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Biomedical subjects

K Murayama

Publications and source records attributed to K Murayama.

At least 37 records · Page 2Linked to original sources
PubMed2001

Crystal structure of omega transcriptional repressor encoded by Streptococcus pyogenes plasmid pSM19035 at 1.5 A resolution.

The 71 amino acid residue omega protein encoded by the Streptococcus pyogenes non-conjugative plasmid pSM19035 is a transcriptional repressor that regulates expression of genes for copy number control and stable maintenance of plasmids. The crystal structure of omega protein has been determined by multiple isomorphous replacement, including anomalous scattering and refined to an R-factor of 21.1 % (R(free)=23.2 %) at 1.5 A resolution. Two monomers related by a non-crystallographic 2-fold axis form a homodimer that occupies the asymmetric unit. Each polypeptide chain is folded into two alpha-helices and one beta-strand forming an antiparallel beta-ribbon in the homodimer. The N-terminal regions (1-23 and 1-22 in subunits I and II, respectively) are not defined in the electron density due to proteolysis of the N-terminal 20 amino acid residues during crystallisation and partial disorder. The omega protein belongs to the structural superfamily of MetJ/Arc repressors featuring a ribbon-helix-helix DNA-binding motif with the beta-ribbon located in and recognizing the major groove of operator DNA; according to a modelled omega protein-DNA complex, residues Arg31 and Arg31' on the beta-ribbon are in positions to interact with a nucleobase, especially guanine.

Amino Acid Sequence↗
PubMed2001

Differential antinociceptive effects induced by intrathecally administered endomorphin-1 and endomorphin-2 in the mouse.

Two highly selective mu-opioid receptor agonists, endomorphin-1 and endomorphin-2, have been identified and postulated to be endogenous ligands for mu-opioid receptors. Intrathecal (i.t.) administration of endomorphin-1 and endomorphin-2 at doses from 0.039 to 5 nmol dose-dependently produced antinociception with the paw-withdrawal test. The paw-withdrawal inhibition rapidly reached its peak at 1 min, rapidly declined and returned to the pre-injection levels in 20 min. The inhibition of the paw-withdrawal responses to endomorphin-1 and endomorphin-2 at a dose of 5 nmol observed at 1 and 5 min after injection was blocked by pretreatment with a non-selective opioid receptor antagonist naloxone (1 mg/kg, s.c.). The antinociceptive effect of endomorphin-2 was more sensitive to the mu (1)-opioid receptor antagonist, naloxonazine than that of endomorphin-1. The endomorphin-2-induced paw-withdrawal inhibition at both 1 and 5 min after injection was blocked by pretreatment with kappa-opioid receptor antagonist nor-binaltorphimine (10 mg/kg, s.c.) or the delta(2)-opioid receptor antagonist naltriben (0.6 mg/kg, s.c.) but not the delta(1)-opioid receptor antagonist 7-benzylidine naltrexone (BNTX) (0.6 mg/kg s.c.). In contrast, the paw-withdrawal inhibition induced by endomorphin-1 observed at both 1 and 5 min after injection was not blocked by naloxonazine (35 mg/kg, s.c.), nor-binaltorphimine (10 mg/kg, s.c.), naltriben (0.6 mg/kg, s.c.) or BNTX (0.6 mg/kg s.c.). The endomorphin-2-induced paw-withdrawal inhibition was blocked by the pretreatment with an antiserum against dynorphin A-(1-17) or [Met(5)]enkephalin, but not by antiserum against dynorphin B-(1-13). Pretreatment with these antisera did not affect the endomorphin-1-induced paw-withdrawal inhibition. Our results indicate that endomorphin-2 given i.t. produces its antinociceptive effects via the stimulation of mu (1)-opioid receptors (naloxonazine-sensitive site) in the spinal cord. The antinociception induced by endomophin-2 contains additional components, which are mediated by the release of dynorphin A-(1-17) and [Met(5)]enkephalin which subsequently act on kappa-opioid receptors and delta(2)-opioid receptors to produce antinociception.

