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Biomedical subjects

K Hiraga

Publications and source records attributed to K Hiraga.

At least 55 records · Page 3Linked to original sources
PubMed1997

Isolation and characterization of chitinase from a flake-chitin degrading marine bacterium, Aeromonas hydrophila H-2330.

A flake-chitin degrading marine bacterium was isolated and identified as Aeromonas hydrophila. This strain secreted five chitinases and an beta-N-acetylglucosaminidase. The main chitinase (Chi-A) was purified and characterized. The optimum pH of Chi-A was 5-8, and the activity was inhibited by Hg2+ and Fe3+. Chi-A was different from chitinases of other Aeromonas species with respect to molecular weight (62,000) and insensitivity to monoiodoacetate. The amino-terminal amino acid sequence showed extensive similarity with chitinases from Gram-negative bacteria.

Aeromonas hydrophila↗
PubMed1996

A thermodynamic analysis of conformational change due to the alpha 2 beta 2 complex formation of tryptophan synthase.

A characteristic property of the tryptophan synthase alpha 2 beta 2 complex is the mutual activation of the alpha and beta subunit upon complex formation. It has been speculated that this mutual activation results from the conformational change due to the alpha/beta subunit interaction. To elucidate this mechanism, we investigated the thermodynamic parameters of association for the various combinations of the alpha and beta subunits from Escherichia coli and Salmonella typhimurium using isothermal titration calorimetry. The negative association enthalpy of the S. typhimurium alpha subunit with the beta subunit from E. coli (or S. typhimurium) was about 20 kJ mol-1 larger than that of the E. coli alpha subunit at 40 degrees C. However, the favorable enthalpy of the S. typhimurium alpha subunit was perfectly compensated by the unfavorable association entropy, therefore, the Gibbs energy of association was similar to that of the E. coli alpha subunit. Furthermore, the site-directed mutagenesis study revealed that a single mutation (K109N; [Asn109] alpha subunit) of the E coli alpha subunit at the subunit interface from E. coli to the S. typhimurium type could change the characteristics of the thermodynamic parameters of association to the S. typhimurium alpha subunit type. The heat-capacity changes of the association of the alpha subunit with the beta subunit were quite great, 6.37-8.21 kJ mol-1 K-1, compared with that due to a decrease in accessible surface area in the subunit interface. The analysis of the thermodynamic parameters of association suggested that the complex formation couples with the folding (rearrangements) of the alpha subunit monomer or/and beta subunit dimer.

Asparagine↗
PubMed1996

Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase. 1. Role in conformational stability.

To eludicate the role in conformational stability of Cys residues buried in the interior of a protein, the thermodynamic properties of denaturation of mutant alpha subunit of Escherichia coli tryptophan synthase, in which Ser, Ala, Val or Gly was substituted for each of the three Cys residues, were analyzed using calorimetry. The mutants were less stable than the wild type, indicating that Cys residues contribute greatly to the stability of the alpha subunit. In most cases, a large decrease in denaturation enthalpy was observed, compensated for by the denaturation entropy to a major extent. The extent of changes in the denaturation Gibbs energy and denaturation enthalpy varied greatly depending on both substituting residues and positions. Of all the mutant proteins, the Cys154Ser mutant showed the greatest decrease in denaturation enthalpy; its denaturation enthalpy was half that of the wild type, and was considerably repaired by adding a ligand of the alpha subunit. Because the enthalpy of ligand binding to Cys154Ser in the native state did not change, it seems that the decrease in the denaturation enthalpy of Cys154Ser and its recovery by ligand binding are caused by conformational changes in the denatured state due to the mutation.

Calorimetry↗
PubMed1996

Study of cysteine residues in the alpha subunit of Escherichia coli tryptophan synthase. 2. Role in enzymatic function.

To understand the functional roles of Cys residues in the alpha subunit of tryptophan synthase from Escherichia coli, single mutants of the alpha subunit, in which each of the three Cys residues was substituted with Ser, Gly, Ala or Val, were constructed by site-directed mutagenesis. The effects of the substitutions on the function of tryptophan synthase were investigated by activity measurements, calorimetric measurements of association with the beta subunit and steady-state kinetic analysis of catalysis. Although the three Cys residues are located away from the apparently important parts for enzymatic activity, substitutions at position 81 by Ser, Ala or Val caused decreases in the intrinsic activity of the alpha subunit. Furthermore, Cys81Ser and Cys81Val reduced stimulation activities in the alpha and beta reactions due to formation of a complex with the beta subunit. The lower stimulation activities of the mutant proteins were not correlated with their abilities to associate with the beta 2 subunit but were correlated with decreases in kcat. The present results suggest that position 81 plays an indirectly important role in the activity of the alpha subunit itself and the mutual activation mechanism of the complex.

