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M Watanabe

Publications and source records attributed to M Watanabe.

At least 271 records · Page 15Linked to original sources
PubMed2001

Two-step regulation of left-right asymmetric expression of Pitx2: initiation by nodal signaling and maintenance by Nkx2.

Pitx2 is left--right (L--R) asymmetrically expressed initially in the lateral plate and later in primordial visceral organs. The transcriptional regulatory mechanisms that underlie L--R asymmetric expression of Pitx2 were investigated. Mouse Pitx2 has a left side-specific enhancer (ASE) that mediates both the initiation and maintenance of L--R asymmetric expression. This element contains three binding sites for the transcription factor FAST. The FAST binding sites function as Nodal-responsive elements and are sufficient for the initiation but not for the maintenance of asymmetric expression. The maintenance requires an Nkx2-5 binding site also present within the ASE. These results suggest that the left-sided expression of Pitx2 is directly initiated by Nodal signaling and is subsequently maintained by Nkx2. Such two-step control may represent a general mechanism for gene regulation during development.

Animals↗
PubMed2001

Cadmium-induced abnormality in strains of Euglena gracilis: morphological alteration and its prevention by zinc and cyanocobalamin.

Euglena gracilis is susceptible to cadmium (Cd) at high concentrations. There are no comparative data on cytotoxicity or abnormality of CdCl2 to E. gracilis Z and its achlorophyllous mutant SMZ. The present study examined the cytotoxicity of CdCl2 under continual exposure at levels ranging from sub-ppm to ppm, and assessed the effects of zinc (Zn) or cyanocobalamin (VB12) supplementation on the suppression of Cd-induced abnormal cell proliferation and hypertrophy. With Zn levels restricted to 1 ppm [as Zn++], cell growth of both E. gracilis strains was reduced in proportion to Cd concentration. More abnormal cells (hypertrophied, V-shape and starfish-shape) were observed in both strains at sub-ppm levels of Cd. ZnSO4 supplementation from 2 to 63 ppm significantly suppressed the incidence of Cd-induced abnormality. However, a significant increase in abnormal cells was observed following Zn supplementation at levels of 125 and 250 ppm, which produced remarkable differences in cell morphology. The incidence of abnormal cells varied with supplemented VB12 levels ranging from 4 to 250 ppb in both E. gracilis strains.

Animals↗
PubMed2001

Induction of CD4+ regulatory T cells by 12-O-tetradecanoylphorbol 13-acetate in mice. Part 1: The strain difference in this induction.

12-O-Tetradecanoylphorbol 13-acetate (TPA), an activator of signal transduction, induced antigen-nonspecific regulatory T (Tr) cells for delayed-type hypersensitivity (DTH) in the spleen. A marked strain difference in the induction of Tr cells was observed when the study was performed by using the several strains of mice at 6-8 weeks old. TPA painting induced CD4+Tr cells in C3H/He (H-2k), C3H/HeN (H-2k), DBA/2 (H-2d) and AKR/N (H-2k) mice, but not in C57BL/6 (H-2b), C57BL/10 (H-2b), BALB/c (H-2d) and A/J (H-2a). Regulatory cells were also induced by incubating spleen cells from unprimed mice with TPA in vitro and were seemed to act by the production of soluble factor(s). A downregulatory activity of the soluble factor(s) was abrogated by SXC-1 (anti-IL-10 monoclonal antibody), but not by SXC-2 (anti-IL-10 monoclonal antibody) and anti-IL-4. For purification of the factor(s), we established the T cell hybridoma 4C5-1 by the fusion of spleen cells from TPA-treated C3H/He mice with AKR thymoma Bw5147 cells. The 4C5-1 cells secrete the factor(s) which can inhibit DTH response. The inhibitory activity of the factor(s) could be neutralized by SXC-1, but not by SXC-2, anti-IL-4, anti-IL-6 and anti-TGF-beta antibodies. The factor(s) could not affect the proliferation and IFN-gamma production of alpha s1-casein-specific 3D20 Th1 cells. The factor(s) termed DIF (DTH Inhibitory Factor) may be a novel cytokine, since they have reduced the footpad swelling response by local injection, and have no immune crossreactivity with the DTH regulatory cytokines and no inhibitory activity for in vitro Th1 response.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed2001

Isolation and structures of haterumadioxins A and B, cytotoxic endoperoxides from the Okinawan sponge Plakortis lita.

