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Biomedical subjects

I Shibuya

Publications and source records attributed to I Shibuya.

At least 109 records · Page 6Linked to original sources
PubMed1992

Metabolic regulations and biological functions of phospholipids in Escherichia coli.

Extensive genetic and biochemical studies in the last two decades have elucidated almost completely the framework of synthesis and turnover of quantitatively major phospholipids in E. coli. The knowledge thus accumulated has allowed to formulate a novel working model that assumes sophisticated regulatory mechanisms in E. coli to achieve the optimal phospholipid composition and content in the membranes. E. coli also appears to possess the ability to adapt phospholipid synthesis to various cellular conditions. Understanding of the functional aspects of E. coli phospholipids is now advancing significantly and it will soon be able to explain many of the hitherto unclear cell's activities on the molecular basis. Phosphatidylglycerol is believed to play the central role both in metabolism and functions of phospholipids in E. coli. The results obtained with E. coli should undoubtedly be helpful in the study of more complicated phospholipid metabolism and functions in higher organisms.

Animals↗
PubMed1992

Effectiveness of GABAB antagonists in inhibiting baclofen-induced reductions in cytosolic free Ca concentration in isolated melanotrophs of rat.

1. The purpose of the present experiments was to assess the activities of GABAB receptor antagonists in mammalian isolated melanotrophs. 2. Cytosolic free Ca concentration ([Ca2+]i) in rat melanotrophs in primary culture was monitored with the fluorescent probe, fura-2. 3. (-)-Baclofen lowered [Ca2+]i in a concentration-dependent manner with an EC50 of 0.96 microM. The reduction in [Ca2+]i produced by (-)-baclofen at a maximally effective concentration (100 microM) was similar to that produced by the classic transmitter inhibitory to melanotroph secretion, dopamine, at a corresponding concentration (100 nM), or by perifusion with a nominally Ca-free solution. 4. The GABAB receptor antagonists, 3-aminopropyl(diethoxymethyl)phosphinic acid (CGP 35348), 2-hydroxy saclofen, phaclofen and 4-amino-3-(5-methoxybenzo[b]furan-2-yl) butanoic acid (9H), had inhibitory effects on the reduction in [Ca2+]i produced by (-)-baclofen (3 microM). Of the antagonists tested, CGP 35348 was the most potent with an IC50 of 60 microM, compared to 120 to 400 microM for the others. CGP 35348 acted competitively. 5. CGP 35348 alone had no effect on basal [Ca2+]i, or on the changes in [Ca2+]i produced by dopamine (10 nM) or the specific GABAA receptor agonist, muscimol (10 microM). 6. The evidence indicates that of the antagonists tested, CGP 35348 offers the greatest promise for pharmacological analysis of the functional significance of the GABAB receptors in melanotrophs.

Animals↗
PubMed1992

Drastic alteration of cycloheximide sensitivity by substitution of one amino acid in the L41 ribosomal protein of yeasts.

Cycloheximide is one of the antibiotics that inhibit protein synthesis in most eukaryotic cells. We have found that a yeast, Candida maltosa, is resistant to the drug because it possesses a cycloheximide-resistant ribosome, and we have isolated the gene responsible for this. In this study, we sequenced this gene and found that the gene encodes a protein homologous to the L41 ribosomal protein of Saccharomyces cerevisiae, whose amino acid sequence has already been reported. Two genes for L41 protein, named L41a and L41b, independently present in the genome of S. cerevisiae, were isolated. L41-related genes were also isolated from a few other yeast species. Each of these genes has an intron at the same site of the open reading frame. Comparison of their deduced amino acid sequences and their ability to confer cycloheximide resistance to S. cerevisiae, when introduced in a high-copy-number plasmid, suggested that the 56th amino acid residue of the L41 protein determines the sensitivity of the ribosome to cycloheximide; the amino acid is glutamine in the resistant ribosome, whereas that in the sensitive ribosome is proline. This was confirmed by constructing a cycloheximide-resistant strain of S. cerevisiae having a disrupted L41a gene and an L41b gene with a substitution of the glutamine codon for the proline codon.

