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Biomedical subjects

Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 127 records · Page 7Linked to original sources
PubMed1996

Nerve growth factor rescues PC12 cells from apoptosis by increasing amount of bcl-2.

Nerve growth factor (NGF) suppressed the decrease in number of viable PC12 cells after serum withdrawal from culture medium. Accordingly, the amount of bcl-2, a suppressive effector of apoptosis, increased in these cells. Bcl-2 antisense oligonucleotide suppressed not only the NGF-induced increase in bcl-2 but also NGF-induced neuronal differentiation. Results of fluorescent DNA staining indicated that NGF inhibited the chromatin condensation of PC12 cells resulting from serum withdrawal and further that the bcl-2 antisense oligonucleotide canceled this effect of NGF. The present results suggest that NGF rescues PC12 cells from apoptosis induced by serum withdrawal via up-regulation of bcl-2.

Animals↗
PubMed1996

A highly expressed 81 kDa protein in immortalized mouse fibroblast: its proliferative function and identity with ezrin.

An 81 kDa protein was found to be highly expressed in spontaneously immortalized CD1-ICR mouse fibroblasts, RS-4, as compared to normal fibroblasts. RS-4 cells have a reduced serum requirement and exhibit multilayered growth in vitro but are not tumorigenic. The protein was purified from RS-4 cell extracts and used to obtain a polyclonal antibody that specifically immunoprecipitated an 81 kDa protein from cell lysates. Immunocloning of its cDNA and sequence analysis revealed its identity with ezrin, an F-actin binding protein that is a component of the cortical cytoskeleton. Microinjection of the purified IgG fraction of the anti-p81 antiserum into the cytoplasm of RS-4 cells blocked their entry into S phase suggesting that the protein has a proliferative function. Immunostaining of normal mouse tissues showed that the expression of p81/ezrin was highest in proliferating cell populations. Unlike RS-4, NIH3T3 cells exhibit contact inhibition and express levels of p81/ezrin similar to those of normal fibroblasts. When NIH3T3 cells were transfected with p81/ezrin cDNA they lost contact inhibition and thus resembled RS-4 cells. The study demonstrates a proliferative function of p81/ezrin and suggests its involvement in pathways that negatively regulate contact inhibition.

3T3 Cells↗
PubMed1996

Modulation of hemopoietic factor production in relation to endothelial cell aging by interleukin-1 induction.

In this study, we examined the modulation of hemopoietic factor production by human umbilical vein endothelial cells in relation to aging and the cell cycle under conditions of interleukin-1 (IL-1) induction and noninduction. Under conditions of IL-1 noninduction, messenger RNA expression levels of macrophage colony-stimulating factor (M-CSF) were three times higher in non-S-phase cells of young cultures than those in S-phase cells. Expression levels decreased in non-S-phase cells of old culture and approached levels similar to that of S-phase cells. The expression of neither E-selectin nor erythropoietin (Epo) was detected in cells from the noninduced state. The expression of granulocyte colony-stimulating factor (G-CSF) was not affected by either cellular aging or the cell cycle; however, the amount of product secreted increased significantly in old cells, suggesting that G-CSF production is under posttranscriptional regulation. Under conditions of IL-1 induction G-CSF and M-CSF expression levels were enhanced in both young and old cells. Expression of Epo was not detected whereas E-selectin was induced. Significant M-CSF product was detected in young cells but not in old cells, whereas G-CSF product increased dramatically in both types of cells. The modulation of these factors is discussed in relation to the maintenance of neutrophil concentration, differentiation, and maturation of leukocytes and their possible effect on atherosclerosis.

Base Sequence↗
PubMed1996

Genetic differences between the pancytosolic and perinuclear forms of murine mortalin.

