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Biomedical subjects

S Murota

Publications and source records attributed to S Murota.

At least 55 records · Page 3Linked to original sources
PubMed1997

A biotinylated perfringolysin O derivative: a new probe for detection of cell surface cholesterol.

theta-Toxin is a cholesterol-binding, pore-forming cytolysin of Clostridium perfringens. To detect cell surface cholesterol, we prepared a theta-toxin derivative, BC theta by biotinylation of a protease-nicked theta-toxin, which has the same binding affinity for cholesterol as theta-toxin without cytolytic activity. Human erythrocytes, V79 cells and human umbilical vein endothelial cells (HUVEC), were stained with BC theta coupled with FITC-avidin, and then the cells were analyzed by either flow cytometry or laser confocal microscopy. The fluorescence intensity increased in both intact and briefly fixed cells when treated with BC theta. BC theta-treated V79 cells were stained by neither trypan blue nor propidium iodide, indicating that BC stained just the outer surface of the plasma membrane of vital cells. Treatment of the cells with digitonin, a cholesterol-sequestering reagent, decreased the fluorescence intensity to the background level, indicating that BC theta staining is specific for cholesterol. The fluorescence intensity of erythrocytes pre-permeabilized with a small amount of theta-toxin increased more than ten-fold, suggesting higher cholesterol contents in the inner layer of the plasma membrane. When cells were cultured with cholesterol-depleted medium, the fluorescence intensity stained by BC theta decreased remarkably in V79 cells, but did not change in HUVEC. This indicates that cell surface cholesterol may be provided in different ways with these two cell lines. These results suggest that BC theta can be a useful probe for visualizing cell surface cholesterol and for evaluating the effects of cellular events on the topology and distribution of cholesterol.

Animals↗
PubMed1997

Involvement of prostaglandin endoperoxide H synthase-2 in osteoclast formation induced by parathyroid hormone.

Prostaglandin (PG)E2 is one of the most important endogenous bone resorbing factors. In the previous study, we demonstrated that osteoclast formation induced by IL-1 beta was mediated by PGE2 produced by induced prostaglandin endoperoxide H synthase-2 (PGHS-2) in osteoclastic cells. In the same bone marrow culture system, indomethacin also suppressed the osteoclast formation induced by PTH. The inhibition was abolished by exogenously added PGE2 at dose as low as 3 x 10(-9) M, which was too low to elevate the intracellular cAMP and calcium levels and also it was too low to cause osteoclast formation by itself. In order to estimate what kind of cell produced such small amount of PGE2 in the PTH treatment, we carried out antibody staining of PGHS-1&PGHS-2 and PGHS activity in the intact bone marrow cells. PTH was found to induce PGHS activity in tartrate-resistant acid phosphatase (TRACP) positive mononuclear cells and the PGHS activity was inhibited by NS-398, a specific inhibitor of PGHS-2. Immunocytochemical staining supported the expression of PGHS-2 in TRACP-positive mononuclear cells. These findings suggest that PGHS-2 induced by PTH may regulate osteoclast formation by different mechanism from that induced by IL-1 beta.

Acid Phosphatase↗
PubMed1997

Effect of IL-6 on tumor cell invasion of vascular endothelial monolayers.

The effect of interleukin-6 (IL-6) on the invasive capacity of B16-F1 mouse melanoma cells into vascular endothelial monolayers was examined, and an in vitro assay system for the quantitative determination of tumor cell invasiveness, using confocal microscopy with a fluorescence image analyzer, was developed. First, the invasive capacity of B16-F1 mouse melanoma cells against bovine vascular endothelial monolayers was estimated; then, the gap junctional intercellular communication (GJIC) of endothelial cells was examined. Treatment of endothelial cells with IL-6 resulted in a remarkable increase in the invasion of tumor cells into the endothelial monolayer, which was found to be significant from 25 ng/ ml, and peaked at levels of more than 50 ng/ml. This stimulatory effect of IL-6, which was observed from 3 h after the initiation of treatment and lasted for up to 24 h, was abolished by the addition of the anti-IL-6 antibody. Although phase-contrast microscopy did not reveal any morphological changes in the endothelial cells following treatment with 25-200 ng/ml IL-6 for 24 h, the GJIC was observed to be significantly decreased. These findings indicate that the invasive capacity of tumor cells into endothelial cells is affected by IL-6.

