Molecular cloning and nucleotide sequence of the superoxide dismutase gene and characterization of its product from Bacillus subtilis

Bacillus subtilis was found to possess one detectable superoxide dismutase (Sod) in both vegetative cells and spores. The Sod activity in vegetative cells was maximal at stationary phase. Manganese was necessary to sustain Sod activity at stationary phase, but paraquat, a superoxide generator, did not induce the expression of Sod. The specific activity of purified Sod was approximately 2, 600 U/mg of protein, and the enzyme was a homodimer protein with a molecular mass of approximately 25,000 per monomer. The gene encoding Sod, designated sodA, was cloned by the combination of several PCR methods and the Southern hybridization method. DNA sequence analysis revealed the presence of one open reading frame consisting of 606 bp. Several putative promoter sites were located in the upstream region of sodA. The deduced amino acid sequence showed high homology with other bacterial manganese Sods. Conserved regions in bacterial manganese Sod could also be seen. The phenotype of double mutant Escherichia coli sodA sodB, which could not grow in minimal medium without supplemental amino acids, was complemented by the expression of B. subtilis sodA.     

Alkyl hydroperoxide reductase, catalase, MrgA, and superoxide dismutase are not involved in resistance of Bacillus subtilis spores to heat or oxidizing agents

Only a single superoxide dismutase (SodA) was detected in Bacillus subtilis, and growing cells of a sodA mutant exhibited paraquat sensitivity as well as a growth defect and reduced survival at an elevated temperature. However, the sodA mutation had no effect on the heat or hydrogen peroxide resistance of wild-type spores or spores lacking the two major DNA protective alpha/beta-type small, acid-soluble, spore proteins (termed alpha(-)beta(-) spores). Spores also had only a single catalase (KatX), as the two catalases found in growing cells (KatA and KatB) were absent. While a katA mutation greatly decreased the hydrogen peroxide resistance of growing cells, as found previously, katA, katB, and katX mutations had no effect on the heat or hydrogen peroxide resistance of wild-type or alpha(-)beta(-) spores. Inactivation of the mrgA gene, which codes for a DNA-binding protein that can protect growing cells against hydrogen peroxide, also had no effect on spore hydrogen peroxide resistance. Inactivation of genes coding for alkyl hydroperoxide reductase, which has been shown to decrease growing cell resistance to alkyl hydroperoxides, had no effect on spore resistance to such compounds or on spore resistance to heat and hydrogen peroxide. However, Western blot analysis showed that at least one alkyl hydroperoxide reductase subunit was present in spores. Together these results indicate that proteins that play a role in the resistance of growing cells to oxidizing agents play no role in spore resistance. A likely reason for this lack of a protective role for spore enzymes is the inactivity of enzymes within the dormant spore.     

Involvement of superoxide dismutase and pyruvate:ferredoxin oxidoreductase in mechanisms of metronidazole resistance in Entamoeba histolytica

Metronidazole resistance has been induced in an axenic strain of Entamoeba histolytica (HTH-56:MUTM) following continuous exposure to steadily increasing drug concentrations. The drug-resistant line is routinely maintained in normally lethal levels of metronidazole (10 microM). Resistance to this concentration of drug was developed over 177 days. Decreased pyruvate:ferredoxin oxidoreductase (PFOR) activity in anaerobic organisms is one mechanism of metronidazole resistance but in entamoeba, PFOR activity was not decreased in metronidazole-resistant parasites as determined by immunofluorescent assays and immunoblotting studies. 2-Oxoacid oxidoreductase activity, which appeared to be due to a single enzyme, PFOR, was evident with pyruvate as well as the alternative substrates, alpha-ketobutyrate, alpha-ketoglutarate and oxaloacetate. A marked increase in superoxide dismutase (SOD) activity was detected in metronidazole-resistant E. histolytica. Increased SOD activity has not previously been documented as a mechanism of drug resistance although SOD has been associated with a range of stress situations in other organisms.     

Elevated superoxide dismutase in black alcoholics

Superoxide dismutase concentrations in lysates of erythrocytes from black alcoholics were higher than those of white alcoholics and of nonalcoholics of both races. Higher concentrations of enzyme protein, as determined by competition radioimmunoassay, correspond to proportionately higher enzyme activity. Elevated superoxide dismutase levels were not related to any other clinical, historical, or demographic variables. Increased superoxide dismutase levels may delay or prevent some of the pathological sequelae of alcoholism and may be a useful biological marker for alcohol abuse.     

