Biomedical subjects

J Nakamura

Publications and source records attributed to J Nakamura.

At least 19 recordsLinked to original sources

PubMed1995

The glutamine hydrolysis function of human GMP synthetase. Identification of an essential active site cysteine.

GMP synthetase (EC 6.3.5.2) is an amidotransferase that catalyzes the amination of xanthosine 5'-monophosphate to form GMP in the presence of glutamine and ATP. Glutamine hydrolysis produces the necessary amino group while ATP hydrolysis drives the reaction. Ammonia can also serve as an amino group donor. GMP synthetase contains two functional domains, which are well coordinated. The "glutamine amide transfer" or glutaminase domain is responsible for glutamine hydrolysis. The synthetase domain is responsible for ATP hydrolysis and GMP formation. Inorganic pyrophosphate inhibits the synthetase and uncouples the two domain functions by allowing glutamine hydrolysis to take place in the absence of ATP hydrolysis or GMP formation. Acivicin, a glutamine analog, selectively abolishes the glutaminase activity. It inhibits the synthetase activity only when glutamine is the amino donor. When ammonia is used in place of glutamine, acivicin has no effect on the synthetase activity. Acivicin inhibits GMP synthetase irreversibly by covalent modification. Enzyme inactivation is greatly facilitated by the presence of substrates. Acivicin labels GMP synthetase at a single site, and a tryptic peptide containing the modified residue was isolated. Mass spectrometry and Edman sequence analysis show that Cys104 is the site of modification. This residue is conserved among GMP synthetases and is located within a predicted glutamine amide transfer domain. These data suggest that Cys104 is an essential residue involved in the hydrolysis of glutamine to produce an amino group and is not needed for the hydrolysis of ATP or amination of xanthosine 5'-monophosphate to produce GMP.

Adenosine Triphosphate↗

PubMed1995

Negative or positive cooperation in calcium binding to detergent-solubilized ATPase of the sarcoplasmic reticulum. Its modulation by a high concentration of ATP.

Two different conformations of chemically equivalent Ca(2+)-ATPase molecules in the sarcoplasmic reticulum have been shown to non- and positive cooperatively bind two calcium ions, respectively (Nakamura, J. (1994) J. Biol. Chem. 269, 30822-30827). At pH 7.40, these ATPase molecules split into E1 (high affinity state for calcium), and E2 (low affinity state for calcium), respectively, before calcium binding. At this pH, calcium binding to the monomeric ATPase, solubilized with dodecyloctaethylenglycol monoether, was studied by examining 45Ca2+ binding to the ATPase and calcium dependence of its phosphorylation, fluorescence intensity, ATP-hydrolysis at a low (5 microM) concentration of ATP, and acetyl phosphate hydrolysis. The results suggest that the solubilized ATPase molecules predominantly preexist in E2 and negative cooperatively (the Hill value (nH) = 0.5-0.6) bind 2 mol of calcium/mol of the ATPase with an apparent calcium affinity (K0.5) of 3-5 microM. The nonequivalences of calcium bindings at the membranous ATPase molecules seem to result from the intermolecular interaction of the molecules. A high concentration (5 mM) of ATP modulated the binding manner so that it became positively cooperative (nH approximately 2) and increased the K0.5 to 0.1 microM.

Adenosine Triphosphate↗

PubMed1995

Effect of chronic L-dopa administration on serum luteinizing hormone levels in male rats.

