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H Sakura

Publications and source records attributed to H Sakura.

At least 37 records · Page 2Linked to original sources
PubMed1995

No evidence for mutations in a putative beta-cell ATP-sensitive K+ channel subunit in MODY, NIDDM, or GDM.

The beta-cell ATP-sensitive K+ (K-ATP) channel has a major role in glucose-induced insulin secretion. Screening the entire coding sequence of the gene for a putative beta-cell K-ATP channel subunit, K-ATP2, with single-strand conformation polymorphism did not show any mutations associated with diabetes in white Caucasian diabetic patients, including five pedigrees with maturity onset diabetes of the young (MODY), 25 patients with noninsulin-dependent diabetes mellitus (NIDDM) selected for marked beta-cell deficiency, 25 selected for mild diabetes presenting before age 50 years with fasting plasma glucose levels < 10 mmol/l, 25 unselected NIDDM patients, and 25 subjects with gestational diabetes mellitus (GDM) and subsequent raised fasting plasma glucose. In five large MODY pedigrees, linkage analysis with simple tandem-repeat polymorphisms (STRPs) near the K-ATP2 gene excluded linkage. In a population association study, no linkage disequilibrium for the STRP was found between 237 unselected white Caucasian NIDDM patients and 104 geographically matched and age-matched white Caucasian nondiabetic subjects. In addition, two silent polymorphisms were found with similar frequency in nondiabetic and diabetic subjects. Mutations in the gene for K-ATP2 are unlikely to be a major cause of MODY, NIDDM, or GDM.

Adenosine Triphosphate↗
PubMed1994

Insulin resistance and growth retardation in mice lacking insulin receptor substrate-1.

Insulin receptor substrate-1 (IRS-1) is the major substrate of insulin receptor and IGF-1 receptor tyrosine kinases; it has an apparent relative molecular mass of 160-190,000 (M(r), 160-190K) on SDS polyacrylamide gel. Tyrosine-phosphorylated IRS-1 binds the 85K subunit of phosphatidylinositol 3-kinase which may be involved in the translocation of glucose transporters and the abundant src homology protein (ASH)/Grb2 which may be involved in activation of p21ras and MAP kinase cascade. IRS-1 also has binding sites for Syp and Nck and other src homology 2 (SH2) signalling molecules. To clarify the physiological roles of IRS-1 in vivo, we made mice with a targeted disruption of the IRS-1 gene locus. Mice homozygous for targeted disruption of the IRS-1 gene were born alive but were retarded in embryonal and postnatal growth. They also had resistance to the glucose-lowering effects of insulin, IGF-1 and IGF-2. These data suggest the existence of both IRS-1-dependent and IRS-1-independent pathways for signal transduction of insulin and IGFs.

Animals↗
PubMed1994

Molecular scanning of the glycogen synthase and insulin receptor substrate-1 genes in Japanese subjects with non-insulin-dependent diabetes mellitus.

We studied a simple tandem repeat DNA polymorphism in the glycogen synthase gene and polymorphisms at codon 513 (Ala-->Pro) and 972 (Gly-->Arg) in the insulin receptor substrate-1 (IRS-1) gene in 197 non-insulin-dependent diabetes mellitus (NIDDM) and 178 control subjects in Japan. Eight alleles (-3G, -2G, -1G, 0G, 1G, 2G, 3G, and 4G) were identified in the tandem repeat polymorphism in the glycogen synthase gene. No difference in the frequencies of these alleles was found between diabetics and controls. The codon 972 polymorphism of IRS-1 gene was observed in 7 diabetics (3.6%) and 8 controls (4.5%), whereas the codon 513 polymorphism was not found in either of the two groups. We conclude that the tandem repeat polymorphism in the glycogen synthase gene and the polymorphisms at codons 513 and 972 of the IRS-1 gene are not associated with a higher risk for the development of NIDDM in Japanese subjects.

Alanine↗
PubMed1994

A subtype of diabetes mellitus associated with a mutation of mitochondrial DNA.

