Kenneth D. Munkres

Ageing of Neurospora crassa. I. Evidence for the free radical theory of ageing from studies of a natural-death mutant.

A recessive mutant of Neurospora crassa, called natural-death, is characterized by a decreasing clonal growth potential under all nutritional conditions and the irreversible cessation of growth. The primary molecular defect of this mutant is not known. Evidence presented here, based upon measurements of the activities and thermolabilities of several enzymes, suggests that faulty protein synthesis is probably not a cause of the senescence and death of the mutant, as suggested by Lewis and Holliday (Nature, 228 (1970) 877). Three lines of evidence indicate that lipid autoxidation and associated free radical reactions contribute to the senescence and death of this mutant: (1) The relative times before the onset of senescence and death of mutant clones in the last 40% of their chronological life-span were prolonged 2 to 3-fold by either dietary antioxidants or selenite and the total life-span was increased by 40% to 80%. These compounds also alleviated the senescent morphology and enhanced biomass production; (2) Senescing clones accumulated a green fluorescent pigment in situ, but dietary antioxidant nordihydroguaiaretic acid prevented this accumulation. The fluorescent pigment exhibited the spectral properties of lipofuscin, an end product of lipid autoxidation; (3) Relative to wild type, mycelial extracts of the mutant exhibited a 2 to 4-fold excess of activities of the antioxygenic enzymes superoxide dismutase, glutathione peroxidase and glutathione reductase. We briefly review: (1) the roles of antioxygenic enzymes and antioxidants in their protection against cellular damage from lipid autoxidation and free radical reactions; and (2) the major lines of evidence which appear to support a form of the free radical theory of ageing, encompassing the interrelated processes of membrane deterioration, lipid autoxidation and deleterious free radical reactions as the major causes of cellular deterioration.

Genetically determined conidial longevity is positively correlated with superoxide dismutase, catalase, glutathione peroxidase, cytochrome c peroxidase, and ascorbate free radical reductase activities in Neurospora crassa

Aging of post-mitotic cells, the conidia, of Neurospora crassa is defined as the time-dependent loss of viability under a constant laboratory environment which probably resembles the organisms tropical habitat; namely, at 30 degrees C, 85-100% relative humidity under white light. Median lifespan is defined as the age at which survival of a conidial population has declined to 50% of that of a fully viable population at birth. A collection of short (age-) and long-lived (age+) mutants were previously selected from the wild-type whose median lifespan is 22 days. Thus, five groups of strains with distinct lifespans of 7, 22, 36, 50 and 60 days were defined. The purposes of the present investigation were to determine if the activities of anti-oxygenic enzymes are correlated with lifespan and to elucidate the function of the cellular longevity determinant genes. The activities of superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) were highly-correlated with lifespan; whereas glutathione reductase and non-specific peroxidase activities were not correlated. The short-lived mutants were also deficient in cytochrome c peroxidase (CPX) and ascorbate free radical reductase (AFR), but not deficient in dehydroascorbate reductase. (These latter three enzymes were not examined in age+ mutants.) By isoelectric focusing analysis, the deficiencies of SOD, CAT, and GPX activities of age- mutants were defined in terms of specific isozymes. The mutants were specifically deficient in a cyanide-resistant mitochondrial isozyme of SOD. Sixteen age- genes, called the age-1 complex, were previously mapped on one arm of the seven chromosomes. On the basis of mapping and complementation data, it was inferred that the genes are spatially and functionally redundant. The hypothesis of functional redundancy is also supported by the enzyme data. Of seven mutants examined, representing seven of the age- genes, all were deficient in SOD, CAT and CPX, and six were deficient in AFR. Of four mutants examined, representing four of the genes, all were deficient in GPX. The results indicate a molecular basis for the previously observed photosensitivity of the mutants.(ABSTRACT TRUNCATED AT 400 WORDS)

Antioxidants prolong life span and inhibit the senescence- dependent accumulation of fluorescent pigment (lipofuscin) in clones of Podospora anserina☆