To determine the genetic relation between the pancytosolic (uniformly distributed in cytoplasm-p66mot-1) and the perinuclear (p66mot-2) mortalin proteins found in normal and immortal mouse cells, respectively, we initiated the present study using PCR cloning, sequencing, and single nucleotide primer extension analyses. The results indicate that the difference between mot-1 and mot-2 at 2 bp in the open reading frame does not arise by mutations in the immortalized cells. Intriguingly, both mot-1 and mot-2 sequences were detected in genomic DNA from normal (CMEF) and immortal (RS-4) cells derived from the CD1-ICR mouse strain. We extended the analyses to four other strains of mouse and detected both mot-1 and mot-2 sequences in genomic DNA from the Balb/c mouse also. Swiss and C57BL/6 mice exhibited only mot-2 and the C3H He mouse only mot-1. Southern analyses on a mouse strain that showed the presence of only mot-2 (Swiss) and one that showed both mot-1 and mot-2 (CD1-ICR) by PCR revealed that there are structural differences between mot-1 and mot-2 loci. Furthermore, there appears to be a correlation between frequency of spontaneous immortalization of cultured mouse fibroblasts and the mot loci present in the mouse strain from which they were derived.

Animals↗
PubMed1996

Sequence and neuronal expression of mouse endothelin-1 cDNA.

We have isolated and sequenced a cDNA that encodes mouse endothelin-1 (ET-1). The putative protein contains 202 amino acids corresponds to the prepro-form of ET-1. Twenty-one amino acids sequence of the putative mature ET-1 was identical with that of rat, porcine, bovine, and human. In situ hybridization histochemistry indicate that ET-1 mRNA was expressed in several hypothalamic nuclei including the suprachiasmatic nucleus (SCN) in rodent brain.

Amino Acid Sequence↗
PubMed1996

Alterations in transcription factor-binding activities to fibronectin promoter during aging of vascular endothelial cells.

Previously, we showed that the expression of the fibronectin (FN) gene is enhanced during aging of human endothelial cells and fibroblasts. To elucidate the mechanism, we explored binding proteins to the FN promoter. The promoter contains sites for the general transcription factors: CAAT-binding transcription factor (CTF), promoter-specific transcription factor-1 (Sp1), and transcription factor-IID (TFIID). The promoter also contains sites for inducible transcription factors, cAMP-responsive element (CRE)-binding protein (CREB), and Activator protein 2 (AP-2). We synthesized 10 different oligonucleotides for these and other potential transcription factor-binding sites. Using these oligonucleotides, we searched for binding proteins in young and old endothelial cells by electrophoretic mobility shift and supershift assays. Our results showed that AP-1 decreased with aging, but Sp1 and CREB1 were unaffected. However, decreased binding activities to CRE at positions -170 and -415 were shown in old cells. This could be explained by the decrease of AP-1 because these CREs bound not only CREB1 but also AP-1. Moreover, we observed that the binding activities of TFIID, CTF, and binding proteins to -40, -120 and -260 regions increased. These differential changes may cause the enhancement of FN expression in senescent cells.

Base Sequence↗
PubMed1996

Crystal structures of the binary and ternary complexes of 7 alpha-hydroxysteroid dehydrogenase from Escherichia coli.

