Search PubMed⌕ Search

Biomedical subjects

Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 91 records · Page 5Linked to original sources
PubMed1998

The solution structure of human endothelin-2 a 1H-NMR and CD study.

The solution structure of the vasoactive endogenous 21-amino-acid human endothelin-2 has been determined by NMR and CD techniques, in a mixed solvent of 100 mM aqueous acetic acid and 25% (by vol.) 1,1,1,3,3,3-hexafluoro-2-propanol. From NMR-derived restraints on upper limit distances and dihedral angles, distance geometry structures were calculated using the program DADAS90, and refined by simulated annealing in X-PLOR. The structure of endothelin-2 consists of an alpha-helix of residues 9 to 17, orientated anti-parallel to a short beta-strand of residues 1 to 3, linked together by a possible beta-turn type I of residues 5-8. These secondary structural elements are stabilised and positioned by two disulphide bonds between residues 1 and 15, and 3 and 11, respectively. The average root mean square deviation over residues 1-17 of 15 accepted low-energy conformers chosen to reflect the solution structure of endothelin-2, was 0.73 A for the backbone and 1.41 A for all heavy atoms. The data on endothelin-2 will be discussed and compared with what has been published on other endothelin/sarafotoxin peptides.

Amino Acid Sequence↗
PubMed1998

Expressional changes in alternative splicing affecting genes during cell passage of human diploid fibroblasts.

Normal human diploid cells have a limited proliferative lifespan in in vitro cultures. Changes in gene expression have been examined for understanding control mechanisms of limited proliferative lifespan. and enhanced expression of growth suppressing genes such as p21 was reported in late-passaged cells. We screened genes which were expressed preferentially in mid-passaged cells by the differential plaque screening of the subtracted cDNA libraries prepared from young, life-extended, and immortalized SV40-transformed human fibroblasts. Among isolated clones, ASF/SF2, which was known to affect alternative splicing, was expressed in normal fibroblasts with a peak at mid-passage. Relative expression levels of SC35 and hnRNPA1, which are also known to affect alternative splicing, was also highest at mid-passage. Changes in alternative splicing at mid-passage, if it occurred, may play a crucial role in the process of cellular senescence.

Alternative Splicing↗
PubMed1998

Malignant transformation of NIH3T3 cells by overexpression of mot-2 protein.

The murine mortalin genes, mot-1 and mot-2, are members of the hsp70 family of proteins and differ from each other by only two amino acid residues. Mot-1 is expressed in normal cells and has pancytosolic cellular distribution whereas mot-2 is found in the perinuclear region of immortal cells. We report here that a high level of expression of mot-2 protein resulted in malignant transformation of cells as analysed by anchorage independent growth and nude mice assays. A high level of protein expression is attributed to the 900 bp 3' untranslated region of the cDNA which does not have any transforming activity per se. Mortalin cDNA clones isolated from human transformed cells were also found to have transforming activity in similar assays and a high level of expression was apparent in some of the human immortalized cells that showed non-pancytosolic mortalin immunofluorescence. Taken together, the data suggest that nonpancytosolic mortalin may have a role in tumorigenesis.

3T3 Cells↗
PubMed1998

Alleviation of apoptosis by serum in Chinese hamster ovary cells ectopically expressing human Fas antigen.

Fas-mediated apoptosis is an important regulatory mechanism for the development of T-cells and prevention of oncogenesis. Here, we establish Chinese hamster ovary (CHO) cell lines which stably express Fas antigen, and analyzed apoptosis induced by anti-Fas IgM. While Fas-transfected hamster cells did not undergo apoptosis when stimulated with anti-Fas antibody in the presence of medium containing 10% serum, in reduced serum concentrations, anti-Fas antibody caused these cells to round up and detach from the culture dish. Analysis of the DNA content by a flow cytometry demonstrated a significant increase of cells with sub-G1 amount of DNA upon Fas stimulation in the low serum concentrations. The increase in the number of apoptosis cells was inhibited by an apopain (CPP32, caspase 3) inhibitor or insulin-like growth factor-I. In contrast, apoptosis in a Fas-transfected mouse T-cell line occurred in the presence of 10% serum. these results suggest that factors including insulin-like growth factor-I in fetal bovine serum protect CHO cells from apopain-dependent apoptosis mediated by Fas-antigen stimulation.