Calorimetry↗
PubMed1996

High-resolution electron microscopy of technegas and pertechnegas.

Once inhaled, technegas remains in the lungs for a long period of time, whereas pertechnegas rapidly disappears. To investigate this difference, the morphology of technegas and of pertechnegas was investigated using high-resolution transmission electron microscopy. The technegas and pertechnegas were generated in an atmosphere of pure argon and in 3% oxygen in argon, respectively, using a commercially available technegas generator. For the technegas, the technetium crystals were observed to be covered with carbon, whereas they were found to have no carbon coating with the pertechnegas. Whether or not the technetium is coated with carbon appears to be responsible for the differences in behaviour of technegas and pertechnegas after deposition on the lung epithelium following inhalation.

Carbon↗
PubMed1995

Protein kinase activity-dependent inhibition of urokinase-type plasminogen activator gene transcription by cyclic AMP in human pre-B lymphoma cell line RC-K8.

We investigated the effects of cAMP on the urokinase-type plasminogen activator (uPA) production in human pre-B lymphoma cell line RC-K8 that is consistently secreting uPA in the conditioned medium. Both Bt2cAMP and PGE1 inhibited the uPA accumulation in a dose-dependent manner. Northern blot analysis and nuclear run-on assay revealed that uPA gene transcription was repressed by Bt2cAMP and the repression was negated by inhibition of de novo protein synthesis by cycloheximide. Pretreatment with H89 (N-[2-(p-bromocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), a specific cAMP-dependent protein kinase (PKA) inhibitor, strongly inhibited both the PKA activation and the supression of uPA mRNA accumulation induced by cAMP. H85 (N-[2-(N-formyl-p-chlorocinnamyl-amino) ethyl]-5-isoquinoline sulfonamide), which closely resembles H89 in its chemical structure but is not a selective inhibitor of PKA, showed little effect on the regulation of uPA gene regulation by Bt2cAMP. These results suggest that cAMP represses uPA gene transcription in human pre-B lymphoma cells through PKA pathway and in which de novo protein synthesis is required.

Alprostadil↗
PubMed1994

Assignment of the true and processed genes for human glycine decarboxylase to 9p23-24 and 4q12.

Southern analysis using a human glycine decarboxylase cDNA probe and genomic DNA preparations from Chinese hamster-human hybridoma cell lines demonstrated that on the segregated human chromosomes, 4 and 9, there are glycine decarboxylase cDNA-related sequences. The finding is confirmatory of the fact that fragments of both the true and processed genes for this protein have been cloned. Since one of four HindIII fragments revealed in the present Southern analysis matched requirements for the property expected from the restriction map reported for the processed gene, this was located on chromosome 4. The three remaining signals were consequently ascribed to fragments from the true gene on chromosome 9. Fluorescence in situ hybridization using the genomic clones assigned the true and processed genes to 9p23-24 and 4q12, respectively.

Amino Acid Oxidoreductases↗
PubMed1993

Identification and characterization of nuclear calmodulin-binding proteins of Saccharomyces cerevisiae.

Nuclear calmodulin-binding proteins of the yeast Saccharomyces cerevisiae were investigated. The soluble fractions after serial treatments of the isolated nuclei with buffers containing the nonionic detergent NP-40 (F1), 0.5 M KCl (F2) and 2.0 M KCl (F3) in this order, and the residual proteins (F4) were obtained. The calmodulin-binding proteins of the nucleus and nuclear subfractions were identified using the gel overlay method using 125I-calmodulin. Each subnuclear fraction contained a large number of components that bound calmodulin in a Ca(2+)-dependent or -independent manners. The calmodulin-binding proteins were isolated from F1 and F2 subnuclear fractions by affinity chromatography. The affinity-purified proteins bound calmodulin in a Ca(2+)-dependent manner when analyzed using the gel overlay method. The major calmodulin-binding components of F1 were 44, 42, 36, 32 and 29 kDa proteins, and those of F2 were 200, 100, 40, 42, 36, 34 and 32 kDa proteins. The isolated proteins also contained several Coomassie-blue stained proteins that did not bind calmodulin and, therefore, may represent the proteins associated with the calmodulin-binding proteins. Antisera raised against the affinity-purified preparation of F1 and F2 recognized almost all of the calmodulin-binding proteins present in the fraction and several other proteins of the nucleus. The presence of Ca(2+)-dependent protein phosphatase (type 2B) in the nucleus was demonstrated by Western blotting. The enzyme was localized predominantly in F1 and F4.

Calmodulin-Binding Proteins↗
PubMed1993

Cloning and characterization of the elongation factor EF-1 beta homologue of Saccharomyces cerevisiae. EF-1 beta is essential for growth.

