Estriol sensitizes rat Kupffer cells via gut-derived endotoxin.
(17/1604)
The relationship between gender and alcohol-induced liver disease is complex; however, endotoxin is most likely involved. Recently, it was reported that estriol activated Kupffer cells by upregulation of the endotoxin receptor CD14. Therefore, the purpose of this work was to study how estriol sensitizes Kupffer cells. Rats were given estriol (20 mg/kg ip), and Kupffer cells were isolated 24 h later. After addition of lipopolysaccharide (LPS), intracellular Ca2+ concentration was measured using a microspectrofluorometer with the fluorescent indicator fura 2, and tumor necrosis factor-alpha was measured by ELISA. CD14 was evaluated by Western analysis. One-half of the rats given estriol intraperitoneally 24 h before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 h, whereas none of the control rats died. Mortality was prevented totally by sterilization of the gut with antibiotics. A similar pattern was obtained with liver histology and serum transaminases. Translocation of horseradish peroxidase was increased about threefold in gut segments by treatment with estriol. This increase was not altered by treatment with nonabsorbable antibiotics. On the other hand, endotoxin levels were increased to 60-70 pg/ml in plasma of rats treated with estriol. As expected, this increase was prevented (<20 pg/ml) by antibiotics. In isolated Kupffer cells, LPS-induced increases in intracellular Ca2+ concentration, tumor necrosis factor-alpha production, and CD14 were increased, as previously reported. All these phenomena were blocked by antibiotics. Therefore, it is concluded that estriol treatment in vivo sensitizes Kupffer cells to LPS via mechanisms dependent on increases in CD14. This is most likely due to elevated portal blood endotoxin caused by increased gut permeability. (+info)
The evolution of liver disease in cystic fibrosis.
(18/1604)
OBJECTIVES: To describe prospectively the evolution of liver abnormalities in cystic fibrosis (CF), and to assess their impact on nutritional status. STUDY DESIGN: 124 children (61 boys) with CF (median age, 5.4 years; range, 0.1-13.9) were followed longitudinally for a median of four years. Annual clinical examination, biochemistry, and ultrasound assessment were performed. Chrispin-Norman score, anthropometry, and bacterial colonisation of airway secretions were measured at each assessment. RESULTS: At initial assessment, 45% of the patients had no liver abnormalities, 42% had biochemical abnormality, 35% ultrasound abnormality, and 6% had clinical abnormality of the liver. In this cross sectional analysis, abnormal biochemistry was present in 40% of children with ultrasound or clinical abnormalities, but when longitudinal follow up data were analysed, abnormal biochemistry preceded or coincided with abnormal ultrasound or clinical hepatosplenomegaly in three quarters of 53 children developing new abnormalities. Eighty four of 124 children (68%) showed ultrasound or clinical evidence of liver abnormality at some point during the four years of follow up. No association was found between liver disease and nutritional status. CONCLUSIONS: Hepatic abnormality was common in this group of children with CF, was often predicted by intermittent biochemical abnormalities, and was not associated with deterioration in nutritional status. (+info)
Carbon and nitrogen repression of arginine catabolic enzymes in Bacillus subtilis.
(19/1604)
Specific activities of arginase and ornithine aminotransferase, inducible enzymes of arginine catabolism in Bacillus subtilis 168, were examined in cells grown with various carbon and nitrogen sources. Levels of these enzymes were similar in arginine-induced cultures whether glucose or citrate was the carbon source (in contrast to histidase), suggesting that carbon source catabolite repression has only limited effect. In media with combinations of nitrogen sources, glutamine strongly repressed induction of these enzymes by proline or arginine. Ammonium, however, only repressed induction by proline and had no effect on induction by arginine. These effects correlate with generation times in media containing these substances as sole nitrogen sources: growth rates decreased in the order glutamine-arginine-ammonium-proline. Similar phenomena were observed when glutamine or ammonium were added to arginine- or proline-grown cultures, or when arginine or proline were added to glutamine- or ammonium-grown cultures. In the latter cases, an additional feature was apparent, namely a surprisingly long transition between steady-state enzyme levels. The results are compared with those for other bacteria and for eucaryotic microorganisms. (+info)
In vitro synthesis of the nucleotide loop of cobalamin by Salmonella typhimurium enzymes.
(20/1604)
In Salmonella typhimurium, the CobU, CobS, CobT, and CobC proteins have been proposed to catalyze the late steps in adenosylcobalamin biosynthesis, which define the nucleotide loop assembly pathway. This paper reports the in vitro assembly of the nucleotide loop of adenosylcobalamin from its precursors adenosylcobinamide, 5, 6-dimethylbenzimidazole, nicotinate mononucleotide, and GTP. Incubation of these precursors with the CobU, CobS, and CobT proteins resulted in the synthesis of adenosylcobalamin-5'-phosphate. This cobamide was isolated by HPLC, identified by UV-visible spectroscopy and mass spectrometry, and shown to support growth of a cobalamin auxotroph. Adenosylcobalamin-5'-phosphate was also isolated from reaction mixtures containing adenosylcobinamide-GDP (the product of the CobU reaction) and alpha-ribazole-5'-phosphate (the product of the CobT reaction) as substrates and CobS. These results allowed us to conclude that CobS is the cobalamin(-5'-phosphate) synthase enzyme in S. typhimurium. The CobC enzyme, previously shown to dephosphorylate alpha-ribazole-5'-phosphate to alpha-ribazole, was shown to dephosphorylate adenosylcobalamin-5'-phosphate to adenosylcobalamin. Adenosylcobinamide was converted to adenosylcobalamin in reactions where all four enzymes were present in the reaction mixture. This in vitro system offers a unique opportunity for the rapid synthesis and isolation of cobamides with structurally different lower-ligand bases that can be used to investigate the contributions of the lower-ligand base to cobalamin-dependent reactions. (+info)
Identification and expression of a cDNA for human hydroxypyruvate/glyoxylate reductase.
