Calcium efflux from sarcoplasmic reticulum vesicles

Ca²⁺ efflux was studied in sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle. In experimental conditions in which the Ca²⁺ pump is reversed, the rate of Ca^2? efflux varies with the ADP, orthophosphate and Mg²⁺ concentrations of the assay medium and is inhibited by Na⁺.     

Effect of compound 48/80 and ruthenium red on the Ca2+-ATPase of sarcoplasmic reticulum

The effects of the condensation product of N-methyl-p-methoxyphenethylamine with formaldehyde (compound 48/80) and ruthenium red on the partial reactions of the catalytic cycle of the sarcoplasmic reticulum Ca2+-ATPase of skeletal muscle were studied. The ATPase activity and both Ca2+ and Sr2+ uptake were inhibited by compound 48/80 when oxalate was used as a precipitating agent. The degree of inhibition decreased when oxalate was replaced by orthophosphate as the precipitating anion. Both the fast Ca2+ efflux and the synthesis of ATP observed during reversal of the Ca2+ pump were inhibited by compound 48/80. Inhibition of the reversal of the Ca2+ pump was caused by a competition between compound 48/80 and orthophosphate for the phosphorylation site of the enzyme. The fast Ca2+ release promoted by arsenate was impaired by compound 48/80. Ruthenium red competes with Ca2+ for the high affinity binding site of the Ca2+-ATPase, but did not interfere with the binding of Ca2+ to the low affinity binding site of the enzyme. In presence of Ca2+ concentrations higher than 5 microM, ruthenium red in concentrations up to 200 microM had no effect on both ATPase activity and Ca2+ uptake. However, the fast Ca2+ efflux promoted by arsenate and the fast Ca2+ efflux coupled with the synthesis of ATP observed during the reversal of the Ca2+ pump were inhibited by ruthenium red, half-maximal inhibition being attained in presence of 10-20 microM ruthenium red. In contrast to the effect of compound 48/80, ruthenium red did not inhibit the phosphorylation of the enzyme by orthophosphate. The ATP in equilibrium with Pi exchange catalyzed by the Ca2+-ATPase in the absence of transmembrane Ca2+ gradient was also inhibited by ruthenium red.     

Ultrastructural observations on the infection of rat liver by Plasmodium berghei sporozoites in vivo

The invasion of liver parenchymal cells by sporozoites of Plasmodium berghei Vincke & Lips, 1948, was studied in vivo using transmission electron microscopy. Livers of Brown Norway rats were examined 30 and 60 min after intraportal injection of 15 million sporozoites each. Sporozoites found after incorporation into vacuoles in hepatocytes were often located near a bile canaliculus at the lateral cell surface, surrounded by hepatocyte lysosomal structures; however, degradation of sporozoites caused by lysosomal digestion inside hepatocytes was never observed. Due to the crescent shape of sporozoites, serial sections were necessary to demonstrate the actual process of invasion of the hepatocyte. The hepatocyte's plasmalemma appeared to invaginate due to the sporozoite's action, thereby creating a parasitophorous vacuole. It was suggested that the sporozoite actively penetrated the hepatocyte; however, no visible depletion of rhoptries and micronemes was observed.     

Pathway for ATP synthesis by sarcoplasmic reticulum ATPase

Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by P_i of the Ca²⁺-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca²⁺ prior to the addition of P_i and Mg²⁺ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca²⁺ prior to the addition to the reaction mixture of P_i, Mg²⁺, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca²⁺ or, in the absence of Ca²⁺, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive P_i. In the presence of Ca²⁺, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca²⁺. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca²⁺-dependent ATPase were used. A transient phosphorylation by P_i was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, Mg²⁺, and excess of Ca²⁺. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by P_i was observed when the enzyme was preincubated with Ca²⁺. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, ADP, Mg²⁺, and 20 mm CaCl₂. This was not observed when the enzyme was preincubated in the presence of Ca²⁺.     

Effect of ADP on the rate of acetyl phosphate hydrolysis by the Ca2+-ATPase of sarcoplasmic reticulum

Hydrolysis of acetyl phosphate is inhibited by high concentrations of Pi and MgCl2, probably due to an increase in the steady-state level of phosphoenzyme formed from Pi in the medium. A dual effect of ADP during steady-state hydrolysis of acetyl phosphate was observed. ADP inhibited hydrolysis in the presence of 5 mM MgCl2 and no added Pi, whereas it stimulated hydrolysis when phosphoenzyme formation by Pi was favored by including 6 mM Pi and 20 mM MgCl2 in the assay medium. ATP inhibited acetyl phosphate hydrolysis in both of these assay media. When phosphoenzyme formation by Pi in the presence of acetyl phosphate was stimulated at Ca2+ concentrations sufficient to saturate the low-affinity Ca2+-binding sites, ADP stimulated acetyl phosphate hydrolysis and also promoted ATP synthesis by reversal of the catalytic cycle. The rate of ATP synthesis was dependent on ADP, Pi and Ca2+. Phosphoenzyme formation by Pi and MgCl2, whether in the absence of Ca2+ and acetyl phosphate, or during acetyl phosphate hydrolysis, was inhibited by ADP and ATP. These results suggest that ADP interacts with different intermediates of the catalytic cycle and that expression of inhibition or activation of acetyl phosphate hydrolysis depends on the steady-state level of phosphoenzyme formed by Pi.     

Plasma cortisol response to feeding in African green vervets

The timing of a single daily meal of monkey chow in African green vervets exerts a synchronizing influence on plasma cortisol concentrations with an initial increase after feeding followed by a decrease to minimal levels five hours after feeding.     

Detection of different developmental stages of malaria parasites by non-radioactive DNAin situ hybridization

Summary A highly sensitive non-radioactive DNAin situ hybridization procedure is described that enables detection and unequivocal identification of various developmental stages of human and rodent malaria parasites. Using biotinylated species-specific DNA probes, erythrocytic parasites can be specifically stained in blood smears. Similarly exoerythrocytic stages can be visualized in cell culture and in sections of paraffin-embedded liver. In blood smears, the hybridization procedure provides a rapid detection of (low) parasitemia and species-determination for experienced microscopists at 100 to 400x magnification. Moreover, the procedure can be applied even after previous Giemsa staining of the preparation, enabling revision of patient smears which were difficult to read after routine Giemsa staining.     

Fine structure of exoerythrocytic merozoite formation of Plasmodium berghei in rat liver

The fine structure of exoerythrocytic merogony of Plasmodium berghei was studied after perfusion-fixation of rat livers from 51 h post-inoculation onwards. Meroblast formation was effected by clefts originating from the parasite plasmalemma and by fusion of vacuoles with each other. Invaginations at the periphery resulted in labyrinthine structures providing the parasites with an enormous increase in surface area, which might facilitate exchange of metabolites. When the parasitophorous vacuole membrane collapsed, the newly formed merozoites were lying free in the hepatocytic cytoplasm, which degenerated until the merozoites were sticking together by a stroma, obviously a remnant of the host hepatocyte. Groups of merozoites, still kept together by the spongy stroma, were subsequently released in the bloodstream. At 53 h most of the developmental stages leading to the release of merozoites could be found and thereafter parasite numbers decreased while large granulomas became apparent.