Biomedical subjects

M Murata

Publications and source records attributed to M Murata.

At least 19 recordsLinked to original sources

PubMed1992

Dopaminergic stimulation up-regulates the in vivo expression of brain-derived neurotrophic factor (BDNF) in the striatum.

We investigated the effect of dopamine on the in vivo expression of brain-derived neurotrophic factor (BDNF) in the striatum of mouse. BDNF mRNA expression in the striatum, which was quantified with the reverse transcriptase polymerase chain reaction, was up-regulated from 2 h after oral administration of levodopa, a precursor of dopamine. The increase was sustained for 16 h. Co-administration of haloperidol partially inhibited dopamine-induced BDNF enhancement. These data suggest that dopaminergic stimulation directly promotes the expression of BDNF in the striatum in vivo.

Amino Acid Sequence↗

PubMed1992

Crystallization and preliminary X-ray study of the cathepsin B complexed with CA074, a selective inhibitor.

Cathepsin B from bovine spleen has been purified and crystallized as a complex with a specific inhibitor CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L- isoleucyl-L-proline], using the hanging-drop method. The complex crystals obtained from 50 mM-citrate buffer (pH 3.5) belong to the tetragonal space group P4(1) (or P4(3)) with a = 73.06 A and c = 141.59 A, and diffract beyond 2.2 A resolution. There are two complex molecules per asymmetric unit giving a packing density of 3.37 A3/Da and indicating a high solvent content of 63.5%.

Animals↗

PubMed1992

Functional signal peptide reduces bilayer thickness of phosphatidylcholine liposomes.

To investigate the interaction between a signal peptide and the lipid bilayer, two kinds of peptides, L8-M5 (L8 = MRL8PLAALG, M5 = KVFER) and L14-M5 (L14 = MRL14PLAALG), were examined in membranes composed of dioleoylphosphatidylcholine (DOPC). Peptides L8 and L14 are artificially designed signal sequences, and M5 is the N-terminal five residues of human lysozyme; L8 mediated effective secretion of human lysozyme in yeast, while L14 did not [Yamamoto, Y., et al. (1987) Biochem. Biophys. Res. Commun. 149, 431-436]. DOPC liposomes incorporating L8-M5 or L14-M5 were observed by electron cryomicroscopy as pairs of concentric circles, and the separation of the bilayer was measured along the membrane. Peptide L8-M5 was found to reduce the bilayer thickness, but L14-M5 did not. CD measurements revealed that L8-M5 adopted an alpha-helical conformation with random coil in the liposome membranes and that L14-M5 adopted a more helical and less random conformation than L8-M5. Fluorescence spectroscopy using both aqueous and membranous probes revealed that L8-M5 destabilized the lipid bilayer more strongly than L14-M5. These results suggest that functional L8-M5 reduces the bilayer thickness and destabilizes the lipid bilayer and that these activities are important for signal peptide function.

Amino Acid Sequence↗

PubMed1992

Determination of cholesterol in sub-nanomolar quantities in biological fluids by high-performance liquid chromatography.

A highly sensitive method for the determination of cholesterol in biological fluids is described. Unsaponifiable lipids from rat serum and thoracic duct lymph chylomicron samples were treated with cholesterol oxidase. The product of the enzymatic reaction, delta 4-cholestenone, was analysed by normal-phase high-performance liquid chromatography (HPLC) using hexane-isopropanol (95:5, v/v) as a mobile phase and detected with a UV spectrophotometer at 240 nm. When the standard samples containing varying amounts of cholesterol (0.15-3 nmol) were treated with cholesterol oxidase and analysed by HPLC (injected amounts 0.09-1.8 nmol of cholesterol), the peak areas increased proportionally with the amounts of authentic cholesterol with a correlation coefficient of 0.996. The values in these biological fluids determined by the HPLC method were identical to those obtained by enzymatic-colorimetric or gas chromatographic methods. Moreover, the detection limit (0.09 nmol) of the present method (0.15 nmol are required for the sample preparation) is lower than those of conventional methods (approximately 30 nmol). Because of the excellent sensitivity and reproducibility, this method is well suited for the determination of cholesterol in biological fluids where cholesterol concentration is low.