7 alpha-Hydroxysteroid dehydrogenase (7 alpha-HSDH;1 EC 1.1.1.159) is an NAD+-dependent oxidoreductase belonging to the short-chain dehydrogenase/reductase (SDR) 1 family. It catalyzes the dehydrogenation of a hydroxyl group at position 7 of the steroid skeleton of bile acids. The crystal structure of the binary (complexed with NAD+) complex of 7 alpha-HSDH has been solved at 2.3 A resolution by the multiple isomorphous replacement method. The structure of the ternary complex [the enzyme complexed with NADH, 7-oxoglycochenodeoxycholic acid (as a reaction product), and possibly partially glycochenodeoxycholic acid (as a substrate)] has been determined by a difference Fourier method at 1.8 A resolution. The enzyme 7 alpha-HSDH is an alpha/beta doubly wound protein having a Rossmann-fold domain for NAD (H) binding. Upon substrate binding, large conformation changes occur at the substrate binding loop (between the beta F strand and alpha G helix) and the C-terminal segment (residues 250-255). The variable amino acid sequences of the substrate-binding loop appear to be responsible for the wide variety of substrate specificities observed among the enzymes of the SDR family. The crystal structure of the ternary complex of 7 alpha-HSDH, which is the only structure available as the ternary complex among the enzymes of the SDR family, indicates that the highly conserved Tyr159 and Ser146 residues most probably directly interact with the hydroxyl group of the substrates although this observation cannot be definite due to an insufficiently characterized nature of the ternary complex. The strictly conserved Lys163 is hydrogen-bonded to both the 2'- and 3'-hydroxyl groups of the nicotinamide ribose of NAD(H). We propose a new catalytic mechanism possibly common to all the enzymes belonging to the SDR family in which a tyrosine residue (Tyr159) acts as a catalytic base and a serine residue (Ser146) plays a subsidiary role of stabilizing substrate binding.

Amino Acid Sequence↗
PubMed1996

Identification of a 79-kDa heparin-binding fibroblast growth factor (FGF) receptor in rat hepatocytes and its correlation with the different growth responses to FGF-1 between hepatocyte subpopulations.

We reported previously that the potency of heparin-binding fibroblast growth factor-1 (FGF-1) as a mitogen for rat hepatocytes in primary culture is as high as that of epidermal growth factor (EGF) and hepatocyte growth factor. To gain insight into the pathophysiological significance of FGF-1 in hepatocyte growth, we analyzed the cooperative mitogenicity of FGF-1 and EGF. Results from a nuclear labeling assay using [3H]thymidine suggest that most hepatocytes in primary culture consist of two cell populations that differ in response to FGF-1; one is an FGF-1-responsive cell population, and the other is an EGF-responsive (but not FGF-1-responsive) cell population. On the other hand, autoradiographic analysis of 125I-FGF-1 binding demonstrated that high affinity FGF receptors were homogeneously distributed on the surface of all hepatocytes. Cross-linking 125I-FGF-1 to the nonstimulated hepatocyte surface indicated that the high affinity FGF receptors comprise two FGF receptors that differ in molecular mass (128 and 79 kDa). Furthermore, the 79-kDa receptor was preferentially down-regulated when the hepatocytes were stimulated with EGF or hepatocyte growth factor. These data suggest that the abundant expression of the 79-kDa FGF receptor on some populations of hepatocytes is involved in their lack of response to FGF-1. The 128- and 79-kDa FGF receptors were assigned as FGFR2 using an antibody specific to the ectodomain of FGFR2, whereas the 79-kDa receptor was not reactive to the antibody against the carboxyl terminus of FGFR2. This 79-kDa FGF receptor was not tyrosine-phosphorylated in response to FGF-1 stimulation, while the 128-kDa FGF receptor was recognized by anti-phosphotyrosine antibody under the same conditions. Also, the heterodimer of 79- and 128-kDa FGF receptors was less tyrosine-phosphorylated than the homodimer of 128-kDa FGF receptors. These data suggest that the 79-kDa FGF receptor inhibits the function of the 128-kDa FGF receptor through their heterodimerization. Thus, we surmise that the difference in response to FGF-1 between the cell populations of normal rat hepatocytes was caused by the different levels of the 79-kDa FGF receptor in each cell population.

Animals↗
PubMed1996

High-resolution NMR study of a GdAGA tetranucleotide loop that is an improved substrate for ricin, a cytotoxic plant protein.

