Biomedical subjects

T Murakami

Publications and source records attributed to T Murakami.

At least 685 records · Page 38Linked to original sources

PubMed1996

Analysis of the interaction of an anti-HIV peptide, T22 ([Tyr5, 12, Lys7]-polyphemusin II), with gp120 and CD4 by surface plasmon resonance.

We have previously found that T22 ([Tyr5, 12, Lys7]-polyphemusin II) exhibits strong anti-human immunodeficiency virus (HIV) activity comparable to that of 3'-azido-2', 3'-dideoxythymidine (AZT). The inhibition mechanism of T22 on HIV-replication has not been elucidated precisely yet, and hence the target molecules of T22 have not been identified. However, our recent research suggested that T22 exerts its effect by blocking virus-cell fusion at an early stage of HIV infection and that T22 might interact with an HIV envelope protein and/or a T-cell surface protein, both of which are critical for HIV infection. In this paper we demonstrated that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) and that both bindings can be inhibited by an anti-T22 antibody, using biosensor technology (BIAcoreTM) based on the principles of surface plasmon resonance. Linearization by the BIAcoreTM system (BIAlogue software) and nonlinear least squares analysis by curve fitting with exponential equations showed that both interactions have close dissociation constants (approximately 10(-7) M). The present study suggests that T22 inhibits the virus-cell fusion process through binding to both gp120 and CD4.

Amino Acid Sequence↗

PubMed1996

Substitution at codon 269 (glutamine --> proline) of the leptin receptor (OB-R) cDNA is the only mutation found in the Zucker fatty (fa/fa) rat.

We recently cloned one of spliced variant forms of rat leptin receptor (OB-R), which contains a short intracellular domain, and found obese-phenotype-linked nucleotide alteration in the extracellular domain of the cDNA from the Zucker (fa/fa) rat, which results in a glutamine269 to proline269 amino acid substitution. Reported herein are the cloning and sequencing of another spliced variant forms of rat OB-R cDNA with a long intracellular domain. Both forms of OB-R cDNA share the same extracellular domain. In the Zucker (fa/fa) rat, no changes in either the gene structure nor in the nucleotide sequence of the long intracellular domain were observed. However, the expression level of OB-R mRNA in the brain of Zucker (fa/fa) rat was higher than for lean littermates. These facts suggest that the substitution at codon 269 of the OB-R cDNA represents the crucial mutation which results in the obese phenotype of Zucker (fa/fa) rat.

Alternative Splicing↗

PubMed1996

Carnitine transport defect in fibroblasts of juvenile visceral steatosis (JVS) mouse.

Juvenile visceral steatosis (JVS) mice are associated with systemic carnitine deficiency (Kuwajima, et al., 1991). In order to investigate the cause of this deficiency, we compared fibroblast carnitine transport activities in normal mice and JVS mice. The kinetic analysis showed that in formal fibroblasts, the Km and Vmax values for saturable uptake was 15.6 microM and 2.56 pmol/min/mg protein, respectively. In JVS fibroblasts, however, saturable uptake was not observed. There was no great difference in the linear component of uptake between normal and JVS fibroblasts. At the physiological concentration (50 microM) of carnitine, the fibroblast carnitine transport activity in JVS mice was decreased to 18% of that in normal mice. Thus there is hardly any carnitine transport activity in the fibroblasts of JVS mice, indicating that the JVS mouse can be regarded as an animal model of primary carnitine deficiency.

Animals↗

PubMed1996

A 1.5-Mb cosmid contig of the CMT1A duplication/HNPP deletion critical region in 17p11.2-p12.

Charcot-Marie-Tooth disease type 1A (CMT1A) is associated with a 1. 5-Mb tandem duplication in chromosome 17p11.2-p12, and hereditary neuropathy with liability to pressure palsies (HNPP) is associated with a 1.5-Mb deletion at this locus. Both diseases appear to result from an altered copy number of the peripheral myelin protein-22 gene, PMP22, which maps within the critical region. To identify additional genes and characterize chromosomal elements, a 1.5-Mb cosmid contig of the CMT1A duplication/HNPP deletion critical region was assembled using a yeast artificial chromosome (YAC)-based isolation and binning strategy. Whole YAC probes were used for screening a high-density arrayed chromosome 17-specific cosmid library. Selected cosmids were spotted on dot blots and assigned to bins defined by YACs. This binning of cosmids facilitated the subsequent fingerprint analysis. The 1.5-Mb region was covered by 137 cosmids with a minimum overlap set of 52 cosmids assigned to 17 bins and 9 contigs.

