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Biomedical subjects

Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 235 records · Page 13Linked to original sources
PubMed1991

Structure of the precursor for vasoactive intestinal contractor (VIC): its comparison with those of endothelin-1 and endothelin-3.

Vasoactive intestinal contractor (VIC) is a member of the endothelin (ET) peptide family, which evokes a strong contractile response in the ileum, its gene being expressed only in the intestine. Using dot blot analysis, we carried out an interspecies comparison of the nucleotide and deduced amino acid sequences of the precursor for VIC with those of ET-1 and ET-3 to investigate the physiological significance of processing of the precursor for VIC. The highly conserved amino acid sequence was observed between the big form region (big VIC, big ET-1, and big ET-3) of about 40 amino acids and the like peptide region (VIC-like peptide, ET-1-like peptide, and ET-3-like peptide) of 15 amino acids downstream from the big form region. Sequence identity of amino acids of the precursors of ET-1 and ET-3 with that of VIC was 29 and 28%, respectively. Thus, the precursors for the three peptides might have arisen from a common progenitor gene. However, apparent cleavage sites of the like peptide regions are rather unique in the VIC-like peptide, i.e., it had dibasic amino acids at the amino and carboxy termini. Therefore, we suggest that the VIC-like peptide might be liberated from its precursor protein and play some role in the intestine in vivo.

Amino Acid Sequence↗
PubMed1991

Endothelin-1 stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium. II.

The effect of endothelin-1 (ET-1) on rat cardiac myocytes cultured in a serum-free medium was determined. Cardiac myocytes cultured in our medium showed an increased rate of protein synthesis, morphological size, contraction rate, and Ca2+ uptake when ET-1 was added. These actions of ET-1 were inhibited by a protein kinase C inhibitor, H-7. Among these four phenomena, contraction and Ca2+ uptake were inhibited by a Ca2+ channel blocker, nicardipine. Therefore, it is likely that the signal for ET-1 to induce these phenomena is transduced by kinase C, and Ca2+ uptake is related to maintaining a high contraction rate.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1991

Contact with basement membrane heparan sulfate enhances the growth of transformed vascular endothelial cells, but suppresses normal cells.

Modulation of vascular endothelial cell growth by basement membrane heparan sulfate was investigated using four lines of normal and transformed cells. The growth of transformed endothelial cells, but not normal cells, on reconstituted basement membrane was severely suppressed when heparan sulfate, one of the components of the membrane, was specifically degraded by an enzyme, heparitinase. Similarly, when cells were grown on surfaces coated with heparan sulfate, as little as 60 pg/cm2 of heparan sulfate caused growth enhancement of transformed cells, but suppression of normal cells. These results together with our previous observations (IMAMURA, T and MITSUI, Y. (1987) Exp. Cell Res., 172: 92-100) argue that transformed cells have reversed a mechanism by which basement membrane heparan sulfate functions as a physiological suppressor for the growth of normal endothelial cells.

Animals↗
PubMed1991

Establishment of vascular endothelial cell lines in a serum-free culture and the discovery of endothelin and a vasoactive intestinal contractor (VIC).

Immortal vascular endothelial cell lines were established and utilized for the production of an endothelium-derived contraction factor (EDCF) in a serum-free medium. After the discovery of Endothelin (21 amino acid peptide, ET) as an EDCF, a prepro ET cDNA isolated from human tissue was used to examine the expression of ET and its regulation in human endothelial cells. A gene family of ET was shown in mouse by using prepro ET cDNA as a probe. Thus, a novel peptide, Vasoactive Intestinal Contractor (VIC) homologous to ET was deduced from the sequence of one of these genes. VIC was confirmed to induce vasocontraction as well as intestinal contraction. Northern blot analysis indicated that this gene was expressed in the intestine but not in endothelial cells. A cloning and sequencing of prepro VIC cDNA from mouse intestine suggest that a VIC-like peptide, as well as VIC, are co-synthesized by cleavage from prepro VIC with 160 amino acids.