Analgesics↗
PubMed2001

Stability and DNA-binding properties of the omega regulator protein from the broad-host range Streptococcus pyogenes plasmid pSM19035.

At the transcriptional level, the pSM19035-encoded omega protein coordinates the expression of proteins required for control of copy number and maintenance of plasmids. Using circular dichroism, fluorescence spectroscopy, ultracentrifugation and an electrophoretic mobility shift assay, the wild-type omega protein and a variant with a C-terminal hexa-histidine tag (omega-H(6)) were characterized. The omega protein is mainly alpha-helical (42%), occurs as homodimer in solution, unfolds thermally with half transition temperatures, T(m), between approximately 43 and approximately 78 degrees C depending on the ionic strength of the buffer, and binds PcopS-DNA with high affinity. The omega-H(6) protein has a modified conformation with lower alpha-helix content (29%), lower thermal stability, and strongly reduced affinity to PcopS-DNA.

Bacterial Proteins↗
PubMed2001

The sarcolemmal proteins dysferlin and caveolin-3 interact in skeletal muscle.

Dysferlin is a surface membrane protein in skeletal muscle whose deficiency causes distal and proximal, recessively inherited, forms of muscular dystrophy designated Miyoshi myopathy (MM) and limb girdle muscular dystrophy type 2B (LGMD2B), respectively. The function of dysferlin is not defined. Caveolin-3 is another skeletal muscle membrane protein which is important in the formation of caveolae and whose mutations cause dominantly inherited limb girdle muscular dystrophy type 1C (LGMD1C). We report that dysferlin co-immunoprecipitates with caveolin-3 from biopsied normal human skeletal muscles. We also describe abnormal localization of dysferlin in muscles from patients with LGMD1C including novel missense mutation (T64P) in the human caveolin-3 gene (CAV3). The immunoprecipitation data are consistent with the parallel observation that dysferlin immunostaining is not normal in LGMD1C muscles. Amino acid sequence analysis of the dysferlin protein reveals seven sites that correspond to caveolin-3 scaffold-binding motifs, and one site that is a potential target to bind the WW domain of the caveolin-3 protein. This is the first description of a possible dysferlin interacting protein; it suggests the hypothesis that one function of dysferlin may be to interact with caveolin-3 to subserve signaling functions of caveolae.

Caveolin 3↗
PubMed2001

Modification of a single tryptophan residue in human Cu,Zn-superoxide dismutase by peroxynitrite in the presence of bicarbonate.

Human recombinant Cu,Zn-SOD was reacted with peroxynitrite in a reaction mixture containing 150 mM potassium phosphate buffer (pH 7.4) 25 mM sodium bicarbonate, and 0.1 mM diethylenetriamine pentaacetic acid. Disappearance of fluorescence emission at 350 nm, which could be attributed to modification of a single tryptophan residue, was observed in the modified enzyme with a pH optimum of around 8.4. A fluorescence decrease with the same pH optimum was also observed without sodium bicarbonate, but with less efficiency. Amino acid contents of the modified enzyme showed no significant difference in all amino acids except the loss of a single tryptophan residue of the enzyme. The peroxynitrite-modified enzyme showed an increase in optical absorption around 350 nm and 30% reduced enzyme activity based on the copper contents. The modified enzyme showed the same electron paramagnetic resonance spectrum as that of the control enzyme. The modified Cu,Zn-SOD showed a single protein band in sodium dodecyl sulfate--polyacrylamide gel electrophoresis (SDS--PAGE) and five protein bands in non-denaturing PAGE. From this evidence, we conclude that nitration and/or oxidation of the single tryptophan 32 and partial inactivation of the enzyme activity of Cu,Zn-SOD is caused by a peroxynitrite-carbon dioxide adduct without perturbation of the active site copper integrity.

Amino Acids↗
PubMed2001

Human biotin-containing subunit of 3-methylcrotonyl-CoA carboxylase gene (MCCA): cDNA sequence, genomic organization, localization to chromosomal band 3q27, and expression.