A Saccharomyces cerevisiae cDNA homologue of the elongation factor EF-1 beta was found among the clones obtained by immunoscreening of a yeast cDNA expression library with an antibody against calmodulin affinity-purified proteins. The cDNA encoded a protein of 206 amino acids which was highly homologous (about 70% homology) with Artemia salina and human EF-1 beta. A protein with an apparent molecular mass of 33,000, significantly larger than that expected from the gene, was identified by Western blotting. Gene disruption experiments indicated that EF-1 beta is essential for growth.

Amino Acid Sequence↗
PubMed1993

The development-associated increase in the hepatic levels of the intrinsic components of the chicken glycine cleavage system.

The hepatic glycine cleavage system in the chicken was examined at different stages of development to study regulation of its biosynthesis. Embryonic levels of polypeptide and mRNA for glycine decarboxylase, one of the three intrinsic components of this enzyme system, were approximately 10% of their adult levels and were rapidly increased following hatching. Those of H-protein, another intrinsic component, were somewhat higher and were more slowly increased throughout the course of development. The change in activities of the two components went with the increase in their polypeptide levels. Moreover, both relative levels of mRNAs for the two components and relative efficiencies of transcription of their genes were not constant during development. T-protein biosynthesis appeared to follow a course similar to that of H-protein. These observations imply that during development, the production of H-protein and T-protein is regulated by similar mechanisms. Further, it is likely that the same mechanisms do not direct the regulatory processes in the biosynthesis of glycine decarboxylase and that embryo and adult livers possess different regulatory modes regarding the biosynthesis of this enzyme system. The shift to the adult mode appeared to occur at birth. The glycine cleavage activity that was three times higher than that in 12-day-old embryo was observed in adult liver mitochondria.

Aging↗
PubMed1993

Bronchospirometry and separate measurement of occlusion pressure before and after lung resection using a double-lumen endotracheal tube.

To assess the distribution of ventilation to each lung, we measured ventilation and maximum negative airway pressure during occluded inspiratory effort (Pmax) of the individual lungs in eight male patients immediately before and after right upper lobectomy using a double-lumen endotracheal tube at a constant depth of enflurane anesthesia (end-tidal concentration 1.7%). Compared with the breathing pattern observed immediately before surgery, minute ventilation, Pmax, and respiratory frequency significantly increased immediately after the surgery, whereas tidal volume was unchanged. Bronchospirometry revealed that tidal volumes on the nonoperated side significantly increased after the operation. In contrast, tidal volumes on the operated side significantly decreased after the operation and were associated with significantly smaller Pmax obtained from unilateral airway than those of the nonoperated side postoperatively. These results indicate that there are considerable differences in ventilatory function between the lungs on the operated side and nonoperated side. The lung on the nonoperated side can partially compensate for the impaired ventilatory function on the operated side.

Adult↗
PubMed1993

Immediate responses of arterial blood pressure and heart rate to sudden inhalation of high concentrations of isoflurane in normotensive and hypertensive patients.

Inhalation of pungent volatile anesthetics elicits respiratory reflex responses. To clarify whether an upper airway irritation produced by pungent anesthetics can also induce circulatory reflex responses that are clinically significant, a sudden administration of 5% isoflurane by mask was performed during continuous measurement of arterial blood pressure (BP) and heart rate (HR) in eight normotensive and eight hypertensive patients anesthetized with thiamylal and paralyzed with vecuronium. The sudden administration of 5% isoflurane caused immediate increases in BP, HR, and rate pressure products (RPP) in both normotensive and hypertensive patients. The responses observed were more pronounced in hypertensive than in normotensive patients. The circulatory changes in hypertensive patients were sufficient to be clinically significant. Our findings suggest that sudden administration of a high concentration of pungent volatile anesthetics may be associated with transient hypertensive responses in hypertensive patients with coronary artery disease.

Administration, Inhalation↗
PubMed1993

[The effect of PaO2 on apneic threshold PCO2 (PATCO2)].

Recovery from posthyperventilation apnea accompanies a rise in PaCO2, and ventilation starts only when the PaCO2 attains the apneic threshold PCO2 (PATCO2). In the present study, we evaluated the effect of decreasing PaO2 on PATCO2 under light enflurane anesthesia in 17 female patients undergoing elective surgery. The apnea was produced by hyperventilating the lungs under two different conditions [with 1.3% (end-tidal) enflurane in 100% O2 or with 1.3% enflurane in 30% O2]. The initiation of ventilation after apnea was identified by the first expiratory act observed in capnogram, at which time an arterial blood sample was taken to determine PATCO2. The values of PaO2 at the initiation of ventilation with 1.3% enflurane in 100% O2 and 1.3% enflurane in 30% O2 were 394.7 +/- 65.0 mmHg (mean +/- SD), and 51.9 +/- 18.2 mmHg, respectively. There was a significant difference between the two values (P < 0.001). The values of PATCO2 with 1.3% enflurane in 100% O2 and 1.3% enflurane in 30% O2 were 48.3 +/- 4.9 mmHg and 47.2 +/- 3.9 mmHg, respectively, and no significant difference was observed between the two values. Our results indicate that a decrease in PaO2 exerts no effect on PATCO2 at 0.7 MAC of enflurane anesthesia. This suggests that hypoxic stimulation does not play an important role in initiation of ventilation following posthyperventilation apnea at a light level of anesthesia.

