Biomedical subjects
Y Oka
Publications and source records attributed to Y Oka.
Acute effects of nicotine content in cigarettes on coronary flow velocity and coronary flow reserve in men.
We investigated the acute effects of smoking on coronary flow reserve in terms of the nicotine content of cigarettes in 21 smokers. Coronary flow velocity was measured with a Doppler flow wire. Subjects smoked cigarettes containing >1 mg nicotine (n = 8, group 1) or <1 mg (n = 6, group 2). Subjects in the control group mimicked smoking without a cigarette (n = 7). Coronary flow reserve decreased after smoking in group 1, but not in group 2 or the control group. This reduction may have mediated nicotine or some other unknown substances influenced by smoking.
Different subcellular distribution and regulation of expression of insulin receptor substrate (IRS)-3 from those of IRS-1 and IRS-2.
Adipocytes contain three major substrate proteins of the insulin receptor, termed IRS-1, IRS-2, and IRS-3. We demonstrated that IRS-1 and IRS-2 are located mainly in the low density microsome (LDM) fraction and are tyrosine phosphorylated in response to insulin stimulation, leading to phosphatidylinositol (PI) 3-kinase activation. In contrast, IRS-3 is located mainly in the plasma membrane (PM) fraction and contributes to PI 3-kinase activation in the PM fraction. The different cellular localizations of IRS proteins may account for the mechanism of insulin resistance induced by a high fat diet, considering that PI 3-kinase activation in the LDM fraction is reportedly essential for the translocation of GLUT4 in adipocytes. High fat feeding in rats increased both protein and mRNA levels of IRS-3 but decreased those of IRS-1 and IRS-2 in epididymal adipocytes. As a result, selective impairment of insulin-induced PI 3-kinase activation was observed in the LDM fraction, whereas PI 3-kinase activation was conserved in the PM fraction. This is the first report showing that different IRS proteins function in different subcellular compartments, which may contribute to determining the insulin sensitivity in adipocytes.
Thrombopoietin induces tyrosine phosphorylation of a common beta subunit of GM-CSF receptor and its association with Stat5 in TF-1/TPO cells.
TF-1/TPO cells are derived from an erythroleukemia cell line, TF-1, and are absolutely dependent on either TPO or granulocyte-macrophage colony-stimulating factor (GM-CSF)/interleukin-3 (IL3) for their continuous growth and survival. To gain insight into the molecular basis of hemopoietic activities shared by TPO and GM-CSF/IL3 in TF-1/TPO cells, we studied the cross-talk between signal transduction pathways elicited by these cytokines. Stimulation of TF-1/TPO cells with TPO resulted in tyrosine phosphorylation of the TPO receptor (c-Mpl) as well as the common beta subunit (beta c) of GM-CSF/IL3 receptor complex. GM-CSF, however, induced tyrosine phosphorylation of beta c but not c-Mpl. TPO-induced tyrosine phosphorylation of beta c was time- and dose-dependent. We next examined whether or not TPO-induced tyrosine phosphorylation of beta c led to recruitment of SH2-containing molecules such as Stat5 and Shc. While GM-CSF caused association of Stat5 and Shc with beta c, TPO caused association of Stat5, but not Shc, with beta c, suggesting that TPO and GM-CSF may not induce phosphorylation of the same sets of tyrosine residues in beta c. These results suggest that activation of c-Mpl affects the signaling pathway of GM-CSF/IL3 but not vice versa.
Interaction of Drosophila inhibitors of apoptosis with thick veins, a type I serine/threonine kinase receptor for decapentaplegic.
Decapentaplegic (Dpp) is a Drosophila member of bone morphogenetic proteins, which belong to the transforming growth factor-beta superfamily. Members of this family regulate a variety of biological processes such as cell proliferation, morphogenesis, immune response, and apoptosis. Dpp plays a critical role in many aspects of Drosophila development. Members of the transforming growth factor-beta superfamily bind to two different types of serine/threonine kinase receptors, termed type I and type II. Type I receptors act as downstream components of type II receptors in the receptor complexes. Therefore, intracellular proteins that interact with the type I receptors are likely to play important roles in signaling. Several proteins have been identified through protein-protein interaction screenings. We identified Drosophila inhibitor of apoptosis (DIAP) 1 as an interacting protein of a Dpp type I receptor, Thick veins (Tkv). DIAP1 associates with Tkv in vivo. The binding region in DIAP1 is mapped to its C-terminal RING finger region. DIAP2, another Drosophila member of the inhibitor of apoptosis protein family, also interacts with Tkv in vivo. These data suggest that DIAP1 and DIAP2 may be involved, possibly as negative regulators, in the Dpp signaling pathway, which leads to cell apoptosis.