3-Methylcrotonyl-CoA carboxylase (MCCase; EC 6.4.1.4) is a mitochondrial biotin enzyme and plays an essential role in the catabolism of leucine and isovalerate in animals, bacterial species, and plants. MCCase consists of two subunits, those that are biotin-containing and non-biotin-containing. The genes responsible for these subunits have been isolated in soybean, Arabidopsis thaliana, and tomatoes, but not in mammals. In humans, MCCase deficiency has been thought to be a rare metabolic disease, but the number of patients with MCCase deficiency appears to be increasing with a wide range of clinical presentations, some that result in a lethal condition and others that are asymptomatic. In this report, we have isolated and carried out chromosomal mapping of the gene for the biotin-containing subunit (A subunit) of the human MCCase gene, MCCA. The cDNA predicts an open reading frame coding for a 725-amino-acid protein with mitochondrial signal peptide, biotin carboxylase, and biotin-carrier domains. The gene is composed of at least 19 exons and covers more than 70 kb of sequence on band q27 of chromosome 3. MCCA was abundantly expressed in mitochondria-rich organs, such as the heart, skeletal muscles, kidney, and liver. In exon 13, we observed a His/Pro polymorphism at codon 464 (an A to C transition at nucleotide position 1391 in the cDNA sequence). Then, we determined the DNA sequences of the 5' untranslated region and entire coding regions in two patients with MCCase deficiency, but no sequence substitution was detected, suggesting that the gene mutations might be in the non-biotin-containing subunit (B subunit) gene, MCCB, in these patients.

Amino Acid Sequence↗
PubMed2001

A genetic factor for age-related cataract: identification and characterization of a novel galactokinase variant, "Osaka," in Asians.

Galactokinase (GALK) deficiency is an autosomal recessive disorder characterized by hypergalactosemia and cataract formation. Through mass screening of newborn infants, we identified a novel and prevalent GALK variant (designated here as the "Osaka" variant) associated with an A198V mutation in three infants with mild GALK deficiency. GALK activity and the amount of immunoreactive protein in the mutant were both 20% of normal construct in expression analysis. The K(m) values for galactose and ATP-Mg(2+) in erythrocytes with homozygous A198V were similar to those of the healthy adult control subjects. A population study for A198V revealed prevalences of 4.1% in Japanese and 2.8% in Koreans, lower incidence in Taiwanese and Chinese, no incidence in blacks and whites from the United States, and a significantly high frequency (7.8%; P < .023) in Japanese individuals with bilateral cataract. This variant probably originated in Japanese and Korean ancestors and is one of the genetic factors that causes cataract in elderly individuals.

Adult↗
PubMed2001

Altered quinone biosynthesis in the long-lived clk-1 mutants of Caenorhabditis elegans.

Mutations in the clk-1 gene of Caenorhabditis elegans result in an extended life span and an average slowing down of developmental and behavioral rates. However, it has not been possible to identify biochemical changes that might underlie the extension of life span observed in clk-1 mutants, and therefore the function of CLK-1 in C. elegans remains unknown. In this report, we analyzed the effect of clk-1 mutation on ubiquinone (UQ(9)) biosynthesis and show that clk-1 mutants mitochondria do not contain detectable levels of UQ(9). Instead, the UQ(9) biosynthesis intermediate, demethoxyubiquinone (DMQ(9)), is present at high levels. This result demonstrates that CLK-1 is absolutely required for the biosynthesis of UQ(9) in C. elegans. Interestingly, the activity levels of NADH-cytochrome c reductase and succinate-cytochrome c reductase in mutant mitochondria are very similar to those in the wild-type, suggesting that DMQ(9) can function as an electron carrier in the respiratory chain. To test this possibility, the short side chain derivative DMQ(2) was chemically synthesized. We find that DMQ(2) can act as an electron acceptor for both complex I and complex II in clk-1 mutant mitochondria, while another ubiquinone biosynthesis precursor, 3-hydroxy-UQ(2), cannot. The accumulation of DMQ(9) and its use in mutant mitochondria indicate, for the first time in any organism, a link between the alteration in the quinone species used in respiration and life span.

