Biomedical subjects

T Oshima

Publications and source records attributed to T Oshima.

At least 289 records · Page 16Linked to original sources

PubMed1995

Formation of amino acid precursors in cometary ice environments by cosmic radiation.

Cometary ices are believed to contain water, carbon monoxide, methane and ammonia, and are possible sites for the formation and preservation of organic compounds relating to the origin of life. Cosmic rays, together with ultraviolet light, are among the most effective energy sources for the formation of organic compounds in space. In order to study the possibility of the formation of amino acids in comets or their precursory bodies (interstellar dust grains), several types of ice mixtures made in a cryostat at 10 K ("simulated cometary ices") were irradiated with high energy protons. After irradiation, the volatile products were analyzed with a quadrupole mass spectrometer, while temperature of the cryostat was raised to room temperature. The non-volatile products remaining in the cryostat at room temperature were collected with water. They were acid-hydrolyzed, and analyzed by ion-exchange chromatography. When an ice mixture of carbon monoxide (or methane), ammonia and water was irradiated, some hydrocarbons were formed, and amino acids such as glycine and alanine were detected in the hydrolyzate. These results suggest the possible formation of "amino acid precursors" (compounds yielding amino acids after hydrolysis) in interstellar dust grains by cosmic radiation. We previously reported that amino acid precursors were formed when simulated primitive planetary atmospheres were irradiated with cosmic ray particles. It will be of great interest to compare the amount of bioorganic compounds that were formed in the primitive earth and that brought by comets to the earth.

Amino Acids↗

PubMed1995

Formation of bioorganic compounds in planetary atmospheres by cosmic radiation.

Simulated planetary atmospheres (mixtures of simple gases) were irradiated with high energy particles to simulate an action of cosmic rays. When a mixture of carbon monoxide, nitrogen and water was irradiated with 2.8-40 MeV protons, a wide variety of bioorganic compounds including amino acids, imidazole, and uracil were identified in the products. The amount of amino acids was proportional to the energy deposit to the system. Various kinds of simulated planetary atmospheres, such as "Titan type" and "Jovian type", were also irradiated with high energy protons, and gave amino acids in the hydrolyzed products. Since cosmic rays are a universal energy source in space, it was suggested that formation of bioorganic compounds in planetary atmospheres is inevitable in the course of cosmic evolution.

Amino Acids↗

PubMed1995

Studies in the search for life on Mars.

The ability of living organisms to survive extraterrestrial conditions has implications for the origins of life in the solar system. We have therefore studied the survival of viruses, bacteria, yeast, and fungi under simulated Martian conditions. The environment on Mars was simulated by low temperature, proton irradiation, ultraviolet irradiation, and simulated Martian atmosphere (CO2 95.46%, N2 2.7%, water vapor 0.03%) in a special cryostat. After exposure to these conditions, tobacco mosaic virus and spores of Bacillus, Aspergillus, Clostridium, and some species of coccus showed significant survival.

Aspergillus niger↗

PubMed1995

Effect of the transmembrane gradient of magnesium and sodium on the regulation of cytosolic free magnesium concentration in human platelets.

1. To clarify the mechanism by which cytosolic free Mg2+ concentrations ([Mg2+]i) are regulated in human platelets, we investigated platelet membrane permeability to Mg2+ by altering extracellular Mg2+ concentrations, and tested the existence of a Na(+)-Mg2+ exchanger by manipulating the transmembrane Na+ gradient. 2. Platelet [Mg2+]i was 421 +/- 52 mumol/l in healthy men. [Mg2+]i remained constant during a 120 min exposure to nominally zero (low) or 5 mmol/l (high) external Mg2+ concentrations. 3. Preincubation of platelets with 10(-4) mol/l ouabain effectively decreased the transmembrane Na+ gradient. The ouabain-induced increase in [Mg2+]i was statistically significant after 30 min of exposure (14.6 +/- 2.0%) and reached 24.6 +/- 4.5% after 60 min. Similarly, the replacement of extracellular Na+ with N-methyl-D-glucamine significantly increased [Mg2+]i by 48.5 +/- 3.9% and 78.8 +/- 12.5%, respectively. 4. These results suggest that [Mg2+]i is well controlled in the presence of large transmembrane Mg2+ concentration gradients, and a Na(+)-Mg2+ exchanger may be involved in the regulation of [Mg2+]i in human platelets.