Wilms' tumor gene (WT1) competes with differentiation-inducing signal in hematopoietic progenitor cells.
The WT1 gene is a tumor-suppressor gene that was isolated as a gene responsible for Wilms' tumor, a childhood kidney neoplasm. We have previously reported that the WT1 gene is strongly expressed in leukemia cells with an increase in its expression levels at relapse and an inverse correlation between its expression levels and prognosis, thus making it a novel tumor marker for leukemic blast cells. Furthermore, WT1 antisense oligomers have been found to inhibit the growth of leukemic cells. These results strongly suggested the involvement of the WT1 gene in human leukemogenesis. The present study was performed to prove our hypothesis that the WT1 gene plays a key role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells, rather than a tumor-suppressor gene function. 32D cl3, an interleukin-3-dependent myeloid progenitor cell line, differentiates into mature neutrophils in response to granulocyte colony-stimulating factor (G-CSF). However, when transfected wild-type WT1 gene was constitutively expressed in 32D cl3, the cells stopped differentiating and continued to proliferate in response to G-CSF. As for signal transduction mediated by G-CSF receptor (G-CSFR), Stat3alpha was constitutively activated in wild-type WT1-infected 32D cl3 in response to G-CSF, whereas, in WT1-uninfected 32D cl3, activation of Stat3alpha was only transient. However, most interesting was the fact that G-CSF stimulation resulted in constitutive activation of Stat3beta only in wild-type WT1-infected 32D cl3, but not in WT1-uninfected 32D cl3. Thus, WT1 expression constitutively activated both Stat3alpha and Stat3beta. A transient activation of Stat1 was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation, but no difference in its activation was found. No activation of MAP kinase was detected in both wild-type WT1-infected and uninfected 32D cl3 after G-CSF stimulation. These results demonstrated that WT1 expression competed with the differentiation-inducing signal mediated by G-CSFR and constitutively activated Stat3, resulting in the blocking of differentiation and subsequent proliferation. Therefore, the data presented here support our hypothesis that the WT1 gene plays an essential role in leukemogenesis and performs an oncogenic function in hematopoietic progenitor cells and represent the first demonstration of an important role of the WT1 gene in signal transduction in hematopoietic progenitor cells.
Type I phosphatidylinositol-4-phosphate 5-kinases. Cloning of the third isoform and deletion/substitution analysis of members of this novel lipid kinase family.
Type I phosphatidylinositol 4-phosphate (PtdIns(4)P) 5-kinases (PIP5K) catalyze the synthesis of phosphatidylinositol 4, 5-bisphosphate, an essential lipid molecule in various cellular processes. Here, we report the cloning of the third member (PIP5Kgamma) and the characterization of members of the type I PIP5K family. Type I PIP5Kgamma has two alternative splicing forms, migrating at 87 and 90 kDa on SDS-polyacrylamide gel electrophoresis. The amino acid sequence of the central portion of this isoform shows approximately 80% identity with those of the alpha and beta isoforms. Northern blot analysis revealed that the gamma isoform is highly expressed in the brain, lung, and kidneys. Among three isoforms, the beta isoform has the greatest Vmax value for the PtdIns(4)P kinase activity and the gamma isoform is most markedly stimulated by phosphatidic acid. By analyzing deletion mutants of the three isoforms, the minimal kinase core sequence of these isoforms were determined as an approximately 380-amino acid region. In addition, carboxyl-terminal regions of the beta and gamma isoforms were found to confer the greatest Vmax value and the highest phosphatidic acid sensitivity, respectively. It was also discovered that lysine 138 in the putative ATP binding motif of the alpha isoform is essential for the PtdIns(4)P kinase activity. As was the case with the alpha isoform reported previously (Shibasaki, Y., Ishihara, H., Kizuki, N., Asano, T., Oka, Y., Yazaki, Y. (1997) J. Biol. Chem. 272, 7578-7581), overexpression of either the beta or the gamma isoform induced an increase in short actin fibers and a decrease in actin stress fibers in COS7 cells. Surprisingly, a kinase-deficient substitution mutant also induced an abnormal actin polymerization, suggesting a role of PIP5Ks via structural interactions with other molecules.
