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J Takeda

Publications and source records attributed to J Takeda.

At least 253 records · Page 14Linked to original sources
PubMed1993

Cloning and functional comparison of kappa and delta opioid receptors from mouse brain.

While trying to identify new members of the somatostatin receptor family of G protein-coupled receptors, we isolated cDNAs from a mouse brain library encoding two related receptor-like proteins, designated msl-1 and msl-2, of 380 and 372 amino acids, respectively. There was 61% identity and 71% similarity between the sequences of msl-1 and msl-2. Among members of the G protein-coupled receptor superfamily, the sequences of both msl-1 and msl-1 were most closely related to those of the somatostatin receptors (SSTRs), having approximately 35% identity with the sequence of SSTR1. Transient expression in COS-1 cells showed that msl-1 and msl-2 did not bind somatostatin. Rather they bound opioids selectively and with high affinity and had the pharmacological properties of kappa and delta opioid receptors, respectively. Indeed, the sequence of msl-2 was identical to that of a delta opioid receptor recently cloned by other workers. Functional characterization of kappa/msl-1 and delta/msl-2 opioid receptors showed that they were coupled to G proteins and mediated opioid receptor class-specific agonist inhibition of forskolin-stimulated cAMP formation. RNA blotting studies and in situ hybridization histochemistry showed that kappa opioid receptor mRNA was expressed at high levels in brain in the neocortex, hippocampus, amygdala, medial habenula, hypothalamus (arcuate and paraventricular nuclei), locus ceruleus, and parabrachial nucleus, suggesting that this receptor may play a role in arousal and regulation of autonomic and neuroendocrine functions.

Amino Acid Sequence↗
PubMed1993

Structure/function studies of human beta-cell glucokinase. Enzymatic properties of a sequence polymorphism, mutations associated with diabetes, and other site-directed mutants.

Glucokinase plays a key role in the regulation of glucose metabolism in insulin-secreting pancreatic beta-cells and in the liver. Recent studies have shown that mutations in this enzyme can lead to the development of a form of non-insulin-dependent diabetes mellitus that is characterized by an autosomal dominant mode of inheritance and onset during childhood. Here, we report the catalytic properties of five additional missense mutations associated with diabetes (Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, Trp257-->Arg and Lys414-->Glu), one polymorphism present in both normal and diabetic subjects (Asp4-->Asn), and three site-directed mutations (Glu177-->Lys, Glu256-->Ala, and Lys414-->Ala). The Trp257-->Arg mutation generated an enzyme that had an activity that was less than 0.5% of that for native human beta-cell glucokinase. By contrast, the Glu70-->Lys, Ser131-->Pro, Ala188-->Thr, and Lys414-->Glu mutations had a Vmax that was 20-100% of normal but a Km for glucose that was 8-14-fold greater than the native enzyme. There was no effect of the Asp4-->Asn polymorphism or the Glu177-->Lys substitution on glucokinase activity. The Lys414-->Ala substitution had no effect on Vmax but increased the Km for glucose 2-fold and the Glu256-->Ala substitution caused a approximately 200-fold decrease in Vmax. These studies have led to the identification of additional residues involved in glucokinase catalysis and substrate binding.

Amino Acid Sequence↗
PubMed1993

Sequence and functional characterization of a third inositol trisphosphate receptor subtype, IP3R-3, expressed in pancreatic islets, kidney, gastrointestinal tract, and other tissues.

Inositol 1,4,5-trisphosphate (IP3) functions as a second messenger for many neurotransmitters, hormones and growth factors. It causes the release of Ca2+ from intracellular stores by binding to specific receptors that are coupled to Ca2+ channels. Recent studies have shown that there is a family of IP3 receptors, and the complete sequences of two members of this family and partial sequences of two others have been reported. We have determined the complete sequence of a third IP3 receptor, designated IP3R-3, and characterized its pharmacological properties and sites of expression. Rat IP3R-3 is 2670 amino acids in size, has 62 and 64% identity with IP3R-1 and IP3R-2, and is predicted to have a similar structure including a region of eight potential membrane-spanning segments at its COOH terminus, which presumably functions as a Ca2+ channel. Expression of recombinant rat IP3R-3 in COS-7 cells showed that it bound IP3 as well as inositol 1,3,4,5-tetrakisphosphate and inositol hexakisphosphate. Immunohistocytochemical studies of cells expressing recombinant IP3R-3 indicated that it has a preferential cellular distribution in the endoplasmic reticulum. RNA and protein blotting studies indicate that IP3R-3 is expressed in a number of different cultured cell lines including insulin-secreting RINm5F cells. The IP3R-3 is also expressed in adult pancreatic islets, kidney, gastrointestinal tract, and brain. Reverse transcriptase-polymerase chain reaction amplification of IP3R-1, -2, and -3 mRNAs in adult rat pancreatic islets indicated that IP3R-3 was the predominant subtype expressed in this tissue and thus may be responsible for mediating the effects of IP3 on insulin secretion.

