Polymorphism of Microsatellite Markers in Papaya (<Emphasis Type="Italic">Carica papaya</Emphasis> L.)

A set of 81 new microsatellite markers for Carica papaya L. previously identified by data mining using freely available sequence information from Genbank were tested for polymorphism using 30 germplasm accessions from the Papaya Germplasm Bank (PGM) at Embrapa Mandioca e Fruticultura Tropical (CNPMF) and 18 landraces. The data were used to estimate pairwise genetic distances between the genotypes. A neighbor-joining based dendrogram was used to define clusters and infer possible genetic structuring of the collection. Most microsatellites were polymorphic (73%), with an observed number of alleles per locus ranging from one to eleven. The levels of observed and expected heterozygosity for 51 polymorphic loci varied from 0.00 to 0.85 and from 0.08 to 0.82, averaging 0.19 and 0.59, respectively. Forty-four percent of microsatellites showed polymorphism information content (PIC) higher than 0.50. The compound microsatellites seem to be more informative than dinucleotide and trinucleotide repeats in average alleles per locus and PIC. Among dinucleotides, AG/TC or GA/CT repeat motifs exhibited more informativeness than TA/AT, GT/CA and TG/AC repeat motifs. The neighbor-joining analysis based on shared allele distance could differentiate all the papaya accessions and landraces as well as differences in their genetic structure. This set of markers will be useful for examining parentage, inbreeding and population structure in papaya.     

Single-molecule Force Spectroscopy Approach to Enzyme Catalysis

Enzyme catalysis has been traditionally studied using a diverse set of techniques such as bulk biochemistry, x-ray crystallography, and NMR. Recently, single-molecule force spectroscopy by atomic force microscopy has been used as a new tool to study the catalytic properties of an enzyme. In this approach, a mechanical force ranging up to hundreds of piconewtons is applied to the substrate of an enzymatic reaction, altering the conformational energy of the substrate-enzyme interactions during catalysis. From these measurements, the force dependence of an enzymatic reaction can be determined. The force dependence provides valuable new information about the dynamics of enzyme catalysis with sub-angstrom resolution, a feat unmatched by any other current technique. To date, single-molecule force spectroscopy has been applied to gain insight into the reduction of disulfide bonds by different enzymes of the thioredoxin family. This minireview aims to present a perspective on this new approach to study enzyme catalysis and to summarize the results that have already been obtained from it. Finally, the specific requirements that must be fulfilled to apply this new methodology to any other enzyme will be discussed.     

Generation of a complement-independent bactericidal IgM against a relapsing fever Borrelia

The spirochetemia of relapsing fever in mice is cleared by a complement-independent, polyclonal IgM response with reactivity to two prominent Ags of 20 and 35 kDa. In this study, we have dissected the polyclonal IgM Ab response against a relapsing fever spirochete to determine the specificity of its complement-independent bactericidal properties. Our experimental approach selectively generated an IgM murine mAb from the early specific immune response to a variable outer membrane protein. This IgM is bactericidal in the absence of complement and is part of the polyclonal Ab response that mediates the clearance of this bacterium from the blood. Purified monoclonal IgM caused direct structural damage to the outer membrane of the spirochete, in the absence of complement, and protected both B cell- and C5-deficient mice from challenge when administered passively. The direct, complement-independent, bactericidal activity of Abs is a critical mechanism of host defense against infection.     

Novel semisynthetic derivatives of betulin and betulinic acid with cytotoxic activity

A series of new imidazole carboxylic esters (carbamates) and N-acylimidazole derivatives of betulin and betulinic acid (14–29) have been synthesized. The new compounds were screened for in vitro cytotoxicity activity against human cancer cell lines HepG2, Jurkat and HeLa. A number of compounds have shown IC₅₀ values lower than 2 μM against the cancer cell lines tested and the vast majority has shown a better cytotoxicity profile than betulinic acid, including the betulin derivatives. N-Acylimidazole derivatives 26 and 27 (IC₅₀ 0.8 and 1.7 μM in HepG2 cells) and the C-3 carbamate derivative 16 (IC₅₀ 2.0 μM in HepG2 cells) were the most promising compounds. Based on the observed cytotoxicity, structure–activity relationships have been established.     

