Encystation of Entamoeba invadens IP-1 is induced by lowering the osmotic pressure and depletion of nutrients from the medium

Trophozoites of Entamoeba invadens IP-1 can be induced to encyst in simple solutions composed of semipermeable constituents (buffer, salts, or sugars) provided that their osmotic pressure is in the range of 60-160 mosmol/kg. Optimal yield of mature cysts was obtained when the osmotic pressure of the medium was 110 mosmol/kg. Encystation could be obtained in the absence of serum although higher yields were obtained in its presence. No difference in the yield of mature cysts was found when either dialyzed or full serum was used. High yields of encystation were obtained (greater than 70%) in the presence of 5% serum in solutions of NaCl, KCl, or MgSO4, suggesting that the mechanism of encystation is not induced via sodium or potassium channels. Cysts were obtained in the presence of 72 mM glucose, indicating that depletion of a carbon source is not the only requirement for encystation. A rapid change in the density of the Entamoeba cells was observed upon transfer of trophozoites (density 1.061-1.073 g/ml) from growth medium to the low osmotic pressure encystation solutions. Within the first 2 min their density decreased (to 1.050 g/ml), but it soon increased, reaching within 30 min a density higher than 1.120 g/ml. As the encystation process continued to completion, the density of the cells gradually decreased, the mature cysts reaching a density of 1.049-1.061 g/ml.     

Na-NMR Studies of the Intracellular Sodium Ion Concentration in the Halotolerant Alga Dunaliella salina

The Intracellular Na⁺ concentration in the halotolerant alga Dunaliella salina was measured in intact cells by ²³Na-NMR spectroscopy, utilizing the dysprosium tripolyphosphate complex as a sodium shift reagent, and was found to be 88 ± 28 millimolar. Intracellular sodium ion content and intracellular volume were the same, within the experimental error, in cells adapted to grow in media containing between 0.1 and 4.0 molar NaCl. These values assume extracellular and intracellular NMR visibilities of the ²³Na nuclei of 100 and 40%, respectively. The relaxation rate of intracellular sodium was enhanced with increasing salinity of the growth medium, in parallel to the intracellular osmosity due to the presence of glycerol, indicating that Na⁺ ions and glycerol are codistribbuted within the cell volume.     

Partial Characterization of K and Ca Uptake Systems in the Halotolerant Alga Dunaliella salina

The uptake of K⁺ and Ca²⁺ in Dunaliella salina is mediated by two distinct carriers: a K⁺ carrier with a high selectivity against Na⁺, Li⁺, and choline⁺ but not towards Rb⁺, K⁺, Cs⁺, or NH₄⁺, and a Ca²⁺ carrier with a high selectivity against Mg²⁺. The latter is specifically blocked by La³⁺ and by Cd²⁺. Apparent K_m values for K⁺ and Ca²⁺ uptake are 2.5 and 0.8 millimolar, respectively, and their maximal calculated fluxes are 22 and 0.8 nanomoles per square meter per second, respectively. Effects of permeable ions and ionophores on K⁺ and Ca²⁺ uptake suggest that the driving force for their uptake is the transmembrane electrical potential. Inhibitors of ATP production, typical inhibitors of plasma membrane H⁺-ATPases and protonionophores inhibit K⁺ and Ca²⁺ uptake and accelerate K⁺ efflux. The results suggest that an H⁺-ATPase in the cell membrane provides the driving force for K⁺ and Ca²⁺ uptake. Efflux measurements from ⁸⁶Rb⁺ and ⁴⁵Ca²⁺ loaded cells suggest that part of the intracellular K⁺ and most of the intracellular Ca²⁺ is nonexchangeable with the extracellular pool. Correlations between phosphate and K⁺ contents and the effect of phosphate on K⁺ efflux suggest intracellular associations between K⁺ and polyphosphates. On the basis of these results, it is suggested that: (a) K⁺ and Ca²⁺ uptake in D. salina is driven by the transmembrane electrical potential which is generated by the action of an H⁺-ATPase of the plasma membrane. (b) Part of the intracellular K⁺ is associated with polyphosphate bodies, while most of the intracellular Ca²⁺ is accumulated in intracellular organelles in the algal cells.     

ATP-induced ΔpH formation in chloroplast ATP synthase proteoliposomes

Summary A procedure to reconstitute CF₀CF₁ proteoliposomes by gel filtration through a Sephadex-column pre-equilibrated with valinomycin and potassium is described. Proteoliposomes reconstituted by this procedure catalyze an ATP-induced pH of 2.5 to 3.5 units. pH was measured with either 9-aminoacridine or with the pH indicator pyranine trapped inside the proteoliposomes. CF₀CF₁ proteoliposomes prepared by conventional techniques catalyzed an ATP-induced formation, but were unable to catalyze an ATP-induced pH even in the presence of valinomycin.The ATP-induced pH was sensitive to uncouplers and energy transfer inhibitors and was increased at low temperatures. It is suggested that ATP-induced pH was observed in these proteoliposomes due to the efficient removal of intravesicular ammonium introduced with the CF₀CF₁ preparation. The ammonium acted as an internal buffer, and thus prevented an observable pH formation.     