Amino Acid Sequence↗
PubMed1992

Calcium channels in rat melanotrophs are permeable to manganese, cobalt, cadmium, and lanthanum, but not to nickel: evidence provided by fluorescence changes in fura-2-loaded cells.

Cobalt (Co), nickel (Ni), manganese (Mn), cadmium (Cd), and lanthanum (La) are commonly used as calcium (Ca) channel blockers, but some of them, besides reducing Ca entry, also traverse Ca channels and can exert effects intracellularly that confound interpretation of functional responses. Because of this and our need to use Ca channel blockers in an ongoing analysis of Ca channel activity in the regulation of the cytosolic free Ca concentration ([Ca2+]i) and secretion in melanotrophs, we assessed whether the cations mentioned enter these cells. This was done by incorporating the fluorescence for changes that would signal the presence of the cations in the cytosol. In cell-free solution, where the probe and cations can interact freely, Mn, Co, and Ni all quench fluorescence, whereas Cd and La act in a Ca-like manner. When tested on fura-2-loaded melanotrophs in basal (unstimulated) conditions, Mn, Co, and Cd each yielded corresponding signals, thereby showing that they had penetrated the cells. By contrast, Ni caused no quenching of fluorescence even in melanotrophs exposed to 100 mM K+ to recruit additional Ca channels. Ni, therefore, did not penetrate the cells. However, as expected, Ni quenched fluorescence when given artificial access to the cytoplasm by ionomycin. Ni blocked spontaneous entry of Mn, Co, and Cd. It also lowered [Ca2+]i in unstimulated melanotrophs, consistent with blockade of spontaneous Ca entry. Like Ni, La lowered basal [Ca2+]i in unstimulated melanotrophs without penetrating the cells; however, unlike Ni, it penetrated when the melanotrophs were exposed to high potassium. We conclude that Ni is the most specific of the Ca channel blockers tested and that results obtained with Mn, Co, Cd, and La must be interpreted with reserve.

Animals↗
PubMed1992

Cloning of the alpha-amylase cDNA of Aspergillus shirousamii and its expression in Saccharomyces cerevisiae.

alpha-Amylase cDNA was cloned and sequenced from Aspergillus shirousamii RIB2504. The putative protein deduced from the cDNA open reading frame (ORF) consisted of 499 amino acids with a molecular weight of 55,000. The amino acid sequence was identical to that of the ORF of the Taka-amylase A gene of Aspergillus oryzae, while the nucleotide sequence was different at two and six positions in the cDNA ORF and 3' non-coding regions, respectively, so far determined. The alpha-amylase cDNA was expressed in Saccharomyces cerevisiae under the control of the yeast ADH1 promoter using a YEp-type plasmid, pYcDE1. The cDNA of glucoamylase, which was previously cloned from the same organism, was also expressed under the same conditions. Consequently, active alpha-amylase and glucoamylase were efficiently secreted into the culture medium. The amino acid sequence of the N-terminal regions of these enzymes purified from the yeast culture medium confirmed that the signal sequences of these enzymes were cleaved off at the same positions as those of the native enzymes of A. shirousamii.

Amino Acid Sequence↗
PubMed1992

Construction of an alpha-amylase/glucoamylase fusion gene and its expression in Saccharomyces cerevisiae.

A fusion gene which encoded a polypeptide comprised of 1116 amino acids was constructed using the alpha-amylase and glucoamylase cDNAs of Aspergillus shirousamii. When the fusion gene was expressed in Saccharomyces cerevisiae using a yeast expression plasmid under the control of the yeast ADH1 promoter, a bifunctional fusion protein (145 kDa) having both alpha-amylase and glucoamylase activities was secreted into the culture medium. The fusion protein had higher raw-starch-digesting activity than those of the original alpha-amylase and glucoamylase, and adsorbed onto raw starch like the glucoamylase. It was suggested that the characteristics are a result of the raw-starch-affinity site in the glucoamylase domain of the fusion protein.

