RESUMO
Recent studies have shown that dietary phospholipids, especially phosphatidylcholine and phosphatidylserine, have various beneficial biological effects. However, there are not enough data concerning the physiological function of dietary phosphatidylinositol (PI). The metabolic syndrome, a cluster of metabolic abnormalities such as dyslipidemia, diabetes mellitus, and hypertension, is widespread and increasingly prevalent diseases in industrialized countries. In the present study, we evaluated that the effect of dietary PI on cholesterol metabolism in metabolic syndrome model Zucker (fa/fa) rats. For 4 weeks, rats were fed semisynthetic diets containing either 7% soybean oil or 5% soybean oil plus 2% PI. Dietary PI prevented the mild hypercholesterolemia and hepatic cholesterol accumulation in Zucker (fa/fa) rats. These effects were attributable to an increased fecal bile acid excretion and to the tendencies of decreased ACAT1 mRNA level and increased CYP7A1 mRNA level in the liver. Additionally, dietary PI markedly increased microsomal PI content in the liver of Zucker (fa/fa) rats. Our study suggests that dietary PI normalizes cholesterol metabolism through the enhancement of fecal bile acid excretion in the metabolic syndrome model rats.
Assuntos
Gorduras na Dieta/farmacologia , Fígado/metabolismo , Síndrome Metabólica/metabolismo , Microssomos Hepáticos/metabolismo , Fosfatidilinositóis/farmacologia , Extratos Vegetais/metabolismo , Acetil-CoA C-Acetiltransferase/biossíntese , Animais , Ácidos e Sais Biliares/metabolismo , Colesterol 7-alfa-Hidroxilase/biossíntese , Modelos Animais de Doenças , Humanos , Hipercolesterolemia/dietoterapia , Hipercolesterolemia/metabolismo , Masculino , Síndrome Metabólica/dietoterapia , RNA Mensageiro/biossíntese , Ratos , Ratos ZuckerRESUMO
Recent studies have shown that dietary phospholipids, especially phosphatidylcholine and phosphatidylserine, have various beneficial biological effects. However, there are not enough data concerning the physiological function of dietary phosphatidylinositol (PI). The metabolic syndrome, a cluster of metabolic abnormalities such as dyslipidemia, diabetes mellitus, and hypertension, is a widespread and increasingly prevalent disease in industrialized countries. Nonalcoholic fatty liver disease (NAFLD) is often associated with features of the metabolic syndrome. NAFLD describes the spectrum of liver damage ranging from hepatic steatosis to steatohepatitis, liver fibrosis, and cirrhosis, and it is emerging as the most common liver disease worldwide. The present study examined whether dietary PI protects Zucker ( fa/ fa) rats from the metabolic syndrome. For 4 weeks, rats were fed semisynthetic diets containing either 7% soybean oil or 5% soybean oil plus 2% PI. Dietary PI markedly prevented the development of hepatomegaly and hepatic steatosis and lowered hepatic injury markers in serum. Additionally, hyperinsulinemia was relieved by the feeding of dietary PI in Zucker rats. These effects were attributable to an increase in serum adiponectin, enhancement of fatty acid beta-oxidation, and suppression of mRNA expression of inflammatory genes in the liver. This is the first report that dietary PI increases serum adiponectin level and prevents the development of NAFLD in a rat model of the metabolic syndrome.
