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Biomedical subjects

Y Mitsui

Publications and source records attributed to Y Mitsui.

At least 163 records · Page 9Linked to original sources
PubMed1995

Identification of urokinase-type plasminogen activator receptor in human endothelial cells and its modulation by phorbol myristate acetate.

Human endothelial cells express antithrombotic properties by producing prostacyclin, heparan sulfate and plasminogen activator (PA). Using an established cell line, TKM-33, from human umbilical vein endothelial cells, the pericellular urokinase-type PA (u-PA) activity and expression of u-PA receptor (u-PAR) were investigated. The endothelial cells produced and secreted large amounts of u-PA and low levels of tissue-type PA (t-PA) and of PA inhibitor-1 (PAI-1), which were identified by immunohistochemical study and electrophoretic enzymography. Diisopropylfluoro-phosphate-treated 125I-u-PA bound specifically to acid-treated monolayered endothelial cells with a Kd of 3.46 +/- 1.17 nM, and Bmax of (0.09 +/- 0.04) x 10(6) sites/cell. mRNA of u-PAR was detected by using Northern blot analysis. Thus, these endothelial cells express u-PAR which bounds u-PA specifically. Phorbol myristate acetate (PMA) stimulation to the endothelial cells altered the Kd value to 3.18 +/- 0.64 nM, and Bmax value to (0.19 +/- 0.10) x 10(6) sites/cell, respectively. PMA treatment of endothelial cells increased u-PAR mRNA. Similarly, H7-treated endothelial cells showed a dose-dependent increase of u-PAR mRNA. However, PMA and H7 did not stimulate the expression of u-PA and t-PA mRNAs significantly. The expression of PAI-1 mRNA was increased by both PMA and H7. These findings suggest that the established endothelial cell line, TKM-33, possesses the character of endothelial cells and expresses u-PAR on their cell surface which is occupied by intrinsic u-PA secreted from the cells. The pericellular u-PA activity and the expression of u-PAR were regulated by protein kinase pathway.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1995

Control of ciliary activities of Schistosoma mansoni miracidia using Triton-extracted parasites.

Extraction with 0.04% (w/v) Triton X-100 for 3 min removed the cell membrane from the locomotory cilia of Schistosoma mansoni miracidia while leaving the motile apparatus apparently intact. Immediately after Triton-extracted miracidia were treated by the reactivating solution containing ATP and magnesium ions (Mg2+) at pH 8.1, nearly 100% of Triton-extracted miracidia showed the ciliary beating and swam forward in a manner resembling that of a normal miracidium. In the standard reactivating solution (2 mM ATP, 2 mM Mg2+, pH 8.1), Triton-extracted miracidia swam at a speed of 580 microns/sec; the comparable value for live miracidia in dechlorinated tap water was 2,200 microns/sec. The swimming velocity of Triton-extracted miracidia was dependent on ATP and Mg2+ concentration, pH, and salinity. In a solution containing 0.9% NaCl, Triton-extracted miracidia were not reactivated. Among the nucleotides tested, only ATP was found to induce a significant amount of ciliary motility. In terms of divalent cation specificity, only Mg2+ was capable of producing normal motility. Barium and calcium ions (at 0.5 mM CaCl2) also were capable of activating ciliary motility but were less effective stimulants than Mg2+. However, in 1 mM CaCl2, no ciliary reactivation was observed and cilia became detached from the body surface of the miracidia. Vanadium inhibited ATP-reactivated ciliary beating of Triton-extracted miracidia.

Adenosine Triphosphate↗
PubMed1995

Epitheloid granuloma formation in type C chronic hepatitis: report of two cases.

Two cases of type C chronic hepatitis with the presence of epitheloid granulomas were reported. Case 1 was a 44-year-old man who presented with a moderate increase in serum ALT and positive anti-HCV. Histological examination of percutaneous liver biopsy specimens showed dense portal inflammation with piecemeal necrosis. Lobular inflammation was present mederately and a large well-organized epitheloid granuloma was found within a liver lobule. Case 2 was a 55-year-old woman, who was presented because of a slight increase in serum ALT and positive anti-HCV. Histologically, liver biopsy specimens showed lymphoid aggregates in the portal area and slight piecemeal necrosis. Moderate steatosis were noted and an epitheloid granuloma was present in the hepatic lobule. Acid-fast stains were negative for both cases and serum ACE and lysozyme were within normal range. These granulomas were composed of epitheloid cells surrounded by lymphocytes. They were not present within the portal tracts, but were found in the hepatic lobule. The incidence of the appearance of epitheloid granuloma in liver biopsy specimens of type C chronic hepatitis was 2 out of 273 cases (0.73%). The role of HCV infection in the genesis of epitheloid granulomatosis is of much interest and should be investigated.