Charcot-Marie-Tooth Disease↗

PubMed1996

Phenotype-linked amino acid alteration in leptin receptor cDNA from Zucker fatty (fa/fa) rat.

The mouse obese (ob) gene product (leptin), expressed specifically in adipose cells, regulates energy balance in mice. Both mouse diabetes (db) and rat fatty (fa) gene products are thought to play major roles in leptin signaling pathways in the hypothalamic area. Mutations of these genes in murines result in marked obesity and type II diabetes as part of a syndrome that resembles morbid obesity in humans. Reported herein are the cloning and sequencing of one of spliced variant forms of rat leptin receptor (OB-R) cDNA with a short intracellular domain. In the Zucker (fa/fa) rat, no changes in either the gene structure or the expression levels were observed. However phenotype-linked nucleotide alteration exists in the cDNA from Zucker (fa/fa) rat, which results in an amino acid substitution.

Amino Acid Sequence↗

PubMed1996

Abnormal cell divisions in leaf primordia caused by the expression of the rice homeobox gene OSH1 lead to altered morphology of leaves in transgenic tobacco.

Transgenic tobacco plants were generated carrying a rice homeobox gene, OSH1, controlled by the promoter of a gene encoding a tobacco pathogenesis-related protein (PR1a). These lines were morphologically abnormal, with wrinkled and/or lobed leaves. Histological analysis of shoot apex primordia indicates arrest of lateral leaf blade expansion, often resulting in asymmetric and anisotrophic growth of leaf blades. Other notable abnormalities included abnormal or arrested development of leaf lateral veins. Interestingly, OHS1 expression was undetectable in mature leaves with the aberrant morphological features. Thus, OSH1 expression in mature leaves is not necessary for abnormal leaf development. Northern blot and in situ hybridization analyses indicate that PR1a-OSH1 is expressed only in the shoot apical meristem and in very young leaf primordia. Therefore, the aberrant morphological features are an indirect consequence of ectopic OSH1 gene expression. The only abnormality observed in tissues expressing the transgene was periclinal (rather than anticlinal) division in mesophyll cells during leaf blade initiation. This generates thicker leaf blades and disrupts the mesophyll cell layers, from which vascular tissues differentiate. The OSH1 product appears to affect the mechanism controlling the orientation of the plane of cell division, resulting in abnormal periclinal division of mesophyll cell, which in turn results in the gross morphological abnormalities observed in the transgenic lines.

Cell Division↗

PubMed1996

Neurotrophin-3 increases the DNA-binding activities of several transcription factors in a mouse osteoblastic cell line.

In the mouse osteoblastic cell line MC3T3-E1, the signaling responses of several DNA-binding proteins induced by the treatment of neurotrophin-3 were examined using electrophoretic mobility shift assay. Neurotrophin-3 increased binding activities in nuclear extracts of MC3T3-E1 cells to TPA-responsive element (TRE), cyclic AMP-responsive element (CRE) and serum-responsive element (SRE), but not binding activity in the nuclear extracts to c-Myc binding DNA element. Competition experiments revealed that the binding activity to TRE in the nuclear extracts of neurotrophin-3-treated MC3T3-E1 cells was entirely inhibited by the both unlabeled TRE and CRE probes. On the other hand, the binding activity to CRE was abolished by the unlabeled CRE probe but not by the same amount of unlabeled TRE probe. Moreover, immunodepletion/supershift assay using antibodies directed to Fos, Jun and CREB proteins, showed that the binding activities to TRE and CRE in the nuclear extracts were derived in part from these proteins.

Animals↗

PubMed1996

The origins of catecholaminergic innervation in the rostral ventromedial medulla oblongata of the rat.

The localization of catecholaminergic neuronal cell bodies which project to the rostral ventromedial medulla oblongata (RVM) were investigated by the combined technique with dopamine beta-hydroxylase (DBH), phenylethanolamine N-methyltransferase (PNMT) or tyrosine hydroxylase (TH) immunocytochemistry and retrograde neuronal tracing method using fluorescent latex microspheres (FluoSpheres) injected into the center of the RVM, the nucleus raphe magnus (NRM). Noradrenaline (NA) neurons in A1, A5, A7 regions, locus coeruleus (LC) and nucleus subcoeruleus (SC) and adrenaline (Ad) neurons in C1 region were double-labeled due to DBH or PNMT and retrogradely transported FluoSpheres, and the ratio of their coexistence was higher in A1, A5, A7 and C1 than in LC and SC. No dopamine neurons in the midbrain and forebrain were double-labeled with TH and FluoSpheres. Thus, it was clarified that the RVM is innervated by the ventral groups of lateral tegmental NA and Ad neurons in the brainstem.

