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M Azuma

Publications and source records attributed to M Azuma.

At least 253 records · Page 14Linked to original sources
PubMed1994

Apoptotic signaling through CD95 (Fas/Apo-1) activates an acidic sphingomyelinase.

Intracellular pathways leading from membrane receptor engagement to apoptotic cell death are still poorly characterized. We investigated the intracellular signaling generated after cross-linking of CD95 (Fas/Apo-1 antigen), a broadly expressed cell surface receptor whose engagement results in triggering of cellular apoptotic programs. DX2, a new functional anti-CD95 monoclonal antibody was produced by immunizing mice with human CD95-transfected L cells. Crosslinking of CD95 with DX2 resulted in the activation of a sphingomyelinase (SMase) in promyelocytic U937 cells, as well as in other human tumor cell lines and in CD95-transfected murine cells, as demonstrated by induction of in vivo sphingomyelin (SM) hydrolysis and generation of ceramide. Direct in vitro measurement of enzymatic activity within CD95-stimulated U937 cell extracts, using labeled SM vesicles as substrates, showed strong SMase activity, which required pH 5.0 for optimal substrate hydrolysis. Finally, all CD95-sensitive cell lines tested could be induced to undergo apoptosis after exposure to cell-permeant C2-ceramide. These data indicate that CD95 cross-linking induces SM breakdown and ceramide production through an acidic SMase, thus providing the first information regarding early signal generation from CD95, and may be relevant in defining the biochemical nature of intracellular messengers leading to apoptotic cell death.

Animals↗
PubMed1994

Identification of EGF as an angiogenic factor present in conditioned medium from human salivary gland adenocarcinoma cell clones with varying degrees of metastatic potential.

We have previously shown that conditioned medium (CM) from metastasizing human salivary gland adenocarcinoma cell clones contains factor(s) that stimulate the proliferation and migration of bovine aortic endothelial (BAE) cells, and inhibit the production of collagenases by BAE cells (Azuma M. et al. (1993) Cancer Lett., 73, 85-93). To further characterize this, we evaluated the expression level of epidermal growth factor (EGF) secreted by a non-metastasizing cell clone (HSGc) and its metastasizing cell clones, and analysed the effect of EGF on the biologic behaviors of BAE cells. When the secretion of EGF by cell clones was estimated by enzyme-linked immunosorbent assay, metastasizing cell clones released a large amount of EGF as compared with HSGc. However, the number of EGF receptor was detected consistently at a level that was similar in all cell clones. With regard to the effect of EGF on the malignant potential of cell clones such as proteolytic aggressiveness, EGF did not affect the secretion of both collagenases and their inhibitor from cell clones. Alternatively, exogenous EGF stimulated the proliferation and migration of BAE cells, and inhibited the secretion of collagenases from BAE cells. Neutralization with a neutralizing antibody of EGF released into CM abolished the inhibitory effect of CM on the secretion of collagenases from BAE cells. Thus, the CM-contained factor, which is responsible for the induction of biologic behaviors of BAE cells, can be attributed to EGF.

Adenocarcinoma↗
PubMed1994

K+/H+ antiport in the tobacco hornworm midgut: the K(+)-transporting component of the K+ pump.

The midgut of the tobacco hornworm secretes K+ across the apical plasma membrane of its goblet cells. This secondary K+ transport results from K+/H+ antiport energized by the proton-motive force generated by a primary, H(+)-transporting plasma membrane V-ATPase. Thus, the lepidopteran midgut constitutes a well-established example of the emerging concept that the proton-motive force is an alternative to the classical sodium-motive force for the energization of animal plasma membranes. K+/H+ antiport in the tobacco hornworm midgut is electrophoretic, exchanging 2H+ for 1K+. Under physiological conditions, it is energized by the voltage component of the proton-motive force. The strong coupling of electrophoretic K+/2H+ antiport with the electrogenic V-ATPase provides, in principle, the minimal device for the alkalization of the midgut lumen to pH values higher than 11. K+/H+ antiport is insensitive to bafilomycin A1, but is inhibited by amiloride or Concanavalin A. Lectin staining of blots after SDS-PAGE revealed several glycosylated polypeptides in the goblet cell apical membrane which are not part of the V-ATPase and thus are candidates for the antiporter protein. Current efforts are focused on the isolation of the K+/H+ antiporter.