Cytotoxic endoperoxides, haterumadioxins A (1) and B (2), were isolated from the Okinawan sponge Plakortis lita. Their structures were determined by spectroscopic analysis. The absolute stereostructure of 1 was determined by degradation reactions and the modified Mosher's method. Haterumadioxins showed significant cytotoxicity against 38 human cancer cell lines.

Animals↗
PubMed2001

Characterization of the amino acid transport of new immortalized choroid plexus epithelial cell lines: a novel in vitro system for investigating transport functions at the blood-cerebrospinal fluid barrier.

PURPOSE: To establish and characterize a choroid plexus epithelial cell line (TR-CSFB) from a new type of transgenic rat harboring the temperature-sensitive simian virus 40 (ts SV 40) large T-antigen gene (Tg rat). METHODS: Choroid plexus epithelial cells were isolated from the Tg rat and cultured on a collagen-coated dish at 37 degrees C during the first period of 3 days. Cells were subsequently cultured at 33 degrees C to activate large T-antigen. At the third passage, cells were cloned by colony formation and isolated from other cells using a penicillin cup. RESULTS: Five immortalized cell lines of choroid plexus epithelial cells (TR-CSFB 1 approximately 5) were obtained from two Tg rats. These cell lines had a polygonal cell morphology, expressed the typical choroid plexus epithelial cell marker, transthyretin, and possessed Na+, K+-ATPase on their apical side. TR-CSFBs cells expressed a large T-antigen and grew well at 33 degrees C with a doubling-time of 35 approximately 40 hr. [3H]-L-Proline uptake by TR-CSFB cells took place in an Na+-dependent, ouabain-sensitive, energy-dependent, and concentration-dependent manner. It was also inhibited by alpha-methylaminoisobutylic acid, suggesting that system A for amino acids operates in TR-CSFB cells. When [3H]-L-proline uptake was measured using the Transwell device, the L-proline uptake rate following application to the apical side was five-fold greater than that following application to the basal side. In addition, both Na+-dependent and Na+-independent L-glutamic acid (L-Glu) uptake processes were present in TR-CSFB cells. CONCLUSIONS: Immortalized choroid plexus epithelial cell lines were successfully established from Tg rats and have the properties of choroid plexus epithelial cells, and amino acid transport activity was observed in vivo.

Amino Acids↗
PubMed2001

Interleukin-2-dependent but not independent T-cell lines infected with human T-cell leukemia virus type 1 selectively express CD45RO, a marker for persistent infection in vivo.

Human T-cell leukemia virus type 1 (HTLV-1) is an etiologic agent of adult T-cell leukemia. HTLV-1 is exclusively detected in CD45RO+ T-cells in infected individuals, but CD45RO is weakly expressed in HTLV-1-transformed T-cell lines in vitro. The aim of this study was to investigate the role of CD45RO in the persistent HTLV-1 infection in vivo. Flow cytometry showed that only two out of eight interleukin(IL)-2-independent HTLV-1-transformed T-cell lines expressed CD45RO, whereas all five IL-2-dependent ones expressed CD45RO, and the level of expression was higher in IL-2-dependent than in IL-2-independent cells. The high CD45RO expression in IL-2-dependent cell lines was not due to IL-2, since IL-2 had little effect on the expression of CD45RO in T-cell lines. Using western blotting, we showed that IL-2-dependent HTLV-1-transformed T-cell lines expressed a lower level of expression of the viral transcriptional regulatory protein Tax than IL-2-independent ones, and that the level of expression correlated inversely with that of CD45RO. However, the expression of Tax in one HTLV-1-negative T-cell line little affected the expression of CD45RO, suggesting that Tax at least alone does not suppress the expression of CD45RO in HTLV-1-infected T-cell lines, and that other viral or cellular factor(s) are probably involved in such suppression. Our results suggest that CD45RO+ Tax-low IL-2-dependent T-cell lines in vitro correspond to the persistent HTLV-1-infected cells in vivo, and HTLV-1-infected cells in vivo are immortalized in IL-2-dependent manner.

Cell Line↗
PubMed2001

HLA class-I-restricted and colon-specific cytotoxic T cells from lamina propria lymphocytes of patients with ulcerative colitis.

