Paul R. Saunders

Evidence that Glyceraldehyde‐3‐Phosphate Dehydrogenase Is Involved in Age‐Induced Apoptosis in Mature Cerebellar Neurons in Culture

Abstract: Under typical culture conditions, cerebellar granule cells die abruptly after 17 days in vitro. This burst of neuronal death involves ultrastructural changes and internucleosomal DNA fragmentations characteristic of apoptosis and is effectively arrested by pretreatment with actinomycin‐D and cycloheximide. The level of a 38‐kDa protein in the particulate fraction is markedly increased during age‐induced cell death and by pretreatment with NMDA, which potentiates this cell death. Conversely, the age‐induced increment of the 38‐kDa particulate protein is suppressed by actinomycin‐D and cycloheximide. N‐terminal microsequencing of the 38‐kDa protein revealed sequence identity with glyceraldehyde‐3‐phosphate dehydrogenase (GAPDH). A GAPDH antisense oligodeoxyribonucleotide blocks age‐induced expression of the particulate 38‐kDa protein and effectively inhibits neuronal apoptosis. In contrast, the corresponding sense oligonucleotide of GAPDH was completely ineffective in preventing the age‐induced neuronal death and the 38‐kDa protein overexpression. Moreover, the age‐induced expression of the 38‐kDa protein is preceded by a pronounced increase in the GAPDH mRNA level, which is abolished by actinomycin‐D, cycloheximide, or the GAPDH antisense, but not sense, oligonucleotide. Thus, our results suggest that overexpression of GAPDH in the particulate fraction has a direct role in age‐induced apoptosis of cerebellar neurons.

Purification and properties of the lethal fraction of the venom of the stonefish Synanceja horrida (Linnaeus).

Abstract The nature and some of the properties of the venom of the tropical stonefish Synanceja horrida (Linnaeus) have been investigated. The fraction lethal to mice was insoluble in deionized water, but was increasingly soluble with increasing ionic strength. The lethal fraction was most stable around neutrality, activity being lost at pH values or 5.6 or less and at pH 10.6. This fraction was heat-labile but could be stored for long periods in the frozen state. Starch-gel electrophoresis in phosphate buffer at pH 8 and ionic strength 0.076–0.1 usually revealed the presence of 7 or 8 components, although as many as 10 appeared with some samples. Material lethal to mice could be recovered from only one of these bands and the active fraction eluted from the gel was approx. twice as potent as the original venom. Re-electrophoresis of this active fraction gave only a single band.

Purification of the lethal fraction of the venom of the stonefish Synanceja horrida (Linnaeus)

Abstract The fraction of the venom of the stonefish Synanceja horrida (Linnaeus) lethal to mice was purified by several methods. The most effective procedure consisted of starch gel electrophoresis of crude venom, followed by recovery of the lethal fraction from one of the zones by a second electrophoresis into buffer solution. High recovery of lethal material which had been purified up to about ten times was obtained by this procedure. Purification was also accomplished by starch block electrophoresis. Ultracentrifugation led to the appearance of two major peaks, and the lethal material appeared only in the more rapidly sedimenting fraction.

Purification of the lethal fraction of the venom of the marine snail Conus californicus

Abstract Gel filtration of the venom of Conus californicus with Sephadex G-200 resulted in a five-fold increase in toxicity per weight of protein. The total activity recovered in the effluent fractions averaged 80 per cent of the initial actitity. Analysis of the purified venom by disc electrophoresis showed a pattern similar to that of the original extract and it was suggested that all of the protein components of the crude venom extract may not appear in the disc electrophoresis pattern. The absorption spectrum of the purified fraction had a maximum at 278 mμ, and the lethal component is thought to be a protein or bound to protein. The ld 50 and 95 per cent confidence limits of the venom in the experiments were calculated with a modification of the up-and-down method enabling the use of relatively small sizes.

Studies on the Venom of the Marine Snail Conus californicus.

Abstract Extracts prepared from the venom duct of the gastropod Conus californicus were lethal in mice following intravenous injection ( LD 50 approximately 2.4 mg protein/kg); symptoms included lethargy, ataxia and convulsions. No activity could be demonstrated in extracts of the venom bulb. The lethal fraction was non-dialyzable, destroyed by heating for five minutes at 100°C and could be lyophilized without loss of activity. Extracts at pH 8 were stable for about a day at 5°C and for weeks at −20°C. Extracts prepared in deionized water were considerably less potent than those prepared in 0.9% NaCl solution containing phosphate buffer at pH 8. In anesthetized rabbits small to moderate doses of venom produced hypotension, increased respiratory rate, bradycardia and with the larger doses changes in the T wave of the electrocardiogram. Lethal doses caused respiratory arrest following severe decline in blood pressure, but artificial respiration was ineffective in preventing death.

Positive inotropic action of ouabain on rat ventricle strips.

Summary A positive inotropic effect of ouabain has been demonstrated on the rat ventricle strip. At a given ouabain concentration the magnitude of the response was quite uniform. The effects of variation in the concentration of ouabain, calcium, and phosphate, and in initial tension on the positive inotropic response have been investigated.

Action of ouabain upon normal and hypodynamic myocardium.

Summary The action of ouabain upon force of contraction of guinea pig ventricular strips in phosphate and bicarbonate media was studied. A positive inotropic response occurred in both “fresh” and hypodynamic strips in phosphate medium, and also in bicarbonate medium.

Effect of Therapeutic and Toxic Concentrations of Ouabain Upon Potassium Content of Myocardium.

Summary Intracellular concentration and total amount of potassium in isolated guinea pig ventricle strips in the presence of both therapeutic and toxic concentrations of ouabain have been investigated. A concentration of the drug which produced an increase in the force of contraction without evidence of toxicity did not alter either intracellular concentration or total amount of this ion in the heart. A toxic concentration of ouabain which produced automaticity and a marked increase in the resting tension, followed by decline in force of contraction to zero, caused a mean decrease of 21% in intracellular potassium concentration.

Comparative effects of ouabain upon contractile force of guinea pig diaphragm and heart.

Summary Ouabain increased the force of contraction of isolated guinea pig diaphragm, stimulated directly or indirectly with supramaximal shocks. At higher concentrations of the drugs, the positive inotropic action was followed by a depressant effect. The concentrations at which these effects occurred were similar to those which produced an increase in force of contraction or toxic effects upon isolated guinea pig ventricular strips. Skeletal muscular paralysis in intact guinea pigs following the administration of ouabain was frequently not accompanied by evidence of cardiac toxicity. The significance of these findings in relation to studies on the mechanism of action of the cardiac glycosides which employ skeletal muscle preparations was discussed.

Energy Sources for Contraction of the Rat Ventricle in Phosphate Media

Various substrates were tested for their ability to restore the amplitude of contraction of hypodynamic electrically-stimulated rat ventricle strips suspended in a phosphate-buffered medium. Greatest recovery of the contractile activity was obtained with pyruvate; lactate, β-hydroxybutyrate and acetate were more effective than succinate and glucose. The finding that glucose, in contrast to pyruvate, was relatively ineffective as an energy source for contraction, when a phosphate-buffered medium was employed, suggests that the defect was due to a rate-limiting step in the conversion of glucose to pyruvate, rather than to an impairment in the functioning of the Krebs cycle.