Human Mn superoxide dismutase (MnSOD) encoded by chromosome 6 is a mitochondrial matrix enzyme positioned to scavenge oxygen radicals produced by the extensive oxidation-reduction and electron transport reactions undergoing in that organelle. cDNA clones containing the entire coding region for human MnSOD were isolated from a T-lymphocyte cDNA library in A gt1O. The cDNA contains a 666 bp coding region followed by a 3' untranslated region which lacks the AATAAA polyadenylation signal. The predicted amino acid sequence is in accordance with the published amino acid sequence of human liver MnSOD (1) with the following exceptions: Glu instead of Gln at positions 42, 88 and 109 and an additional Gly-Trp after amino acids 123. The deduced protein sequence extends 24 amino acids upstream from the N-terminal Lys of human MnSOD, suggesting a pre-peptide. Hence human MnSOD is composed of 222 amino acids, 24 of which are removed during processing and maturation of the enzyme.
In many pathological situations, tissue damage is caused by cellular generation of superoxide free radicals (O2-). These active species are generated during post-ischemic reperfusion of organs, in hyperoxic tissue, during acute and chronic inflammation and during exposure to ionizing radiation. Exogenous superoxide dismutase (SOD) was shown to significantly prevent such damage. The genes for human cytosolic Cu/ZnSOD and mitochondrial MnSOD were cloned and introduced into an E. coli expression system. The proteins were expressed in high yields and purified to homogeneity, yielding pharmaceutical-grade materials. These enzymes were used in a variety of in vivo animal models for the demonstration of their protective effects against oxidative damage. Comparative pharmacokinetic studies in rats have revealed that the half-life of Cu/ZnSOD was 6-10 min., while that of MnSOD was 5-6 hours, thus indicating that MnSOD may be superior to Cu/ZnSOD for the treatment of chronic diseases. Indeed, MnSOD was found to be effective as an anti-inflammatory agent in the rat carrageenan induced paw edema acute inflammation model. Both enzymes were also effective in ameliorating post-irradiation damage in mice exposed to whole-body or localized chest X-ray radiation.
R plasmids R702., R711b, R1, D, Rip69, R447b, R471 andiR394, belonging to different incompatibility groups, mobilized the Proteus rnorganii 28 15 chromosome. Matings employing plasmids K711 b or R702 as sex factors with doubly auxotrophic recipients produced recombinants characterized by the obligatory inheritance of ser-I+, irrespective of the selected marker. I N T R O D U C T I O NProteus rnorganii occupies a unique position in the Proteus group. It differs in biochemical aspects as well as in its G + C mole percentage (see Coetzee, 1972). The chromosome of P. rnirabilis is mobilized by various R plasmids (Coetzee, 1978a(Coetzee, , b, 1979a) and a map of the chromosome is available (Coetzee, 1979 b). This paper reports attempts to mobilize the P. morganii chromosome with R plasmids. METHODSBacteria undplasmids. These are listed in Table 1. Media. Nutrient broth and agar were as described by Coetzee ( 1 9 7 8~) .MacConkey agar was from Difco. Minimal medium (MM) was that of Grabow & Smit (1967) containing methionine (4.0 pg ml-l) and calcium pantothenate (5 pg ml-l) and was supplemented with amino acids (40 pg ml-l) when necessary. Incubation temperature was 37 "C.Antibacterial drugs. Ampicillin, chloramphenicol, tetracycline and kanamycin (each 30 pg ml-l), nalidixic acid (75 ,ug ml-l) and streptomycin ( 1 mg ml-l) were added to MM or MacConkey agar when required.Plate matings. These were done by the method of Coetzee ( 1 9 7 8~) . Briefly, equal volumes (0.4 ml) of the partners were mixed and filtered through Millipore membranes which were then placed on nutrient agar and incubated overnight. Growth was harvested and washed with saline and suitable dilutions were plated on selective media and incubated for 4 d. Recombinants were tested for expression of plasmid antibiotic resistance markers by the method of Coetzee (1978a, 19796).Umelected marker analysis. Recombinants (50 to 500) selected for the transfer of a single marker on appropriately supplemented MM were replicated to MM to score prototrophs. R E S U L T S A N D D I S C U S S I O NPlasmids R702, R711b, R1, D, Rip69, R447b, R471 and R394 were stably maintained in P. morganii 28115, had intra-strain transfer frequencies of per donor (not shown) and mobilized the P. morganii chromosome. Plasmids Rip69, R447b, R471 or
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