Amino Acid Sequence↗
PubMed1991

[A case of eosinophilic granuloma of the cervical lymph nodes associated with pulmonary cystic lesions].

A 33-year-old man was referred to our department because of left cervical lymphadenopathy for one month which did not respond to antibiotics. Histological examination of the lymph node biopsy revealed eosinophilic granuloma. However, repeated bronchoscopic examinations performed to evaluate the pulmonary lesions appearing on chest roentgenograms and chest CT scans revealed no evidence of eosinophilic granuloma. Steroid therapy resulted in resolution of the cervical lymphadenopathy, but the pulmonary lesions showed no response. The possible relationship between eosinophilic granuloma and pulmonary cystic lesions in our patient is discussed.

Adult↗
PubMed1991

Establishment of human endothelial cell lines in a serum-free culture and its application for expression of transfected prepro endothelin gene.

Endothelial cells cover the inner layer of vascular tube and are involved in the pathogenesis of some diseases in heart and brain. A development of endothelial cell lines in a serum-free culture lead to our discovery of endothelin (ET) and is expected to be breakthrough for other new fields of medical science. In this paper, we report establishment of vascular endothelial cell lines in serum-free and protein-free culture, transfection of prepro ET cDNA into them, and it's expression and processing to mature ET. A human endothelial cell line, ECV304, has been subcultivated in medium with 10% fetal bovine serum. We tested serum-free culture of this cell line and a several months later, established a cell line, t-HUE2 which would show stable growth in serum-free medium ASF301 containing EGF, insulin, transferrin and albumin as protein components. After additional 12-months' struggle for obtaining a new cell line which can grow without any protein components, we established t-HUE4 cell line which can be subcultivated in basal medium of HamF-12. Although the cell line was derived from human umbilical vein endothelial cells, production and secretion of ET by the cell line into culture medium was not detected with radioimmunoassay method. By transfecting prepro ET cDNA into t-HUE2 and Raji (a human B cell line), we examined the regulation of gene expression in the transformed t-HUE2 and Raji cell lines. The results indicated that all the transformants expressed high level of prepro ET mRNA but that Raji transformant did not produce detectable amounts of ET nor ER precursor protein (big ET). In addition, conversion ratio of ET to big ET in t-HUE2 transformants was much higher than those in normal endothelial cells. Thus, post-transcriptional regulation of prepro ET gene in human B lymphocyte cell line and induction of ET converting enzyme in human vascular endothelial cell line were suggested. In addition to the present study, usefulness of t-HUE2 cell line in serum-free culture for the production of human-type glycoproteins was discussed.

Aspartic Acid Endopeptidases↗
PubMed1991

Effect of glucose and an aldose reductase inhibitor on myo-inositol uptake by cultured human endothelial cells.

The effects of an aldose reductase (AR) inhibitor, elevated glucose and other compounds were evaluated on in vitro 2-[3H] myo-inositol (MI) uptake in cultured human endothelial cells (ECs). Significant AR activity was present in ECs (1,373 +/- 170 mumol/mg.min: incubated with 28 mM glucose for 48 hr). Since Na(+)-deprivation and the addition of Ouabain (5 mM) significantly reduced MI uptake, MI incorporation into ECs might be dependent on an active transport system via Na(+)-K+ ATPase activity. MI uptake was reduced significantly (21 +/- 6, 39 +/- 7% reduction) in the presence of excess glucose (27.5, 55 mM). However, addition of the AR inhibitor (ONO-2235 100 microM) prevented the glucose mediated inhibition of MI uptake (15 +/- 5, 21 +/- 6% reduction). These results suggest that inhibition of AR might prevent glucose-mediated toxicity via an increment of MI uptake.

Aldehyde Reductase↗
PubMed1991

[Study on measurement and significance of erythrocyte deformability by electron spin resonance (ESR) method].