Ricin is a cytotoxic plant protein that inactivates ribosomes by hydrolyzing the N-glycosidic bond at position A4324 in eukaryotic 28S rRNA. Recent studies showed that a four-nucleotide loop, GAGA, can function as a minimum substrate for ricin (the first adenosine corresponds to the site of depurination). We previously clarified the solution structure of this loop by NMR spectroscopy [Orita et al. (1993) Nucleic Acids Res. 21, 5670-5678]. To elucidate further details of the structural basis for recognition of its substrate by ricin, we studied the properties of a synthetic dodecanucleotide, r1C2U3C4A5G6dA7G8A9U10G11A12G (6dA12mer), which forms an RNA hairpin structure with a GdAGA loop and in which the site of depurination is changed from adenosine to 2'-deoxyadenosine. The N-glycosidase activity against the GdAGA loop of the A-chain of ricin was 26 times higher than that against the GAGA loop. NMR studies indicated that the overall structure of the GdAGA loop was similar to that of the GAGA loop with the exception of the sugar puckers of 6dA and 7G. Therefore, it appears that the 2'-hydroxyl group of adenosine at the depurination site (6A) does not participate in the recognition by ricin of the substrate. Since the 2'-hydroxyl group can potentially destabilize the developing positive charge of the putative transition state intermediate, an oxycarbonium ion, the electronic effect may explain, at least in part, the faster rate of depurination of the GdAGA loop compared to that of GAGA loop. We also show that the amino group of 7G is essential for substrate recognition the ricin A-chain.

Base Sequence↗
PubMed1996

Three-dimensional structures of free form and two substrate complexes of an extradiol ring-cleavage type dioxygenase, the BphC enzyme from Pseudomonas sp. strain KKS102.

The crystal structure of an enzyme having polychlorinated-biphenyl degrading activity, the BphC enzyme from Pseudomonas sp. strain KKS102, has been solved as a free form at 1.8 A resolution. This is the first three-dimensional structure among the extradiol-type dioxygenases. Based on 34,387 reflections (10.0 to 1.8 A, completeness 87.8%), a current R-factor of 20.4% (with a free R-factor of 24.3%) was obtained with a model obeying standard geometry within 0.011 A in bond lengths and 1.91 degrees in bond angles. The BphC enzyme is a homo-octamer and each subunit is composed of two domains: Domain 1 (N-terminal part) and Domain 2 (C-terminal part). Each domain contains two repetitions of a novel folding motif (the "beta alpha beta beta beta" motif) each consisting of ca 55 amino acid residues. A single Fe ion in the active site coordinates the side-chains of three amino acid residues (His145, His209 and Glu260) and two solvent molecules. The coordination geometry is that of a square pyramid. In addition to the free form of the BphC enzyme, we have solved two three-dimensional structures of the BphC enzyme complexed with its substrates, 2,3-dihydroxybiphenyl (2,3-DHBP) or 3-methylcatechol (3-MCT). These substrates were found intact in the active site probably because of the oxidation of the Fe ion into ferric form (as judged by EPR spectra) in the present crystals. In both of the two substrate complexes, the two hydroxyl groups of the substrate, together with the three enzymatic side-chain ligands, were found to form a penta-coordinated system around the Fe ion roughly arranged in a trigonal bipyramidal configuration. The active site structures appear to be essentially consistent with the reaction mechanism proposed so far.

Amino Acid Sequence↗
PubMed1996

The crystal structure of ribonuclease Rh from Rhizopus niveus at 2.0 A resolution.

The three-dimensional structure of ribonuclease Rh (RNase Rh), a new class of microbial ribonuclease from Rhizopus niveus, has been determined at 2.0 A resolution. The overall structure of RNase Rh is completely different from those of other previously studied RNases, such as RNase A from bovine pancreas and RNase T1 from Aspergillus oryzae. In the structure of RNase Rh, two histidine residues (His46 and His109) and one glutamic acid residue (Glu105), which were predicted to be critical to the activity from the chemical modification and mutagenesis experiments, are found to be located close together, constructing the active site. The indole ring of Trp49 plays an important role in preserving the active site structure by its stacking interactions with the imidazole ring of His 109, and by hydrogen bonding with the carboxyl group of Glu105. There exists a hydrophobic pocket around the active site, which contains the aromatic side-chain of Trp49 and Tyr57. The results of mutagenesis studies suggest that this pocket is the base binding site of the substrate.