Animals↗

PubMed1992

Selective stimulation of Ca2+ flux in cells by maitotoxin.

Maitotoxin elicits a dose-dependent stimulation of 45Ca2+ influx in glioma C6, pheochromocytoma PC12, insulinoma HIT and human blood cells, while having no effect in liposomes. In HIT cells maitotoxin also elicited influx of 86Rb+ greater than 22Na+ greater than 54Mn2+, but the stimulation was far less than for 45Ca2+. Stimulation of 45Ca2+ influx was blocked by Ni2+, Co2+, Cd2+ and Mn2+, and markedly reduced by Ba2+. Divalent cations, in particular Ca2+, Ba2+, Mn2+ and Cd2+, enhanced influx of the monovalent cations 22Na+ and 86Rb+.

Animals↗

PubMed1992

Inhibitory effect of okadaic acid derivatives on protein phosphatases. A study on structure-affinity relationship.

The effect of structural modifications of okadaic acid (OA), a polyether C38 fatty acid, was studied on its inhibitory activity toward type 1 and type 2A protein phosphatases (PP1 and PP2A) by using OA derivatives obtained either by isolation from natural sources or by chemical processes. The dissociation constant (Ki) for the interaction of OA with PP2A was estimated to be 30 (26-33) nM [median (95% confidence limits)]. The OA derivatives used and their affinity for PP2A, expressed as Ki (in brackets) were as follows: 35-methyl-OA (DTX1) [19 (12-25) pM], OA-9,10-episulphide (acanthifolicin) [47 (25-60) pM], 7-deoxy-OA [69 (31-138) pM], 14,15-dihydro-OA [315 (275-360) pM], 2-deoxy-OA [899 (763-1044) pM], 7-O-palmitoyl-OA [greater than 100 nM], 7-O-palmitoyl-DTX1 [greater than 100 nM], methyl okadate [much greater than 100 nM], 2-oxo-decarboxy-OA [much greater than 100 nM] and the C-15-C-38 fragment of OA [much greater than 100 nM]. The sequence of the affinity of these derivatives for PP1 was essentially the same as that observed with PP2A, although the absolute values of Ki were very different for the enzymes. The inhibitory effect of OA on PP2A was reversed by applying a murine monoclonal antibody against OA, which recognizes modifications of the 7-hydroxyl group of the OA molecule. It has been shown by n.m.r. spectroscopy and X-ray analysis that one end (C-1-C-24) of the OA molecule assumes a circular conformation. The present results suggest the importance of the conformation for the inhibitory action of OA on the protein phosphatases. The ratios of the Ki values for PP1 to that for PP2A, which were within the range 10(3)-10(4), tended to be smaller for the derivatives with lower affinity, indicating that the structural changes in OA impaired the affinity for PP2A more strongly than that for PP1.

Antibodies, Monoclonal↗

PubMed1992

Genetic and structural characterization of an amino acid dimorphism in glycoprotein Ib alpha involved in platelet transfusion refractoriness.

The platelet-specific alloantigen, Siba, located within the alpha-subunit of the glycoprotein (GP) Ib-IX membrane receptor, has been found to be involved in the pathogenesis of platelet transfusion refractoriness. We have identified the existence of a naturally occurring threonine/methionine dimorphism at position 145 of the GPIb alpha sequence, and determined that the Siba antigen corresponds to the molecule containing methionine145. The diallelic codons can be detected by restriction enzyme analysis of amplified genomic DNA fragments from the GPIb alpha gene. Evaluation of 61 healthy blood donors showed that the allele frequencies are 89% and 11% for the threonine145 and methionine145 codons, respectively. A positive correlation exists between platelet reactivity with the anti-Siba antibody and the presence of a methionine145-encoding allele. Moreover, recombinant expression of two soluble GPIb alpha fragments differing only at residue 145, provided definitive evidence that the human anti-Siba antibody reacts only with the molecule containing methionine145. These results explain the structural basis of the Siba human alloantigen system and define screening methodologies useful in transfusion medicine to match donor and recipient platelets accordingly.