We examined whether the repeated oral administration with a high dose of L-3-(3,4-dihydroxyphenyl)-alanine (L-DOPA) in 0.5% carboxymethyl cellulose increases serum luteinizing hormone (LH) levels in male rats. Serum LH levels were increased 4 h after a single administration of 1000 mg/kg L-DOPA to male rats, and returned to control levels within 8 h after administration. Four hours after a single administration, serum LH levels were significantly increased by L-DOPA at 1000 mg/kg, but not at 20, 100 or 200 mg/kg. Decreases in body weight and relative weight of the prostate were observed after 7 and 14 days of administration of 1000 mg/kg per day L-DOPA, but no changes were observed in weight of the testis, epididymis or seminal vesicle. The administration of L-DOPA at 500 or 1000 mg/kg per day for 7 or 14 days resulted in increased basal serum LH levels and decreased basal serum prolactin levels 24 h after the last administration. Serum testosterone levels tended to be higher in treated than in control rats. The levels of two metabolites of dopamine (DA), 3,4-dihydroxyphenylacetic acid (DOPAC) and homovanillic acid (HVA), in rats treated with 500 mg/kg per day tended to be slightly higher than those in control rats after 7 days of administration. Levels of DA, DOPAC and HVA were significantly increased after 7 and 14 days of administration of 1000 mg/kg per day and after 14 days of administration of 5000 mg/kg per day. The level of norepinephrine, but not its metabolite 3-methoxy-4-hydroxyphenylglycol, was significantly increased after only 7 days of administration of 1000 mg/kg per day. No significant changes were observed in levels of 5-hydroxytryptamine or its metabolite 5-hydroxyindole-3-acetic acid with administration of 500 or 1000 mg/kg per day. These findings suggest that a prolonged treatment with a high dose of L-DOPA in male rats induces release of LH from the pituitary, resulting in sustained elevation of LH levels in peripheral circulation.

3,4-Dihydroxyphenylacetic Acid↗

PubMed1995

Biochemical characterization of human GMP synthetase.

GMP synthetase (EC 6.3.5.2) plays a key role in the de novo synthesis of guanine nucleotides. It is a potential target for immunosuppressive therapy. Recently, the human enzyme was purified to homogeneity (Hirst, M., Haliday, E., Nakamura, J., and Lou, L. (1994) J. Biol. Chem. 269, 23830-23837). We now report the characterization of this enzyme in terms of its biochemical and kinetic properties. We found that there are distinct features of the human enzyme that has not been reported for GMP synthetase from other sources. There are two variant forms of human GMP synthetase. Their catalytic properties are very similar, although their isoelectric points are different. They most likely represent post-translational modification variants. Magnesium ion is required for enzyme activity, and the requirement is beyond levels needed for ATP chelation. Magnesium appears to be an essential activator and there may be more than one binding site. Interaction of GMP synthetase with xanthosine 5'-monophosphate (XMP), a substrate, exhibits sigmoidal kinetics with a Hill coefficient of 1.48 +/- 0.07. This positive cooperativity is not due to ligand-induced oligomerization, since GMP synthetase remains a monomer in the presence of XMP and other substrates. Decoyinine, a selective inhibitor of GMP synthetase, inhibits the human enzyme reversibly with uncompetitive inhibition kinetics toward glutamine and XMP and non-competitive kinetics toward ATP.

Adenosine Triphosphate↗

PubMed1995

Analysis of molluscum contagiosum virus genomes isolated in Japan.

The genomes of 477 Japanese strains of molluscum contagiosum virus (MCV) were analyzed using an in-gel digestion method with the restriction enzyme BamHI, and classified into four types, including a newly detected type (MCV type 4). All type 1 (MCV-1) genomes examined so far in Japan showed a common difference from the genome of the MCV-1 prototype (MCV-1p), the type reported to be most prevalent in Europe. The common markers of the variants of MCV-1 were 24-kbp fusion fragments generated by the loss of a BamHI site between the D2 and F fragments of MCV-1p. These variants of MCV-1 were classified into three groups (MCV-1va, MCV-1vb, MCV-1vc), with the variability among them being due to additions and losses of BamHI sites located in the right terminus and around the E and I fragments of MCV-1va. The restriction map of MCV-4 was generated and lined up with those of the other types. Cross-hybridization analysis revealed that the organization of all types of MCV genomes were essentially colinear. Considerable numbers of BamHI restriction sites were conserved between MCV-2 and 4, indicating a close analogy between them. The overall prevalence of MCV, as shown by the ratios of MCV-1 (MCV-1p):MCV-2:MCV-3:MCV-4, was 436(0):13:24:4. Thus, the molecular epidemiology of MCV in Japan is characterized by the absence of the European prototype of MCV-1, the exclusive occurrence and abundance of variants of MCV-1, a greater prevalence of MCV-3 over MCV-2, and the presence of MCV-4.

