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Biomedical subjects

Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 199 records · Page 11Linked to original sources
PubMed1993

Structural, functional and evolutionary implications of the three-dimensional crystal structure of murine interferon-beta.

The crystal structure of recombinant murine interferon-beta as elucidated by Senda et al. (Proc. Jap. Acad. 66B: 77-80 (1990); EMBO J. 11: 3193-3201 (1992)) appears to represent the basic structural framework of all Type I interferons including interferons-beta and all subtypes of interferons-alpha of various mammalian origin. Now the huge accumulated data on the structure-activity relationship of Type I interferons using various chemical and genetic techniques can be systematically evaluated in terms of the three-dimensional structure. Structural comparison with other cytokines, for which three-dimensional structures have been established, including interferon-gamma and considerations on the evolution of cytokines and cytokine receptors are also given.

Amino Acid Sequence↗
PubMed1993

Changes of hemorheologic properties during normal human pregnancy.

To investigate alterations of hemorheologic properties during pregnancy, we measured the hematocrit (Hct) and fibrinogen levels, blood viscosity, and erythrocyte deformability in 10 healthy nonpregnant women and in 10 women with uncomplicated pregnancies. The Hct decreased during pregnancy, while the plasma fibrinogen concentration was elevated. No significant changes in blood viscosity were observed throughout pregnancy. Erythrocyte deformability was significantly decreased in pregnancy when compared to the nonpregnant state, but returned to the nonpregnant level by the 7th postpartum day. These findings suggest that adequate microcirculation is maintained in the uteroplacental unit during gestation by hemodilution, which prevents an elevation of blood viscosity despite the increase in fibrinogen and the decrease of erythrocyte deformability. These changes are thus thought to be among the physiological maternal adaptations to fetal growth and development.

Adult↗
PubMed1993

New mutation of the myelin P0 gene in a pedigree of Charcot-Marie-Tooth neuropathy 1.

P0, the major structural protein of peripheral myelin, is a homophilic adhesion molecule with a single immunoglobulin (Ig) domain, which contains a single N-linked glycosylation site and two cysteines. We have previously reported four different mutations of the myelin P0 gene in four families of Charcot-Marie-Tooth neuropathy type 1 (CMT1). In this study we found a new mutation of the myelin P0 gene in a small family of CMT1. The affected persons had an A - to - G substitution of nucleotide 245 of the myelin P0 gene in one allele, leading to a cysteine substitution for tyrosine82 in the extracellular Ig-domain. An additional cysteine in the extracellular domain may form a disulfide bond and cause an inappropriate change in the tertiary structure of the functional Ig-domain of P0.

Adolescent↗
PubMed1992

P2 purinoceptor-mediated cyclic AMP accumulation in bovine vascular smooth muscle cells.

Extracellular ATP has been shown to induce intracellular Ca2+ mobilization and adenylate cyclase inhibition via P2 purinoceptors in several species of cells. Now we found that in calf vascular smooth muscle cells the addition of ATP to the medium did not induce inhibition but stimulation of cyclic AMP accumulation, in addition to stimulation of inositol phosphate production. Adenosine and AMP also induced cyclic AMP accumulation but their efficacy was much less than that of ATP. The ATP action was not influenced by the presence of either adenosine deaminase or of an ATP regenerating system, whereas the AMP action was increased by the regenerating system. The results indicate that the cyclic AMP accumulation by ATP is due to ATP itself but neither to adenosine nor to AMP, both of which are produced from ATP. ATP receptor coupled to the cyclic AMP generation was shown to be different from that coupled to phospholipase C based on the difference in the potency order of the receptor agonists and in the sensitivity of P2 receptor agonists to 8-cyclopentyl-1,3-dipropylxanthine (CPX)- and suramin-induced antagonism. We conclude that in the aortic smooth muscle cells a novel P2-type receptor directly coupled to adenylate cyclase activation exists in addition to the previously known P2 receptor linked to phospholipase C activation.

Adenosine Triphosphate↗
PubMed1992

Identification of the hepatocyte mitogen in bovine spleen as heparin-binding growth factors.

Growth promoting activity for rat hepatocytes in bovine spleen was identified as three heparin-binding growth factors. All the features tested, such as heparin affinity, molecular mass, cross reactivity with antibody, and partial amino acid sequence, indicated that one of the three factors was identical to FGF-1 (fibroblast growth factor-1, acidic FGF), another one was related to FGF-2 (fibroblast growth factor-2, basic FGF), whereas it was more potent for hepatocytes than the FGF-2 purified from bovine brain. The third one was eluted from heparin-Sepharose column at 0.75M NaCl, of which activity was not abolished by anti-FGF-1 or FGF-2 antibodies. In addition, the mitogenic effect of this factor was synergistic with that of HGF (hepatocyte growth factor), a known potent hepatocyte mitogen, suggesting that it is a novel growth factor for hepatocytes.

