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it may represent a pore-forming subunit of a brain and fl-cell Abstract The ATP-sensitive K-channel plays a central role in KAT P channel (5). insulin release from pancreatic/3-cells. We report here the don- In this study, we report the cloning and genomic structure ing of the gene (KCNJ6) encoding a putative subunit of a human of the human BIR1 gene, identification of its chromosomal ATP-sensitive K-channel expressed in brain and /)-cells, and characterisation of its exon-intron structure. Screening of a location and determination of the frequency of polymorphisms somatic cell mapping panel and fluorescent in situ hybridization in this gene. place the gene on chromosome 21 (21q22.1-22.2). Analysis of single-stranded conformational polymorphisms revealed the pres- 2. Materials and methods ence of two silent polymorphisms (Pro-149: CCG-CCA and Asp- 328: GAC-GAT_.) with similar frequencies in normal and non- 2.1. Isolation of the human BIR1 genomic DNA insulin-dependent diabetic patients. The human BIR1 gene was isolated from a human genomic library (Clonetech and ATCC) using the full-length rat BIR1 cDNA (5) as a

The ATP-sensitive K-channel plays a central role in insulin release from pancreatic beta cells. This channel consists of two subunits: a sulphonyl-urea receptor, SUR1, and an inwardly rectifying K-channel subunit, Kir6.2. We screened 135 white Caucasian patients with non-insulin-dependent diabetes mellitus (NIDDM) and 90 non-diabetic subjects for mutations in the Kir6.2 gene by single-stranded conformational polymorphism (SSCP) analysis. We identified one silent mutation (A190A) and four missense mutations (E23K, L270V, I337V and S385C) in normal and diabetic individuals. In a single diabetic subject, we identified a two-amino acid insertion (380KP). We also screened 39 Afro-Caribbean diabetic subjects and identified one additional missense (L355P) and one more silent (S363S) mutation. The E23K and I337V variants were completely linked. The common variants (E23K, 1337V and L270V) were found with similar frequency in diabetic and normal subjects. Diabetic subjects with the variants responded normally to sulphonylurea therapy. When mutant Kir6.2 subunits were coexpressed with SUR1 in Xenopus oocytes, there was no difference in the sensitivity of the whole-cell currents to metabolic inhibition or to the sulphonylurea tolbutamide. We therefore conclude that mutations in Kir6.2 are unlikely to be a major cause of NIDDM.

Insulin secretion is stimulated by glucose, hormones and neurotransmitters. Both activation of a non-selective cation current and activation of a Ca2+ current in response to depletion of intracellular Ca2+ stores have been suggested to play a role in this stimulation. The properties of these currents resemble those reported for the Drosophila genes trp and trpl. Using the reverse transcription polymerase chain reaction and Northern blot analysis we found that of the six mammalian trp-related genes (trp1-6), only trp1 was expressed at high levels in the mouse insulinoma cell line MIN6. We cloned the murine homologue of human trp1 from MIN6 cells and identified four variants (alpha, beta, gamma and delta), generated by alternative splicing near the N-terminus of the protein. In vitro translation showed that only the alpha and beta splice variants are efficiently expressed. The beta variant is the dominant form in MIN6 cells (and probably in mouse pancreatic islets), whereas the alpha variant is the major type in the mouse brain. The beta variant showed 99% identity to the human homologue at the amino acid level.

The β-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 β-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 β-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.

This preliminary investigation, involving 422 patients, tested the hypothesis that rate of eating is associated with obesity in patients with type 2 diabetes or hyperlipidaemia at all ages. The patients' eating habits were determined using a questionnaire, and the patients were classified as quick, normal or slow eaters. The body mass indices of the three groups were compared. The body mass indices of the male patients who ate quickly (25.4 ± 0.2 kg/m2) were significantly higher than those of the patients who ate at a normal rate (24.4 ± 0.3 kg/m2) or slowly (24.1 ± 0.5 kg/m2). No difference between body mass indices in the female groups was found. It was speculated that rate of eating affects body weight in male patients with type 2 diabetes or hyperlipidaemia.