Chronic graft-versus-host disease and seizure.
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Isolation of full-length cDNA of mouse PAX4 gene and identification of its human homologue.
Recent genetic studies have suggested that PAX4, a member of the paired box (PAX) gene family, is involved in the mechanism regulating the fate of pancreatic islet endocrine progenitor cells. Murine PAX4 was originally identified by genomic screening and, to date, only a partial sequence of PAX4 has been reported. In this study, we cloned the full-length cDNA of mouse PAX4 by RACE (rapid amplification of cDNA ends) using RNA from MIN6 cells, a mouse insulinoma cell line. The full length of cDNA was 1.38 kb, consistent with the estimated size of the transcript by Northern blot. The deduced mouse PAX4 protein was 349 amino acids and had the predicted molecular weight of 38 kDa. Two DNA binding motifs, a 128-amino acid paired domain and a 61-amino acid paired-type homeodomain exhibit the highest amino acid homology with PAX6 (71.2%, 65.0%, respectively), another member of the PAX gene family. However, the sequence of the C-terminal segment of PAX4 diverged and showed no significant homology with any other known PAX genes. As to the genomic DNA, the coding region of the mouse PAX4 gene spanned approximately 5.5 kb and was composed of 10 exons. In the public DNA database, a human cosmid (g1572c264), which was localized on human chromosome 7q31.3, was found to contain a gene homologous to PAX4. The nucleotide and protein sequence homologies between mouse PAX4 and its human homologue were 83.1% and 80.0%, respectively. Interestingly, the ARP5 (ADP-ribosylation factor 5) gene was also found in the same cosmid g1572c264, suggesting the ARP5 gene to be adjacent to the human PAX4 homologue. The human cosmid g1572c264 contains at least four SSRPs (simple sequence repeat polymorphism), which could be used for genetic linkage studies of the locus. The results of this study, i.e. isolation of the full-length cDNA sequence of PAX4 and identification of the homologous human gene, will facilitate further functional and genetic studies of the PAX4 gene.
Gonadotropin-releasing hormone neurons in the gourami midbrain: a double labeling study by immunocytochemistry and tracer injection.
There are three groups of gonadotropin-releasing hormone (GnRH) neurons in the teleost brain. Midbrain GnRH neurons in the dwarf gourami send axons to various areas of the central nervous system. However, it is not clear whether midbrain GnRH neurons form a cell cluster separate from the nucleus of the medial longitudinal fasciculus (nMLF), which has been reported to project to the spinal cord. Thus, we performed a double labeling study. GnRH neurons were immunostained but were very faintly labeled with biocytin injected into the spinal cord. In contrast, nMLF neurons were strongly labeled with biocytin but were GnRH-immunonegative. GnRH neurons are distributed at almost the same rostrocaudal levels as nMLF neurons, but they constitute a separate cell group dorsocaudal to nMLF neurons.
GnRH-immunoreactive neuronal system in the presumptive ancestral chordate, Ciona intestinalis (Ascidian).
Gonadotropin-releasing hormone (GnRH) of the vertebrate brain, which has originally been identified as a peptidergic hypophysiotropic hormone, is now believed to act also as a neuromodulator. It has recently been shown that a vertebrate-like GnRH-gonadotropin system occurs in the urochordates, which are considered to be the presumptive ancestral chordate. In this paper, we examined the morphology of the GnRH neuronal system of ascidian, Ciona intestinalis, by immunocytochemistry and found many GnRH-immunoreactive neuronal cells and fibers in a specific surface area of the cerebral ganglion, along the inner wall of the dorsal blood sinus, as well as on the anterior surface of the ovary.
Repetitive mitochondrial Ca2+ signals synchronize with cytosolic Ca2+ oscillations in the pancreatic beta-cell line, MIN6.