Amino Acid Sequence↗
PubMed1994

Ca2+ inactivation of voltage-dependent Na+ channels in cultured bovine adrenal chromaffin cells: further studies on inhibition of veratridine-induced catecholamine secretion by external Ca2+.

The stimulatory actions of veratridine (VTD) on catecholamine secretion and Na+ influx in cultured bovine adrenal chromaffin cells were studied in the presence of high concentrations of Ca2+ in the incubation mixture. Catecholamine secretion evoked by VTD was reduced by elevating the external Ca2+ concentration to higher than 2 mM. Under the same conditions, VTD-stimulated 22Na+ uptake into the cells was also reduced by elevating the extracellular Ca2+ concentration. In contrast, the secretory action of VTD was not significantly suppressed by preloading Ca2+ to the cells. Furthermore, the effects of extracellular Ca2+ on the sensitivities of the cells to VTD and tetrodotoxin (TTX) were examined, and neither their sensitivities to VTD nor those to TTX were shown to be significantly altered by elevating the Ca2+ concentration in the incubation mixture. These results seem to indicate that the elevation of extracellular Ca2+ concentration may cause the inhibition of VTD-induced catecholamine secretion as a consequence of the inactivation of voltage-dependent Na+ channels, and suggest that Ca2+ may directly act on the cell surface, and the site of Ca2+ action is presumably distinct from the sites of both VTD and TTX actions in the plasma membranes of adrenal chromaffin cells.

Adrenal Medulla↗
PubMed1994

Stimulatory effect of pituitary adenylate cyclase-activating polypeptide on catecholamine synthesis in cultured bovine adrenal chromaffin cells: involvements of tyrosine hydroxylase phosphorylation caused by Ca2+ influx and cAMP.

In cultured bovine adrenal chromaffin cells, pituitary adenylate cylase-activating polypeptide (PACAP) stimulated [14C]catecholamine synthesis from [14C]tyrosine (but not from [14C]DOPA) in a concentration-dependent manner, causing maximal stimulation at 10(-7) M. The stimulatory action of PACAP was not affected by staurosporine (an inhibitor of protein kinase C) or in the cells in which protein kinase C was down-regulated by prolonged exposure to TPA (an activator of protein kinase C), whereas it was partially attenuated in Ca(2+)-free medium. PACAP (10(-7) M) increased the formation of [3H]inositol phosphates, [Ca2+]i and 45Ca2+ uptake as well as cAMP. The peptide also stimulated the phosphorylation of tyrosine hydroxylase, the enzyme catalyzing the rate-limiting step in catecholamine synthesis. Catecholamine synthesis and tyrosine hydroxylase phosphorylation stimulated by the maximal effective concentration of dibutyryl cAMP or high K+, which activates Ca2+ uptake, were further enhanced by PACAP, suggesting that both cAMP- and Ca(2+)-dependent protein kinases may be involved in the stimulation of tyrosine hydroxylase phosphorylation and catecholamine synthesis caused by PACAP.

Adrenal Medulla↗
PubMed1994

Formation of anti-sense RNA to IFN mRNA in poly (rI):(rC) induced HEL cells.