Adult↗
PubMed1995

[Measurement of electrooculogram in the cat].

In humans, the light peak/dark trough ratio (L/D ratio or Arden ratio) of the electrooculogram (EOG) is conventionally used as an index to estimate the function of the retinal pigment epithelium, but it is rarely in animals. We obtained stable EOG recording in the cat under general anesthesia during light and dark adaptations by applying mechanical forced duction of approximately 3.0 mm in width to one eye of an animal. In five animals, voltages of the dark baseline, the light peak, and dark trough were 580-1,800 (mean, 1,312 microV), 825-2,660 microV (mean, 1,881 microV) and 415-1,480 microV (mean, 1,030 microV), respectively. The L/D ratio in the five animals was 1.70-1.99 (mean, 1.83), similar to that in humans. The present results suggest that the EOG recording by forced duction in experimental animals can be developed into another method to evaluate the retinal pigment epithelial function, particularly to evaluate its potential disorder induced by fluoroquinolones.

Animals↗
PubMed1994

Enhanced expression of multiple forms of VEGF is associated with spontaneous immortalization of murine fibroblasts.

The mechanism(s) involved in immortalization that constitute the first step during malignant transformation has been the subject of our interest. By the use of spontaneously immortalized mouse embryonic fibroblasts we have earlier identified two stages of immortalization which are characterized by growth characteristics of the cells, their conditioned medium and the protein markers such as p53, p81 and mortalin (Kaul et al. (1994) Biochim. Biophys. Acta, in press). The present study was planned to purify the mitogenic factors from the conditioned medium of stage II cells. Sequential purification by chromatography followed by peptide sequencing has characterized one of these as vascular endothelial growth factor (VEGF). Further analysis by RT-PCR suggests that the spontaneously immortalized stage II fibroblasts have enhanced synthesis and secretion of VEGF as compared to their mortal parent cells. Expression of a novel 304 bp long form of VEGF is identified in immortal fibroblasts in addition to the three known alternatively spliced forms. The study points to the involvement of VEGF function during spontaneous immortalization of mouse embryonic fibroblasts.

Alternative Splicing↗
PubMed1994

Identification of genetic events involved in early steps of immortalization of mouse fibroblasts.

The spontaneously immortalized early passaged fibroblasts from three different strains of mouse are observed to represent two distinct stages of immortalization. The cells at stage I are characterized by slow growth rate, contact inhibition and requisition of serum factors for their growth and proliferation. Stage II cells are marked by fast, multilayer growth that is independent of serum supplementation in growth medium and by the elevated levels of the two marker proteins, i.e., p53 and p81. The change from cytosolic distribution of mortalin, a senescence inducing protein (J. Biol. Chem. (1993) 268, 6615-6621; 22239-22242) to the perinuclear locale is detected as an early event during cellular immortalization. Furthermore, the distinct stages could be characterized by thermal analysis of intact cells, that to the best of our knowledge is employed for the first time for the analysis of cellular mortal and immortal phenotypes. The study characterizes at least two distinct end points in rodent transformation suggesting that there are multiple routes to immortalization.

Animals↗
PubMed1994

Fluorescent differential display: arbitrarily primed RT-PCR fingerprinting on an automated DNA sequencer.

We established robust, reliable protocols for 'Differential Display (DD),' an RNA fingerprinting method originally developed by Liang and Pardee [(1992) Science 257, 967-971] using RT-PCR with arbitrary primers. Our protocols are optimized so that reliable DD analysis can be performed on a fluorescent DNA sequencer to ensure high throughput as well as improved operational safety, compared with the original one using radioactive compounds. Such 'Fluorescent Differential Display (FDD)' techniques will accelerate the identification of differentially expressed as well as polymorphic transcripts to address various biological questions.

Base Sequence↗
PubMed1994

Low oxygen enhances endothelin-1 (ET-1) production and responsiveness to ET-1 in cultured cardiac myocytes.