Culture of clones of Podospora anserina s+ with either nordihydroguaiaretic acid or reduced glutathione (GSH) at concentrations that were not inhibitory to growth significantly prolonged the average time to onset of senscence. GSH also prolonged the average time to onset of clonal death. The specific concentration of chloroform-methanol soluble fluorescent pigment was larger in senescent than in pre-senescent cells. The pigment exhibited fluorescence excitation and emission spectra and fluorescence polarization numbers characteristic of lipofuscin, an end-product of lipid peroxidation. Analyses of the lipofuscin concentration in either sub-clonal fractions of different times of origin from senescent clones, or in sub-clonal fractions of identical age in time of origin from parent clones of different age, revealed a similar concentrations distribution. Although pre-senscent cells contained rather large concentrations, a massive increase occurred during senescence prior to the time of onset of clonal death. Culture with GSH not only prolonged clonal life span but aslo inhibited the formation of lipofuscin by an average factor of 30. Furthermore, unlike untreated clones, the sub-clonal distribution of the pigment was not only low but was also independent of their age.

Preparation of mitochondrial membrane proteins fromNeurospora crassa: prevention of lipid autoxidation damage by an antioxidant

Abstract Lipid autoxidation products, such as malonaldehyde, react with proteins, cross-linking them and decreasing their solubility. These reactions are of practical importance in experiments with membranes where lipids and proteins are closely associated. When no precautions against lipid autoxidation were taken, both aged and freshly prepared mitochondrial membrane proteins fromNeurospora crassa contained 1-amino-3-iminopropene groups formed by reaction of protein amino groups with malonaldehyde. This conclusion was derived by analysis of fluorescence emission, fluorescence polarization, the effect of porohydride reduction upon the fluorescence, and qualitative and quantitative determination of malonaldehyde with 4,4′-sulfonyldianiline after hydrolysis of the proteins. The efficiency of antioxidants in the prevention of lipid autoxidation and consequent modification of protein was tested in a model system consisting of an aerated, aqueous solution of albumin, and methyl linolenate. The antioxidant, 3,5-ditert-butyl-4-hydroxybenzylalcohol, a sterically hindered phenol, appeared to be highly efficient in either the model system or in the isolation of membrane proteins. Mitochondrial membrane proteins, prepared in parallel procedure in the presence and absence of this antioxidant, differed in malonaldehyde concentration, iminopropene fluorescence and electrophoretic mobility. Several unusual properties of aged membrane proteins, such as low solubility, resistance to trypsin hydrolysis, and changes in isoelectric points and electrophoretic mobilities can be explained as consequences of lipid autoxidation processes. We suggest that antioxidants, such as sterically hindered phenols, be employed in the preparation and storage of proteins from membranes or other systems containing large amounts of lipids or unsaturated fatty acids in order to prevent artifactual modification of the proteins by lipid autoxidation products.

Cytoplasmic and mitochondrial malate dehydrogenases of Neurospora: Regulatory and enzymic properties

Abstract The metabolic regulation of Neurospora crass cytoplasmic malate dehydrogenase ( l -malate:NAD oxidoreductase, EC 1.1.1.37) is described. Synthesis of the cytoplasmic isozyme is subject to glucose repression whereas the mitochondrial forms are constitutive. Mycelia upon transfer from glucose to acetate as sole carbon source differentially synthesize cytoplasmic malate dehydrogenase, other enzymes of the glyoxylate cycle, and cytoplasmic aspartate aminotransferase ( l -aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1). The kinetics of cytoplasmic malate dehydrogenase synthesis indicate that the apparent increase in activity is 7-fold above the basal level of mitochondrial malate dehydrogenase activity and the absolute increase is several hundred-fold; maximal derepression occurs at the onset of growth on acetate; glucose repression after maximal derepression is not a rapid process; inhibition of derepression by antibiotics indicates that the isozymes are synthesized by cytoplasmic and not mitochondrial ribosomes; mineral starvation does not release glucose repression of either cytoplasmic malate dehydrogenase or aspartate aminotransferase but leads to gross cytological abnormalities. A purification procedure for the cytoplasmic and mitochondrial isozymes is described. The enzymes are demonstrated to differ in stability, ethanol solubility, isoelectric point, kinetics with substrate inhibition, coenzyme and substrate analogs, and inhibition by either low-molecular-weight compounds, antiserum, or mitochondrial structural protein. Differential association of the two isozymes of Neurospora and porcine heart with homologous structural protein is described.