Amino Acids↗

PubMed1992

pH-dependent membrane fusion and vesiculation of phospholipid large unilamellar vesicles induced by amphiphilic anionic and cationic peptides.

We studied fusion induced by a 20-amino acid peptide derived from the amino-terminal segment of hemagglutinin of influenza virus A/PR/8/34 [Murata, M., Sugahara, Y., Takahashi, S., & Ohnishi, S. (1987) J. Biochem. (Tokyo) 102, 957-962]. To extend the study, we have prepared several water-soluble amphiphilic peptides derived from the HA peptide; the anionic peptides D4, E5, and E5L contain four and five acidic residues and the cationic peptide K5 has five Lys residues in place of the five Glu residues in E5. Fusion of egg phosphatidylcholine large unilamellar vesicles induced by these peptides is assayed by two different fluorescence methods, lipid mixing and internal content mixing. Fusion is rapid in the initial stage (12-15% within 20 s) and remains nearly the same or slightly increasing afterward. The anionic peptides cause fusion at acidic pH lower than 6.0-6.5, and the cationic peptide causes fusion at alkaline pH higher than 9.0. Leakage and vesiculation of vesicles are also measured. These peptides are bound and associated with vesicles as shown by Ficoll discontinuous gradients and by the blue shift of tryptophan fluorescence. They take an alpha-helical structure in the presence of vesicles. They become more hydrophobic in the pH regions for fusion. When the suspension is made acidic or alkaline, the vesicles aggregate, as shown by the increase in light scattering. The fusion mechanism suggests that the amphiphilic peptides become more hydrophobic by neutralization due to protonation of the carboxyl groups or deprotonation of the lysyl amino groups, aggregate the vesicles together, and interact strongly with lipid bilayers to cause fusion. At higher peptide concentrations, E5 and E5L cause fusion transiently at acidic pH followed by vesiculation.

Amino Acid Sequence↗

PubMed1992

Deletion mapping on the short arm of chromosome 3 in squamous cell carcinoma and adenocarcinoma of the lung.

We examined loss of heterozygosity in 49 adenocarcinomas and 18 squamous cell carcinomas of the lung with 19 RFLP markers on the short arm of chromosome 3. Although no interstitial deletions were observed in any squamous cell carcinomas, interstitial or partial deletions were detected in 23 adenocarcinomas. Identification of two common regions of deletion in adenocarcinomas, at 3p21.3 and 3p14.1-21.1, suggested the presence of at least two tumor suppressor genes on 3p within the same regions commonly deleted in renal cell carcinomas. Correlation between the frequency of loss of heterozygosity on 3p and histopathological grade of adenocarcinoma also was observed. These results imply an etiological difference between two major types of non-small cell lung cancers, adenocarcinoma and squamous cell carcinoma.

Adenocarcinoma↗

PubMed1992

Tumor-reactive T-cells accumulate in lung cancer tissues but fail to respond due to tumor cell-derived factor.

The purpose of this study was to elucidate a possible immune response to tumor cells mediated by tumor-infiltrating lymphocytes (TIL) in lung cancer. In flow cytometry, the majority of T-cells of TIL were CD45RA-, CD45RO+, and CDw29high, and expressed HLA-DR. The expression of interleukin 2 receptor beta chain increased in both CD4+ and CD8+ TIL compared with both types of T-cells in peripheral blood. These results indicate that the major population of TIL is activated memory T-cells. The TIL preparation, which was usually contaminated with 5 to 10% tumor cells, did not exhibit any response in autologous mixed lymphocyte-tumor culture even in the presence of interleukin 2 (IL-2) in all five cases tested. Although purified T-cells from TIL showed the positive response in only 1 of 10 cases tested without addition of IL-2, it occurred in 7 of 10 cases in the addition of a low concentration of IL-2. The IL-2-dependent response to irradiated autologous tumor cells was suppressed when nonirradiated autologous tumor cells were added to the culture. Culture supernatants of four lung cancer cell lines and freshly prepared lung cancer cells obtained from 6 cases exhibited suppressive activity against anti-CD3 antibody-induced mitogenesis of peripheral blood mononuclear cells from healthy donors. We suggest that, taken together, (a) the major population of TIL in lung cancer are activated memory T-cells, and they include tumor-reactive ones, and that (b) the function of the TIL is impaired by unavailability of IL-2 and/or by suppression due to lung cancer cell-derived factor(s).