Macrolide antibiotics inhibit 50S ribosomal subunit assembly in Bacillus subtilis and Staphylococcus aureus

Macrolide antibiotics are clinically important antibiotics which are effective inhibitors of protein biosynthesis in bacterial cells. We have recently shown that some of these compounds also inhibit 50S ribosomal subunit formation in Escherichia coli. Now we show that certain macrolides have the same effect in two gram-positive organisms, Bacillus subtilis and Staphylococcus aureus. Assembly in B. subtilis was prevented by erythromycin, clarithromycin, and azithromycin but not by oleandomycin. 50S subunit formation in S. aureus was prevented by each of seven structurally related 14-membered macrolides but not by lincomycin or two streptogramin antibiotics. Erythromycin treatment did not stimulate the breakdown of performed 50S subunits in either organism. The formation of the 30S ribosomal subunit was also unaffected by these compounds. Assembly was also inhibited in a B. subtilis strain carrying a plasmid with the ermC gene that confers macrolide resistance by rRNA methylation. These results suggest that ribosomes contain an additional site for the inhibitory functions of macrolide antibiotics.     

Structural roles of the spore coat proteins in Dictyostelium discoideum

The integrity of spores formed by mutant strains of Dictyostelium discoideum lacking the major spore coat proteins, SP96, SP70, or SP60, was compared to that of wild-type strains. Single, double, and triple knock-out strains developed normally and produced spores which were indistinguishable from wild-type spores by light or electron microscopy. However, the mutant strains were susceptable to staining with the lectin, ricin A, which recognizes a galactose-rich polysaccharide that is normally hidden by overlying spore coat proteins. The intensity of staining with fluorescently labeled ricinA increased as the spore coat proteins were incrementally lost. While these results indicate that the major outer spore coat proteins are not essential for the construction of a multi-layered spore coat in Dictyostelium, they show that the spores are more porous which might make them at risk to predators before germination.     

Expression of superoxide dismutase following axotomy

Genomic clones for a chitinolytic enzyme were isolated from a library of Sau 3A digested DNA from the tobacco hornworm, Manduca sexta, using a previously isolated chitinase cDNA clone as a probe [Kramer et al., Insect Biochem. Molec. Biol. 23, 691-701 (1993)]. Restriction enzyme mapping and Southern blot analysis of four genomic clones suggested that these are overlapping clones. Sequence analysis of the genomic clones and Southern blot analysis of total genomic DNA also suggest that the M. sexta genome has only one chitinase gene detectable by the cDNA probe. This gene is organized into at least 11 exons in a region spanning > 11 kb. The sequenced M. sexta chitinase gene has a series of exons corresponding to identifiable structural/functional regions of the protein. Similarities in structure and organization between the M. sexta chitinase gene and chitinase genes from other sources are described.     

Spectral and physical properties of human extracellular superoxide dismutase: a comparison with CuZn superoxide dismutase

A mouse monoclonal IgG1 antibody, referred to as NI-58, has been produced. In immunofluorescence assay, this antibody reacted with myelomonocytes, EBV-B cells, Burkitt's lymphoma cells, T cell leukemia cells and peripheral blood mononuclear cells, but not with erythroid cells. The surface antigen on U937 cells recognized by NI-58 had a molecular size of 65 kDa as determined by immunoblotting analysis. As a biological function, NI-58 strongly inhibited the homotypic cell adhesion of LPS-stimulated U937 cells. It was found that the antigen defined by NI-58 was distinct from CD54 (intercellular adhesion molecule-1) in it's pattern of cellular expression and molecular weight, suggesting that NI-58 recognizes a new adhesion molecule and inhibits the homotypic cell adhesion of LPS-stimulated U937 cells.     

Identification of superoxide dismutase activity in Borrelia burgdorferi

Infective and noninfective strains of Borrelia burgdorferi, along with Borrelia afzelii and Borrelia garinii, possessed a single iron-containing superoxide dismutase (SOD). None of the Lyme disease spirochetes tested possessed catalase or peroxidase activities. The borrelial SOD was not inducible by growth with increased oxygen concentrations and thus appeared to be produced constitutively.     