Amino Acid Sequence↗
PubMed1993

Deficiency of the GPI anchor caused by a somatic mutation of the PIG-A gene in paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria is an acquired hematopoietic disease characterized by abnormal blood cell populations in which the biosynthesis of the glycosylphosphatidylinositol (GPI) anchor is deficient. Deficiency of surface expressions of GPI-anchored complement inhibitors leads to complement-mediated hemolysis. Here we report that PIG-A, which participates in the early step of GPI anchor biosynthesis, is the gene responsible for paroxysmal nocturnal hemoglobinuria. Affected granulocytes and B lymphocytes had the same somatic mutation of PIG-A, indicating their clonal origin from a multipotential hematopoietic stem cell. We localized PIG-A to the X chromosome, which accounts for expression of the recessive phenotype of the somatic mutation and the fact that the same one of the multiple biosynthetic steps is affected in all patients so far characterized.

B-Lymphocytes↗
PubMed1993

Cloning of a human gene, PIG-F, a component of glycosylphosphatidylinositol anchor biosynthesis, by a novel expression cloning strategy.

The glycosylphosphatidylinositol (GPI)-anchored proteins are widely distributed in eukaryotic cells, from yeasts to mammals. A number of proteins, such as glycosyltransferases, are necessary for GPI anchor biosynthesis. Cloning of genes encoding these proteins is required for analyses of their nature and the biosynthetic pathway. Here we report a new method of expression cloning that is applicable to many mutant rodent and human cells, and its application for cloning a human cDNA termed PIG-F (for Phosphatidyl-Inositol-Glycan class F) using a Thy-1-negative mutant murine thymoma cell line of complementation class F. PIG-F takes a part in the step of transfer of ethanolamine phosphate to the GPI intermediate containing three residues of mannose. This expression cloning strategy is applicable to the identification of not only other genes involved in GPI anchor biosynthesis but also human disease-associated genes using mutant mammalian cell lines.

Amino Acid Sequence↗
PubMed1993

Differential targeting of glucose transporter isoforms heterologously expressed in Xenopus oocytes.

We have examined the subcellular distribution of three members of the human glucose transporter family expressed in oocytes from Xenopus laevis. Following injection of in vitro-transcribed mRNA encoding the transporter isoform to be studied, we have determined the subcellular localization of the expressed protein by immunofluorescence and by subcellular fractionation coupled with immunoblotting using specific anti-peptide antibodies. We have shown that both the liver-type (GLUT 2) and brain-type (GLUT 3) glucose transporters are expressed predominantly in the plasma membranes of oocytes, and in both cases high levels of glucose transport activity are exhibited. In contrast, the insulin-regulatable glucose transporter (GLUT 4) is localized predominantly to an intracellular membrane pool, and the levels of transport activity recorded in oocytes expressing GLUT 4 are correspondingly lower. The localization of the different transporter isoforms to distinct subcellular fractions mirrors the situation observed in their native cell type and thus demonstrates that oocytes may prove to be a useful system with which to study the targeting signals for this important class of membrane proteins. In addition, the determination of the amounts of the transporters expressed per oocyte together with a knowledge of their Km values has allowed us to estimate the turnover numbers of these transporters. Insulin was without effect on glucose transport in oocytes expressing any of these transporter isoforms. Microinjection of guanosine 5'-[gamma-thio]triphosphate into oocytes expressing GLUT 4 was also without effect on the transport rate.

Animals↗
PubMed1993

Familial hyperglycemia due to mutations in glucokinase. Definition of a subtype of diabetes mellitus.