To elucidate the mechanism of super induction of IFN, mRNA and anti-sense RNA were analyzed by using reverse transcription-polymerase chain reaction (RT-PCR) method, and following results were obtained. (1) When human embryonic lung fibroblast cells (HEL cells) were exposed to poly (rI):(rC) for 1 hr, treated with cycloheximide for 3 hr, treated with actinomycin-D for 30 min at the final period of cycloheximide treatment, then washed and replenished with maintenance medium, (HEL-I:C/CH/Act. D), they produced much higher amount of IFN and IFN production continued for longer period when compared to the HEL cells which were not treated with actinomycin-D (HEL-I:C/CH). In agreement with these IFN production kinetics, HuIFN-beta mRNA was stabilized in HEL-I:C/CH/Act. D. In HEL-I:C/CH/Act.D, the mRNA was detected at the end of cloheximide treatment (4 hr after induction) and 6 hr after induction, however, it was not detected at 6 hr after induction in HEL-I:C/CH. (2) Anti-sense RNA was detected in HEL-I:C/CH/Act.D at 4 hr and 6 hr after induction, and also in HEL-I:C/CH at 4 hr after induction, however, it was not detected at 6 hr after induction in HEL-I:C/CH. These results indicated that an anti-sense RNA to HuIFN-beta mRNA was formed in poly (rI):(rC) induced HEL cells, and that aRNA had the same fate as mRNA, suggesting it's role in the regulation of IFN production. Possible role of aRNA in the regulation mechanism of IFN production was discussed.

Base Sequence↗
PubMed1994

[Enterocele].

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Female↗
PubMed1994

[Enterocele].

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Female↗
PubMed1994

Morphogenesis of normal human salivary gland cells in vitro.

Primary cultured human salivary gland cells were transfected with ori-defective mutant DNA of SV40. Following 2-3 weeks of transfection, slowly expanding colonies consisting of small compact cells emerged, while mock-transfected cells did not grow any more and eventually entered crisis, followed by cell death. Using limited dilution technique, we isolated 4 cell clones with distinct morphology from a single colony. Morphological observation of cells cultured on plastic dishes precisely revealed the characteristics of the constituent cells of salivary gland; i.e., three cell clones showing cuboidal- (NS-SV-DC), spindle- (NS-SV-MC), and flattened morphology (NS-SV-SC) were similar to duct-, myoepithelial-, and squamous phenotype, respectively. A remaining cell clone, polygonal in shape and with numerous secretory granules (NS-SV-AC), resembled an acinar cell. Characterization of cell clones by ultrastructural examination and search for specific antigens showed the similarity of NS-SV-DC, NS-SV-MC, NS-SV-AC, and NS-SV-SC to duct, myoepithelial, acinar, and squamous cells, respectively. Anchorage-independent growth in soft agar and tumorigenicity in nude mice were not recognized in all cell clones. These results demonstrate that establishment of cell clones with duct-, myoepithelial-, acinar-, or squamous phenotype was accomplished in the in vitro system, and that based on the evaluation of colony-forming ability in soft agar and tumorigenicity in nude mice these cell clones can be considered to be non-tumorigenic. Using the above in vitro system, we examined the effect of a reconstituted basement membrane extract, Matrigel, on the morphogenesis of cultured normal human salivary gland cells. When NS-SV-DC or NS-SV-MC were seeded on Matrigel in serum-free culture conditions, they formed round or zonal clusters on day 1; failing however, to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, while on days 2 and 3 these cells lost both cell-cell and cell-Matrigel interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we have investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators (PAs) as compared with a neoplastic human salivary gland cell line (HSG), which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression level of tissue inhibitor of metalloproteinases-1 (TIMP-1) was not observed in these cells.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

Hepatitis B virus carrier with low titer of antibody to hepatitis B core antigen.

Of 798 hepatitis B virus (HBV) carriers, 22 had low titers (lower than 80% inhibition in 200- or 400-fold diluted serum) of antibody to hepatitis B core antigen (anti-HBc). Among these 22 patients, there were 12 (1.50%) with viremia who were positive for hepatitis B e antigen and had a high titer of HBV-associated DNA polymerase activity. Among these 12 patients, four showed no significant change in the anti-HBc titer, while four others showed significant increases in the anti-HBc titer during the follow-up periods. The former all remained asymptomatic carriers (ASCs), while the latter all developed chronic hepatitis (CH). The liver histology of four patients (ASC: 2, CH: 2) showed mild inflammation, and the localization of hepatitis B core antigen (HBcAg) in the liver specimens showed a nuclear pattern in the two ASCs, but a nuclear plus cytoplasmic pattern in the two CH cases. In HBV carriers, the increase in anti-HBc titer seems to be closely correlated to the change in HBcAg localization from the nucleus to the cytoplasm in the liver. Therefore, rising titers of anti-HBc were assumed to be associated with increased expression of HBcAg on the hepatocyte, and hence increased immune-mediated hepatic damage and the onset of hepatitis in ASC with low titer of anti-HBc.