Mitochondrial disease in superoxide dismutase 2 mutant mice

Oxidative stress has been implicated in many diseases. The chief source of reactive oxygen species within the cell is the mitochondrion. We have characterized a variety of the biochemical and metabolic effects of inactivation of the mouse gene for the mitochondrial superoxide dismutase (CD1-Sod2(tm1Cje)). The Sod2 mutant mice exhibit a tissue-specific inhibition of the respiratory chain enzymes NADH-dehydrogenase (complex I) and succinate dehydrogenase (complex II), inactivation of the tricarboxylic acid cycle enzyme aconitase, development of a urine organic aciduria in conjunction with a partial defect in 3-hydroxy-3-methylglutaryl-CoA lyase, and accumulation of oxidative DNA damage. These results indicate that the increase in mitochondrial reactive oxygen species can result in biochemical aberrations with features reminiscent of mitochondrial myopathy, Friedreich ataxia, and 3-hydroxy-3-methylglutaryl-CoA lyase deficiency.     

Constitutive overexpression of Cu/Zn superoxide dismutase exacerbates kainic acid-induced apoptosis of transgenic-Cu/Zn superoxide dismutase neurons

Cu/Zn superoxide dismutase (Cu/Zn SOD) is a key enzyme in the metabolism of oxygen free radicals. The gene resides on chromosome 21 and is overexpressed in patients with Down syndrome. Cultured neurons of transgenic Cu/Zn SOD (Tg-Cu/Zn SOD) mice with elevated activity of Cu/Zn SOD were used to determine whether constitutive overexpression of Cu/Zn SOD creates an indigenous oxidative stress that predisposes the Tg-Cu/Zn SOD neurons to added insults. Neurons from three independently derived Tg-Cu/Zn SOD strains showed higher susceptibility than nontransgenic neurons to kainic acid (KA)-mediated excitotoxicity, reflected by an earlier onset and enhanced apoptotic cell death. This higher susceptibility of transgenic neurons to KA-mediated apoptosis was associated with a chronic prooxidant state that was manifested by reduced levels of cellular glutathione and altered [Ca2+]i homeostasis. The data are compatible with the thesis that overexpression of Cu/Zn SOD creates chronic oxidative stress in the transgenic neurons, which exacerbates their susceptibility to additional insults such as KA-mediated excitotoxicity.     

Two proteins of the Dictyostelium spore coat bind to cellulose in vitro

The spore coat of Dictyostelium contains nine different proteins and cellulose. Interactions between protein and cellulose were investigated using an in vitro binding assay. Proteins extracted from coats with urea and 2-mercaptoethanol could, after removal of urea by gel filtration, efficiently bind to particles of cellulose (Avicel), but not Sephadex or Sepharose. Two proteins, SP85 and SP35, were enriched in the reconstitution, and they retained their cellulose binding activities after purification by ion exchange chromatography under denaturing conditions to suppress protein--protein interactions. Neither protein exhibited cellulase activity, though under certain conditions SP85 copurified with a cellulase activity which appeared after germination. Amino acid sequencing indicated that SP85 and SP35 are encoded by the previously described pspB and psvA genes. This was confirmed for SP85 by showing that natural M(r) polymorphisms correlated with changes in the number of tetrapeptide-encoding sequence repeats in pspB. Using PCR to reconstruct missing elements from the recombinogenic middle region of pspB, SP85 was shown to consist of three sequence domains separated by two groups of the tetrapeptide repeats. Expression of partial pspB cDNAs in Escherichia coli showed that cellulose-binding activity resided in the Cys-rich COOH-terminal domain of SP85. This cellulose-binding activity can explain SP85's ultrastructural colocalization with cellulose in vivo. Amino acid composition and antibody binding data showed that SP35 is derived from the Cys-rich N-terminal region of the previously described psvA protein. SP85 and SP35 may link other proteins to cellulose during coat assembly and germination.     