Adult↗
PubMed1992

Prostacyclin administration suppresses the increase in hepatic levels of COL1A(I) and glyceraldehyde-3-phosphate dehydrogenase mRNAs in the rat treated with carbon tetrachloride.

Northern analysis using total RNAs from the component cells of normal rat liver indicated that COL1A(I) mRNA is present in fat-storing cells (Ito cells) and sinusoidal endothelial cells. A fraction for Kupffer cells also contained this mRNA. When CCl4 was given, COL1A(I) mRNA was increased in a factor of 1.5 in the fractions of these component cells. After 48 h of the drug administration, hepatocytes appeared to possess over 60% of liver COL1A(I) mRNA, although in normal hepatocytes its level was below the range detectable by our procedures. Under this injured condition of liver, glyceraldehyde-3-phosphate dehydrogenase (GAPDH) mRNA level was elevated, while activity of this enzyme was lowered by 50% of the control value. All the changes were obviously suppressed by the simultaneous administration of prostacyclin.

Animals↗
PubMed1992

Origin of the mutual activation of the alpha and beta 2 subunits in the alpha 2 beta 2 complex of tryptophan synthase. Effect of alanine or glycine substitutions at proline residues in the alpha subunit.

To understand how the alpha and beta 2 subunits of tryptophan synthase from Escherichia coli interact to form an alpha 2 beta 2 complex and undergo mutual activation, we have investigated alpha subunits with single amino acid replacements at conserved proline residues. Although the activities of alpha 2 beta 2 complexes that contain wild type alpha subunit or alpha subunits substituted at positions 28, 62, 96, and 207 are similar, the activities of alpha 2 beta 2 complexes that contain alpha subunits substituted at positions 57 and 132 are remarkably altered. Whereas the latter enzymes have greatly reduced activities in the individual half-reactions, they have considerably higher activities in the overall reaction. These remarkable activity results are explained by a decrease in the affinity of these mutant alpha subunits for the beta 2 subunit and by an increase in the affinity in the combined presence of ligands of both the alpha subunit and the beta 2 subunit. Isothermal calorimetric titrations of wild type beta 2 subunit with wild type alpha subunit and a mutant alpha subunit containing a substitution of glycine for proline at position 132 show that both the affinity and the exothermic association enthalpy are greatly reduced in the mutant alpha subunit although the stoichiometry of association is unchanged. The affinity of the mutant alpha subunit for the beta 2 subunits is greatly increased in the presence of an alpha subunit ligand, alpha-glycerol phosphate. We conclude that proline 132 plays a critical role in subunit interaction and in mutual subunit activation.

Alanine↗
PubMed1992

The antithrombin III gene polymorphism in Japan: examination for haplotypes relevant to disordered antithrombin III biosynthesis.

In the human antithrombin III (AT III) gene of Caucasian, two restriction fragment length polymorphism (RFLPs) have been identified and used for the linkage analysis of many congenital AT III abnormality and deficiency. In the present study, we attempted to examine the existence and distribution of these RFLPs in Japanese and utilize them for the molecular survey of the members in the AT III Toyama kindred and 4 type Ia deficient families. An AT III cDNA clone was isolated by ourselves and served as a hybridization probe. In Japanese, the intragenic polymorphism, which is referred to + and - alleles, was evenly distributed (.49: .51), and the 5'-length polymorphism, designated as S and F alleles, was also conserved at a ratio of .4 to .6. However, the combined genotypes of both polymorphisms revealed disproportionate, and + and S, and - and F alleles seemed mainly to coexist. AT III genes of the AT III Toyama kindred showed the homozygous genotype of -/F, and all affected members of the deficient families demonstrated no distinguishable alterations on Southern blots, suggesting that a subtle defect in the AT III gene or the "trans-acting" disordered mechanism is responsible for the decreased AT III levels. According to some reports that a defective AT III gene is the cause of inherited AT III deficiency, it was implied that the abnormal AT III gene was located on the haplotype of -/F in 3 families and +/F in one. In two deficient families with heterozygous genotypes, the RFLPs were considered to bring a clue to determine the structural changes.

Alleles↗