To study changes in hemorheological factors during normal pregnancy and pregnancy induced hypertension (PIH), erthrocyte deformability was measured by the electron spin resonance (ESR) method. The results obtained by this new method showed that erythrocyte deformability in normal pregnant women decreases significantly in comparison with nonpregnant women and in PIH decreases even more. By also utilizing one of the most remarkable advantages of this method which is good reproducibility at high hematocrit, an attempt was made to determine the influence on erythrocyte deformability by changing hematocrit and extracellular viscosity of suspension in order to investigate hemorheological significance of hemodilution in hydremia in normal pregnancy and the hemoconcentration in PIH. It seems that deterioration of erythrocyte deformability is compensated for by hydremia in normal pregnancy. Ultimately in order to lessen the deterioration of erythrocyte deformability, the effect of alpha-tocopherol and isoxsuprine are investigated in vitro. These drugs alleviate erythrocyte deformability in vitro, suggesting that these drugs lessen the disturbance of microcirculation in PIH.

Electron Spin Resonance Spectroscopy↗
PubMed1990

Receptor-linked early events induced by vasoactive intestinal contractor (VIC) on neuroblastoma and vascular smooth-muscle cells.

Vasoactive intestinal contractor (VIC) caused a series of biochemical events, including the temporal biphasic accumulation of 1,2-diacylglycerol (DAG), transient formation of Ins(1,4,5)P3, and increase in intracellular free Ca2+ [( Ca2+]i) in neuroblastoma NG108-15 cells. In these cellular responses, VIC was found to be much more potent in NG108-15 cells than in cultured rat vascular smooth-muscle cells. The single cell [Ca2+]i assay revealed that in the presence of nifedipine (1 microM) or EGTA (1 mM), the peak [Ca2+]i declined more rapidly to the resting level in VIC-stimulated NG108-15 cells, indicating that the receptor-mediated intracellular Ca2+ mobilization is followed by Ca2+ influx through the nifedipine-sensitive Ca2+ channel. Pretreatment with pertussis toxin only partially decreased Ins(1,4,5)P3 generation as well as the [Ca2+]i transient induced by VIC, whereas these events induced by endothelin-1 were not affected by the toxin, suggesting involvement of distinct GTP-binding proteins. The VIC-induced transient Ins(1,4,5)P3 formation coincident with the first early peak of DAG formation suggested that PtdIns(4,5)P2 is a principal source of the first DAG increase. Labelling studies with [3H]myristate, [14C]palmitate and [3H]choline indicated that in neuroblastoma cells phosphatidylcholine (PtdCho) was hydrolysed by a phospholipase C to cause the second sustained DAG increase. Down-regulation of protein kinase C (PKC) by prolonged pretreatment with phorbol ester markedly prevented the VIC-induced delayed DAG accumulation. Furthermore, chelation of intracellular CA2+ completely abolished the second sustained phase of DAG production. These findings suggest that PtdCho hydrolysis is responsible for the sustained production of DAG and is dependent on both Ca2+ and PKC.

Animals↗
PubMed1990

Endothelin stimulates hypertrophy and contractility of neonatal rat cardiac myocytes in a serum-free medium.

The effect of endothelin (ET) on rat cardiac myocytes cultured in a serum-free, defined medium was determined. ET simulated cardiac myocyte hypertrophy in a dose-dependent manner as determined by the protein synthesis and cell surface area. Since the myocyte hypertrophy was abolished by H-7, a protein kinase C inhibitor, ET-receptor mediated protein kinase C activation may be involved in cardiac myocyte hypertrophy. At the same time, ET also stimulated myocyte contractility in this medium, and this stimulatory effect was inhibited by nicardipine. This result indicates that the influx of extracellular calcium ion is necessary for the stimulation of contractility induced by ET.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1990

Characterization of endothelin converting enzyme activities in soluble fraction of bovine cultured endothelial cells.

