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H Hirano

Publications and source records attributed to H Hirano.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of GLUT3 protein expressed in Chinese hamster ovary cells.

We have expressed GLUT3 protein, an isoform of a facilitative glucose transporter, in Chinese hamster ovary cells by transfection of its cDNA using an expression vector. The expressed GLUT3 protein was detected by Western-blot analysis as a broad band of 45-65 kDa, indicating intensive glycosylation of the protein. The expressed GLUT3 protein was observed, by immunofluorescence staining, to be located mainly at the plasma membrane, and its expression was associated with a marked increase in glucose-transport activity. Kinetic analysis revealed that the Km value of GLUT3 protein for 3-O-methylglucose uptake was approx. 35% of that of GLUT1 protein, whereas the Km value of GLUT3 protein for 2-deoxy-D-glucose uptake was very similar to that of GLUT1 protein. The Vmax. value of GLUT3 protein for 3-O-methylglucose and 2-deoxyglucose uptake was approx. 20-50% of that of GLUT1 protein. GLUT3 protein was well photolabelled with [3H]cytochalasin B or a mannose derivative, 2-N-4-[3H](1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-(D-mannos -4-yloxy)-2- propylamine. Thus GLUT3 protein has very similar characteristics to GLUT1 protein including its subcellular localization, but exhibits lower Km and Vmax. values for 3-O-methylglucose uptake.

3-O-Methylglucose↗
PubMed1992

CD45 expression by B cells. Expression of different CD45 isoforms by subpopulations of activated B cells.

To determine the effect of distinct activation stimuli on CD45 expression by B cells, we have examined the expression of CD45 molecules on murine B cells stimulated with LPS or the Th cell cytokine IL-5. Analysis of CD45 by flow cytometry revealed that unstimulated and stimulated B cells expressed homogeneous amounts of total CD45 but that stimulation with IL-5 resulted in a CD44hi, hyaluronate-adherent subpopulation of activated B cells that expressed a markedly altered pattern of expression of exon-specific CD45R or B220 determinants. The predominant CD45 immunoprecipitated from either unstimulated or LPS-stimulated B cells was of the high molecular mass form (approximately 220 kDa) usually associated with B cells. In contrast, the CD45 proteins immunoprecipitated from the hyaluronate-adherent subpopulation of IL-5-activated B cells were predominantly lower m.w. forms. PCR analysis of amplified CD45 cDNA also showed distinct expression profiles characteristic of each B cell population. The highest molecular size PCR product, corresponding to expression of all three variably expressed CD45 exons (A, B, and C) was prominent in resting B cells and in LPS-activated B cells but was selectively reduced in hyaluronate-adherent IL-5-activated B cells, where lower molecular size PCR products predominated, corresponding to expression of one or two of the variable exons. In contrast, LPS-activated B cells expressed reduced levels of these one- or two-exon forms. In addition, all B cell populations expressed a lower m.w. PCR product corresponding in size to the product expected when exons A, B, and C are spliced out of CD45 mRNA. Thus, analysis of alternative splicing of CD45 mRNA, as well as cell surface expression of CD45 provides a novel parameter for analysis of B cell activation by different stimuli.

Animals↗
PubMed1992

[Clinical evaluation of lumbosacral nerve root and lateral stenosis using coronal MR imaging].

MR imaging is being used more frequently to study the lumbar spine and is becoming the modality of choice in the assessment of patient with low back pain. Using a new technique of coronal and half coronal scan with MR imaging, it was possible to visualize L4, L5 and S1 nerve roots accurately. We described the MR findings of lateral stenosis using this technique. Several characteristic MR findings were identified, and the most important one was nerve root impingement in the intervertebral foramen. We consider that coronal and half coronal scan with MR imaging is useful in diagnosis of lateral stenosis.

Adult↗
PubMed1992

Domains responsible for the differential targeting of glucose transporter isoforms.