Animals↗
PubMed2001

One-step subcellular fractionation of rat liver tissue using a Nycodenz density gradient prepared by freezing-thawing and two-dimensional sodium dodecyl sulfate electrophoresis profiles of the main fraction of organelles.

In the present study, we describe a new procedure using freezing-thawing to density gradient solution of Nycodenz for one-step separation of organelles from the rat liver and subsequent proteome analysis of subcellular fractions. To prepare two-dimensional electrophoresis (2-D PAGE) profiles of tissue organelles, we performed one-step subcellular fractionation of rat liver homogenate using a density gradient of Nycodenz solution, which resulted in the separation of the cytosolic fraction from the postnuclear supernatant. The density gradient of Nycodenz was prepared from a 20% solution in a centrifuge tube by freezing-thawing overnight at -20 degrees C and at room temperature for a few hours without the initial centrifugation procedure. The shape of the gradient density curve was dependent on Nycodenz concentration and tube size. After fractionation, the protein profiles were examined using one-dimensional sodium dodecyl sulfate (SDS)-PAGE. The organelles were confirmed using Western blotting. Our results indicate that our procedure provides a simple method for the separation of organelle fractions from the rat liver tissue.

Animals↗
PubMed2001

Cesarean section in a patient with syringomyelia.

PURPOSE: To describe the anesthetic management of Cesarean section in a patient with syringomyelia. CLINICAL FEATURES: A 27-yr-old pregnant woman with syringomyelia was scheduled to undergo elective Cesarean section. At the age of 25 yr, she had begun to experience headaches, and at the age of 26 yr, a diagnosis of syringomyelia of the upper spinal cord was made on the basis of magnetic resonance imaging findings. No symptoms other than headache were noted preoperatively. General anesthesia was used for the Cesarean section. After the administration of 1 mg vecuronium as a priming dose, 5 mg vecuronium were injected. At the onset of clinical muscle weakness, 225 mg thiamylal were promptly administered as the induction agent and the patient was intubated (timing principle with priming method) and pressure on the cricoid cartilage applied to prevent regurgitation of stomach contents. Anesthesia was maintained with oxygen, nitrous oxide and isoflurane at a low concentration. Mild hyperventilation was used throughout the procedure. Anesthesia and surgery proceeded without any problem, response to vecuronium was clinically normal and recovery was uneventful. Neurological status remained normal. CONCLUSION: We report the safe use of general anesthesia for Cesarean section in a patient with syringomyelia. Precautions were taken to avoid increases in intracranial pressure and our patient experienced no untoward neurologic event.

Adult↗
PubMed2001

Quantitative Analysis of Indocyanine Green Angiographic Image in Central Serous Chorioretinopathy.

Purpose: To disclose the possible involvement of choroidal vascular change in developement of central serous chorioretinopathy (CSC).Methods: Indocyanine green (ICG) angiography was performed in 31 eyes with acute CSC, and 21 eyes from normal subjects. The maximum diameter of the choroidal veins and intensity of background fluorescence in the posterior fundus with ICG video images were measured for further analysis using IMAGEnet((R)) (Topcon). Then the results from CSC affected eyes, their fellow eyes, and normal eyes were compared. Aging factors were taken into consideration when we analyzed the data.Result: The maximum diameters of the choroidal veins were larger in both affected and fellow eyes than in the normal eyes (P <.001), and had a positive correlation with aging particularly in fellow eyes (r = 36). Both in the affected and fellow eyes, the background fluorescein intensity in the posterior pole of the late phase images was lower than in the normal eyes (P <.001), and was correlated with aging (r = 0.28, r = 0.43).Conclusion: This quantitative study showed that choroidal venous dilatation and the residual background fluorescence in the posterior fundus might be positive findings reflecting the pathogenesis of CSC.

