature cortex bathed with low Mg(2+) medium, insular stimulation elicited both the initial solitary potential and the subsequent parietal oscillation, which were both prevented by thapsigargin or AP5. Theses results suggest that the insular and parietal cortices are anatomically connected but functionally unlinked at an early postnatal stage, and that a functional linkage, dependent both on NMDA and ryanodine receptors, is formed during the first postnatal month.

2-Amino-5-phosphonovalerate↗

PubMed2003

Peroxisome degradation requires catalytically active sterol glucosyltransferase with a GRAM domain.

Fungal sterol glucosyltransferases, which synthesize sterol glucoside (SG), contain a GRAM domain as well as a pleckstrin homology and a catalytic domain. The GRAM domain is suggested to play a role in membrane traffic and pathogenesis, but its significance in any biological processes has never been experimentally demonstrated. We describe herein that sterol glucosyltransferase (Ugt51/Paz4) is essential for pexophagy (peroxisome degradation), but not for macroautophagy in the methylotrophic yeast Pichia pastoris. By expressing truncated forms of this protein, we determined the individual contributions of each of these domains to pexophagy. During micropexophagy, the glucosyltransferase was associated with a recently identified membrane structure: the micropexophagic apparatus. A single amino acid substitution within the GRAM domain abolished this association as well as micropexophagy. This result shows that GRAM is essential for proper protein association with its target membrane. In contrast, deletion of the catalytic domain did not impair protein localization, but abolished pexophagy, suggesting that SG synthesis is required for this process.

Autophagy↗

PubMed2003

Photochemical reactions of 2-bromotropone and 2,7-dibromotropone with 9,10-dicyanoanthracene.

The photochemical reactions of 2-bromotropone and 2,7-dibromotropone with 9,10-dicyanoanthracene gave products with anthracene, anthracenone, and dihydroanthracene skeletons both in polar and non-polar solvents. These products were formed by attack of water contaminated in the solvent, by attack of the troponoid, and by attack of the solvent used in the reactions, respectively, on a reaction intermediate. In a mixed solvent of benzene and methanol, a benzaldehyde derivative with a tribenzo-2-oxabicyclo[3.2.2]nonane system was obtained. This result was informative about the reaction mechanism, and suggested the formation of an [8 + 4]pi cycloadduct with a tribenzo-2-oxabicyclo[3.2.2]nonane system between the troponoid as the 8 pi component and the 9,10-dicyanoanthracene as the 4 pi component. In non-polar benzene, a new tetrabromodihydroanthracene derivative was obtained together with anthracenone and anthracene derivatives. It was proved by the reaction in benzene-d6 that the new product was formed by attack of benzene-d6.

Journal Article↗

PubMed2003

Wax ester production by bacteria.

The enzymological and genetic aspects of microbial metabolism of hydrocarbons have been extensively revealed. Such molecular information is useful for understanding the bioremediation of oil spill environments and production of hydrocarbon-specific fine chemicals.

Bacteria↗

PubMed2003

Physiological role of S-formylglutathione hydrolase in C(1) metabolism of the methylotrophic yeast Candida boidinii.

The methylotrophic yeast Candida boidinii exhibits S-formylglutathione hydrolase activity (FGH, EC 3.1.2.12), which is involved in the glutathione-dependent formaldehyde oxidation pathway during growth on methanol as the sole carbon source. The structural gene, FGH1, was cloned from C. boidinii, and its predicted amino acid sequence showed more than 60 % similarity to those of FGHs from Paracoccus denitrificans and Saccharomyces cerevisiae, and human esterase D. FGH from C. boidinii contained a C-terminal tripeptide, SKL, which is a type I peroxisome-targeting signal, and a bimodal distribution of FGH between peroxisomes and the cytosol was demonstrated. The FGH1 gene was disrupted in the C. boidinii genome by one-step gene disruption. The fgh1Delta strain was still able to grow on methanol as a carbon source under methanol-limited chemostat conditions with low dilution rates (D<0.05 h(-1)), conditions under which a strain with disruption of the gene for formaldehyde dehydrogenase (another enzyme involved in the formaldehyde oxidation pathway) could not survive. These results suggested that FGH is not essential but necessary for optimal growth on methanol. This is believed to be the first report of detailed analyses of the FGH1 gene in a methylotrophic yeast strain.

Base Sequence↗

PubMed2003

Formaldehyde fixation contributes to detoxification for growth of a nonmethylotroph, Burkholderia cepacia TM1, on vanillic acid.