Facilitative glucose transporter isoforms, GLUT1 and GLUT4, have different intracellular distributions despite their very similar structure. In insulin-responsive tissues such as adipose tissues and muscle, GLUT4 protein resides mainly in the intracellular region in a basal condition and is translocated to the plasma membrane upon stimulation of insulin. In contrast, GLUT1 protein was distributed about equally between plasma membranes and low density microsomal membranes in 3T3-L1 adipocytes. Furthermore, GLUT1 and GLUT4 were reported to be differentially targeted to the plasma membrane and intracellular region, respectively, when expressed in Chinese hamster ovary cells and HepG2 cells. To elucidate the differential intracellular targeting mechanisms, several chimeric glucose transporters in which portions of GLUT4 are replaced with corresponding portions of GLUT1 have been stably expressed in Chinese hamster ovary cells. Immunofluorescence and immunoelectron microscopy as well as measurement of glucose transport activity revealed that two domains of GLUT4, which are not the NH2- or COOH-terminal domain, determine its targeting to the intracellular vesicles. The first domain contains the consensus sequence of the leucine zipper structure, suggesting that a dimer-forming structure of the glucose transporter might be required for its proper targeting. The other domain contains 28 amino acids, nine of which are different between GLUT1 and GLUT4. Immunoelectron microscopy revealed that the chimeric transporters containing both of these two domains of GLUT1, only the first domain of GLUT1, and none of the domains, exhibited a different cellular distribution with approximately 65, 30, and 15% of the transporters apparently on the plasma membrane, respectively. The addition of insulin did not alter the apparent cellular distributions of these chimeric transporters. These domains would be specifically recognized by intracellular targeting mechanisms in Chinese hamster ovary cells.

Animals↗
PubMed1992

Molecular cloning and bacterial expression of cDNA encoding a plant cysteine synthase.

Cysteine synthase (CSase) [O-acetyl-L-serine acetate-lyase (adding hydrogen sulfide), EC 4.2.99.8] catalyzes the formation of L-cysteine, the key step in sulfur assimilation in plants, from O-acetyl-L-serine and hydrogen sulfide. We report here the isolation and characterization of cDNA clones encoding cysteine synthase from spinach (Spinacia oleracea L.). Internal peptide sequences were obtained from V8 protease-digested fragments of purified CSase. A lambda gt10 cDNA library was constructed from poly(A)+ RNA of young green leaves of spinach. Screening with two synthetic mixed nucleotides encoding the partial peptide sequences revealed 19 positively hybridized clones among 2 x 10(5) clones. Nucleotide sequence analysis of two independent cDNA clones revealed a continuous open reading frame encoding a polypeptide of 325 amino acids with a calculated molecular mass of 34,185 Da. Sequence comparison of the deduced amino acids revealed 53% identity with CSases of Escherichia coli and Salmonella typhimurium. Sequence homology was also observed with other metabolic enzymes for amino acids in bacteria and yeast and with rat hemoprotein H-450. A bacterial expression vector was constructed and could genetically complement an E. coli auxotroph that lacks CSases. The accumulation of functionally active spinach CSase in E. coli was also demonstrated by immunoblotting and assaying enzymatic activity. Southern hybridization analysis showed the presence of two to three copies of the cDNA sequence in the genome of spinach. RNA blot hybridization suggested constitutive expression in leaves and roots of spinach.

Amino Acid Sequence↗
PubMed1992

An osteonectin-like protein in the matrix of cultured osteogenic cell-line MC3T3-E1, which is associated with calcification.

Time-dependent changes of the [3H]-proline-labeled noncollagenous proteins synthesized by the osteogenic cell-line MC3T3-E1 were analyzed over a range starting from cell confluency to 13 days after confluency during which time cells formed a bone-like structure. It was found that the 40 kDa protein on SDS-polyacrylamide gel (SDS-PAGE) remarkably increased in the cell-matrix layer at about 9 days after cell confluence, just before calcification. This protein was highly purified and was found to contain high amounts of glutamic acid, glycine, and serine. An internal amino acid sequence of this protein was revealed to be K-X-M-A-P-E-E-X-P, which showed homology with the sequence of the EF-hand domain in osteonectin/SPARC (secreted protein, acidic, and rich in cysteine). This protein co-migrated with collagen in gel filtration, and ion-exchange chromatography. Furthermore, it showed high affinity to type I collagen.

Amino Acid Sequence↗
PubMed1992

Immunohistochemical localization of Na(+)-dependent glucose transporter in rat jejunum.