Blood Platelets↗

PubMed1995

The crystal structures of mutated 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 and their relationship to the thermostability of the enzyme.

The structures of two mutant forms (G240A and L246E/V249M) of 3-isopropylmalate dehydrogenase from Thermus thermophilus HB8 were studied by X-ray crystallography. In the case of G240A, the replacement of glycine by alanine at residue 240 was expected to decrease the thermostability as a result of abnormal contacts between the methyl group of alanine and the peptide chain. However, the normal van der Waals' contacts were achieved owing to a shift in a bundle of beta-strands that yielded a vacant space for the alanine residue. The extended hydrogen bonds within the beta-sheet are the major reason for the decreased thermostability of G240A. The mutations in L246E/V249M are located in an alpha-helix region which is involved in subunit-subunit contact via hydrophobic interaction. Loosening of the subunit-subunit contact owing to ionic repulsion was the major cause of the lower heat stability of L246E/V249M.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Crystallization and preliminary X-ray studies of isocitrate dehydrogenase from Thermus thermophilus HB8.

Isocitrate dehydrogenase from the thermophilic bacterium, Thermus thermophilus HB8, was crystallized by the vapor diffusion hanging drop method with polyethylene glycol 6000 as the precipitant. Pillar-like crystals of about 0.6 x 0.6 x 0.3 mm3 were obtained. Analysis of a series of oscillation photographs indicated that the orthorhombic crystals belonged to the I222 or I2(1)2(1)2(1) space group with unit cell dimensions of a = 100.1 A, b = 150.4 A, and c = 87.4 A. Intensity data were collected up to 2.5 A resolution.

Crystallization↗

PubMed1995

Ligand-induced changes in the conformation of 3-isopropylmalate dehydrogenase from Thermus thermophilus.

The structures of 3-isopropylmalate dehydrogenase (IPMDH) from Thermus thermophilus in complexes with its substrate, cofactor, and a cofactor analog were investigated by X-ray diffraction in a crystalline state and by small-angle X-ray scattering (SAXS) in solution. The structures at 2.8 A resolution of the complexes with the substrate, 3-isopropylmalate (IPM), and with an analog of NAD, ADP-ribose, were both very close to the structure of the free enzyme, which adopts an open conformation. However, the binding of a ligand induced a small conformational change near the binding site. This result contrasts with results for NADP(+)-bound and isocitrate-bound isocitrate dehydrogenase (ICDH) from Escherichia coli, which adopts a closed conformation. The SAXS analysis in solution clearly showed that IPMDH without a ligand adopts two distinct intermediate conformations, between the open and closed states, upon binding of NADH and IPM respectively, and adopts a fully closed conformation when in a ternary complex with NADH and IPM together.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Orotate phosphoribosyltransferase from Thermus thermophilus: overexpression in Escherichia coli, purification and characterization.