To understand the pathophysiological role of ET-1 in heart, the ET-1 production ability and the responsiveness to ET-1 in cultured neonatal rat cardiac myocytes were compared between high (50%) and low (21%) oxygen atmosphere culture conditions. The amount of immunoreactive ET-1 secreted by the cardiac myocytes into the culture medium was much higher at the low oxygen condition. An analysis of ET-1 binding capacity to the cardiac myocytes revealed that the receptor number of ET-1 was about two-fold at low oxygen culture condition. In fact, an addition of ET-1 (1nM) increased in protein synthesis at the low but not the high oxygen culture condition. Thus, the increase in ET-1 production and responsiveness to ET-1 in the cardiac myocytes at the low oxygen culture condition suggests that autocrine ET-1 might be involved in the maintenance of beating ability in heart at hypoxia.

Animals↗
PubMed1994

Circadian expression of NMDA receptor mRNAs, epsilon 3 and zeta 1, in the suprachiasmatic nucleus of rat brain.

The presence of the N-methyl-D-aspartate (NMDA) receptor channel subunit epsilon 3 and zeta 1 mRNAs in the rat suprachiasmatic nucleus (SCN) was detected by sensitive in situ hybridization. The daily fluctuations in the epsilon 3 and zeta 1 subunit mRNAs in their abundance were found in the SCN to be high during the day and lower during the night under 12 h light:12 h dark conditions (LD 12:12). Under constant darkness for 15 days, both the epsilon 3 and the zeta 1 mRNA levels in the SCN remained cyclic. Furthermore, after exposure of rats to light, the epsilon 3 and zeta 1 subunit mRNAs increased during the subjective night, but not during the subjective day. These results implicate the involvement of the epsilon 3 and zeta 1 subunits in neuronal signaling in the SCN and suggest that these subunits of the NMDA receptor channel are regulated by light and a circadian clock.

Animals↗
PubMed1994

Aging- and growth-dependent modulation of endothelin-1 gene expression in human vascular endothelial cells.

Earlier we reported the undetectable level of endothelin (ET)-1 mRNA in aorta from young donors, in contrast to the detectable levels in older donors (Lab. Invest. 67, 210-217, 1992). We also found that the synthesis of ET-1 peptide is elevated in cultured endothelial cells from the aorta of over-50-year-old donors. In the present report, we show by in situ hybridization that the level of ET-1 mRNA is not so different in aortic endothelial cells from 5- and 50-year-old donors, but increases in cells from 76-year-old donors. The parallel results are obtained from Northern and in situ hybridization analyses by using serially passaged human umbilical vein endothelial cells. Thus, the increase of ET-1 peptide synthesis is achieved mainly at the level of mRNA. Furthermore, these results suggest that the increased expression of ET-1 in elderly people is due to the exhaustion of cell division potential of endothelial cells in vivo. We have also analyzed whether the expression of ET-1 is affected by the growth state. The data show that sparsely growing cells secrete more ET peptide than confluent and stationary cells. In situ hybridization also shows that S-phase cells express more ET-1 mRNA than non-S-phase cells. Thus, it is suggested that there are, at least, two ways for upregulation of ET-1 expression.

Aged↗
PubMed1994

Magnetic resonance imaging and brain-stem auditory evoked potentials in neuro-Behçet's disease.

We studied central nervous system lesions in patients with neuro-Behcet's disease using magnetic resonance imaging (MRI) of the brain and recording of brain-stem auditory evoked potentials (BAEPs). MRI revealed abnormal findings in seven of eight patients. MRI studies demonstrated extensive regions with high intensity signal in the brain stem and/or basal ganglia on T2-weighted images obtained during the acute stage of the disease in three patients. One of these patients had a strongly gadolinum-enhanced round lesion in the lower pons. In four of the other five patients with chronic disease, brain-stem atrophy was observed on T1-weighted images. Atrophic changes were more severe in the brain stem than in the cerebellum. Abnormal BAEPs were observed in three patients and consisted of prolongation of interpeak latency of waves III-V and defects of wave III or V. Abnormal BAEPs were recorded in patients with severe inflammatory changes or progression of atrophic changes in the brain stem. Our findings show that MRI and BAEPs are useful in detecting the presence and assessing the degree of neurological involvement in patients with neuro-Behçet's disease.

Adult↗
PubMed1994

A novel in vitro assay system for transendothelial tumor cell invasion: significance of E-selectin and alpha 3 integrin in the transendothelial invasion by HT1080 fibrosarcoma cells.