Animals↗

PubMed1996

Alteration of extracellular matrix in dilated cardiomyopathic hamster heart.

The purpose of this study was to characterize the collagen in hereditary dilated cardiomyopathic hamster hearts, and to examine the participation of the collagen in the occurrence and progression of cardiomyopathy. BIO 53.58 hamsters (5, 10, 20 weeks old) were used as the model of dilated cardiomyopathy. Flb hamsters were used as controls. The collagen content was almost constant at any age in the Flb hamsters, but increased with age in BIO 53.58 hamsters. Type III collagen increased significantly in BIO 53.58 hamsters at 10 weeks. The acetic acid solubility of collagen decreased in BIO 53.58 hamsters as the fibrosis progressed, but was unchanged in controls. Reducible crosslinks showed a tendency to decrease progressively in BIO 53.58 hamsters. There were no differences between Flb and BIO 53.58 hamsters at 5 weeks, but its expression in BIO 53.58 hamsters at 10 and 20 weeks of age increased compared to Flb controls. These findings indicate that in the early phase of cardiomyopathy the extracellular matrix of the myocardium is rich in type III collagen. In the later phase, the matrix resembles that of hard tissues, whose collagen is mainly of type I collagen and is insoluble. These data suggest that the increased collagen synthesis may impair the cardiac function in the development of cardiomyopathy.

Animals↗

PubMed1996

Interaction of an anti-HIV peptide, T22, with gp120 and CD4.

T22 ([Tyr5,12, Lys7]-polyphemusin II) has been shown to have strong anti-human immunodeficiency virus (HIV) activity. The precise mechanism of action of T22 on HIV-replication has not been elucidated yet, nor have the targets of T22 been identified. However, our previous research suggested that T22 exerts its effect by blocking virus-cell fusion and that T22 might interact with an HIV envelope protein and/or a T-cell surface protein. Herein we use a novel biosensor based on the principles of surface plasmon resonance (BIAcore) to demonstrate that T22 binds specifically to both gp120 (an envelope protein of HIV) and CD4 (a T-cell surface protein) and that both bindings can be inhibited by an anti-T22 antibody. The data obtained suggest that T22 inhibits virus-cell fusion through the double binding to the above two proteins.

Amino Acid Sequence↗

PubMed1996

Molecular cloning of a 74-kDa regulatory subunit (B" or delta) of human protein phosphatase 2A.

Based on amino acid sequence data of a 74-kDa regulatory subunit (B" or delta) of a human heterotrimeric protein phosphatase 2A, a cDNA encoding the subunit was isolated from a human cerebral cortex library. The cDNA had an open reading frame encoding an M(r) 66,138 protein of 570 amino acids. Bacterial expression of the cDNA yielded a protein immunoreactive with antisera specific to the 74-kDa subunit. The predicted primary structure of the subunit had no similarity to already reported sequences of PP2A regulatory subunits including A, B, and PR72. Potential phosphorylation sites for protein kinases A and C, a bipartite motif of putative nuclear localization signal, and SH3 accessible proline-rich domain, and a unique PQ repeat were found in the sequence. The subunit mRNA of about 2.9 kb was ubiquitously expressed in rat tissues.

Amino Acid Sequence↗

PubMed1996

Mutations in the nonstructural protein 5A gene and response to interferon in patients with chronic hepatitis C virus 1b infection.

BACKGROUND: A region associated with sensitivity to interferon has been identified in the nonstructural protein 5A (NS5A) of hepatitis C virus (HCV) genotype 1b. The region spans amino acid residues 2209 to 2248 (NS5A2209-2248) of HCV-J, a strain of HCV-1b whose complete genomic sequence has been identified. We examined whether the NS5A2209-2248 sequence present before therapy could be used as a predictor of the response to interferon therapy in patients with chronic HCV-1b infection. METHODS: We retrospectively analyzed 84 patients with chronic HCV-1b infection who had received interferon alfa (total dose, 516 million to 880 million units) for six months. Pretreatment serum samples were analyzed. The amino acid sequence of NS5A2209-2248 was determined by direct sequencing of the HCV genome amplified by the polymerase chain reaction (PCR) and was compared with the established sequence for HCV-J. RESULTS: A complete response, as evidenced by the absence of HCV RNA in serum on nested reverse-transcription PCR for six months after therapy, did not occur in any of the 30 patients whose NS5A2209-2248 sequences were identical to that of HCV-J (wild type). Five of 38 patients (13 percent) with 1 to 3 changes in NS5A2209-2248 (intermediate type) had complete responses, as did all 16 patients with 4 to 11 amino acid substitutions (mutant type), indicating that the mutant type was significantly associated with a complete response (P < 0.001). Although baseline serum HCV RNA levels, as measured by a branched-chain DNA assay, were lower in patients with the mutant type of NS5A2209-2248 than in those with the other types (P < 0.001), multivariate analyses revealed that the number of amino acid substitutions in NS5A2209-2248 was the only variable associated with an independent effect on the outcome of interferon therapy (odds ratio, 5.3; 95 percent confidence interval, 1.6 to 18; P = 0.007). CONCLUSIONS: In patients with chronic HCV-1b infection, there is a substantial correlation between responses to interferon and mutations in the NS5A gene.

