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S Takasawa

Publications and source records attributed to S Takasawa.

At least 55 records · Page 3Linked to original sources
PubMed1996

Enhancement of glucose-induced insulin secretion in transgenic mice overexpressing human VIP gene in pancreatic beta cells.

Using transgenic mice technology, it has now become possible to test directly whether VIP and PHM-27 can enhance glucose-induced insulin secretion and reduce blood glucose in vivo. By microinjecting the entire human VIP gene ligated to the rat insulin II promoter, we have established a mouse model that overproduces VIP and PHM-27 in pancreatic beta cells. VIP was secreted from transgenic islets in a glucose-dependent manner. Analyses of these VIP-transgenic mice indicated that the transgene efficiently enhances glucose-induced insulin secretion and significantly reduces blood glucose as compared with control mice. The transgene also ameliorated glucose intolerance of 70% depancreatized mice. The present results suggest that somatic cell gene therapy directed to diabetic islets by human VIP/PHM-27 gene introduction may provide a means to improve the secretory function of the diabetic islets.

Animals↗
PubMed1996

Identification of the five essential histidine residues for peptidylglycine monooxygenase.

Peptidylglycine monooxygenase (PGM) is a copper-containing monooxygenase that plays a key role in the peptide C-terminal alpha-amidation. Comparative analysis of the amino acid sequences of rat, human, bovine and frog PGMs revealed that ten histidines (residues 107, 108, 172, 235, 242, 244, 279, 364, 366 and 367 in rat PGM) are conserved among the four species. We introduced site-directed mutations to the ten histidines of rat PGM and found that the mutation of His- 107-->Ala, His-108-->Ala, His-172-->Ala, His-242-->Arg or His-244-->Ala abolished the enzyme activity. The five mutant proteins lacking the enzyme activity bound to a substrate, Phe-Gly-Phe-Gly, as did the wild type PGM. These results along with available evidence indicate that the five histidine residues (His-107, 108, 172, 242 and 244) are essential for PGM activity, acting as copper ligands.

Amino Acid Sequence↗
PubMed1996

Reg gene expression is increased in rat gastric enterochromaffin-like cells following water immersion stress.

BACKGROUND & AIMS: Reg gene has been isolated from regenerating rat pancreatic islets, and subsequent studies have shown a trophic effect of Reg protein on islet cells. However, little is known about the role of Reg protein in the stomach. The aim of this study was to clarify the localization of Reg messenger RNA (mRNA) and its product in the stomach and to examine changes in the level of their expression during regeneration of gastric mucosal cells. METHODS: Gastric lesions were experimentally induced in Sprague-Dawley rats by water immersion stress. Northern blot analysis and in situ hybridization studies were performed to examine changes in mRNA levels. Immunohistochemical studies were performed to identify the cellular localization and to investigate the change in Reg protein level. RESULTS: Reg mRNA and its product were distributed in the basal part of the oxyntic mucosa and were expressed mainly in enterochromaffin-like cells. Levels of both Reg mRNA and its product were markedly increased in the water immersion-induced gastric lesions. CONCLUSIONS: Reg mRNA and its product are expressed in gastric enterochromaffin-like cells, and their levels are increased during the healing process of water immersion-induced gastric lesions.

Animals↗
PubMed1995

Human REG family genes are tandemly ordered in a 95-kilobase region of chromosome 2p12.

Reg, first isolated from a rat regenerating islet cDNA library, is expressed in regenerating islet beta-cells. Recently, it has been revealed that Reg and Reg-related genes constitute a multigene family, the Reg family. In human, the four REG family genes, i.e., REG 1 alpha, REG 1 beta, REG-related sequence (RS) and HIP/PAP, have so far been isolated. In this study, we analyzed YAC clones containing the four genes and performed two-color FISH to determine the map order of the genes. The human REG family genes are tandemly ordered in the 95-kbp DNA region of chromosome 2p12 as follows: 2cen-HIP/PAP-RS-REG I alpha-REG I beta-ptel.

Acute-Phase Proteins↗
PubMed1995

Requirement of calmodulin-dependent protein kinase II in cyclic ADP-ribose-mediated intracellular Ca2+ mobilization.