Animals↗
PubMed1998

The regulation of cyclin D1 expression in senescent human fibroblasts.

To clarify the molecular mechanisms of cyclin D1 expression during in vitro cellular aging, we investigated the binding of nuclear protein factors to the cyclin D1 gene promoter domain in young and senescent normal human fibroblasts. The cyclin D1 promoter binding activities of nuclear protein factors from young and senescent cells were examined by the gel mobility shift assay. Our findings revealed that (i) the binding of a specific nuclear factor to the enhancer element was very weak in senescent cells; (ii) the binding of a specific nuclear factor to the CRE, which is independent of cell growth, was unchanged between young and senescent cells; (iii) nuclear factors from senescent cells did not bind to the presumptive silencer element; (iv) the binding of specific factors to the Inr (transcription initiation region) and E2F increased with growth stimulation in young cells and was weakly detectable in senescent cells; and (v) the binding of Sp1 to its promoter element occurred only in senescent cells. The analysis of the silencer element by the gel mobility shift assay revealed that the essential sequence required for binding of specific factors to the silencer element was TTTAAT. The molecular weight of the binding factor to the silencer element was determined to be approximately 35 kDa by the Southwestern blotting and UV cross-linking assay. Thus, we postulated that the observed increase of cyclin D1 expression during cellular aging is due to an increase in the binding activity of specific nuclear protein factors to an enhancer element, Sp1, and a decrease in binding to a silencer element in senescent cells.

Cell Division↗
PubMed1998

Enhanced expression of mitochondrial genes in senescent endothelial cells and fibroblasts.

It has been suggested that some mitochondrial genes are important in cellular senescence. In order to identify the mitochondrial genes that are involved in cellular senescence, we have constructed a cDNA library from senescent human vascular endothelial cells and isolated 86 senescence-specific cDNA clones by differential screening. Among the clones, we identified four distinct mitochondrial genes including NADH dehydrogenase subunit 2 (ND2), ND3, ATPase 6 and 16S ribosomal RNA. We then compared the levels of expression of these genes in young and senescent cells by using two endothelial and two fibroblast cell strains. Northern blot and slot blot hybridization confirmed that the expression levels of ND3, ATPase 6 and 16S rRNA were elevated in senescent cells of all four strains. The expression level of ND2 was also elevated during cellular senescence in three of the four strains. Because mitochondria are actively involved in oxidative phosphorylation and respiratory functions, the altered expression levels of these genes may participate in aging processes.

Adenosine Triphosphatases↗
PubMed1998

Triplet repeat-containing ribosomal protein L14 gene in immortalized human endothelial cell line (t-HUE4).

A cDNA encoding human 60S ribosomal subunit protein L14 (hRL14) was isolated from a human immortal endothelial cell line, t-HUE4. This cell line was established via a series of cell lines cultured in a serum-free and a protein-free medium, and a directional cDNA library has been constructed and screened in search for the genes modulating protein synthesis machinery in cell proliferation. A putative full-length clone with an open reading frame of 220 amino acids; predicted molecular weight of 23.6 kDa. A significant identity for hRL14 was observed with rat RL14 (85% identity), with exception of COOH-terminal region, but not with any eukaryote amino acid sequences so far deposited to database. The typical features of ribosomal proteins were observed in hRL14, as seen in nuclear targeting sequences necessary for the transport from cytoplasm to nucleolus, a bZIP like (basic region-leucine zipper) element for the binding to rRNA, and the internal repeat sequences; the pentapeptide QKA(A/S)X. The COOH-terminal region of the transcripts contained fifteen triplet repeats (GCT; alanine) at nucleotide 465 to 509, which is significantly expanded compared to the rat RL14. However, the repeat number was all the same among the normal human endothelial cell line and the cell lines established in the course of t-HUE4 establishment. A single band with about 800 bases was identified by Northern blot analysis without tissue specificity. This GCT repeat was found to be one of the longest uninterrupted repeats in a coding sequence, which were associated with the highest degree of polymorphism.