Amino Acid Sequence↗

PubMed1996

Substitution of glycine for arginine-213 in extracellular-superoxide dismutase impairs affinity for heparin and endothelial cell surface.

Extracellular-superoxide dismutase (EC-SOD) levels in sera divide into two discontinuous groups: a low-level group below 400 ng/ml and a high-level group above 400 ng/ml [Adachi, Nakamura, Yamada, Futenma, Kato and Hirano (1994) Clin. Chim. Acta 229, 123-131]. Molecular genetic studies have shown that the donors in the high-level group have a single base substitution generating the exchange of glycine for arginine-213 (R213G) in the heparin-binding domain of EC-SOD [Sandström, Nilsson, Karlsson and Marklund (1994) J. Biol. Chem. 269, 19163-19166; Yamada, Yamada, Adachi, Goto, Ogasawara, Futenma, Kitano, Hirano and Kato (1995) Jpn. J. Hum. Genet. 40, 177-184]. The serum EC-SOD level in homozygote subjects was significantly higher than that in heterozygotes and in normal subjects. Serum EC-SOD from heterozygotes and homozygotes had equally decreased affinity for heparin, as judged by heparin-HPLC, as compared with that from normal donors. This result suggests that the serum EC-SOD in heterozygotes was mainly composed of the mutant form which has reduced heparin affinity. On the other hand, fibroblast cells derived from heterozygote subjects generated mRNA of both normal and mutant EC-SOD (m-EC-SOD), and expressed the corresponding proteins. EC-SOD is a tetrameric enzyme, and in heterozygote donors would be heterogeneous with regard to the constitution of normal and mutant subunits. The enzyme form consisting of only mutant subunits, the form with the weakest heparin affinity, can be preferentially driven out to the plasma phase, because EC-SOD in the vasculature exists in equilibrium between plasma and the endothelial cell surface. The binding of m-EC-SOD to bovine aortic endothelial cells was about 50-fold less than that of normal EC-SOD. This result suggests that the binding of m-EC-SOD to vascular endothelial cells is much decreased in vivo, which causes a high level of serum EC-SOD.

Animals↗

PubMed1996

Digoxin reduces beta-adrenergic contractile response in rabbit hearts. Ca(2+)-dependent inhibition of adenylyl cyclase activity via Na+/Ca2+ exchange.

Whereas mobilization of intracellular Ca2+ stimulates neuronal adenylyl cyclase via Ca2+/calmodulin, mobilized Ca2+ directly inhibits adenylyl cyclase in other tissues. To determine the physiologic role of the Ca(2+)-dependent interaction between Na+/Ca2+ exchange and beta-adrenergic signal transduction in the intact heart, digoxin (0.3 mg/kg) was administered intravenously in rabbits. 30 min after the administration, digoxin impaired the peak left ventricular dP/dt response to dobutamine infusions by up to 38% as compared with control rabbits. This impairment was not caused by changes in either beta-adrenergic receptor number or in the functional activity of stimulatory guanine nucleotide-binding protein. It was associated with 33-36% reductions in basal and stimulated adenylyl cyclase activities. Animals treated with calcium gluconate (20 mg/kg/min for 30 min) also demonstrated similar reductions in adenylyl cyclase activities. In addition, increasing the free Ca2+ concentration progressively inhibited adenylyl cyclase activity in the control, digoxin-treated, and calcium gluconate-treated sarcolemma preparations in vitro. Moreover, digoxin and calcium gluconate pretreatment blunted the increase in cAMP in myocardial tissue after dobutamine infusion in vivo. Thus, digoxin rapidly reduces beta-adrenergic contractile response in rabbit hearts. This reduction may reflect an inhibition of adenylyl cyclase by Ca2+ mobilized via Na+/Ca2+ exchange.