BACKGROUND AND METHODS: Non-insulin-dependent diabetes mellitus (NIDDM) is a genetically heterogeneous disorder. Maturity-onset diabetes of the young, a form of NIDDM with an early age of onset and autosomal dominant inheritance, can result from mutations in glucokinase, a key enzyme of glucose metabolism in beta cells and the liver. We studied 32 French families with maturity-onset diabetes of the young as well as 21 families with late-onset NIDDM to determine the frequency and clinical features of mutations of glucokinase. Fasting plasma glucose concentrations and oral glucose-tolerance tests were used to determine metabolic status. DNA was isolated from lymphocytes, and DNA polymorphisms in the glucokinase gene were tested for linkage with diabetes. Individual exons of the glucokinase gene from one affected member in each family were amplified by the polymerase chain reaction and screened for mutations by analysis of the conformation-dependent polymorphisms of single-stranded DNA and by DNA sequencing. RESULTS: We found substantial evidence of linkage between the glucokinase locus and maturity-onset diabetes of the young but not between this locus and late-onset NIDDM: Sixteen mutations were identified in 18 of the 32 families with maturity-onset diabetes of the young, but none were found in families with late-onset NIDDM: They included 10 mutations that resulted in an amino acid substitution, 3 that resulted in the synthesis of a truncated protein, and 3 that affected RNA processing. The affected subjects with glucokinase mutations usually had mild hyperglycemia that began during childhood, whereas in subjects with maturity-onset diabetes of the young not due to glucokinase mutations, hyperglycemia usually appeared after puberty. CONCLUSIONS: Mutations in glucokinase are the primary cause of hyperglycemia in a substantial fraction of French patients with maturity-onset diabetes of the young and result in a relatively mild form of NIDDM that can be diagnosed in childhood.

Adult↗
PubMed1993

Glucokinase mutations associated with non-insulin-dependent (type 2) diabetes mellitus have decreased enzymatic activity: implications for structure/function relationships.

The glycolytic enzyme glucokinase plays an important role in the regulation of insulin secretion and recent studies have shown that mutations in the human glucokinase gene are a common cause of an autosomal dominant form of non-insulin-dependent (type 2) diabetes mellitus (NIDDM) that has an onset often during childhood. The majority of the mutations that have been identified are missense mutations that result in the synthesis of a glucokinase molecule with an altered amino acid sequence. To characterize the effect of these mutations on the catalytic properties of human beta-cell glucokinase, we have expressed native and mutant forms of this protein in Escherichia coli. All of the missense mutations show changes in enzyme activity including a decrease in Vmax and/or increase in Km for glucose. Using a model for the three-dimensional structure of human glucokinase based on the crystal structure of the related enzyme yeast hexokinase B, the mutations map primarily to two regions of the protein. One group of mutations is located in the active site cleft separating the two domains of the enzyme as well as in surface loops leading into this cleft. These mutations usually result in large reductions in enzyme activity. The second group of mutations is located far from the active site in a region that is predicted to undergo a substrate-induced conformational change that results in closure of the active site cleft. These mutations show a small approximately 2-fold reduction in Vmax and a 5- to 10-fold increase in Km for glucose. The characterization of mutations in glucokinase that are associated with a distinct and readily recognizable form of NIDDM has led to the identification of key amino acids involved in glucokinase catalysis and localized functionally important regions of the glucokinase molecule.

Amino Acid Sequence↗
PubMed1993

The cloning of PIG-A, a component in the early step of GPI-anchor biosynthesis.

The glycosylphosphatidylinositol (GPI) anchor is a membrane attachment structure of many proteins and occurs in a wide variety of eukaryotes from yeasts to mammals. The structure of the core of the GPI anchor is conserved in protozoa and mammals and so is its biosynthetic pathway. A complementary DNA encoding a human protein termed PIG-A (phosphatidylinositol glycan-class A) was cloned. PIG-A was necessary for synthesis of N-acetylglucosaminyl-phosphatidylinositol, the very early intermediate in GPI-anchor biosynthesis.

Amino Acid Sequence↗
PubMed1993

Deficient biosynthesis of N-acetylglucosaminyl-phosphatidylinositol, the first intermediate of glycosyl phosphatidylinositol anchor biosynthesis, in cell lines established from patients with paroxysmal nocturnal hemoglobinuria.