Alcohol Dehydrogenase↗
PubMed1991

Studies on pituitary melanotrophs reveal the novel GABAB antagonist CGP 35-348 to be the first such compound effective on endocrine cells.

One obstacle to understanding the action and physiological significance of the responsiveness of various endocrine cells to gamma-aminobutyric acid (GABA) has been that previously available substances, all active as GABAB antagonists in the nervous system, are ineffective on endocrine cells. The introduction of a potent new member of this class, CGP 35-348, of very different chemical structure, encouraged us to examine its effect on endocrine cells. For this purpose, we studied melanotroph secretion from pituitary neurointermediate lobes. We found that CGP 35-348, in contrast to previously available members of this class, suppressed completely, in rat and toad, secretory responses to baclofen, the classic GABAB agonist. Analysis, in toad, showed CGP 35-348 did not affect responses to GABAA agonists (muscimol; isoguvacine), dopamine, or neuropeptide Y. When tested against GABA, the physiological ligand present in the innervation of melanotrophs (along with dopamine and neuropeptide Y), CGP 35-348 completely suppressed the secretory response, which, in toad, is purely inhibitory and unaffected by bicuculline, the specific GABAA antagonist. In addition, CGP 35-348 unmasked a stimulant effect that bicuculline blocked. In CGP 35-348, we thus have a new tool with which to analyse responses to GABA and their physiological involvement in endocrine cells.

Animals↗
PubMed1991

Amplification and substantial purification of cardiolipin synthase of Escherichia coli.

A simple, specific, and sensitive assay procedure for cardiolipin synthase of Escherichia coli has been developed. This measures the radioactivity of glycerol formed from phosphatidyl [2-3H]glycerol and is mainly based on the findings that 400 mM phosphate and 0.015% Triton X-100 markedly activate the enzyme. Cardiolipin synthase was amplified 760-fold upon induction with isopropyl beta-D-thiogalactoside in cells harboring a pBR322 derivative in which the cls gene encoding this enzyme was preceded by the tac promoter. Under these conditions, cardiolipin content increased, membrane potential decreased, spheroplasts became fragile, cells lost viability, and inducer-resistant mutants appeared at a high frequency. The amplification enabled the isolation of an enzyme preparation with a specific activity approximately 10,000-times higher than that of wild-type whole cell lysate. This purification was simply achieved by extraction of the crude membrane fraction with Triton X-100 and a single phosphocellulose column chromatography. This preparation, together with the crude envelope fraction, was used to characterize the basic properties of E. coli cardiolipin synthase, some of which were utilized in setting up the assay conditions.

Detergents↗
PubMed1991

Why are several inhibitory transmitters present in the innervation of pituitary melanotrophs? Actions and interactions of dopamine, GABA and neuropeptide Y on secretion from neurointermediate lobes of Xenopus laevis.

The aim of the present experiments was to determine whether some rationale for the presence of the several inhibitory neurotransmitters in the innervation to the toad melanotroph might be found in differences in their individual effects or in possible cooperative interactions affecting secretion. Measurements of peptide release from isolated, perifused neurointermediate lobes of the toad Xenopus laevis showed that each of the three identified inhibitory transmitters, dopamine, GABA and NPY, was able to inhibit secretion profoundly and no less effectively than omission of Ca. Moreover, the inhibitory effects were rather similar in onset, duration and recovery. Furthermore, there was no evidence of any cooperative interactions when the transmitters were given in various combinations. And finally, the inhibitory response to each of the transmitters was abolished by pretreatment with pertussis toxin. While not excluding differential postsynaptic effects on other parameters of melanotroph function, the similarities observed have encouraged alternative speculations on the significance of the apparent redundancy of inhibitory transmitters.

Animals↗
PubMed1991

A microfluorometric method for simultaneous measurement of changes in cytosolic free calcium concentration and pH in single cardiac myocytes.