Cyclic ADP-ribose (cADPR) is generated in pancreatic islets by glucose stimulation, serving as a second messenger for Ca2+ mobilization from the endoplasmic reticulum for insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373). In the present study, we observed that the addition of calmodulin (CaM) to rat islet microsomes sensitized and activated the cADPR-mediated Ca2+ release. Inhibitors for CaM-dependent protein kinase II (CaM kinase II) completely abolished the glucose-induced insulin secretion as well as the cADPR-mediated and CaM-activated Ca2+ mobilization. Western blot analysis revealed that the microsomes contain the alpha isoform of CaM kinase II but do not contain CaM. When the active 30-kDa chymotryptic fragment of CaM kinase II was added to the microsomes, fully activated cADPR-mediated Ca2+ release was observed in the absence of CaM. These results along with available evidence strongly suggest that CaM kinase II is required to phosphorylate and activate the ryanodine-like receptor, a Ca2+ channel for cADPR as an endogenous activator, for the cADPR-mediated Ca2+ release.

Adenosine Diphosphate Ribose↗
PubMed1995

Regulatory role of CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase) in insulin secretion by glucose in pancreatic beta cells. Enhanced insulin secretion in CD38-expressing transgenic mice.

Cyclic ADP-ribose (cADPR) serves as a second messenger for Ca2+ mobilization in insulin secretion, and CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Here, we produced transgenic mice overexpressing human CD38 in pancreatic beta cells. The enzymatic activity of CD38 in transgenic islets was greatly increased, and ATP efficiently inhibited the cADPR hydrolase activity. The Ca2+ mobilizing activity of cell extracts from transgenic islets incubated in high glucose was 3-fold higher than that of the control, suggesting that ATP produced by glucose metabolism increased cADPR accumulation in transgenic islets. Glucose- and ketoisocaproate-induced but not tolbutamide- nor KCl-induced insulin secretions from transgenic islets were 1.7-2.3-fold higher than that of control. In glucose-tolerance tests, the transgenic serum insulin level was higher than that of control. The present study provides the first evidence that CD38 has a regulatory role in insulin secretion by glucose in beta cells, suggesting that the Ca2+ release from intracellular cADPR-sensitive Ca2+ stores as well as the Ca2+ influx from extracellular sources play important roles in insulin secretion.

ADP-ribosyl Cyclase↗
PubMed1995

Nicotinamide inhibits IRF-1 mRNA induction and prevents IL-1 beta-induced nitric oxide synthase expression in pancreatic beta cells.

Nitric oxide produced by inducible nitric oxide synthase in islets exerts inhibitory and cytotoxic effects on pancreatic beta cells and is therefore thought to be a potent mediator in the pathogenesis of Type I diabetes mellitus. Here, using isolated rat pancreatic islets, we show that high-concentration nicotinamide (20 mM), but not low-concentration nicotinamide (5 mM), attenuates the interleukin-1 beta-evoked inhibition of glucose-induced insulin secretion by preventing the induction of interferon regulatory factor-1, a transcriptional factor which plays an essential role in inducible nitric oxide synthase gene expression, and the interleukin-1 beta-induced nitric oxide formation. High-concentration nicotinamide also restored an interleukin-1 beta-induced decrease in ATP content in pancreatic beta cells, suggesting that interleukin-1 beta-induced nitric oxide inhibits the mitochondrial function. The present results show the molecular basis of the preventive effect of high-dose nicotinamide on Type I diabetes mellitus.

Adenosine Triphosphate↗
PubMed1995

NAD(+)-glycohydrolase from Streptococcus pyogenes shows cyclic ADP-ribose forming activity.

NAD(+)-glycohydrolase from Streptococcus pyogenes was purified by successive chromatography on CM Sepharose CL-6B, Sephacryl S-200 HR and hydroxyapatite. The purified enzyme possessed synthesis and hydrolysis activities of cyclic ADP-ribose (cADPR), a newly found second messenger for Ca2+ mobilisation, along with cleavage activity of the ribose-nicotinamide bond in NAD+.

