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Yusei Miyamoto

Publications and source records attributed to Yusei Miyamoto.

9 recordsLinked to original sources
PubMed2003

Decrease in expression of alpha 5 beta 1 integrin during neuronal differentiation of cortical progenitor cells.

Neuronal differentiation of embryonic neural progenitor cells is regulated by both intrinsic and extrinsic signals. Since dynamic changes in cell shape typify neuronal differentiation, cell adhesion molecules could be relevant to this process. Although it has been reported that fibronectin-integrin interactions are important for the proliferation of neural progenitor cells, little is known about the contribution of integrins to neuronal differentiation. In order to address this shortfall, we examined integrin expression on cortical progenitor cells by using immunohistochemistry and FACS analysis of cells in which GFP expression was driven by regulatory (promoter) regions of the nestin gene (nestin-GFP(+)). We here report that high levels of nestin promoter activity correlated with high expression levels of alpha(5)beta(1) integrin (alpha(5)beta(1)(high) cells). FACS analysis of nestin-GFP(+) cortical cells revealed an additional subpopulation with reduced expression of alpha(5)beta(1) integrin (alpha(5)beta(1)(low) cells). The size of the alpha(5)beta(1)(low) subpopulation increased during cortical development. To investigate the correlation between integrin and neuronal differentiation, nestin-GFP(+) cortical progenitor cells were sorted into alpha(5)beta(1)(high) or alpha(5)beta(1)(low) populations, and each potential to differentiate was analyzed. We show that the nestin-GFP(+) alpha(5)beta(1)(high) population corresponded to broadly multipotential neural progenitor cells, whereas nestin-GFP(+) alpha(5)beta(1)(low) cells appeared to be committed to a neuronal fate. These findings suggest that alpha(5)beta(1) expression on cortical progenitor cells is developmentally regulated and its downregulation is involved in the process of neuronal differentiation.

Animals↗
PubMed2003

Nonrenewal of neurons in the cerebral neocortex of adult macaque monkeys.

The concept that, after developmental periods, neocortical neurons become numerically stable and are normally nonrenewable has been challenged by a report of continuous neurogenesis in the association areas of the cerebral cortex in the adult Macaque monkey. Therefore, we have reexamined this issue in two different Macaque species using the thymidine analog bromodeoxyuridine (BrdU) as an indicator of DNA replication during cell division. We found several BrdU+/NeuN+ (neuronal nuclei) double-labeled cells, but cortical neurons, distinguished readily by their size and cytological and immunohistochemical properties, were not BrdU positive. We examined in detail the frontal cortex, where it is claimed that the largest daily addition of neurons has been made, but did not see migratory streams or any sign of addition of new neurons. Thus, we concluded that, in the normal condition, cortical neurons of adult primates, similar to other mammalian species, are neither supplemented nor renewable.

Age Factors↗
PubMed2003

NMDA receptors in cortical development are essential for the generation of coordinated increases in [Ca2+](i) in "neuronal domains".

Spontaneous correlated activity regulates the precision of developing neural circuits. A synchronized elevation of intracellular calcium ion concentration, [Ca(2+)](i), occurred in 5-50 adjacent neurons--known as a "neuronal domain"--in developing neocortex. This coordinated response of neuronal cells is mediated by the diffusion of inositol trisphosphate (IP(3)) via gap-junction channels. In this study, we utilized the N-methyl-D-aspartate (NMDA)-type glutamate receptor epsilon 2 (GluR epsilon 2/NR2B)(-/-) mouse, which does not possess any functional NMDA receptors in the developing neocortex, and showed that NMDA receptors are essential for the generation of "neuronal domains". First, the frequency of spontaneously occurring neuronal domains in brain slices from GluR epsilon 2(-/-) mice was significantly reduced compared to that seen in brain slices from wild-type mice. Secondly, IP(3) injection into a single neuron in a cortical slice from a GluR epsilon 2(-/-) brain resulted in very few neuronal domains being observed, but an injection similarly made into a neuron in a wild-type slice promptly resulted in neuronal domains. Even in the GluR epsilon 2(-/-) brain, the elevation of intracellular [Ca2+](i) was observed frequently in single neurons and microinjection of IP(3) produced an elevation of [Ca2+](i) in the injected cells. These results suggest that the diffusion of IP(3) into the surrounding neurons via gap junctions is almost completely absent in the GluR epsilon 2(-/-) brain. Our results may reflect the critical role of NMDA receptors in the formation of cortical circuitry, probably via the regulation of gap-junction channels between immature cortical neurons.

