Influence of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxylic acid and diphenyliodonium chloride on ruminal protein metabolism and ruminal microorganisms.
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The effects of 1-[(E)-2-(2-methyl-4-nitrophenyl)diaz-1-enyl]pyrrolidine-2-carboxy lic acid (LY29) and diphenyliodonium chloride (DIC) on the degradation of protein to ammonia were determined in a mixed rumen microbial population taken from sheep on a grass hay-concentrate diet. Both compounds decreased NH3 production by inhibiting deamination of amino acids. LY29, but not DIC, inhibited growth of the high-activity ammonia-producing species, Clostridium aminophilum and Clostridium sticklandii. (+info)
Effect of the ratio between essential and nonessential amino acids in the diet on utilization of nitrogen and amino acids by growing pigs.
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In 36 growing pigs (30 to 60 kg), N balance and amino acid (AA) composition of weight gain were measured to evaluate the interactive effect of the ratio between N from essential amino acids (EAA(N)) to nonessential amino acids (NEAA(N)) and total N level (T(N)) in the diet on N retention and utilization of N, EAA(N), NEAA(N), and AA. Nine diets composed from ordinary feedstuffs and supplemented with crystalline AA were used (three EAA(N):NEAA(N) ratios of 38:62, 50:50, and 62:38 at three T(N) levels of 18.8, 22.9, and 30.0 g/kg). Pigs were fed restrictedly, at a level of 2.8 x energy for maintenance. In all diets, EAA (including arginine) supply was according to or slightly above the recommended ratios to lysine. Measurements were done in four blocks of nine pigs each. In a concomitant slaughter experiment, the AA composition of deposited body protein was determined to estimate AA utilization. The effects of T(N) and EAA(N):NEAA(N) and their interaction for N retention and utilization were significant. Nitrogen retention increased with higher T(N) in the diet. Increasing EAA(N):NEAA(N) from 38:62 to 50:50 improved N retention only at the two lower T(N) levels. Increasing EAA(N): NEAA(N) above 50:50 failed to improve N retention significantly at any of the three T(N) levels. Lowering T(N) improved the utilization of total and digested N and of EAA(N) and NEAA(N). The increase in EAA(N): NEAA(N) consistently resulted in a lower utilization of EAA(N), but this was compensated by a higher utilization of NEAA(N). The utilization of T(N) was improved by increasing EAA(N):NEAA(N) from 38:62 to 50:50 at the two lower T(N) levels and was relatively unaffected by EAA(N):NEAA(N) at the highest T(N). However, a lower utilization of N was observed at a ratio of 62:38 at a T(N) level of 22.9 g/kg. The effects were similar for utilization of individual EAA and NEAA. Utilization of alanine, aspartic acid, and glycine was close to or >100% at the highest EAA(N):NEAA(N), which was expected because all of these AA are synthesized in pigs. Also, the utilization of arginine was >100% in most of the treatments, which confirms the semiessential character of this AA for maintenance. We concluded that the required ratio of EAA(N):NEAA(N) for optimal N retention and utilization is approximately 50:50. The EAA(N):NEAA(N) is more important at lower dietary protein levels. This study indicates that EAA(N): NEAA(N) can be increased up to 70:30 without lowering the utilization of N. Thus, deaminated EAA(N) was efficiently utilized for the synthesis of NEAA(N). (+info)
Nitric oxide-induced damage to mtDNA and its subsequent repair.
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Mutations in mitochondrial DNA (mtDNA) have recently been associated with a variety of human diseases. One potential DNA-damaging agent to which cells are continually exposed that could be responsible for some of these mutations is nitric oxide (NO). To date, little information has been forthcoming concerning the damage caused by this gas to mtDNA. Therefore, this study was designed to investigate damage to mtDNA induced by NO and to evaluate its subsequent repair. Normal human fibroblasts were exposed to NO produced by the rapid decomposition of 1-propanamine, 3-(2-hydroxy-2-nitroso-1-propylhydrazino) (PAPA NONOate) and the resultant damage to mtDNA was determined by quantitative Southern blot analysis. This gas was found to cause damage to mtDNA that was alkali-sensitive. Treatment of the DNA with uracil-DNA glycosylase or 3-methyladenine DNA glycosylase failed to reveal additional damage, indicating that most of the lesions produced were caused by the deamination of guanine to xanthine. Studies using ligation-mediated PCR supported this finding. When a 200 bp sequence of mtDNA from cells exposed to NO was analyzed, guanine was found to be the predominantly damaged base. However, there also was damage to specific adenines. No lesions were observed at pyrimidine sites. The nucleotide pattern of damage induced by NO was different from that produced by either a reactive oxygen species generator or the methylating chemical, methylnitrosourea. Most of the lesions produced by NO were repaired rapidly. However, there appeared to be a subset of lesions which were repaired either slowly or not at all by the mitochondria. (+info)
AMP deamination and purine exchange in human skeletal muscle during and after intense exercise.