Genetic Variation↗

PubMed1995

A possible mechanism for the increase in serum luteinizing hormone levels in male rats by oxolinic acid.

Oxolinic acid (1-ethyl-1,4-dihydro-6,7-methylenedioxy-4-oxo-3- quinolinecarboxylic acid), an antimicrobial agent, raises the serum levels of luteinizing hormone (LH) and increases the incidence of testicular Leydig cell tumors in male rats. In the present study the mechanism by which serum LH levels are raised in male rats receiving oxolinic acid was investigated. Aged Wistar rats were fed a diet containing oxolinic acid at 0 or 3000 ppm for more than 4 weeks. There was no effect of oxolinic acid on either the maximal levels of serum LH after castration nor on the serum levels of LH stimulated with 1 microgram/rat of luteinizing hormone-releasing hormone (LHRH). The concentrations of testosterone in serum and testis were not changed by the treatment of oxolinic acid. In the in vitro organ culture, the testes of rats receiving oxolinic acid released testosterone in the same manner as the controls, in the presence or the absence of human chorionic gonadotropin (100 mIU/ml). The oxolinic acid-stimulated serum LH was not increased further by the daily administration of L-dopa (500 mg/kg/day, po, 7 days) and was blocked by the injection of a dopamine antagonist, haloperidol (2 mg/kg, ip). In a microdialysis study, oxolinic acid increased the extracellular concentration of dopamine in the preoptic area of hypothalamus. These findings suggest that a high dietary level of oxolinic acid elevates LH release from the anterior pituitary with an increase in LHRH, in part, by the excitatory input of a dopaminergic system in the preoptic area of rat hypothalamus.

Animals↗

PubMed1995

Neurotropin prevents neurophysiological abnormalities and ADP-induced hyperaggregability in rats with streptozotocin-induced diabetes.

Neurotropin, a non-proteinaceous extract from the inflamed dermis of rabbits inoculated with vaccinia virus, was administered for 8 weeks to rats with streptozotocin-induced diabetes. The physiological and biochemical changes of the nerves were studied as well as ADP-induced platelet aggregation. Neurotropin improved the caudal motor nerve conduction velocity, R-R variability, sciatic nerve blood flow, and platelet hyperaggregability in diabetic rats, despite having no effect on sorbitol and fructose accumulation or myoinositol depletion in the sciatic nerve. The correlation between nerve conduction velocity, R-R variability, nerve blood flow, and platelet aggregation were significant between each two parameters (p < 0.0001). Thus, the mechanism of action of neurotropin differed from that of aldose reductase inhibitors. These findings suggest that vascular factors may play an important role in the development of diabetic neuropathy, and that neurotropin may be useful for the treatment of this condition.

Adenosine Diphosphate↗

PubMed1995

Gamma knife surgery for Cushing's disease.

BACKGROUND: The efficacy of gamma knife surgery on Cushing's disease is not well known to date. In most reported cases of Cushing's disease treated with gamma knife, the area to be irradiated was determined with computed tomography or pneumoencephalography. We report two cases of recurrent pituitary-dependent Cushing's disease treated with gamma knife using stereotactic magnetic resonance imaging (MRI). MATERIALS AND METHODS: Recurrent microadenomas were visualized as hypointense areas using gadolinium-enhanced MRI after two transsphenoidal surgeries in both cases. The doses of irradiation given were 35 Gy and 20 Gy to the margin of the tumors, and less than 8 Gy and 21 Gy to the optic apparatus and cranial nerves in the cavernous sinus, respectively. RESULTS: Both patients had clinical remission with normal serum cortisol and adrenocorticotrophic hormone levels, during 2-year follow-up after radiosurgery, without endocrinologic deficiency or neurologic deterioration. CONCLUSIONS: Gamma knife surgery can be an alternative therapy for Cushing's disease when pituitary adenomas are apart from the optic apparatus and can be visualized clearly by MRI, even if tumors are recurrent after microsurgery.

Adenoma↗

PubMed1995

Prevention of abnormalities in motor nerve conduction and nerve blood-flow by a prostacyclin analog, beraprost sodium, in streptozotocin-induced diabetic rats.