Effect of Inhibitors on the Formation of Stereoisomers in the Biosynthesis of {beta}-Carotene in Dunaliella bardawil

An assay system was developed in which the effects of inhibitorsof ß-carotene biosynthesis in Dunaliella bardawilcould be tested. Since D. bardawil can be induced to accumulateover 10% of its dry weight as ß-carotene, it is particularlysuitable for such studies. Norflurazon a desaturation inhibitor,caused the accumulation of phytoene, or of phytoene and phytofluene,depending on the concentration employed. J-334, a substituted6-methylpyrimidine which also inhibits desaturation, causedthe accumulation of ß-zeacarotene, -carotene and phytoenein different proportions, depending on the concentration employed.The cyclization inhibitors, nicotine, CPTA and MPTA, severelyaffected the growth and survival of the alga, and their effectscould therefore not be studied directly. However, their actionwas observed indirectly by following the transformation of phytoenein norflurazon-pretreated phytoene-rich algae. Under these conditions,presence of the cyclase inhibitors caused the transformationof phytoene to lycopene, rather than to ß-carotene.The accumulated ß-carotene or the intermediates ß-zeacarotene,lycopene, -carotene, phytofluene and phytoene in D. bardawilwere all composed of two stereoisomers, tentatively assignedas the all-trans stereoisomer (55%) and the 9-cis stereoisomer(45%). This suggests that the isomerization reaction which leadsto the production of the presumed 9-cis isomers occurs earlyin the pathway of carotene biosynthesis, at or before phytoene,with no isomerization during the further transformations leadingto ß-carotene. (Received January 29, 1990; Accepted May 9, 1990)     

Are active oxygen species involved in induction of β-carotene in Dunaliella bardawil?

The purpose of this work was to test whether induction of massive -carotene synthesis in the alga Dunaliella bardawil is triggered by oxygen radicals. The following results were obtained: (i) The induction of -carotene synthesis is preceded by a lag period of about 4 h during which the cells swell and photosynthesis is partially inhibited, (ii) Addition of promoters of oxygen radicals or of azide (an inhibitor of catalase and superoxide dismutase) during the induction period, under conditions which are suboptimal for massive -carotene accumulation, greatly enhances -carotene synthesis, photodegradation of chlorophyll and inhibition of photosynthesis, (iii) High irradiance, which induces massive -carotene accumulation, also induces a high catalase activity. It is suggested that photosynthetically produced oxygen radicals are involved in triggering massive -carotene accumulation in D. bardawil.     

Dihydroxyacetone Kinase Activity in Dunaliella parva

An enzyme catalyzing the phosphorylation of dihydroxyacetone has been identified in the halophilic alga, Dunaliella parva. Since glycerol and glyceraldehyde are not substrates, the enzyme is referred to as dihydroxyacetone kinase. Dihydroxyacetone kinase was purified 9-fold by ammonium sulfate fractionation followed by DEAE-cellulose chromatography.     

Metabolic studies with NMR spectroscopy of the alga Dunaliella salina trapped within agarose beads

A technique for the entrapment of the unicellular algae Dunaliella salina in agarose beads and their perfusion during NMR measurements is presented. The trapped cells maintained their ability to proliferate under normal growth conditions, and remained viable and stable under steady-state conditions for long periods during NMR measurements. Following osmotic shock in the dark, prominent changes were observed in the intracellular level of ATP and polyphosphates, but little to no changes in the intracellular pH or orthoposphate content. When cells were subjected to hyperosmotic shock, the ATP level decreased. The content of NMR-visible polyphosphates decreased as well, presumably due to the production of longer, NMR-invisible structures. Following hypoosmotic shock, the ATP content increased and longer polyphosphates were broken down to shorter, more mobile polymers.     

Site of action of inhibitors of carbon dioxide assimilation by whole lettuce chloroplasts

The sites of action of several compounds, reported to inhibit CO(2) fixation by chloroplast preparations were located by developing assays in lettuce chloroplasts to test their effect on partial reactions of the carbon cycle and on carbonic anhydrase. The results indicated that: d, l-glyceral-dehyde and 5'-AMP inhibit phosphoribulose kinase or isomerase. 3-Phosphoglyceric acid and 6-phosphogluconate inhibit ribulose diphosphate carboxylase. Azide, Mg(2+), and nitrite inhibit the activity of carbonic anhydrase of lettuce chloroplasts and light-dependent CO(2) fixation by intact chloroplasts with similar sensitivities. None of these inhibited CO(2) fixation in ruptured chloroplasts. It is suggested that the inhibition by azide, nitrite, and magnesium ions of CO(2) fixation by intact chloroplasts is due to their inhibition of the activity of carbonic anhydrase.