Orotate phosphoribosyltransferase (OPRTase, EC2.4.2.10) plays a role in de novo synthesis of pyrimidine nucleotide and transfers orotate to 5-phosphoribosyl-1-pyrophosphate (PRPP) to form orotidine-5'-monophosphate (OMP). To obtain heat-stable OPRTase and to elucidate the mechanism of heat stability, this enzyme from Thermus thermophilus was expressed in Escherichia coli and purified. The pyrE gene of T. thermophilus which encodes OPRTase, contains an open reading frame of 549 base pairs with 69% G+C content. Since this gene expressed itself inefficiently in E. coli, the 5' and 3' ends of the coding regions were replaced with synonymous codons which contain more A+T and corresponds to major codons for E. coli. Introduction of the modified gene fragments into a plasmid having a tac promoter resulted in production of a polypeptide of molecular weight (M(r)) 20,000 in the presence of isopropyl-beta-D-thiogalactopyranoside (IPTG) in E. coli. This protein represented as much as 16% of the bacterial total protein and showed the OPRTase activity. Three purification steps, consisting of heat treatment at 65 degrees C, 40% ammonium sulfate fractionation, and KCl gradient elution from DEAE-Sephadex A-50, resulted in highly purified single polypeptide. The optimum activity of the purified OPRTase was observed at 150 mM KCl, pH 9.0, 75-80 degrees C, and in the presence of 100 microM PRPP. The activation energy of this enzyme reaction was 20.3 kJ/mol. The Km of this enzyme for orotate as a substrate was 75 microM and the maximum specific activity was 300 units/mg protein under the optimum conditions. The purified OPRTase was stable for 20 min at 85 degrees C.

Amino Acid Sequence↗

PubMed1995

Thermal stability of chimeric isopropylmalate dehydrogenase genes constructed from a thermophile and a mesophile.

Chimeric isopropylmalate dehydrogenases were constructed by connecting the genes isolated from an extreme thermophile, Thermus thermophilus, and a mesophile, Bacillus subtilis. These genes were expressed in Escherichia coli. The enzymes were purified and analysed. Enzymes of T.thermophilus and B.subtilis and chimeric enzymes showed similar enzymological characteristics except for thermal stability. The stability of each enzyme was approximately proportional to the content of the amino acid sequence from the T.thermophilus enzyme. The results suggested that amino acid residues contributing the thermal stability distribute themselves, in general, evenly at least in the N-terminal half of the amino acid sequence of T.thermophilus isopropylmalate dehydrogenase.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

The crystal structure of thermostable mutants of chimeric 3-isopropylmalate dehydrogenase, 2T2M6T.

A chimeric 3-isopropylmalate dehydrogenase (IPMDH), 2T2M6T, was produced by replacing the amino acid sequences of the Thermus thermophilus enzyme with those of the Bacillus subtilis enzyme from residues 75 to 113. Decreased thermostability of the chimeric enzyme was recovered by either evolutionary engineering (I93L) or site-directed mutagenesis (S82R). The 3-D structures of the mutants have been determined by X-ray diffraction at 2.1 A resolution. Although S82R was refined routinely, I93L required the preliminary rigid-body refinement of each domain. The R-factors were reduced to 0.18 for both mutants. Removal of the unfavorable torsion angle at isoleucine 93 may have made I93L more thermostable than 2T2M6T. In the case of S82R, the replaced arginine residue contributed to the extra hydrogen bond with water molecules. The large replaced residue decreased the entropy of the solvent, which may have caused the improvement in enzyme thermostability. Denaturation by heating may be interpreted from these structural results.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Screening of stable proteins in an extreme thermophile, Thermus thermophilus.

The leuB gene codes for 3-isopropylmalate dehydrogenase of the leucine biosynthetic pathway in an extreme thermophile, Thermus thermophilus. The leuB gene of the thermophile was replaced with a temperature-sensitive chimeric leuB gene. The resultant transformant was adapted to high temperature, a thermostable mutant strain being obtained. A single base substitution that replaces isoleucine at 93 with leucine was found in the chimeric leuB gene of the thermostable mutant. The resultant amino acid residue coincided with the corresponding residue of the T. thermophilus enzyme. It was confirmed that the mutant enzyme is more stable than the original chimeric enzyme. This system can be used to produce stabilized mutants of other enzymes without structural knowledge of them.

3-Isopropylmalate Dehydrogenase↗

PubMed1995

Cu, Zn superoxide dismutase in patients with dementia of the Alzheimer type.