Amino Acid Sequence↗
PubMed1996

Crystal structure of the ternary complex of mouse lung carbonyl reductase at 1.8 A resolution: the structural origin of coenzyme specificity in the short-chain dehydrogenase/reductase family.

BACKGROUND: Mouse lung carbonyl reductase (MLCR) is a member of the short-chain dehydrogenase/reductase (SDR) family. Although it uses both NADPH and NADH as coenzymes, the structural basis of its strong preference for NADPH is unknown. RESULTS: The crystal structure of the ternary complex of MLCR (with NADPH and 2-propanol) has been determined at 1.8 A resolution. This is the first three-dimensional structure of a carbonyl reductase, and MLCR is the first member of the SDR family to be solved in complex with NADPH (rather than NADH). Comparison of the MLCR ternary complex with three structures reported previously for enzymes of the SDR family (all crystallized as complexes with NADH) reveals a pair of basic residues (Lys17 and Arg39) making strong electrostatic interactions with the 2'-phosphate group of NADPH. This pair of residues is well conserved among the NADPH-preferring enzymes of the SDR family, but not among the NADH-preferring enzymes. In the latter, an aspartate side chain occupies the position of the two basic side chains. The aspartate residue, which would come into unacceptably close contact with the 2'-phosphate group of the adenosine moiety of NADPH, is replaced by a threonine or alanine in the primary sequences of NADPH-preferring enzymes of the SDR family. CONCLUSIONS: The cofactor preferences exhibited by the enzymes of the SDR family are mainly determined by the electrostatic environment surrounding the 2'-hydroxyl (or phosphate) group of the adenosine ribose moiety of NADH (or NADPH). Thus, positively charged and negatively charged environments correlate with preference for NADPH and NADH respectively.

1-Propanol↗
PubMed1996

Crystallization and preliminary X-ray crystallographic studies of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli.

Crystals of 7alpha-hydroxysteroid dehydrogenase from E. coli, which is a tetramer in its active form, have been obtained by a hanging-drop vapor-diffusion method in the presence of the coenzyme NAD(+). Crystals as large as 0.25 x 0.25 x 0.75 mm could be grown within a month at pH 8.5 with polyethylene glycol as precipitating agent. Preliminary X-ray crystallographic analysis revealed that they belong to one of the enantiomorphic space groups P4(1)2(1)2 or P4(3)2(1)2 with dimensions a = b = 81.66 and c = 214.6 A, having two subunits in an asymmetric unit. The crystals diffract to at least 2.3 A resolution.

Journal Article↗
PubMed1996

Fluorescence behavior of tryptophan residues of bovine and human serum albumins in ionic surfactant solutions: a comparative study of the two and one tryptophan(s) of bovine and human albumins.

The fluorescence behavior of two tryptophans (Trp-134, Trp-213) in bovine serum albumin (BSA) and a single tryptophan (Trp-214) in human serum albumin (HSA) was examined. The maximum emission wavelength (lambda max) was 340.0 nm for both proteins. In a solution of sodium dodecyl sulfate (SDS), the lambda max of BSA abruptly shifted to 332 nm at 1 mM SDS and then reversed to 334 nm at 3 mM SDS. The lambda max of HSA gradually shifted to 330 nm below 3 mM SDS, although it returned to 338 nm at 10 mM SDS. In contrast to this, in a solution of dodecltrimethylammonium bromide, the lambda max positions of BSA and HSA gradually shifted to 334.0 and 331.5 nm, respectively. Differences in the fluorescence behavior of the proteins are attributed to the fact that Trp-134 exists only in BSA, with the assumption that Trp-213 of BSA behaves the same as Trp-214 of HSA. The Trp-134 behavior appears to relate to the disruption of the helical structure in the SDS solution.