We established cytotoxic T-lymphocyte (CTL) lines against colonic epithelial cell line from LPLs of patients with ulcerative colitis by continuous stimulation with human lymphocyte antigen A (HLA-A) matched colonic epithelial cell lines and clones from LPLs using polyclonal stimulation with phytohemagglutinin. The established CTL lines and clones showed cytotoxicity against HLA-A-matched colonic epithelial cell line but not against HLA-mismatched colonic epithelial cell lines, and HLA-A-matched lung and esophagus cell lines. The CTL response was HLA class I-restricted and mediated by CD8-positive T-lymphocytes. Moreover, the CTL line showed cytotoxicity against autologous B-LCLs pulsed with peptides extracted from HLA-A-matched colonic epithelial cell line but not against other organ-derived peptides pulsed and unpulsed autologous B-LCLs. CTL lines and clones established from LPLs of patients with ulcerative colitis showed colon-specific and HLA class I-restricted killing of human colonic epithelial cell line, suggesting that these CTLs may play a role in colonic epithelial cell damage in some patients with ulcerative colitis.

Adult↗
PubMed2001

Possible involvement of ERK 1/2 in UVA-induced melanogenesis in cultured normal human epidermal melanocytes.

UV-induced melanogenesis is a well known physiological response of human skin exposed to solar radiation; however, the signaling molecules involved in the stimulation of melanogenesis in melanocytes following UV exposure remain unclear. In this study we induced melanogenesis in vitro in normal human epidermal melanocytes using a single irradiation with UVA at 1 kJ/m2 and examined the potential involvement of mitogen-activated protein kinases (MAPK) as UVA-responsive signaling molecules in those cells. UVA irradiation did not affect the proliferation of melanocytes, but it did increase tyrosinase mRNA expression, which reached a maximum level 4 hr after UVA irradiation. The amount of tyrosinase protein, as quantitated by immunoblotting, was also increased at 24 hr following UVA irradiation. Among the MAPK examined, extracellular signal-related kinase (ERK) 1/2 was phosphorylated within 15 min of UVA irradiation, but no such phosphorylation was observed for c-Jun N-terminal kinases (JNK) or p38. Accordingly, the activity of ERK1/2 was also increased shortly after UVA irradiation. These responses of ERK1/2 to UVA irradiation were markedly inhibited when cells were pre-treated with N-acetyl-L-cysteine, an antioxidant, or with suramin, a tyrosine kinase receptor inhibitor. The formation of (6-4)photoproducts or cyclobutane pyrimidine dimers was not detected in cellular DNA after UVA irradiation. These findings suggest that a single UVA irradiation-induced melanogenesis is associated with the activation of ERK1/2 by upstream signals that originate from reactive oxygen species or from activated tyrosine kinase receptors, but not from damaged DNA.

Acetylcysteine↗
PubMed2001

The role of epigenetic modifications in retinoic acid receptor beta2 gene expression in human prostate cancers.

The retinoic acid receptor (RAR) beta gene is a putative tumor suppressor gene on chromosome 3p24, where a high incidence of loss of heterozygosity is detected in many types of tumors. Retinoic acid suppresses cancer cell growth through binding to RARs, especially RARbeta, indicating a critical role in mediating anticancer effects. Selective loss or down-regulation of RARbeta mRNA and protein has been reported in prostate cancers (PCas), although the mechanisms remain unclear. We investigated the role of epigenetic modification in RARbeta2 gene silencing. Aberrant methylation was detected in 11 of 14 (79%) primary PCas, 9 of 10 (90%) hormone-refractory PCas, and 2 of 4 (50%) PCa cell lines, but not in any normal prostate samples. Chromatin immunoprecipitation assay showed that all RARbeta2-negative cells (LNCaP, PC3, and DU145) were hypoacetylated at both histones H3 and H4. After exposure to 5-aza-2prime;-deoxycytidine treatment, Trichostatin A and all-trans retinoic acid induced partial demethylation, increased accumulation of acetylated histones, and markedly restored the expression of RARbeta2 in RARbeta2-negative cells. These data suggest that the RARbeta2 gene may be one of the frequently silenced genes by epigenetic modifications in PCa.

Base Sequence↗
PubMed2001

The gene mutated in ataxia-ocular apraxia 1 encodes the new HIT/Zn-finger protein aprataxin.

The newly recognized ataxia-ocular apraxia 1 (AOA1; MIM 208920) is the most frequent cause of autosomal recessive ataxia in Japan and is second only to Friedreich ataxia in Portugal. It shares several neurological features with ataxia-telangiectasia, including early onset ataxia, oculomotor apraxia and cerebellar atrophy, but does not share its extraneurological features (immune deficiency, chromosomal instability and hypersensitivity to X-rays). AOA1 is also characterized by axonal motor neuropathy and the later decrease of serum albumin levels and elevation of total cholesterol. We have identified the gene causing AOA1 and the major Portuguese and Japanese mutations. This gene encodes a new, ubiquitously expressed protein that we named aprataxin. This protein is composed of three domains that share distant homology with the amino-terminal domain of polynucleotide kinase 3'- phosphatase (PNKP), with histidine-triad (HIT) proteins and with DNA-binding C2H2 zinc-finger proteins, respectively. PNKP is involved in DNA single-strand break repair (SSBR) following exposure to ionizing radiation and reactive oxygen species. Fragile-HIT proteins (FHIT) cleave diadenosine tetraphosphate, which is potentially produced during activation of the SSBR complex. The results suggest that aprataxin is a nuclear protein with a role in DNA repair reminiscent of the function of the protein defective in ataxia-telangiectasia, but that would cause a phenotype restricted to neurological signs when mutant.

