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H Inokuchi

Publications and source records attributed to H Inokuchi.

At least 145 records · Page 8Linked to original sources
PubMed1988

Mischarging mutants of Su+2 glutamine tRNA in E. coli. I. Mutations near the anticodon cause mischarging.

In order to select the mischarging mutants of Su+2 glutamine tRNA, auxotrophic amber mutants of E. coli K12 which cannot be suppressed particularly by Su+2 were screened. By utilizing these mutants, cysam235 and metam3, several tens of mischarging mutants of Su+2 were isolated, as those conferring altered suppression patterns for a set of tester amber mutants of bacteria and phages. Nucleotide sequence analysis revealed that the mutation sites were found to be exclusively at psi 37 residue located at the 3'-end of anticodon loop, changing it to either A37 or C37. These mutants were obtained as those suppressing cysam235, and not metam3. From these, secondary mutants were selected. In these mutants suppression patterns were further altered by the additional base substitutions, capable of suppressing metam3. Such mutants were obtained exclusively from A37 and not from C37 mutant tRNA. Additional mutations to A37 were found to be either A29 or C38, which are located at the lowermost two base pairs in anticodon stem. The mischarging sites in Su+2 glutamine tRNA locate in the newly detected region of tRNA, differing from the previous case of Su+3 tyrosine or Su+7 tryptophan tRNAs. Implication of this finding is discussed on L-shaped tRNA molecule in relation to aminoacyl-tRNA synthetase recognition. Suppression patterns given by the double-mutants, A37A29 and A37C38, were consistent with the observation that the mutant tRNAs interact with tryptophanyl-tRNA synthetase.

Anticodon↗
PubMed1988

Mischarging mutants of Su+2 glutamine tRNA in E. coli. II. Amino acid specificities of the mutant tRNAs.

Among the mischarging mutants isolated from strains with Su+2 glutamine tRNA, two double-mutants, A37A29 and A37C38, have been suggested to insert tryptophan at the UAG amber mutation site as determined by the suppression patterns of a set of tester mutants of bacteria and phages (Yamao et al., 1988). In this paper, we screened temperature sensitive mutants of E. coli in which the mischarging suppression was abolished even at the permissive temperature. Four such mutants were obtained and they were identified as the mutants of a structural gene for tryptophanyl-tRNA synthetase (trpS). Authentic trpS mutations, such as trpS5 or trpS18, also restricted the mischarging suppression. These results strongly support the previous prediction that the mutant tRNAs of Su+2, A37A29 and A37C38, are capable of interacting with tryptophanyl-tRNA synthetase and being misaminoacylated with tryptophan in vivo. However, in an assay to determine the specificity of the mutant glutamin tRNAs, we detected predominantly glutamine, but not any other amino acid, being inserted at an amber codon in vivo to any significant degree. We conclude that the mutant tRNAs still accept mostly glutamine, but can accept tryptophan in an extent for mischarging suppression. Since the amber suppressors of Su+7 tryptophan tRNA and the mischarging mutants of Su+3 tyrosine tRNA are charged with glutamine, structural similarity among the tRNAs for glutamine, tryptophan and tyrosine is discussed.

Base Sequence↗
PubMed1987

Single crystals of hydrogenase from Desulfovibrio vulgaris Miyazaki F.

