Biomedical subjects
Y Mori
Publications and source records attributed to Y Mori.
2-Aminoethoxydiphenyl borate (2-APB) inhibits capacitative calcium entry independently of the function of inositol 1,4,5-trisphosphate receptors.
Capacitative calcium entry (CCE), the mechanism that replenishes intracellular calcium stores after depletion, is essential to intracellular calcium signaling. CCE is mediated by the channels in the plasma membrane generally referred to as "store operated channels (SOCs)". However, the molecular identity of the SOCs has never been determined, and the mechanism of the activation of SOCs remains to be elucidated. Recent studies have demonstrated that 2-aminoethoxydiphenyl borate (2-APB), which has been found to be an antagonist of inositol 1,4,5-trisphosphate receptors (IP3Rs), inhibits CCE, suggesting that IP3Rs channel activity is essential to the generation of CCE. However, CCE has also been reported to occur normally in IP3R-deficient cells. In order to resolve this discrepancy, we investigated the effect of 2-APB on CCE in IP3Rs-deficient cells. In response to store depletion with thapsigargin or N,N,N',N'-tetrakis (2-pyridylmethyl) ethylene diamine (TPEN), CCE was generated in IP3Rs-deficient cells the same as in wild-type cells, however, 2-APB abolished CCE in IP3Rs-deficient cells, despite the fact that this cell line does not possess functional IP3Rs. We also examined the effect of 2-APB on several types of TRP Ca2+ channels, which exhibit properties similar to those of SOCs. 2-APB had a different inhibitory effect on spontaneous and thapsigargin-induced Ba2+ influx in cells that transiently expressed individual TRP subtypes. These results suggest that the channel activity of IP3Rs is not essential to the generation of CCE in this cell line and that 2-APB inhibits CCE independently of the function of IP3Rs.
[Dynamic instability of biological system under natural and experimental gravity condition].
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A secure method of nasal endotracheal tube stabilization with suture and rubber tube.
A new method of stabilizing the nasal endotrascheal tube was described. The tube was secured to the anterior portion of the nasal septum with braided silk thread, which was tightened over the rubber tube to keep air route of the cuff open. There found no complications such as unplanned extubation, necrosis and infections of the nasal septum.
[Structure and function of Ca2+ channels].
Ca(2+) influx across plasma membrane is critical to evoke increase in intracellular Ca(2+) concentration ([Ca(2+)](i)) that controls various physiological and pathological responses. Recently, diverse Ca(2+) channels responsible for Ca(2+) influx have been identified. In this review, we attempt to overview molecular entities of Ca(2+) channels, and their structure, function, and biological roles.
Antagonistic regulation of type I collagen gene expression by interferon-gamma and transforming growth factor-beta. Integration at the level of p300/CBP transcriptional coactivators.
Among the extracellular signals that modulate the synthesis of collagen, transforming growth factor-beta (TGF-beta) and interferon-gamma (IFN-gamma) are preeminent. These two cytokines exert antagonistic effects on fibroblasts, and play important roles in the physiologic regulation of extracellular matrix turnover. We have shown previously that in normal skin fibroblasts, TGF-beta positively regulates alpha2(I) procollagen gene (COL1A2) promoter activity through the cellular Smad signal transduction pathway. In contrast, IFN-gamma activates Stat1alpha, down-regulates COL1A2 transcription, and abrogates its stimulation induced by TGF-beta. The level of integration of the two pathways mediating antagonistic collagen regulation is unknown. We now report that IFN-gamma abrogates TGF-beta-stimulated COL1A2 transcription in fibroblasts by inhibiting Smad activities. IFN-gamma appears to induce competition between activated Stat1alpha and Smad3 for interaction with limiting amounts of cellular p300/CBP. Overexpression of p300 restored COL1A2 stimulation by TGF-beta in the presence of IFN-gamma, and potentiated IFN-gamma-dependent positive transcriptional responses. In contrast to fibroblasts, in U4A cells lacking Jak1 and consequently unable to activate Stat1alpha-mediated responses, IFN-gamma failed to repress TGF-beta-induced transcription. These results indicate that as essential coactivators for both Smad3 and Stat1alpha, nuclear p300/CBP integrate signals that positively or negatively regulate COL1A2 transcription. The findings implicate a novel mechanism to account for antagonistic interaction of Smad and Jak-Stat pathways in regulation of target genes. In fibroblasts responding to cytokines with opposing effects on collagen transcription, the relative levels of cellular coactivators, and their interaction with regulated transcription factors, may govern the net effect.