Journal Article↗
PubMed2001

Expression of a newly defined tumor-rejection antigen SART3 in musculoskeletal tumors and induction of HLA class I-restricted cytotoxic T lymphocytes by SART3-derived peptides.

We recently reported that a SART3 tumor-rejection antigen possessing tumor epitopes is capable of inducing HLA class 1-restricted and tumor-specific cytotoxic T lymphocytes in cancer patients. We studied the expression of the SART3 protein in musculoskeletal tumors to find a molecule for potential use in tumor-specific immunotherapy. The SART3 was detected at protein levels in 100% of the osteosarcoma cell lines (n = 20), in 50% of the musculoskeletal tumor tissue specimens (n = 32), and at notable levels in 67% of osteosarcoma tissues (n = 9) and malignant fibrous histiocytosis tissues (n = 9), respectively. SART3-derived peptides at positions 109-118 and 315-323 induced HLA-A24-restricted tumor-specific cytoxic T lymphocytes from peripheral blood mononuclear cells of patients with osteosarcoma or malignant fibrous histiocytosis. These peptide-induced cytotoxic T lymphocytes recognized HLA-A24+ SART3+ osteosarcoma cells but not HLA-A24 or SART3 cells. These results suggest that the SART3 protein and its derived peptides could be molecules appropriate for use in specific immunotherapies for approximately 60% of HLA-A24+ patients with osteosarcoma or malignant fibrous histiocytosis.

Antigens, Neoplasm↗
PubMed2001

Calpain 3 gene mutations: genetic and clinico-pathologic findings in limb-girdle muscular dystrophy.

Mutations in the calpain 3 gene have been proven to be responsible for limb-girdle muscular dystrophy (LGMD) type 2A. To determine the incidence and genotypes of the calpain 3 (p94) gene mutations in Japanese LGMD patients, we sequenced the gene in 80 patients with clinical characteristics of autosomal recessive or sporadic LGMD. We identified 13 distinct pathogenic mutations in 21 patients (26%), including seven missense mutations, four splice-site mutations and two insertions in which six were novel mutations. Among the 21 patients, 15 (71%) had three types of the common missense (G233V, R461C, D707G) and one insertion (1795-1796insA) mutation. The patients had slowly progressive muscle weakness with age of onset of the disease varying from 6 to 52 years, averaging 20.9. The most striking pathologic findings were the presence of lobulated fibers in 14 patients, especially in the advanced stages. Differing from Duchenne and Becker muscular dystrophy, opaque (hypercontracted) fibers were very rarely seen. These findings may be helpful in establishing diagnostic screening strategies in Japanese LGMD patients.

Adolescent↗
PubMed2001

[Clinical effects of meropenem on infectious diseases in obstetrics and gynecology].

The efficacy and the safety of meropenem (MEPM) were examined in obstetric and gynecological infections and the results shown below were obtained. 1. The subjects were patients with infectious diseases during a non-pregnant period (n = 14), a pregnant period (n = 13) and a puerperal period (n = 11). After intravenous drip infusion of MEPM at 1-2 g/day, the response rate was 38/39 (97.4%) (Excellent: 13/39 (33.3%), Effective: 25/39 (64.1%)). 2. The response rates in a group receiving 1 g/day, a group receiving 2 g/day and a group receiving a combination of both were, respectively, 25/26 (96.2%), 9/9 (100%) and 4/4 (100%). 3. In cases that did not respond to previous treatments, the response rate to MEPM was 20/20 (100%). 4. In clinical efficacy classified by causative organisms, the rate of Excellent was 10/23 (43.5%) and the rate of Effective was 13/23 (56.5%), and the bacterial eradication rate was 21/23 (91.3%). 5. No subjective and objective adverse reactions or abnormalities in clinical laboratory tests due to administration of MEPM were observed.

Adult↗
PubMed2001

[Clinical investigation of cefpirome for perinatal infection and infection prevention].