The hydrogenase solubilized from the particulate fraction from Desulfovibrio vulgaris Miyazaki F (IAM 12604) has been crystallized. Although the solubilized hydrogenase purified by the previous method (Yagi, T., Kimura, K., Daidoji, H., Sakai, F., Tamura, S., and Inokuchi, H. (1976) J. Biochem. (Tokyo) 79,661-671) revealed a single band upon disc electrophoresis, it could not be crystallized. The apparently homogeneous hydrogenase has been separated into three components of similar molecular weights by high performance liquid chromatography on DEAE-Toyopearl. Each hydrogenase component was successfully crystallized by means of the vapor diffusion method with polyethylene glycol or 2-methyl-2,4-pentanediol as a precipitating agent. Seeding procedure is necessary to grow an x-ray grade crystal. Preliminary x-ray experiments reveal that crystals grown from one component are in space group of P2(1)2(1)2(1) with a = 102.1(1), b = 126.8 (3), and c = 66.9(1) A. The unit cell volume of 8.66 X 10(5) A3 suggests that it contains one molecule/asymmetric unit (Vm = 2.43). The crystals grown from another component are in the same space group with a = 99.6(1), b = 126.8(3), c = 66.9(1) A, and the unit cell volume is 8.45 X 10(5) A3 (Vm = 2.37). The crystals diffract more than 2.5 A and are suitable for complete crystal analysis. Up to 4 A resolution native data have been collected on a diffractometer.

Chromatography, High Pressure Liquid↗
PubMed1986

Effects of exogenous and endogenous bombesin on gastrin secretion from rat antral mucosa in tissue culture.

Effects of exogenous and endogenous bombesin on gastrin secretion were examined using rat antral mucosa in tissue culture. Gastrin secretion was significantly stimulated by exogenous bombesin at a dose of 10(-8) M. Atropine 10(-6) M, which abolished the action of the cholinergic agent carbachol to stimulate gastrin secretion, had no effect on bombesin-stimulated gastrin secretion. In addition, gastrin secretion was significantly inhibited by anti-bombesin antiserum used to block the effect of endogenous bombesin by immunoneutralization. These findings suggest that the stimulation of gastrin secretion by bombesin does not involve cholinergic neural pathways and that endogenous bombesin exerts a continuous stimulation on gastrin secretion in the basal state.

Animals↗
PubMed1986

Occurrence and neonatal development of N-terminal (1-15) immunoreactivity of big gastrin in the rat antrum.

G34N (1-15) immunoreactive cells and C-terminal tetrapeptide immunoreactive cells in the antrum of rats of various ages were studied immunocytochemically in both naturally weaned and non-weaned conditions. Some of the C-terminal immunoreactive cells were found to lack G34N (1-15) immunoreactivity while the remainder showed both types of immunoreactivity. G34N (1-15)/C-terminal immunoreactive cell rates increased with age and reached adult values at 3 weeks after birth. The rates in a non-weaned condition were, however, significantly lower than those in a naturally weaned rats. These findings suggest that the development of G34N (1-15) immunoreactivity in rat antral gastrin cells is closely related to maturation of the gastrin cells, and weaning may enhance the maturation process.

Aging↗
PubMed1986

Neural and paracrine regulation of gastrin release using rat antral mucosa in tissue culture--the effect of carbachol, bombesin, and anti-somatostatin antibody on gastrin release.

Gastrin release was significantly stimulated by the cholinergic agent carbachol at doses of 10(-4) M, 10(-5) M, and 10(-6) M. Peak stimulation was observed at 10(-5) M. Gastrin release was also significantly stimulated by bombesin at a dose of 10(-8) M, and 10(-6) M atropine which abolished the effect of carbachol in stimulating gastrin release had no effect on the bombesin-stimulated gastrin release. In addition, anti-somatostatin antiserum significantly stimulated gastrin release. These findings suggest that gastrin release is regulated by cholinergic and noncholinergic neurons the latter being thought to be a bombesin-containing neuron, and that antral somatostatin exerts a continuous restraint on gastrin release by the paracrine mechanism.

Animals↗
PubMed1986

A case of primary amyloidosis confined to the small intestine.

A 60 year-old man with primary amyloidosis confined to the small intestine was reported. Multiple polyps of the small intestine were found by an upper GI series and enteroscopic polypectomy revealed massive deposition of amyloid in the lamina propria and the submucosa. No predisposing disorder or other sites of deposition were found, and the diagnosis of primary amyloidosis of the small intestine was confirmed.

Amyloidosis↗