Paroxysmal nocturnal hemoglobinuria (PNH) is a hemolytic disorder caused by a deficiency of biosynthesis of the glycosyl phosphatidylinositol (GPI) anchor, but the biochemical defect is not completely understood. In the present study, we have analyzed affected cell lines established recently from two Japanese patients with PNH. Two lines of evidence indicate that these cells do not synthesize N-acetylglucosaminyl-phosphatidylinositol, the first intermediate in the GPI anchor biosynthesis. First, somatic cell hybridization analysis using Thy-1-deficient murine thymoma cell lines with known biochemical defects as fusion partners showed that the PNH cell lines belong to complementation class A, which is known not to synthesize N-acetylglucosaminyl-phosphatidylinositol. Second, analysis of in vitro glycolipid biosynthesis demonstrated that cell lysates of these PNH cell lines in fact did not support biosynthesis of N-acetylglucosaminyl-phosphatidylinositol. Thus, we have characterized for the first time the exact biochemical defect leading to PNH.

Cells, Cultured↗
PubMed1993

Morphological aspects of Achacin-treated bacteria.

1. The morphology of bacteria treated with the bactericidal glycoprotein, Achacin, purified from the giant African snail, Achatina fulica Férussac, has been studied. 2. Achacin lengthens the bodies of Escherichia coli by three to seven times. 3. Achacin damages the surface of Staphylococcus aureus and sinks the cytoplasmic membranes into the cytoplasm. 4. Achacin causes neither the leakage nor the destruction of cells.

Escherichia coli↗
PubMed1993

A molecular inventory of human pancreatic islets: sequence analysis of 1000 cDNA clones.

The islets of Langerhans play a central role in glucose homeostasis by secreting the polypeptide hormones insulin and glucagon. They are comprised primarily of four endocrine cell types: insulin-secreting beta-cells which represent about 70% of the cells in the islet along with smaller number of cells secreting glucagon, somatostatin and pancreatic polypeptide. Diabetes mellitus results from the specific loss or dysfunction of the beta-cells. Because of the central role of the islets of Langerhans in the regulation of glucose homeostasis, we are preparing a database of genes expressed in this tissue. One thousand cDNA clones randomly isolated from a human pancreatic islet library were partially sequenced yielding 280 kilobases of sequence. Database searches indicated that 397 of the cDNAs represented known human genes or human homologs of genes identified in other species and a further 58 sequences corresponded to expressed sequence tags identified in other tissues or cells (contamination by exocrine pancreatic tissue was estimated to be less than 10%). 545 of the cDNAs were not related to any other sequences in the databases. The islet cDNA collection provides a unique source of genes for genetic studies of diabetes as well as for molecular studies of islet function in normal and diabetic states.

Animals↗
PubMed1993

Ligand specificities of mouse complement receptor types 1 (CR1) and 2 (CR2) purified from spleen cells.

Murine complement receptor type 2 (MCR2) is homologous to human CR2, whereas murine CR1 (MCR1) is structurally and evolutionary different from human CR1. Since ligand specificities of MCR1 and MCR2 are not completely clarified, we analyzed their functional characteristics to correlate them with structural information obtained in molecular biological studies. MCR1 and MCR2 purified from spleen cells were incubated with thiol-Sepharose bearing murine C3b, iC3b, or C3d in the presence or absence of various anti-MCR1 or -MCR1/MCR2 mAbs. Bound and free MCR1 and MCR2 were quantitated by Western blotting or two-site immunoradiometric assay. MCR2 bound to C3d and iC3b similarly efficiently, and 5-fold less efficiently to C3b. These bindings were completely inhibited by MCR1/MCR2-crossreactive antibodies 7G6 and 4E3. MCR1 bound to C3b efficiently and this was partially inhibited by MCR1-monospecific antibody 8C12, but not by 7G6 and 4E3. Combinations of 8C12 and 7G6 or 4E3 completely inhibited MCR1 binding to C3b. Therefore, two binding sites, one unique to MCR1 and the other shared with MCR2, are involved in MCR1 binding to C3b. MCR1 bound also to C3d and this was completely inhibited by 7G6 and 4E3. The binding of this solubilized MCR1 to C3d was as efficient as that of MCR2 to C3d. It seems, therefore, that the site shared by MCR1 and MCR2 that is recognized by both 7G6 and 4E3 binds to C3d and less efficiently to C3b. These results clarify the ligand specificities of MCR1 and MCR2.

Animals↗
PubMed1993

Contribution of rib cage and abdominal movement to ventilation for successful weaning from mechanical ventilation.