The study was performed to indicate the ADPase activity of 5'-nucleotide phosphodiesterase (PDEase) from human umbilical cord blood serum and demonstrates the effect of this enzyme on ADP-induced platelet aggregation. The PDEase was purified by using p-nitrophenyl-5'-TMP as a substrate. The PDEase had a molecular weight of 128,000 daltons, and activity of 103 nmol/min/mg protein. The PDEase activity was inhibited by 5'-AMP, ADP, ATP. But 2'-AMP, 3'-AMP, 3':5' cAMP, and adenosine had no inhibiting effects. Kinetic analysis indicated that ADP was a competitive inhibitor with a Ki value of 4.05x10(-5) M. The enzyme was markedly inhibited by 1 mM EDTA. The ADPase activity of the PDEase was 7.79 nmol/min/mg protein. The hydrolized products of ADP by the PDE ase were AMP and phosphoric acid. The platelet aggregation by ADP was inhibited by the addition of the PDEase in the platelet-rich plasma.

Mice carrying a null mutation in the glucokinase (GK) gene in pancreatic beta-cells, but not in the liver, were generated by disrupting the beta-cell-specific exon. Heterozygous mutant mice showed early-onset mild diabetes due to impaired insulin-secretory response to glucose. Homozygotes showed severe diabetes shortly after birth and died within a week. GK-deficient islets isolated from homozygotes showed defective insulin secretion in response to glucose, while they responded to other secretagogues: almost normally to arginine and to some extent to sulfonylureas. These data provide the first direct proof that GK serves as a glucose sensor molecule for insulin secretion and plays a pivotal role in glucose homeostasis. GK-deficient mice serve as an animal model of the insulin-secretory defect in human non-insulin-dependent diabetes mellitus.

The ATP-sensitive K channel (K-ATP channel) in pancreatic β cells is believed to play a crucial role in glucose stimulated insulin release. We investigated whether defects in the recently cloned gene for a putative subunit of this channel (KATP-2) could be a cause of diabetes in Japanese patients. The coding region of this β-cell type channel gene was investigated in 192 diabetics with a family history of the disorder by single-stranded conformational polymorphism (SSCP) analysis. Two silent polymorphisms were found and confirmed by sequencing, but no missense or nonsense mutations were detected. The allele frequency of the polymorphisms was compared with 96 control subjects without a family history of the disease, and no clear difference was found. These results indicate that genetic defects of the KATP-2 channel may not be a major cause of diabetes in Japan.

A cDNA clone encoding an inwardly-rectifying K-channel (BIR1) was isolated from insulinoma cells. The predicted amino acid sequence shares 72% identity with the cardiac ATP-sensitive K-channel rcKATP (KATP-1; [6]). The mRNA is expressed in the brain and insulinoma cells. Heterologous expression in Xenopus oocytes produced currents which were K+-selective, time-independent and showed inward rectification. The currents were blocked by external barium and caesium, but insensitive to tolbutamide and diazoxide. In inside-out patches, channel activity was not blocked by 1 mM internal ATP. The sequence homology with KATP-1 suggests that BIR1 is a subunit of a brain and β-cell KATP channel. However, pharmacological differences and the lack of ATP-sensitivity, suggest that if, this is the case, heterologous subunits must exert strong modulatory influences on the native channel.

We studied a simple tandem repeal 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.

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 85590. 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

We have recently identified a diabetic patient with marked fasting hyperinsulinemia. Family study revealed that the abnormality was an autosomal dominant trait. High-performance liquid chromatography (HPLC) profile of the patient's serum insulin showed that she had an abnormal insulin in addition to a normal insulin. We have purified her insulin(s) from the specimen of her pancreas, which was biopsied during an operation of cholelithiasis. Insulin was also immunologically purified from the serum of her portal vein. The reverse-phase HPLC analysis revealed that the ratios of normal to abnormal insulin in the pancreas, portal vein, and peripheral vein were 5:4, 4:5, and 1:7, respectively. Radioreceptor assay for insulin using guinea pig kidney membrane revealed that the binding activities of the normal component insulin, the abnormal component insulin and her pancreatic insulin containing both components were 100, 5, and 50% of standard human insulin, respectively. The biological activities of the normal component, the abnormal component and her pancreatic insulin to stimulate glucose oxidation in rat adipocytes were found to be 100, 8, and 60% of standard human insulin, respectively. Analysis of amino acid sequences of the abnormal insulin purified from her pancreas strongly suggested the substitution of leucine for valine at the third position of the A chain, A3 (Val----Leu).