The interaction of tumor cells with endothelial cells is a key event in tumor metastasis. We established an in vitro invasion assay system, in which the invasion of tumor cells after interaction with endothelial cells can be examined. Two chamber culture wells separated by porous membrane were used. Human umbilical vein endothelial cells (HUVEC) were placed on porous membranes coated with matrix components. The invasion by HT1080 fibrosarcoma cells was determined in this system by counting the number of cells that moved through the membranes from upper to lower chambers. HUVEC cells did not migrate through the membranes as judged by the staining with UEA-I. Observation by scanning electron microscopy revealed that HT1080 cells bound to HUVEC surfaces and migrated underneath the HUVEC monolayer. Effects of antibodies specific for cell surface adhesion molecules on the migration of HT1080 cells were examined. Invasion of uncoated membranes and membranes coated with HUVEC cells was compared. Antibody against E-selectin significantly suppressed an increase of HT1080 cell invasion of HUVEC monolayers stimulated by IL-1 beta or TNF alpha. Antibody against integrin alpha 3 subunit remarkably inhibited the invasion of HUVEC cell-coated membranes, suggesting that integrins with the alpha 3 subunit may play an important role in the transendothelial invasion by HT1080 cells.

Cell Adhesion Molecules↗
PubMed1994

Diurnal regulation of per repeat family in the suprachiasmatic nucleus of rat brain.

We have recently reported fluctuations in the expression of the period repeat sequence, pp2.5, during light-dark cycles in the suprachiasmatic nucleus (SCN) of rat. Presently, we performed in situ hybridization which shows that the fluctuation of pp2.5 expression continues during constant darkness conditions in the SCN of rat. The light exposure during subjective night but not subjective day triggered its elevated expression in a time-dependent manner which is parallel to that of c-fos expression. In this review, the cloning and characterization of multiple per repeat sequences from mouse genom and rat brain mRNA were summarized. The abundance of a novel per repeat mRNA (designated as RB15) fluctuates during a light-dark cycle in the SCN. These findings suggest that per repeat sequence may have a role for the mammalian circadian rhythms. The evolutionary relationship between the mammarian per repeat sequence and the Drosophila period gene is also discussed.

Animals↗
PubMed1994

Molecular cloning of a gene under control of the circadian clock and light in the rodent SCN.

We recently found a mouse unusual per repeat genomic gene showing circadian expression in the suprachiasmatic nucleus (SCN) of rat brain. As an initial step to the better understanding of biological functions of mammalian per repeat family, we isolated a new cDNA clone that encodes for the putative open reading frame of 133 amino acids, designating as mp41, having a per repeat sequence of (ACAGC)32 which lacks one base pair from a mouse unusual per repeat sequence (ACAGGC)n. In situ hybridization showed that the mRNA of mp41 gene expression is detected in the rat pancreas, uterus, ovary, liver, adrenal glands, kidney, intestine, spleen and brain. In brain, daily fluctuations of mp41 mRNA levels were found in the SCN under light and dark cycles--high during the day time and lower during the night time, even in constant darkness for 15 days. After exposing rats to light, mp41 mRNA increased only during the subjective night of the circadian cycle when light also induced the c-fos mRNA expression in the SCN. These results suggest that the transcriptional control of mp41 gene is regulated by light and a circadian clock and indicate that mp41 is a new marker gene for a cycling transcript in the SCN.

Amino Acid Sequence↗
PubMed1994

pH profile of kinetic constants of RNase Rh from Rhizopus niveus and its mutant enzymes towards UpU, and possible mechanisms of RNase Rh.

In order to elucidate the mechanism of action of Rhizopus niveus RNase Rh, we investigated the pH profiles of the kinetic parameters of RNase RNAP Rh, a derivative of RNase Rh, and its mutant enzymes, i.e., RNase RNAP Rh H104F, RNase RNAP Rh E105Q, and RNase RNAP Rh D51N. Based on comparisons of their profiles we concluded that protonation of His104 is indispensable for the enzymatic activity and Glu105 accelerates the enzymatic activity, especially at acid pH centered at pH 3.5. Based on these data and the previous data on the chemical modification and enzymatic properties of other mutant enzymes, we propose the following as a possible mechanisms of RNase Rh action. (i) His109 participates in enzymatic action as a general base catalyst which removes the hydrogen of the 2'-OH of the ribose moiety. (ii) His46 participates in the reaction as a general acid catalyst which interacts with the 5'-oxygen atom of the scissile phosphodiester bond and becomes a proton donor to the departing nucleoside or nucleotide. (iii) His104 interacts with phosphate anion and its protonation is favorable for the enzymatic activity. (iv) Since the protonated form of Glu105 is more favorable for activity, we postulate two possible roles for Glu105: (a) its stabilizes the intermediate, and (b) it interacts with the oxygen atom of P = O and polarizes the phosphorus atom.

Dinucleoside Phosphates↗