We describe a new system to measure changes in cytosolic free Ca concentration ([Ca2+]i) and pH (pHi) simultaneously using the dyes fura-2 and BCECF, and a random access camera with computer-operated synchronously-changing excitation and emission filters. Fluorescence intensities for fura-2 and BCECF were recorded from rat cardiac myocytes and calibrated in vivo using multiple-point determinations. [Ca2+]i and pHi were 88.6 +/- 32.8 nM and 7.16 +/- 0.12, respectively.

Animals↗
PubMed1990

Etching of dentin surface with ammonium citrate aqueous solution.

New dentin etchants AC (ammonium citrate) were examined. The pH of the AC solution was 7.3-7.4, and the concentration of the solutions varied from 5% to 30%. The etching of a 10% AC solution was the most effective in improving the adhesion between the composite resin and dentin among the AC solutions employed when MTYA.G.H was used as a dentin primer. The smear layer was removed and the tubules were not generally opened with AC etching. The bond strengths to dentin etched with 10% AC were comparable with those to dentin etched with neutralized 0.5 M EDTA or EDTA 3.2. The bond strengths increased with primers MTYA.H and MTYA.G.H, in that order.

Acid Etching, Dental↗
PubMed1990

Molecular cloning of the glucoamylase gene of Aspergillus shirousami and its expression in Aspergillus oryzae.

The glucoamylase enzyme (GAase) gene from Aspergillus shirousami was cloned and sequenced from genomic and cDNA libraries. The genomic gene was located in the 5.4 kb EcoRI fragment. The deduced amino acid sequence of GAase contained 639 amino acid residues with a relative molecular mass of approximately 68,000 daltons (non-glycosylated form). The genomic gene of A. shirousami GAase was introduced into Aspergillus oryzae. These transformants had increased GAase and raw starch degradation (RSD) activity in culture media and in rice-koji extracts. Analysis by Southern, Northern, SDS-PAGE, and Western blot techniques confirmed the foreign gene was correctly transcribed, translated, and expressed in A. oryzae.

Amino Acid Sequence↗
PubMed1989

Angioscopic observation of the coronary luminal changes induced by percutaneous transluminal coronary angioplasty.

Coronary luminal changes induced by percutaneous balloon or laser coronary angioplasty were examined angioscopically in 10 patients with ischemic heart disease. A fiberscope (2.6F to 5.3F) was introduced into the coronary artery with the use of a guiding catheter with or without a balloon at the tip. Angioscopy was successful before angioplasty in nine and after angioplasty in 10 of 11 coronary segments. Before angioplasty, the stenotic segments were composed of yellowish or whitish atheromatous plaques with smooth surfaces or spiral folds in patients with stable angina pectoris. In one patient with unstable angina pectoris, the stenotic segment was scalloped angiographically and showed an irregular and whitish plaque angioscopically. After angioplasty, endothelial exfoliation with scattered thrombi (10 segments), bellows-like folds (two), longitudinal cleft (one), plaque rupture (two), and intimal dissection (two) could be identified angioscopically. These changes, with the exception of dissection, could not be identified angiographically. In one patient who underwent thermal argon laser angioplasty, the dilated segment appeared brownish, which indicated carbonization. In one patient with acute myocardial infarction, the occlusive thrombi were vaporized by thermal laser angioplasty. The results demonstrate the clinical feasibility of percutaneous angioscopy for pathologic documentation of coronary luminal changes before and after angioplasty.

Aged↗
PubMed1988

Estimation of the transfer coefficients of oxygen and carbon monoxide in the boundary of human and chicken red blood cells by a microphotometric method.