Antigens, CD↗

PubMed1992

Expressivity of a common fragile site, fra(3)(p14.2), in patients with cancer and other diseases.

A population survey of a common folate-sensitive fragile site, fra(3)(p14.2), has been carried out on PHA-stimulated peripheral lymphocytes of patients with cancer and other diseases, under both culture conditions of folate deprivation and aphidicolin treatment. Overall findings regarding variability of expressivity due to age and sex were very similar to those obtained in a healthy population. The expression of fra(3)(p14.2) by folate deficient condition appeared hardly influenced by such exogenous factors as tobacco smoking habit, past histories of radiotherapy and chemotherapy, while it was associated with the unfavorable prognosis of cancers. Furthermore, proportion of those with higher expression was slightly but significantly larger in both lung and breast cancer patients. These findings suggest that some factors relevant to the expression of fra(3)(p14.2) may be associated with development and progression of certain kinds of cancer.

Adolescent↗

PubMed1992

Chromosome abnormalities and rare fragile sites detected in azoospermia patients.

We have examined constitutional chromosome abnormalities and fragile sites in 40 patients with azoospermia. Chromosome abnormalities were found in four cases. Three cases showed a deletion of the long arm of the Y chromosome 46,X,del(Yq) and the other case had a ring of G group chromosome 46,XY,r(G). In a rare fragile sites test, four fragile site carriers were detected and three rare autosomal fragile sites were identified; fra(8)(q24.1), fra(11)(p15.1), and fra(17)(p12). The expression of these fragile sites were induced specifically by AT-specific DNA ligands, such as distamycin A and Hoechst 33258. In addition, one patient was found to be the case of double ascertainment of fragile sites, fra(8)(q24.1) and fra(17)(p12). The overall frequency of distamycin A-inducible fragile sites in azoospermia patients appeared to be higher than those reported for Japanese healthy subjects and cancer patients. However, no significant relation among fragile sites, clinical and histological findings has been detected so far.

Chromosome Aberrations↗

PubMed1992

CA074 methyl ester: a proinhibitor for intracellular cathepsin B.

The specificity of compound CA074 [N-(L-3-trans-propylcarbamoyloxirane-2-carbonyl)-L-isoleucyl-L-pro line] for the inactivation of cathepsin B was quantified in in vitro measurements with cysteine endopeptidases from cattle, it being found that the compound is a very rapid inactivator of cathepsin B (rate constant 112,000 M-1.s-1), with barely detectable action on cathepsins H, L, and S or m-calpain. Conversion of the proline carboxyl group of the inhibitor to the methyl ester virtually abolished the effect on cathepsin B, and a possible explanation for the importance of the carboxyl is presented on the basis of the tertiary structure of cathepsin B. It was found that CA074 methyl ester (1 microM, 3 h) caused selective inactivation of the intracellular cathepsin B of human gingival fibroblasts in culture, in contrast to other available agents, and we suggest that CA074 methyl ester will be of value in the elucidation of the biological functions of cathepsin B.

Binding Sites↗

PubMed1992

Acute myelocytic leukemia associated with thrombocytosis and inv 3(q21.3; q26.2) in a case of Grönbland-Strandberg syndrome.

Grönbland-Strandberg syndrome is an inherited connective tissue disorder with cutaneous, ophthalmologic, and systemic manifestations. Here we report a case of AML with thrombocytosis arising in a patient with this syndrome. Karyotypic analysis of bone marrow cells revealed the inversion 3 (q21.3;q26.2). To our knowledge, this is the first report of the association of AML with thrombocytosis and Grönbland-Strandberg syndrome. This might be a coincidental association, but it suggests some interesting speculations regarding the pathogenesis of these diseases.

