Organization and dynamics of NBD-labeled lipids in lipid bilayer analyzed by FRET using the small membrane fluorescent probe AHBA as donor

Fluorescent probes are employed to investigate natural and model membranes. It is important to know probe location and extent of perturbations they cause into the lipid bilayer. Förster Resonance Energy Transfer (FRET) is a useful tool to investigate phenomena involving plasma membranes, and reports in literature used relatively large fluorophores like 1,6-diphenylhexatriene, located at the center of the hydrophobic region, 4-aminophthalimide-based molecules located at lipid/water interfaces and BODIPY-labeled phosphatidylcholine. In this work we explored FRET process in 1,2-dimyristoyl-L-α-GPC large unilamellar vesicles, in gel and fluid phase, using as donor the very small group o-Abz bound to hexadecyl chain (2-amino-N-hexadecyl-benzamide - AHBA) and 7-nitro-2-1,3-benzoxadiazol-4-yl (NBD) labeled lipids as acceptor. From the intensity decay of donor in presence of acceptors, the FRET efficiency was calculated, and used to fit the model proposed by Fung and Stryer to that efficiency. Using lipid bilayer structural data, the procedure allowed the determination of Förster distance for each donor-acceptor pair in vesicles, without imposing any value for the orientational factor κ². From distance distributions between o-Abz in AHBA and NBD in lipid bilayer obtained using the program CONTIN, we obtained donor-acceptor populations having different separation distances. The populations reflect the occurrence of FRET involving probes in the same or in opposite leaflet. A dynamic picture emerged showing how relative position of the probes is dependent on the structural thermal phase of the DMPC bilayer. The results emphasize the need of careful analysis in order to understand processes involving fluorescent probes in model membranes.     

Molecular dynamics simulation of α-melanocyte stimulating hormone in a water-membrane model interface

The conformation of the tridecapeptide α-melanocyte stimulating hormone in the presence of a double water-membrane interface was studied by molecular dynamics simulation, using the computational package THOR. In this program the solvent is represented by a continuous medium with dielectric constant ɛ, and the interface between different media is simulated by a surface of discontinuity of the dielectric constant. The electrostatic image method was used to write down the terms, added to the force field, that describe the polarisation effects induced in the interface by the atomic charges. The program was further improved by the introduction of a second surface, parallel to the first one, to mimic the membrane. A conformational search using the software Prelude was employed to find an initial geometry for the peptide in water. The molecular dynamics simulation performed during 10 ns showed that the peptide structure is flexible in water, without stabilisation of any preferential conformation. In the presence of the model membrane, the peptide moved to the medium representing the interior of the membrane. Inside the low dielectric constant medium, the structure of the peptide showed a turn in the central sequence of amino acids and a packed conformation remained stabilised during more than 7.0 ns of simulation. Received: 27 November 1998 / Revised version: 11 March 1999 / Accepted: 8 April 1999     

Isolation and kinetic properties of pyruvate kinase activated by fructose-1,6-biphosphate from Salmonella typhimurium LT-2.I

Pyruvate kinase, activated by fructose-1,6-biphosphate from Salmonella typhimurium LT-2, has been isolated and purified to homogeneity. The enzyme, similar to that from Escherichia coli, is a tetramer with an approximate molecular weight of 240,000. The native enzyme shows optimum pH 6.8 (T = 30 degrees C). The enzymatic reaction does not require K+ ions; while Mg2+ or Mn2+ are essential for its activity. The non-activated enzyme shows sigmoid kinetics to phosphoenolpyruvate with a Hill coefficient of 2.73; the activated enzyme becomes michaelian with KSADP y KSPEP 0.25 and 0.08 mM, respectively. Both substrates excess and ATP cause enzyme inhibition. In agreement with the experimental results a steady-state random-ordered hybrid Bi-Bi mechanism with two dead-end complexes is proposed.     

Toxin and molecular analysis of Gymnodinium catenatum (Dinophyceae) strains from Galicia (NW Spain) and Andalucia (S Spain)

Nineteen strains of Gymnodinium catenatum were isolated fromone bloom in Andalucía (S Spain) and from different bloomsin Galicia (NW Spain). The PSP toxin profiles of 16 of the strainswere analyzed, and although the saxitoxin was exclusive to theGalician strains, the corresponding dendogram showed no clusteringof the isolates from this location. However, nine out of elevenAndalusian strains were included in the same cluster. In orderto compare toxin with molecular analysis, a fragment of thelarge subunit ribosomal (LSU) RNA gene was partially sequencedfor all of the strains and fully sequenced for the five strainsthat had shown most different growth curves. Since all the strainswere identical in the LSU sequenced region, another fragmentcomprising the internal transcribed spacer 1 (ITS 1), 5.8S rRNAgene and ITS 2, was sequenced and compared among all the strains.Although this region has been used before for the detectionof intraspecific variability, it was similar in all our strains.Finally, to detect molecular differences in the strains, a randomamplified polymorphic DNA (RAPD) analysis was performed. Thecorresponding cluster analysis grouped the strains in threeclusters: one of them comprised all the Galician strains plusthree from Andalucía, another one included eight Andalusianstrains, and the last one, more separated from the two previous,was constituted by two Andalucía isolates. Although theresults of the toxin and RAPD analysis were different, sevenAndalusian strains were clustered together in both dendograms.Since neither the toxin nor the RAPD analysis brought out aclear geographic signal, we can conclude that differences intoxin content and RAPD profile between the isolates of G. catenatumare probably not linked to the location in which the strainswere collected.     