The reaction rates of O2 and CO with the human and chicken red blood cell (RBC) were measured by using a microphotometric apparatus. In the experiments on the human RBC, a small amount of RBCs were put in an air-tight reaction cuvette. Gas mixtures containing various concentrations of O2 and CO were sequentially injected into the cuvette and the change in O2 and CO saturation of hemoglobin was measured from the change in transmission of the RBCs at 402 and 416.5 nm. The reaction rate of CO with RBCs was significantly influenced by photodissociation of carboxyhemoglobin (COHb). To eliminate this, a short-pass filter (400 to 435 nm) and a sector (100 Hz) were used. By comparing the measured reaction rates of O2 and CO with the theoretical rates obtained from the numerical solutions of the partial differential equations of the diffusions of O2 and CO, the transfer coefficients of O2 and CO (eta O2 and eta CO) in the RBC boundary, including the RBC membrane and water layer around the RBC, were estimated. Both the values showed good agreement, ranging from 0.3 to 2.5 x 10(-6) cm.sec-1.Torr-1. Furthermore, the chorioallantoic capillary of chicken embryo was used for the measurements of the reaction rates of O2 and CO with RBC through the capillary membrane. The reaction rates of O2 and CO in the chorioallantoic capillary were slower than those obtained in the human RBC. By comparing the measured reaction rates and the numerical solutions, the eta O2 and eta CO in the boundary, including the capillary membrane, plasma, and RBC membrane, were estimated. These two values ranged from 0.1 to 0.4 x 10(-6) cm.sec-1.Torr-1 and showed good agreement. These results suggest that the diffusion rates for O2 and CO across the capillary and RBC membrane are similar.

Animals↗
PubMed1988

Disruption of the CHO1 gene encoding phosphatidylserine synthase in Saccharomyces cerevisiae.

A Saccharomyces cerevisiae mutant that lacked phosphatidylserine synthase [EC 2.7.8.8] (CDP-1,2-diacyl-sn-glycerol: L-serine O-phosphatidyltransferase) completely was constructed by disrupting its structural gene, CHO1. Over two-thirds of its coding region, from the starting to the 200th codon, was replaced with a LEU2 DNA fragment. This new cho1 mutant showed no detectable synthesis of phosphatidylserine but grew slowly in a medium that contained either ethanolamine or choline. These results indicate that phosphatidylserine synthase and most probably phosphatidylserine are dispensable in S. cerevisiae but necessary for its optimal growth. Additional supplementation with myo-inositol raised the cellular content of phosphatidylinositol and improved the growth of the mutant, suggesting the importance of the negative charges of the membrane surface. The CHO1-disrupted mutant, when grown on choline, accumulated phosphatidylethanolamine to a significant level even after extensive dilution of the initial culture. It segregated prototrophic revertants that could synthesize phosphatidylethanolamine without recovery of phosphatidylserine synthesis. These results imply the presence of a route(s) for the formation of ethanolamine or its phosphorylated derivative in S. cerevisiae.

Blotting, Southern↗
PubMed1988

Disruption of the Escherichia coli cls gene responsible for cardiolipin synthesis.

The cls gene of Escherichia coli is responsible for the synthesis of a major membrane phospholipid, cardiolipin, and has been proposed to encode cardiolipin synthase. This gene cloned on a pBR322 derivative was disrupted by either insertion of or replacement with a kanamycin-resistant gene followed by exchange with the homologous chromosomal region. The proper genomic disruptions were confirmed by Southern blot hybridization and a transductional linkage analysis. Both types of disruptants had essentially the same properties; cardiolipin synthase activity was not detectable, but the strains grew well, although their growth rates and final culture densities were lower than those of the corresponding wild-type strains and strains with the classical cls-1 mutation. A disruptant harboring a plasmid that carried the intact cls gene grew normally. The results indicate that the cls gene and probably the cardiolipin synthase are dispensable for E. coli but may confer growth or survival advantages. Low but definite levels of cardiolipin were synthesized by all the disruptants. Cardiolipin content of the cls mutants depended on the dosage of the pss gene, and attempts to transfer a null allele of the cls gene into a pss-1 mutant were unsuccessful. We point out the possibilities of minor cardiolipin formation by phosphatidylserine synthase and of the essential nature of cardiolipin for the survival of E. coli cells.

Alleles↗