Mutations in the hepatocyte nuclear factor-1alpha/MODY3 gene in Japanese subjects with early- and late-onset NIDDM

Recent studies have shown that mutations in the hepatocyte nuclear factor (HNF)-1alpha gene are the cause of maturity-onset diabetes of the young type 3 (MODY3). We have screened 193 unrelated Japanese subjects with NIDDM for mutations in this gene: 83 with early-onset NIDDM (diagnosis at <30 years of age) and 110 with late-onset NIDDM (diagnosis > or = 30 years of age). All of the members of the latter group also had at least one sibling with NIDDM. The 10 exons, flanking introns, and promoter region were amplified using polymerase chain reaction and were sequenced directly. Mutations were found in 7 of the 83 (8%) unrelated subjects with early-onset NIDDM. The mutations were each different and included four missense mutations (L12H, R131Q, K205Q, and R263C) and three frameshift mutations (P379fsdelCT, T392fsdelA, and L584S585fsinsTC). One of the 110 subjects with late-onset NIDDM was heterozygous for the missense mutation G191D. This subject, who was diagnosed with NIDDM at 64 years of age, also had a brother with NIDDM (age at diagnosis, 54 years) who carried the same mutation, suggesting that this mutation contributed to the development of NIDDM in these two siblings. None of these mutations were present in 50 unrelated subjects with normal glucose tolerance (100 normal chromosomes). Mutations in the HNF-1alpha gene occur in Japanese subjects with NIDDM and appear to be an important cause of early-onset NIDDM in this population. In addition, they are present in about 1% of subjects with late-onset NIDDM.     

Human hepatocyte nuclear factor-4 (hHNF-4) gene maps to 20q12-q13.1 between PLCG1 and D20S17

Male Hartley guinea pigs (480-610 g; n=5) were treated intratracheally with saline, cadmium (Cd, 0.3 mg) as cadmium chloride, selenium (Se, 0.3 or 0.06 mg) as sodium selenite or Se (0.06 mg) and Cd (0.3 mg). After 24 hours, lungs were collected and analyzed for prostaglandin (PGE2), thromboxane (TXB2) and leukotriene (LTC4) levels. Results indicated that, 0.3 mg Se and 0.06 mg Se in combination with 0.3 mg Cd increased PGE2 significantly. Selenium and Cd alone or in combination, decreased LTC4 and TXB2 significantly.     

Site-directed mutagenesis and steady-state kinetic analysis of mutant enzymes of human adenylate kinase

Infection of rats with the enteric, lumen-dwelling tapeworm Hymenolepis diminuta causes electric changes in host intestinal smooth muscle and decreased luminal transit. The mechanisms that stimulate host intestinal alterations during this nontissue invasive infection may include the tapeworm's biomass, its diurnal migratory behavior, a host immune-mediated response, or direct parasite stimulation of host motor activity. In vivo intestinal myoelectric activity was monitored to evaluate the following: (1) that reinfection with H. diminuta is influenced by host immune regulation and (2) that administration of tapeworm fractions to never-before-infected rats initiates an alteration of enteric smooth muscle activity. To address the first hypothesis, we determined that altered intestinal myoelectric activity patterns were no different and did not occur earlier in a second infection with H. diminuta than in a primary infection. The lack of either a change in myoelectric pattern or an earlier onset of intestinal myoelectric changes indicates that tapeworm-induced myoelectric activity is not anamnestically stimulated by host immunomodulatory mechanisms. Consistent with the second hypothesis, administration of either H. diminuta carcass homogenate or tegument-enriched fractions directly into the intestinal lumen of tapeworm-naive rats initiated myoelectric patterns previously characteristic of chronic H. diminuta infection. Additionally, the appearance of characteristic nonmigrating myoelectric patterns in uninfected rats administered tapeworm fractions indicates that a substance from H. diminuta acts as the triggering signal molecule for intestinal myoelectric alterations. These findings also indicate that neither the tapeworm's biomass nor its diurnal movement is required for initiation of H. diminuta-altered myoelectric patterns. We have shown that H. diminuta possess a signal molecule(s) that alters host enteric electric activity, and we suggest that these alterations may play an important role in the symbiotic rat-tapeworm interrelationship.     