Estradiol and neuropeptide Y (intra-lateral septal) reduce anxiety-like behavior in two animal models of anxiety

Anxiolytic-like effects of intra-lateral septal nuclei (LSN) infusions of the neuropeptide Y (NPY) alone or combined with estradiol benzoate were assessed in ovariectomized Wistar rats in two animal models of anxiety-like behavior. In a conflict test, immediately punished responses were assessed: 17-beta-estradiol (50.0microg/rat, P<0.05) plus vehicle (intra-LSN) or intra-LSN infusions of NPY (2.5microg/microl, P<0.05; 3.0microg/mul, P<0.05) plus vehicle (systemic route) or the combination of subthreshold doses of 17-beta-estradiol (25.0microg/kg) plus intra-LSN infusions of NPY (2.0microg/mul, P<0.05) increased the amount of immediately punished reinforcers. In the elevated plus-maze test several spatial-temporal variables were evaluated: 17-beta-estradiol (50.0microg/kg, P<0.05) plus vehicle (intra-LSN) or intra-LSN infusions of NPY (2.5microg/mul, P<0.05; 3.0microg/mul, P<0.05) plus vehicle (systemic route) or the combination of subthreshold doses of 17-beta-estradiol (25.0microg/kg) plus intra-LSN infusions of NPY (2.0microg/mul, P<0.05) produced anxiolytic-like actions without affecting locomotion. It is concluded that estradiol or NPY may produce anxiolytic-like actions and that subthreshold doses of estradiol and subthreshold doses of NPY when combined produced anxiolytic-like actions.     

Control in vitro tick (Boophilus microplus; Acari: Ixodidae) through plant extracts

Control in vitro tick (Boophilus microplus; Acari: Ixodidae) through plant extracts. Hydro-alcohol extracts of ten tropical plants were tested by the immersion method on mature ticks (Boophilus microplus Canestrini 1887). The biological evaluation was made with raw hydro alcoholic extracts (R-OH) to determine the most promissory compounds, which later were fractioned in their polar and non-polar parts. The polar parts showed a significantly inferior effect. The species that produced the highest mortality were Zizigium aromaticum, Morus alba, Piper nigrum and the mixture of Allium sativum- Z. aromaticum (all non-polar); on oviposition: M. alba, P. nigrum and the mixture of A. sativum - Z. aromaticum (all non-polar); on eclosion: P. nigrum, Z. aromaticum, Echinacea angustiofilia, Poligonum punctatum, M. alba and the mixture of A. sativum-Z. aromaticum (all non-polar): on control percentage: P. nigrum, E. angustifolia, P. punctatum and M. alba (all non-polar, except for M. alba in which both fractions showed important activity); and finally, on the percentage of oviposition inhibition: P. nigrum, E. angustifolia, P. punctatum, Gliricidia sepium and M. alba.     

Selection of an antibody library identifies a pathway to induce immunity by targeting CD36 on steady-state CD8 alpha+ dendritic cells

Improvement of the strategy to target tumor Ags to dendritic cells (DCs) for immunotherapy requires the identification of the most appropriate ligand/receptor pairing. We screened a library of Ab fragments on mouse DCs to isolate new potential Abs capable of inducing protective immune responses. The screening identified a high-affinity Ab against CD36, a multi-ligand scavenger receptor primarily expressed by the CD8alpha+ subset of conventional DCs. The Ab variable regions were genetically linked to the model Ag OVA and tested in Ag presentation assays in vitro and in vivo. Anti-CD36-OVA was capable of delivering exogenous Ags to the MHC class I and MHC class II processing pathways. In vivo, immunization with anti-CD36-OVA induced robust activation of naive CD4+ and CD8+ Ag-specific T lymphocytes and the differentiation of primed CD8+ T cells into long-term effector CTLs. Vaccination with anti-CD36-OVA elicited humoral and cell-mediated protection from the growth of an Ag-specific tumor. Notably, the relative efficacy of targeting CD11c/CD8alpha+ via CD36 or DEC205 was qualitatively different. Anti-DEC205-OVA was more efficient than anti-CD36-OVA in inducing early events of naive CD8+ T cell activation. In contrast, long-term persistence of effector CTLs was stronger following immunization with anti-CD36-OVA and did not require the addition of exogenous maturation stimuli. The results identify CD36 as a novel potential target for immunotherapy and indicate that the outcome of the immune responses vary by targeting different receptors on CD8alpha+ DCs.     

