Response of chloride efflux from skeletal muscle ofRana pipiens to changes of temperature and membrane potential and diethylpyrocarbonate treatment

Summary Efflux of³⁶Cl^– from frog sartorius muscles equilibrated in two depolarizing solutions was measured. Cl^– efflux consists of a component present at low pH and a pH-dependent component which increases as external pH increases.For temperatures between 0 and 20°C, the measured activation energy is 7.5 kcal/mol for Cl^– efflux at pH 5 and 12.6 kcal/mol for the pH-dependent Cl^– efflux. The pH-dependent Cl^– efflux can be described by the relationu=1/(1+10n(pK _a -pH)), whereu is the Cl^– efflux increment obtained on stepping from pH 5 to the test pH, normalized with respect to the increment obtained on stepping from pH 5 to 8.5 or 9.0. For muscles equilibrated in solutions containing 150mm KCl plus 120mm NaCl (internal potential about –15 mV), the apparent pK _a is 6.5 at both 0 and 20°C, andn=2.5 for 0°C and 1.5 for 20°C. For muscles equilibrated in solutions containing 7.5mm KCl plus 120mm NaCl (internal potential about –65 mV), the apparent pK _a at 0°C is 6.9 andn is 1.5. The voltage dependence of the apparent pK _a suggests that the critical pH-sensitive moiety producing the pH-dependent Cl^– efflux is sensitive to the membrane electric field, while the insensitivity to temperature suggests that the apparent heat of ionization of this moiety is zero. The fact thatn is greater than 1 suggests that cooperativity between pH-sensitive moieties is involved in determining the Cl^– efflux increment on raising external pH.The histidine-modifying reagent diethylpyrocarbonate (DEPC) applied at pH 6 reduces the pH-dependent Cl^– efflux according to the relation, efflux=exp(–k·[DEPC]·t), wheret is the exposure time (min) to DEPC at a prepared initial concentration of [DEPC] (mm). At 17°C,k ^–1=188mm·min. For temperatures between 10 and 23°C,k has an apparent Q₁₀ of 2.5. The Cl^– efflux inhibitor SCN^– at a concentration of 20mm substantially retards the reduction of the pH-dependent Cl^– efflux by DEPC. The findings that the apparent pK _a is 6.5 in depolarized muscles, that DEPC eliminates the pH-dependent Cl^– efflux, and that this action is retarded by SCN^– supports the notion that protonation of histidine groups associated with Cl^– channels is the controlling reaction for the pH-dependent Cl^– efflux.     

The role of malic acid in the metabolism of Schizosaccharomyces pombe: substrate consumption and cell growth

Summary The effect of initial concentrations of malate varying from 0 to 28.6 g/l was studied. The acid was found to be inhibitory for growth of Schizosaccharomyces pombe but not for its deacidification activity. Malate was never integrated into biomass but partly transformed into ethanol if the aeration rate was weak (oxygen limitation). In the absence of glucose, resting cells of S. pombe were able to degrade malic acid if their concentration was sufficient, but their viability gradually decreased. However, for 0.15 g/l of growing cells (inoculum) 6 g/l of glucose was necessary to consume 8 g/l of malate. When the medium did not contain sugar no growth was observed despite the partial consumption of malate, showing that the acid was neither a carbon source nor an energy source. Offprint requests to: P. Strehaiano     

Study of properties of cholinephosphotransferase from fetal guinea pig lung mitochondria and microsomes

Summary We have reported earlier that cholinephosphotransferase (EC 2.7.8.2) is present in both mitochondria and microsomes of fetal guinea pig lung. This study was designed to compare the properties of mitochondrial and microsomal cholinephosphotransferase in fetal guinea pig lung. Various parameters, such as substrate specificity, K_m values, sensitivity to N-ethylmaleimide, dithiothreitol and trypsin were measured. Both showed significant preference for unsaturated diacylglycerols over saturated diacylglycerols. Data on K_m and V_max indicate that the affinity of this enzyme for different diacylglycerols varies between the two forms. The ID₅₀ values for N-ethylmaleimide were 20 mM and 12.5 mM for the mitochondrial and microsomal form of the enzyme, respectively. Dithiothreitol showed an inhibitory effect on both; however, the mitochondrial form was inhibited less than the microsomal form. The effects of N-ethylmaleimide and dithiothreitol on both forms of enzyme indicated that the microsomal cholinephosphotransferase requires a higher concentration of -SH for its activity than the mitochondrial enzyme does. The enzyme was inhibited by trypsin in both mitochondria and microsome under isotonic condition suggesting that this enzyme is on the outside of the membrane in both endoplasmic reticulum and mitochondria.     

