Glucocorticoids mediate the enhanced expression of intestinal type II arginase and argininosuccinate lyase in postweaning pigs.
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Arginine metabolism is enhanced in the small intestine of weanling pigs, but the molecular mechanism(s) involved is not known. The objectives of this study were to determine the following: 1) whether glucocorticoids play a role in induction of intestinal arginine metabolic enzymes during weaning; 2) whether the induction of enzyme activities was due to increases in corresponding mRNA levels; and 3) the identity of the arginase isoform(s) expressed in the small intestine. Jejunum was obtained from 29-d-old weaned pigs that were or were not treated with 17-beta-hydroxy-11beta-(4-dimethylaminophenyl)17alpha-(prop- 1-ynyl)es tra-4,9-dien-3-one (RU486, an antagonist of glucocorticoid receptors), or from age-matched suckling pigs. Activities and mRNA levels for type I and type II arginases, argininosuccinate synthase (ASS) and argininosuccinate lyase (ASL) were determined. Activities of arginase, ASL and ASS increased by 635, 56 and 106%, respectively, in weanling pigs, compared with suckling pigs. RU486 treatment attenuated the increase in arginase activity by 74% and completely prevented the ASL induction in weanling pigs, but had no effect on ASS activity. Pig intestine expresses both type I and type II arginases. On the basis of immunoblot analyses, there was no significant difference in levels of intestinal type I arginase among these three groups of pigs, indicating that changes in arginase activity were due only to type II arginase. The mRNA levels for type II arginase and ASL increased by 135 and 198%, respectively, in weanling pigs compared with suckling pigs, and this induction was completely prevented by RU486. In contrast, ASS mRNA levels did not differ between suckling and weanling pigs. These results suggest that intestinal type II arginase, ASS and ASL are regulated differentially at transcriptional and post-translational levels and that glucocorticoids play a major role in the induction of type II arginase and ASL mRNAs in the small intestine of weanling pigs. (+info)
Competitive inactivation of a double-strand DNA break site involves parallel suppression of meiosis-induced changes in chromatin configuration.
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In Saccharomyces cerevisiae, DNA double-strand breaks (DSBs) initiate meiotic recombination at open sites in chromatin, which display a meiosis-specific increase in micrococcal nuclease (MNase) sensitivity. The arg4 promoter contains such a DSB site. When arg4 sequences are placed in a pBR322-derived insert at HIS4 (his4 :: arg4 ), the presence of strong DSB sites in pBR322 sequences leads to an almost complete loss of breaks from the insert-borne arg4 promoter region. Most of the MNase-sensitive sites occurred at similar positions in insert-borne and in normal ARG4 sequences, indicating that hotspot inactivation is not a consequence of changes in nucleosome positioning. However, a meiosis-specific increase in MNase hypersensitivity was no longer detected at the inactive insert-borne arg4 DSB site. Elimination of pBR322 sequences restored DSBs to the insert-borne arg4 promoter region and also restored the meiotic induction of MNase hypersensitivity. Thus, the meiotic induction of MNase hypersensitivity at the DSB sites is suppressed and activated in parallel to DSBs themselves, without changes in the underlying DNA sequence or nucleosome positioning. We suggest that meiosis-specific changes in chromatin at a DSB site are a signal reflecting a pivotal step in DSB formation. (+info)
Expression of Escherichia coli K-12 arginine genes in Pseudomonas fluorescens.
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Escherichia coli argE and argH gene products were detected in Pseudomonas fluorescens argH122 carrying the E. coli F110 plasmid. (+info)
Use of a recombination reporter insert to define meiotic recombination domains on chromosome III of Saccharomyces cerevisiae.
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In Saccharomyces cerevisiae, meiotic recombination is initiated by DNA double-strand breaks (DSBs). DSBs usually occur in intergenic regions that display nuclease hypersensitivity in digests of chromatin. DSBs are distributed nonuniformly across chromosomes; on chromosome III, DSBs are concentrated in two "hot" regions, one in each chromosome arm. DSBs occur rarely in regions within about 40 kb of each telomere and in an 80-kb region in the center of the chromosome, just to the right of the centromere. We used recombination reporter inserts containing arg4 mutant alleles to show that the "cold" properties of the central DSB-deficient region are imposed on DNA inserted in the region. Cold region inserts display DSB and recombination frequencies that are substantially less than those seen with similar inserts in flanking hot regions. This occurs without apparent change in chromatin structure, as the same pattern and level of DNase I hypersensitivity is seen in chromatin of hot and cold region inserts. These data are consistent with the suggestion that features of higher-order chromosome structure or chromosome dynamics act in a target sequence-independent manner to control where recombination events initiate during meiosis. (+info)
(CA/GT)(n) microsatellites affect homologous recombination during yeast meiosis.
