Pol32, a subunit of Saccharomyces cerevisiae DNA polymerase delta, suppresses genomic deletions and is involved in the mutagenic bypass pathway.
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The Pol32 subunit of S. cerevisiae DNA polymerase (Pol) delta plays an important role in replication and mutagenesis. Here, by measuring the CAN1 forward mutation rate, we found that either POL32 or REV3 (which encodes the Pol zeta catalytic subunit) inactivation produces overlapping antimutator effects against rad mutators belonging to three epistasis groups. In contrast, the msh2Delta pol32Delta double mutant exhibits a synergistic mutator phenotype. Can(r) mutation spectrum analysis of pol32Delta strains revealed a substantial increase in the frequency of deletions and duplications (primarily deletions) of sequences flanked by short direct repeats, which appears to be RAD52 and RAD10 independent. To better understand the pol32Delta and rev3Delta antimutator effects in rad backgrounds and the pol32Delta mutator effect in a msh2Delta background, we determined Can(r) mutation spectra for rad5Delta, rad5Delta pol32Delta, rad5Delta rev3Delta, msh2Delta, msh2Delta pol32Delta, and msh2Delta rev3Delta strains. Both rad5Delta pol32Delta and rad5Delta rev3Delta mutants exhibit a reduction in frameshifts and base substitutions, attributable to antimutator effects conferred by the pol32Delta and rev3Delta mutations. In contrast, an increase in these two types of alterations is attributable to a synergistic mutator effect between the pol32Delta and msh2Delta mutations. Taken together, these observations indicate that Pol32 is important in ensuring genome stability and in mutagenesis. (+info)
Inhibition of inducible nitric oxide synthase attenuates interleukin-1 beta induced vascular hyporeactivity in the rabbit.
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Inhibition of nitric oxide (NO) synthesis has been indicated to improve vasopressor-responsiveness and to increase blood pressure in most septic models. However, numerous adverse effects of non-selective NO synthase (NOS) inhibition have been reported, and the effect of NOS inhibition on vascular responsiveness to vasodilators has not been well studied. Using an isometric tension measurement system of vascular rings, we evaluated the effects of an inducible NOS (iNOS) inhibitor, L-canavanine (L-CAN) and a non-selective NOS inhibitor, NG-nitro-L-arginine methyl ester (L-NAME) on interleukin-1 beta (IL-1 beta)-induced vascular hyporeactivity in the four different rabbit arteries. Pretreatment of IL-1 beta inhibited phenylephrine (Phe)-induced vascular constriction in the carotid artery (CA, 49% of control), pulmonary artery (PA, 66%), femoral artery (FA, 71%) and in the renal artery (RA, 83%). A combination of NOS inhibitors attenuated the vascular hyporeactivity to Phe in all arteries. Pretreatment of IL-1 beta also inhibited acetylcholine (Ach)-induced vascular relaxation in FA, RA and CA. In PA, the rings were inversely constricted after Ach administration. The combination of IL-1 beta with L-CAN, but not with L-NAME, attenuated the Ach-induced vasorelaxation to the control level in all arteries. These data suggest that the selective inhibition of iNOS attenuates the direct endothelial damage induced by IL-1 beta in vitro. (+info)
Misfolded proteins are competent to mediate a subset of the responses to heat shock in Saccharomyces cerevisiae.
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Cells may sense heat shock via the accumulation of thermally misfolded proteins. To explore this possibility, we determined the effect of protein misfolding on gene expression in the absence of temperature changes. The imino acid analog azetidine-2-carboxylic acid (AZC) is incorporated into protein competitively with proline and causes reduced thermal stability or misfolding. We found that adding AZC to yeast at sublethal concentrations sufficient to arrest proliferation selectively induced expression of heat shock factor-regulated genes to a maximum of 27-fold and that these inductions were dependent on heat shock factor. AZC treatment also selectively repressed expression of the ribosomal protein genes, another heat shock factor-dependent process, to a maximum of 20-fold. AZC treatment thus strongly and selectively activates heat shock factor. AZC treatment causes this activation by misfolding proteins. Induction of HSP42 by AZC treatment required protein synthesis; treatment with ethanol, which can also misfold proteins, activated heat shock factor, but treatment with canavanine, an arginine analog less potent than AZC at misfolding proteins, did not. However, misfolded proteins did not strongly induce the stress response element regulon. We conclude that misfolded proteins are competent to specifically trigger activation of heat shock factor in response to heat shock. (+info)
Multiple associated proteins regulate proteasome structure and function.
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We have identified proteins that are abundant in affinity-purified proteasomes, but absent from proteasomes as previously defined because elevated salt concentrations dissociate them during purification. The major components are a deubiquitinating enzyme (Ubp6), a ubiquitin-ligase (Hul5), and an uncharacterized protein (Ecm29). Ecm29 tethers the proteasome core particle to the regulatory particle. Proteasome binding activates Ubp6 300-fold and is mediated by the ubiquitin-like domain of Ubp6, which is required for function in vivo. Ubp6 recognizes the proteasome base and its subunit Rpn1, suggesting that proteasome binding positions Ubp6 proximally to the substrate translocation channel. ubp6Delta mutants exhibit accelerated turnover of ubiquitin, indicating that deubiquitination events catalyzed by Ubp6 prevent translocation of ubiquitin into the proteolytic core particle. (+info)
The small ubiquitin-like modifier (SUMO) protein modification system in Arabidopsis. Accumulation of SUMO1 and -2 conjugates is increased by stress.