We examined the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial matrix Ca2+ concentration ([Ca2+]m) in the pancreatic beta-cell line, MIN6. [Ca2+]c was monitored in a single or a group (30 cells) of fura-2-loaded MIN6 cells, and [Ca2+]m was measured in a group (1 x 10[6] cells) of MIN6 cells stably transfected with aequorin targeted at the mitochondria. Exogenous ATP (0.25 mmol/l) produced a single transient increase in [Ca2+]c whereas 22 mmol/l KCl produced a sustained plateau increase. ATP and KCl evoked transient increases in [Ca2+]m but with distinct time courses of [Ca2+]m decline: the [Ca2+]m increase induced by ATP decreased more rapidly than that induced by KCl. Nitrendipine (3 micromol/l), a blocker of L-type Ca2+ channels, inhibited both [Ca2+]c and [Ca2+]m signals in response to KCl and tolbutamide, but not those to ATP. Peak levels of [Ca2+]m increase (around 2 micromol/ l) exceeded those of [Ca2+]c increase (around 500 nmol/l). A rise in glucose concentration from 3 to 30 mmol/l induced oscillations of [Ca2+]c that overlay the sustained increases in [Ca2+]c in single cells. An oscillatory increase in [Ca2+]m was similarly observed in response to glucose. Addition of 10 mmol/l 2-ketoisocaproic acid at 20 mmol/l glucose further increased the plateau level of [Ca2+]c and the frequency of [Ca2+]c oscillations, which were correlated with a further increase in [Ca2+]m. In response to pulsatile exposure to KCl, [Ca2+]c and [Ca2+]m increased synchronously. These data suggest that an oscillatory increase in [Ca2+]m in beta cells, the signal which is thought to be necessary for continuous stimulation of mitochondrial metabolism, is produced synchronously with the [Ca2+]c oscillations.
Role of JTT-501, a new insulin sensitiser, in restoring impaired GLUT4 translocation in adipocytes of rats fed a high fat diet.
JTT-501 is an insulin-sensitising compound with an isoxazolidinedione rather than a thiazolidionedione structure. Sprague-Dawley rats fed a high fat diet for 2 weeks were used as an animal model of insulin resistance, and JTT-501 was administered for the final week of the diet. An euglycaemic glucose clamp study showed that the glucose infusion rate (GIR) required to maintain euglycaemia was 57% lower in rats fed a high fat diet than in control rats, and that JTT-501 treatment restored the reduction in GIR produced by the high fat diet. To explain the mechanisms underlying the effects of a high fat diet and JTT-501 treatment, epididymal fat pads were excised and used in the analysis of insulin action. The high fat diet caused: (1) a 58% decrease in insulin receptor substrate-1 (IRS-1) content with a 58% decrease in IRS-1 tyrosine phosphorylation; (2) reductions of 56% and 73% respectively in insulin-induced maximal PI 3-kinase activation in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates; (3) a 46% reduction in the glucose transporter protein, GLUT4 content and, consequently, (4) severely impaired insulin-induced GLUT4 translocation to the plasma membrane and glucose uptake in adipocytes. JTT-501 treatment restored appreciably the protein content and tyrosine phosphorylation level of IRS-1. Insulin-stimulated PI 3-kinase activation was also restored in anti-phosphotyrosine and anti-IRS-1 antibody immunoprecipitates. As reflected by these improvements in insulin signalling, JTT-501 treatment improved considerably insulin-induced GLUT4 translocation to the plasma membrane as well as insulin-induced glucose uptake. However, JTT-501 had no effect on the decrease in GLUT4 content produced by the high fat diet. These observations suggest that JTT-501 enhances insulin signalling and may be effective in reducing insulin resistance.
Overexpression of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase does not correct glucose-stimulated insulin secretion from diabetic GK rat pancreatic islets.