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1. The present study examined the regulation of human skeletal muscle AMP deamination during intense exercise and quantified muscle accumulation and release of purines during and after intense exercise. 2. Seven healthy males performed knee extensor exercise at 64.3 W (range: 50-70 W) to exhaustion (234 s; 191-259 s). In addition, on two separate days the subjects performed exercise at the same intensity for 30 s and 80 % of exhaustion time (mean, 186 s; range, 153-207 s), respectively. Muscle biopsies were obtained from m.v. lateralis before and after each of the exercise bouts. For the exhaustive bout femoral arterio-venous concentration differences and blood flow were also determined. 3. During the first 30 s of exercise there was no change in muscle adenosine triphosphate (ATP), inosine monophosphate (IMP) and ammonia (NH3), although estimated free ADP and AMP increased 5- and 45-fold, respectively, during this period. After 186 s and at exhaustion muscle ATP had decreased (P < 0.05) by 15 and 19 %, respectively, muscle IMP was elevated (P < 0. 05) from 0.20 to 3.65 and 5.67 mmol (kg dry weight)-1, respectively, and muscle NH3 had increased (P < 0.05) from 0.47 to 2.55 and 2.33 mmol (kg d.w.)-1, respectively. The concentration of H+ did not change during the first 30 s of exercise, but increased (P < 0.05) to 245.9 nmol l-1 (pH 6.61) after 186 s and to 374.5 nmol l-1 (pH 6. 43) at exhaustion. 4. Muscle inosine and hypoxanthine did not change during exercise. In the first 10 min after exercise the muscle IMP concentration decreased (P < 0.05) by 2.96 mmol (kg d.w.)-1 of which inosine and hypoxanthine formation could account for 30 %. The total release of inosine and hypoxanthine during exercise and 90 min of recovery amounted to 1.07 mmol corresponding to 46 % of the net ATP decrease during exercise or 9 % of ATP at rest. 5. The present data suggest that AMP deamination is inhibited during the initial phase of intense exercise, probably due to accumulation of orthophosphate, and that lowered pH is an important positive modulator of AMP deaminase in contracting human skeletal muscle in vivo. Furthermore, formation and release of purines occurs mainly after intense exercise and leads to a considerable loss of nucleotides. (+info)
Helicobacter pylori rocF is required for arginase activity and acid protection in vitro but is not essential for colonization of mice or for urease activity.
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Arginase of the Helicobacter pylori urea cycle hydrolyzes L-arginine to L-ornithine and urea. H. pylori urease hydrolyzes urea to carbon dioxide and ammonium, which neutralizes acid. Both enzymes are involved in H. pylori nitrogen metabolism. The roles of arginase in the physiology of H. pylori were investigated in vitro and in vivo, since arginase in H. pylori is metabolically upstream of urease and urease is known to be required for colonization of animal models by the bacterium. The H. pylori gene hp1399, which is orthologous to the Bacillus subtilis rocF gene encoding arginase, was cloned, and isogenic allelic exchange mutants of three H. pylori strains were made by using two different constructs: 236-2 and rocF::aphA3. In contrast to wild-type (WT) strains, all rocF mutants were devoid of arginase activity and had diminished serine dehydratase activity, an enzyme activity which generates ammonium. Compared with WT strain 26695 of H. pylori, the rocF::aphA3 mutant was approximately 1, 000-fold more sensitive to acid exposure. The acid sensitivity of the rocF::aphA3 mutant was not reversed by the addition of L-arginine, in contrast to the WT, and yielded a approximately 10, 000-fold difference in viability. Urease activity was similar in both strains and both survived acid exposure equally well when exogenous urea was added, indicating that rocF is not required for urease activity in vitro. Finally, H. pylori mouse-adapted strain SS1 and the 236-2 rocF isogenic mutant colonized mice equally well: 8 of 9 versus 9 of 11 mice, respectively. However, the rocF::aphA3 mutant of strain SS1 had moderately reduced colonization (4 of 10 mice). The geometric mean levels of H. pylori recovered from these mice (in log(10) CFU) were 6.1, 5.5, and 4.1, respectively. Thus, H. pylori rocF is required for arginase activity and is crucial for acid protection in vitro but is not essential for in vivo colonization of mice or for urease activity. (+info)
UV filter compounds in human lenses: the origin of 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside.
