cDNA and deduced amino acid sequence of murine Cu - Zn superoxide dismutase

Bewley, Glenn C.; Stott, R. A. W.

doi:10.1093/nar/16.6.2728

Cited by 36 publications

(15 citation statements)

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“…HPRT was amplified using 37 cycles, inducible NO synthase (iNOS) was amplified using 41 cycles, and catalase, MnSOD, and CuZnSOD were amplified using 27 cycles. Primers used for catalase, CuZnSOD, and Mn-SOD amplifications are as follows: catalase (forward, 5Ј-CCACCGGAG-GCGGGAACC-3Ј; reverse, 5Ј-GCAATAGGGGTCCTCTTTCC-3Ј) (16), CuZnSOD (forward, 5Ј-GATTAACTGAAGGCCAGCATG-3Ј; reverse, 5Ј-GTCATCTTGTTTCTCATGGACC-3Ј) (17), and MnSOD (forward, 5Ј-CCCAGACCTGCCTTACGACT-3Ј; reverse, 5Ј-CGACCTTGCTCCT-TATTGAA-3Ј) (18). Primers for HPRT and iNOS amplification were described previously (15).…”

Section: Rt-pcrmentioning

confidence: 99%

IL-4 Plays a Crucial Role in Regulating Oxidative Damage in the Liver During Schistosomiasis

Flamme

Patton

Bauman

et al. 2001

The Journal of Immunology

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Liver enlargement and hepatocyte proliferation, normal responses in wild-type (WT) mice infected with the parasitic helminth Schistosoma mansoni, were found to be severely impaired in infected IL-4−/− mice. Compared with WT mice, increased levels of O2−, NO, and the more highly reactive ONOO− were detected in the liver and produced by lesional cells isolated from liver granulomas of infected IL-4−/− mice. Concurrently, antioxidant defenses in the liver, specifically catalase levels, diminished dramatically during the course of infection in these animals. This contrasted to the situation in infected WT mice, where catalase levels remained as high as those in normal mice. Actual levels of reactive oxygen and nitrogen intermediates in the livers of infected IL-4−/− animals are thus likely to be considerably higher than those in the livers of infected WT mice. To determine whether these changes contributed to the development of the more severe disease that characterizes infection in the IL-4−/− animals, we treated infected IL-4−/− mice with uric acid, a potent scavenger of ONOO−. This resulted in significantly increased hepatocyte proliferation, decreased morbidity, and prolonged survival. Taken together, these data indicate that IL-4 is playing a protective role during schistosomiasis by controlling the tight regulation of the generation of reactive oxygen and nitrogen intermediates in the liver.

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Section: Rt-pcrmentioning

confidence: 99%

IL-4 Plays a Crucial Role in Regulating Oxidative Damage in the Liver During Schistosomiasis

Flamme

Patton

Bauman

et al. 2001

The Journal of Immunology

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“…The mouse cDNA probes for γ-glutamyl cysteine synthetase heavy chain (γ-GCS), heme oxygenase-1 (HO-1) and Cu,Zn superoxide dismutase (Cu,Zn-SOD) were generated by PCR. Primer design was based on the published cDNA sequences in the literature (Shibahara et al, 1985;Bewley, 1993;Kang et al, 1997). The β-actin cDNA probe is described in Oakes et al (1997).…”

Section: Materials and Probesmentioning

confidence: 99%

gamma-Glutamyl transferase (GGT) deficiency in the GGTenu1 mouse results from a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA

Jean¹

1999

Mutagenesis

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GGT enu1 , a recently described genetic murine model of γ-glutamyl transferase (GGT) deficiency, was induced by the point mutagen N-ethyl-N-nitrosourea and is inherited as an autosomal recessive trait. The phenotype of systemic GGT deficiency suggested a mutation site within the cDNA coding region which is common in all GGT transcripts. To identify this site, total lung and kidney RNA was isolated from normal and mutant mice, amplified by RT-PCR using GGT-specific primers, cloned as two overlapping~1 kb GGT cDNA fragments, sequenced and compared with that in the literature. A single base pair substitution was identified in the coding region at position 237, where thymidine became adenine, and this mutation replaced a leucine codon, TTG, with a termination codon, TAG. This mutation site was confirmed in mutant genomic DNA by PCR using primers that flanked the predicted site and spanned the intron between the common GGT non-coding exon and the first GGT coding exon. This PCR product was sequenced directly with the secondary 3Ј PCR primer, the mutation site identified and the protocol then utilized to genotype animals. In addition to this mutation, the steady-state level of GGT mRNA in mutant kidney is reduced 3-fold compared with the control. Heterodimeric GGT protein is not detectable by western blot in either whole kidney homogenate or a microsomal membrane fraction. The steady-state mRNA level of γ-glutatmyl cysteinyl synthetase was unchanged in mutant mice compared with normal, but that of heme oxygenase-1 and Cu,Zn-SOD was induced 4-and 3-fold, respectively. Hence, the GGT enu1 mouse model of GGT deficiency results from a single point mutation in the first coding exon of GGT mRNA and the resulting impairment in glutathione turnover induces oxidative stress in the kidney. IntroductionThe GGT enu1 mouse is a genetic model of γ-glutamyl transferase (GGT, EC 2.3.2.2) deficiency with an autosomal recessive mode of inheritance. Its glutathionuric phenotype was one of several aminoacidurias that was selected after mutagenesis of the murine genome with N-ethyl-N-nitrosourea (ENU) (Harding et al., 1997). This model of GGT deficiency also exhibits glutathionemia, growth retardation, neurological abnormalities, infertility, cataract formation and premature mortality in common with the GGT null mutant mouse, GGT m1 /GGT m1 (Lieberman et al., 1996). The two models differ in that not all GGT enu1 mice develop cataracts, they live almost twice as long and