Amino Acid Sequence↗
PubMed2001

BK channel activation by NS-1619 is partially mediated by intracellular Ca2+ release in smooth muscle cells of porcine coronary artery.

1. Effects of NS-1619, an opener of large conductance Ca2+-activated K+ (BK) channel, on intracellular Ca2+ concentration ([Ca2+]i) and membrane potential were examined in single myocytes freshly isolated from porcine coronary artery. 2. Under current clamp mode, the application of 1-30 microM NS-1619 hyperpolarized the membrane in concentration-dependent manner. The NS-1619-induced hyperpolarization was abolished by the presence of 100 nM iberiotoxin. 3. Application of 1-10 microM NS-1619 hyperpolarized the membrane by approximately 6 mV or less but did not change significantly the [Ca2+]i. When membrane hyperpolarization of 12 mV or so was caused by 30 microM NS-1619, [Ca2+]i was unexpectedly increased by approximately 200 nM. This increase in [Ca2+]i and the concomitant outward current activation were also observed under voltage-clamp at holding potential of -40 mV. 4. The increase in [Ca2+]i by 30 microM NS-1619 occurred mainly in peripheral regions than in the centre of the myocytes. The removal of extracellular Ca2+ affected neither the membrane hyperpolarization nor the increase in [Ca2+]i. 5. In the presence of 10 mM caffeine and 10 microM ryanodine, the increase in [Ca2+]i by 30 microM NS-1619 was not observed and the membrane hyperpolarization was reduced to approximately 67% of the control. 6. These results indicate that the opening of BK channels by NS-1619 at 30 microM, which is the most frequently used concentration of this agent, is partly due to Ca2+ release from caffeine/ryanodine-sensitive intracellular storage sites but is mainly due to the direct activation of the channels.

Animals↗
PubMed2001

Effects of KRN2391 on ionic currents in rabbit femoral arterial myocytes.

The effects of KRN2391, an ATP-sensitive K+ channel opener (KCO) which also acts as a nitrate, on ionic membrane currents in rabbit femoral arterial myocytes were examined. Under whole-cell clamp conditions where cells were superfused with physiological salts solution containing 5.9 mM K+, KRN2391 elicited an outward current at a holding potential of -30 mV. KRN2391-induced current had a reversal potential of -78 mV and was abolished by glibenclamide (glib). KRN2391 was approximately 25 times more potent than nicorandil to activate an ATP-sensitive K+ current (I:(KATP)). On the other hand, 10 microM KRN2391 did not affect either voltage-dependent Ca(2+) or delayed rectifier K+ channel currents. In the inside-out patch configuration, KRN2391 activated 47 pS K+ channels in the presence of nucleotide diphosphates (NDPs) under the symmetrical 140 mM K+ conditions. Glib and intracellular ATP reversibly inhibited the activity of the 47 pS K+ channels. The 47 pS K+ channels activated by KRN2391 are similar in their conductance and other properties to NDP-sensitive K+ channels (K(NDP) channels) described in other smooth muscles and the cloned channels. KRN2391 is a potent activator of the 47 pS K+ channels and the activation can contribute to the KRN2391-induced vasodilation in arterial muscles.

Adenosine Diphosphate↗
PubMed2001

Multiple founder effects in spinal and bulbar muscular atrophy (SBMA, Kennedy disease) around the world.