The effects of the prostacyclin analog beraprost sodium on motor nerve function and nerve blood-flow were examined in streptozotocin-induced diabetic rats. Oral administration of beraprost sodium 0.1 mg/kg/day for 8 wks significantly (P < 0.001) improved caudal motor nerve conduction velocity and sciatic nerve blood-flow, both of which are impaired in diabetic rats. Beraprost sodium did not affect glucose, sorbitol, or fructose levels in the sciatic nerve. However, a decreased content of cyclic AMP in the sciatic nerve and higher level of thromboxane B2 in the thoracic aorta of diabetic rats, as compared with those in normal rats, were reversed by the treatment with beraprost sodium (P < 0.01). Results suggest that beraprost sodium may have value in treating diabetic neuropathy, mainly by improving endoneurial blood-flow.

Animals↗

PubMed1995

Effect of a potent new aldose reductase inhibitor, (5-(3-thienyltetrazol-1-yl)acetic acid (TAT), on diabetic neuropathy in rats.

(5-(3-Thienyl)tetrazol-1-yl)acetic acid (TAT), a novel potent aldose reductase inhibitor, was administered for 4 weeks to rats with streptozotocin-induced diabetes. Physiological and biochemical studies were subsequently conducted on rat nerve tissue and erythrocyte sorbitol content was estimated. Sciatic nerve blood flow (SNBF) was markedly lower (about 43.4%) in untreated diabetic (DC) rats than in non-diabetic controls (NC). A significant delay in caudal motor nerve conduction velocity (MNCV) and significantly higher glucose, sorbitol and fructose values were observed in the sciatic nerve, accompanied by a markedly higher sorbitol concentration in erythrocytes. In contrast, TAT-treated diabetic groups (DT-10, DT-40 and DT-200) had significantly higher SNBF, MNCV and sciatic nerve myo-inositol values and lower sciatic nerve sorbitol and fructose levels and erythrocyte sorbitol concentration than the DC group. There were good correlations between SNBF and MNCV (r = 0.672, P < 0.001) and between SNBF and erythrocyte sorbitol (r = 0.455, P < 0.003). These findings suggest that both vascular and metabolic factors play an important role in diabetic neuropathy and the effect of aldose reductase inhibitors on diabetic neuropathy may be mediated by at least these two factors.

Aldehyde Reductase↗

PubMed1995

In vivo modification of GABAA receptor with a high dose of pyridoxal phosphate induces tonic-clonic convulsion in immature mice.

The biologic cofactor, pyridoxal-5'-phosphate (PLP), is responsible for tonic-clonic convulsion in immature mice. The mechanisms underlying such convulsive fits induced by administration of a single high dose of PLP were studied. The administration of PLP resulted in a 13% increase of PLP in the P2 fraction compared to control P2, and the calculated data suggested that membrane bound PLP increased over 31% (approximately 1 microM). The P2 fraction of administered mice was treated with [3H]NaBH4 and analyzed by SDS-polyacrylamide gel electrophoresis. The radioactivity was mainly incorporated into a 52 kDa protein which corresponded to a GABAA receptor subunit. The addition of PLP in vitro competitively inhibited [3H]GABA binding as well as [3H]flunitrazepam binding to synaptic membranes in a concentration-dependent manner, and 50% inhibition was achieved with 1 mM PLP. The results obtained in the present study demonstrate that PLP was rapidly permeable into the brain through the immature blood-brain barrier and then bound directly to GABAA receptor. It is probable that specific amino groups of lysine residues on the GABAA receptor react in vivo with PLP to form Schiff bases, and that the in vivo modification of the receptor produces a degeneration of GABAergic neurotransmission leading to the onset of a convulsive fit.

Animals↗

PubMed1995

Different effects of absorption promoters on corneal and conjunctival penetration of ophthalmic beta-blockers.

