Molecular systematics of the frog genus Leptodactylus (Amphibia: Leptodactylidae) / Linda R. Maxson, W. Ronald Heyer.

n.s. no.41(1988)

Molecular phylogenies, chromosomes and dispersion in Brazilian akodontines (Rodentia, Sigmodontinae)

A new molecular phylogeny for akodontine rodents from Brazil was proposed. The phylogenetic tree was enriched with the area of occurrence and with information on the karyotype of the samples. Based on this enriched tree, and with a described methodology, hypotheses were proposed on the karyotype and area of occurrence of the ancestors of each Clade. Thus it was possible to discuss hypotheses on chromosome evolution of the group, and on dispersion events from the "area of original differentiation" of akodontines in the Andes. Chromosome evolution started with high diploid numbers (2n=52) and showed a tendency to reduction (until 2n=14 in more recent clades). Independent side-branches of the tree showed 2n reduction and in one case the 2n increased. At least four dispersion events from the Andes down to South-eastern Brazil were proposed. The results should suggest the direction of new studies on comparative karyology.

A molecular contribution to the controversial taxonomical status of some freshwater snails (Caenogastropoda: Rissooidea, Cochliopidae) from the Central Andes desert to Patagonia

For over 40 years malacologists have been discussing the taxonomical status of Heleobia species, an enigmatic genus from Cochliopidae family (Caenogastropoda: Rissooidea). As with other rissooidean families, the considerable character convergence and the paucity of anatomical synapomorphies has proved to be a problem in resolving cochliopid phylogenetic relations and establishing the validity of several nominal cochliopid species. Here we present a molecular contribution to solve the taxonomical status of one of the most abundant Southern South America cochliopid genera which has many endemic species. We report molecular evidence that supports three of the four Heleobia groups described for this region, the "australis", "parchappii" and "piscium" groups. The fourth, the "hatcheri" group, belongs not to Heleobia but to a different genus which itself should not be considered as part of the family Cochliopidae but closely related to genus Potamolithus Pilsbry & Rush, 1896.

Caracterización agronómica, fenológica, cualitativa y molecular de la colección nuclear de cebadas españolas

El presente proyecto planteaba el paso siguiente a la construcción, por nosotros mismos, de la Colección Nuclear de Cebadas Españolas, que es una representación esquemática de la variabilidad genética de las cebadas ancestralmente cultivadas en nuestro país. Para la explotación completa de estos materiales autóctonos en el Programa Nacional de Mejora de Cebadas, que estamos llevando a cabo los grupos integrantes de este proyecto, se realizó el mismo, que ha comprendido los objetivos siguientes: - Caracterización agronómica, mediante ensayos de campo en ambientes contrastantes y representativos, incluyendo la evaluación de respuestas a factores productores de estreses bióticos y abióticos. - Caracterización fenológica, mediante ensayos en invernadero con protocolos desarrollados por nosotros, para identificar la respuesta de estos genotipos a la vernalización y el fotoperiodo. - Caracterización maltero-cervecera/pienso, mediante análisis de cebada y malta. - Caracterización molecular, mediante el uso de marcadores SSRs y STS.

The fourth extracellular loop of the alpha subunit of Na,K-ATPase. Functional evidence for close proximity with the second extracellular loop.

Na,K-ATPase is the main active transport system that maintains the large gradients of Na(+) and K(+) across the plasma membrane of animal cells. The crystal structure of a K(+)-occluding conformation of this protein has been recently published, but the movements of its different domains allowing for the cation pumping mechanism are not yet known. The structure of many more conformations is known for the related calcium ATPase SERCA, but the reliability of homology modeling is poor for several domains with low sequence identity, in particular the extracellular loops. To better define the structure of the large fourth extracellular loop between the seventh and eighth transmembrane segments of the alpha subunit, we have studied the formation of a disulfide bond between pairs of cysteine residues introduced by site-directed mutagenesis in the second and the fourth extracellular loop. We found a specific pair of cysteine positions (Y308C and D884C) for which extracellular treatment with an oxidizing agent inhibited the Na,K pump function, which could be rapidly restored by a reducing agent. The formation of the disulfide bond occurred preferentially under the E2-P conformation of Na,K-ATPase, in the absence of extracellular cations. Using recently published crystal structure and a distance constraint reproducing the existence of disulfide bond, we performed an extensive conformational space search using simulated annealing and showed that the Tyr(308) and Asp(884) residues can be in close proximity, and simultaneously, the SYGQ motif of the fourth extracellular loop, known to interact with the extracellular domain of the beta subunit, can be exposed to the exterior of the protein and can easily interact with the beta subunit.