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PURPOSE: To investigate UV filter synthesis in the human lens, in particular the biosynthetic origin of the second most abundant UV filter compound, 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside. METHODS: Human lenses were analyzed by high-performance liquid chromatography (HPLC) after separate incubation with 3H-tryptophan (3H-Trp), beta-benzoylacrylic acid, D,L-alpha-amino-beta-benzoylpropionic acid, or D,L-3-hydroxykynurenine O-beta-D-glucoside. The effect of pH on the model compound D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside was also investigated. RESULTS: UV filters were not detected in fetal lenses, despite a 5-month postnatal lens displaying measurable levels of UV filters. In adults no radiolabel was incorporated into 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside after 3H-Trp incubations. Beta-benzoylacrylic acid was readily reduced in lenses. D,L-alpha-amino-beta-benzoylpropionic acid and D,L-3-hydroxykynurenine O-beta-D-glucoside slowly deaminated at physiological pH and were converted to beta-benzoylpropionic acid and 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside, respectively, after lens incubations. CONCLUSIONS: UV filter biosynthesis appears to be activated at or near birth. Compounds containing the kynurenine side chain slowly deaminate, and in the lens, the newly formed double bond is rapidly reduced. These findings suggest that 4-(2-amino-3-hydroxyphenyl)-4-oxobutanoic acid O-beta-D-glucoside is derived from L-3-hydroxykynurenine O-beta-D-glucoside through this deamination-reduction process. The slowness of the deamination presumably accounts for the absence of incorporation of radiolabel from 3H-Trp into 4(2-amino-3-hydroxyphenyl)4-oxobutanoic acid O-beta-D-glucoside. (+info)
Characterization of human lens major intrinsic protein structure.
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PURPOSE: To determine the primary covalent structure of human lens major intrinsic protein (MIP) in lenses of varying age. METHODS: MIP was isolated from single human lenses of various ages (7- 86 years) by homogenization of the lenses, followed by centrifugation and urea washes of the membranes. Proteins present in the membrane preparation were reduced, alkylated, and cleaved by CNBr. Peptide fragments were fractionated by reverse-phase high-performance liquid chromatography, and the primary structures of the peptides were determined by tandem mass spectrometry and Edman sequencing. RESULTS: Complete coverage of the human MIP sequence was observed in the form of CNBr fragments. In addition, peptide structures resulting from in vivo heterogeneous N- and C-terminal cleavage were characterized. The amount of intact MIP decreased with lens age; however, the pattern of truncation did not change from 7 to 86 years. The major site of phosphorylation was identified as serine 235. Asparagine residues 246 and 259 were completely deamidated by age 7 years. CONCLUSIONS: The major structural modifications of human lens MIP have been determined. Human MIP is heterogeneously modified in lenses ranging in age from 7 to 86 years of age by N- and C-terminal truncation, phosphorylation, and deamidation, resulting in decreased levels of native intact MIP with age. (+info)
Effect of UV-A light on the chaperone-like properties of young and old lens alpha-crystallin.
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PURPOSE: To study the damaging effect of UV-A irradiation on the chaperone-like properties of alpha-crystallin and the subsequent recovery process of young and old bovine lenses. METHODS: Young and old bovine lenses were kept in organ culture. After 24 hours of incubation they were irradiated with UV-A at 365 nm, and optical quality measurements were performed during the experiments (192 hours). alpha-Crystallin and alpha1-, alphaA2-, alphaB1-, and alphaB2-crystallin subunits were analyzed, separated by gel filtration and cation exchange chromatography, respectively, after different culture times. Protein patterns were obtained after two-dimensional (2-D) gel electrophoresis. Chaperone-like activity was determined on the basis of insulin B-chain and betaL-crystallin aggregation assays. Aggregation of alpha-crystallin was analyzed, tryptophan fluorescence measurements were performed, and alpha-crystallin mRNA levels were determined. RESULTS: The water-soluble alpha-crystallin obtained from old lenses compared with young lenses after UV irradiation had decreased chaperone activity, a higher molecular weight, and increased loss of tryptophan fluorescence. Moreover, alpha-crystallin mRNA virtually disappeared, whereas extra spots on the 2-D protein pattern appeared, possibly because of deamidation. CONCLUSIONS: alpha-Crystallin obtained from old lenses is more affected by irradiation than alpha-crystallin derived from young lenses. Moreover, it appeared that alphaB-crystallin from UV-treated old lenses compared with control lenses was less susceptible to UV-A than alphaA-crystallin. It may well be that alphaB-crystallin protects alphaA-crystallin in vivo. (+info)