In Vitro Culture of Phytomonas Sp. Isolated from Euphorbia characias. Metabolic Studies by 1H NMR

ABSTRACT. We describe the in vitro culture of Phytomonas species isolated from Euphorbia characias . The best choice between tested media was SDM-79, in which promastigotes, after 6 days of culture, reached cell densities as high as 4 × 10⁷ cells/ml. Cells growing in LIT or MTL medium showed longer division times and lower cell densities. We succeeded in obtaining Phytomonas sp. amastigote and spheromastigote forms in modified GRACE's medium, yielding transformation rates of up to 70%. Electron microscopy studies were performed in order to characterize the ultrastructural features of these forms obtained in vitro. On the other hand, metabolic studies based on qualitative (nuclear magnetic resonance spectroscopy) and quantitative metabolic methods (enzymatic assays) showed that promastigote forms secreted mainly ethanol, acetate, glycine, glycerol, piruvate and succinate in SDM-79 medium, whereas the major metabolites found after transformation in modified Grace's medium were ethanol, acetate, glycine, piruvate and smaller amounts of glycerol.     

The Different Behavior of Diphtheria Toxin, Modeccin and Ricin in HeLa Cells Infected with Trypanosoma cruzi

ABSTRACT. We have studied the action of diphtheria toxin, modeccin and ricin on HeLa cells infected by Trypanosoma cruzi . Parasitized HeLa cells were resistant to diphtheria toxin and modeccin, whereas non-parasitized cells from the same cultures and control cultures showed cytopathological alterations. Protein synthesis, assayed by the incorporation of labelled methionine, diminished in toxin-treated control cultures but remained unaltered in the infected ones, compared to synthesis by untreated infected cells. Ricin, on the other hand, is a toxin that enters the cytoplasm by endocytosis. It has greater cytopathological effects in parasitized cells than in non-parasitized ones from the same cultures or uninfected control cells. Protein synthesis was inhibited in infected cultures treated with ricin.     

Species-specific phytoplankton responses to nutrients and zooplankton manipulations in enclosure experiments

1. In situ enclosure experiments were performed in the mesotrophic Bermejales reservoir to evaluate the algal response to changes in the nutrient supply and in the zooplankton size structure and density in a 2 × 2 factorial design. The experiments were conducted during the spring bloom of nanoplanktonic diatoms in 1989. 2. Nutrient enrichment promoted a great increase of phytoplankton biomass indicating a strong nutrient limitation on phytoplankton growth. Total phytoplankton biomass was significantly lower in the Daphina-added enclosures at a given nutrient level and strong direct an indirect effect of zooplankton on phytoplankton community structure and nutrient availability were observed. 3. Most of the nanoplanktonic species were effectively grazed but species with protective coverings and large size colonies were favoured by grazers and small chlorococcales were unaffected probably because of their compensatory high growth rates. The decrease in total biomass imposed by grazers is attributable mainly to the decrease of Cyclotella ocellata, the most abundant species. This taxon suffers two net effects of zooplankton: direct grazing and the indirect decrease of Si availability caused by the growth of C. ocellata which was promoted by P excretion by zooplankton. Indirect effects of grazers on Si availability should, therefore, be taken into account in explaining phytoplankton succession and community structure. 4. In this experiment grazers affected considerably the nanoplanktonic community in Bermejales reservoir. The extent which they were affected, however, depended not only on the algal size as a determinant of edibility but also greatly on the specific nutrient requirements and taxonomic features of the algal species.     