By screening a lambda gt11 library with the multimerized sequence of the cAMP response element (CRE), we isolated human clones encoding the CRE binding protein, CRE-BP1, from a human brain cDNA library. CRE-BP1 expressed in Escherichia coli bound not only to the CRE element of the somatostatin and fibronectin genes, but also to the CRE element of the adenovirus E4 gene, suggesting that the protein was not distinguishable from the adenovirus transcription factor, ATF. The human CRE-BP1 clone encoded a 54.5 kd protein similar at its carboxy terminus to the leucine zipper motifs found in other enhancer binding proteins such as C/EBP and c-jun/AP-1. CRE-BP1 mRNA was expressed in all of the cells examined and was abundant in brain. The structure of CRE-BP1 and its recognition elements suggest that cellular response to extracellular stimuli is controlled by a family of transcription factors that bind to related cis-active elements and that contain several highly conserved domains.

The inactivation of oxytocin and angiotensin II by retroplacental serum and placental leucine aminopeptidase (P-LAP), which had been highly purified from retroplacental serum, was demonstrated using biological methods.

We determined Placental-Leucine Aminopeptidase (P-LAP) activity, one of the oncodevelopmental antigens, in sera and in tissues of patients with gynecological cancers. The incidence of P-LAP activity and clinical usefulness of the determination of serum P-LAP activity were studied. The mean level in healthy non-pregnant sera used as controls was 6.0 +/- 2.4mg/dl/h. The mean level of P-LAP activity in patients with benign tumors such as myoma uteri and benign ovarian tumor did not increase in comparison with the controls. The mean level of P-LAP activity in patients with malignant tumor increased with advancing stages, and especially in advanced cervical and ovarian cancer, serum P-LAP levels were significantly higher than in the controls. Serum P-LAP activity correlated with the serum ferritin concentration (r=0.613), but not with the serum alpha-fetoprotein and serum carcinoembryonic antigen concentration. Serial measurements of serum P-LAP activities in patients with gynecological cancer showed that serum P-LAP activity might reflect the progress of cervical and ovarian cancer. Tissue P-LAP activities in 29 ovarian cancers were compared with those in normal tissues. Tissue P-LAP activities in 26 cases out of 29 increased to twice as high as the mean activities in 10 normal ovaries. Our present results suggest the possibility of using P-LAP activity as one of tumor markers for gynecological malignant tumors.

Plasma renin activity (PRA) and relative changes in angiotensin I compared with non-pregnant women were determined by radioimmunoassay (RIA) at different stages of normal gestation. Their levels showed increases in the early and late stages, and decreases at the period of 25 to 28 weeks of pregnancy in the middle stage, although their levels were higher than those in non-pregnant women throughout pregnancy. The increase in angiotensin I was less striking than that of PRA. Placental leucine aminopeptidase (P-LAP) activity increased progressively with advancing gestation, reaching the highest value at the 38th week of pregnancy. Simultaneous estimations of changes in angiotensin I and placental leucine aminopeptidase (P-LAP) were performed longitudinally in 12 normal pregnant women. The significance of the increase in P-LAP activity and the relationships among PRA, angiotensin I and P-LAP during normal pregnancy are discussed.

Aminopeptidase A ( l -α-aspartyl( l -α-glutamyl)-peptide hydrolase, EC 3.4.11.7) was found in human placenta, partially purified from it and briefly characterized in comparison with the placental leucine aminopeptidase. The aminopeptidase A could be separated from leucine aminopeptidase after trypsin digestion followed by Sephacryl S-300 chromatography. The angiotensinase (EC 3.4.99.3) activity of aminopeptidase A in human placenta was confirmed by a biological method.