Characterization of mouse Ire1 alpha: cloning, mRNA localization in the brain and functional analysis in a neural cell line.
In yeast, an endoplasmic reticulum (ER)-associated protein, Ire1p, is believed to initiate the unfolded protein response (UPR), that is responsible for protein folding in the ER under stressed conditions. Two mammalian homologs of Ire1p have been identified, Ire1 alpha and Ire1 beta. We have previously reported that familial Alzheimer's disease linked presenilin-1 variants downregulate the signaling pathway of the UPR by affecting the phosphorylation of Ire1 alpha. In the present study, we cloned the mouse homolog of Ire1 alpha for generating genetically modified mice. Ire1 alpha was ubiquitously expressed in all mouse tissues examined, and was expressed preferentially in neuronal cells in mouse brain. This led us to investigate the effects of the downregulation of the UPR on the survival of neuronal cells under conditions of ER stress. Morphological and biochemical studies using a dominant-negative form of mouse Ire1 alpha have revealed that cell death caused by ER stress can be attributed to apoptosis, and that the downregulation of the UPR enhances the apoptotic process in the mouse neuroblastoma cell line, Neuro2a. Our results indicate that genetically modified mice such as transgenic mice with a dominant-negative form of Ire1 alpha might provide further understanding of the pathogenic mechanisms of Alzheimer's disease and other neurodegenerative disorders.
Effects of both the emotional behavior and feeding conditions on the circulating plasma volume and plasma glucose levels in cats.
Influence of hypothalamically induced emotional behavior on the circulating plasma volume, plasma levels of glucose, epinephrine (E), norepinephrine (NE), dopamine (DA) and cortisol were examined in awake cats under both fasted and fed conditions. Restlessness was evoked intermittently for 6 h by electrical stimulation of the anteromedial hypothalamus (AMH). Blood was sampled immediately before, 1 h after and 6 h after the start of stimulation. Changes in the plasma volume was calculated by changes of hemoglobin (Hb) and hematocrit (Ht). As the control group, another 7 cats with electrodes implanted but unstimulated were identically treated under both fasted and fed conditions. Both E and glucose levels in restlessness group once markedly increased after 1 h and then tended to decrease after 6 h, whereas NE levels in restlessness group increased after 1 h and further increased after 6 h, whether cats were fasted or fed. DA levels increased under the fasted condition of restlessness. The cortisol level markedly increased in both fasted and fed restlessness groups. The plasma volume in control group increased under the fed condition, while in restlessness group it decreased remarkably and tended to decrease more in a fasted state than in a fed state. These results indicated that AMH induced restlessness elicited marked sympatho-adrenal activation, hyperglycemia and hemoconcentration, whether cats were fasted or fed. Relationship among such responses, and the difference in responses between fasted and fed conditions were also discussed in the paper.
Serine 6 of Lck tyrosine kinase: a critical site for Lck myristoylation, membrane localization, and function in T lymphocytes.
Lck is a member of the Src family kinases expressed predominantly in T cells, and plays a pivotal role in TCR-mediated signal transduction. Myristoylation of glysine 2 in the N-terminal Src homology 4 (SH4) domain of Lck is essential for membrane localization and function. In this study, we examined a site within the SH4 domain of Lck regulating myristoylation, membrane localization, and function of Lck. A Lck mutant in which serine 6 (Ser6) was substituted by an alanine was almost completely cytosolic in COS-7 cells, and this change of localization was associated with a drastic inhibition of myristoylation in this mutant. To assess the role of Ser6 of Lck in T cell function, we established stable transfectants expressing various Lck mutants using Lck-negative JCaM1 cells. The Lck mutant of Ser6 to alanine, most of which did not target to the plasma membrane, was not able to reconstitute TCR-mediated signaling events in JCaM1 cells, as analyzed by tyrosine phosphorylation of intracellular proteins and CD69 expression. These results demonstrate that Ser6 is a critical factor for Lck myristoylation, membrane localization, and function in T cells, presumably because the residue is important for N-myristoyl transferase recognition.