Glucose is actively absorbed via a Na(+)-dependent active glucose transporter (Na-GT) in the small intestine. We raised a polyclonal antibody against the peptide corresponding to amino acids 564-575 of rabbit intestinal Na-GT, and localized it immunohistochemically in the rat jejunum. By means of immunofluorescence staining, Na-GT was located at the brush border of the absorptive epithelial cells of the intestinal villi. Electron-microscopic examination showed that Na-GT was localized at the plasma membrane of the apical microvilli of these cells. Little Na-GT was found at the basolateral plasma membrane. Along the crypt-villus axis, all of the absorptive epithelial cells in the villus were positive for Na-GT. In addition to the brush border staining, the supranuclear positive staining, which was shown to be the Golgi apparatus by use of electron microscopy, was seen in cells located between the base to the middle of the villus. Cells in crypts exhibited little or no staining for Na-GT. Goblet cells scattered in the intestinal epithelium were negative for Na-GT staining. These observations show that Na-GT is specific to the apical plasma membrane of the absorptive epithelial cells, and that the onset of Na-GT synthesis may occur near the crypt-villus junction.

Animals↗
PubMed1992

Localization of erythrocyte/HepG2-type glucose transporter (GLUT1) in human placental villi.

The syncytiotrophoblast covering the surface of the placental villi contains the machinery for the transfer of specific substances between maternal and fetal blood, and also serves as a barrier. Existence of a facilitated-diffusion transporter for glucose in the syncytiotrophoblast has been suggested. Using antibodies to erythrocyte/HepG2-type glucose transporter (GLUT1), one isoform of the facilitated-diffusion glucose transporters, we detected a 50 kD protein in human placenta at term. By use of immunohistochemistry, GLUT1 was found to be abundant in both the syncytiotrophoblast and cytotrophoblast. Endothelial cells of the fetal capillaries also showed positive staining for GLUT1. Electron-microscopic examination revealed that GLUT1 was concentrated at both the microvillous apical plasma membrane and the infolded basal plasma membrane of the syncytiotrophoblast. Plasma membrane of the cytotrophoblast was also positive for GLUT1. GLUT1 at the apical plasma membrane of the syncytiotrophoblast may function for the entry of glucose into its cytoplasm, while GLUT1 at the basal plasma membrane may be essential for the exit of glucose from the cytoplasm into the stroma of the placental villi. Thus, GLUT1 at the plasma membranes of syncytiotrophoblast and endothelial cells may play an important role in the transport of glucose across the placental barrier.

Biological Transport↗
PubMed1992

Changes in lectin binding patterns of Leydig cells during fetal and postnatal development in mice.

Changes in the lectin binding of mouse Leydig cells during fetal and postnatal development were examined by light- and electron-microscopy using eight different biotinylated lectins (ConA, WGA, RCA-I, UEA-I, GS-I, PNA, SBA and GS-II). At the light-microscopic level, ConA, WGA, RCA-I, UEA-I and GS-I showed the same binding pattern in which all five lectins bound to the plasma membrane and cytoplasm of Leydig cells from the 13th day post coitum (p.c.) to the 8th postnatal week. PNA, SBA and GS-II reactions were positive in the plasma membrane and cytoplasm of Leydig cells from the 13th day p.c. to 15th day post partum (p.p.) but disappeared completely by day 20. At the electron-microscopic level, gold particles representing the GS-I or GS-II binding sites were distributed primarily along the cell surface membrane, including that of microvilli, as well as in the cytoplasm. These results indicate that certain glycoconjugates bearing D-galactose, N-acetyl-D-galactosamine, and N-acetyl-D-glucosamine residues are expressed on the cell surface and in the cytoplasm of Leydig cells during the period from the 13th day p.c. to around the 20th day p.p. The results suggest that these glycoconjugates might play some role in modulating hormone-receptor interaction in the Leydig cells before the 20th day. Furthermore, these results may indicate that sugar residues expressed on the cell surface and in the cytoplasm of Leydig cells are different from those in the fetal-neonatal and adult phases.

Animals↗
PubMed1992

The relation between amniotic fluid surfactant concentration in preterm labour and histological evidence of chorioamnionitis.