Amino Acid Sequence↗
PubMed1998

A novel alternatively spliced form of murine vascular endothelial growth factor, VEGF 115.

Murine immortal fibroblasts express a form of vascular endothelial growth factor (VEGF) that was cloned, characterized and named VEGF 115. It differs from VEGF 120 by 37 amino acids at the carboxyl terminus. VEGF 115-specific sequence reacted to a single transcript in mouse tissues. Reverse transcription-polymerase chain reaction was performed in mouse tissues and in fibroblasts of normal and immortal divisional phenotypes. The data from mouse tissues suggested that VEGF 115 is not a tissue-specific isoform of VEGF 120, whereas a functional relevance with immortalization is indicated from the latter. The novel cDNA was expressed in Escherichia coli, and the His-tagged VEGF 115 (17.2 kDa) thus obtained was recognized by anti-VEGF antibody. A mammalian expression plasmid, pCMVneo+, encoding for VEGF 115 was transfected to NIH 3T3 cells, and the conditioned medium of stable transfectants was found to have fibroblast growth factor-replacing activity for human umbilical vein endothelial cells. Two independent genomic P1 clonings with primers specific for VEGF 164 and VEGF 115, respectively, resulted in isolation of identical P1 clones. We analyzed these three P1 clones on Southern blots with common and specific probes for VEGF 164 and VEGF 115. The results support the hypothesis that VEGF 115 is a new alternatively spliced form of mouse VEGF.

Alternative Splicing↗
PubMed1998

Efficacy of ivermectin against Strongyloides stercoralis infection in jirds (Meriones unguiculatus).

The activity and distribution of ivermectin (IVM), a broad spectrum anthelmintic for various nematodes and arthropods, was tested against Strongyloides stercoralis infection in the jird (Meriones unguiculatus.) The pattern of IVM concentration in the serum of jirds after either low- (200 microg/kg) or high-dose (1000 microg/kg) treatment by intraperitoneal injection showed a typical single-peak profile with the maximum drug levels detected at 1 h followed by a rapid decline to undetectable values by 48 h posttreatment. With equivalent IVM doses, the serum levels in male jirds were significantly higher than those observed in female jirds at 1 and 12 h posttreatment (P < 0.05). Low-dose IVM treatment of either male or female jirds, in comparison with that of the respective control groups, produced no observable effect on the adult worm burden or the parasite fecundity. The curative dose for S. stercoralis in both sexes of animals was achieved when using the high dose of 1000 microg/kg of IVM. The area under the curve calculated from the serum-concentration profiles in males was significantly higher than that for females after the high-dose treatment of IVM (P < 0.05). By low-dose treatment, a significantly lower adult worm burden was observed in males compared with that in females (P < 0.05), and this difference correlated with the higher serum levels of IVM in male animals. The distribution of IVM in the jird after high-dose treatment was characterized by initial appearances at 1 h of maximum levels of the drug in the serum and intestinal content. The maximum level in the feces was reached at 12 h posttreatment and rapidly declined thereafter. The level in the intestinal tissue was comparatively much lower than that in the intestinal content and the feces. The half-life of IVM in the intestinal content (49.5 h) was longer than those in the serum, feces, and intestinal tissue (6.53-13.07 h). Analyses of the relationship between the peak serum IVM occurring at 1 h posttreatment and the adult worm recovery revealed that the minimal serum concentration associated with clearance of the adult worm from the jird was approximately 0.65 microg/ml. The jird-S. stercoralis model has not only made studies on various aspects of parasite biology possible, but also provided an in vivo system to examine the efficacy as well as the mode of action of IVM against this intestinal nematode.

Animals↗
PubMed1998

A direct competitive ELISA for the simple, sensitive and accurate determination of diethylcarbamazine concentration in serum.

