Amino acid sequence of toxin VII,A β-toxin from the venom of the scorpton Tityus serrulatus

The sequence of the 61 amino acids of toxin VII, a β-toxin from the venom of the South American scorpion Tityus serrulatus, has been determined by automatic sequencing of the reduced and S-[¹⁴C] car?ymethylated protein and of tryptic peptides obtained before or after citraconylation of this protein. This toxin, the most active β-toxin from this venom, is the first Tityus toxin to be fully sequenced. The results clearly show that toxin VII belongs to the structural group of scorpion toxins originating from Central and North America.     

Effect of N,N-Dicyclohexylcarbodiimide on Acetylcholine Release from Torpedo Synaptosomes and Proteoliposomes Reconstituted with the Proteolipid Mediatophore

The mediatophore is a presynaptic membrane protein that has been shown to translocate acetylcholine (ACh) under calcium stimulation when reconstituted into artificial membranes. The mediatophore subunit, a 15-kDa proteolipid, presents a very high sequence homology with the N,N'-dicyclohexylcarbodiimide (DCCD)-binding proteolipid subunit of the vacuolar-type H(+)-ATPase. This prompted us to study the effect of DCCD, a potent blocker of proton translocation, on calcium-dependent ACh release. The present work shows that DCCD has no effect on ACh translocation either from Torpedo synaptosomes or from proteoliposomes reconstituted with purified mediatophore. However, using [14C]DCCD, we were able to demonstrate that the drug does bind to the 15-kDa proteolipid subunit of the mediatophore. These results suggest that although the 15-kDa proteolipid subunits of the mediatophore and the vacuolar H(+)-ATPase may be identical, different domains of these proteins are involved in proton translocation and calcium-dependent ACh release and that the two proteins have a different membrane organization.     

Ontogenetic reaction norm for binary traits: the timing of phallus development in the snail Bulinus truncatus

The ontogenetic trajectory of plastic binary traits may provide valuable insights into their evolutionary rate of change. In this paper, the timing of the plastic response of a temperature-dependent sexual polymorphism, aphally, is investigated in the freshwater snail Bulinus truncatus. Aphally is defined as the loss of the male copulatory organ in otherwise hermaphroditic animals. Individuals from two inbred lines were switched at various times during their early development between 25 and 30 degrees C, and their phally status ascertained, in order to evaluate the parameters characterising the ontogenetic reaction norm of aphally to temperature. A series of nested models including parameters for the onset, offset, and the intensity of the response to temperature were fitted to the data, allowing for a wide range of reaction norms. One genotype did not show any variation in aphally ratio with switching temperature, while a switch-point model (onset and offset corresponding to the same developmental point in time) best fitted the second genotype. The results suggest that the plasticity of aphally is expressed before eggs hatch. Their consequences on the evolution of aphally are discussed. More generally, the methodology proposed here can be used to analyse variation in ontogenetic parameters of discrete traits.     

Antibodies directed against L and D isovaline using a chemical derivatizing reagent for the measurement of their enantiomeric ratio in extraterrestrial samples: first step-production and characterization of antibodies

Determining the enantiomeric ratio of amino acids in meteorites requires very sensitive and precise measurements. In this study, an immunochemical approach, combined with new chemical derivatizing agents, was investigated for the measurement of the enantiomeric ratio of isovaline. In the initial step, L and D isovaline were derivatized with epsilon-benzyloxycarbonyl-L-lysine-(t-butyl ester)-chloroethylnitrosourea (Z-L-Lys-(OtBu)-CENU). The Z group was hydrolyzed and the resulting isovaline derivatives (L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) were conjugated with protein using glutaraldehyde and reduced with sodium borohydride. Rabbits were immunized with the immunogenic conjugates thus obtained. Antibodies were characterized using many compounds, both derivatized and underivatized, in competitive ELISA tests. These competition experiments performed enabled us to establish the following results: 1) unconjugated L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline were poorly recognized; 2) all related L-Lys(OtBu)-alpha-hydrogenated amino acids (L and D) were not recognized at all, which eliminates the possibility of the measurements being distorted by contamination; 3) only conjugated L-Lys(OtBu)-alpha-amino-isobutyric acid (AIB) was recognized by the antibody, 4) the enantiomeric discrimination of L and D isovaline through their derivatives (diastereoisomeric L-Lys(OtBu)-L-isovaline and L-Lys(OtBu)-D-isovaline) was in accordance with the measurement of their enantiomeric ratio. Immunopurification was shown to enhance antibody specificity. The strategy employed shows potential for the quantification of meteoritic amino acids.     

Influence of sward structure on daily intake and foraging behaviour by horses

The spatial heterogeneity of grasslands determines the abundance and quality of food resources for grazing animals. As plants mature, they increase in mass, which allows greater instantaneous intake rates, but the cell wall concentrations increase too, reducing diet quality. In ruminants, daily intake rates are often constrained by the time needed for the ingesta to pass through the rumen, which is influenced by the rate of digestion. It has been suggested that the digestive constraint should have much less effect on hindgut fermenters such as equids. Horses play an increasing role in the management of grasslands in Europe, but the data on the influence of the heterogeneity of the vegetation on their daily intake and foraging behaviour are sparse. We report here the results of a preliminary study concerning the effects of sward structure on nutrient assimilation and the use of patches of different heights by horses grazing successively a short immature, a tall mature and a heterogeneous pastures (with short and tall swards). Daily nutrient assimilation was higher in the heterogeneous pasture compared to the short (+35%) and the tall (+55%) ones. The digestive constraints may have limited voluntary intake by horses on the tall swards. In the heterogeneous pasture, the mean height used for feeding (6 to 7 cm) by horses was intermediate between the heights used in the short (4 to 5 cm) and tall pastures (22 to 23 cm), and the animals may thus have benefited from both short swards of high quality and tall swards offering a higher instantaneous intake rate.     