Concentration of amniotic fluid disaturated phosphatidylcholine (DSPC), factors related to cervical ripening, and histopathological evidence of chorioamnionitis were studied in 38 patients in preterm labour with intact membranes; all of them delivered spontaneously before 37 weeks. There was no correlation between the amniotic fluid DSPC level and gestational age at the time of amniocentesis. However, a significant inverse correlation was found between the amniotic fluid DSPC level and the interval between the onset of labour and delivery. The amniotic fluid DSPC level in cases with onset-delivery interval of less than 48 h was significantly higher than that in cases with an onset-delivery interval of 48 h or more. The gestational age in the former group was significantly lower than in the latter (28.6 vs 32.0 weeks). The amniotic fluid DSPC level in the patients with chorioamnionitis was significantly higher than that in the patients without chorioamnionitis, although the gestational age did not differ between the two groups. All 3 infants with RDS were associated with cervical incompetence. Patients in preterm labour with chorioamnionitis may be refractory to tocolysis and have higher amniotic fluid surfactant levels.

Adult↗
PubMed1992

Identification of Tetrahymena 14-nm filament-associated protein as elongation factor 1 alpha.

Tetrahymena 14-nm filament-forming protein has dual functions as a citrate synthase in mitochondria and as a cytoskeletal protein involved in oral morphogenesis and in pronuclear behavior during conjugation. By immunoblotting using monoclonal and polyclonal antibodies following two-dimensional gel electrophoresis, we demonstrated that the 14-nm filament protein fraction contained two 49-kDa proteins whose isoelectric points were 8.0 and 9.0; a monoclonal antibody (MAb) 26B4 and a polyclonal antibody 49KI reacted only to a pI 8.0 protein, while two other MAbs, 11B6 and 11B8, reacted only to a pI 9.0 protein. From the N-terminal amino acid sequences, the pI 8.0 protein was identified as the previously reported 14-nm filament-forming protein/citrate synthase, but the pI 9.0 protein N-terminal sequence had no similarity with that of the pI 8.0 protein. The pI 9.0 protein is considered to be a 14-nm filament-associated protein since the pI 9.0 protein copurifies with the pI 8.0 protein during two cycles of an assembly and disassembly purification protocol. Cloning and sequencing the pI 9.0 protein gene from a Tetrahymena pyriformis cDNA library, we identified the pI 9.0 protein as elongation factor 1 alpha (EF-1 alpha) based on it sharing 73-76% sequence identity with EF-1 alpha from several species.

Actin Cytoskeleton↗
PubMed1992

Changes in expression of the endogenous beta-galactoside-binding 14-kDa lectin of chick embryonic skin during epidermal differentiation.

Changes in the expression pattern of the gene for the endogenous beta-galactoside-binding 14-kDa lectin of chick embryo were examined immunohistochemically during epidermal differentiation in vivo and in vitro with special reference to detailed localization of the 14-kDa lectin. The gene expression was visualized by the HRP-staining method following in situ hybridization, in which sulfonated cDNA was employed as a probe. The 14-kDa lectin gene expression (mRNA) was detected mainly in the intermediate layer of the epidermis: it was faint in 13-day-old embryos, gradually increased in intensity during epidermal differentiation, and became intensely positive in 17-day-old embryo. The expression of the gene in skin explants was suppressed by vitamin A, which induces mucous metaplasia of the epidermis in vitro. The anti-14-kDa lectin reaction was positive mainly in the intermediate layer of the differentiating epidermis, coinciding chronologically with expression of the gene at the light microscopic level. Immunoelectron microscopy revealed that the positive reaction was primarily localized in desmosomes, in tonofilament bundles anchored to the desmosomes, along the outer surface of the plasma membrane, and in the intercellular space. Essentially the same staining pattern was observed in differentiating epidermis in vitro. The positive reaction was markedly reduced in the epidermis in which differentiation had been suppressed in vitro by the addition of vitamin A.

Animals↗
PubMed1992

Characterization of proteins released from legume seeds in hot water.

When immersed in water at 50-60 degrees, mature soybean seeds release a large amount of protein. The major protein released was basic 7S globulin (Bg), which is present in the cotyledons of soybean seeds. The released Bg consisted of the 27,000 and 16,000 subunits which were linked by disulphide bonding and glycosylated. The released Bg exhibited an identical structure with the mature Bg which was synthesized in the normal developing seeds. Proteins like Bg were also found to be released into hot water from the seeds of legume species such as azuki-bean, cowpea, mung-bean and winged-bean. Besides Bg and Bg-like proteins, a few proteins including the 9,000 hydrophobic protein in soybean, ubiquitin in cowpea and mung-bean, and Kunitz trypsin inhibitor in winged-bean, were released from the seeds in hot water.