Effectiveness and safety of cefpirome (CPR) were studied in perinatal infection and prevention of postoperative infections. 1. Enrolled in the study of perinatal infections were the total 62 cases comprising 47 of chorioamnionitis, 10 of puerperal intrauterine infections, 1 of infectious abortion and 4 of others. The effective rate of CPR 2 g/day given in drip infusion was 61/62 (98.4%). In bacteriological studies, the bacterial elimination rate was 57/61 (93.4%). Unchanged and remained were 3 strains of Gram-positive bacteria and 1 strain of Bacteroides spp. 2. Enrolled in the study of prevention of postoperative infections during perinatal period were the total 88 cases comprising 74 who underwent cesarean section and 14 others. The effective rate of CPR 2 g/day given in drip infusion was 87/88 (98.9%). 3. With respect to subjective and objective adverse affects due to the agent, drug eruption was observed in 1/150 (0.7%). No abnormal result of clinical laboratory tests was found. The above results suggested the usefulness of CPR for treatment of perinatal infections and prevention of postoperative infections.

Adult↗
PubMed2001

[Embolization of the bilateral internal pudendal arteries for intractable priapism in a child with chronic myelogenous leukemia].

Priapism is an uncommon clinical symptom in children with chronic myelogenous leukemia (CML). Here we report a 14-year-old boy with this symptom, which had appeared 4 days prior to hospitalization. Peripheral blood examination revealed a leukocyte count of 510,000/microliter, (87% neutrophils, 3% eosinophils, 6% basophils, and 1.6% lymphocytes), a hemoglobin level of 6.5 g/dl and a platelet count of 640,000/microliter. Karyotype analysis revealed the Ph1 chromosome and myeloid hyperplasia in the bone marrow. The patient was diagnosed as having chronic myelogenous leukemia (CML) complicated by priapism. In an unsuccessful attempt to alleviate and improve the priapism, urokinase was injected and hydroxyurea was administered for the CML. Angiography confirmed the presence of venous return from the scrotum, and embolization of the bilateral internal pudendal arteries was performed to reduce the amount of inflow. Although this relieved the patient of his pain and prevented penile necrosis, the patient's future sexual potency was sacrificed. Selective embolization of the pudendal arteries can be one of the most effective ways of treating intractable priapism, if angiography confirms the presence of venous return from the penis.

Adolescent↗
PubMed2000

Differential antagonism of endomorphin-1 and endomorphin-2 spinal antinociception by naloxonazine and 3-methoxynaltrexone.

To determine the role of spinal mu-opioid receptor subtypes in antinociception induced by intrathecal (i.t.) injection of endomorphin-1 and -2, we assessed the effects of beta-funaltrexamine (a selective mu-opioid receptor antagonist) naloxonazine (a selective antagonist at the mu(1)-opioid receptor) and a novel receptor antagonist (3-methoxynaltrexone) using the paw-withdrawal test. Antinociception of i.t. endomorphins and [D-Ala(2), MePhe(4), Gly(ol)(5)]enkephalin (DAMGO) was completely reversed by pretreatment with beta-funaltrexamine (40 mg/kg s.c.). Pretreatment with a variety of doses of i.t. or s.c. naloxonazine 24 h before testing antagonized the antinociception of endomorphin-1, -2 and DAMGO. Judging from the ID(50) values of naloxonazine, the antinociceptive effect of endomorphin-2 was more sensitive to naloxonazine than that of endomorphin-1 or DAMGO. The selective morphine-6beta-glucuronide antagonist, 3-methoxynaltrexone, which blocked endomorphin-2-induced antinociception at each dose (0.25 mg/kg s.c. or 2.5 ng i.t.) that was inactive against DAMGO, did not affect endomorphin-1-induced antinociception but shifted the dose-response curve of endomorphin-2 3-fold to the right. These findings may be interpreted as indicative of the existence of a novel mu-opioid receptor subtype in spinal sites, where antinociception of morphine-6beta-glucuronide and endomorphin-2 are antagonized by 3-methoxynaltrexone. The present results suggest that endomorphin-1 and endomorphin-2 may produce antinociception through different subtypes of mu-opioid receptor.

Analgesics, Opioid↗