Nickel and Cadmium-related Changes in Growth, Plasma Membrane Lipid Composition, ATPase Hydrolytic Activity and Proton-pumping of Rice (Oryza sativa L. cv. Bahia) Shoots

Six-day-old rice plants (Oryza sativa L. cv. Bahia) were grownin the presence of 0.5 mol m^–3 Ni or Cd for 5 or 10 d.Five days after treatment, some plants were transferred to amedium containing no heavy metal for 5 or 10 d. Plasmalemmavesicles from shoots of treated, transferred (recovery experiments)and control plants were isolated, ATPase activity and proton-pumpingwere assessed, and free sterols and phospholipids determined.The ATPase hydrolytic activity was increased by 37% and 34%in 5 and 10 d Cd-treated plants, respectively; and by 66% in5 and 10 d Ni-treated plants. However, neither the initial rateof H⁺ transport nor the proton-pumping rate at steady-statewere significantly affected by the treatments. The relativeproportion (%) of the plasmalemma sterols campesterol and ⁵-avenasteroldecreased while sitosterol increased during heavy metal treatment.The overall plasmalemma phospholipid fatty acyl chain lengthand degree of unsaturation were also reduced. In experimentswhere plants recovered from Cd and Ni treatment, differencesbetween treated and control plants were reverted, particularlyin 10 d Ni-recovered plants. The possible involvement of lipidsin the regulation of the plasmalemma ATPase as well as the relationshipbetween growth, ATPase and adaptations to stress conditionsare discussed. Key words: Cadmium, nickel, sterols, phospholipids, ATPase     

Energy storage during the transition from endogenous to exogenous feeding in Australian redclaw crayfish Cherax quadricarinatus (Von Martens, 1898)

Summary

The Australian redclaw crayfish Cherax quadricarinatus hatches at an advanced stage. Two post-hatching stages rely on yolk reserves before the organism becomes a juvenile. After a juvenile appears, the yolk is depleted and young crayfish might spend a few days among female pleopods before starting to feed independently. During this phase, the juvenile organism needs alternative energy sources. Offspring from three females were analyzed with histological and histochemical techniques, from hatching until reaching the 2-week juvenile stage, to trace digestive system development and internal organ lipid and glycogen accumulation. Neutral lipids are stored in specialized cells of the hepatopancreatic tissue until the juvenile stage is reached. An increasing rate of accumulation was found. Glycogen was stored in hepatopancreas and tail muscle cells until post-embryo II, and no increasing or decreasing rate was found in storage functions.     

The K+/H+ antiporter LeNHX2 increases salt tolerance by improving K+ homeostasis in transgenic tomato

The endosomal LeNHX2 ion transporter exchanges H⁺ with K⁺ and, to lesser extent, Na⁺. Here, we investigated the response to NaCl supply and K⁺ deprivation in transgenic tomato (Solanum lycopersicum L.) overexpressing LeNHX2 and show that transformed tomato plants grew better in saline conditions than untransformed controls, whereas in the absence of K⁺ the opposite was found. Analysis of mineral composition showed a higher K⁺ content in roots, shoots and xylem sap of transgenic plants and no differences in Na⁺ content between transgenic and untransformed plants grown either in the presence or the absence of 120 mm NaCl. Transgenic plants showed higher Na⁺/H⁺ and, above all, K⁺/H⁺ transport activity in root intracellular membrane vesicles. Under K⁺ limiting conditions, transgenic plants enhanced root expression of the high‐affinity K⁺ uptake system HAK5 compared to untransformed controls. Furthermore, tomato overexpressing LeNHX2 showed twofold higher K⁺ depletion rates and half cytosolic K⁺ activity than untransformed controls. Under NaCl stress, transgenic plants showed higher uptake velocity for K⁺ and lower cytosolic K⁺ activity than untransformed plants. These results indicate the fundamental role of K⁺ homeostasis in the better performance of LeNHX2 overexpressing tomato under NaCl stress.     

Regulation by light of ammonium transport systems in Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii is able to take up methylammonium/ammonium from the medium at different stages of its sexual life cycle. Vegetative cells and pre‐gametes mostly used a low‐affinity system (LATS) component, but gametes obtained after light treatment of N‐deprived pre‐gametes expressed both LATS and high‐affinity system (HATS) components for the uptake of methylammonium/ammonium. The activity of the LATS component was stimulated by light in only 5 min in a process independent of protein synthesis. By using the lrg6 mutant that produces sexually competent gametes in the dark, light effects on ammonium transport and gamete differentiation have been separately analysed. We have found light regulation of four Amt1 genes: Amt1; 1, Amt1; 2, Amt1; 4 and Amt1; 5. Whereas light‐dependent expression of Amt1; 1, Amt1; 2 and Amt1; 4 was independent of gametogenesis, and that of Amt1; 5 was activated in the lrg6 mutant, suggesting a connection between this transporter and the subsequent events taking place during gametogenesis.     