PURPOSE: The purpose of this study was to investigate the improvement in corneal penetration of ophthalmic beta-blockers of various lipophilicities afforded by absorption promoters and to compare the corneal against conjunctival penetration in response to absorption promoters. METHODS: The penetration of the beta-blockers, atenolol, carteolol, tilisolol, timolol, and befunolol, in the presence of absorption promoters, across the isolated corneal and conjunctival membranes of albino rabbits was measured using a two-chamber glass diffusion cell. EDTA, taurocholic acid, capric acid, and saponin were used as the absorption promoters. RESULTS: The absorption promoters significantly increased the corneal permeability of most beta-blockers, especially the hydrophilic agents. The absorption promoters also enhanced the conjunctival permeability of beta-blockers, although their effect in promoting conjunctival penetration was less than that on corneal penetration. There was a differing penetration of instilled beta-blockers in the cornea and conjunctiva in response to absorption promoters. Capric acid and saponin showed significant promoting action on corneal penetration, but not on conjunctival penetration. Taurocholic acid had a significant effect on conjunctival penetration but not on corneal penetration. Saponin caused slight irritation. CONCLUSIONS: Absorption promoters can improve the ocular delivery of beta-blockers and a selective use of absorption promoter can improve the extent and pathway of drug ocular absorption.

Absorption↗

PubMed1995

Disulfide bond-forming reaction using a dimethyl sulfoxide/aqueous HCl system and its application to regioselective two disulfide bond formation.

Disulfide bond formation in S-acetamidomethyl (Acm) cysteine-containing peptides by successive treatments with silver trifluoromethanesulfonate (AgOTf) and dimethyl sulfoxide (DMSO)/aqueous HCl is described. An S-Acm cysteine was found to be quantitatively converted into cysteine by deprotection of the Acm group with AgOTf followed by DMSO/aqueous HCl treatment. Under these reaction conditions, no significant side reactions were observed with oxidation-sensitive amino acids such as Met, Tyr and Trp. Oxytocin and a Trp-containing peptide, urotensin II, were prepared by this method. Furthermore, regioselective two disulfide bond formation was found to be feasible by the combination of air oxidation and the AgOTf-DMSO/HCl system. This strategy has been successfully applied to the syntheses of tachyplesin I and endothelin I, which have two disulfide bonds and a Trp residue in the molecule.

Amino Acid Sequence↗

PubMed1995

Mechanism for drug absorption from rat-liver surface membrane: effect of dose and transport inhibitors on the pharmacokinetics of phenol red.

We examined the effect of dose and transport inhibitors on the pharmacokinetics of phenol red as a model drug after application to rat liver surface in-vivo, employing a cylindrical glass cell (i.d. 9 mm, area 0.64 cm2), to elucidate the mechanism for drug absorption from liver surface membrane. Absorption ratios of phenol red in 6 h were determined to be 91.1, 91.8 and 89.9% at a dose of 0.3, 1 and 3 mg, respectively. The AUC value for plasma concentration profile of phenol red was proportional to the dose. It is thus suggested that the absorption process of phenol red from rat liver surface does not approach saturability. The time course of the remaining amount of phenol red in the glass cell obeyed first-order kinetics at a dose of 0.3 mg, and its rate constant Ka was calculated to be 0.0069 min-1. Moreover, no significant difference was seen in the Ka value within the dose range of 0.3-3 mg, which was estimated by curve fitting of the plasma concentration profile of phenol red after application to rat liver surface in the two-compartment model with first-order absorption. 2,4-Dinitrophenol (0.3 mg) and probenecid (0.5 and 1 mg), inhibitors of metabolic energy and anion transport, respectively, had no significant effect on the pharmacokinetics of phenol red after application to rat liver surface. These data demonstrate that a specific transport mechanism such as active transport is not involved in phenol red absorption from rat liver surface membrane.

2,4-Dinitrophenol↗

PubMed1995

[Eight cases of infection caused by the Streptococcus milleri group--significance of serum antibody titer and a comparative investigation of the backgrounds and factors of infections caused by Streptococcus milleri and Streptococcus pneumoniae].