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“…Compared with other mammalian species, an 83% homology was found in the Cu/Zn-SOD cDNA sequence for both equine genes with humans [23] and bovines [10], while an 81% homology was found with mice [3] and rats [11]. In amino acids, the homology was approximately 81% with humans [23], bovines [10], mice [3], and rats [11].…”

mentioning

confidence: 82%

“…In amino acids, the homology was approximately 81% with humans [23], bovines [10], mice [3], and rats [11]. For Mn-SOD, on the other hand, an 89% homology was found in the cDNA sequence with humans [2], while an 84% homology was found with mice [9] and rats [12].…”

mentioning

confidence: 94%

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The cDNA Sequences of Equine Antioxidative Enzyme Genes Cu/Zn-SOD and Mn-SOD, and These Expressions in Equine Tissues

Ishida

Katayama

Sato

et al. 1999

The Journal of Veterinary Medical Science

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ABSTRACT. The entire cDNA sequences were determined by reverse transcription-polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends (RACE) techniques for equine copper/zinc superoxide dismutase (Cu/Zn-SOD) and manganese superoxide dismutase (Mn-SOD) through the use of total RNA extracted from the testis of an adult Thoroughbred. The results revealed a protein coding region for equine Cu/Zn-SOD with bases totaling 465 bp, accompanied by an estimated 154 residues of amino acids. As for equine Mn-SOD, its coding region contained a total of 669 bp and an estimated 222 residues of amino acids. Further, the expression of Cu/Zn-SOD and Mn-SOD genes were confirmed in the equine tissues by RT-PCR and in situ hybridization. -KEY WORDS: equine, in situ hybridization, RT-PCR, SOD.J. Vet. Med. Sci. 61(3): 291-294, 1999 confirmed by RT-PCR and in situ hybridization. Total RNAs for RT-PCR were extracted from the nine tissues; testis, adrenal gland, spleen, cerebrum, cerebellum, heart, thyroid gland, kidney, and liver. For in situ hybridization, the clones of equine Cu/Zn-SOD and Mn-SOD genes were digested with the restriction enzyme Spe I. In vitro transcription was conducted to produce RNA probes using a DIG-RNA Labeling Kit SP6/T7 (Boehringer Mannheim, Tokyo). In situ hybridization was conducted using a deparaffinated pieces of spleen tissue and these probes. Compared with other mammalian species, an 83% homology was found in the Cu/Zn-SOD cDNA sequence for both equine genes with humans [23] [7]. In mammals, the presence of the following three types of SOD has been confirmed: Cu/Zn-SOD found in the cytoplasm [17], Mn-SOD in the mitochondria [20], and EC-SOD in serum [16]. The cDNA sequences of these enzymes in humans have already been determined [2,6,23]. In this study, to allow us to carry out our basic research and enable clinical application regarding the role of active oxygen and antioxidative enzymes in equine biological functions, the cDNA sequences of Cu/Zn-SOD and Mn-SOD genes were first determined. Based on this data, the sequences of amino acids were then deduced. At the same time, the expressions of SOD genes in equine tissues were investigated by RT-PCR and in situ hybridization.Total RNA from the testis of an adult Thoroughbred was extracted by guanidinium/cesium chloride ultracentrifugation [4,21], and used in cDNA synthesis with RNA PCR Kit (Takara, Kyoto) using random primer. For both Cu/Zn-SOD and Mn-SOD, RT-PCR was performed using primers designed by referring to highly conserved regions of cDNA sequences between humans [2, 23] and mice [3,9]. The PCR products were cloned to T-vector (pCR TM II, Invitrogen, San Diego), and these cDNA sequences were determined by the dideoxy chain termination method [22]. Based on the cDNA sequences identified using the RT-PCR products, new PCR primers were designed. The entire cDNA sequences of equine Cu/Zn-SOD and Mn-SOD genes were then determined by 5' RACE and 3' RACE methods [8] using a 5'/3' RACE Kit (Boehringer Mannheim, Tokyo). The expressions of ...

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cDNA and deduced amino acid sequence of murine Cu - Zn superoxide dismutase

Cited by 36 publications

References 4 publications

IL-4 Plays a Crucial Role in Regulating Oxidative Damage in the Liver During Schistosomiasis

IL-4 Plays a Crucial Role in Regulating Oxidative Damage in the Liver During Schistosomiasis

gamma-Glutamyl transferase (GGT) deficiency in the GGTenu1 mouse results from a single point mutation that leads to a stop codon in the first coding exon of GGT mRNA

The cDNA Sequences of Equine Antioxidative Enzyme Genes Cu/Zn-SOD and Mn-SOD, and These Expressions in Equine Tissues

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