Animals↗
PubMed2002

A glycine receptor antagonist, strychnine, blocked NMDA receptor activation in the neonatal mouse neocortex.

The NMDA receptor (NMDAR) is a Ca (2+)-permeable cation channel that plays a critical role in neural network formation during brain development. Since it is blocked in a voltage-dependent manner by extracellular Mg(2+), in order for the NMDA to be activated, the membrane must be strongly depolarized. Immature neurons in the developing neocortex can be depolarized by ligand-gated Cl(-) channels, such as the glycine receptor (GlyR) or GABA(A) receptor (GABA(A) R). We here assess the contribution of GlyRs to Ca(2+) influx via NMDARs in neonatal mouse cortical neurons. The GlyR antagonist, strychnine, was more effective in suppressing postsynaptic Ca(2+) influx than the GABA(A) R antagonist, picrotoxin, suggesting greater potentiation of NMDARs by GlyRs than by GABA(A) Rs. The GlyR, known to be endogenously activated at this stage, may play a critical role in neocortical development.

Animals↗
PubMed2002

Oxidation of Ikappa Balpha at methionine 45 is one cause of taurine chloramine-induced inhibition of NF-kappa B activation.

A band shift of IkappaBalpha was observed in Western blots with Jurkat cells treated with 1 mm taurine chloramine (TauCl) for 1 h. TauCl treatment inhibited tumor necrosis factor alpha (TNFalpha)-initiated nuclear factor kappaB (NF-kappaB) activation. TauCl did not inhibit either the upstream of IkappaB kinase (IKK) activation or IKK itself but did inhibit NF-kappaB activation induced by IKK overexpression. Deletion experiments showed that a TauCl modification site causing the band shift of IkappaBalpha is Met45. High performance liquid chromatography and mass spectrometry analyses of a small peptide containing Met45 revealed that TauCl oxidizes Met45. A mutant of IkappaBalpha whose Met45 was converted to alanine did not generate a band shift upon TauCl treatment and degraded in response to TNFalpha stimulation. However, a reporter assay revealed that NF-kappaB-dependent luciferase expression was not fully recovered in cells transfected with this mutant. These results indicate that Met45 oxidation of IkappaBalpha is a molecular mechanism underlying the TauCl-induced inhibition of NF-kappaB activation. A similar band shift was observed when HL-60 cells expressing myeloperoxidase were treated with 100 microm hydrogen peroxide for 5 min. When rat neutrophils were incubated with bacteria, intracellular taurine decreased interleukin-8 production. Therefore, taurine may help suppress excessive inflammatory reaction in neutrophils.

Amino Acid Substitution↗
PubMed2002

Characteristics of lysophosphatidylcholine in its inhibition of taurine uptake by human intestinal Caco-2 cells.

The characteristics of lysophosphatidylcholine (LPC) in its inhibition of the taurine uptake by human intestinal Caco-2 cells were investigated. By treating the cells with 200 microM of LPC, the taurine uptake was rapidly decreased by approximately 60%. This decrease was accompanied by an increase in the Km value for the uptake. A rapid uptake of LPC itself by the cells was also observed. The inhibitory activity of LPC was specific to the uptake of taurine and certain amino acids, while the uptake of glucose, glutamic acid and peptide (glycylglutamine) was not affected by LPC. The activity was dependent on the structure of a polar head and the bound fatty acid. The phosphorylcholine residue was likely to have played an important role, and surface active LPC with fatty acids of C14 or longer was highly inhibitory. These results suggest that the interaction of LPC with the taurine transporter in the intestinal cell membrane was the cause of the reduced taurine uptake.