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One of the most common microsatellites in eukaryotes consists of tandem arrays of the dinucleotide GT. Although the study of the instability of such repetitive DNA has been extremely fruitful over the last decade, no biological function has been demonstrated for these sequences. We investigated the genetic behavior of a region of the yeast Saccharomyces cerevisiae genome containing a 39-CA/GT dinucleotide repeat sequence. When the microsatellite sequence was present at the ARG4 locus on homologous chromosomes, diploid cells undergoing meiosis generated an excess of tetrads containing a conversion of the region restricted to the region of the microsatellite close to the recombination-initiation double-strand break. Moreover, whereas the repetitive sequence had no effect on the frequency of single crossover, its presence strongly stimulated the formation of multiple crossovers. The combined data strongly suggest that numerous recombination events are restricted to the initiation side of the microsatellite as though progression of the strand exchange initiated at the ARG4 promoter locus was impaired by the repetitive sequence. This observation corroborates in vitro experiments that demonstrated that RecA-promoted strand exchange is inhibited by CA/GT dinucleotide tracts. Surprisingly, meiotic instability of the microsatellite was very high (>0.1 alterations per tetrad) in all the spores with parental and recombinant chromosomes. (+info)
Guanidine hydrochloride induced reversible dissociation and denaturation of duck delta2-crystallin.
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The tetrameric delta2-crystallin from duck lens exhibits a reversible dissociation-denaturation process in solutions containing guanidine hydrochloride (GdnHCl). Sigmoidal or biphasic curves for the dissociation/denaturation processes, obtained using different methods of structural analysis, as a function of GdnHCl concentration were not coincidental with each other. delta2-crystallin in 0.91 M GdnHCl existed primarily as a monomer, which had no endogenous argininosuccinate lyase activity. After dilution of the GdnHCl-treated protein, the monomers reassociated into tetramers with concomitant recovery of enzyme activity. The sigmoidal recovery of enzyme activity demonstrates a cooperative hysteretic reactivation process. When the concentration of GdnHCl was higher than 1.2 M, various partially unfolded soluble forms of delta2-crystallin were produced from the dissociated monomers as shown by size-exclusion chromatography. The formation of a partially unfolded intermediate during the dissociation-denaturation process is proposed. (+info)
Regulation of diaphragmatic nitric oxide synthase expression during hypobaric hypoxia.
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Nitric oxide (NO) is normally synthesized inside skeletal muscle fibers by both endothelial (eNOS) and neuronal (nNOS) nitric oxide synthases. In this study, we evaluated the influence of hypobaric hypoxia on the expression of NOS isoforms, argininosuccinate synthetase (AS), argininosuccinate lyase (AL), and manganese superoxide dismutase (Mn SOD) in the ventilatory muscles. Rats were exposed to hypobaric hypoxia ( approximately 95 mmHg) from birth for 60 days or 9-11 mo. Age-matched control groups of rats also were examined. Sixty days of hypoxia elicited approximately two- and ninefold increases in diaphragmatic eNOS and nNOS protein expression (evaluated by immunoblotting), respectively, and about a 50% rise in diaphragmatic NOS activity. In contrast, NOS activity and the expression of these proteins declined significantly in response to 9 mo of hypoxia. Hypoxia elicited no significant alterations in AS, AL and Mn SOD protein expression. Moreover, the inducible NOS (iNOS) was not detected in normoxic and hypoxic diaphragmatic samples. We conclude that diaphragmatic NOS expression and activity undergo significant adaptations to hypobaric hypoxia and that iNOS does not participate in this response. (+info)
Accumulation of arginine precursors in Escherichia coli: effects on growth, enzyme repression, and application to the forward selection of arginine auxotrophs.
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The accumulation or ornithine, citrulline, and possibly acetylornithine by Escherichia coli K-12 arginineless mutants provided with acetylarginine as source of arginine causes severe growth inhibition. This occurs under conditions where comparable derivatives of E. coli W (Bollon and Vogel, 1973) show little or no growth inhibition. The same conditions, which have been reported to cause noncorrelative synthesis of acetylornithinase and argininosuccinase in E. coli W (Bollon and Vogel, 1973), do not alter the correlative pattern of enzyme synthesis observed in E. coli K-12. Moreover, previously reported effects of ornithine and citrulline on repression of the arginine regulon in E. coli W are not observed in the K-12 strains examined. The bearing of these observations on possible differences between the mechanism of enzyme repression operating in the two types of strains cannot yet be fully evaluated; it is, however, clear that considerable care should be exercised before extrapolating the results obtained with one type of strain to the other one. The particularly strong inhibition of acetylarginine utilization exerted by ornithine in E. coli K-12 allows the forward selection of several classes of arginine auxotrophs from strains deficient in carbamoylphosphate biosynthesis and thus capable of ornithine accumulation. Possible applications of this technique to the genetic analysis of the bipolar argECBH operon are discussed. (+info)