Molecular and cellular mechanisms involved in the Trypanosoma cruzi/host cell interplay

The protozoan parasite Trypanosoma cruzi has a complex biological cycle that involves vertebrate and invertebrate hosts. In mammals, the infective trypomastigote form of this parasite can invade several cell types by exploiting phagocytic-like or nonphagocytic mechanisms depending on the class of cell involved. Morphological studies showed that when trypomastigotes contact macrophages, they induce the formation of plasma membrane protrusions that differ from the canonical phagocytosis that occurs in the case of noninfective epimastigotes. In contrast, when trypomastigotes infect epithelial or muscle cells, the cell surface is minimally modified, suggesting the induction of a different class of process. Lysosomal-dependent or -independent T. cruzi invasion of host cells are two different models that describe the molecular and cellular events activated during parasite entry into nonphagocytic cells. In this context, we have previously shown that induction of autophagy in host cells before infection favors T. cruzi invasion. Furthermore, we demonstrate that autophagosomes and the autophagosomal protein LC3 are recruited to the T. cruzi entry sites and that the newly formed T. cruzi parasitophorous vacuole has characteristics of an autophagolysosome. This review summarizes the current knowledge of the molecular and cellular mechanisms of T. cruzi invasion in nonphagocytic cells. Based on our findings, we propose a new model in which T. cruzi takes advantage of the upregulation of autophagy during starvation to increase its successful colonization of host cells.     

Promiscuous mating produces offspring with higher lifetime fitness

In many species, each female pairs with a single male for the purpose of rearing offspring, but may also engage in extra-pair copulations. Despite the prevalence of such promiscuity, whether and how multiple mating benefits females remains an open question. Multiple mating is typically thought to be favoured primarily through indirect benefits (i.e. heritable effects on the fitness of offspring). This prediction has been repeatedly tested in a variety of species, but the evidence has been equivocal, perhaps because such studies have focused on pre-reproductive survival rather than lifetime fitness of offspring. Here, we show that in a songbird, the dark-eyed junco (Junco hyemalis), both male and female offspring produced by extra-pair fertilizations have higher lifetime reproductive success than do offspring sired within the social pair. Furthermore, adult male offspring sired via extra-pair matings are more likely to sire extra-pair offspring (EPO) themselves, suggesting that fitness benefits to males accrue primarily through enhanced mating success. By contrast, female EPO benefited primarily through enhanced fecundity. Our results provide strong support for the hypothesis that the evolution of extra-pair mating by females is favoured by indirect benefits and shows that such benefits accrue much later in the offspring's life than previously documented.     

DESICCATION AND STARVATION TOLERANCE OF ADULT DROSOPHILA: OPPOSITE LATITUDINAL CLINES IN NATURAL POPULATIONS OF THREE DIFFERENT SPECIES

Desiccation and starvation tolerance were measured along latitudinal transects in three Drosophilid species (Drosophila ananassae, D. melanogaster, and Zaprionus indianus) of the Indian subcontinent. In each case, significant latitudinal clines were observed; desiccation tolerance increased with latitude while starvation tolerance decreased. Such field observations suggest that desiccation and starvation tolerance are fitness related traits that are independently selected in nature and genetically independent. It was, however, difficult to relate these genetic changes with precise climatic variables, except winter temperature. The overall negative correlation between the two traits, which was evidenced in natural populations, contrasts with a positive correlation generally observed in various laboratory selection experiments and that also seems to exist between different species. These observations point to the difficulty of interpreting correlations among fitness-related traits when different evolutionary levels are compared, and also different sets of data, that is, field versus laboratory studies.     

