Search PubMed⌕ Search

Biomedical subjects

I Shibuya

Publications and source records attributed to I Shibuya.

At least 127 records · Page 7Linked to original sources
PubMed1987

Primary structure and product characterization of the Saccharomyces cerevisiae CHO1 gene that encodes phosphatidylserine synthase.

An open reading frame of 828 base pairs was found in the CHO1 gene region of Saccharomyces cerevisiae by nucleotide sequencing analysis. Its enhanced expression with the aid of the PHO5 regulatory sequence resulted in an overproduction of a protein with a molecular weight of approximately 30,000, which in turn was converted by proteolysis to active phosphatidylserine synthase, whose molecular weight was approximately 23,000. The larger protein was concluded to be the primary product of the CHO1 gene, since its amino-terminal sequence was identical to that deduced from the nucleotide sequence of the above open reading frame, except for the terminal methionine residue. A partial homology in primary structures was noticed between this yeast enzyme and phosphatidylglycerophosphate synthase of Escherichia coli which also uses CDP-diacylglycerol as a substrate. The overproduced phosphatidylserine synthase in both microsomal and extensively purified fractions displayed two different Km values for L-serine, i.e., 0.14 mM at low L-serine concentrations and 9.5 mM at high L-serine concentrations. This may indicate a negatively cooperative regulation of this enzyme activity or the presence of two active components with different affinities for L-serine.

Amino Acid Sequence↗
PubMed1987

Difference in the mode of acute cold-induced hypothermia between rat and hamster.

The present work was undertaken to elucidate the underlying causative factors limiting survival in hypothermic rats (non-hibernator) and hamsters (hibernator). A variety of physiological and biochemical parameters were measured for 28 male Wistar rats (205-286 g) and 26 male golden hamsters (85-118 g) before and during induction of hypothermia to colonic temperature (Tco) of 18 degrees C. With progressive decreases in Tco, parallel falls in VO2, VCO2, and heart rate were observed. In rats, plasma pH (pHp) in arterial blood was significantly lowered from 7.453 at Tco of 38 degrees C to 7.327 at Tco of 18 degrees C (p less than 0.001). From the observations of increased arterial PCO2 and bound CO2 (bicarbonate ion) concentration, together with the unchanged blood lactate, respiratory acidosis caused by hypoventilation was suggested to be responsible for the decrease in pHp. In contrast to rats, in hamsters pHp was almost unchanged during the induction of hypothermia, while nearly complete depletion of blood glucose (-84.6%) and liver glycogen (-99.5%) were observed when Tco of 38 degrees C was lowered to 18 degrees C. It is concluded that the decrease in pHp is a limiting factor for rats to survive the deep hypothermia at Tco of 18 degrees C and the exhaustion of carbohydrates as an energy supply for hamsters.

Animals↗
PubMed1987

A method for estimating contact time of red blood cells through lung capillary from O2 and CO2 concentrations in rebreathing air in man.

The gas exchange ratio (R) obtained from O2 and CO2 concentrations measured in rebreathing air usually shows a linear relation to the PCO2. By referring to this relation and the R which equals the Haldane effect coefficient and zero, the true- and oxygenated-venous PCO2 are obtained in addition to the alveolar PCO2. When these PCO2 are evaluated, the R-PCO2 line can also be computed theoretically from a numerical solution on the overall O2 and CO2 diffusions in the red blood cell. By comparing both the experimental and theoretical R-PCO2 lines with each other, we derived a contact time equation. Since the linear approximation of the R-PCO2 relation gave rise to an error in the contact time (tc), first a factor to correct the linearity of the R-PCO2 line was derived. Next, using these parameter values, tc was quantitatively determined from experimental data obtained during rebreathing in 5 normal subjects and it was compared with that estimated from the pulmonary diffusing capacity for CO in the same rebreathing experiment. When the extracellular HCO3- dehydration rate was taken to be 0.1 s in time constant, the tc, being ca. 0.7 and 0.4 s at rest and during exercise, respectively, showed good agreement with those obtained from the diffusing capacity for CO.

Capillaries↗
PubMed1987

Experimental analyses of pulmonary gas exchange in a standing position at rest and during treadmill exercise.