Analysis of Variance↗
PubMed1997

Re-expression of functional P-selectin molecules on the endothelial cell surface by repeated stimulation with thrombin.

P-selectin (GMP-140, PADGEM, CD62P) is a cell adhesion receptor which is believed to play an important role in inflammatory diseases by supporting leucocyte rolling. P-selectin is located on the granule membrane of Weibel-Palade bodies in resting endothelial cells and is expressed on the cell surface during cellular activation with various stimulators such as thrombin. Thereafter, P-selectin is internalized and sorted to the Golgi region and Weibel-Palade bodies again. However, whether P-selectin is re-expressed upon subsequent cellular stimulation has, to date, been unclear. To address this question, we measured the cellular content and surface expression of P-selectin, using indirect immunofluorescence and confocal laser cytometry. Surface expression of P-selectin reached a maximum < 2 min after thrombin stimulation and declined to basal levels after 180 min. Rechallenge with thrombin induced rapid surface re-expression of P-selectin, which was independent of de novo protein synthesis, since cycloheximide did not inhibit re-expression. Moreover, re-expressed P-selectin supported the adherence of HL60 promyelocytic cells. These results clearly demonstrated that functional P-selectin molecule was recycled after repeated stimulation with thrombin, raising the possibility that P-selectin is involved in chronic inflammation.

Endothelium, Vascular↗
PubMed1997

[Studies on 5-lipoxygenase inhibitors. Synthesis of and structure--activity relationship in p-hydroxyarylalkenylbenzoazoles].

In a previous paper we reported that 2-(p-hydroxyarylbutadienyl)benzoxazoles are highly potent 5-lipoxygenase inhibitors. We synthesized their ethenyl homologues and benzothiazole derivatives, and evaluated their 5-lipoxygenase inhibitory activity in vitro with cell-free rat basophilic leukemia (RBL-1). In most cases the replacement of benzoxazolyl with benzothiazolyl resulted in an enhancement of the activity. All compounds with butadienyl spacers tested herein exhibited strong inhibitory activities. While most of the ethenyl homologues showed weaker activities than their corresponding butadienyl homologues, some ethenyl compounds in the benzothiazole derivatives were found to be as potent as their corresponding butadienyl homologues. The inhibitory activity was also affected by the variation in the p-hydroxyaryl functionality.

Animals↗
PubMed1997

Enhancement of membrane fluidity in cholesterol-poor endothelial cells pre-treated with simvastatin.

We examined the membrane fluidity of the cholesterol-poor bovine carotid artery endothelial cells (BAEC). Cholesterol-poor BAEC were obtained by treating the cells with 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors under 10% low density lipoprotein (LDL)-deficient serum condition for 2 days. Simvastatin reduced the intracellular cholesterol content significantly at a concentration of 0.1 microgram/ml. The reduction in the cholesterol content was accompanied by the enhancement of the cell membrane fluidity which was measured by a photobleaching technique. Additional data suggested that the reduction in cholesterol content referred to the reduction in the proliferation of BAEC.

Animals↗
PubMed1996

Simvastatin inhibits the division and induces neurite-like outgrowth in PC12 cells.

Simvastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor. inhibited cell division in a dose dependent fashion and induced neurite-like outgrowth in PC12 cells. The neurite-like outgrowth was detectable at 0.5 microg/ml of simvastatin 24 h after the treatment. The responses to simvastatin were completely prevented by incubating the cells with mevalonate. In contrast to simvastatin, pravastatin, a similar HMG-CoA reductase inhibitor but lipophobic, had no effect on the cells. The results provide new possibilities for the central nervous system (CNS) side effects of simvastatin therapy.

Acyl Coenzyme A↗
PubMed1996

Purkinje cells express neuronal nitric oxide synthase after methylmercury administration.

