Hematologic, protein electrophoresis, biochemistry, and cholinesterase values of free-living black stork nestlings (Ciconia nigra)

Hematologic, protein electrophoresis, serum biochemistry, and cholinesterase values were determined in 36 free-living black stork nestlings (Ciconia nigra) between 25 and 53 days of age in order to establish normal reference values for this population. The following values were evaluated: white blood cell counts, red blood cell counts, packed cell volume, hemoglobin, heterophils, lymphocytes, monocytes, eosinophils, prealbumin, albumin, alpha-globulin, beta-globulin, gamma-globulin, total protein, aspartate aminotransferase, alkaline phosphatase, lactate dehydrogenase, creatine kinase, calcium, phosphorus, iron, cholesterol, glucose, triglycerides, uric acid, urea, creatinine, total solids, bile acids, and butyrylcholinesterase. Sex-dependent differences were observed in hemoglobin, prealbumin, albumin, gamma-globulin, total protein, alkaline phosphatase, and triglycerides. Packed cell volume, butyrylcholinesterase, aspartate aminotransferase, creatine kinase, and creatinine increased with age, whereas albumin, mean cell volume, calcium, phosphorus, cholesterol, and total solids decreased with age. These hematologic and serum biochemistry values can be used as reference ranges in free-living black stork nestlings.     

Structural and evolutionary analyses of the Ty3/gypsy group of LTR retrotransposons in the genome of Anopheles gambiae

The recent availability of the genome of Anopheles gambiae offers an extraordinary opportunity for comparative studies of the diversity of transposable elements (TEs) and their evolutionary dynamics between two related species, taking advantage of the existing information from Drosophila melanogaster. To this goal, we screened the genome of A. gambiae for elements belonging to the Ty3/gypsy group of long-terminal repeat (LTR) retrotransposons. The A. gambiae genome displays a rich diversity of LTR retrotransposons, clearly greater than D. melanogaster. We have characterized in detail 63 families, belonging to five of the nine main lineages of the Ty3/gypsy group. The Mag lineage is the most diverse and abundant, with more than 30 families. In sharp contrast with this finding, a single family belonging to this lineage has been found in D. melanogaster, here reported for the first time in the literature, most probably consisting of old inactive elements. The CsRn1 lineage is also abundant in A. gambiae but almost absent from D. melanogaster. Conversely, the Osvaldo lineage has been detected in Drosophila but not in Anopheles. Comparison of structural characteristics of different families led to the identification of several lineage-specific features such as the primer-binding site (PBS), the gag-pol translational recoding signal (TRS), which is extraordinarily diverse within the Ty3/gypsy retrotransposons of A. gambiae, or the presence/absence of specific amino acid motifs. Interestingly, some of these characteristics, although in general well conserved within lineages, may have evolved independently in particular branches of the phylogenetic tree. We also show evidence of recent activity for around 75% of the families. Nevertheless, almost all families contain a high proportion of degenerate members and solitary LTRs (solo LTRs), indicative of a lower turnover rate of retrotransposons belonging to the Ty3/gypsy group in A. gambiae than in D. melanogaster. Finally, we have detected significant overrepresentations of insertions on the X chromosome versus autosomes and of putatively active insertions on euchromatin versus heterochromatin.     

The RETINOBLASTOMA-RELATED gene regulates stem cell maintenance in Arabidopsis roots

The maintenance of stem cells in defined locations is crucial for all multicellular organisms. Although intrinsic factors and signals for stem cell fate have been identified in several species, it has remained unclear how these connect to the ability to reenter the cell cycle that is one of the defining properties of stem cells. We show that local reduction of expression of the RETINOBLASTOMA-RELATED (RBR) gene in Arabidopsis roots increases the amount of stem cells without affecting cell cycle duration in mitotically active cells. Conversely, induced RBR overexpression dissipates stem cells prior to arresting other mitotic cells. Overexpression of D cyclins, KIP-related proteins, and E2F factors also affects root stem cell pool size, and genetic interactions suggest that these factors function in a canonical RBR pathway to regulate somatic stem cells. Expression analysis and genetic interactions position RBR-mediated regulation of the stem cell state downstream of the patterning gene SCARECROW.     