In order to test the hypothesis whether the breathing pattern is helpful in predicting weaning outcome in patients being weaned from mechanical ventilation, 38 patients who underwent operation for esophageal cancer were evaluated at weaning from mechanical ventilation (19 unsuccessful weanings, group U, and 19 successful weanings in age-matched patients, group S). Since all patients initially fulfilled our weaning criteria, ventilatory parameters such as tidal volume, respiratory frequency, minute ventilation, and arterial blood gas analysis showed no significant differences between the groups. The breathing pattern was registered quantitatively by means of respiratory inductive plethysmography at 3 cmH2O (0.3 kPa) of CPAP prior to weaning. The contribution of rib cage movement to tidal volume (%RC) was significantly greater in group U than in group S (P < 0.05). Indeed, 84% of the patients in group S showed %RC less than 50%, compared to only 16% of the patients in group U (P < 0.05). The results suggest that the breathing pattern is one important factor in predicting the outcome of weaning in patients after thoraco-abdominal surgery. Diaphragmatic fatigue is suspected to be the mechanism for the increase in the RC component in patients with unsuccessful weaning outcome.

Abdomen↗
PubMed1993

Organization of the human GLUT2 (pancreatic beta-cell and hepatocyte) glucose transporter gene.

The gene encoding the predominant facilitative glucose transporter expressed in pancreatic beta-cells and hepatocytes, termed GLUT2, has been cloned and characterized. The human GLUT2 gene is composed of 11 exons spanning approximately 30 kilobases. The sequence of the promoter region and all exons and adjacent intron regions has been determined and deposited in the GenBank database. Two highly polymorphic simple tandem repeat DNA polymorphisms useful for linkage studies were localized in introns 1 and 4a. In addition, a 168-base pair insertion/deletion polymorphism was identified in intron 3. The characterization of the human GLUT2 gene will facilitate studies of its role in the development of diabetes mellitus.

Amino Acid Sequence↗
PubMed1993

Identification of glucokinase mutations in subjects with gestational diabetes mellitus.

Recent studies have shown that mutations in the glucokinase gene on chromosome 7 can cause an autosomal dominant form of NIDDM with a variable clinical phenotype and onset during childhood. The variable clinical phenotype includes mild fasting hyperglycemia (i.e., a plasma glucose value of > 110 mg/dl, a value that is at least 2-3 SDs above normal), impaired glucose tolerance, gestational diabetes mellitus, as well as overt NIDDM as defined using National Diabetes Data Group or World Health Organization criteria. Because gestational diabetes mellitus was a clinical feature associated with glucokinase mutations, we have screened a group of women with gestational diabetes who also had a first-degree relative with diabetes mellitus for the presence of mutations in this gene. Among 40 subjects, we identified two mutations, suggesting a prevalence of approximately 5% in this group. Extrapolating from this result, the prevalence of glucokinase-deficient NIDDM among Americans may be approximately 1 in 2500.

Amino Acid Sequence↗
PubMed1993

Expression of calcium channel mRNAs in rat pancreatic islets and downregulation after glucose infusion.

Recent studies have shown that two different voltage-dependent Ca2+ channels are expressed in pancreatic islets, the beta-cell/neuroendocrine-brain and the cardiac subtypes. The effects of chronic hyperglycemia on the levels in pancreatic islets of the mRNAs encoding the alpha 1-subunits of the beta-cell and cardiac subtype Ca2+ channels were studied in rats made hyperglycemic by infusion of glucose for 48 h. A competitive reverse transcriptase-polymerase chain reaction procedure was used to obtain quantitative data on the levels of these two transcripts in islets obtained from individual rats. The quantitative polymerase chain reaction data indicate that the levels of mRNA encoding the alpha 1-subunit of the beta-cell Ca2+ channel are 2.5-fold greater than those for the cardiac subtype. The levels of beta-cell Ca2+ channel mRNA were 72.9% lower in the glucose-infused animals when compared with the saline-infused animals (P < 0.005) and those of the cardiac channel were 72.1% lower in the animals infused with glucose (P < 0.02). In contrast, glucose infusion resulted in a twofold increase in insulin mRNA levels and did not significantly alter levels of beta-actin mRNA. In situ hybridization studies revealed that the mRNAs for these two Ca2+ channels are expressed at higher levels in normal rat islets than in the surrounding acinar tissue, which suggests that the observed changes in mRNA levels occur within cells of the pancreatic islet. To assess the possible functional consequences of this reduction in expression of mRNA for the Ca2+ channels, the insulin secretory responses of perfused pancreases to the Ca2+ channel agonist Bay K8644 were studied.(ABSTRACT TRUNCATED AT 250 WORDS)

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