Glucose utilization and production in patients with maturity-onset diabetes of the young caused by a mutation of the hepatocyte nuclear factor-1alpha gene

Mutations of the hepatocyte nuclear factor (HNF)-1alpha gene cause impaired insulin secretion and hyperglycemia in patients with maturity-onset diabetes of the young (MODY)3. Whether these mutations also affect glucose metabolism in tissues other than the beta-cell has not yet been documented. We therefore assessed, in five MODY3 patients and a dozen healthy control subjects, insulin secretion, oxidative and nonoxidative glucose disposal, and glucose production during a two-step hyperglycemic clamp and a euglycemic hyperinsulinemic (0.4 mU x kg(-1) x min(-1)) clamp. Compared with healthy control subjects, MODY3 patients had higher fasting plasma glucose (+100%) but similar rates of fasting glucose production and oxidation. Both the early and late phases of insulin secretion were virtually abolished during the hyperglycemic clamp, and glucose production was suppressed by only 43% in MODY3 patients vs. 100% in healthy control subjects. The rate of glucose infusion required to produce a 5 mmol/l increase above basal glycemia was reduced by 30%, net nonoxidative glucose disposal (which is equal to net glycogen deposition) was inhibited by 39%, and net carbohydrate oxidation during hyperglycemia was 25% lower in MODY3 patients compared with control subjects. Insulin-stimulated glucose utilization and oxidation measured during the hyperinsulinemic clamp (at approximately 200 pmol/l insulin) were identical in MODY3 patients and in healthy control subjects, indicating that peripheral insulin sensitivity was not altered. Suppression of endogenous glucose production was, however, mildly impaired. It is concluded that MODY3 patients have severely depressed glucose-induced insulin secretion. The development of hyperglycemia in these patients appears to be caused by a decreased stimulation of glucose utilization, oxidation, and nonoxidative glucose disposal as well as by a blunted suppression of endogenous glucose output. These phenomena are essentially secondary to insulinopenia, whereas insulin sensitivity remains intact.     

A novel Phe75fsdelT mutation in the hepatocyte nuclear factor-4alpha gene in a Danish pedigree with maturity-onset diabetes of the young

Mutations in 5 different genes [the hepatocyte nuclear factor (HNF)-4alpha), glucokinase, HNF-1alpha, insulin promoter factor-1, and HNF-1beta genes] have been shown to cause maturity onset diabetes of the young (MODY). About 50% of all known MODY in Danish Caucasian MODY probands can be explained by mutations in the HNF-1alpha gene (MODY3). To estimate the prevalence of MODY caused by mutations in the HNF-4alpha gene (MODY1), we screened 10 non-MODY3 probands for mutations in the minimal promoter and the 12 exons of the HNF-4alpha gene. One of the probands had a novel frameshift mutation (Phe75fsdelT) in exon 2 of the HNF-4alpha gene, resulting in a premature termination of translation after 117 amino acids of the messenger RNA encoded by that allele. The mutation cosegregated with diabetes in the pedigree and was not detected in 84 unrelated Danish Caucasian healthy glucose-tolerant control subjects or in 84 type 2 diabetic patients. At the time of examination, 4 of 6 mutation carriers were treated with insulin and 2 with oral hypoglycemic medication. Two mutation carriers had late-diabetic complications. Even though the HNF-4alpha protein is known to be important in the regulation of genes involved in lipid metabolism, carriers of the mutation did not differ from age and sex-matched control subjects, in regard to levels of fasting serum total cholesterol, serum high-density lipoprotein-cholesterol, and serum triglyceride. In conclusion, by screening 10 non-MODY3 probands for mutations in the HNF-4alpha gene, we identified 1 diabetes-associated frameshift mutation (Phe75fsdelT), suggesting that defects in HNF-4alpha are a rare cause of MODY in Denmark.     