Animals↗
PubMed1996

Effects of lipopolysaccharide on the expression of fibrinolytic factors in an established cell line from human endothelial cells.

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulphate and plasminogen activator (PA). Bacterial extract, such as lipopolysaccharide (LPS), damaged the blood vessels and destroyed the balance between the antithrombotic and thrombotic functions of endothelial cells. The fibrinolytic system is involved in antithrombotic functions. The TKM-33 cell line was established from human endothelial cells. In order to determine whether TKM-33 is a good fibrinolytic system endothelial cell expression model, the expression of fibrinolytic factors in TKM-33 cells treated with or without LPS was studied. The endothelial cells which had not been treated with LPS produced and secreted a large amount of urokinase-type PA (u-PA), and small amounts of tissue-type PA (t-PA) and PA inhibitor-1 (PAI-1), which were identified immunohistochemically and by electrophoretic enzymography. Diisopropylfluorophosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 2.83 +/- 0.61 nM, and Bmax of (0.11 +/- 0.01) x 10(6) sites/cell. u-PAR expression was detected in endothelial cells by Northern blot analysis. Thus, endothelial cells was shown to express u-PAR which binds u-PA specifically. In the binding assay, the stimulation of endothelial cells with 0.1, 1.0 and 10 micrograms/ml of LPS altered the Kd values to 6.04 +/- 0.71, 7.03 +/- 1.55 and 7.38 +/- 1.03 nM, respectively. However the Bmax values did not change significantly. Although LPS treatment increased u-PAR expression in endothelial cells in a dose-dependent manner, the expression of u-PA and t-PA mRNAs was not altered significantly. LPS stimulation (10 micrograms/ml) increased the expression of PAI-1 mRNA, significantly. The PA activity recovered from the cell surface fraction was not affected by LPS stimulation, but the PAI-1 activity was increased. These findings suggest that the established endothelial cell line, TKM-33, possesses the characteristics of endothelial cells and they express u-PAR on their cell surface, which is occupied by intrinsic u-PA secreted from the cells, and that treatment of endothelial cells with LPS changes the cell surface characteristics and inhibited the u-PAR expression thus promoting the prothrombotic function concomitantly with increased PAI-1 activity.

Binding, Competitive↗
PubMed1996

Presenilin 1 mRNA expression in hippocampi of sporadic Alzheimer's disease patients.

Missense mutations in presenilin 1 (PSNL1) are known to cause familial Alzheimer's disease which is a subtype of Alzheimer's disease (AD). Our investigation, using an in situ hybridization technique, indicated that the localization of PSNL1 mRNA is similar in sporadic AD affected and normal human brains. Furthermore, the amounts of PSNL1 mRNA in neurofibrillary tangle (NFT)-bearing neurons and those without NFTs did not differ, and the clinical severity of AD was not related to PSNL1 mRNA expression level.

Aged↗
PubMed1996

Release of interleukin-1 beta by dermatan sulfate suppresses hepatocyte growth.

Among glycosaminoglycans, dermatan sulfate (DS) is the strongest inhibitor of DNA synthesis in adult rat hepatocytes in primary culture stimulated with insulin and epidermal growth factor. Hyaluronate also inhibited DNA synthesis, whereas chondroitin-6 sulfate, 4-sulfate or heparin had no effect on DNA synthesis in hepatocytes. Analysis of growth regulatory factors in hepatocyte culture medium treated with DS revealed that interleukin-1 (IL-1) was released into the medium. IL-1 beta mRNA was detected in DS-treated hepatocytes by reverse transcriptase-polymerase chain reaction, but not in untreated hepatocytes. For a marked inhibition of DNA synthesis, more than 10 hr of exposure to DS before cultured hepatocytes started DNA synthesis, was required. Similarly, more than 10 hr was required after the addition of DS before IL-1 beta mRNA was detected. These findings suggest that DS in the medium induced the production of IL-1 beta which, in turn, reduced DNA synthesis in hepatocytes.

Animals↗