Biological Transport↗
PubMed2002

Osmosensitive taurine transporter expression and activity in human corneal epithelial cells.

PURPOSE: To characterize in SV40-immortalized human corneal epithelial cells (tHCEC) osmosensitive taurine transporter gene and protein expression as well as its functional activity. To evaluate whether medium supplementation with taurine improves cell viability during a hypertonic challenge. METHODS: tHCEC were preincubated for up to 48 hours in hypertonic DMEM medium (i.e., up to 500 mosmol/kg). Taurine uptake was monitored through measurements of intracellular [3H]taurine accumulation. Gene and protein expression was detected by Northern and Western blot analyses, respectively. An amino acid analyzer measured intracellular cold taurine content. The live/dead assay evaluated with confocal microscopy determined cell viability. RESULTS: Na+-dependent taurine uptake occurred in an isotonic (310 mosmol/kg) medium. The apparent Michaelis-Menten constant, K(t), for taurine was 4.6 micro M, and uptake increased as a function of exposure time and rises in osmolality. Exposure for 12 hours to a 450 mosmol/kg medium increased uptake by 4.1-fold. However, after 48 hours of exposure to this medium, taurine uptake returned to its isotonic level. With time, biphasic changes occurred in taurine transporter gene and protein expression and taurine transport activity as well as elevating intracellular taurine content by 4.5-fold. Taurine medium supplementation for 48 hours improved cell viability. CONCLUSIONS: tHCEC express Na+-dependent osmosensitive taurine transport activity. The hypertonic-induced biphasic effects on gene and protein expression as well as transport activity suggest feedback regulation of these responses. Rises in intracellular taurine do not appear to be essential for osmoregulation, but instead enhance cell survival perhaps through a membrane stabilizer or an antioxidant effect.

Blotting, Northern↗
PubMed2002

Suppression of the TNFalpha-induced increase in IL-1alpha expression by hypochlorite in human corneal epithelial cells.

PURPOSE: In response to injury, activated neutrophils release tumor necrosis factor (TNF)-alpha and myeloperoxidase (MPO). TNFalpha in turn causes human corneal epithelial cells to secrete interleukin (IL)-1alpha, whereas MPO results in formation of HClO/OCl(-). The effect of HClO/OCl(-) on the expression of the IL-1alpha gene and protein is unknown. The current study was undertaken to examine in immortalized human corneal epithelial cells whether NaOCl alters TNFalpha-induced increases in expression of IL-1alpha gene and protein. METHODS: Semiquantitative RT-PCR and ELISA characterized IL-1alpha gene and protein expression, respectively. TNFalpha-induced nuclear transfer of nuclear factor (NF)-kappaB was measured by electrophoretic mobility shift assay (EMSA). The alpha isoform of inhibitory protein kappaB (IkappaBalpha) was identified by Western blot analysis. RESULTS: Exposure to NaOCl (0.75 mM) for 10 minutes caused suppression of TNFalpha-induced increases in IL-1alpha mRNA and protein, declines in NFkappaB nuclear transfer, and a modification of IkappaBalpha, based on a bandshift detected by Western blot analysis. Modified IkappaBalpha became resistant to TNFalpha-induced proteolysis. Methionine sulfoxide reductase A (MsrA, 10 micro M) eliminated the NaOCl-induced IkappaBalpha bandshift. CONCLUSIONS; NaOCl oxidizes IkappaBalpha at methionine residues and thereby suppresses dissociation of IkappaBalpha from NFkappaB. Decreased dissociation could in turn suppress TNFalpha-induced activation of NFkappaB, resulting in declines in expression of IL-1alpha gene and protein. These effects suggest that release of HClO/OCl(-) in vivo by activated neutrophils may counterbalance TNFalpha-induced NFkappaB-dependent secretion if IL-1alpha and suppress an excessive inflammatory reaction.

Alkaline Phosphatase↗