BACKGROUND: Several families have been described in which a mutation of mitochondrial DNA, the substitution of guanine for adenine (A-->G) at position 3243 of leucine transfer RNA, is associated with diabetes mellitus and deafness. The prevalence, clinical features, and pathophysiology of diabetes with this mutation are largely undefined. METHODS: We studied 55 patients with insulin-dependent diabetes mellitus (IDDM) and a family history of diabetes (group 1), 85 patients with IDDM and no family history of diabetes (group 2), 100 patients with non-insulin-dependent diabetes mellitus (NIDDM) and a family history of diabetes (group 3), and 5 patients with diabetes and deafness (group 4) for the mutation. We also studied the prevalence and characteristics of diabetes in 39 patients with a syndrome consisting of mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes who were known to have the mutation and 127 of their relatives (group 5). RESULTS: We identified 16 unrelated patients with diabetes associated with the A-->G mutation: 3 patients from group 1 (6 percent), 2 patients from group 3 (2 percent), 3 patients from group 4 (60 percent), and 8 patients from group 5 (21 percent). We also identified 16 additional subjects who had diabetes and the mutation among 42 relatives of the patients with diabetes and the mutation in groups 1, 2, 3, and 4 and 20 affected subjects among the 127 relatives of the patients in group 5. Diabetes cosegregated with the mutation in a fashion consistent with maternal transmission, was frequently (in 61 percent of cases) associated with sensory hearing loss, and was generally accompanied by impaired insulin secretion. CONCLUSIONS: Diabetes mellitus associated with the A-->G mutation at position 3243 of mitochondrial leucine transfer RNA represents a subtype of diabetes found in both patients with IDDM and patients with NIDDM in Japan.

Adolescent↗
PubMed1994

Cloning of a complete protein-coding sequence of human platelet-type phosphofructokinase isozyme from pancreatic islet.

We have cloned a full length protein-coding sequence of human platelet-type phosphofructokinase (PFK) from pancreatic islet cDNA library. The platelet-type PFK was composed of 784 amino acids and had a deduced molecular weight of 85,590. Homologies in the primary structure with muscle- and liver-type PFK were 71 and 67%. Clear similarities of the amino and carboxyl halves with a prokaryotic PFK indicated an evolutionary event that duplicated genes of a prototype PFK fused into larger genes of eukaryotic PFKs. Amino acid residues constituting the binding sites for various allosteric modulators were well conserved, while a couple of different residues at the inhibitory ATP sites among three isozymes may partly explain their varied degree of sensitivities to ATP. Considerable amount of platelet-type PFK expression was demonstrated in brain, heart, kidney, colon and testis.

Amino Acid Sequence↗
PubMed1994

The high prevalence of the diabetic patients with a mutation in the mitochondrial gene in Japan.

Recently, an A to G transition at position 3243 in transfer ribonucleic acidLeu(UUR) [the 3243 base-pair (bp) mutation] originally found in patients with mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes has been identified in patients with diabetes and deafness. To determine the prevalence of the diabetic patients with this mutation in Japan, we screened 550 randomly selected cohorts of diabetic patients without prior information about clinical features such as type of diabetes, family history of diabetes, age of onset, and mode of therapy. We have identified 5 patients with this mutation, suggesting that approximately 0.9% of diabetic patients have the 3243 bp mutation. However, there were no subjects with this mutation in 250 controls with normal glucose tolerance. The percentage of mutant DNA in whole mitochondrial DNA did not correlate to the degree of symptoms. We conclude that the 3243 bp mutation in the mitochondrial gene plays an important part as a cause of diabetes in Japan.

Adolescent↗
PubMed1994

Analysis of the glucokinase gene promoter in Japanese subjects with noninsulin-dependent diabetes mellitus.