MHC class I genes in the owl monkey: mosaic organisation, convergence and loci diversity

The MHC class I molecule plays an important role in immune response, pathogen recognition and response against vaccines and self- versus non-self-recognition. Studying MHC class I characteristics thus became a priority when dealing with Aotus to ensure its use as an animal model for biomedical research. Isolation, cloning and sequencing of exons 1–8 from 27 MHC class I alleles obtained from 13 individuals classified as belonging to three owl monkey species (A. nancymaae, A. nigriceps and A. vociferans) were carried out to establish similarities between Aotus MHC class I genes and those expressed by other New and Old World primates. Six Aotus MHC class I sequence groups (Ao-g1, Ao-g2, Ao-g3, Ao-g4, Ao-g5 and Ao-g6) weakly related to non-classical Catarrhini MHC were identified. An allelic lineage was also identified in one A. nancymaae and two A. vociferans monkeys, exhibiting a high degree of conservation, negative selection along the molecule and premature termination of the open reading frame at exon 5 (Ao-g5). These sequences high conservation suggests that they more likely correspond to a soluble form of Aotus MHC class I molecules than to a new group of processed pseudogenes. Another group, named Ao-g6, exhibited a strong relationship with Catarrhinis classical MHC-B-C loci. Sequence evolution and variability analysis indicated that Aotus MHC class I molecules experience inter-locus gene conversion phenomena, contributing towards their high variability.     

Structural and functional characterisation of the Toll like receptor 9 of Aotus nancymaae, a non-human primate model for malaria vaccine development

In the absence of suitable rodent animal models for Plasmodium falciparum malaria, the efficacy testing of asexual blood-stage vaccine candidates in Aotus nancymaae represents a tool to select between different formulations before conducting expensive human clinical trials. CpG oligonucleotides (ODN) specifically promote the production of pro-inflammatory and Th1-type cytokines and they enhance the immunogenicity of co-administered antigens. Toll like receptor 9 (TLR-9) binds directly and sequence-specifically to single-stranded un-methylated CpG-DNA mediating the biological effects of CpG ODN. We cloned and functionally characterised the TLR-9 cDNA of A. nancymaae. The cDNA encompassed 3,099 bp predicted to code for 1,032 amino acid residues. Results of homology searches to human TLR-9 suggested that the receptor is 93 and 94% identical at the nucleotide and amino acid sequence levels, respectively. Stimulation of splenocytes of A. nancymaae with CpG ODN resulted in proliferative responses in all animals analysed. FACS analysis of cultures incubated with CpG ODN 2006 indicated that the B cell marker CD20 was up-regulated consistent with B cell activation. The high level of sequence conservation of Aona-TLR-9 reinforces the suitability of A. nancymaae as animal model for malaria subunit vaccine development.The nucleotide sequence has been submitted to the GenBank nucleotide sequence database under the accession number AY788894.     

Thermal Stability and Activity Regain of Horseradish Peroxidase in Aqueous Mixtures of Imidazolium-Based Ionic Liquids

Thermal deactivation kinetics of horseradish peroxidase (HRP) were studied from 45 to 90 °C in phosphate buffer and 5–25% (v,w/v) 1-butyl-3-methylimidazolium tetrafluoroborate [BMIM][BF₄] and 1-butyl-3-methylimidazolium chloride [BMIM][Cl]. HRP activity at 25 °C was not affected by the presence of ionic liquids up to 20% (v,w/v). Increasing the ionic liquids concentration up to 25% (v,w/v) changed the biphasic character of deactivation kinetics to an apparent single first-order step. The presence of 5–10% (v/v) [BMIM][BF₄] significantly improved HRP thermal stability with lower activation energies for the deactivation second phase (83–87 kJ mol⁻¹). After deactivation, enhanced activity regain of the enzyme, up to 70–80% of the initial activity, was found in 25% (v/v) [BMIM][BF₄] and 10% (w/v) [BMIM][Cl] and correlated to prevalence of the deactivation first phase.     