PET Molecular Imaging of Hypoxia in Ischemic Stroke: An Update.

Hypoxia, a condition of insufficient oxygen availability to support metabolism, occurs when the vascular supply is interrupted, as in stroke. The identification of the hypoxic and viable tissue in stroke as compared with irreversible lesions (necrosis) has relevant implications for the treatment of ischemic stroke. Traditionally, imaging by positron emission tomography (PET), using 15O-based radiotracers, allowed the measurement of perfusion and oxygen extraction in stroke, providing important insights in its pathophysiology. However, these multitracer evaluations are of limited applicability in clinical settings. More recently, specific tracers have been developed, which accumulate with an inverse relationship to oxygen concentration and thus allow visualizing the hypoxic tissue non invasively. These belong to two main groups: nitroimidazoles, and among these the 18F-Fluoroimidazole (18F-FMISO) is the most widely used, and the copper-based tracers, represented mainly by Cu-ATSM. While these tracers have been at first developed and tested in order to image hypoxia in tumors, they have also shown promising results in stroke models and preliminary clinical studies in patients with cardiovascular disorders, allowing the detection of hypoxic tissue and the prediction of the extent of subsequent ischemia and clinical outcome. These tracers have therefore the potential to select an appropriate subgroup of patients who could benefit from a hypoxia-directed treatment and provide prognosis relevant imaging. The molecular imaging of hypoxia made important progress over the last decade and has a potential for integration into the diagnostic and therapeutic workup of patients with ischemic stroke.

Mutational analysis of the highly conserved arginine within the Glu/Asp-Arg-Tyr motif of the alpha(1b)-adrenergic receptor: effects on receptor isomerization and activation.

We have suggested previously that both the negatively and positively charged residues of the highly conserved Glu/Asp-Arg-Tyr (E/DRY) motif play an important role in the activation process of the alpha(1b)-adreneric receptor (AR). In this study, R143 of the E/DRY sequence in the alpha(1b)-AR was mutated into several amino acids (Lys, His, Glu, Asp, Ala, Asn, and Ile). The charge-conserving mutation of R143 into lysine not only preserved the maximal agonist-induced response of the alpha(1b)-AR, but it also conferred high degree of constitutive activity to the receptor. Both basal and agonist-induced phosphorylation levels were significantly increased for the R143K mutant compared with those of the wild-type receptor. Other substitutions of R143 resulted in receptor mutants with either a small increase in constitutive activity (R143H and R143D), impairment (R143H, R143D), or complete loss of receptor-mediated response (R143E, R143A, R143N, R143I). The R413E mutant displayed a small, but significant increase in basal phosphorylation despite being severely impaired in receptor-mediated response. Interestingly, all the arginine mutants displayed increased affinity for agonist binding compared with the wild-type alpha(1b)-AR. A correlation was found between the extent of the affinity shift and the intrinsic activity of the agonists. The analysis of the receptor mutants using the allosteric ternary complex model in conjunction with the results of molecular dynamics simulations on the receptor models support the hypothesis that mutations of R143 can drive the isomerization of the alpha(1b)-AR into different states, highlighting the crucial role of this residue in the activation process of the receptor.

Ab initio modeling and molecular dynamics simulation of the alpha 1b-adrenergic receptor activation.

This work describes the ab initio procedure employed to build an activation model for the alpha 1b-adrenergic receptor (alpha 1b-AR). The first version of the model was progressively modified and complicated by means of a many-step iterative procedure characterized by the employment of experimental validations of the model in each upgrading step. A combined simulated (molecular dynamics) and experimental mutagenesis approach was used to determine the structural and dynamic features characterizing the inactive and active states of alpha 1b-AR. The latest version of the model has been successfully challenged with respect to its ability to interpret and predict the functional properties of a large number of mutants. The iterative approach employed to describe alpha 1b-AR activation in terms of molecular structure and dynamics allows further complications of the model to allow prediction and interpretation of an ever-increasing number of experimental data.

Disaggregating chaperones: an unfolding story.