Glucokinase plays an important role in glucose metabolism in pancreatic beta-cells and liver. Recently, several mutations responsible for noninsulin-dependent diabetes mellitus (NIDDM) have been identified within the coding regions of the glucokinase gene. We screened the promoter regions using polymerase chain reaction followed by single strand conformation polymorphisms in 240 Japanese NIDDM and 111 control subjects. In the beta-cell promoter, two kinds of sequence variations were detected. One variation, in which 2 nucleotides at position -282 (C-->T) plus -194 (A-->G) were changed simultaneously, was found in 23 NIDDM (9.6%) and 12 control (10.8%) subjects. The other variation [e.g. -30 (G-->A)] was identified in 87 NIDDM (36.3%) and 40 control (36.0%) subjects. In the liver promoter, in addition to the -603 (G-->T) substitution in 1 NIDDM (0.4%) and 2 control (1.8%) subjects, the -120 (G-->T) substitution in 1 control (0.9%) subject was found. However, there were no differences in these allele frequencies between NIDDM and control subjects. We conclude that the prevalence of mutations in the promoter of the glucokinase gene responsible for NIDDM is rare among Japanese patients.

Adolescent↗
PubMed1993

Site-directed mutagenesis of the juxtamembrane domain of the human insulin receptor.

We have studied the functions of the juxtamembrane domain (941-989) of the human insulin receptor by site-directed mutagenesis. Tyrosine phosphorylation of pp185 was impaired in Chinese hamster ovary cells expressing the receptors with the alteration of Tyr960, but not of Tyr953 or Tyr972, to Phe (CHO-Y960F cells) as compared with cells expressing the normal receptors. In CHO-Y960F cells, tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1), the activation of phosphatidylinositol 3-kinase in the anti-phosphotyrosine and anti-IRS-1 immunoprecipitates, the activation of mitogen-activated protein (MAP) kinase, and biological actions were also impaired. In addition, although the deletion of residues 954-965 severely impaired insulin internalization, the deletion of NPXY (957-960), the internalization signal of the low density lipoprotein receptor, did not affect internalization. Moreover, neither the deletions around Tyr953 nor the alterations of the tyrosines (953, 960, or 972) significantly reduced internalization. These data suggest that: 1) Tyr960 is important for the recognition of pp185/IRS-1, the association of phosphatidylinositol 3-kinase with pp185/IRS-1, and the activation of MAP kinase; 2) MAP kinase may lie downstream of pp185/IRS-1 in insulin's signal transduction; and 3) the juxtamembrane domain, but not NPXY or individual tyrosines, is important for insulin internalization.

Amino Acid Sequence↗
PubMed1993

Sequence variations of the glucokinase gene in Japanese subjects with NIDDM.

Mutations in the glucokinase gene have been identified recently in patients with maturity-onset diabetes of the young, a subtype of NIDDM. The proposed role of glucokinase as a glucose sensor, combined with the low insulin response to glucose found in most Japanese with NIDDM, prompted us to speculate that mutations in the glucokinase gene might be one of the major causes of NIDDM in Japanese subjects. To determine the prevalence of mutations and sequence variations in the glucokinase gene, we screened all 12 exons of the glucokinase gene, including exon/intron junctions, by polymerase chain reaction followed by single-strand conformation polymorphism in 209 Japanese NIDDM subjects. In addition to the mutation in exon 7, which substituted Arg (AGG) for Gly (GGG) at codon 261 (10), a silent mutation of Pro (CCC-->CCG) in exon 4 at codon 145 and several new sequence variations in intervening sequences and the 5'-untranslated region of exon 1 beta (beta-cell-specific exon 1) were identified. Because we identified only one subject who had a structurally abnormal glucokinase molecule, we conclude that the prevalence of structural mutations in the glucokinase gene responsible for NIDDM appears to be rare among Japanese patients. To our knowledge, this is the first thorough study describing the ethnic prevalence of mutations and sequence variations in the glucokinase gene in NIDDM.

Adolescent↗
PubMed1992

Site-directed mutagenesis of GLUT1 in helix 7 residue 282 results in perturbation of exofacial ligand binding.