Glucose-stimulated insulin secretion is impaired in GK (Goto-Kakizaki) rats, perhaps because of abnormalities in glucose metabolism in pancreatic islet beta cells. The glycerol phosphate shuttle plays a major role in glucose metabolism by reoxidizing cytosolic NADH generated by glycolysis. In the pancreatic islets of GK rats, the activity of mitochondrial FAD-linked glycerol-3-phosphate dehydrogenase (mGPDH), the key enzyme of the glycerol phosphate shuttle, is decreased and this abnormality may be responsible, at least in part, for impaired glucose-stimulated insulin secretion. To investigate this possibility, we overexpressed mGPDH in islets isolated from GK rats via recombinant adenovirus-mediated gene transduction, and examined glucose-stimulated insulin secretion. In islets isolated from diabetic GK rats at 8 to 10 weeks of age, glucose-stimulated insulin secretion was severely impaired, and mGPDH activity was decreased to 79 % of that in non-diabetic Wistar rats. When mGPDH was overexpressed in islets from GK rats, enzyme activity and protein content increased 2- and 6-fold, respectively. Basal (3 mmol/l glucose) and glucose-stimulated (20 mmol/l) insulin secretion from the Adex1CAlacZ-infected GK rat islets were, respectively, 4.4 +/- 0.7 and 8.1 +/- 0.7 ng. x islet(-1) x 30 min(-1), and those from mGPDH-overexpressed GK rat islets 4.7 +/- 0.3 and 9.1 +/- 0.8 ng x islet(-1) x 30 min(-1), in contrast to those from the AdexlCAlacZ-infected non-diabetic Wistar rat islets (4.7 +/- 1.6 and 47.6 +/- 11.9 ng x islet(-1) x 30 min(-1)). Thus, glucose-stimulated insulin secretion is severely impaired in GK rats even in the stage when mGPDH activity is modestly decreased, and at this stage, overexpression of mGPDH cannot restore glucose-stimulated insulin secretion. We conclude that decreased mGPDH activity in GK rat islets is not the defect primarily responsible for impaired glucose-stimulated insulin secretion.
Ontogenic development of salmon GnRH and chicken GnRH-II systems in the brain of masu salmon (Oncorhynchus masou).
Ontogenic development of salmon gonadotropin-releasing hormone (GnRH) and chicken GnRH-II systems in masu salmon (Oncorhynchus masou) was examined. Salmon GnRH was first detected by radioimmunoassay in the embryo on day 36 after fertilization. Salmon GnRH-immunoreactive fibers were detected initially by immunocytochemistry in the vicinity of the olfactory placode of the embryo (day 36) and were distributed widely in the brain as well as in the pituitary gland of fish just after hatching (day 80). Salmon GnRH-immunoreactive neuronal somata were first detected about 6 months after fertilization in the rostroventral brain area, ranging from the olfactory nerve to the preoptic area. Salmon GnRH neuronal somata were detected earlier by in situ hybridization than by immunocytochemistry. Neuronal somata expressing salmon GnRH mRNA were first detected in the vicinity of the olfactory epithelium on day 40 and then were seen to be migrating from the olfactory epithelium, along the olfactory nerve, on day 60 and in the transitional area between olfactory nerve and olfactory bulb on day 80. In the brain, these neurons were first detected in the ventral olfactory bulb on day 80, and thereafter they were also detected in the caudal brain regions. The chicken GnRH-II system was detected later than the salmon GnRH system; chicken GnRH-II was first detected by radioimmunoassay on day 57, and chicken GnRH-II-immunoreactive fibers were first detected on day 67. Chicken GnRH-II-immunoreactive neuronal somata were not detected during the observation period. These results suggest that salmon GnRH neurons derive from the olfactory placode and then migrate into the brain and that salmon GnRH is synthesized before chicken GnRH-II.
Suppression of Wilms' tumor gene (WT1) expression induces G2/M arrest in leukemic cells.
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Preoptic gonadotropin-releasing hormone (GnRH) neurons innervate the pituitary in teleosts.
In most teleosts, there are three groups of gonadotropin-releasing hormone (GnRH) neurons. In this study we addressed the question of GnRH neuronal innervation of the pituitary in the dwarf gourami and the tilapia using immunocytochemistry combined with biocytin tract tracing. Biocytin was applied to the pituitary attached to the brain in vitro. Similar results were obtained in both species. GnRH neurons retrogradely labeled with biocytin were observed only in the preoptic area. These results indicate that preoptic GnRH neurons innervate the pituitary. Negative labeling of biocytin in the terminal-nerve and midbrain GnRH neurons suggests that these two GnRH neuronal populations do not project to the pituitary. Biocytin-positive but GnRH-negative neurons were also observed in the preoptic area and the ventromedial parts of the hypothalamus, suggesting neuropeptidergic and aminergic innervation of the pituitary besides GnRH.