Temporal variation in hepatic superoxide dismutase activity in mice

Intestinal ischemia is a common clinical event and reperfusion results in further tissue damage exceeding that of ischemia alone. The present study was designed to test this and to assess the role of pentoxifylline, (administered intravenously as a bolus dose of 25 mg/kg in 1 ml normal saline, followed by continuous infusion of 0.2 mg/kg/minute for 95 minutes), in ischemia-reperfusion injury of the rat intestine. Intestinal ischemia was produced by occlusion of the superior mesenteric artery (SMA) with interruption of the collateral flow for 30 minutes. Reperfusion was established by declamping the (SMA) for 1 hour and evaluation of the mucosal damage was determined using a grading scale from 0 to 5, with estimation of mean mucosal thickness, villous height and crypt depth. The grade of mucosal damage, mucosal thickness, villous height and crypt depth were 2.2, 407 microns, 210 microns, and 196 microns respectively in the ischemia group, and 3.6, 327 microns, 156 microns, and 171 microns respectively in the ischemia reperfusion group, while these values in ischemia reperfusion with administration of pentoxifylline group were 2.5, 505 microns, 294 microns, and 200 microns respectively. The severity of the tissue injury increased considerably after reperfusion of the ischemic intestine and pentoxifylline was effective in attenuating the reperfusion injury significantly.     

Selegiline and lymphocyte superoxide dismutase activities in Parkinson''s disease

Health care: public, private or both? In Great Britain, about 13% of the population is covered by private health insurance, and everyone else is served by the public health care system known as the National Health Service, or NHS. Caroline Richmond, who examined the impact of private medical practice in Britain, says people become private patients for one compelling reason: to avoid the NHS's notoriously long waiting lists for surgery. According to Professor Alan Maynard, a health care researcher, the mainstays of the private sector are the "three h's" --hips, hernias and hemorrhoids-- along with some elective surgery, particularly in gynecology and opthalmology. Another small sector focuses on fertility regulation and cosmetic surgery. Although the levels are not monitored closely, physician consultants are not permitted to earn more than 10% of their income from private practice.     

Maternal administration of superoxide dismutase and catalase in phenytoin teratogenicity

Embryonic bioactivation and formation of reactive oxygen species (ROS) are implicated in the mechanism of phenytoin teratogenicity. This in vivo study in pregnant CD-1 mice evaluated whether maternal administration of the antioxidative enzymes superoxide dismutase (SOD) and/or catalase conjugated with polyethylene glycol (PEG) could reduce phenytoin teratogenicity. Initial studies showed that pretreatment with PEG-SOD alone (0.5-20 KU/kg i.p. 4 or 8 h before phenytoin) actually increased the teratogenicity of phenytoin (65 mg/kg i.p. on gestational days [GD] 11 and 12, or 12 and 13) (p < .05), and appeared to increase embryonic protein oxidation. Combined pretreatment with PEG-SOD and PEG-catalase (10 KU/kg 8 or 12 h before phenytoin) was not embryo-protective, nor was PEG-catalase alone, although PEG-catalase alone reduced phenytoin-initiated protein oxidation in maternal liver (p < .05). However, time-response studies with PEG-catalase (10 KU/kg) on GDs 11, or 11 and 12, showed maximal 50-100% increases in embryonic activity sustained for 8-24 h after maternal injection (p < .05), and dose-response studies (10-50 KU/kg) at 8 h showed maximal respective 4-fold and 2-fold increases in maternal and embryonic activities with a 50 KU/kg dose (p < .05). In controls, embryonic catalase activity was about 4% of that in maternal liver, although with catalase treatment, enhanced embryonic activity was about 2% of enhanced maternal activity (p < .05). PEG-catalase pretreatment (10-50 KU/kg 8 h before phenytoin) also produced a dose-dependent inhibition of phenytoin teratogenicity, with maximal decreases in fetal cleft palates, resorptions and postpartum lethality at a 50 KU/kg dose (p < .05). This is the first evidence that maternal administration of PEG-catalase can substantially enhance embryonic activity, and that in vivo phenytoin teratogenicity can be modulated by antioxidative enzymes. Both the SOD-mediated enhancement of phenytoin teratogenicity, and the inhibition of phenytoin teratogenicity by catalase, indicate a critical role for ROS in the teratologic mechanism, and the teratologic importance of antioxidative balance.