Improved methods for measurement of the anti-filariasis drug diethylcarbamazine in serum would assist in the design of effective therapy. The method evaluated in the present paper is a direct competitive ELISA which is sensitive, specific and accurate. Horseradish peroxidase-labelled diethylcarbamazine conjugate was incubated with diethylcarbamazine and anti-diethylcarbamazine antiserum over a bound second antibody. The enzyme activity of the remaining diethylcarbamazine-horseradish peroxidase conjugate was measured. The intra- and inter-assay coefficient of variation was < 10% in the range of 1.0-30 ng ml-1 and the limit of detection was 0.3 ng ml-1. The cross reactivities of anti-diethylcarbamazine antibodies with diethylcarbamazine metabolites and ivermectin were < 0.09%. Using the ELISA, the serum levels of diethylcarbamazine were successfully determined in mongolian jirds (Meriones unguiculantus) up to 4h following a single dose of 50 mg kg-1 of body weight. The values of pharmacokinetic parameters of diethylcarbamazine in jirds were determined.

Animals↗
PubMed1998

cGMP production is coupled to Ca(2+)-dependent nitric oxide generation in rabbit parotid acinar cells.

We investigated the mechanism of guanosine 3',5'-monophosphate (cGMP) production in rabbit parotid acinar cells. Methacholine, a muscarinic cholinergic agonist, stimulated cGMP production in a dose-dependent manner but not isoproterenol, a beta-adrenergic receptor stimulant. Methacholine-stimulated cGMP production has been suggested to be coupled to Ca2+ mobilization, because intracellular Ca2+ elevating reagents, such as thapsigargin and the Ca2+ ionophore A23187, mimicked the effect of methacholine. The cGMP production induced by Ca2+ mobilization has also been suggested to be coupled to nitric oxide (NO) generation because the effects of methacholine, thapsigargin and A23187 on cGMP production were blocked by NG-nitro-L-arginine methyl ester (L-NAME), a specific inhibitor of nitric oxide synthase (NOS), and hemoglobin, a scavenger of nitric oxide (NO). Sodium nitroprusside (SNP), a NO donor, stimulated cGMP production. Furthermore, methacholine stimulated NO generation, and NOS activity in the cytosolic fraction in rabbit parotid acinar cells was exclusively dependent on Ca2+. These findings suggest that cGMP production induced by the activation of muscarinic cholinergic receptors is coupled to NO generation via Ca2+ mobilization.

Aminoquinolines↗
PubMed1998

Roles of the Ser146, Tyr159, and Lys163 residues in the catalytic action of 7alpha-hydroxysteroid dehydrogenase from Escherichia coli.

The Escherichia coli 7alpha-hydroxysteroid dehydrogenase (7alpha-HSDH; EC 1.1.1.159) has been the subject of our studies, including the cloning of its gene, and determination of the crystal structures of its binary and ternary complexes [J. Bacteriol. 173, 2173-2179 (1991); Biochemistry 35, 7715-7730 (1996)]. Through these studies, the Ser146, Tyr159, and Lys163 residues were found to be involved in its catalytic action. In order to clarify the roles of these residues, we constructed six single mutants of 7alpha-HSDH, Tyr159-Phe (Y159F), Tyr159-His (Y159H), Lys163-Arg (K163R), Lys163-Ile (K163I), Ser146-Ala (S146A), and Ser146-His (S146H), by site-directed mutagenesis. These mutants were overexpressed in E. coli WSD, which is a 7alpha-HSDH null strain, and the expressed enzymes were purified to homogeneity. The kinetic constants of the mutant enzymes were determined, and the structures of the Y159F, Y159H, and K163R mutants were analyzed by X-ray crystallography. The Y159F mutant showed no activity, while the Y159H mutant exhibited 13.3% of the wild-type enzyme activity. No remarkable conformational change between the Y159F (or Y159H) and wild-type proteins was detected on X-ray crystallography. On the other hand, the K163I mutant showed just 5.3% of the native enzyme activity, with a 8. 5-fold higher Kd. However, the K163R mutant retained 64% activity, and no remarkable conformational change was detected on X-ray crystallography. In the cases of the S146A and S146H mutants, the activities fairly decreased, with 20.3 and 35.6% of kcat of the wild-type, respectively. The data presented in this paper confirm that Tyr159 acts as a basic catalyst, that Lys163 binds to NAD(H) and lowers the pKa value of Tyr159, and that Ser146 stabilizes the substrate, reaction intermediate and product in catalysis.