Angiotensin II initiates tyrosine kinase Pyk2-dependent signalings leading to activation of Rac1-mediated c-Jun NH2-terminal kinase.
Ca(2+)-sensitive tyrosine kinase Pyk2 was shown to be involved in angiotensin (Ang) II-mediated activation of extracellular signal-regulated kinase (ERK) via transactivation of epidermal growth factor receptor (EGF-R). In this study, we tested the involvement of Pyk2 and EGF-R in Ang II-induced activation of JNK and c-Jun in cardiac fibroblasts. Ang II markedly stimulated JNK activities, which were abolished by genistein and intracellular Ca(2+) chelators but partially by protein kinase C depletion. Inhibition of EGF-R did not affect Pyk2 and JNK activation by Ang II. Stable transfection with a dominant negative (DN) mutant for Pyk2 (PKM) completely blocked JNK activation by Ang II. DN mutants of Rac1 (DN-Rac1) and MEK kinase (DN-MEKK1) also abolished it, whereas those of Cdc42, RhoA, and Ha-Ras had no effect. Induction of c-Jun gene transcription by Ang II was abolished in PKM, DN-Rac1, and DN-MEKK1, in which Ang II-induced binding of ATF2/c-Jun heterodimer to the activator protein-1 sequence at -190 played a key role. These results suggest that 1) in cardiac fibroblasts activation of JNK and c-Jun by Ang II is initiated by Pyk2-dependent signalings but not by downstream signals of EGF-R or Ras, 2) Rac1 but not Cdc42 is required for JNK activation by Ang II upstream of MEKK1, and 3) ATF-2/c-Jun binding to the activator protein-1 sequence at -190 plays a key role for induction of c-Jun gene by Ang II.
[Efficacy of the extra-corporeal lung assist (ECLA) on the alveolar hemorrhage in a case with microscopic polyarteritis nodosa].
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Preparation of a monoclonal antibody to N(epsilon)-(Hexanonyl)lysine: application to the evaluation of protective effects of flavonoid supplementation against exercise-induced oxidative stress in rat skeletal muscle.
The monoclonal antibody to N(epsilon)-(hexanonyl)lysine (HEL), a novel adduct formed by the reaction of linoleic acid hydroperoxide and lysine, has been prepared and characterized. The obtained antibody specifically recognized the HEL moiety. Using the monoclonal antibody, we evaluated the protective effects of feeding eriocitrin, which is one of flavonoids in lemon fruit, on oxidative modification induced by exercise in rats. The supplementation of eriocitrin significantly suppressed the increase in HEL in the skeletal muscle by exercise. The result suggests that the determination of HEL may be a good method for evaluation of the protective effect of beneficial food factors against oxidative stress.
Modulation of endogenous Smad expression in normal skin fibroblasts by transforming growth factor-beta.