Development of an <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation method for <Emphasis Type="Italic">Taxus</Emphasis> suspension cultures

The FDA-approved anti-cancer compound paclitaxel is currently produced commercially by Taxus plant cell suspension cultures. One major limitation to the use of plant cell culture as a production platform is the low and variable product yields. Therefore, methods to increase and stabilize paclitaxel production are necessary to ensure product security, especially as the demand for paclitaxel continues to rise. Although a stable transformation method for Taxus suspension cultures has been developed, stable transformant yields are low (around 1% of experiments) and the method does not translate to the Taxus cuspidata Siebold and Zucc. and Taxus canadensis Marshall cell lines utilized in this study. Therefore, a new method for Agrobacterium-mediated transformation of Taxus callus and suspension cultures was developed through identification of the optimal Agrobacterium strain, inclusion of an anti-necrotic cocktail (silver nitrate, cysteine, and ascorbic acid) and increased recovery time for cells after cocultivation, the time following infection with Agrobacterium tumefaciens. Application of the increased recovery time to transformation of T. cuspidata line PO93XC resulted in 200 calluses staining positive for GUS. Additionally, two transgenic lines have been maintained with stable transgene expression for over 5 yr. This method represents an improvement over existing transformation methods for Taxus cultures and can be applied for future metabolic engineering efforts.     

Organellar DNA restriction fragment length polymorphism (RFLP) and nuclear random amplified polymorphic DNA (RAPD) analyses of morphotypes of Gracilaria (Gracilariales,Rhodophyta) from Chile

The extreme phenotypic variability recognized among the species of Gracilaria has highlighted the need for the application of refined methods to help solve taxa identifications. In Chile, there still exists uncertainty about the exact number of Gracilaria species. Our investigations are centered on DNA analyses of morphotypes collected from different geographical locations, namely Lenga and Isla Santa María, Region VIII (36°00 S to 38°00 S), and Maullín, Region X (39°30 S to 43°40 S). These two regions of Chile are considered as areas of confluence of G. chilensis, G. verrucosa, and a species of Gracilariopsis. In this study four morphotypes, from a natural bed located in Maullín, were analyzed for RFLP of plastid DNA and the results compared with data of four morphotypes from a bed in Lenga. The DNA banding patterns from each enzyme digest were identical irrespective of morphotypes and/or locations. In an attempt to unravel the nature of the morphological differences found among Lenga and Maullín morphotypes, RAPD analyses of nuclear DNA were also performed; however, no polymorphism has been found yet. Therefore, the data of this study, as well as concurrent data from preliminary interfertility tests, suggest that all morphotypes belong to a single taxon, Gracilaria chilensis.Departamento de Botánica, Facultad de Ciencias Naturales y Oceanográficas     

Physiographic and climatic events in the Chihuahuan Desert lead to the speciation and distinct demographic patterns of two sister Sceloporus lizards

The biogeographic history of the Chihuahuan Desert is known to be complex, and there is evidence of the effects of physiographic and climatic events in species diversification and demographic population changes in many taxa. Here, using DNA sequence data, we studied the influence of the physiographic and climatic events that occurred in the Chihuahuan Desert during the Pliocene–Pleistocene transition on the speciation and evolutionary history of the sister lizard species Sceloporus cyanostictus and S. gadsdeni. First, based on mtDNA and nDNA sequences, we estimated the divergence times of the sister species. Then, based on mtDNA sequences, we investigated the demographic history of both species within a phylogeographic framework. The divergence time was inferred to be 1.48 Mya, date that corresponds to the existence of a large lake in the Mapimian subprovince, between the current geographic locations of S. cyanostictus and S. gadsdeni. This lake could have acted as a barrier, leading to the speciation of both species. For the demographic history of the two species, we identified two distinct patterns: the population expansion of S. gadsdeni within the Last Glacial Maximum and the potential population decline of S. cyanostictus. Our results can be used as a guide for the study of other aspects that could be critical to developing conservation actions that ensure the survival of not only S. gadsdeni and S. cyanostictus, but also other co‐occurring lizard species.