Serum and tissue ferritins were quantitated by a radioimmunoassay kit (SPAC KIT, Daiichi Radioisotope Lab.) and a diagnostic implication of serum ferritins in patients with gynecological diseases was evaluated. In order to investigate the potential use of tumor marker as a feto-placental antigen (protein), ferritin from ovarian cancer was compared with ferritins from normal adult and feto-placental organs. Serum ferritin levels were significantly higher (p less than 0.01) in patients with ovarian adenocarcinoma, Krukenberg's tumor, cervical squamous cell carcinoma and other malignant diseases than in normal women. Among adult organs the kidney and spleen showed the highest and the heart the lowest ferritin content. The ferritin contents of the kidney and spleen were 78.4 micrograms and 76.2 micrograms/g wet weight, respectively and that of the heart was 5.7 micrograms/g wet weight. The ferritin contents of other adult organs ranged from 10 to 25 micrograms/g wet weight. On the other hand the placenta showed the highest and the heart and stomach the lowest ferritin content among feto-placental organs. The ferritin content of the placenta was 7 micrograms/g wet weight. The ferritin contents of other fetal organs were only half as in the placenta. The ferritin contents of ovarian cancers ranged 6 to 8 micrograms/g wet weight and was almost identical to that of the placenta.

The region containing two copies of the sequence GGGACTTTCC in the human immunodeficiency virus type 1 (HIV-1) long terminal repeat, that is an NF-kappa B binding site, functions as an enhancer element for HIV transcriptional regulation. By a Southwestern method we have isolated a cDNA encoding the HIV-1 enhancer binding protein (HIV-EP1) from a human B-cell lambda gt11 library. DNase I footprinting analysis using the HIV-EP1 protein expressed in Escherichia coli showed that HIV-EP1 specifically bound to the HIV-1 enhancer. HIV-EP1 protein contains a domain with two tandem "zinc finger" sequences initially described in the Xenopus transcription factor IIIA. This represents the first demonstration of the structural feature of the protein that binds to the HIV-1 enhancer.

Purified placental aminopeptidase A cleaved N-terminal aspartic acid of Angiotensin II, and it was inhibited by amastatin. Amastatin and various angiotensin analogs having N-terminal dicarboxylic acid were potent inhibitors of the enzyme. Kinetic analysis indicated that amastatin, angiotensin II, the N-terminal tripeptide of angiotensin II, and aspartic acid were competitive inhibitors, with Ki values 1.25 X 10(-7) M, 2.40 X 10(-5) M, 2.67 X 10(-4) M, and 1.2 X 10(-3)M respectively. The enzyme was also inhibited by transition metals, such as Zn2+, Cu2+, Cd2+ and Ni2+. Serum aminopeptidase A activity progressively increased during the course of normal pregnancy.

The acute effect of cigarette smoking on the gastric mucosal blood volume index and the oxygen saturation of hemoglobin (SO2) in the gastric mucosa was investigated in 12 young male volunteers using reflectance spectrophotometry during endoscopy. Six of these volunteers were habitual smokers who had smoked more than 20 cigarettes a day for more than five years. The others were non-habitual smokers who smoked less than 20 cigarettes a year. The indices of mucosal blood volume and the mucosal blood SO2 level were calculated from the spectra obtained at the lesser curvature of the lower corpus of the stomach before and after cigarette smoking. The indices of mucosal blood volume and mucosal blood SO2 decreased significantly after one to three puffs of cigarette smoking in all subjects as compared to the value before smoking, and the degree of decrease in these parameters was significantly greater in the non-habitual smokers than in the habitual smokers. These results suggest that only one to three puffs of cigarette smoking causes a decrease in the mucosal blood volume and the mucosal blood SO2 which might be related to weakening of mucosal defensive factors.

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.

The administration of leucine aminopeptidase purified from human placenta was found to be effective in lowering the blood pressure in rats with experimental hypertension induced by the infusion of angiotensin II or renin.