Transforming growth factor-beta (TGF-beta) stimulation of collagen synthesis plays a fundamental role in physiological tissue repair as well as pathological fibrosis. Members of the SMAD family of intracellular proteins are phosphorylated by TGF-beta receptors and convey signals to specific TGF-beta-inducible genes. Ligand binding initiates signaling through the SMAD pathway, but it is unknown how signaling is terminated. The expression and regulation of Smads have been characterized mostly in transformed cells using transient expression systems. In this study, we investigated the physiological regulation of endogenous Smads by TGF-beta in nontransformed normal skin fibroblasts in vitro. Treatment with TGF-beta resulted in time- and dose-dependent translocation of SMAD3 and SMAD4 from the cytoplasm to the nucleus. The levels of SMAD3 and Smad3 mRNA were profoundly down-regulated by TGF-beta1 or TGF-beta3 in a time-dependent manner, whereas expression of antagonistic Smad7 was rapidly and transiently induced. The stability of Smad3 mRNA transcripts was unaffected by TGF-beta. Cycloheximide prevented the inhibition of Smad3, but not the induction of Smad7, mRNA expression by TGF-beta1, identifying Smad7 as an immediate-early gene target of TGF-beta in fibroblasts. In Smad4-deficient breast cancer cells, TGF-beta failed to modulate Smad expression, suggesting that SMADs mediate their own regulation in response to ligand. These results demonstrate that TGF-beta not only triggers functional activation of the SMAD signaling cascade in primary skin fibroblasts, but also simultaneously exerts potent effects on endogenous SMAD expression and intracellular trafficking. Taken together with recent reports implicating ubiquitination in SMAD turnover, these findings indicate the existence of multiple levels of control for modulating SMAD-mediated TGF-beta signaling in fibroblasts.
Recognition of chitooligosaccharides and their N-acetyl groups by putative subsites of chitin deacetylase from a deuteromycete, Colletotrichum lindemuthianum.
The reaction pattern of an extracellular chitin deacetylase from a Deuteromycete, Colletotrichum lindemuthianum ATCC 56676, was investigated by use of chitooligosaccharides [(GlcNAc)(n)(), n = 3-6] and partially N-deacetylated chitooligosaccharides as substrates. When 0.5% of (GlcNAc)(n)() was deacetylated, the corresponding monodeacetylated products were initially detected without any processivity, suggesting the involvement of a multiple-chain mechanism for the deacetylation reaction. The structural analysis of these first-step products indicated that the chitin deacetylase strongly recognizes a sequence of four N-acetyl-D-glucosamine (GlcNAc) residues of the substrate (the subsites for the four GlcNAc residues are defined as -2, -1, 0, and +1, respectively, from the nonreducing end to the reducing end), and the N-acetyl group in the GlcNAc residue positioned at subsite 0 is exclusively deacetylated. When substrates of a low concentration (100 microM) were deacetylated, the initial deacetylation rate for (GlcNAc)(4) was comparable to that of (GlcNAc)(5), while deacetylation of (GlcNAc)(3) could not be detected. Reaction rate analyses of partially N-deacetylated chitooligosaccharides suggested that subsite -2 strongly recognizes the N-acetyl group of the GlcNAc residue of the substrate, while the deacetylation rate was not affected when either subsite -1 or +1 was occupied with a D-glucosamine residue instead of GlcNAc residue. Thus, the reaction pattern of the chitin deacetylase is completely distinct from that of a Zygomycete, Mucor rouxii, which produces a chitin deacetylase for accumulation of chitosan in its cell wall.
Enzymatic synthesis of 1,3,6,8-tetrahydroxynaphthalene solely from malonyl coenzyme A by a fungal iterative type I polyketide synthase PKS1.
The Colletotrichum lagenarium PKS1 gene encoding iterative type I polyketide synthase of 1,3,6,8-tetrahydroxynaphthalene (T4HN) was overexpressed in Aspergillus oryzae. SDS-PAGE analysis of the cell-free extract prepared from the transformant showed an intense band of 230000 which corresponded to the molecular weight of the deduced PKS1 protein. By using this cell-free extract, in vitro synthesis of T4HN was successfully confirmed as the first example of the fungal multi-aromatic ring polyketide synthase activity ever detected. To identify the starter unit for T4HN synthesis, (14)C-labeled acetyl CoA and/or (14)C-labeled malonyl CoA were used as substrates for T4HN synthase reaction. Observed was the incorporation of (14)C label into T4HN solely from malonyl CoA even in the absence of acetyl CoA and not from acetyl CoA. This in vitro result unambiguously identified that malonyl CoA serves as the starter as well as extender units in the formation of T4HN by fungal polyketide synthase PKS1.