Cu, Zn superoxide dismutase (SOD) levels in the serum, cerebrospinal fluid (CSF) and skin fibroblasts of patients with dementia of the Alzheimer type (DAT) were estimated using enzyme immunoassay. The SOD mRNA level in the skin fibroblasts was also determined by the Northern blot analysis. As compared with the age-matched control groups of neurological patients without dementia, the AD group consisting of patients with DAT at ages under 65 years of age as well as the SDAT group of patients with DAT at ages over 65 years of age showed no significant changes in serum or CSF SOD levels. However, the skin fibroblast SOD- and SOD mRNA levels, which were correlate each other, were significantly higher in the AD group (p < 0.05), while lower in the SDAT group (p < 0.05). These results suggest that the determination of the SOD level of skin fibroblast may be useful for diagnosis of DAT and that the abnormality of SOD may play an important role in developing DAT.

Alzheimer Disease↗

PubMed1995

Characterization of the endosymbiont of a deep-sea bivalve, Calyptogena soyoae.

We have purified DNA from gill tissue of a marine bivalve, Calyptogena soyoae, collected from the deep-sea cold seep communities in Sagami Bay, Japan. An rRNA gene was amplified, cloned, and sequenced. In situ hybridization revealed that the sequence is that of a bacterial endosymbiont within the gill of C. soyoae.

Animals↗

PubMed1995

Morphological variation of new Thermoplasma acidophilum isolates from Japanese hot springs.

We isolated 12 strains of Thermoplasma acidophilum from hot springs in Hakone, Japan. T. acidophilum strains showed morphological variation in the crystal-like structure in the cell and the fibrous structure on the cell surface. Two strains tested were sensitive to novobiocin. However, a novobiocin-resistant mutant was obtained by spontaneous mutation.

Base Composition↗

PubMed1995

Sulredoxin: a novel iron-sulfur protein of the thermoacidophilic archaeon Sulfolobus sp. strain 7 with a Rieske-type [2Fe-2S] center.

A novel pink [2Fe-2S] protein has been purified from the cytosol fraction of the thermoacidophilic archaeon Sulfolobus sp. strain 7 (originally named Sulfolobus acidocaldarius 7) and called "sulredoxin." Its absorption, circular dichroism, and electron paramagnetic resonance spectra suggest the presence of a Rieske-type [2Fe-2S] cluster (g-factors of 2.01, 1.91, and 1.79; average g-factor [gav] = 1.90) which is remarkably similar to that of Thermus thermophilus respiratory Rieske FeS protein (J. A. Fee, K. L. Findling, T. Yoshida, R. Hille, G. E. Tarr, D. O. Hearshen, W. R. Dunham, E. P. Day, T. A. Kent, and E. Münck, J. Biol. Chem. 259:124-133, 1984) and distinctively different from those of the plant-type ferredoxins (gav = 1.96). Sulredoxin, which is the first Rieske-type [2Fe-2S] protein isolated from an archaeal species, does not function as an electron acceptor of the cognate 2-oxoacid:ferredoxin oxidoreductase. Whether sulredoxin is derived from the archaeal membrane-bound respiratory Rieske-type FeS center (gy = 1.91) is the subject of further investigation.

Amino Acid Sequence↗

PubMed1995

Dietary salt decreases cytosolic calcium in platelets from Dahl salt-sensitive rats.

To determine whether abnormal cellular Ca2+ handling is involved in salt-induced hypertension of Dahl salt-sensitive rats (DS), we investigated Ca2+ handling in fura 2-loaded platelets of DS and Dahl salt-resistant rats (DR) fed a high-NaCl (8%) or a low-NacL (0.3%) diet for 4 wk from 5 wk of age. At 5 wk of age, blood pressure, resting cytosolic Ca2+ concentration ([Ca2+]i), the thrombin-evoked increase in [Ca2+]i and the size of internal Ca2+ stores of DS were comparable with those of DR. After 4 wk on the diets, resting [Ca2+]i of DS on high-NaCl diet was lower than that of DS on low-NaCl diet, and there was no effect of high salt intake on resting [Ca2+]i in DR. In DS, high salt intake attenuated the [Ca2+]i response to thrombin in the presence of external Ca2+. In contrast, the [Ca2+]i response to thrombin in the absence of external Ca2+ was enhanced by high salt intake in DS. The size of internal Ca2+ stores was increased by high salt intake in DS but not in DR. These data suggest that it is not obligatory for hypertension to be accompanied by an increase in platelet [Ca2+]i.