Quantitative relations between O2 uptake (Vo2), arterial-venous O2 content difference [a-v)CO2), and contact time (tc) were measured at rest and during exercise in 7 normal subjects, using a rebreathing technique. By injecting a known amount of pure O2 into a rebreathing circuit, the VO2 was measured from the time interval during which the injected O2 volume was consumed. From the O2 and CO2 concentrations in rebreathing air the gas exchange ratio (R), which was linearly related to the PCO2, was determined. By using the slope of the R-PCO2 relation, the (a-v)CO2 was evaluated from the slope of the CO2 dissociation curve between the true- and oxygenated-venous PCO2. These two venous PCO2 were estimated from the R-PCO2 relation by use of the Haldane effect coefficient and by extrapolating it to the abscissa, where R = O. Furthermore, tc was evaluated by inserting the above two venous PCO2 together with the alveolar PCO2 into a contact time equation. The mean (a-v)CO2 and tc had consistent relations to the VO2 and (a-v)CO2, respectively. (a-v)CO2 = 9.5 X VO2 0.35, (vol%), and tc = 4.86 X (a-v)CO2 -1.025, (s).

Adult↗
PubMed1986

Overproduction of proteins encoded by lambda replicon-integrated plasmid: effect of partial inactivation of cI repressor.

Composite plasmids containing the 7.2-kb fragment of bacteriophage lambda cI857cro27, essential for lambda DNA replication and its control, have been constructed. The plasmids contained, in addition, pBR322, a part of Co1E1, and the Escherichia coli pss gene, coding for phosphatidylserine synthase. When the plasmid-harboring cells were induced at different temperatures, the phosphatidylserine synthase production was maximum at 37 degrees C, and the specific activities at 37 degrees C continuously increased until the culture reached saturation. The results suggest that, when the cro gene is defective, only a transient temperature for inactivation of cI repressor provides both the increase in copy number of the plasmids and cellular activities that allow overproduction of the plasmid-encoded protein.

Bacteriophage lambda↗
PubMed1986

Membrane phospholipid synthesis in Escherichia coli: alteration by glycerol and physiological consequences in a pss mutant.

Escherichia coli mutants harboring the pss-1 allele (coding for a temperature-sensitive phosphatidylserine synthase) are temperature sensitive for growth and synthesize less phosphatidylethanolamine at higher temperatures, giving rise to abnormal membrane phospholipid compositions. To obtain information concerning the determinant for the phospholipid polar headgroup composition and the lethal factor in the defective membranes, we have examined the effect of increased supply of sn-glycerol 3-phosphate on the phospholipid synthesis and the growth ability of a pss-1 mutant. For this purpose, a pair of E. coli K-12 derivatives isogenic except for the pss-1 allele was constructed from strain BB26-36 to harbor the mutations related to glycerol metabolism (glpD3, glpR2, glpKi, and phoA8). Pulse- and uniform-labeling of phospholipids with 32P at 42 degrees C in a synthetic medium with (0.2%) or without glycerol showed that glycerol further lowered the temperature-sensitive formation of phosphatidylethanolamine, removed the phosphatidate and CDP-diacylglycerol accumulated in the absence of glycerol, and resulted in an increase in cardiolipin content in the pss-1 mutant. The phospholipid synthesis and contents in the pss+ strain were not significantly affected by glycerol. Glycerol in the medium markedly enhanced the growth defect of the pss-1 mutant, which was remediable by sucrose. The results indicate that the intracellular pool of sn-glycerol 3-phosphate is the limiting factor for acidic phospholipid synthesis in the pss-1 mutant, and cardiolipin unusually accumulated is injurious to the functional E. coli membranes. Possible determinants for the phospholipid composition of the wild-type E. coli cells are also discussed on the basis of the present observations.

CDPdiacylglycerol-Serine O-Phosphatidyltransferase↗
PubMed1986

Construction of a host-vector system in Candida maltosa by using an ARS site isolated from its genome.