3T3 Cells↗
PubMed1992

Crystal and molecular structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus.

The crystal structure of RNase Rh, a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.5 A resolution by the multiple isomorphous replacement method. The crystal structure was refined by simulated annealing with molecular dynamics. The current crystallographic R-factor is 0.200 in the 10-2.5 A resolution range. The molecular structure which is completely different from the known structures of RNase A and RNase T1 consists of six alpha-helices and seven beta-strands, belonging to the alpha+beta type structure. Two histidine and one glutamic acid residues which were predicted as the most probably functional residues by chemical modification studies are found to be clustered. The steric nature of the active site taken together with the relevant site-directed mutagenesis experiments (Irie et al.) indicates that: (i) the two histidine residues are the general acid and base; and (ii) an aspartic acid residue plays a role of recognizing adenine moiety of the substrate.

Amino Acid Sequence↗
PubMed1992

Crystal structure of an engineered subtilisin inhibitor complexed with bovine trypsin.

Proteinase specificity of a proteinaceous inhibitor of subtilisin (SSI; Streptomyces subtilisin inhibitor) can be altered so as to strongly inhibit trypsin simply by replacing P1 methionine with lysine (with or without concomitant change of the P4 residue) through site-directed mutagenesis. Now the crystal structure of one such engineered SSI (P1 methionine converted to lysine and P4 methionine converted to glycine) complexed with bovine trypsin has been solved at 2.6 A resolution and refined to a crystallographic R factor of 0.173. Comparing this structure with the previously established structure of the native SSI complexed with subtilisin BPN', it was found that (i) P1 lysine of the mutant SSI is accommodated in the S1 pocket of trypsin as usual, and (ii) upon complex formation, considerable conformation change occurs to the reactive site loop of the mutant SSI. Thus, in this case, flexibility of the reactive site loop seems important for successfully changing the proteinase specificity through mere replacement of the P1 residue.

Amino Acid Sequence↗
PubMed1992

Secretion of endothelin-1 in human endothelial cell line but not in B cell line by transfection of preproendothelin-1 cDNA.

Stable transformants with preproendothelin-1 (preproET-1) cDNA were established for the study of the regulation of endothelin-1 (ET-1) biosynthesis in human cells. ET-1, a potent vasoconstrictor peptide, is produced by endothelial cells and is secreted into the blood at a low level. Human preproET-1 cDNA was introduced into two immortal human cell lines, t-HUE2, an endothelial cell line, and Raji, a B cell line, with Ecogpt selection. Several stable transformants of t-HUE2 expressed extraordinarily high levels of preproET-1 specific mRNA and secreted ET-1 into serum-free culture medium, while the transformants of Raji cells expressed high levels of ET-1 mRNA, but secreted a negligible amount of ET-1. Immunocytochemical studies of intracellular ET-1 content revealed that there were some defects in the translation or processing of preproET-1 in the B cell line transformants. In addition, the ratio of ET-1 to ET-1 precursor (big ET-1) was much higher in the t-HUE2 transformants than in normal endothelial cells, suggesting that t-HUE2 transformants (for example t-HUE2-1) possess high levels of endothelin converting enzyme (ECE). The establishment of stable transformants producing high levels of ET-1 in serum-free medium will be useful for the study of cell-type-specific translation and processing to mature ET-1, and of the regulatory factors of ECE.

B-Lymphocytes↗
PubMed1992

Identification of a heparin-binding growth factor-1 nuclear translocation sequence by deletion mutation analysis.

We have shown previously that a deletion mutant of human heparin-binding growth factor (HBGF)-1, HBGF-1U, lacking the sequence Asn-Tyr-Lys-Lys-Pro-Lys-Leu is capable of initiating c-fos mRNA expression and polypeptide phosphorylation on tyrosine residues at concentrations that do not induce either DNA synthesis or cell proliferation (1). The fact that addition of the nuclear translocation signal from the yeast histone 2B protein to the HBGF-1U mutant caused reconstitution of the biological activity of HBGF-1 indicated that nuclear translocation may be an important component of the mitogenic signal induced by HBGF-1. In order to examine the nuclear translocation potential of HBGF-1 alpha, the deletion mutant HBGF-1U, and the yeast histone 2B-HBGF-1 chimera, HBGF-1U2, we expressed these forms of HBGF-1 in murine endothelial cells. Western blot and two-dimensional Western blot analysis of cytosol and nuclei demonstrate that although the three forms of HBGF-1 are readily detectable in the cytosol of the individual transfectants, HBGF-1 alpha and HBGF-1U2 but not HBGF-1U was detected in the nucleus. Furthermore, murine endothelial cells expressing HBGF-1 alpha and HBGF-1U2 exhibited an atypical cellular phenotype in vitro that was absent in the HBGF-1U transfectants. These data suggest that HBGF-1 contains a functional nuclear translocation sequence that may be responsible for the initiation of DNA synthesis, and these data further correlate the presence of the nuclear translocation sequence with an abnormal endothelial cell phenotype in vitro.