The structure-function relationship of the HepG2/erythrocyte-type glucose transporter (GLUT1) has been studied by in vitro site-directed mutagenesis. Chinese hamster ovary clones in which glucose transporters were transfected were shown by Western blotting with a GLUT1 anti-COOH-terminal peptide antibody to have expression levels of Gln282----Leu, Asn288----Ile, and Asn317----Ile mutations that were comparable with the wild type. All three mutant GLUT1 clones had high 2-deoxy-D-glucose transport activity compared with a nontransfected clone, suggesting that these residues are not absolutely required for the transport function. We have examined the possibility that the inner and outer portions of the transport pathway are structurally separate by measuring the interaction of the mutant transporters with the inside site-specific ligand cytochalasin B and the outside site-specific ligand 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis(D-mannos-4 -yloxy)-2- propylamine (ATB-BMPA). All three mutant GLUT1 clones showed high levels of cytochalasin B labeling, and the N288I and N317I mutants showed high levels of ATB-BMPA labeling. In contrast to the transport and cytochalasin B labeling results, the transmembrane helix 7 Gln282----Leu mutant was labeled by ATB-BMPA to a level that was only 5% of the level observed in the wild type. We have confirmed that this mutant was defective in the outer site by comparing the inhibition of wild-type and mutant 2-deoxy-D-glucose transport by the outside site-specific ligand 4,6-O-ethylidene-D-glucose. 4,6-O-Ethylidene-D-glucose inhibited wild-type transport with a Ki of approximately 12 mM, but this was increased to greater than 120 mM in the Gln282----Leu mutant. Thus, of the 3 residues mutated in this study, only glutamine 282 substitution causes a major perturbation in function, and this is a specific and striking reduction in the affinity for the outside site-specific ligands ATB-BMPA and 4,6-O-ethylidene-D-glucose.

Amino Acid Sequence↗
PubMed1992

Enhanced insulin-induced mitogenesis and mitogen-activated protein kinase activities in mutant insulin receptors with substitution of two COOH-terminal tyrosine autophosphorylation sites by phenylalanine.

We have studied the function of a mutant human insulin receptor in which two COOH-terminal autophosphorylation sites (Tyr-1316 and -1322) were replaced by phenylalanine (F/Y COOH-terminal 2 tyrosines (CT2)). In addition, we have also constructed a mutant receptor in which Lys-1018 in the ATP-binding site was changed to arginine (R/K 1018). Both the wild type insulin receptor (HIR) and the mutant receptors were expressed in Chinese hamster ovary (CHO) cells by stable transfection. Autophosphorylation of solubilized and partially purified F/Y CT2 was decreased by approximately 30% compared with the HIR. Tyrosine kinase activities of F/Y CT2 and HIR toward exogenous substrates were almost equal. When CHO cells transfected with F/Y CT2 (CHO-F/Y CT2) were stimulated with insulin, autophosphorylation of the beta-subunit of the insulin receptor and the phosphorylation of an endogenous substrate (pp185) in the intact cell were normal compared with cells expressing HIR (CHO-HIR). CHO-F/Y CT2 exhibited the same insulin sensitivity as CHO-HIR with respect to 2-deoxyglucose uptake. However, the dose-response curve of insulin-stimulated thymidine incorporation in CHO-F/Y CT2 was shifted to the left (approximately 5-7-fold) compared with that in CHO-HIR. There was no significant difference in insulin-like growth factor 1-stimulated thymidine incorporation between CHO-F/Y CT2 and CHO-HIR. Furthermore, the dose-response curve of insulin-stimulated kinase activity toward myelin basic protein in CHO-F/Y CT2 was also shifted to the left (approximately 5-fold) compared with that in CHO-HIR. Kinase assays in myelin basic protein-containing gels revealed that both species of MAP kinases (M(r) 44,000, 42,000) were more sensitive to activation by insulin in CHO-F/Y CT2 than in CHO-HIR. This observation was confirmed in immune complex kinase assays toward microtubule-associated protein 2 (MAP2) using specific antibodies against mitogen-activated protein (MAP) kinase. R/K 1018 mutant insulin receptors showed an absence of insulin-stimulated kinase activity and CHO cells transfected with R/K 1018 (CHO-R/K 1018) failed to enhance 2-deoxyglucose uptake or thymidine incorporation in response to insulin. In addition, R/K 1018 kinase-defective insulin receptors were unable to mediate insulin-stimulated MAP kinase activation. These data suggest that: 1) tyrosine kinase activity of the insulin receptor is required for activation of insulin-stimulated MAP kinases and 2) phosphorylation of COOH-terminal tyrosine residues may play an inhibitory role in mitogenic signaling through regulation of MAP kinases.