Assuntos
Dieta , Fígado Gorduroso/prevenção & controle , Fosfatidilinositóis/administração & dosagem , Adiponectina/sangue , Animais , Hiperinsulinismo/tratamento farmacológico , Masculino , Síndrome Metabólica/prevenção & controle , Ratos , Ratos Zucker , Óleo de Soja/administração & dosagemRESUMO
Fructosyl-amino acid oxidase (FOD-F) from Fusarium oxysporum f. sp. raphani (NBRC 9972) is the enzyme catalyzing the oxidative deglycation of fructosyl-amino acids such as N(epsilon)-fructosyl N(alpha)-benzyloxycarbonyl-lysine (FZK) and fructosyl valine (FV), which are model compounds of the glycated proteins in blood. Wild-type FOD-F has high activities toward both substrates. We obtained a mutant FOD-F, which reacts with FZK but not with FV by random mutagenesis. One amino-acid substitution (K373R) occurred in the mutant FOD-F. In addition to K373R, K373W, K373M, K373T, and K373V, which were selected for optimization of the substitution at position K373, were purified and characterized. Kinetic analysis showed that the catalytic turnover for FV greatly decreased, whereas that for FZK did not. In consequence, the specificities toward FZK were increased in the mutant FOD-Fs. The relation between the substrate specificity of the mutant FOD-Fs and the position of the carboxyl group of the substrates was demonstrated using a series of the substrates having the carboxyl group at the different position. The mutant FOD-Fs are attractive candidates for developing an enzymatic measurement method for glycated proteins such as glycated albumin in serum. This study will be helpful to establish an easier and rapid clinical assay system of glycated albumin.
Assuntos
Aminoácido Oxirredutases/genética , Aminoácido Oxirredutases/metabolismo , Fusarium/enzimologia , Especificidade por Substrato/genética , Aminoácido Oxirredutases/isolamento & purificação , Sequência de Aminoácidos , Substituição de Aminoácidos , Cinética , Lisina/análogos & derivados , Lisina/metabolismo , Dados de Sequência Molecular , Mutagênese , Mutação de Sentido Incorreto , Valina/análogos & derivados , Valina/metabolismoRESUMO
We showed by random mutagenesis that one-amino-acid substitution at Arg94 in fructosyl-amino acid oxidase from Ulocladium sp. JS-103 enhanced substrate specificity toward fructosyl valine (FV), a model compound of hemoglobin A(1c). Kinetic analysis showed that the specificity of the R94W mutant enzyme toward FV was 14-fold that of the wild-type enzyme. The mutant enzyme obtained will be useful in developing an enzymatic measurement method for hemoglobin A(1c).
Assuntos
Aminoácido Oxirredutases/genética , Substituição de Aminoácidos , Ascomicetos/genética , Proteínas Fúngicas/genética , Mutagênese/genética , Mutação de Sentido Incorreto , Aminoácido Oxirredutases/química , Aminoácido Oxirredutases/metabolismo , Ascomicetos/química , Ascomicetos/enzimologia , Bioensaio/métodos , Proteínas Fúngicas/química , Proteínas Fúngicas/metabolismo , Hemoglobinas Glicadas/análise , Humanos , Especificidade por Substrato/genética , Valina/análogos & derivados , Valina/química , Valina/metabolismoRESUMO
ADP-dependent kinases are used in the modified Embden-Meyerhoff pathway of certain archaea. Our previous study has revealed a mechanism for ADP-dependent phosphoryl transfer by Thermococcus litoralis glucokinase (tlGK), and its evolutionary relationship with ATP-dependent ribokinases and adenosine kinases (PFKB carbohydrate kinase family members). Here, we report the crystal structure of glucokinase from Pyrococcus furiosus (pfGK) in a closed conformation complexed with glucose and AMP at 1.9A resolution. In comparison with the tlGK structure, the pfGK structure shows significant conformational changes in the small domain and a region around the hinge, suggesting glucose-induced domain closing. A part of the large domain next to the hinge is also shifted accompanied with domain closing. In the pfGK structure, glucose binds in a groove between the large and small domains, and the electron density of O1 atoms for both the alpha and beta-anomer configurations was observed. The structural details of the sugar-binding site of ADP-dependent glucokinase were firstly clarified and then site-directed mutagenesis analysis clarified the catalytic residues for ADP-dependent kinase, such as Arg205 and Asp451 of tlGK. Homology search and multiple alignment of amino acid sequences using the information obtained from the structures reveals that eucaryotic hypothetical proteins homologous to ADP-dependent kinases retain the residues for the recognition of a glucose substrate.