SBMA (spinal and bulbar muscular atrophy), also called Kennedy disease, is an X-chromosomal recessive adult-onset neurodegenerative disorder caused by death of the spinal and bulbar motor neurones and dorsal root ganglia. Patients may also show signs of partial androgen insensitivity. SBMA is caused by a CAG repeat expansion in the first exon of the androgen receptor (AR) gene on the X-chromosome. Our previous study suggested that all the Nordic patients with SBMA originated from an ancient Nordic founder mutation, but the new intragenic SNP marker ARd12 revealed that the Danish patients derive their disease chromosome from another ancestor. In search of relationships between patients from different countries, we haplotyped altogether 123 SBMA families from different parts of the world for two intragenic markers and 16 microsatellites spanning 25 cM around the AR gene. The fact that different SBMA founder haplotypes were found in patients from around the world implies that the CAG repeat expansion mutation has not been a unique event. No expansion-prone haplotype could be detected. Trinucleotide diseases often show correlation between the repeat length and the severity and earlier onset of the disease. The longer the repeat, the more severe the symptoms are and the onset of the disease is earlier. A negative correlation between the CAG repeat length and the age of onset was found in the 95 SBMA patients with defined ages at onset.

Exons↗
PubMed2001

Fluorimetric determination of isatin in human urine and serum by liquid chromatography postcolumn photoirradiation.

For the fluorimetric determination of isatin in human urine and serum, HPLC-postcolumn photoirradiation using a mobile phase has been developed. Isatin in the urine or serum sample was separated on a Capcell Pak C1 column (250 x 4.6 mm id). The mobile phase consisted of 70 mmol l-1 phosphate buffer (pH 7.2)-tetrahydrofuran (85 + 15% v/v) containing 5 mmol l-1 hydrogen peroxide, which was irradiated with germicidal light to induce fluorescence (lambda ex 302 nm, lambda em 418 nm). The addition of tetrahydrofuran to the mobile phase led to the peaks showing good separation as well as increased sensitivity. The calibration graph for isatin was linear over the range of 0.16-10.7 ng. The pretreatment of the acidified urine or serum samples consisted of diluting steps or deproteinizing steps using perchloric acid, respectively. The mean recovery of isatin from urine and serum was greater than 94%.

Biomarkers↗
PubMed2001

Enhancement of hippocampal LTP, reference memory and sensorimotor gating in mutant mice lacking a telencephalon-specific cell adhesion molecule.

Telencephalin (TLCN) is a cell adhesion molecule selectively expressed in the telencephalon of the mammalian brain. The mutant mice lacking TLCN had no detectable abnormalities in their neural development and synaptic structures. Ablation of TLCN increased the hippocampal long-term potentiation and its saturation level. The TLCN mutation selectively enhanced the performance of the radial maze and water-finding tasks, learning tasks with appetitive reinforcers, but not the contextual fear conditioning and Morris water maze tasks with aversive stimuli for conditioning. Furthermore, the TLCN mutant mice showed an increase of prepulse inhibition of the acoustic startle response. These results suggest that TLCN is a determinant of the dynamic range of synaptic plasticity and plays roles in reward-motivated learning and memory and sensorimotor gating.

Animals↗
PubMed2001

NMDA receptor subunits GluRepsilon1, GluRepsilon3 and GluRzeta1 are enriched at the mossy fibre-granule cell synapse in the adult mouse cerebellum.

Cerebellar N-methyl-D-aspartate (NMDA) receptors are concentrated in the granular layer and are involved in motor coordination and the induction of long-term potentiation at mossy fibre-granule cell synapses. In the present study, we used immunohistochemistry to examine the distribution of NMDA receptor subunits in the adult mouse cerebellum. We found that appropriate pepsin pretreatment of sections greatly enhanced the sensitivity and specificity of immunohistochemical detection. As a result, intense immunolabelling for GluRepsilon1 (NR2A), GluRepsilon3 (NR2C), and GluRzeta1 (NR1) all appeared in synaptic glomeruli of the granular layer. Double immunofluorescence showed that these subunits were colocalized in individual synaptic glomeruli. Within the glomerulus, NMDA receptor subunits were located between centrally-located huge mossy fibre terminals and peripherally-located tiny Golgi axon terminals. By immunoelectron microscopy, all three subunits were detected at the postsynaptic junction in granule cell dendrites, forming synapses with mossy fibre terminals. Consistent with the known functional localization, GluRepsilon1, GluRepsilon3, and GluRzeta1 are, thus, anatomically concentrated at the mossy fibre-granule cell synapse. By contrast, immunohistochemical signals were very low in Purkinje cell somata and dendrites in the molecular layer. The lack of GluRzeta1 immunolabelling in Purkinje cells was unexpected because the cells express GluRzeta1 mRNA at high levels and high levels of GluRzeta1 protein in the molecular layer were revealed by immunoblot. As Purkinje cells are exceptionally lacking GluRepsilon expression, the discrepant result may provide in vivo evidence suggesting the importance of accompanying GluRepsilon subunits in synaptic localization of GluRzeta1.

Animals↗