To investigate the functional role of renal intrinsic antioxidant enzymes (AOEs), the levels of AOE activities in isolated glomeruli and the changes in renal function to oxidant insults were assessed in normal control rats (NC, N = 23) and rats subjected to 30-minutes of complete renal ischemia for three days (day-3, N = 20) or six days (day-6, N = 23) prior to study. When compared to NC, the activities of total and manganese (cyanide-insensitive) superoxide dismutase, glutathione peroxidase, and catalase were increased more than twofold in day-6 animals, on average, from 36 +/- 4 U/mg protein, 9 +/- 1 U/mg protein, 129 +/- 21 U/mg protein and 1.32 +/- 0.20 k/mg protein, respectively, to 80 +/- 5, 27 +/- 3, 283 +/- 41 and 3.20 +/- 0.20, respectively (P less than 0.05 for all). There were no changes in AOE activities in day-3 animals. In day-6 animals, however, the activities of non-AOEs, LDH and fumarase were found to be unaffected. Separate groups of NC (N = 12), day-3 (N = 5) and day-6 (N = 12) rats were subjected to either 30 minutes of ischemia plus 60 minutes of reperfusion (I/R) or unilateral i.a. infusion of hydrogen peroxide (H2O2, 35 mu moles in 1 hr). The degree of reduction in inulin and para-amino hippurate clearance rates following I/R were significantly less in day-6 (-21 +/- 3% and -12 +/- 2, respectively) compared to NC (-69 +/- 9% and -59 +/- 11, respectively) or day-3 rats (-73 +/- 7% and -62 +/- 10, respectively).(ABSTRACT TRUNCATED AT 250 WORDS)
Cultured rat glomerular mesangial and epithelial cells and bovine glomerular endothelial cells were exposed to various concentrations of hydrogen peroxide (H2O2). Mesangial cells treated with 10 to 100 microM H2O2 for 24 hours showed a two- to ninefold increase in Mn-SOD mRNA expression associated with significantly (P < 0.005) increased Mn-SOD activity (22.2 +/- 1.2 and 12.2 +/- 0.7 mu/mg protein for H2O2 100 microM treated and untreated cells, respectively). In contrast, expression of Cu-Zn SOD and beta-actin mRNA was not affected by H2O2. Induction of Mn-SOD mRNA by H2O2 was inhibited by actinomycin-D (4 microM) treatment. Glomerular endothelial cells also showed an increase in Mn-SOD mRNA expression following 100 microM H2O2 treatment, as did glomerular epithelial cells following treatment with 500 and 1000 microM H2O2 but not with 100 microM. Transcriptional activity of the Mn-SOD gene was assessed with a fusion reporter gene consisting of a luciferase gene (pGL2P) and a 1.2 kb fragment from the rat Mn-SOD genomic DNA (-806 to +408 bp of the transcription initiation site, -806:+408). The construct was transfected into rat glomerular mesangial and epithelial cells. Mesangial and epithelial cells transfected with pGL2P (-806:+408) and treated with H2O2 (100 microM and 1 mM for mesangial and epithelial cells, respectively) demonstrated some threefold increase in luciferase activity, whereas cells transfected with pGL2P lacking the Mn-SOD fragment did not show changes in luciferase activity following H2O2 treatment.(ABSTRACT TRUNCATED AT 250 WORDS)
The present study describes a genome-wide method for biallelic mutagenesis in mammalian cells. Novel poly(A) gene trap vectors, which contain features for direct cloning vector–cell fusion transcripts and for post-entrapment genome engineering, were used to generate a library of 979 mutant ES cells. The entrapment mutations generally disrupted gene expression and were readily transmitted through the germline, establishing the library as a resource for constructing mutant mice. Cells homozygous for most entrapment loci could be isolated by selecting for enhanced expression of an inserted neomycin-resistance gene that resulted from losses of heterozygosity (LOH). The frequencies of LOH measured at 37 sites in the genome ranged from 1.3 × 10−5 to 1.2 × 10−4 per cell and increased with increasing distance from the centromere, implicating mitotic recombination in the process. The ease and efficiency of obtaining homozygous mutations will (i) facilitate genetic studies of gene function in cultured cells, (ii) permit genome-wide studies of recombination events that result in LOH and mediate a type of chromosomal instability important in carcinogenesis, and (iii) provide new strategies for phenotype-driven mutagenesis screens in mammalian cells.
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