To study the effects of chemical injury on the cerebellar nitric oxide synthase (NOS), we administered methylmercury chloride subcutaneously to mice, 10 mg/kg/day for 9 days. In the methylmercury-treated cerebellum. Purkinje cells were positive both for NADPH-diaphorase and for neuronal NOS. Calcium-dependent NOS activity was increased to 160% of the controls. The present study suggests the ability of Purkinje cells to produce NO through the expression of neuronal NOS.

Animals↗
PubMed1996

Nuclear disintegration as a leading step of glutamate excitotoxicity in brain neurons.

Recent studies on ischemic brain disease in vivo and glutamate excitotoxicity in vitro suggest that apoptosis may play a role in excitotoxic neuronal death. To examine the possible involvement of apoptosis in glutamate excitotoxicity, we studied an early process of morphological changes in rat cortical neurons exposed to 1 mM glutamate. Observations under Nomarski optics combined with a digital image processor revealed a rapid change in the nucleus followed by a cellular swelling. The nucleus increased in granularity and swelled in 5 min, then became liquefied in 30 min. The cell body swelled slowly in 15-45 min. These changes could be prevented by treatment of the neuron with MK-801 (dizocilpine maleate), a blocker of N-methyl-D-aspartate (NMDA) receptor-coupled ion channel. However, treatment of the neurons with N(G)-nitro-L-arginine (N-NORG), a nitric oxide synthase inhibitor, had no significant effect. Use of the in situ end-labeling technique for the demonstration of free 3'-hydroxyl ends revealed that DNA fragmentation took place within 1 hr after glutamate exposure. A change in intracellular Ca(2+) concentration was examined with fluo-3 under a confocal laser microscope. Application of 1 mM glutamate induced rapid Ca transients in the nucleus as well as in the cytoplasm. Both of these Ca responses were blocked by MK-801. These results indicate that glutamate excitotoxicity in the brain neuron does not fulfill morphological criteria of apoptosis, but suggest that the nuclear disintegration associated with DNA fragmentation is involved as a leading step in glutamate excitotoxicity.

Animals↗
PubMed1996

Lipopolysaccharide-induced nitric oxide synthase activity in cultured cerebellar granule neurons.

We examined the inducible form of nitric oxide synthase (iNOS) activity in cerebellar primary cultures enriched with granule neurons. Treatment with lipopolysaccharide (LPS) for 24 h was performed in two different cultures in which the survival of neurons was controlled by the levels of extracellular K+. Treatment of the granule neurons with the contaminating non-neuronal cells (< 5%) in a high K+ (25 mM) -containing culture medium with LPS induced four-fold NOS activity compared to that of the control, and the induced NOS activity was calcium-independent. LPS did not induce the NOS activity at all in the contaminating non-neuronal cells alone which were obtained by eliminating all the granule neurons from the culture by lowering the K+ concentration (5 mM). We conclude that the LPS-activated granule neurons can express iNOS activity and that this induction is not attributable to the contaminating nonneuronal cells.

Animals↗
PubMed1996

Prostaglandin production via induction of cyclooxygenase-2 by human gingival fibroblasts stimulated with lipopolysaccharides.

The purpose of the present study was to investigate the involvement of cyclooxygenase-1(COX-1) and cyclooxygenase-2 (COX-2) in PGE2 production by human gingival fibroblasts stimulated with lipopolysaccharides (LPS) from periodondopathogenic bacteria. LPS were isolated from Porphyromonas gingivalis (P. gingivalis), Actinobacillus actinomycetemcomitans (A. actinomycetemcomitans) and Eschericia coli (E coli) by the phenol-water procedure. The three LPS preparations produced PCE2 up to 48 h in a time-dependent manner in human gingival fibroblasts. P. gingivalis-LPS was the most potent stimulator of PGE2 production and, to a lesser extent, A actinomycetemcomitans- and E coli-LPS. Treatment of the cells with indomethacin, a non selective COX-1/COX-2 inhibitor and NS-398, a selective COX-2 inhibitor, completely depressed PGE2 production. Treatment of dexamethasone, known to inhibit COX-2 expression, also significantly prevented PGE2 production. Immunohistochemical staining of COX-2 protein demonstrated that expression of COX-2 protein was increased at 24 h after P gingivalis-LPS stimulation, while expression of COX-1 protein was not affected by P. gingivalis-LPS. In order to investigate the regulation of PGE2 production. P. gingivalis-LPS-stimulated cells were treated with herbimycin A and genistein, both inhibitors of tyrosine kinases. Both the inhibitors significantly inhibited PGE2 production. Herbimycin A treatment depressed expression of COX-2 protein. These data suggest that human gingival fibroblasts stimulated with LPS from periodontopathogenic bacteria mainly produce PGE2 not by COX-1, but by COX-2, induction of which may be regulated by tyrosine kinase and that the produced PGE2 may be involved in the pathogenesis of periodontal diseases.

