Biomedical subjects

Tatsuaki Nishiyama

Publications and source records attributed to Tatsuaki Nishiyama.

5 recordsLinked to original sources

PubMed2007

Functional analysis of an established mouse vascular endothelial cell line.

BACKGROUND: In vitrostudies using cell lines are useful for the understanding of cellular mechanisms. The purpose of our study is to develop a new immortalized aortic vascular endothelial cell (EC) line that retains endothelial characteristics and can facilitate the study of ECs. METHODS: A mouse aortic vascular EC line (MAEC) was established from p53-deficient mouse aorta and cultured for over 100 passages. The expression of endothelial markers was assessed, and the function of this cell line was analyzed by tube formation and binding assays. RESULTS: MAEC retained many endothelial properties such as cobblestone appearance, contact-inhibited growth, active uptake of acetylated low-density lipoprotein, existence of Weibel-Palade bodies and several EC markers. MAECs exhibited tube formation activity both in vitro and in vivo. Furthermore, crucially, tumor necrosis factor alpha, an inflammatory cytokine, promoted lymphocyte adhesion to MAECs, suggesting that MAECs may facilitate the study of atherosclerosis and local inflammatory reactions in vitro. CONCLUSION: We describe the morphological and cell biological characteristics of MAEC, providing strong evidence that it retained endothelial properties. This novel cell line can be a useful tool for studying the biology of ECs.

Animals↗

PubMed2006

Up-regulated PAR-2-mediated salivary secretion in mice deficient in muscarinic acetylcholine receptor subtypes.

Protease-activated receptor-2 (PAR-2) is expressed in the salivary glands and is expected to be a new target for the treatment of exocrine dysfunctions, such as dry mouth; however, the salivary secretory mechanism mediated by PAR-2 remains to be elucidated. Therefore, mechanism of the PAR-2-mediated salivary secretion was investigated in this study. We found that a PAR-2 agonist peptide, SLIGRL-OH, induced salivary flow in vivo and dose-dependent increase in [Ca(2+)](i) submandibular gland (SMG) acinar cells in wild-type (WT) mice and mice lacking M(3) or both M(1) and M(3) muscarinic acetylcholine receptors (mAChRs), whereas secretions in PAR-2 knockout (PAR-2KO) mice were completely abolished. The saliva composition secreted by SLIGRL-OH was similar to that secreted by mAChR stimulation. Ca(2+) imaging in WT acinar cells and beta-galactosidase staining in PAR-2KO mice, in which the beta-galactosidase gene (LacZ) was incorporated into the disrupted gene, revealed a nonubiquitous, sporadic distribution of PAR-2 in the SMG. Furthermore, compared with the secretion in WT mice, PAR-2-mediated salivary secretion and Ca(2+) response were enhanced in mice lacking M(3) or both M(1) and M(3) mAChRs, in which mAChR-stimulated secretion and Ca(2+) response in acinar cells were severely impaired. Although the mechanism underlying the enhanced PAR-2-mediated salivary secretion in M(3)-deficient mice is not clear, the result suggests the presence of some compensatory mechanism involving PAR-2 in the salivary glands deficient in cholinergic activation. These results indicate that PAR-2 present in the salivary glands mediates Ca(2+)-dependent fluid secretion, demonstrating potential usefulness of PAR-2 as a target for dry mouth treatment.

Animals↗

PubMed2006

Involvement of cAMP response element-binding protein activation in salivary secretion.

OBJECTIVE: Saliva secretion is mediated by cAMP and the calcium signaling pathway in salivary acinar cells. The PKA signaling pathway plays an important role in protein secretion through the activation of cAMP, in fluid secretion through the elevation of intracellular calcium and in the activation of cAMP response element-binding protein (CREB), which is involved in these signaling cascades. In this study, we investigated whether the activation of CREB plays a part in the salivary secretion in mice. METHODS: We examined CREB activation by assessing phosphorylation at the serine-133 position using Western blotting. RESULTS: Carbachol (a muscarinic acetylcholine agonist) and isoproterenol (a beta-adrenergic agonist) markedly activated CREB in parotid acinar cells. Carbachol and isoproterenol-induced CREB phosphorylation was blocked by atropine (a muscarinic acetylcholine antagonist) and propranolol (a beta-adrenergic antagonist), respectively. The PKA inhibitor H89 inhibited CREB activation, but the PLC inhibitor U73122 did not. Moreover, carbachol- and isoproterenol-stimulated amylase secretion from parotid acinar cells was inhibited by H89 and adenoviral dominant-negative CREB. CONCLUSION: These results indicate that the muscarinic and beta-adrenergic activation of CREB was mediated through the PKA pathway and that CREB is involved in protein secretion from parotid acinar cells.

Adrenergic beta-Agonists↗

PubMed2004

Expression profiles of polyhydroxyalkanoate synthesis-related genes in Paracoccus denitrificans.

A facultative methylotrophic bacterium, Paracoccus denitrificans can synthesize polyhydroxyalkanoate acids (PHA) from various alcohols. Recently, six genes, phaA, B, C, P, R, and Z, related to PHA synthesis have been cloned and characterized. PHA synthesis and the expression of phaA, B, C, P, R, and Z in P. denitrificans were examined at the transcriptional and translational levels under both nitrogen-sufficient and nitrogen-deficient conditions. The results showed that PHA synthesis is not regulated at the mRNA or protein level in phaA, B, and C. We also observed the condensation of acetyl-coenzyme A (acetyl-CoA) in the cells by high-performance liquid chromatography (HPLC). The results suggest that the amount of acetyl-CoA would regulate PHA synthesis. Finally, we discuss a possible regulation mechanism for PHA synthesis in P. denitrificans.

Journal Article↗

PubMed2002

A repressor protein, PhaR, regulates polyhydroxyalkanoate (PHA) synthesis via its direct interaction with PHA.

Phasins (PhaP) are predominantly polyhydroxyalkanoate (PHA) granule-associated proteins that positively affect PHA synthesis. Recently, we reported that the phaR gene, which is located downstream of phaP in Paracoccus denitrificans, codes for a negative regulator involved in PhaP expression. In this study, DNase I footprinting revealed that PhaR specifically binds to two regions located upstream of phaP and phaR, suggesting that PhaR plays a role in the regulation of phaP expression as well as autoregulation. Many TGC-rich sequences were found in upstream elements recognized by PhaR. PhaR in the crude lysate of recombinant Escherichia coli was able to rebind specifically to poly[(R)-3-hydroxybutyrate] [P(3HB)] granules. Furthermore, artificial P(3HB) granules and 3HB oligomers caused the dissociation of PhaR from PhaR-DNA complexes, but native PHA granules, which were covered with PhaP or other nonspecific proteins, did not cause the dissociation. These results suggest that PhaR is able to sense both the onset of PHA synthesis and the enlargement of the granules through direct binding to PHA. However, free PhaR is probably unable to sense the mature PHA granules which are already covered sufficiently with PhaP and/or other proteins. An in vitro expression experiment revealed that phaP expression was repressed by the addition of PhaR and was derepressed by the addition of P(3HB). Based on these findings, we present here a possible model accounting for the PhaR-mediated mechanism of PHA synthesis. Widespread distribution of PhaR homologs in short-chain-length PHA-producing bacteria suggests a common and important role of PhaR-mediated regulation of PHA synthesis.

Bacterial Proteins↗