Wolbachia detection: an assessment of standard PCR protocols

Wolbachia is a large monophyletic genus of intracellular bacteria, traditionally detected using PCR assays. Its considerable phylogenetic diversity and impact on arthropods and nematodes make it urgent to assess the efficiency of these screening protocols. The sensitivity and range of commonly used PCR primers and of a new set of 16S primers were evaluated on a wide range of hosts and Wolbachia strains. We show that certain primer sets are significantly more efficient than others but that no single protocol can ensure the specific detection of all known Wolbachia infections.     

Allozyme variation of oleaster populations (wild olive tree) (Olea europaea L.) in the Mediterranean Basin

As a result of the early domestication and extensive cultivation of the olive tree throughout the Mediterranean Basin, the wild-looking forms of olive (oleasters) presently observed constitute a complex, potentially ranging from wild to feral forms. Allozyme variation was analysed at 10 loci in 31 large and 44 small oleaster populations distributed in various habitats of the Mediterranean Basin and in two populations of the wild subspecies Olea europaea subsp (ssp) guanchica, endemic to the Canary islands and closely related to oleasters. At eight polymorphic loci, 25 alleles were identified. Genetic evidence that nondomesticated oleasters still survive locally was provided by the occurrence of four and one alleles shared exclusively by the eight western and two eastern oleaster populations, respectively, which were collected in forests potentially containing genuinely wild forms according to environmental, historical and demographic criteria. As reported previously from cytoplasmic and RAPDs analysis, substantial genetic differentiation was observed between the eastern oleaster populations genetically close to most olive clones cultivated in the Mediterranean Basin, and the western populations that are related to the wild Canarian populations. In addition, the occurrence of significantly lower heterozygosity in cultivated olive than in oleasters, whatever their origin, suggests that intensive selection involving inbreeding has taken place under cultivation to obtain particular characteristics in the olive cultivars.     

The identification of cutin synthase: formation of the plant polyester cutin

A hydrophobic cuticle consisting of waxes and the polyester cutin covers the aerial epidermis of all land plants, providing essential protection from desiccation and other stresses. We have determined the enzymatic basis of cutin polymerization through characterization of a tomato extracellular acyltransferase, CD1, and its substrate, 2-mono(10,16-dihydroxyhexadecanoyl)glycerol. CD1 has in vitro polyester synthesis activity and is required for cutin accumulation in vivo, indicating that it is a cutin synthase.     

Cerebrospinal fluid hypocretin (orexin) levels are elevated by play but are not raised by exercise and its associated heart rate, blood pressure, respiration or body temperature changes

Hypocretin (Hcrt) has been implicated in the control of motor activity and in respiration and cardiovascular changes. Loss of Hcrt in narcolepsy is linked to sleepiness and to cataplexy, a sudden loss of muscle tone which is triggered by sudden strong emotions. In the current study we have compared the effects of treadmill running, to yard play on cerebrospinal fluid (CSF) Hcrt level in normal dogs. We find that treadmill locomotion, at a wide range of speeds, does not increase Hcrt level beyond baseline, whereas yard play produces a substantial increase in Hcrt, even though both activities produce comparable increases in heart rate, respiration and body temperature. We conclude that motor and cardiovascular changes are not sufficient to elevate CSF levels of Hcrt and we hypothesize that the emotional aspects of yard play account for the observed increase in Hcrt.     

Mg-ATPase and Torpedo Cholinergic Synaptic Vesicles

The reported presence of Mg-ATPase activity in cholinergic synaptic vesicles from the electric organ of Torpedo marmorata was reinvestigated in view of possible contamination of vesicles by other subcellular fractions. After dilution in concentrated sucrose, the vesicular fraction isolated on a sedimentation sucrose gradient was purified further on a flotation density gradient. It appears that this treatment allows separation of the vesicles according to their content. The two vesicular content markers, acetylcholine and ATP, are recovered as sharp coincident peaks at a density close to 0.48 M sucrose. Empty vesicles are identified in denser regions by the protein pattern on gel electrophoresis which is identical to the pattern obtained for filled vesicles. Refractionation of vesicles depleted of their acetylcholine content by valinomycin leads to an extreme picture, with a massive shift of the vesicles toward denser regions. We have then shown that a ouabain-insensitive Mg-ATPase is indeed associated with the vesicle membrane, but the activity is fully apparent only when vesicles are permeabilized either as the result of the fractionation procedure or after detergent treatment. The relative insensitivity of the Mg-ATPase associated with the synaptic vesicles to oligomycin, N,N'-dicyclohexylcarbodiimide, and azide indicates that this enzyme differs from the classic F1F0 mitochondrial enzyme. The most striking finding is the sensitivity to vanadate of the vesicular Mg-ATPase, which suggests the involvement of a phosphorylated intermediate. On the basis of both the difference in inhibitor sensitivity between untreated and detergent-treated vesicles and of the pronase experiments, the possibility that the enzyme has an inward orientation is discussed.