Selection of conidial longevity mutants of Neurospora crassa

Conidial survival was measured after incubation in white light at 30 degrees C and 85-100% relative humidity. The heritable median lifespan (MLS) of the wild-type ( age0 ) was 22 days. Spontaneous short-lived mutants (age-) with MLS about 7 days occurred among both sexual and asexual progeny of wild-type at a frequency of about 10%. Radiation of conidia with near ultraviolet light increased the mutation frequency about 5-9-fold above spontaneous. The apparent spontaneous reversion frequency was about 0.1% or less. Four lines of evidence indicated that conditional defective conidiophorogenesis is a pleiotropic expression of the age- mutations. Of five age- mutants tested, all were photosensitive and remedial by vitamin E, exhibiting wild-type survival in darkness or after preculture with vitamin E. Selective breeding yielded variants (age+) with about 14-day increments of MLS at each of three generations beyond the F1. The segregation of either age- or age+ in crosses to age0 was Mendelian. A survey of mutants of 61 genes with other biochemical or morphological phenotypes indicated, as a conservative estimate, that 9 or 15% may exert a pleiotropic adverse effect on conidial longevity.

Ageing of Neurospora crassa. VIII. Lethality and mutagenicity of ferrous ions, ascorbic acid, and malondialdehyde

Ferrous ions were highly lethal and mutagenic to germinated conidia of Neurospora crassa. At comparable survival, treatment with 0.2 mM ferrous ions was 14- and 50-fold more mutagenic than ultra-violet irradiation or X-rays, respectively, in the reversion of an inositol auxotroph. Ascorbic acid alone (2 mM) was not reproducibly lethal and inhibited both the lethality and mutagenicity of ferrous ions. Bovine superoxide dismutase (SOD) completely inhibited the residual lethality of ferrous ascorbate. Protection by ascorbic acid and SOD indicates that superoxide radicals, generated by oxidation of Fe(II), are directly or indirectly mutagenic and lethal. Malondialdehyde (MDA) was lethal and appeared to be mutagenic; however, its action is probably different from that of superoxide. Therefore, superoxide-mediated production of endogenous MDA by way of peroxidation of polyunsaturated fatty acids is probably not an alternate mutagenic pathway, at least in the reversion of the allele of the inl locus examined. These results and the demonstration of superoxide-mediated decrease in the synthetic fidelity of DNA polymerase in vitro (Rana and Munkres, in preparation) warrant additional exploration of the hypothesis that endogenous cellular free radicals, generated by pre- and post-senescent metabolism, may enter into lethal and mutagenic reactions.

Ageing of Neurospora crassa. II. Organic hydroperoxide toxicity and the protective role of antioxidant and the antioxygenic enzymes.