T4 ribonucleotide reductase. Physical and kinetic linkage to other enzymes of deoxyribonucleotide biosynthesis

This laboratory has described a multienzyme aggregate from T4 phage-infected Escherichia coli which seems to participate in deoxyribonucleotide biosynthesis and efficient delivery of DNA precursors to the replication apparatus. This paper describes improved methodology for isolation of this aggregate, and we present three lines of evidence supporting a role for ribonucleoside diphosphate reductase in functioning of the presumed complex. 1) Ribonucleoside diphosphates are readily incorporated into DNA as deoxyribonucleotides in an in situ DNA-synthesizing system from T4 phage-infected cells. 2)Ribonucleotide reductase is associated with the complex, as shown by co-sedimentation of reductase activity with other activities in the multienzyme aggregate we have described. 3)Ribonucleotide reductase is kinetically coupled to at least four other enzymes involved in a sequential pathway. The aggregated enzymes catalyze the five-step conversion of uridine diphosphate to deoxythymidine triphosphate with but a brief lag before dTTP production reaches its maximal rate. These studies have also confirmed the existence of dCTPase-dUTPase and dCMP deaminase activities in the putative complex.     

Processing by CD26/dipeptidyl-peptidase IV reduces the chemotactic and anti-HIV-1 activity of stromal-cell-derived factor-1alpha

A method for simultaneous determination of glucosamine (GlcN) and glucosamine 4-phosphate (GlcN-4-P) in Lipid A with high-performance anion-exchange chromatography (HPAEC) is described. Lipid A is hydrolyzed with 4 M HCl for 16 h at 100 degrees C, and the peaks of glucosamine and glucosamine 4-phosphate were measured. The true GlcN value can be computed from the GlcN value after correction for the incomplete hydrolysis of GlcN-4-P, or by the combined yield of GlcN and GlcN-4-P.     

The effect of two frequent amino acid variants of the hepatocyte nuclear factor-1alpha gene on estimates of the pancreatic beta-cell function in Caucasian glucose-tolerant first-degree relatives of type 2 diabetic patients

The objective of the present study was to investigate whether the frequent amino acid polymorphisms, Ile/Leu27 and Ser/Asn487, of the hepatocyte nuclear factor-1alpha gene were associated with alterations in glucose-induced serum C-peptide and serum insulin responses among glucose-tolerant first-degree relatives of type 2 diabetic patients. The study comprised 2 independent Danish cohorts. Among 74 unrelated type 2 diabetic relatives, 12 homozygous carriers of the Ile/Leu27 polymorphism had a 32% decrease in the 30-min serum C-peptide level (P = 0.01), as well as a 39% decrease in the 30-min serum insulin level (P = 0.02) during an oral glucose tolerance test. Ten homozygous carriers of the Ile/Leu27 variant did, however, not differ from wild-type carriers, with respect to the acute circulating insulin and serum C-peptide responses during an i.v. glucose tolerance test in the same study cohort. In a larger (more than 3-fold) study group of 230 glucose tolerant offspring of 62 type 2 diabetic probands, 33 homozygous carriers of the Ile/Leu27 variant did not differ, with respect to either serum insulin and serum C-peptide levels during an oral glucose tolerance test or acute serum insulin and serum C-peptide responses during an i.v. glucose tolerance test. We therefore consider the former positive finding as a statistical type I error. There were no differences in the above mentioned variables between carriers of the Ser/Asn487 polymorphism and wild-type carriers within any of the 2 study populations. Nor did carriers of combined genotypes, i.e. carriers of both the Ile/Leu27 and the Ser/Asn487 variants, show any associations with the examined variables. In conclusion, the Ile/Leu27 and Ser/ Asn487 polymorphisms of the hepatocyte nuclear factor-1alpha gene have apparently no major impact on the pancreatic beta-cell function, after an oral and i.v. glucose challenge, in Caucasian first-degree relatives of type 2 diabetic patients.     