Endothelin converting enzyme activities in the soluble fraction of cultured bovine aortic endothelial cells were characterized. The two major endothelin converting enzyme activities were eluted from a hydrophobic chromatography column and the elution profile of the endothelin converting enzyme activities was the same as that of cathepsin D activities. These activities had a same pH optimum at pH 3.5 and were effectively inhibited by pepstatin A. Furthermore, anti-cathepsin D antiserum absorbed these activities as well as cathepsin D activity. Immunoblotting analysis using the antiserum showed the major active fractions have immunostainable components of identical molecular weights with cathepsin D. From these results, we concluded that the major endothelin converting activities in the soluble fraction of endothelial cells are due to cathepsin D. In addition to these cathepsin D activities, a minor endothelin converting enzyme activity with an optimum pH at 3.5 was found, which does not have angiotensin I generating (cathepsin D) activity from renin substrate and needs much higher concentrations of pepstatin A to inhibit the activity than cathepsin D.

Animals↗
PubMed1990

Appearance of the terminal senescent cell population in human diploid fibroblasts analyzed by flow cytometry.

We studied changes in the distribution pattern of relative RNA content during the in vitro aging of TIG-3 cells by flow cytometry (FACS III). Propidium iodide (PI) does not stain total cellular RNA, but it intercalates specifically into double-helical regions of both DNA and RNA. In applying this principle to RNA, we stained double-stranded RNA (dsRNA) in whole cells with PI after DNA digestion with DNase. The results showed that dsRNA distribution patterns were relatively constant at 7-75 population doublings (PD) but were significantly altered after 77 PD. The distribution patterns were similar as those for cell volume measured with a Coulter Counter. The total cellular dsRNA contents increased linearly at the senescent phase of their in vitro life span. In contrast, the mean dsRNA contents (50% dsRNA contents) rapidly increased to 77-79 PD, but decreased somewhat at 81-83 PD. Two-dimensional histograms of the dsRNA contents versus cell size were little altered from 25 PD to 75 PD. However, a population with relatively larger cell volume and weaker fluorescence intensity appeared and increased after 79 PD. This cell population group may be categorized as "terminal senescent cells" that no more divide in respect that the dsRNA content decreases in spite of the increase of total RNA content.

Cell Survival↗
PubMed1990

Possible direct effect of diethylcarbamazine on the infective larvae of Brugia pahangi.

The direct action of diethylcarbamazine (DEC) on the infective larvae of Brugia pahangi was studied. The larvae were cultured in RPMI 1640 supplemented with foetal bovine serum and antibiotics for 22 days. Most of the larvae remained alive for 8 days, but survival rate of larvae decreased rapidly from day 10 onwards. The larvae did not grow in the culture system. The addition of DEC did not affect the morbidity of the larvae and no difference was observed in the morphological characteristics between the larvae cultured in the presence or absence of DEC. The infective larvae were cultured in vitro for 5 days in the presence or absence of DEC, and inoculated into jirds. The animals were necropsied at intervals, and developing larvae and adult worms were recovered. When the larvae were cultured without DEC and then inoculated subcutaneously into jirds, 29.8% of the inoculum was recovered 3-15 days, and 25% 19-22 weeks, post-inoculation. However, when the larvae were exposed to DEC in vitro and inoculated into jirds, the rate of recovery was reduced to 25% 3-15 days post-inoculation and 2% after 19-22 weeks. When the control larvae cultured in vitro were inoculated intraperitoneally into jirds, 41.3% of inoculum was recovered 3-15 days, and 42.8% 19-22 weeks, post-inoculation. Again the corresponding value for larvae exposed to DEC in vitro was reduced to 19.8% 3-15 days, and 8% 19-22 weeks, post-inoculation. It was observed that the larvae exposed to DEC in vitro were retarded in their development in jirds.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Effects of vasoactive intestinal contractor (VIC) and endothelin on intracellular calcium level in neuroblastoma NG108-15 cells.