During bacterial degradation of methoxylated lignin monomers, such as vanillin and vanillic acid, formaldehyde is released through the reaction catalyzed by vanillic acid demethylase. When Burkholderia cepacia TM1 was grown on vanillin or vanillic acid as the sole carbon source, the enzymes 3-hexulose-6-phosphate synthase (HPS) and 6-phospho-3-hexuloisomerase (PHI) were induced. These enzymes were also expressed during growth on Luria-Bertani medium containing formaldehyde. To understand the roles of these enzymes, the hps and phi genes from a methylotrophic bacterium, Methylomonas aminofaciens 77a, were introduced into B. cepacia TM1. The transformant strain constitutively expressed the genes for HPS and PHI, and these activities were two- or threefold higher than the activities in the wild strain. Incorporation of [14C]formaldehyde into the cell constituents was increased by overexpression of the genes. Furthermore, the degradation of vanillic acid and the growth yield were significantly improved at a high concentration of vanillic acid (60 mM) in the transformant strain. These results suggest that HPS and PHI play significant roles in the detoxification and assimilation of formaldehyde. This is the first report that enhancement of the HPS/PHI pathway could improve the degradation of vanillic acid in nonmethylotrophic bacteria.

Aldehyde-Lyases↗

PubMed2003

Propane monooxygenase and NAD+-dependent secondary alcohol dehydrogenase in propane metabolism by Gordonia sp. strain TY-5.

A new isolate, Gordonia sp. strain TY-5, is capable of growth on propane and n-alkanes with C(13) to C(22) carbon chains as the sole source of carbon. In whole-cell reactions, significant propane oxidation to 2-propanol was detected. A gene cluster designated prmABCD, which encodes the components of a putative dinuclear-iron-containing multicomponent monooxygenase, including the large and small subunits of the hydroxylase, an NADH-dependent acceptor oxidoreductase, and a coupling protein, was cloned and sequenced. A mutant with prmB disrupted (prmB::Kan(r)) lost the ability to grow on propane, and Northern blot analysis revealed that polycistronic transcription of the prm genes was induced during its growth on propane. These results indicate that the prmABCD gene products play an essential role in propane oxidation by the bacterium. Downstream of the prm genes, an open reading frame (adh1) encoding an NAD(+)-dependent secondary alcohol dehydrogenase was identified, and the protein was purified and characterized. The Northern blot analysis results and growth properties of a disrupted mutant (adh1::Kan(r)) indicate that Adh1 plays a major role in propane metabolism. Two additional NAD(+)-dependent secondary alcohol dehydrogenases (Adh2 and Adh3) were also found to be involved in 2-propanol oxidation. On the basis of these results, we conclude that Gordonia sp. strain TY-5 oxidizes propane by monooxygenase-mediated subterminal oxidation via 2-propanol.

Alcohol Dehydrogenase↗

PubMed2003

(+)-Menthol and its hydroxy derivatives, novel fungal monoterpenols from the fusicoccin-producing fungi, Phomopsis amygdali F6a and Niigata 2.

In our search for new fusicoccins of unique diterpene glucosides from Phomopsis amygdali, we found that a fragrant substance was formed in the early stage of fusicoccin fermentation. This fragrant constituent was isolated and identified as (+)-menthol, which is a novel fungal metabolite as the enantiomer of well-known peppermint (-)-menthol. (+)-7-Hydroxymenthol and new (+)-(6S)-hydroxymenthol were also isolated and identified as fungal metabolites. In addition, p-menthanetriol, which has been reported as the first fungal monoterpene from the fungus, was also isolated. The possible biosynthetic relationship of these metabolites is discussed.

Carbon Isotopes↗

PubMed2002

Physiological role of the second alcohol oxidase gene MOD2 in the methylotrophic growth of Pichia methanolica.

The methylotrophic yeast Pichia methanolica has nine multiple alcohol oxidase (AOD) isozymes, which can be detected on native electrophoretic polyacrylamide gel and are encoded by two genes, MOD1 and MOD2. The aim of this work is to reveal the physiological roles of these AOD subunits, especially that of Mod2p, encoded by the second AOD-encoding gene, MOD2. A strain expressing only MOD2 showed severe growth inhibition with a low concentration of methanol (0.1%), but its growth was restored with an increase in the methanol concentration (up to 3%). The expression of MOD2 using the CbAOD1 promoter in the Candida boidinii alcohol oxidase-depleted strain was more advantageous for methylotrophic growth with high methanol concentrations than that of MOD1. The expression of MOD2 was not observed under derepression conditions (0% methanol), and the expression level increased with an increase in the methanol concentration used for induction. The expression of MOD1 was observed under derepression conditions and was rather constant throughout the tested methanol concentration range. Therefore, the ratio of Mod2p to Mod1p in an active AOD octamer was proved to be mainly controlled by changes in the MOD2 mRNA level. These and other results show that Mod2p is a unique AOD subunit more adapted to methylotrophic growth with high methanol concentrations (3%) than Mod1p.

Alcohol Oxidoreductases↗