Cumene hydroperoxide and tert-butyl hydroperoxide at sublethal concentrations initially prevent growth of mycelia of wild-type Neurospora crassa, but after a time the cells grow at a subnormal steady-state rate. The antioxidant nordihydroguaiaretic acid protects unadapted cells from hydroperoxide inhibition, leading to a decrease in the time before growth begins, an increase in steady-state growth rate and an increase in biomass production. The results of growth transfer experiments and enzyme measurements indicated that the acquired resistance to the hydroperoxides is physiological and most likely involves the induction of the synthesis of the antioxygenic enzymes superoxide dismutase, glutathione peroxidase and glutathione reductase. Nordihydroguaiaretic acid normalizes the levels of activities of glutathione peroxidase and glutathione reductase during culture with hydroperoxide. Molecular-induced homolysis of the hydroperoxides, a process that is induced by unsaturated fatty acids of membrane lipids, leads to lipid autoxidation in a chain reaction which produces lipid hydroperoxides, which in turn decomposes to form more free radicals. Nordihydroguaiaretic acid, a well-known free radical scavenger, probably serves to minimize hydroperoxide decomposition, lipid autoxidation and molecular damage from free radicals, whereas the coupled enzyme system glutathione peroxidase and glutathione reductase minimizes these processes by decomposing the hydroperoxides to harmless alcohols. We suggest that either free radicals derived from these processes or some consequent non-radical products may serve as the inducers of this enzyme system, rather than the hydroperoxide substrates.

Ageing of Neurospora crassa. V. Lipid peroxidation and decay of respiratory enzymes in an inositol auxotroph.

Abstract An inositol auxotroph of Neurospora crassa was grown in liquid culture with various inositol concentrations. Although the initial exponential growth rate was independent of the initial inositol concentration and equivalent to that of wild type, growth ceased prematurely with limiting concentrations. The period from the time of premature decline of growth rate until the time when cells begin to die at an exponential rate is defined as the senescence period. Comparisons of age-dependent changes in clones cultured with optimal and limiting inositol revealed that the latter accumulate more fluorescent pigment (lipofuscin) and that their mitochondria contained excessive concentrations of malondialdehyde, exhibited excessive rates of non-enzymatic lipid peroxidation in vitro and an age-dependent decline of specific activities of respiratory enzymes of the inner membrane. Cellular respiratory capacity was abnormal at an early age and deteriorated with increasing age. The in vitro rate of non-enzymatic lipid peroxidation was inversely correlated with the activities of the enzymes at various culture ages. Most of these abnormalities were evident in 9 h-old clones prior to the onset of senescence, becoming more severe during senescence. Culture with either nordihydroguaiaretic acid or hydrocortisone completely or partly prevented the occurrence of the biochemical abnormalities. Since these drugs also alleviate deterioration of clonal growth rates and cellular death, the observation that they protect mitochondria from the development of symptoms related to abnormal membranes and lipid peroxidation offers additional support to the hypothesis that their action is that of antioxidant and membrane stabilizer, respectively, and therefore, provides a molecular basis for their therapeutic role in vivo .

[24] Purification of exocellular superoxide dismutases

Publisher Summary This chapter discusses a technology used for conducting comparative exocellular forms of the enzyme extracellular superoxide dismutase (EC-SOD) therapy of the physiological diseases of the mutants. Human EC-SOD is a tetrameric glycoprotein, whose molecular weight is 135,000. The chapter describes purification procedures for selecting proteins of this nature. In SOD assay, the spectrophotometric method of Misra and Fridovich is used. The method is based on the inhibition by the enzyme of the spontaneous, alkali-catalyzed autoxidation of epinephrine to norepinephrine. In protein assay, the spectrophotometric method of Murphy and Kies is used. The course of dialysis to remove tetraborate is monitored by a colorimetric assay. A sample of the dialysate (0.9 ml) is mixed with 0.1 ml of 1% silver nitrate and incubated at room temperature for about 10 minutes or until the absorbancy at 500 nm becomes constant. The extinction coefficient of tetraborate is 0.255. The assay detects as little as 10–100 μ g sodium tetraborate per milliliter. The purification procedures are based on the assumption that all EC-SODs are large, highly glycosylated proteins. The literature on highly glycosylated proteins indicates that their properties resemble that of polysaccharides and sugars. Solubility and resistance to denaturation in organic solvents is one feature of highly glycosylated proteins exhibited by EC-SODs. They are soluble and remain active in 80–95% ethanol. The yeast enzyme is also soluble and fully active after extraction in 80% phenol. Furthermore, unlike ordinary protein, EC-SODs are soluble in aqueous saturated ammonium sulfate.