Reduced voltage sensitivity of activation of P/Q-type Ca2+ channels is associated with the ataxic mouse mutation rolling Nagoya (tg(rol)).
Recent genetic analyses have revealed an important association of the gene encoding the P/Q-type voltage-dependent Ca(2+) channel alpha(1A) subunit with hereditary neurological disorders. We have identified the ataxic mouse mutation, rolling Nagoya (tg(rol)), in the alpha(1A) gene that leads to a charge-neutralizing arginine-to-glycine substitution at position 1262 in the voltage sensor-forming segment S4 in repeat III. Ca(2+) channel currents in acutely dissociated Purkinje cells, where P-type is the dominant type, showed a marked decrease in slope and a depolarizing shift by 8 mV of the conductance-voltage curve and reduction in current density in tg(rol) mouse cerebella, compared with those in wild-type. Compatible functional change was induced by the tg(rol) mutation in the recombinant alpha(1A) channel, indicating that a defect in voltage sensor of P/Q-type Ca(2+) channels is the direct consequence of the tg(rol) mutation. Furthermore, somatic whole-cell recording of mutant Purkinje cells displayed only abortive Na(+) burst activity and hardly exhibited Ca(2+) spike activity in cerebellar slices. Thus, in tg(rol) mice, reduced voltage sensitivity, which may derive from a gating charge defect, and diminished activity of the P-type alpha(1A) Ca(2+) channel significantly impair integrative properties of Purkinje neurons, presumably resulting in locomotor deficits.
Smad-dependent stimulation of type I collagen gene expression in human skin fibroblasts by TGF-beta involves functional cooperation with p300/CBP transcriptional coactivators.
Transforming growth factor-beta (TGF-beta) stimulation of Type I collagen gene (COL1A2) transcription involves the Smad signal transduction pathway, but the mechanisms of Smad-mediated transcriptional activation are not fully understood. We now demonstrate that the ubiquitous transcriptional coactivators p300 and CREB-binding protein (CBP) enhanced basal as well as TGF-beta- or Smad3-induced COL1A2 promoter activity, and stimulated the expression of endogenous Type I collagen. The adenoviral E1A oncoprotein abrogated stimulation of COL1A2 activity in transfected fibroblasts, and reduced the basal level of collagen gene expression. This effect was due to specific interaction of E1A with cellular p300/CBP because (a) a mutant form of E1A defective in p300 binding failed to abrogate stimulation, and (b) forced expression of p300/CBP restored the ability of TGF-beta to stimulate COL1A2 promoter activity in the presence of E1A. The effect of p300 on COL1A2 transcription appeared to be due, in part, to its intrinsic acetyltransferase activity, as stimulation induced by a histone acetyltransferase-deficient mutant p300 was substantially reduced. Transactivation of COL1A2 by p300 involved the Smad signaling pathway, as Smad4-deficient cells failed to respond to p300, and stimulation was rescued by overexpression of Smad4. Furthermore, minimal constructs containing only the Smad-binding CAGACA element of COL1A2 were transactivated by p300 in the presence of TGF-beta. These results indicate, for the first time, that the multifunctional p300/CBP coactivators play a major role in Smad-dependent TGF-beta stimulation of collagen gene expression in fibroblasts. Oncogene (2000) 19, 3546 - 3555
Anisotropic superconducting gap in the spin-triplet superconductor Sr2RuO4: evidence from a Ru-NQR study
We have investigated a gap structure in the spin-triplet superconductor Sr2RuO4 through the measurement of the 101Ru nuclear spin-lattice relaxation rate (101)(1/T1) down to 0.09 K at zero magnetic field. In the superconducting state, 1/T1 in a high-quality sample with T(c) approximately 1.5 K exhibits a sharp decrease without the coherence peak, followed by a T3 behavior down to 0.15 K. This result is in marked contrast to the behavior observed below approximately 0.4 K in samples with lower T(c), where T1T is a constant. This behavior is demonstrated to be not intrinsic. We conclude that the gap structure in Sr2RuO4 is significantly anisotropic, consistent with line-node-like models.