Animals↗
PubMed1992

Three-dimensional crystal structure of recombinant murine interferon-beta.

The crystal structure of recombinant murine interferon-beta (IFN-beta) has been solved by the multiple isomorphous replacement method and refined to an R-factor of 20.5% against 2.6 A X-ray diffraction data. The structure shows a variant of the alpha-helix bundle with a new chain-folding topology, which seems to represent a basic structural framework of all the IFN-alpha and IFN-beta molecules belonging to the type I family. Functionally important segments of the polypeptide chain, as implied through numerous gene manipulation studies carried out so far, are spatially clustered indicating the binding site(s) to the receptor(s). Comparison of the present structure with those of other alpha-helical cytokine proteins, including porcine growth hormone, interleukin 2 and interferon gamma, indicated either a topological similarity in chain folding or a similar spatial arrangement of the alpha-helices.

Amino Acid Sequence↗
PubMed1992

Crystal growth of ribonuclease S under microgravity.

Expecting that protein crystal growth in space would show us a new aspect of its crystallization process and might result in the improvement of crystal quality, we performed a series of growth experiments for bovine pancreatic ribonuclease S crystals under microgravity. For this purpose, the COSIMA2 system launched in September 1989 was utilized. In one of the eight crystallization chamber setups in the COSIMA2 experiment, a large "Z-form" single crystal appeared and we found that (a) the space-grown single crystal exhibited a higher fraction of significant X-ray diffraction spots than ground-grown crystals of approximately the same size and (b) the corresponding Wilson plots also tended to show a slightly lower overall temperature factor for the space-grown crystals, although the quality of the plot was not sufficient for quantitative evaluation. Thus, mainly based on the above observation (a), it is most likely that the internal order of the ribonuclease S crystal was in fact improved through the crystallization under microgravity.

Crystallization↗
PubMed1992

Effect of heparin on pulmonary fibroblasts and vascular cells.

BACKGROUND: There is a large increase in mast cell numbers in fibrotic lung tissue, suggesting that mast cells may play a part in the pathogenesis of pulmonary fibrosis. Glycosaminoglycans, such as heparan sulphate, that are structurally related to heparin (a mast cell product) are part of the extracellular matrix and known to regulate cell growth. Basic fibroblast growth factor is a heparin binding growth factor produced by endothelial cells. METHODS: A study was carried out to examine the effect of heparin, basic fibroblast growth factor, and mast cell products on the proliferation of normal human lung fibroblasts and the effect of adding heparin on the proliferation of lung fibroblasts and pulmonary vascular cells incubated with basic fibroblast growth factor. RESULTS: Heparin at low concentration (0.03, 0.3-1.0 micrograms/ml) stimulated the proliferation of normal human lung fibroblasts in culture whereas a higher concentration (100 micrograms/ml) had an inhibitory effect. Mast cell products also stimulated the proliferation of fibroblasts, and the effect was decreased by pretreatment with heparinase or protamine. Heparin enhanced the growth of both fibroblasts and pulmonary vascular cells induced by low concentrations of basic fibroblast growth factor. CONCLUSIONS: Mast cells in fibrotic lung tissue may regulate fibroblast proliferation by releasing heparin. These results suggest that endothelial cells may interact with mast cells and modulate fibroblast growth by release of basic fibroblast growth factor.

Animals↗
PubMed1992

Developmental changes of glutamate dehydrogenase activity in rat liver mitochondria and its enhancement by branched-chain amino acids.