Chromosome Inversion↗

PubMed1992

Two chicken monoclonal antibodies specific for heterophil Hanganutziu-Deicher antigens.

Two chicken monoclonal antibodies (MAbs), HU/Ch2-7 and HU/Ch6-1, against heterophil Hanganutziu-Deicher (HD) antigens with N-glycolylneuraminic acid (NeuGc) at a terminal carbohydrate were established by cell fusions using chicken B cell lines lacking thymidine kinase and spleen cells from chickens immunized with II3NeuGc alpha-LacCer (HD3). The reactivities of these MAbs against several gangliosides including NeuGc-containing glycosphingolipids were examined by a thin-layer chromatography/immunostaining method. MAb HU/Ch2-7 (IgG) reacted strongly with HD3 and IV3NeuGc alpha-nLc4Cer (HD5) and weakly with VI3NeuGc alpha-nLc6Cer (HD7) and 4-O-acetyl-HD3. HU/Ch6-1 (IgG) reacted with HD3 and HD5, but did not react with the other HD antigens. The reactivities of these MAbs against HD antigen were greatly reduced by pre-treatment of the antigen with neuraminidase. These MAbs did not react with N-acetylneuraminic acid-containing gangliosides (GM1 and GM3). These results indicate that these two chicken MAbs are directed toward the antigenic epitope containing the NeuGc.

Animals↗

PubMed1992

Interaction of the Golgi membranes isolated from rabbit liver with microtubules in vitro.

We have developed a reconstituted model system to study the interaction of the Golgi membranes isolated from rabbit liver with taxol-stabilized bovine-brain microtubules without microtubule-associated proteins (MAPs). The Golgi membranes are associated with microtubules. The sheets of vesicles and the membranous tubules are observed along microtubules by direct visualization using differential-interference-contrast, dark field, or fluorescence microscopy. The monoclonal antibody against Golgi membranes suggests that the Golgi membranes, but not the contaminating vesicles, are interacting with microtubules. The degree of association is assayed quantitatively using rhodamine-labeled microtubules after separation of the complex from unbound microtubules by centrifugation upon sucrose gradient. The association is inhibited by crude MAPs, purified MAP2, or 1.0 mM ATP. However, the association neither requires the cytosol from rat liver or bovine brain nor N-ethylmaleimide, brefeldin A, or GTP-gamma-S. The association is mediated by trypsin-sensitive peripheral protein(s) on the Golgi membranes.

Adenosine Triphosphate↗

PubMed1992

Comparison of bone matrix-derived bone morphogenetic proteins from various animals.

Bone matrix-derived bone morphogenetic protein (BMP) was extracted from bovine, porcine, rabbit, Sprague-Dawley rat, and Wistar rat bone and purified. The purified fractions all had similar molecular weights and induced new bone in 3 weeks when implanted into muscle pouches of Wistar rats. Bone matrix-derived BMP is believed to consist of subunits and that of different animal origin to contain the same fraction with BMP activity.

Alkaline Phosphatase↗

PubMed1992

Detailed mapping around the breakpoint of (3;8) translocation in familial renal cell carcinoma and FRA3B.

As a critical step toward cloning the breakpoint of the (3;8) translocation observed in familial renal cell carcinoma and a common fragile site on chromosome 3p (FRA3B), we have characterized the 3p14 region containing the breakpoint and FRA3B by fluorescence in situ hybridization (FISH), pulsed-field gel electrophoresis (PFGE), and genetic linkage analysis. Of 23 cosmids mapped by FISH, 14 cosmids were distal to the breakpoint of t(3;8) and 9 were proximal. Analyses of FRA3B by FISH were identical to those for the (3;8) breakpoint and indicated that the breakpoint of t(3;8) occurred at or very close to the fragile site. We have also constructed a genetic linkage map and a preliminary long-range restriction map using PFGE of the 3p14 region. The linkage results were consistent with the physical data. The combined results of FISH, PFGE, and linkage analysis establish a basis for further experiments to clone the (3;8) breakpoint and FRA3B.

Animals↗