Effects on [Ca2+]i levels of endothelin-l (ET) and vasoactive intestinal contractor peptide (VIC), which is a novel member of the endothelin family, were examined in fura 2-loaded neuroblastoma NG108-15 cells. VIC was found to be a very effective stimulus for intracellular Ca2+ mobilization and to be more potent than ET. Intracellular calcium response to sequential addition of two stimulants exhibited the homologous desensitization of either ET or VIC, but no heterologous desensitization between ET and VIC. This indicates evidence suggesting that these two peptides act through distinct receptors.

Calcium↗
PubMed1989

The human preproendothelin-1 gene. Complete nucleotide sequence and regulation of expression.

Endothelin-1 is a 21-amino acid potent vasoconstrictor peptide produced by vascular endothelial cells. We have cloned the whole length of the human preproendothelin-1 (PPET-1) gene and the corresponding cDNA and determined the complete nucleotide sequences. The 2026-nucleotide human mRNA for PPET-1 (excluding the polY(A) tail) is encoded in five exons distributed over 6836 base pairs of the genome. The 5'-flanking region of the gene contains (i) octanucleotide sequences for the phorbol ester-responsive elements, also known as the binding elements for FOS.JUN complex; (ii) consensus motifs for the binding site of nuclear factor 1, which may mediate the induction described previously of PPET-1 mRNA by transforming growth factor-beta; (iii) hexanucleotide sequences for the acute phase reactant regulatory elements that may be involved in the induction of endothelin-1 under acute physical stress in vivo. Further, the 3'-nontranslated sequence of human PPET-1 mRNA contains three AUUUA motifs, which may mediate selective translation-dependent destabilization of the mRNA. Northern blot analysis in cultured endothelial cells from human umbilical veins shows that PPET-1 mRNA is in fact rapidly induced by the active phorbol ester 12-O-tetradecanoylphorbol 13-acetate within 10 min. Analysis of mRNA life span by using actinomycin D demonstrates that PPET-1 mRNA has a short intracellular half-life of about 15 min and is superinduced by cycloheximide. This superinduction is found to be due to the stabilization of the mRNA by cycloheximide, as in the case of other known AUUUA-containing mRNAs. These findings suggest that the regulation of expression of PPET-1 mRNA may be mediated in part by these sequence elements.

Amino Acid Sequence↗
PubMed1989

A novel peptide, vasoactive intestinal contractor, of a new (endothelin) peptide family. Molecular cloning, expression, and biological activity.

A new peptide family (endothelin (ET] consisting of three members in mammals appears to be present in mice according to genomic Southern blot analysis. Two ET-related genes were identified by cloning and sequence analysis of a mouse genome. One encoded a peptide identical to porcine and human vasoconstrictor peptide ET, and the other encoded a novel peptide differing from ET in 3 amino acid residues, with 4 cysteines in the same positions as in ET. This novel peptide was synthesized and confirmed to have in vivo pressor activity similar to that of ET. Northern blot analysis, however, indicated the gene of this novel peptide to be expressed only in the intestine, and not in other tissues or cell lines, or endothelial cells. Furthermore, the peptide evoked a strong contractile response in the guinea pig ileum. This peptide may thus be reasonably classified as a gastrointestinal peptide, vasoactive intestinal contractor.

Amino Acid Sequence↗
PubMed1989

New crystal form of recombinant murine interferon-beta.

Although we have reported (Matsuda, S., Kawano, G., Itoh, S., Mitsui, Y., and Iitaka, Y. (1986) J. Biol. Chem. 261, 16207-16209) that recombinant murine interferon-beta produced in Escherichia coli was crystallized in an orthorhombic space group C222(1) using polyethyleneglycol 8000 as precipitant, the crystals had an insufficient resolution and a marked tendency for orientational disorder around the c axis. We now report that another form of murine interferon-beta crystals with little disorder was obtained in the presence of dioxane using ammonium sulfate as precipitant. The new crystals belong to a hexagonal space group P6(1) or P6(5) with a = b = 71.4 A and c = 79.6 A having only one murine interferon-beta molecule in an asymmetric unit. The crystals are reasonably stable to x-rays and significantly diffract up to 2.2 A resolution when a synchrotron beam is used.

Animals↗