Amino Acid Sequence↗
PubMed1992

Rice seed globulin: a protein similar to wheat seed glutenin.

The globulin of the seed endosperms of rice has an isoelectric point of 6.4 and a M(r) of 26,000. There is one intra-disulphide bond, but no N-linked oligosaccharide chain. The amino acid sequence of the N-terminal and internal regions of the globulin was determined and found to be homologous with that of glutenin, the storage protein in the seed endosperms of wheat. Rice globulin and wheat glutenin were rich in glycine, and glutamic acid or glutamine, and in addition, wheat glutenin cross-reacted with antibody raised against rice globulin. These results suggest that rice seed globulin represents a protein similar to wheat seed glutenin.

Amino Acid Sequence↗
PubMed1992

Electrophysiological study of four patients with Fisher syndrome: a mechanism of areflexia.

Electrophysiological studies were performed on four patients with the Fisher syndrome (two 4-year-old boys, a 5-year-old boy and a 9-year-old girl). Motor nerve conduction velocity (MNCV), nerve action potential, F wave, H reflex and T reflex were measured at the stage when areflexia was present but ataxia and ophthalmoplegia had recovered. MNCV and SNCV by compound mixed nerve potential were normal in all our patients. The amplitude of the muscle responses (M-responses) and compound mixed nerve action potentials were also within normal limits. The latencies of the F waves and F wave conduction velocities were normal. However, H-reflexes and T-reflexes were absent in all four cases. Our results suggested that areflexia in the Fisher syndrome is attributed to the desynchronization of the impulses or the partial conduction block of GIa fibers.

Ataxia↗
PubMed1992

Comparison of Ki-ras gene mutation among simultaneously occurring multiple urethan-induced lung tumors in individual mice.

Mouse lung tumors were induced in C57BL/6J(female) x A/J(male) F1 mice by a single s.c. injection of urethan. About 6 months later, multiple small-sized lung tumors were detectable in almost all mice. After a further 6 months, some of these tumors became larger than the rest. We examined whether there were any mutational differences among multiple lung tumors in a single mouse. Direct DNA sequencing of a separately amplified Ki-ras gene by polymerase chain reaction (PCR) was carried out with 25 DNA samples from multiple tumors in four mice. Twenty-four of 25 tumors (96%) had mutations at the codon 61 of the Ki-ras gene. The major mutations involved were either AT to GC transition (44%) or AT to TA transversion (44%) at the second base of codon 61. We compared the types of these gene mutations among the tumors from each of two mice from two different groups of siblings and then compared the two groups. Interestingly, in the first group of siblings, we detected CTA in 5/6 tumors in the first mouse and again CTA in 4/6 tumors in the second one. In the second group of siblings, we detected CGA in 5/7 tumors in one mouse and CGA again in 3/5 tumors in the second mouse. These results show that the pattern of Ki-ras codon 61 mutations in urethan-induced lung tumors is similar in tumors developing in siblings, suggesting that host factors have an effect on the carcinogen-induced mutational pattern. There was no major mutational difference between small and large tumors. The results suggested that other event(s) in addition to the mutation of the Ki-ras gene might play a role during the development of large-sized tumors.

Amino Acid Sequence↗
PubMed1992

Deblocking and subsequent microsequence analysis of N alpha-blocked proteins electroblotted onto PVDF membrane.

A method was developed for direct microsequencing of N alpha-acetylated proteins electroblotted onto polyvinylidene difluoride membranes from polyacrylamide gels. N alpha-Acetylated proteins (greater than 32 pmol), including horse heart cytochrome c, five mutants of yeast cytochrome c, and bovine erythrocyte superoxide dismutase, were separated by SDS-PAGE and electroblotted onto polyvinylidene difluoride membranes. The portions of the membrane carrying the bands were cut out and treated with 0.5% polyvinylpyrrolidone in acetic acid solution at 37 degrees C for 30 min. The protein was digested on the membrane with 5-10 micrograms of trypsin at 37 degrees C for 24 h. During tryptic digestion, the resultant peptides were released from the membrane and the N-terminal peptide was efficiently deblocked with 50 mU of acylamino acid-releasing enzyme at 37 degrees C for 12 h. Picomole levels of the deblocked proteins could be sequenced directly by use of a gas-phase protein sequencer.

Acetylation↗