Adenosine Diphosphate Ribose↗
PubMed1995

The structure of the Aplysia kurodai gene encoding ADP-ribosyl cyclase, a second-messenger enzyme.

The complete nucleotide (nt) sequences of the cDNA and gene encoding the marine mollusk Aplysia kurodai (Ak) ADP-ribosyl cyclase (ADRC) which synthesizes cyclic ADP-ribose (cADP-ribose), a second messenger for Ca2+ mobilization from endoplasmic reticulum, were determined. Ak ADRC consists of 258 amino acids (aa) (29 kDa). It shares 86% aa sequence homology with that from A. californica, and 31-32% homology with the human, rat and mouse cluster of differentiation 38 (CD38) that has both ADRC and cADP-ribose hydrolase activities. The Ak ADRC-encoding gene (ADRC) spans approx. 7 kb and contains eight exons and seven introns. The transcription start point (tsp) determined by primer extension analysis and S1 mapping is 28 bp downstream from the TATA box. This gene is expressed specifically in the ovotestis, although the mammalian CD38-encoding gene is expressed in many kinds of tissues and cells. The 5'-flanking region contains several consensus sequences responsible for the germ-cell-specific expression of the mouse zona pellucida 3 (ZP3) and Drosophila melanogaster chorion genes. The existence of the consensus sequences located at nt -1649, -1161, -234 and -90 may account for the ovotestis-specific expression of the Ak ADRC gene.

ADP-ribosyl Cyclase↗
PubMed1995

Nicotinamide-adenine dinucleotide regulates muscarinic receptor-coupled K+ (M) channels in rodent NG108-15 cells.

1. The possible role of nicotinamide-adenine dinucleotide (NAD+) and cyclic adenosine diphosphate ribose (cADPR) as regulators of M-type K+ currents (IK(M)) has been studied in whole-cell patch-clamped NG108-15 mouse neuroblastoma x rat glioma cells that had been transformed to express m1 muscarinic acetylcholine receptors (mAChRs). 2. Pre-incubation of NG108-15 cells for 6-8 h with streptozotocin (2-5 mM) reduced NAD+ levels by 40-50%. Nicotinamide (2-5 mM) increased NAD+ levels and prevented depletion by streptozotocin. 3. Streptozotocin pretreatment reduced the inhibition of IK(M) produced by 100 microM acetylcholine (ACh) from 51.6 +/- 7.0 to 29.1 +/- 7.5%. This was prevented by simultaneous pre-incubation with 2 mM nicotinamide or by adding 2 mM NAD+ to the pipette solution. Neither procedure significantly affected the initial amplitude of IK(M). 4. Inclusion of 2 microM cADPR in the pipette solution induced a slow loss of IK(M) with a time constant of about 20 min. 5. It is concluded that mAChR-induced inhibition of IK(M) requires intracellular NAD+. This might be needed for the formation of cADPR as a regulator or messenger for IK(M) inhibition.

Adenosine Diphosphate Ribose↗
PubMed1995

Assignment of CD38, the gene encoding human leukocyte antigen CD38 (ADP-ribosyl cyclase/cyclic ADP-ribose hydrolase), to chromosome 4p15.

CD38 has been used as a phenotype marker of lymphocyte differentiation. Recently, we have demonstrated that cyclic ADP-ribose can be synthesized and hydrolyzed by CD38 and acts as a second messenger in insulin secretion from pancreatic beta-cells. We have mapped the CD38 gene to human chromosome 4p15 by fluorescence in situ hybridization.

ADP-ribosyl Cyclase↗
PubMed1994

ADP ribosyl cyclase activity of a novel bone marrow stromal cell surface molecule, BST-1.

Human BST-1, a bone marrow stromal cell surface molecule, is a GPI-anchored protein that facilitates the growth of pre-B cells. The deduced amino acid sequences of human and mouse BST-1 show around 30% homology with those of CD38 and Aplysia ADP ribosyl cyclase. Therefore, like CD38, BST-1 might possess ADP ribosyl cyclase activity. Here, we report the establishment of a stable transformant CHO cell line, which secretes truncated human soluble BST-1, and show that purified soluble BST-1 displays both ADP ribosyl cyclase and cADPR hydrolase activities.