Eight cases of infection caused by Streptococcus milleri were studied clinically, and a comparative study was made of the backgrounds and factors influencing the development of infectious diseases caused by Streptococcus milleri and Streptococcus pneumoniae. The patients (six males and two females) with infectious diseases caused by Streptococcus milleri ranged in age from 21 to 81 years old with an average age of 62.0 years. The infectious diseases encountered among the patients were pyothorax in three patients, and in one patient each pyothorax and lung abscess, pleurisy, a secondary infection of pulmonary emphysema, skin infection related to a tracheostomy, a subdural abscess and a brain abscess, respectively. Six out of the eight cases had infections which formed pus. An underlying disease existed in seven cases, and, in five of these seven cases, it influenced the general condition of these patients. Six serum antibody titers for the S. milleri groups were examined in the First Department of Internal Medicine, University of the Ryukyus. Three cases for S. anginosus, two cases for S. intermedius, and one case for S. constellatus showed high titers of X1024 or X2048. Therefore, the examination of the serum antibody titer seems useful for the diagnosis of infections caused by the S. milleri group. We also carried out a comparative investigation of the backgrounds and factors of infectious diseases caused by S. milleri and S. pneumoniae. There are more underlying diseases in infections caused by S. milleri than in those caused by S. pneumoniae. There were more smokers among the patients with infections caused by S. milleri than among those with S. pneumonia infections.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗

PubMed1995

Anucleate cell production by Escherichia coli delta hns mutant lacking a histone-like protein, H-NS.

Normal-sized anucleate cells were observed in the cultures of a delta hns mutant strain. Even in nucleate cells, some populations showed irregular intracellular localization of the nucleoids. The delta hns mutant showed reduced ploidy, although initiation of chromosome replication was essentially synchronous as defined by flow cytometric analysis. These results indicate that the delta hns mutant is defective in the mechanisms of chromosome partitioning and chromosome replication.

Bacterial Outer Membrane Proteins↗

PubMed1995

Isolation and characterization of chemotaxis mutants and genes of Pseudomonas aeruginosa.

Two chemotaxis-defective mutants of Pseudomonas aeruginosa, designated PC1 and PC2, were selected by the swarm plate method after N-methyl-N'-nitro-N-nitrosoguanidine mutagenesis. These mutants were fully motile but incapable of swarming, suggesting that they had a defect in the intracellular signalling pathway. Computer-assisted capillary assays confirmed that they failed to show behavioral responses to chemical stimuli, including peptone, methyl thiocyanate, and phosphate. Two chemotaxis genes were cloned by phenotypic complementation of PC1 and PC2. From nucleotide sequence analysis, one gene was found to encode a putative polypeptide that was homologous to the enteric CheZ protein, while the other gene was cheY, which had been previously reported (M. N. Starnbach and S. Lory, Mol. Microbiol. 6:459-469, 1992). Deletion and complementation analysis showed that PC1 was a cheY mutant, whereas PC2 had a double mutation in the cheY and cheZ genes. A chromosomal cheZ mutant, constructed by inserting a kanamycin resistance gene cassette into the wild-type gene, changed its swimming direction much more frequently than did wild-type strain PAO1. In contrast, cheY mutants were found to rarely reverse their swimming directions.

Amino Acid Sequence↗

PubMed1995

Effect of ophthalmic preservatives on serum concentration and local irritation of ocularly applied insulin.

We previously compared hypoglycemic responses after the instillation of insulin formulations. A hypoglycemic response was actually observed after an instillation of insulin with ophthalmic preservatives. In the present study, in order to evaluate the usefulness of insulin formulation containing ophthalmic preservatives, a serum concentration of insulin and an irritation to the eye were investigated after instillation of the insulin formulation in albino rabbits. The ophthalmic preservatives used were benzalkonium chloride, paraben, 2-phenylethanol, benzyl alcohol and sorbic acid. As a result, ophthalmic preservatives, especially benzalkonium chloride and paraben, increased the serum concentration of insulin. The insulin concentration showed a significant correlation with the hypoglycemic response reported previously. This result indicates that ophthalmic preservatives increase the absorption of ocularly applied insulin, and the absorbed insulin decreases serum glucose concentration. The insulin formulation with preservatives showed little irritation on rabbit eyes according to blinking measurements. These results indicate that ophthalmic preservatives are useful for the systemic delivery of ocularly applied insulin.

Administration, Topical↗