Animals↗

PubMed1995

Effects of L-arginine infusion on renal hemodynamics in patients with mild essential hypertension.

Previous studies have shown that endothelium-derived relaxing factor/nitric oxide plays an important role in the regulation of systemic and renal hemodynamics. The purpose of the present study was to determine whether endothelium-dependent renovascular relaxation was impaired in patients with mild essential hypertension who had normal renal plasma flow and glomerular filtration rate. We evaluated the effects of intravenous administration of L-arginine on blood pressure and renal hemodynamics in 13 patients with mild essential hypertension and 15 normotensive control subjects. L-Arginine infusion (500 mg/kg over 30 minutes) reduced mean blood pressure (from 82.5 +/- 2.5 to 76.3 +/- 2.6 mm Hg in hypertensive patients and from 106.1 +/- 3.0 to 97.5 +/- 2.9 mm Hg in control subjects; P < .001) and renovascular resistance (from 0.084 +/- 0.009 to 0.067 +/- 0.009 mm Hg.mL-1.min-1.[1.48 m2]-1 and from 0.105 +/- 0.010 to 0.093 +/- 0.011 mm Hg.mL-1.min-1.[1.48 m2]-1, respectively; P < .001). L-Arginine infusion increased renal plasma flow (from 602 +/- 36 to 698 +/- 40 mL.min-1.[1.48 m2]-1, P < .05) in normotensive subjects but not in hypertensive subjects, and glomerular filtration rate was unaffected in both groups. Although the L-arginine-induced reduction in mean blood pressure was similar in both groups, the decline in renovascular resistance was smaller in hypertensive subjects. The response of renal plasma flow was also smaller in hypertensive subjects. These findings suggest that dysfunction of the L-arginine-nitric oxide pathway exists in the renal circulation even in mild essential hypertension with normal renal plasma flow and glomerular filtration rate.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Intravenous administration of L-arginine inhibits angiotensin-converting enzyme in humans.

The iv administration of L-arginine, a precursor of endothelium-derived relaxing factor/nitric oxide, is known to decrease blood pressure in humans by its direct vasodilatory effects. The purpose of the present study was to determine whether L-arginine infusion modifies the renin-angiotensin (Ang)-aldosterone system as well as blood pressure and renal hemodynamics. L-Arginine and saline vehicle were iv administered to 10 healthy male subjects in random order on different days. L-Arginine infusion (500 mg/kg over 30 min) decreased mean blood pressure (from 81.2 +/- 2.7 to 74.0 +/- 2.5 mm Hg; P < 0.001) and renal vascular resistance (from 0.085 +/- 0.007 to 0.074 +/- 0.006 mm Hg/mL.min; P < 0.01) and increased heart rate (from 60.3 +/- 2.7 to 69.7 +/- 2.1 beats/min; P < 0.001) and renal plasma flow (from 616.6 +/- 37.8 to 701.0 +/- 49.2 mL/min; P < 0.05). L-Arginine reduced serum Ang-converting enzyme activity (from 10.4 +/- 0.6 to 8.9 +/- 0.5 nmol/mL.min; P < 0.05) and plasma Ang-II (from 19.3 +/- 3.3 to 12.7 +/- 2.8 pg/mL; P < 0.001), but had no effect on PRA or the glomerular filtration rate. The saline vehicle did not alter any of these parameters. The iv administration of L-arginine (endothelium-derived relaxing factor/nitric oxide) may reduce the plasma Ang-II concentration by inhibiting Ang-converting enzyme. The mechanism by which L-arginine infusion decreases blood pressure can be at least in part explained by inhibition of the renin-Ang system.

Adult↗