To construct a host-vector system in an n-alkane-assimilating yeast, Candida maltosa, the isolation of an ARS site from its genome which replicates autonomously in C. maltosa was attempted. Leu- mutants of C. maltosa were transformed with a gene library prepared by using YEp13 (LEU2+) as a vector, and Leu+ transformants were obtained at a high frequency. A plasmid named pCS1 was isolated from the recipient cells. pCS1 contained a 6.3-kilobase (kb) fragment of the C. maltosa genome, and a 3.8-kb fragment with ARS activity was subcloned and designated the TRA (transformation ability) region. Vectors (pTRA1 and pTRA11) for C. maltosa J288 were constructed that contained this 3.8-kb fragment, pBR322, and the LEU2 gene of Saccharomyces cerevisiae. Transformation of C. maltosa J288 with these plasmids was successful by both spheroplast and lithium acetate methods. Southern blot analysis suggested that the copy number of pTRA1 in C. maltosa was between 10 and 20, and it was stably maintained during growth without selective pressure in the medium. It was also found that these vectors could transform S. cerevisiae leu2- to LEU2+, suggesting that the TRA region contained an ARS site(s) that was specific not only for C. maltosa but also for S. cerevisiae.

Candida↗
PubMed1986

Cloning in Saccharomyces cerevisiae of a cycloheximide resistance gene from the Candida maltosa genome which modifies ribosomes.

We have previously shown that cycloheximide resistance can be induced in a strain of Candida maltosa by modifying ribosomes (M. Takagi, S. Kawai, Y. Takata, N. Tanaka, M. Sunairi, M. Miyazaki, and K. Yano, J. Gen. Appl. Microbiol. 31:267-275, 1985). The present paper describes the cloning of the gene involved in this resistance (designated RIM-C for ribosome modification by cycloheximide) by using a host-vector system of Saccharomyces cerevisiae.

Candida↗
PubMed1986

A replica method useful in the isolation of mutants of FM3A, a non-adherent cell line from mouse mammary carcinoma.

Existing replica methods designed for adherent somatic cells are not applicable to non-adherent cells of tumor origin. We have developed a new, efficient replica method for the negative selection of mutants of non-adherent mouse FM3A cells. Mutagenized cells are grown clonally on soft-agar plates then allowed to proliferate upwards through layered polyester disks, thus producing replicas with the same colony patterns. Isolation of mutants with increased requirements for myo-inositol by this method is described.

Agar↗
PubMed1986

Simultaneous measurement of cardiac output and pulmonary diffusing capacity for CO by a rebreathing method.

A rebreathing method was developed to obtain the pulmonary diffusing capacity for CO, DL(CO) and the cardiac output, Q, simultaneously at rest and during exercise. From DL(CO) and Q, contact time of the red blood cell (RBC) through the lung capillary, tc, was estimated by referring to a reaction rate factor of CO with RBC, Fc(CO). The DL(CO) and Q were calculated from the changes in CO, O2, and CO2 concentrations in rebreathing air. The Fc(CO) was evaluated so as to attain a proportional relation between the DL(CO) and Fc(CO) by varying a transfer coefficient for CO across the RBC boundary including the capillary wall. The PO2 dependence of Q was also taken into account in the above analyses. The tc was determined by dividing the DL(CO) by Fc(CO), Q, and fractional hematocrit. The DL(CO) and Q measured in five normal subjects in a sitting position were 26.6 +/- 2.9 ml X min-1 X Torr-1 and 5.9 +/- 0.6 l X min-1 (mean +/- S.D.), respectively, which increased during moderate exercise up to 34.6 +/- 3.9 ml X min-1 X Torr-1 and 13.0 +/- 1.2 l X min-1. The tc was 0.70 +/- 0.06 s at rest and 0.40 +/- 0.05 s during exercise. The rebreathing method for measuring DL(CO) was more advantageous than the single breath method, because the tc could be obtained as well as the Q and the influence of the dead space and ventilation-perfusion uneveness on DL(CO) was reduced.

Carbon Monoxide↗
PubMed1986

Change in O2 uptake during rebreathing in hyperoxia in man.

