MODY 2: Mutation identification and molecular ancestry in a Brazilian family

Maturity Onset Diabetes of the Young (MODY) is a heterogeneous group of genetic diseases characterized by a primary defect in insulin secretion and hyperglycemia, non-ketotic disease, monogenic autosomal dominant mode of inheritance, age at onset less than 25 years, and lack of auto-antibodies. It accounts for 2–5% of all cases of non-type 1 diabetes. MODY subtype 2 is caused by mutations in the glucokinase (GCK) gene. In this study, we sequenced the GCK gene of two volunteers with clinical diagnosis for MODY2 and we were able to identify four mutations including one for a premature stop codon (c.76C>T). Based on these results, we have developed a specific PCR-RFLP assay to detect this mutation and tested 122 related volunteers from the same family. This mutation in the GCK gene was detected in 21 additional subjects who also had the clinical features of this genetic disease. In conclusion, we identified new GCK gene mutations in a Brazilian family of Italian descendance, with one due to a premature stop codon located in the second exon of the gene. We also developed a specific assay that is fast, cheap and reliable to detect this mutation. Finally, we built a molecular ancestry model based on our results for the migration of individuals carrying this genetic mutation from Northern Italy to Brazil.     

Hepatic DNAJB9 Drives Anabolic Biasing to Reduce Steatosis and Obesity

1. Download : Download high-res image (127KB)
2. Download : Download full-size image

     

A novel galactosyl-binding lectin from the plasma of the blood clam,Anadara granosa (L) and a study of its combining site

Control Analysis has been carried out in the first steps of a rat liver glycolytic system. Attention has been focused on the effect of several glucose concentrations on the control, particularly regarding the role of glucokinase. From kinetic studies of the whole metabolic system we have obtained information on the flux variation under different glucose concentrations. This information together with the kinetics of glucokinase has allowed us to calculate Flux Control and Elasticity Coefficients for glucokinase and the Response Coefficient of the system with respect to glucose. The changes in of the value of Flux Control Coefficients demonstrates that in conditions of low glucose concentration, glucokinase is the main enzyme in controlling the flux through the pathway, but at high glucose concentration the control moves to phosphofructokinase. Next, we have compared our results with those obtained with the shortening and titration method, previously described (Torres, N.V., Mateo, F., Mélendez-Hevia, E. and Kacser, H., (1986) Biochem. J. 234, 169–174; Torres, N.V. and Meléndez-Hevia, E. 1991. Molec. Cell. Biochem. 101, 1–10). Furthermore, from knowledge of the enzyme kinetics of the system we have been able to build a model of the pathway that allows us computer similation of its behavior and calculation of the Flux Control Coefficient profile at different glucose concentrations. By the three methods the results correlate, supporting the use of the pathway substrate as external modulator of the metabolic system as a tool for practical application of Control Analysis.     

Development of PEGylated serum albumin with multiple reduced thiols as a long-circulating scavenger of reactive oxygen species for the treatment of fulminant hepatic failure in mice

Reactive oxygen species (ROS) are involved in the pathophysiology of fulminant hepatic failure. Therefore, we developed polyethylene glycol-conjugated bovine serum albumin with multiple reduced thiols (PEG-BSA-SH) for the treatment of fulminant hepatic failure. As a long-circulating ROS scavenger, PEG-BSA-SH effectively scavenged highly reactive oxygen species and hydrogen peroxide in buffer solution. PEG-BSA-SH showed a long circulation time in the plasma after intravenous injection into mice. Fulminant hepatic failure was induced by intraperitoneal injection of lipopolysaccharide and d-galactosamine (LPS/d-GalN) into mice. The LPS/d-GalN-induced elevation of plasma alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels was significantly inhibited by a bolus intravenous injection of PEG-BSA-SH. Furthermore, the changes in hepatic lipid peroxide and hepatic blood flow were effectively suppressed by PEG-BSA-SH. In contrast, l-cysteine, glutathione, and dithiothreitol, three traditional reduced thiols, had no statistically significant effects on the serum levels of ALT or AST. These findings indicate that PEG-BSA-SH is a promising ROS scavenger and useful in the treatment of fulminant hepatic failure.     

Investigations on the mechanism of hypercholesterolemia observed in copper deficiency in rats

The mechanism of hypercholesterolemia effect of Cu²⁺ deficiency was studied in rats. There was increased activity of hepatic hydroxymethylglutaryl-coenzyme A reductase and increased incorporation of labelled acetate into free cholesterol of liver in the Cu²⁺ deficient rats. Incorporation of label into ester cholesterol was however decreased in the liver. Concentration of bile acids in the liver was not significantly altered. Increase in the incorporation of labelled acetate into serum cholesterol and increase in the concentration of cholesterol and apo B in the low density lipoproteins + very low density lipoproteins fractions were observed. Activity of lipoprotein lipase of the extrahepatic tissues decreased in the Cu²⁺ deficient rats.     