Aggregatibacter actinomycetemcomitans↗
PubMed1996

Docosapentaenoic acid (22:5, n-3), an elongation metabolite of eicosapentaenoic acid (20:5, n-3), is a potent stimulator of endothelial cell migration on pretreatment in vitro.

Endothelial cell (EC) migration plays an important role in wound repair of blood vessels. We have previously reported that eicosapentaenoic acid (EPA; 20:5, n-3) pretreatment stimulates migration of ECs but not smooth muscle cells. In the present study, we used the modified Boyden chamber technique to investigate whether the stimulative effect of EPA pretreatment on EC migration is caused by EPA itself or by some metabolites of EPA. When ECs were treated with EPA (5 micrograms/ml) for 2 days, EPA was predominantly elongated to docosapentaenoic acid (DPA; 22:5, n-3), with little docosahexaenoic acid (DHA; 22:6, n-3) being formed. Direct pretreatment of ECs with DPA (0.01-1.0 microgram/ml) resulted in a dose-dependent increase in migration in response to fetal bovine serum. Moreover, maximum stimulation of EC migration by DPA pretreatment (0.5 microgram/ml) was achieved at a concentration one-tenth of that required for maximal stimulation by EPA pretreatment (5.0 micrograms/ml), indicating that DPA is a potent stimulator of EC migration. We have demonstrated by lipid analysis that direct DPA pretreatment (0.5 microgram/ml) sufficiently increased the absolute quantity of phospholipids of ECs. Cyclooxygenase inhibitor and lipoxygenase inhibitor did not abolish the stimulative effect of DPA pretreatment on EC migration. In contrast to EC migration, DPA pretreatment had no effect on smooth muscle cell migration. Together these data suggest that the stimulative effect of EPA on EC migration occurs via DPA, and that DPA may act as a powerful anti-atherogenic factor.

Animals↗
PubMed1996

Cell adhesion molecule mediates endothelial cell injury caused by activated neutrophils.

Addition of PMA (phorbol myristate acetate)-stimulated neutrophils to an endothelial cell monolayer caused a significant increase in the intracellular peroxide level of the endothelial cells after 15 minutes and endothelial cell injury after 5 hours. Both the early and the late events were abolished in the presence of specific antibodies against CD (cluster of differentiation) 11a, CD11b, CD18 and ICAM (intercellular adhesion molecule) 1, but not CD11c. These antibodies affected neither the production of active oxygen species by the neutrophils nor the rate of adhesion of neutrophils to endothelial cells. Pretreatment of endothelial cells with allopurinol caused significant inhibition of both the early and the late events, suggesting that the binding of adhesion molecules may trigger the activation of XO (xanthine oxidase) of endothelial cells, and have the cells produce more hydrogen peroxide and ferrous ions, followed by producing more hydrogen peroxide. The hydrogen peroxide produced by endothelial cells themselves and by neutrophils may be converted to hydroxyl radicals by ferrous ions, which may cause lethal cell damage. Examination of XO activity in endothelial cells showed that the enzyme activity increased double within 15 minutes after the addition of PMA activated neutrophils. Monoclonal antibodies against CD11a and CD18 significantly inhibited the increased conversion of XD (xanthine dehydrogenase) to XO induced by PMA-activated neutrophils. Moreover, tyrosine kinase inhibitors also inhibited the increased conversion of XD to XO. These results indicate that the adhesion of activated neutrophils to endothelial cells via CD11a/CD18-ICAM-1 is involved in the conversion of XD to XO in endothelial cells, which results in endothelial cell injury.

Animals↗