Assuntos
Glucoquinase/química , Glucose/farmacologia , Pyrococcus furiosus/enzimologia , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Sequência de Aminoácidos , Sítios de Ligação , Catálise , Cristalografia por Raios X , Estabilidade Enzimática , Glucoquinase/genética , Glucoquinase/metabolismo , Glucose/metabolismo , Magnésio/metabolismo , Modelos Moleculares , Dados de Sequência Molecular , Fosfofrutoquinase-1/química , Fosfofrutoquinase-1/genética , Estrutura Quaternária de Proteína/efeitos dos fármacos , Estrutura Terciária de Proteína/efeitos dos fármacosRESUMO
Bacillus stearothermophilus H-804 isolated from a hot spring in Beppu, Japan, produced an ammonia-specific NAD synthetase (EC 6.3.1.5). The enzyme specifically used NH3 as an amide donor for the synthesis of NAD as it formed AMP and pyrophosphate from deamide-NAD and ATP. None of the l-amino acids tested, such as l-asparagine or l-glutamine, or other amino compounds such as urea, uric acid, or creatinine was used instead of NH3. Mg2+ was needed for the activity, and the maximum enzyme activity was obtained with 3 mM MgCl2. The molecular mass of the native enzyme was 50 kDa by gel filtration, and SDS-PAGE showed a single protein band at the molecular mass of 25 kDa. The optimum pH and temperature for the activity were from 9.0 to 10.0 and 60 degrees C, respectively. The enzyme was stable at a pH range of 7.5 to 9.0 and up to 60 degrees C. The Km for NH3, ATP, and deamide-NAD were 0.91, 0.052, and 0.028 mM, respectively. The gene encoding the enzyme consisted of an open reading frame of 738 bp and encoded a protein of 246 amino acid residues. The deduced amino acid sequence of the gene had about 32% homology to those of Escherichia coli and Bacillus subtilis NAD synthetases. We caused the NAD synthetase gene to be expressed in E. coli at a high level; the enzyme activity (per liter of medium) produced by the recombinant E. coli was 180-fold that of B. stearothermophilus H-804. The specific assay of ammonia and ATP (up to 25 microM) with this stable NAD synthetase was possible.
Assuntos
Amida Sintases/metabolismo , Amônia/metabolismo , Geobacillus stearothermophilus/enzimologia , Trifosfato de Adenosina/metabolismo , Algoritmos , Amida Sintases/genética , Amida Sintases/isolamento & purificação , Sequência de Aminoácidos , Clonagem Molecular , Sondas de DNA , Inibidores Enzimáticos/farmacologia , Genes Bacterianos/genética , Geobacillus stearothermophilus/genética , Geobacillus stearothermophilus/ultraestrutura , Concentração de Íons de Hidrogênio , Focalização Isoelétrica , Cinética , Metais/farmacologia , Dados de Sequência Molecular , Peso Molecular , TemperaturaRESUMO
The method of National Committee for Clinical Laboratory Standards (NCCLS) is widely used for the daily quality control of the antimicrobial susceptibility test. This method, however, cannot detect the accidental error, although it is useful to detect the systematic error in the examination. We developed a computer program using the correlation between the various antimicrobial susceptibility test results to detect an accidental error. The combinations of the MIC results determined for two antimicrobial agents which showed a high correlation coefficient (> or = 0.7), were selected from 98 bacterial species (2122 strains) isolated from January 2000 to December 2000 at Oita Medical University Hospital. Subsequently, a total of 127 combinations of antimicrobial agents for 13 species were selected on the basis of acceptable correlation ranges. Then, the method were verified with 666 strains (5753 combinations) isolated during the period of January to June, 2001. Twenty-six strains (47 combinations) were identified as an unexpected result, and the occurrence of error were confirmed in 3 strains (12 combinations). These results suggest that this method which evaluated the correlation between MICs against different antimicrobial agents is applicable for the quality control of antimicrobial susceptibility testings.
Assuntos
Antibacterianos/farmacologia , Técnicas de Laboratório Clínico/normas , Testes de Sensibilidade Microbiana/métodos , Testes de Sensibilidade Microbiana/normas , Análise de Componente Principal , Controle de QualidadeRESUMO
A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.