Lactococcus lactis as a vehicle for the heterologous expression of fungal ribotoxin variants with reduced IgE-binding affinity

Fungal ribotoxins are a family of extracellular ribonucleases which inhibit protein biosynthesis by inactivating the ribosomes. This inactivation results in the induction of cell death by apoptosis. Ribotoxins show antitumoral properties based on their ability to cross the membrane of some transformed cells. Unfortunately, they also show an unspecific cytotoxicity which has greatly impaired their potential clinical uses. alpha-Sarcin, produced by Aspergillus giganteus, is the best-characterized ribotoxin. Asp f 1, another ribotoxin produced by A. fumigatus, is indeed one of its major allergens. In this work, the Lactococcus lactis MG1363 strain has been engineered to produce and secrete not only wild-type Asp f 1 and alpha-sarcin but also three different mutants with reduced cytotoxicity and/or IgE-binding affinity. The proteins were secreted in native and active form when the extracellular medium employed was buffered at pH values around 8.0. Strains producing the wild-type natural alpha-sarcin were proved to be innocuous when administered intragastrically to mice for a period of 14 days. Overall, the results presented are discussed in terms of its potential application as a vehicle of oral delivery of hypoallergenic variants as well as a starting point to approach the design of strategies to accomplish the safe delivery of these proteins as antitumoral agents.     

Mother — Offspring Conflict in the Saharan Arrui,Ammotragus lervia sahariensis: Relation to Weaning and Mother's Sexual Activity

Behavioural conflict between mother and offspring was investigated in a captive population of the Saharan arrui (Ammotragus lervia sahariensis, Artiodactyla: Bovidae). The frequency of agonistic behaviours (mother's aggressiveness and calf's retreats) and unsuccessful suckling attempts, Hinde's proximity index and the distances between mother and calf have been analysed in relation to weaning and sexual activity of the mother. Mothers that conceive in the following mating season accelerate the weaning process of their current offspring; furthermore, high-ranking females wean their male calves earlier. Behavioural conflict occurs during the resumption of the mother's sexual activity; however, no conflict appears to happen during weaning.     

Satellite tracking of gulls and genomic characterization of faecal bacteria reveals environmentally mediated acquisition and dispersal of antimicrobial‐resistant Escherichia coli on the Kenai Peninsula,Alaska

Gulls (Larus spp.) have frequently been reported to carry Escherichia coli exhibiting antimicrobial resistance (AMR E. coli); however, the pathways governing the acquisition and dispersal of such bacteria are not well described. We equipped 17 landfill‐foraging gulls with satellite transmitters and collected gull faecal samples longitudinally from four locations on the Kenai Peninsula, Alaska to assess: (a) gull attendance and transitions between sites, (b) spatiotemporal prevalence of faecally shed AMR E. coli, and (c) genomic relatedness of AMR E. coli isolates among sites. We also sampled Pacific salmon (Oncorhynchus spp.) harvested as part of personal‐use dipnet fisheries at two sites to assess potential contamination with AMR E. coli. Among our study sites, marked gulls most commonly occupied the lower Kenai River (61% of site locations) followed by the Soldotna landfill (11%), lower Kasilof River (5%) and upper Kenai River (<1%). Gulls primarily moved between the Soldotna landfill and the lower Kenai River (94% of transitions among sites), which were also the two locations with the highest prevalence of AMR E. coli. There was relatively high spatial and temporal variability in AMR E. coli prevalence in gull faeces and there was no evidence of contamination on salmon harvested in personal‐use fisheries. We identified E. coli sequence types and AMR genes of clinical importance, with some isolates possessing genes associated with resistance to as many as eight antibiotic classes. Our findings suggest that gulls acquire AMR E. coli at habitats with anthropogenic inputs and subsequent movements may represent pathways through which AMR is dispersed.