Characterization of a soluble IL-6 receptor alpha mutant C277D/H280I expressed in E.coli

BACKGROUND: Cyclo-oxygenase-1 (COX-1) is believed to produce prostaglandins vital to mucosal defence, whereas cyclo-oxygenase-2 (COX-2) is induced at sites of inflammation. Little is known about the regulation of COX-2 in the stomach, particularly during the period following mucosal injury. In this study, we examined COX-1 and COX-2 expression shortly after administration of NSAIDs or ethanol. METHODS: Fasted rats were given aspirin, salicylate, indomethacin or ethanol (20% or 40%) orally. Three hours later the stomach was excised, the severity of damage scored and samples taken for RT-PCR of COX-1 and COX-2 mRNA and immunohistochemistry. Nitric oxide synthase mRNA (iNOS and eNOS) and activity were also measured. RESULTS: Aspirin, indomethacin and the higher concentration of ethanol produced widespread mucosal damage, whereas salicylate and 20% ethanol caused only superficial epithelial damage. Aspirin caused a significant increase in COX-2 mRNA expression and a marked increase in COX-2 immunoreactivity, particularly in the superficial mucosa. Expression of COX-1 (mRNA and protein) was unaffected by aspirin, as were NOS mRNA expression and enzyme activity. Pre-treatment with prostaglandin E2 prevented the induction of COX-2 by aspirin. Salicylate and indomethacin caused modest increases in COX-2 immunoreactivity but no change in COX-2 mRNA. Neither concentration of ethanol affected COX-2 mRNA or protein expression, suggesting that this was a specific response to the aspirin, rather than to injury. CONCLUSIONS: These results demonstrate a rapid up-regulation of COX-2 expression in response to aspirin, possibly representing a compensatory response to inhibition of gastric prostaglandin synthesis.     

Human alpha1,3/4-fucosyltransferases. I. Identification of amino acids involved in acceptor substrate binding by site-directed mutagenesis

In a previous study (Xu, Z., Vo, L., and Macher, B. A. (1996) J. Biol. Chem. 271, 8818-8823), a domain swapping approach demonstrated that a region of amino acids found in human alpha1, 3/4-fucosyltransferase III (FucT III) conferred a significant increase in alpha1,4-FucT acceptor substrate specificity into alpha1, 3-fucosyltransferase V (FucT V), which, under the same assay conditions, has extremely low alpha1,4-FucT acceptor substrate specificity. In the current study, site-directed mutagenesis was utilized to identify which of the eight amino acids, associated with alpha1,4-FucT acceptor substrate specificity, is/are responsible for conferring this new property. The results demonstrate that increased alpha1,4-FucT activity with both disaccharide and glycolipid acceptors can be conferred on FucT V by modifying as few as two (Asn86 to His and Thr87 to Ile) of the eight amino acids originally swapped from FucT III into the FucT V sequence. Neither single amino acid mutant had increased alpha1,4-FucT activity relative to that of FucT V. Kinetic analyses of FucT V mutants demonstrated a reduced Km for Galbeta1,3GlcNAc (type 1) acceptor substrates compared with native FucT V. However, this was about 20-fold higher than that found for native FucT III, suggesting that other amino acids in FucT III must contribute to its overall binding site for type 1 substrates. These results demonstrate that amino acid residues near the amino terminus of the catalytic domain of FucT III contribute to its acceptor substrate specificity.     

A dominant-negative cyclin D1 mutant prevents nuclear import of cyclin-dependent kinase 4 (CDK4) and its phosphorylation by CDK-activating kinase