Stress, molecular crowding and mutations may jeopardize the native folding of proteins. Misfolded and aggregated proteins not only loose their biological activity, but may also disturb protein homeostasis, damage membranes and induce apoptosis. Here, we review the role of molecular chaperones as a network of cellular defenses against the formation of cytotoxic protein aggregates. Chaperones favour the native folding of proteins either as "holdases", sequestering hydrophobic regions in misfolding polypeptides, and/or as "unfoldases", forcibly unfolding and disentangling misfolded polypeptides from aggregates. Whereas in bacteria, plants and fungi Hsp70/40 acts in concert with the Hsp100 (ClpB) unfoldase, Hsp70/40 is the only known chaperone in the cytoplasm of mammalian cells that can forcibly unfold and neutralize cytotoxic protein conformers. Owing to its particular spatial configuration, the bulky 70 kDa Hsp70 molecule, when distally bound through a very tight molecular clamp onto a 50-fold smaller hydrophobic peptide loop extruding from an aggregate, can locally exert on the misfolded segment an unfolding force of entropic origin, thus destroying the misfolded structures that stabilize aggregates. ADP/ATP exchange triggers Hsp70 dissociation from the ensuing enlarged unfolded peptide loop, which is then allowed to spontaneously refold into a closer-to-native conformation devoid of affinity for the chaperone. Driven by ATP, the cooperative action of Hsp70 and its co-chaperone Hsp40 may thus gradually convert toxic misfolded protein substrates with high affinity for the chaperone, into non-toxic, natively refolded, low-affinity products. Stress- and mutation-induced protein damages in the cell, causing degenerative diseases and aging, may thus be effectively counteracted by a powerful network of molecular chaperones and of chaperone-related proteases.

Molecular estimation of dispersal for ecology and population genetics

The dispersal process, by which individuals or other dispersing agents such as gametes or seeds move from birthplace to a new settlement locality, has important consequences for the dynamics of genes, individuals, and species. Many of the questions addressed by ecology and evolutionary biology require a good understanding of species' dispersal patterns. Much effort has thus been devoted to overcoming the difficulties associated with dispersal measurement. In this context, genetic tools have long been the focus of intensive research, providing a great variety of potential solutions to measuring dispersal. This methodological diversity is reviewed here to help (molecular) ecologists find their way toward dispersal inference and interpretation and to stimulate further developments.

Aplicación de técnicas de citogenética molecular para la detección de aneuploidía y clastogenicidad en células humanas expuestas a arsénico

Los objetivos de esta investigación son: a) Determinar la potencialidad genotóxica de diversos compuestos de arsénico (incluyendo inorgánicos y sus formas metiladas) con el ensayo de micronúcleos utilizando cultivos de células humanas; y b)Estudiar la posible acción aneugénica y clastogénica de los compuestos analizados, determinando el origen de los micronúcleos inducidos mediante la técnica FISH, utilizando sondas pancentroméricas.

Molecular variation at a candidate gene implicated in the regulation of fire ant social behavior

The fire ant Solenopsis invicta and its close relatives display an important social polymorphism involving differences in colony queen number. Colonies are headed by either a single reproductive queen (monogyne form) or multiple queens (polygyne form). This variation in social organization is associated with variation at the gene Gp-9, with monogyne colonies harboring only B-like allelic variants and polygyne colonies always containing b-like variants as well. We describe naturally occurring variation at Gp-9 in fire ants based on 185 full-length sequences, 136 of which were obtained from S. invicta collected over much of its native range. While there is little overall differentiation between most of the numerous alleles observed, a surprising amount is found in the coding regions of the gene, with such substitutions usually causing amino acid replacements. This elevated coding-region variation may result from a lack of negative selection acting to constrain amino acid replacements over much of the protein, different mutation rates or biases in coding and non-coding sequences, negative selection acting with greater strength on non-coding than coding regions, and/or positive selection acting on the protein. Formal selection analyses provide evidence that the latter force played an important role in the basal b-like lineages coincident with the emergence of polygyny. While our data set reveals considerable paraphyly and polyphyly of S. invicta sequences with respect to those of other fire ant species, the b-like alleles of the socially polymorphic species are monophyletic. An expanded analysis of colonies containing alleles of this clade confirmed the invariant link between their presence and expression of polygyny. Finally, our discovery of several unique alleles bearing various combinations of b-like and B-like codons allows us to conclude that no single b-like residue is completely predictive of polygyne behavior and, thus, potentially causally involved in its expression. Rather, all three typical b-like residues appear to be necessary.