The developmental changes of glutamate dehydrogenase activity in the fetal and neonatal rat liver were investigated, as well as the effects of branched-chain amino acids on this enzyme. Hepatic glutamate dehydrogenase activity showed a marked increase at the end of the fetal period and peaked on the 5th day of neonate at approximately 3 times higher than the adult level. Glutamate dehydrogenase was activated by leucine, isoleucine, and valine in this order when they were added to isolated intact liver mitochondria in vitro. The enhancement of enzyme activity was more marked in fetal rats than in adults. In contrast, when branched-chain amino acids were added after disrupting the mitochondrial membrane by sonication, only leucine slightly activated glutamate dehydrogenase, while isoleucine and valine slightly inhibited its activity. Our findings suggest that glutamate may be actively synthesized in the developing rat liver mitochondria and then transaminated to other nonessential amino acids for protein synthesis, and that increased intramitochondrial branched-chain amino acid concentrations may enhance glutamate dehydrogenase activity. This anabolic metabolism will contribute to the fetal growth and development.

Amino Acids, Branched-Chain↗
PubMed1992

Development changes of polyamine biosynthesis in rat liver.

To investigate the role of polyamine metabolism in fetal development, we determined the hepatic polyamine content and the activity of enzymes related to polyamine metabolism in fetal, newborn and adult rats. In the fetuses, spermidine was detected at the highest level, followed by spermine and then putrescine. Putrescine and spermidine levels were higher in the fetuses than in adult rats, while the spermine level was significantly lower in the fetuses. The activities of the enzymes investigated were also higher in the fetuses than in adult rats. These findings indicate that polyamine synthesis was enhanced in the fetal liver, and that degradation of spermidine via spermidine N1-acetyltransferase contributes to the increase in putrescine levels. Thus, our study suggests that an increase in putrescine is important for fetal growth and that putrescine per se has a significant role in fetal cellular proliferation.

Acetyltransferases↗
PubMed1992

Histamine decreases the permeability of an endothelial cell monolayer by stimulating cyclic AMP production through the H2-receptor.

To determine if histamine acts directly on the vascular endothelium, the effect of histamine on the permeability of cultured human endothelial cell monolayers and the role of second messengers were examined. The addition of 10(-6) to 10(-4) M histamine to the culture medium decreased the endothelial cell monolayer permeability and increased both cyclic AMP and free-calcium levels. The decrease in permeability and the increase in cyclic AMP mediated by histamine were prevented by an H2-blocker (famotidine) while the increase in free-calcium was inhibited by an H1-blocker (diphenhydramine). These results suggest that histamine decreases the permeability of endothelial cell monolayers through the H2-receptor, and cyclic AMP plays a more important role than calcium ion as a second messenger.

Animals↗
PubMed1992

Inhibitory action of dilazep on histamine-stimulated cytosolic Ca2+ increase in cultured human endothelial cells.

Using a fluorescent Ca(2+)-sensitive dye, fura-2, and photometric fluorescence microscopy, we measured changes in cytosolic Ca2+ concentration ([Ca2+]i) in cultured human endothelial cells and studied the effect of dilazep on [Ca2+]i elevation induced by histamine. Histamine (1 microM) caused a rapid transient peak in the average [Ca2+]i of a group of cells (approximately 10(2) cells), followed by a decrease to a sustained elevation. Dilazep as well as diltiazem (1.0 to 100 microM) concentration-dependently inhibited the latter sustained elevation, which was eliminated by removal of extracellular Ca2+, while the initial transient response was not changed by dilazep at concentrations up to 100 microM. The IC50 values of dilazep and diltiazem were 16 and 58 microM, respectively. The patterns of the [Ca2+]i elevation responses to histamine were variable among individual cells. Some single cells showed a transient peak and a sustained elevation as observed in a group of cells. Some single cells caused a repetitive spikelike elevation of [Ca2+]i. Dilazep lowered the sustained elevation to the resting level and in some single cells, changed the sustained elevation to the spikelike elevation. The frequency of the spikelike [Ca2+]i elevation was also decreased by dilazep. Decrease in extracellular [Ca2+] showed the same pattern of inhibitory actions as dilazep did. These results indicate that dilazep inhibits the extracellular Ca2+ influx in endothelial cells.

Calcium↗
PubMed1992

Changes in plasma human atrial natriuretic peptide (hANP) level in normal pregnancy and pregnancy induced hypertension.

We determined plasma human atrial natriuretic peptide (hANP) levels in normal pregnancy and pregnancy induced hypertension (PIH). The plasma hANP levels slightly decreased in the first trimester of normal pregnancy and tended to recover as pregnancy advanced, although these changes were slight. However, the plasma hANP level in puerperium was higher than that in the third trimester of normal pregnancy. The plasma hANP level in mild PIH was not significantly higher than that in the third trimester of normal pregnancy. In contrast, the plasma hANP level in three cases of severe PIH was approximately 200% higher than those in the normal third trimester and mild PIH.

Atrial Natriuretic Factor↗