Adolescent↗
PubMed1994

Cataract and the acceleration of calpain-induced beta-crystallin insolubilization occurring during normal maturation of rat lens.

PURPOSE: To determine if limited proteolysis of beta-crystallins is associated with insolubilization of proteins in rats lens during maturation and to test if the protease, calpain II, is involved. METHODS: Soluble and insoluble lens proteins from 4-day-old to 4-month-old rat lens cortexes and nuclei were separated by two-dimensional electrophoresis. The insoluble proteins from 4-month-old nuclei were electroblotted and the NH2 termini of proteins sequenced. Cleavage sites appearing at 4 months of age were compared to cleavage sites produced by purified calpain II and to cleavage sites appearing in cataracts induced by selenite in vivo or in lenses cultured with calcium ionophore A23187 or diamide. RESULTS: In solubilization of more than 50% of proteins occurred in the nucleus of the transparent rat lens by 4 months of age. The insoluble protein that formed contained an abundance of partially degraded beta-crystallin polypeptides missing portions of their NH2 terminal extensions. In contrast, these truncated beta-crystallins were largely absent from both the cortex and soluble fraction of the nucleus. The cleavage sites in the insoluble beta-crystallins appearing during maturation in the lens nucleus were similar to cleavage sites produced by purified calpain II and also similar to cleavage sites appearing in the insoluble protein of cataractous lenses. CONCLUSIONS: These results suggest that proteolysis of beta-crystallins by the protease calpain II contributes to protein insolubilization during lens maturation and that acceleration of this insolubilization process is associated with cataract formation in rodent lenses.

Amino Acid Sequence↗
PubMed1994

Enhanced proteolytic activity is responsible for the aberrant morphogenetic development of SV40-immortalized normal human salivary gland cells grown on basement membrane components.

BACKGROUND: A reconstituted basement membrane extract, Matrigel, is a potent inducer of cell growth and differentiation in vitro in a number of cell types. In this study, we examined the effect of Matrigel on the morphogenesis of cultured normal human salivary gland cells. EXPERIMENTAL DESIGN: SV40-immortalized normal human salivary gland-cell clones with duct- (NS-SV-DC) or myoepithelial phenotype (NS-SV-MC), which had been established in our laboratory (Azuma M, et al., Lab Invest, 1993;69:24-42), were cultured on Matrigel in serum-free culture conditions, and then morphogenetic behavior of cell clones was examined. RESULTS: When cell clones were seeded on Matrigel, they formed round or zonal clusters on day 1; however, they failed to develop into a salivary gland morphogenesis. Semithin sections of cell clones cultured on Matrigel exhibited multicellular aggregates on day 1, whereas on days 2 and 3, these cells lost both cell-Matrigel and cell-cell interactions and eventually entered crisis. In an attempt to understand the mechanism involved in this phenomenon, we investigated proteolytic enzymes and their inhibitors secreted by cell clones. Although cell clones produced almost identical levels of gelatinases, they released increased amounts of plasminogen activators as compared with a neoplastic human salivary gland cell line, which had already been demonstrated to differentiate into acinar cells when cultured on Matrigel. Obvious difference of expression level of tissue inhibitor of metalloproteinases-1 was not observed in these cells, however, NS-SV-DC produced a relatively small amount of endothelial-type plasminogen activator inhibitor as compared with NS-SV-MC. Neutralization of excess plasminogen activators by exogenously added serine protease inhibitors corrected the aberrant in vitro morphogenesis of NS-SV-DC, but not of NS-SV-MC, and allowed NS-SV-DC to form glandular-like structures.

Animals↗