Decavanadate effects in biological systems

Vanadium biological studies often disregarded the formation of decameric vanadate species known to interact, in vitro, with high-affinity with many proteins such as myosin and sarcoplasmic reticulum calcium pump and also to inhibit these biochemical systems involved in energy transduction. Moreover, very few in vivo animal studies involving vanadium consider the contribution of decavanadate to vanadium biological effects. Recently, it has been shown that an acute exposure to decavanadate but not to other vanadate oligomers induced oxidative stress and a different fate in vanadium intracellular accumulation. Several markers of oxidative stress analyzed on hepatic and cardiac tissue were monitored after in vivo effect of an acute exposure (12, 24 h and 7 days), to a sub-lethal concentration (5 mM; 1 mg/kg) of two vanadium solutions ("metavanadate" and "decavanadate"). It was observed that "decavanadate" promote different effects than other vanadate oligomers in catalase activity, glutathione content, lipid peroxidation, mitochondrial superoxide anion production and vanadium accumulation, whereas both solutions seem to equally depress reactive oxygen species (ROS) production as well as total intracellular reducing power. Vanadium is accumulated in mitochondria in particular when "decavanadate" is administered. These recent findings, that are now summarized, point out the decameric vanadate species contributions to in vivo and in vitro effects induced by vanadium in biological systems.     

A phenological mid-domain effect in flowering diversity

In this paper, we test the mid-domain hypothesis as an explanation for observed patterns of flowering diversity in two sub-alpine communities of insect-pollinated plants. Observed species richness patterns showed an early-season increase in richness, a mid-season peak, and a late-season decrease. We show that a mid-domain null model can qualitatively match this pattern of flowering species richness, with R² values typically greater than 60%. We find significant or marginally significant departures from expected patterns of diversity for only 3 out of 12 year-site combinations. On the other hand, we do find a consistent pattern of departure when comparing observed versus null-model predicted flowering diversity averaged across years. Our results therefore support the hypothesis that ecological factors shape patterns of flowering phenology, but that the strength or nature of these environmental forcings may differ between years or the two habitats we studied, or may depend on species-specific characteristics of these plant communities. We conclude that mid-domain null models provide an important baseline from which to test departure of expected patterns of flowering diversity across temporal domains. Geometric constraints should be included first in the list of factors that drive seasonal patterns of flowering diversity.     

Hydrogel substrate amendment alleviates drought effects on young citrus plants

Water deficits affect citrus physiology, yield, fruit size and quality. Citrus can respond to drought stress conditions through endogenous hormonal regulation of water status and leaf abscission. In this work, we assayed the efficiency of an amendment to soilless media in delaying the drought stress effect in young citrus seedlings and trees. Substrate amendment promoted plant survival of citrus seedlings subjected to several cycles of drought stress and rehydration. In budded trees, the amendment increased substrate water content, leaf water potential, leaf number, root biomass, CO₂ assimilation and stomatal conductance over that of control plants growing in non-amended substrates. We conclude that the substrate amendment reduced the damaging effects of drought stress in citrus plants. The longer survival of seedlings in the amended treatment together with the reduction in leaf abscission and the improvement of physiological parameters, can account for a higher vigour of citrus grown under water stress conditions.     

dUTPase as a platform for antimalarial drug design: structural basis for the selectivity of a class of nucleoside inhibitors

Pyrimidine metabolism is a major route for therapeutic intervention against malaria. Here we report inhibition and structural studies on the deoxyuridine nucleotidohydrolase from the malaria parasite Plasmodium falciparum (PfdUTPase). We have identified a series of triphenylmethane derivatives of deoxyuridine with antimalarial activity in vitro which inhibit specifically the Plasmodium dUTPase versus the human enzyme. A 2.4 Angstrom crystal structure of PfdUTPase in complex with one of these inhibitors reveals an atypical trimeric enzyme in which the triphenylmethane derivative can be seen to select for PfdUTPase by way of interactions between the trityl group and the side chains of residues Phe46 and Ile117. Immunofluorescence microscopy studies of parasitized red blood cells reveal that enzyme concentrations are highest during the trophozoite/schizont stages, suggesting that PfdUTPase has a major role in DNA replication. Taken together the data show that PfdUTPase may be considered as an antimalarial drug target.