Intraspecific genome size variation and morphological differentiation of Ranunculus parnassifolius (Ranunculaceae), an Alpine–Pyrenean–Cantabrian polyploid group

The aim of this study was to assess genome size variation and multivariate morphometric analyses to ascertain cytotype distribution patterns and the morphological differentiation within the Ranunculus parnassifolius group in the Pyrenees and the Alps. Although divergences in nuclear DNA content among different species within a genus are widely acknowledged, intraspecific variation is still a somewhat controversial issue. Holoploid and monoploid genome sizes (C‐ and Cx‐values) were determined using propidium iodide flow cytometry in 125 plants of R. parnassifolius s.l. distributed across four European countries. Three different DNA ploidy levels were revealed in the study area: diploid (2n ～ 2x, 57.14%), triploid (2n ～ 3x, 1.19%), and tetraploid (2n ～ 4x, 41.67%). The mean population 2C‐values ranged from 8.15 pg in diploids to 14.80 pg in tetraploids, representing a ratio of 1 : 1.8. Marked intraspecific/interpopulation differences in nuclear DNA content were found. Diploid populations prevail in the Pyrenees, although tetraploid cytotypes were reported throughout the distribution area. In general, mixed‐cytotype populations were not found. The Spearman correlation coefficient did not reveal significant correlations between genome size and altitude, longitude, or latitude. Morphometric analyses and cluster analyses based on genome size variation revealed the presence of three major groups, which exhibited a particular biogeographical pattern. A new cytotype, DNA triploid, was found for the first time. Tetraploid populations showed constant nuclear DNA levels, whereas diploid populations from the Pyrenees, in which introgressive hybridization is suggested as a presumable trigger for genome size variation, did not. Scenarios for the evolution of geographical parthenogenesis in R. parnassifolius s.l. are discussed. Finally, the different levels of effectiveness between plant and animal reference standards are analysed. © 2010 The Linnean Society of London, Biological Journal of the Linnean Society, 2010, 101 , 251–271.     

Ecological rather than geographical isolation dominates Quaternary formation of Mediterranean Cistus species

The lack of a comparative approach makes it impossible to determine the main factors influencing colonization and evolution in plants. Here we conducted the first comparative study of a characteristic Mediterranean lineage (white‐flowered Cistus) taking advantage of its well‐known phylogenetic relationships. A two‐scale approach was applied to address the hypothesis of higher levels of isolation in mountain than in lowland species. First, a time‐calibrated phylogeny using plastid sequences of Cistaceae suggested that the origin of Cistus species postdated both the refilling of the Mediterranean Sea (5.59–5.33 Ma) and the onset of the Mediterranean climate (3.2 Ma). Two hundred and sixty‐three additional, plastid sequences from 111 populations showed different numbers of haplotypes in C. laurifolius (7), C. monspeliensis (2) and C. salviifolius (7). Although haplotype sharing among disjunct populations was observed in all species, phylogeographic analyses revealed haplotype lineages exclusive to Europe or Africa only in the mountain species (C. laurifolius). Isolation by either geographical distance or sea barriers was not significantly supported for the lowland species (C. monspeliensis; C. ladanifer from a previous study). The same is true for the less habitat‐specific species of the lineage (C. salviifolius). Comparative phylogeography of the Cistus species leads us to interpret a general pattern of active colonization surpassing Mediterranean barriers. In contrast, ecological conditions (precipitation, temperature, soils) appear to have determined the distribution of the Cistus species of Mediterranean mountains. This study further provides molecular evidence for multiple colonization patterns in the course of successful adaptation of Cistus species to Mediterranean habitats.