Cyclins contain two characteristic cyclin folds, each consisting of five alpha-helical bundles, which are connected to one another by a short linker peptide. The first repeat makes direct contact with cyclin-dependent kinase (CDK) subunits in assembled holoenzyme complexes, whereas the second does not contribute directly to the CDK interface. Although threonine 156 in mouse cyclin D1 is predicted to lie at the carboxyl terminus of the linker peptide that separates the two cyclin folds and is buried within the cyclin subunit, mutation of this residue to alanine has profound effects on the behavior of the derived cyclin D1-CDK4 complexes. CDK4 in complexes with mutant cyclin D1 (T156A or T156E but not T156S) is not phosphorylated by recombinant CDK-activating kinase (CAK) in vitro, fails to undergo activating T-loop phosphorylation in vivo, and remains catalytically inactive and unable to phosphorylate the retinoblastoma protein. Moreover, when it is ectopically overexpressed in mammalian cells, cyclin D1 (T156A) assembles with CDK4 in the cytoplasm but is not imported into the cell nucleus. CAK phosphorylation is not required for nuclear transport of cyclin D1-CDK4 complexes, because complexes containing wild-type cyclin D1 and a CDK4 (T172A) mutant lacking the CAK phosphorylation site are efficiently imported. In contrast, enforced overexpression of the CDK inhibitor p21Cip1 together with mutant cyclin D1 (T156A)-CDK4 complexes enhanced their nuclear localization. These results suggest that cyclin D1 (T156A or T156E) forms abortive complexes with CDK4 that prevent recognition by CAK and by other cellular factors that are required for their nuclear localization. These properties enable ectopically overexpressed cyclin D1 (T156A), or a more stable T156A/T286A double mutant that is resistant to ubiquitination, to compete with endogenous cyclin D1 in mammalian cells, thereby mobilizing CDK4 into cytoplasmic, catalytically inactive complexes and dominantly inhibiting the ability of transfected NIH 3T3 fibroblasts to enter S phase.     

The Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene contributes to the interindividual variation in serum C-peptide response during an oral glucose tolerance test: evidence from studies of 231 glucose-tolerant first degree relatives of type 2 diabetic probands

The third form of maturity-onset diabetes of the young is caused by mutations in the hepatocyte nuclear factor-1alpha gene. Recently, we demonstrated an association between a prevalent polymorphism at codon 98, Ala/Val98, of this gene and a 20% decreased insulin release during an oral glucose tolerance test (OGTT) in middle-aged glucose-tolerant Danish Caucasian subjects. The major objective of the present study was to replicate this finding among glucose-tolerant first degree relatives of type 2 diabetic patients of the same ethnic origin. All participants, 231 glucose-tolerant offspring of 62 type 2 diabetic probands, underwent an OGTT with measurements of plasma glucose, serum insulin, and serum C peptide during the test. Thirty-three heterozygous carriers of the Ala/Val variant were identified, whereas no subjects had the variant in its homozygous form. Ala/Val carriers had a 20% reduction in serum C peptide at 30 min during the OGTT (1225+/-636 vs. 1507+/-624 pmol/L; P=0.02) compared to wild-type carriers. No significant differences in serum insulin levels during the OGTT were observed between carriers of the variant and Ala/Ala homozygotes. In conclusion, among Danish glucose-tolerant first degree relatives of type 2 diabetic patients the Ala/Val98 polymorphism of the hepatocyte nuclear factor-1alpha gene is associated with a decreased serum C-peptide secretion during an OGTT. This finding confirms our previously reported observation of the functional importance of the variant to insulin secretion during an OGTT among middle-aged healthy subjects.     

Nucleoside 2-deoxyribosyltransferase. Pre-steady-state kinetic analysis of native enzyme and mutant enzyme with an alanyl residue replacing Glu-98

Nucleoside 2-deoxyribosyltransferase catalyzes cleavage of a 2'-deoxyribosylnucleoside (A) to a nucleobase (P) with deoxyribosylation of the enzyme. Substrates quenched the intrinsic fluorescence of native enzyme (E) and a catalytically inactive mutant enzyme (E98A enzyme). The time courses of these reactions were analyzed in terms of the following scheme where EX is the 2-deoxyribosyl ester of Glu-98. [formula: see text] The initial complexes between E and dAdo, dGuo, dIno, and dCyd or those between EX and the corresponding nucleobases were formed in a rapid equilibrium step. Native enzyme and E98A enzyme bound 2'-deoxyribosylnucleosides with similar affinities (k-1/k1). From a comparison of the time-dependent fluorescence changes associated with the reaction of native enzyme or E98A enzyme with these substrate, the kinetic step for 2-deoxyribosylation of Glu-98 was identified (k2 and k-2). dThd and dUrd quenched the fluorescence of native enzyme in a biphasic process. The late phase of this reaction was associated with 2-deoxyribosylation of Glu-98. The pre-steady-state kinetic constants calculated from fluorescence quenching data for dAdo and Cyt were consistent with the experimental values for the steady-state kinetic coefficients and the equilibrium constant of the reaction.