In pertaining to PO2 dependency of the pulmonary CO diffusing capacity during rebreathing, the O2 uptake (VO2) and cardiac output (Q) were measured at three different PO2 levels between 100 and 500 Torr. Since the VO2 measured by an O2 injection method is strongly influenced in hyperoxia by a gas exchange ratio (R), a simulation method using a R-PCO2 relation during rebreathing was developed. Gas volume in the lung-bag-system needed in the computation was measured from the difference in O2 concentration between before and after injecting a known amount of O2 into the rebreathing circuit. The accuracy of the volume was checked by comparing it with the volume measured successively with a body box. The VO2 was determined by comparing the simulated O2 and CO2 concentrations in rebreathing gas with the measured ones. The VO2 significantly increased by rebreathing in hyperoxia. To analyze the VO2 increase, the Q was computed by dividing the VO2 by the arteriovenous O2 content difference, which in turn was obtained by dividing the slope of the CO2 dissociation curve by that of the R-PCO2 line. The Q was almost linearly related to the VO2. Since there was no difference in VO2 in steady state breathing between normoxia and hyperoxia, the increase in VO2 and Q seemed to occur transiently. This finding is very important in evaluating the PO2 dependency of the pulmonary diffusing capacity for CO.

Adult↗
PubMed1985

Genetic manipulation of membrane phospholipid composition in Escherichia coli: pgsA mutants defective in phosphatidylglycerol synthesis.

Unique mutants of Escherichia coli K-12, defective in phosphatidylglycerol synthesis, have been isolated from a temperature-sensitive strain incubated at its nonpermissive temperature. The parent strain had excess phosphatidylglycerol by harboring both the pss-1 allele [coding for a temperature-sensitive phosphatidylserine synthase (EC 2.7.8.8)] and the cls- allele (responsible for a defective cardiolipin synthase). The newly acquired mutations caused better growth at higher temperatures. One of the mutations (pgsA3) has been identified in the structural gene for phosphatidylglycerophosphate synthase [glycerophosphate phosphatidyltransferase (EC 2.7.8.5)]. Phospholipid compositions of these mutants were remarkable; phosphatidylethanolamine was the sole major lipid. In media with low osmotic pressures, these cells grew more slowly than the wild-type cells. They grew normally without recovering from the phospholipid abnormality in media appropriately supplemented with sucrose and MgCl2. Formation of cardiolipin and phosphoglycerol derivatives of membrane-derived oligosaccharides was reduced in a pgsA3 mutant. E. coli strains having the pgsA3, pss-1, and cls- mutations, either individually or in combination, constitute an empirical system in which the molar ratio of three major membrane phospholipids can be variously altered.

Cardiolipins↗
PubMed1985

Biosynthesis of novel acidic phospholipid analogs in Escherichia coli.

When cultured in the presence of 600 mM D-mannitol, Escherichia coli K-12 cells synthesized two novel phospholipids. The identities of these compounds are postulated to be phosphatidylmannitol and diphosphatidylmannitol, the sugar alcohol analogs of phosphatidylglycerol and cardiolipin, respectively. The nonacylated glycerol moieties of the normal acidic phospholipids were substituted by D-mannitol. The formation of the analogs was significantly enhanced when strains harboring the pss-1 allele, a temperature-sensitive mutation in phosphatidylserine synthase (Ohta and Shibuya, J. Bacteriol. 132:434-443, 1977), were grown at 42 degrees C, and the accumulation of the analogs was maximum in late stationary phase; more than 90% of the total cellular lipids were these novel phospholipids. Strains with a defective cardiolipin synthase (Pluschke et al., J. Biol. Chem. 253:5048-5055, 1978) failed to form the analog lipids, whereas cells with increased cardiolipin synthase activity due to the presence of a pBR322-derived recombinant plasmid containing the structural gene for cardiolipin synthase produced more mannitol lipids than wild-type strains. These observations and the structures of the analog lipids indicated that cardiolipin synthase participates in the formation of these novel phospholipids. We suggest that reversible alcoholysis and condensation, in addition to low substrate specificity of the enzyme, are the mechanisms involved in this process. Addition to the medium of other straight-chain alditols, D-arabitol, ribitol, xylitol, erythritol, and L-threitol also yielded pairs of novel phospholipids, whereas sorbitol or galactitol produced only one analog in small quantities. These acidic phospholipid analogs have not been reported in any living system. They should be useful in the study of structure-function relationships of phospholipids and in manipulating the structures of various membrane systems.

Escherichia coli↗