4-Nitrophenylphosphatase↗

PubMed1996

Pancreatoblastoma: case report and review of treatment in the literature.

A case of pancreatoblastoma arising from the body-to-tail of the pancreas in a 5-year-old boy is presented. The patient underwent exploratory laparotomy and, 11 days later, resection of the tumor (partial pancreatectomy, pyloroplasty, and splenectomy). Before resection, cyclophosphamide and vincristine were administered. Because of tumor spillage during resection, a combination of chemotherapy (administration of cyclophosphamide and adriamycin on that day) and postoperative radiotherapy was given. Nine months after resection, partial hepatectomy was performed for liver metastasis and consolidated by a more intensive chemotherapy regimen using cisplatin, adriamycin, vincristine, and cyclophosphamide. After completion of the chemotherapy, the patient had a 14-month uneventful course, and a locally recurrent tumor was treated by the fourth surgery (extirpation of the recurrent tumor, partial hepatectomy, partial colectomy, and partial gastrectomy) and intraoperative radiation. Thereafter, the boy has shown no evidence of disease at 3 years 8 months. The literature of pancreatoblastoma is reviewed from the therapeutic viewpoint.

Child, Preschool↗

PubMed1996

Association of tissue factor with a gamma chain homodimer of the IgE receptor type I in cultured human monocytes.

Tissue factor (TF) is a high-affinity receptor for coagulation factors VII (F VII) and VIIa. The F VII/VIIa/TF complex is the major cellular initiator of the extrinsic coagulation cascade. We found that the occupancy of TF by its ligand, F VIIa, is involved with signal transduction and that TF is associated with the gamma chain homodimer identified as a component of IgE receptor type I (Fc epsilon RI). When 4-day cultured human monocytes were incubated with F VIIa, several polypeptides, especially a 70-kDa polypeptide, were transiently phosphorylated on tyrosine, residues. These phosphorylation events were inhibited by prior binding of anti-TF monoclonal antibody (mAb) HTF-K14, but not anti-TF mAb HTF-K180 to intact cultured monocytes. HTF-K14 blocked the binding of F VII/ VIIa to cell surface TF, whereas HTF-K180 did not. Anti-TF immunoprecipitates prepared from 1% digitonin lysates of cultured human monocytes incorporated phosphate in a gamma chain homodimer when incubated with [gamma-32P] ATP. The identity of the T-associated structures as gamma chains was established by immunoblot analysis of anti-TF mAb immunoprecipitates with anti-gamma chain mAb. In addition, anti-TF immunoblot analysis showed that TF co-precipitated with anti-gamma chain mAb. Our data suggest that gamma chains may play an important role in signaling via TF in human monocytes/macrophages.

Cells, Cultured↗

PubMed1996

Interaction of tobramycin and pH in cultured chick tibiae.

The toxicity of tobramycin at concentrations released from antibiotic-impregnated polymethylmethacrylate beads was determined using cultured embryonic chick tibiae. Because previous results from this laboratory have shown that osteoblast metabolism is inhibited at low pH and because of the potential for a low local pH in infected bone, the antibiotic was tested in medium with pH values from 6.8 to 7.4. Bone metabolism was evaluated by measuring the rates of glycolysis (lactate production), protein synthesis ([3H]proline uptake), and collagen synthesis ([3H]proline hydroxylation). Tobramycin at the concentrations released from the beads (1.0-1.5 mg/ml) contributed to lowering the pH of the medium. At pH 7.4, the antibiotic produced as much as a 30, 39, and 48% decrease in glycolysis, protein synthesis, and collagen synthesis, respectively. Tibiae exposed to tobramycin for 3 days, followed by 2 days without the antibiotic, showed only a partial recovery from its toxic effects. Although tobramycin was relatively less toxic in an acidic environment, the overall metabolic activity of the bones was poorest when the tobramycin concentration was high (2.0 mg/ml) and pH was low (6.8). The results of this study support the following conclusions: (a) tobramycin at high concentrations is toxic to bone, (b) a combination of high tobramycin concentration and low pH in the bone microenvironment may greatly inhibit bone metabolism, and (c) treatment and prevention of osteomyelitis by means of tobramycin-impregnated beads may be augmented by preventing the pH from decreasing in traumatized areas.

Animals↗

PubMed1996

Gradenigo's syndrome: CT and MRI findings.

We report the case of an 8-year-old girl with Gradenigo's syndrome. Involvement of the petrous portion of the left temporal bone was demonstrated by CT and an inflammatory lesion of the left petrous apex was clearly shown by MRI, which is useful in diagnosis and management of apical petrositis.

Abducens Nerve↗