Animals↗
PubMed1992

Detection of mutations in the insulin receptor gene in patients with insulin resistance by analysis of single-stranded conformational polymorphisms.

We analyzed single-stranded conformational polymorphisms to screen for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Using this new technique, we demonstrated the existence of mutations in the insulin receptor gene which we had identified previously. In addition, a new mutation was found in exon 20 of the insulin receptor gene in a patient with moderate insulin resistance associated with morbid obesity, acanthosis nigricans, and polycystic ovary syndrome. The patient was heterozygous for a mutation substituting Leu (CTG) for Pro (CCG) at codon 1178. Pro1178 is a part of a characteristic sequence motif (D1150 F1151 G1152---A1177 P1178 E1179) common to many protein kinases. Analysis of single-stranded conformational polymorphisms was also used to estimate the frequency of a polymorphism at codon 1058. The two codons CAC (1058 His) and CAT (1058 His) both had a prevalence of 50% in 30 Japanese subjects. These data demonstrate that analysis of single-stranded conformational polymorphisms is a simple and sensitive screening method for mutations and polymorphisms in the insulin receptor gene in subjects with or without insulin resistance. Identification of a mutation in the insulin receptor gene in a patient with a moderate degree of insulin resistance associated with morbid obesity suggests that insulin receptor mutations may exist in patients with Type 2 (non-insulin-dependent) diabetes mellitus associated with a moderate degree of insulin resistance.

Amino Acid Sequence↗
PubMed1992

Detection of mutations in the human insulin gene by single strand conformation polymorphisms.

Single strand conformation polymorphisms (SSCP) is the method by which mutations can be detected in DNA amplified by the polymerase chain reaction (PCR). This method utilizes the fact that the electrophoretic mobility of single stranded DNA under nondenaturing condition depends not only on its size but also on its conformation. We examined whether this technique could detect five mutations that had previously been identified in the insulin genes of patients with abnormal insulin or familial hyperproinsulinemia. Five mutant insulin genes were constructed by site directed mutagenesis, and the cloned mutant insulin genes were amplified by PCR. All five mutations were detected because they were associated with shifts in the electrophoretic mobility of the DNA fragments. In addition, we analyzed amplified genomic DNA from a patient who is heterozygotes for the Insulin Wakayama mutation A3(Val----Leu). This mutation was also detected by the SSCP technique. We conclude that PCR-SSCP method is a simple, fast, and efficient method for detection of mutations in the human insulin gene.

Base Sequence↗
PubMed1992

Structure of the human glucokinase gene and identification of a missense mutation in a Japanese patient with early-onset non-insulin-dependent diabetes mellitus.

Glucokinase is thought to play a glucose-sensor role in the pancreas, and abnormalities in its structure, function, and regulation can induce diabetes. We isolated the human glucokinase gene, and determined its genomic structure including exon-intron boundaries. Structure of the glucokinase gene in human was very similar to that in rat. Then, by screening Japanese diabetic patients using polymerase chain reaction--single strand conformation polymorphism (PCR-SSCP) and direct-sequencing strategies, we identified a missense mutation substituting arginine (AGG) for glycine (GGG) at position 261 in exon 7 of the glucokinase gene in a patient with early-onset non-insulin-dependent diabetes (NIDDM).

Base Sequence↗
PubMed1991

A study of human calcitonin in an ovarian carcinoid and ovarian cancers.

High concentration of human calcitonin (hCT) was found in an ovarian carcinoid by radioimmunoassay. The hCT value was not affected by the presence of protease inhibitors. To confirm the presence of hCT in an ovarian carcinoid, hCT was isolated by the Baghdiantz method. The molecular weight of the ovarian hCT was determined using Sephadex G-75 gel filtration. Though the molecular weight of the ovarian hCT was variable, 90% corresponded to that of the authentic hCT. The carcinoid cells were examined by immunoperoxidase techniques. Those composed of strumal and trabecular structure were all argyrophilic, but hCT was only found in strumal structure. Significant concentrations of hCT were also found in ovarian cancers.

Aprotinin↗