In Vivo measurement of unscheduled DNA synthesis and S-phase synthesis as an indicator of hepatocarcinogenesis in rodents

Measurement of chemically induced DNA repair as unscheduled DNA synthesis in rodent liver following in vivo treatment is a useful screen for potential hepatocarcinogens. In addition to measurement of unscheduled DNA synthesis, examination of S-phase synthesis provides an indicator of chemically induced cell proliferation in the liver, which may be a basis for hepatic tumor promotion. Several chemicals and classes of chemicals have been examined using these endpoints. The pyrrolizidine alkaloid riddelline is a potent genotoxic agent in vitro, and in vivo studies confirm this response as riddelline induces significant elevations in unscheduled DNA synthesis and S-phase synthesis in rat liver. Conversely, H. C. Blue dyes #1 and #2 are both potent genotoxic agents in vitro but fail to express this genotoxicity in vivo. H. C Blue #1 induces significant increases in S-phase synthesis in B6C3F1 mouse liver, which correlates with the observed carcinogenicity of this compound. Halogenated hydrocarbons likewise fail to induce unscheduled DNA synthesis in vivo, but many of these compounds do increase hepatic cell proliferation in mice, which may be the principal mechanism of hepatocarcinogenesis in this species.Abbreviations BCMEE bis(2-chloro-l-methylethyl)ether - dThd thymidine - HCB1 H.C. Blue #1 - HCB2 H.C. Blue #2 - UDS unscheduled DNA synthesis     

The stimulation of metallothionein synthesis in neuroblastoma IMR-32 by zinc and cadmium but not dexamethasone

Metallothioneins are a class of cysteine-rich and low molecular weight, metal-binding proteins that are inducible by a wide variety of agents, including metal ions, such as cadmium and zinc, glucocorticoid hormones, interferon, and tumor promoters. In an effort to delineate the regulation of the synthesis of the recently identified brain metallothionein-like protein, a study was undertaken to compare the induction of metallothionein in human neuroblastoma IMR-32 cells by zinc, cadmium, and dexamethasone using the human Chang liver cells as a control. Both cadmium (1 microM) and zinc (100 microM) significantly enhanced the incorporation of [35S]cysteine into metallothioneins isolated from both neuroblastoma and Chang liver cells. Dexamethasone in concentrations of 10 microM stimulated the synthesis of metallothionein in the Chang cells, whereas it had no effects on the synthesis of metallothionein in the neuroblastoma cells at concentrations ranging from 2.5--100 microM. The degree of stimulation of metallothionein synthesis in the Chang cells by cadmium and zinc was significantly higher than seen in neuroblastoma cells. The neuroblastoma IMR-32 exhibited less tolerance to the toxicity of both cadmium and zinc than the Chang cells, which may correlate with the inherent ability of these ions to induce metallothioneins in these dissimilar cells. The results of these studies are interpreted to indicate that the factors regulating the synthesis of metallothioneins in the Chang and neuroblastoma cells are not identical, suggesting also of the presence of dissimilar regulatory mechanisms in the liver and brain.     

Regional brain GABA metabolism and release during hepatic coma produced in rats chronically treated with carbon tetrachloride

Hepatic coma was induced in rats chronically treated with CCl₄, by means of a single injection of ammonium acetate. The activities of glutamate decarboxylase (GAD) and GABA transaminase (GABA-T), as well as the synaptosomal uptake and release of [³H]GABA, were measured in the following brain areas of the comatose rats: cortex, striatum, hypothalamus, hippocampus, midbrain and cerebellum. Hepatic coma was associated with a general decrease of GAD activity, whereas GABA-T activity was diminished only in the hypothalamus, striatum and midbrain. During hepatic coma, the K⁺-stimulated [³H]GABA release was notably diminished in the striatum and cerebellum, whereas a significant increase was observed in the hippocampus. [³H]GABA uptake increased in most regions after CCl₄ treatment, independently of the presence of coma. The results indicate that GABAergic transmission seems to be decreased in most cerebral regions during hepatic coma.     

Comparison of glucokinase in C3H/He and C58 mice that differ in their hepatic activity

Partially purified preparations of the hepatic glucokinase from C3H/He and C58 inbred mice have been used to explore the molecular basis for the observed twofold difference in activity between the strains. The single codominant gene that appears to regulate activity, the alleles of which are designated Gk^a and Gk^b, respectively, for the two strains, could represent a structural gene change. This now seems unlikely because the mouse enzyme, although showing small differences from rat glucokinase, appeared to be identical in the two strains with respect to thermal stability, electrophoretic mobility in agarose gels, and kinetic properties such as the apparent K _m values for MgATP^2– and glucose and the unique cooperative interaction with the latter substrate. The enzymes also reacted identically in a range of immunological tests (double-diffusion, immunoelectrophoresis, immune precipitation and immune inhibition assays) and ELISA immune inhibition assays indicated that the twofold difference in activity was due to a similar difference in antigenically active enzyme. Genetic control over the physiologically significant regulation of enzyme amount is therefore probable.This work has been supported in part by a grant from the British Diabetic Association and a Training Studentship to PAJ from the Medical Research Council (U.K.).