The genetic location of the self-incompatibility locus in white clover (Trifolium repens L.)

White clover (Trifolium repens L.) is a forage legume of considerable economic importance in temperate agricultural systems. It has a strong self-incompatibility system. The molecular basis of self-incompatibility in T. repens is unknown, but it is under the control of a single locus, which is expressed gametophytically. To locate the self-incompatibility locus (S locus) in T. repens, we carried out cross-pollination experiments in an F₁ mapping population and constructed a genetic linkage map using amplified fragment length polymorphism and simple sequence repeat markers. As the first step in a map-based cloning strategy, we locate for the first time the S locus in T. repens on a genetic linkage map, on the homoeologous linkage group pair 1 (E), which is broadly syntenic to Medicago truncatula L. chromosome 1. On the basis of this syntenic relationship, the possibility that the S locus may or may not possess an S-RNase gene is discussed.     

Paraquat-induced oxidative stress response during amphibian early embryonic development

In addition to the endogenous production of reactive oxygen species (ROS) as a result of normal development, amphibian external development often forces embryos to deal with oxidative stress-producing agents present in the environment. Embryos should therefore develop protective systems to reduce ROS toxicity and achieve successful development. The present work was aimed to characterize the effects produced by the widespread-used ROS-generator pesticide Paraquat during early embryonic development in the toad Chaunus arenarum, as well as to get insights into the defense response elicited by amphibian embryos. The approach consisted in generating a sharp and brief oxidative stress condition early during embryonic development to stimulate the cellular mechanisms involved in ROS-antioxidant response. Results revealed that Paraquat-treatment reduced the ability of embryos to develop normally, leading to arrests of development and severe malformations such as tail abnormalities, abdominal edema, reduced head development and curved dorsal structures. Although Paraquat effects were morphologically evident from gastrula stage on, alterations such as chromatin condensation were observed even at blastula stage by histological examinations. Regarding detoxifying enzymes, a significant induction of Mn-superoxide dismutase activity was detected at stages beyond gastrula in embryos surviving Paraquat treatment, suggesting a major role of this enzyme in the antioxidant response during early embryonic development.     

Computational Prediction and Experimental Assessment of Secreted/Surface Proteins from Mycobacterium tuberculosis H37Rv

The mycobacterial cell envelope has been implicated in the pathogenicity of tuberculosis and therefore has been a prime target for the identification and characterization of surface proteins with potential application in drug and vaccine development. In this study, the genome of Mycobacterium tuberculosis H37Rv was screened using Machine Learning tools that included feature-based predictors, general localizers and transmembrane topology predictors to identify proteins that are potentially secreted to the surface of M. tuberculosis, or to the extracellular milieu through different secretory pathways. The subcellular localization of a set of 8 hypothetically secreted/surface candidate proteins was experimentally assessed by cellular fractionation and immunoelectron microscopy (IEM) to determine the reliability of the computational methodology proposed here, using 4 secreted/surface proteins with experimental confirmation as positive controls and 2 cytoplasmic proteins as negative controls. Subcellular fractionation and IEM studies provided evidence that the candidate proteins Rv0403c, Rv3630, Rv1022, Rv0835, Rv0361 and Rv0178 are secreted either to the mycobacterial surface or to the extracellular milieu. Surface localization was also confirmed for the positive controls, whereas negative controls were located on the cytoplasm. Based on statistical learning methods, we obtained computational subcellular localization predictions that were experimentally assessed and allowed us to construct a computational protocol with experimental support that allowed us to identify a new set of secreted/surface proteins as potential vaccine candidates.     

Fast- or Slow-inactivated State Preference of Na+ Channel Inhibitors: A Simulation and Experimental Study

Sodium channels are one of the most intensively studied drug targets. Sodium channel inhibitors (e.g., local anesthetics, anticonvulsants, antiarrhythmics and analgesics) exert their effect by stabilizing an inactivated conformation of the channels. Besides the fast-inactivated conformation, sodium channels have several distinct slow-inactivated conformational states. Stabilization of a slow-inactivated state has been proposed to be advantageous for certain therapeutic applications. Special voltage protocols are used to evoke slow inactivation of sodium channels. It is assumed that efficacy of a drug in these protocols indicates slow-inactivated state preference. We tested this assumption in simulations using four prototypical drug inhibitory mechanisms (fast or slow-inactivated state preference, with either fast or slow binding kinetics) and a kinetic model for sodium channels. Unexpectedly, we found that efficacy in these protocols (e.g., a shift of the “steady-state slow inactivation curve”), was not a reliable indicator of slow-inactivated state preference. Slowly associating fast-inactivated state-preferring drugs were indistinguishable from slow-inactivated state-preferring drugs. On the other hand, fast- and slow-inactivated state-preferring drugs tended to preferentially affect onset and recovery, respectively. The robustness of these observations was verified: i) by performing a Monte Carlo study on the effects of randomly modifying model parameters, ii) by testing the same drugs in a fundamentally different model and iii) by an analysis of the effect of systematically changing drug-specific parameters. In patch clamp electrophysiology experiments we tested five sodium channel inhibitor drugs on native sodium channels of cultured hippocampal neurons. For lidocaine, phenytoin and carbamazepine our data indicate a preference for the fast-inactivated state, while the results for fluoxetine and desipramine are inconclusive. We suggest that conclusions based on voltage protocols that are used to detect slow-inactivated state preference are unreliable and should be re-evaluated.     