ADP-ribosyl Cyclase↗
PubMed1994

Essential cysteine residues for cyclic ADP-ribose synthesis and hydrolysis by CD38.

We have recently demonstrated that cyclic ADP-ribose (cADPR) serves as a second messenger for glucose-induced insulin secretion (Takasawa, S., Nata, K., Yonekura, H., and Okamoto, H. (1993) Science 259, 370-373) and that human leukocyte antigen CD38 has both ADP-ribosyl cyclase and cADPR hydrolase activities (Takasawa, S., Tohgo, A., Noguchi, N., Koguma, T., Nata, K., Sugimoto, T., Yonekura, H., and Okamoto, H. (1993) J. Biol. Chem. 268, 26052-26054). Although the amino acid sequence of Aplysia ADP-ribosyl cyclase exhibits a high degree of amino acid sequence identity with that of CD38, the Aplysia enzyme shows only ADP-ribosyl cyclase but not cADPR hydrolase. In the present study, we introduced site-directed mutations to CD38 and found that C119K- and/or C201E-CD38 exhibited only ADP-ribosyl cyclase activity. Furthermore, Aplysia ADP-ribosyl cyclase into which we introduced the mutations K95C and E176C, which correspond to residues 119 and 201 of human CD38, exhibited not only ADP-ribosyl cyclase activity but also cADPR hydrolase. These results indicate that cysteine residues 119 and 201 in CD38 have crucial roles in the synthesis and hydrolysis of cADPR.

ADP-ribosyl Cyclase↗
PubMed1994

Characterization of the 5'-regulatory region of rat Reg I gene.

We report here the characterization of the 5'-regulatory region of rat Reg I gene encoding a growth stimulating factor for pancreatic beta-cells. Transient expression assays of the 5'-flanking region/luciferase fusion gene in AR4-2J cells showed that the -304/-237 region contained positive cis-acting elements. Gel shift assays using AR4-2J and rat pancreas nuclear extracts showed the formation of a specific complex with the -256/-237 oligonucleotide.

Animals↗
PubMed1994

Transgenic mice overexpressing human vasoactive intestinal peptide (VIP) gene in pancreatic beta cells. Evidence for improved glucose tolerance and enhanced insulin secretion by VIP and PHM-27 in vivo.

Vasoactive intestinal peptide (VIP), a 28-amino acid peptide hormone, plays many physiological roles in the peripheral and central nervous systems. It has been proposed that endogenous VIP released from VIP-containing nerves is involved in the regulation of the secretory function of the endocrine pancreas. To test this hypothesis in vivo, we produced transgenic mice carrying the human VIP/peptide histidine methionine 27 (PHM-27) gene under the control of insulin promoter. In immunohistochemical analyses of islets, all the islet beta cells of transgenic mice were intensely stained for both VIP and PHM-27, consistent with the fact that these two peptides are encoded in a single mRNA (Itoh, N., Obata, K., Yanaihara, N., and Okamoto, H. (1983) Nature 304, 547-549). VIP was efficiently secreted from isolated transgenic islets in vitro. The blood glucose assays in free-fed mice indicated that the transgene lowered the blood glucose levels of transgenic mice (128 +/- 4 mg/dl) by about 20% below control levels (155 +/- 6 mg/dl). In the glucose tolerance test, at 60 min after glucose administration, the transgenic blood glucose levels (129 +/- 12 mg/dl) were much lower than control levels (175 +/- 13 mg/dl). The transgenic serum insulin levels at 15 min after glucose administration were 2.5-3.0-fold higher than control levels. The transgene was also effective in ameliorating glucose intolerance of 70% depancreatized mice. These results indicate that VIP and PHM-27 produced from the transgenic beta cells efficiently enhance glucose-induced insulin secretion from beta cells by an autocrine mechanism. These results also suggest that genetic manipulation of islet beta cells by the human VIP/PHM-27 gene or delivery of VIP to beta cells may ultimately provide a valuable approach to enhancing insulin secretion in clinical diabetes.

Animals↗