Claude Nations

Oxygen free radicals play a role in cellular differentiation: An hypothesis

Evidence from a variety of sources supports the view that oxygen free radicals play a role in cellular differentiation. It is postulated that cellular differentiation is accompanied by changes in the redox state of cells. Differentiated cells have a relatively more prooxidizing or less reducing intracellular environment than the undifferentiated or dedifferentiated cells. Changes in the redox balance during differentiation appear to be due to an increase in the rate of O2- generation. Differentiated cells, in general, exhibit higher rates of cyanide-resistant respiration, cyanide-insensitive SOD activity, and peroxide concentration and lower levels of GSH as compared to undifferentiated cells. The effects of free radicals on cellular differentiation may be mediated by the consequent changes in ionic composition.

Superoxide dismutase induces differentiation in microplasmodia of the slime mold Physarum polycephalum

Evidence is presented that supports a role for the enzyme superoxide dismutase (SOD) in the differentiation of the slime mold, Physarum polycephalum. SOD activity increases 46-fold during differentiation. A strain of Physarum that does not differentiate exhibits no change in SOD activity. Addition of SOD, via liposomes, to the nondifferentiating strain induces differentiation; this effect is enhanced by an inhibitor of glutathione synthesis. Other antioxidants selected for study failed to induce differentiation. Conversely, oxidative treatments including introduction of D-amino acid oxidase, via liposomes, induced differentiation. Cellular oxidation is the probable cause of the SOD effect.

Superoxide dismutase activity during the plasmodial life cycle ofPhysarum polycephalum

Superoxide dismutase activity was slow throughout the cell cycle of surface cultures ofPhysarum polycephalum. This activity increased markedly when the organism was induced to spherulate. Glutathione (GSH) and hydrogen peroxide (H2O2) concentrations changed very little during the cell cycle. During spherulation GSH decreased; H2O2 and the cyanide-resistant respiration of plasmodial homogenates increased.

Electron microscopic and microprobe analysis of calciuminduced differentiation of the white mutant (LU887 × LU897) strain of Physarum polycephalum

Differentiation of the white mutant (LU887 x LU897) strain of Physarum polycephalum leading to spherule formation can be induced by CaCl(2) if the concentration in the nutrient medium is increased by 5mM prior to the transfer to a non-nutrient salts medium. All stages previously reported for the typical (M(3)cVII) strain of Physarum polycephalum from microplasmodia to spherules are seen but the mutant lacks the synchrony that the replacement technique induces in the typical strain. X-ray microanalyses locate calcium and phosphorus in granules in mitochondria and in the cytoplasm of specimens fixed without osmium. Mitochondria accumulate calcium-containing granules during early differentiation and appear to be essentially without granules in mature spherules. Mobilization of mitochondrial calcium is implicated in the initiation of differentiation. A longitudinally striated cytoplasmic inclusion is abundant in microplasmodia grown in media that have not been supplemented with additional calcium and is seen more rarely during calcium-induced spherulation. Whether or not this inclusion represents cytoplasmic contractile elements is unknown. The calcium-treated mutant strain, previously considered non-differentiating, may prove to be a good alternate model for the study of factors influencing differentiation. It was employed earlier as a control in studies of strains that readily spherulate in response to routine procedures.

Activity of some dehydrogenase enzymes in mitochondria from Physarum polycephalum.

1. Coupled mitochondria were isolated from exponentially growing Physarum polycephalum. 2. Activity of malate dehydrogenase (oxalacetate reduction) was 10.9 mumol/min/mg protein; the apparent Km was 64 microM. 3. The activity of NADP-isocitric dehydrogenase (IDH) was 110 nmol/min/mg with apparent Km of 35 microM. 4. NAD-IDH showed allosteric properties with AMP as a positive modulator. The apparent Km for the unmodulated activity, 2 mM, was decreased to 0.95 mM by 0.13 mM AMP. 5. Succinic dehydrogenase activity was estimated as three times higher than that of alpha-glycerophosphate dehydrogenase. 6. Mitochondria contained significant amounts of phenolic compounds. Protein estimation by the Bradford method is recommended.

Growth of white microplasmodia of Physarum polycephalum

Abstract 1. 1. Both yellow (M3c VII) and white (LU887 × LU897) microplasmodia of Physarum polycephalum follow a sigmoidal growth pattern; the yellow strain proliferates more rapidly. 2. 2. The white strain exhibits a rapid increase in wet: dry weight ratio, probably accounted for by glycoprotein slime generation. 3. 3. The yellow strain accumulates protein more rapidly than the white; a difference in the pattern of energy expenditure for the two strains is suggested. 4. 4. The glucose content of the culture media decreases linearly from 90 mg/ml to zero, the pH changes from 4.6 to greater than 5.3 and lactic acid remains approximately constant after 6 days of culture.

Inhibition of glutamate decarboxylase in extracts of the slime mold physarum polycephalum

Abstract Ammonium sulfate fractions of supernatants from Physarum polycephalum browned rapidly at room temp. This browning was accompanied by complete inactivation of glutamate decarboxylase. Fractions exhibiting low activity and poor yields were obtained by decreasing plasmodium-buffer ratios of homogenates. Total activity of these fractions was increased by gel-filtration with Sephadex G-150. Samples of a crude slime mold pigment preparation inhibited supernatant activity by 42%. Since the enzyme was being inactivated either by phenols or their oxidation products, EDTA, cysteine, and Polyclar AT were added to extracting media to increase enzyme recovery. With their aid the enzyme was purified more than 15-fold by ammonium sulfate fractionation and gel-filtration with Sephadex G-200.

Electron microscopic observations of an unusual cytoplasmic inclusion in the calcium-starved white mutant (LU887 × LU897) of Physarum polycephalum

Abstract Electron micrographs of Physarum polycephalum microplasmodia (LU887 × LU897) reveal cytoplasmic inclusions that appear “striated” at low magnifications; at higher magnifications these exhibit a structure that we have interpreted as microtubule bundles. The light and dark regions in the inclusions are due to the affinity of some microtubules for osmic acid; these appear to have dense regions while other microtubules remain electron lucent. The diameters of the microtubules are about 32–33nm; the subunits forming the tubule walls measure about 8–9nm in diameter. The diameter measurements are slightly larger than the dimensions assigned to vertebrate microtubules (28nm); however, the diameter of the subunits in the microtubule wall measures about 8–9nm which is essentially the same measurement reported for vertebrate tubulin dimers.

Developmental changes in protein generation by cultured porcine granulosa cells

1. The gross complement of proteins from porcine ovarian follicular fluid does not change as the follicles increase in size. 2. The electrophoretic profile of proteins from porcine follicular fluid is strikingly different from that of plasma proteins from the same animals. 3. Cultured granulosa cells from different sized follicles synthesize and release L-leucine-[3H]labeled proteins at different rates. 4. Cultured porcine granulosa cells appear capable of synthesizing most follicular fluid proteins.