Bill harnesses on nestling Tufted Puffins influence adult provisioning behavior

ABSTRACT.   For burrow-nesting seabirds, investigators have examined nestling diet by attaching harnesses to the bills of nestlings to intercept food delivered by the parent. To determine whether this method provides an unbiased estimate of nestling diet, we evaluated its effect on the provisioning behavior of Tufted Puffins ( Fratercula cirrhata ) nesting on Triangle Island, British Columbia. Adults delivering food to nestlings with bill harnesses always hesitated before entering a burrow with food, increasing their susceptibility to kleptoparasitism by gulls, and did not always leave the food intended for the nestling. These responses by adult puffins could lead to underestimates of energy intake rates of nestlings and unreliable comparisons with other species if prey left by adults in nest burrows were the only source of data. We also compared estimates of the species, number, and size of prey delivered by adult puffins as determined by direct observation from blinds to samples of prey collected directly from nest burrows and found that the two sampling techniques produced similar results. However, identifying rare prey species and gathering precise information about prey length, mass, and condition require collection of prey, and we recommend using a combination of techniques to obtain the most reliable estimates of nestling diet.     

Development of experimental genetic tools for Campylobacter fetus

Molecular analysis of the virulence mechanisms of the emerging pathogen Campylobacter fetus has been hampered by the lack of genetic tools. We report the development and functional analysis of Escherichia coli-Campylobacter shuttle vectors that are appropriate for C. fetus. Some vectors were constructed based on the known Campylobacter coli plasmid pIP1455 replicon, which confers a wide host range in Campylobacter spp. Versatility in directing gene expression was achieved by introducing a strong C. fetus promoter. The constructions carry features necessary and sufficient to detect the expression of phenotypic markers, including molecular reporter genes in both subspecies of C. fetus, while retaining function in C. jejuni. The capacity to express several gene products from different vectors in a single host can be advantageous but requires distinct plasmid replicons. To this end, replication features derived from a cryptic plasmid of C. fetus subsp. venerealis strain 4111/108, designated pCFV108, were adapted for a compatible series of constructions. The substitution of the C. coli replication elements reduced vector size while apparently limiting the host range to C. fetus. The complementation of a ciprofloxacin-resistant mutant phenotype via vector-driven gyrA expression was verified. Cocultivation demonstrated that shuttle vectors based on the pCFV108 replicon were compatible with pIP1455 replication functions, and the stable maintenance of two plasmids in a C. fetus subsp. venerealis host over several months was observed. The application of both vector types will facilitate the investigation of the genetics and cellular interactions of the emerging pathogen C. fetus.     

TNT transformation products are affected by the growth conditions of Raoultella terrigena

High concentrations of 2,4,6-trinitrotoluene (TNT) and related nitroaromatic compounds are commonly found in soil and groundwater at former explosive plants. The bacterium, Raoultella terrigena strain HB, isolated from a contaminated site, converts TNT into the corresponding amino products. Radio-HPLC analysis with [¹⁴C]TNT identified aminodinitrotoluene, diaminonitrotoluene and azoxy-dimers as the main metabolites. Transformation rate and the type of metabolites that predominated in the culture medium and within the cells were significantly influenced by the culture conditions. The NAD(P)H-dependent enzymatic reduction of nitro-substituted compounds by cell-free extracts of R. terrigena was evaluated in vitro.     

Molecular structure, bioavailability and bioactivity of [Cu(o-phen)2(cnge)](NO3)2.2H2O and [Cu(o-phen)(cnge)(H2O)(NO3)2] complexes

Two Cu(II) complexes with cyanoguanidine (cnge) and o-phenanthroline, [Cu(o-phen)(2)(cnge)](NO(3))(2).2H(2)O (1) and [Cu(o-phen)(cnge)(H(2)O)(NO(3))(2)] (2), have been synthesized using different experimental techniques and characterized by elemental analyses, FTIR, diffuse and UV-vis spectra and EPR and magnetic moment measurements techniques. The crystal structures of both complexes were solved by X-ray diffraction methods. Complex (1) crystallizes in the monoclinic space group C2/c with a=12.621(5), b=31.968(3), c=15.39(1)A, beta=111.68(4) degrees, and Z=8 and complex (2) in the monoclinic space group P2(1)/n with a=10.245(1), b=13.923(2), c=12.391(2)A, beta=98.07(1) degrees, and Z=4. The environments of the copper(II) center are trigonal bipyramidal (TBP) for [Cu(o-phen)(2)(cnge)](2+) and an elongated octahedron for [Cu(o-phen)(cnge)(H(2)O)(NO(3))(2)]. Solution studies have been performed to determine the species distribution. The superoxide dismutase (SOD) activities of both complexes have also been tested in order to determine if these compounds mimic the enzymatic action of the enzyme SOD that protects cells against peroxide radicals.