Binding Sites↗
PubMed1998

The cloning and sequencing of ribosomal protein S18 of parasitic protozoa, Entamoeba histolytica.

A cDNA clone encoding ribosomal protein S18 has been isolated from parasitic protozoa, Entamoeba histolytica. The cDNA insert was 495 nucleotides long and an open reading frame (468 nucleotides including the stop codon) coded for 156 amino acid protein which was highly homologous with human, plant and yeast counterparts. PROSITE analysis of the ribosomal protein S18 in eukaryote revealed homology with prokaryotic ribosomal protein S13 signature, which is known to be involved in the initiation of translation.

Amino Acid Sequence↗
PubMed1998

Isolation and structure of an antimitotic cyclic peptide, ustiloxin F: chemical interrelation with a homologous peptide, ustiloxin B.

Ustiloxin F, a microtubule inhibitor, was isolated as a minor metabolite of Ustilaginoidea virens. The structure was determined from the spectral data and by chemical interrelation to ustiloxin B through reductive removal of the sulfoxide-containing side chain of ustiloxin B to give ustiloxin F. Ustiloxin F inhibited microtubule assembly with an IC50 value of 10.3 microM.

Anti-Bacterial Agents↗
PubMed1998

Synthesis and anti-tubulin activity of ustiloxin D derivatives.

Ustiloxin D, produced by the rice plant pathogen Ustilaginoidea virens, exhibits potent anti-tubulin activity. In order to elucidate the effects of functional groups in ustiloxin D on its activity, several derivatives were synthesized and their anti-tubulin activities were estimated. The N,N-dimethylamino derivative and the 14-O-methyl derivative were inactive (IC50 > 50 microM). 20-Hydroxymethylated ustiloxin D showed decreased inhibitory activity compared with ustiloxin D.

Animals↗
PubMed1998

mdm2 and bax, downstream mediators of the p53 response, are degraded by the ubiquitin-proteasome pathway.

Upon activation in response to cellular stress or DNA damage, the p53 tumor suppressor induces the expression of gene products involved in cell cycle arrest and apoptosis. Using the proteasome-specific inhibitors, MG132 (N-acetyl-L-leucinyl-L-leucinal-L-leucinal) and lactacystin, here we show that the p53-response proteins, bax and mdm2 as well as p21, are degraded by the ubiquitin-proteasome pathway in HeLa cells. MG132 also increased expression of the three proteins in cells that lack p53, showing that stabilization of the p53 response proteins is not due to increased levels of p53 itself. Increases in mdm2 protein levels by MG132 was accompanied by increases in polyubiquitinated forms of the proteins. Our results indicate that ubiquitin-dependent protein degradation influences the turnover of downstream targets of p53, therefore suggesting that the proteasome plays a role in regulating apoptosis and cell cycle arrest in response to p53.

Animals↗
PubMed1998

[Percutaneous ethanol injection for unresectable pancreatic cancer-report of two cases].

Two cases of unresectable pancreatic cancer underwent percutaneous ethanol injection (PEI). The first was a 65-year-old man with a 43 x 39 mm mass in the head of the pancreas. He received PEI (total 38.5 ml of ethanol) 6 times. Serum CA19-9 decreased from 3,800 U/ml to 1,700 U/ml. The patient was dead 6 months after PEI. Autopsy revealed coagulative necrosis at the site of ethanol injection. The second case was a 66-year-old man with a 70 x 50 mm mass in the body of the pancreas. He was treated by PEI (total 10 ml). After PEI, his abdominal pain was relieved considerably. He died 4 months after PEI treatment. These findings suggest that PEI may be useful not only for pain control but also for tumor reduction of unresectable pancreatic cancer.

Aged↗