Recovery of the algae and macroinvertebrate benthic community after <Emphasis Type="Italic">Didymosphenia geminata</Emphasis> mass growths in Spanish rivers

This study aims to assess the ecological profile of the invasive alga Didymosphenia geminata in NW Spain, analysing the biotic and abiotic factors related to the presence of massive colonies and their effect on river benthos. Physical and chemical parameters were measured in three infested rivers during 2009 and 2010, and biological samples of benthic community were taken according to standard protocols. Collected data was compared with that of control stations located in the same rivers but presenting no evident infestations. The autoecology shown by D. geminata in this study supports former observations reporting the expansion of its ecological niche, with current velocity and nutrients concentrations the environmental variables that best explain the establishment and development of mats in the studied rivers. Regarding its impact, it was observed that the mass growths of this diatom produced a dramatic change in the composition of the algae and macroinvertebrate benthic community which persisted after their disappearance. These results led to the development of a geostatistical predictive model to identify potential risk of dispersion of this diatom in Spanish rivers. The expected distribution of this algae according to this model agreed in general with the actual observed infestations, thus validating it as a tool to prevent and manage future infestations.     

Degradation of 3- and 4-hydroxybenzoate byKlebsiella pneumoniae

Summary Two different hydroxylases involved in benzenoid degradation have been isolated from Klebsiella pneumoniae and they can be differentially induced in the same strain. 3-Hydroxybenzoate-6-hydroxylase has an interesting biochemical mechanism and kinetic properties. 4-Hydroxybenzoate-1-hydroxylase activity is reported for the first time. The kinetic parameters of both enzymes have been studied. Offprint requests to: A. Garrido-Pertierra     

Phylogeography of African fruitbats (Megachiroptera)

Joint sequences from the mitochondrial cytochrome b and 16S rRNA genes of a wide representation of Megachiroptera were employed to evaluate the traditional taxonomic arrangement of African fruitbats and to examine their origins and evolutionary relationships. The resulting phylogenetic hypotheses are inconsistent with the previously established morphology-based subdivisions of Megachiroptera at the suprageneric level. Findings indicate the existence of an African clade, which appears to be formed by two endemic clades: the epomophorines and the myonycterines. According to our topologies, the genus Rousettus is monospecific in mainland Africa. Its traditional subgenera Stenonycteris and Lissonycteris appear closer to the myonycterines than to Rousettus. Topologies also indicate that the African genus Eidolon is not phylogenetically related to any other African fruitbat. It would seem that the arrival of fruitbats in Africa was a complex process involving at least three independent colonization events. One event took place probably in the Miocene via forested corridors that connected the African and Asian rain forest blocks, as for other groups of mammals. The resulting lineage diversified into most of the extant African fruitbats. Related to this clade, the Rousettus species group is thought to have arrived in Africa in more recent times, possibly by progressive displacement from the East through India. Finally, the present topologies suggest an independent colonization of Africa by ancestors of Eidolon.     

Trophic specialization and morphological divergence between two sympatric species in Lake Catemaco,Mexico

The association of morphological divergence with ecological segregation among closely related species could be considered as a signal of divergent selection in ecological speciation processes. Environmental signals such as diet can trigger phenotypic evolution, making polymorphic species valuable systems for studying the evolution of trophic‐related traits. The main goal of this study was to analyze the association between morphological differences in trophic‐related traits and ecological divergence in two sympatric species, Astyanax aeneus and A. caballeroi, inhabiting Lake Catemaco, Mexico. The trophic differences of a total of 70 individuals (35 A. aeneus and 35 A. caballeroi) were examined using stable isotopes and gut content analysis; a subset of the sample was used to characterize six trophic and six ecomorphological variables. In our results, we recovered significant differences between both species in the values of stable isotopes, with higher values of δ¹⁵N for A. caballeroi than for A. aeneus. Gut content results were consistent with the stable isotope data, with a higher proportion of invertebrates in A. caballeroi (a consumption of invertebrates ten times higher than that of A. aeneus, which in turn consumed three times more vegetal material than A. caballeroi). Finally, we found significant relationship between ecomorphology and stable isotopes (r = .24, p < .01), hence, head length, preorbital length, eye diameter, and δ¹⁵N were all positively correlated; these characteristics correspond to A. caballeroi. While longer gut and gill rakers, deeper bodies, and vegetal material consumption were positively correlated and corresponded to A. aeneus. Our results are consistent with the hypothesis that morphological divergence in trophic‐related traits could be associated with niche partitioning, allowing the coexistence of closely related species and reducing interspecific competition.