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Small ubiquitin-like modifier (SUMO) is a member of the superfamily of ubiquitin-like polypeptides that become covalently attached to various intracellular target proteins as a way to alter their function, location, and/or half-life. Here we show that the SUMO conjugation system operates in plants through a characterization of the Arabidopsis SUMO pathway. An eight-gene family encoding the SUMO tag was discovered as were genes encoding the various enzymes required for SUMO processing, ligation, and release. A diverse array of conjugates could be detected, some of which appear to be SUMO isoform-specific. The levels of SUMO1 and -2 conjugates but not SUMO3 conjugates increased substantially following exposure of seedlings to stress conditions, including heat shock, H(2)O(2), ethanol, and the amino acid analog canavanine. The heat-induced accumulation could be detected within 2 min from the start of a temperature upshift, suggesting that SUMO1/2 conjugation is one of the early plant responses to heat stress. Overexpression of SUMO2 enhanced both the steady state levels of SUMO2 conjugates under normal growth conditions and the subsequent heat shock-induced accumulation. This accumulation was dampened in an Arabidopsis line engineered for increased thermotolerance by overexpressing the cytosolic isoform of the HSP70 chaperonin. Taken together, the SUMO conjugation system appears to be a complex and functionally heterogeneous pathway for protein modification in plants with initial data indicating that one important function may be in stress protection and/or repair. (+info)
The biosynthesis of guanidinosuccinic acid by perfused rat liver.
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The metabolic pathway for the synthesis of guanidinosuccinic acid was studied in the rat. Labeled guanidinosuccinic acid was isolated from the urine of animals given L-[guanidino-14C]arginine intraperitoneally but did not appear in the urine after administration of D,L-[guanidino-14C]canavanine. Radioactive arginine and nonradioactive aspartic acid and arginine were infused in the isolated, perfused rat liver. After 20 min, small amounts of both labeled and unlabeled guanidinosuccinic acid and large amounts of urea were detected in radiochromatograms of the perfusate. These results support the theory that guanidinosuccinic acid is formed in the liver from transamidination of arginine to aspartic acid. (+info)
Disseminated cryptococcosis in an AIDS patient caused by a canavanine-resistant strain of Cryptococcus neoformans var. grubii.
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A case of disseminated cryptococcosis caused by Cryptococcus neoformans var. grubii is presented in a male diabetic who had AIDS. The diagnosis was based upon the isolation and identification of the aetiological agent from a lymph-node biopsy, cerebrospinal fluid and sputum. The isolate formed spherical, encapsulated yeast cells, produced cherry-brown colonies on niger-seed agar, grew on canavanine-glycine-bromothymol blue (CGB) medium, changing its colour from greenish yellow to blue, and hydrolysed urea weakly in the presence of 100 microM EDTA. The strain was unable to assimilate D-proline and, serologically, it was untypable. The identity of the isolate as C. neoformans var. grubii, serotype A, possessing a mating-type allele A alpha, was confirmed by crossing with standard laboratory test strains and by performing PCR with the mating-type alpha allele-specific primer of the STE12 gene and with serotype (A and D)- and mating type (a and alpha)-specific primers of the STE20 gene. To the best of our knowledge, this is the first report of disseminated cryptococcosis in an AIDS patient caused by a canavanine-resistant strain of C. neoformans var. grubii, serotype A, possessing mating type allele A alpha; the strain is probably a hybrid. The report suggests that, in the absence of a clear-cut serotyping result, a positive CGB reaction alone is not sufficient for intervarietal discrimination and additional confirmatory evidence is required. (+info)
The heterogeneity of arginases in rat tissues.
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Arginase reactions in rat tissues were shown to be catalysed by three isoenzymes which can be separated by bidirectional electrophoresis on polyacrylamide gels. Anodic electrophoresis reveals a migrating band (isoenzyme I) present in all-non-hepatic tissues except submaxillary gland and a non-migrating band found in all tissues. The latter is resolved by cathodic electrophoresis into isoenzymes III (characteristic of liver and submaxillary gland) and a non-moving band (isoenzyme II), present in kidney, intestine and pancreas. Sequential electrophoresis, in the two directions, of mixture of liver and kidney extracts in the same gel columns separated all three isoenzymes. Differences in the solubilization properties, heat-sensitivity and substrate specificity of arginases from different tissues could be correlated with their electrophoretic behaviour. L-Canavanine could replace arginine as substrate in extracts of kidney but not of liver. Both kidney isoenzymes hydrolysed L-canavanine equally well, whereas isoenzyme III from submaxillary gland showed only very low activity. Antiserum against liver arginase interacted with the enzyme with submaxillary gland, but did not inactivate or adsorb arginase from kidney, intestine or pancreas. The distribution of arginase among 16 normal adult rat tissues is presented; the improved, sensitive, assay method was applicable to tissues containing as little as 0.1% of the hepatic activity. (+info)