(21/1604)
The isolation and expression of a human liver cDNA encoding a 40-kDa protein with glyoxylate and hydroxypyruvate reductase activities is described. The cDNA (GLXR) is 1235 bp and consists of a predicted open reading frame of 987 bp with a 225-bp 3'-untranslated region. The 328-amino acid protein has partial sequence similarity to hydroxypyruvate and glyoxylate reductases from a variety of plant and microbial species. (+info)
Genetic characterization of the major lactococcal aromatic aminotransferase and its involvement in conversion of amino acids to aroma compounds.
(22/1604)
In lactococci, transamination is the first step of the enzymatic conversion of aromatic and branched-chain amino acids to aroma compounds. In previous work we purified and biochemically characterized the major aromatic aminotransferase (AraT) of a Lactococcus lactis subsp. cremoris strain. Here we characterized the corresponding gene and evaluated the role of AraT in the biosynthesis of amino acids and in the conversion of amino acids to aroma compounds. Amino acid sequence homologies with other aminotransferases showed that the enzyme belongs to a new subclass of the aminotransferase I subfamily gamma; AraT is the best-characterized representative of this new aromatic-amino-acid-specific subclass. We demonstrated that AraT plays a major role in the conversion of aromatic amino acids to aroma compounds, since gene inactivation almost completely prevented the degradation of these amino acids. It is also highly involved in methionine and leucine conversion. AraT also has a major physiological role in the biosynthesis of phenylalanine and tyrosine, since gene inactivation weakly slowed down growth on medium without phenylalanine and highly affected growth on every medium without tyrosine. However, another biosynthesis aromatic aminotransferase is induced in the absence of phenylalanine in the culture medium. (+info)
Alanine aminotransferase and glycine aminotransferase from maize (Zea mays L.) leaves.
(23/1604)
Alanine aminotransferase (AlaAT, EC 2.6.1.2) and glycine aminotransferase (GlyAT, EC 2.6.1.4), two different enzymes catalyzing transamination reactions with L-alanine as the amino-acid substrate, were examined in maize in which alanine participates substantially in nitrogen transport. Preparative PAGE of a partially purified preparation of aminotransferases from maize leaves gave 6 fractions differing in electrophoretic mobility. The fastest migrating fraction I represents AlaAT specific for L-alanine as amino donor and 2-oxoglutarate as amino acceptor. The remaining fractions showed three aminotransferase activities: L-alanine-2-oxoglutarate, L-alanine-glyoxylate and L-glutamate-glyoxylate. By means of molecular sieving on Zorbax SE-250 two groups of enzymes were distinguished in the PAGE fractions: of about 100 kDa and 50 kDa. Molecular mass of 104 kDa was ascribed to AlaAT in fraction I, while the molecular mass of the three enzymatic activities in 3 fractions of the low electrophoretic mobility was about 50 kDa. The response of these fractions to: aminooxyacetate, 3-chloro-L-alanine and competing amino acids prompted us to suggest that five out of the six preparative PAGE fractions represented GlyAT isoforms, differing from each other by the L-glutamate-glyoxylate:L-alanine-glyoxylate:L-alanine-2-oxoglutarate activity ratio. (+info)
Cloning two genes for nicotianamine aminotransferase, a critical enzyme in iron acquisition (Strategy II) in graminaceous plants.
(24/1604)
Nicotianamine aminotransferase (NAAT), the key enzyme involved in the biosynthesis of mugineic acid family phytosiderophores (MAs), catalyzes the amino transfer of nicotianamine (NA). MAs are found only in graminaceous plants, although NA has been detected in every plant so far investigated. Therefore, this amino transfer reaction is the first step in the unique biosynthesis of MAs that has evolved in graminaceous plants. NAAT activity is dramatically induced by Fe deficiency and suppressed by Fe resupply. Based on the protein sequence of NAAT purified from Fe-deficient barley (Hordeum vulgare) roots, two distinct cDNA clones encoding NAAT, naat-A and naat-B, were identified. Their deduced amino acid sequences were homologous to several aminotransferases, and shared consensus sequences for the pyridoxal phosphate-binding site lysine residue and its surrounding residues. The expression of both naat-A and naat-B is increased in Fe-deficient barley roots, while naat-B has a low level of constitutive expression in Fe-sufficient barley roots. No detectable mRNA from either naat-A or naat-B was present in the leaves of either Fe-deficient or Fe-sufficient barley. One genomic clone with a tandem array of naat-B and naat-A in this order was identified. naat-B and naat-A each have six introns at the same locations. The isolation of NAAT genes will pave the way to understanding the mechanism of the response to Fe in graminaceous plants, and may lead to the development of cultivars tolerant to Fe deficiency that can grow in calcareous soils. (+info)