Potentiation by ammonia of the metabolic effects of pent-4-enoate in isolated rat hepatocytes.

The metabolic effects of pent-4-enoate were studied in isolated rat hepatocytes; 1 mM-pent-4-enoate did not significantly inhibit gluconeogenesis from lactate, alanine and glycerol, but significantly decreased glucose synthesis from pyruvate. The addition of 1 mM-NH4Cl led to a drastic inhibition of glucose synthesis from all these substrates. In hepatocytes incubated with 10 mM-alanine and 1 mM-oleate, pent-4-enoate at 0.05-1 mM slightly inhibited glucose synthesis and ketogenesis. The addition of ammonia resulted in a dramatic potentiation of the metabolic effects of pent-4-enoate. Half-maximum effect of ammonia was observed at 0.2 mM concentration. Concomitant cellular concentrations of ATP and acetyl-CoA were also decreased by the addition of ammonia, as were lactate/pyruvate ratio and beta-hydroxybutyrate/acetoacetate ratio. These data suggest that ammonia seriously interferes with the cellular metabolism of pent-4-enoate and leads to a dramatic potentiation of its effects.     

Characterization of enzymatic deficiencies of branched chain amino-acid catabolism in human fibroblasts by genetic complementation

Leucine and Isoleucine metabolism in cultured skin fibroblasts from patients with leucinosis, beta-Ketothiolase deficiency, propionic, methylmalonic and isovaleric acidemia, was compared with that in normal fibroblasts. A simple assay was developed using (U14C) Isoleucine and (U14C) Leucine as substrates. Radioactive incorporation into protein aminoacids were measured. The (U14C) branched chain aminoacid incorporation into proteins provides an estimation of the protein synthesis and the incorporation ratio (14C) Aspartate + (14C) Glutamate/(14C) branched chain aminoacid, measures the integrity of the metabolic pathway. Complementation tests permits to characterize the genetic defect. The incorporation ratio was significantly decreased in blocked pathways, namely in leucinosis and isovaleric acidemia in the presence of (U14C) Leucine and in Leucinosis, beta-Ketothiolase deficiency, propionic and methylmalonic acidemia in the presence of (U14C) Isoleucine. There was a significant restoration of activity in mutant strains with distinct genetic defects after polyethylene-glycol fusion. This assay provides a valuable tool to screen for enzymatic deficiencies of branched chain aminoacid catabolism.     

Genotypic Characterization of Bradyrhizobium Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses

We examined the genotypic diversity of 64 Bradyrhizobium strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera Abrus, Alysicarpus, Bryaspis, Chamaecrista, Cassia, Crotalaria, Desmodium, Eriosema, Indigofera, Moghania, Rhynchosia, Sesbania, Tephrosia, and Zornia, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different Bradyrhizobium species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the Bradyrhizobium strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in Bradyrhizobium, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.     

Swine and poultry pathogens: the complete genome sequences of two strains of Mycoplasma hyopneumoniae and a strain of Mycoplasma synoviae

This work reports the results of analyses of three complete mycoplasma genomes, a pathogenic (7448) and a nonpathogenic (J) strain of the swine pathogen Mycoplasma hyopneumoniae and a strain of the avian pathogen Mycoplasma synoviae; the genome sizes of the three strains were 920,079 bp, 897,405 bp, and 799,476 bp, respectively. These genomes were compared with other sequenced mycoplasma genomes reported in the literature to examine several aspects of mycoplasma evolution. Strain-specific regions, including integrative and conjugal elements, and genome rearrangements and alterations in adhesin sequences were observed in the M. hyopneumoniae strains, and all of these were potentially related to pathogenicity. Genomic comparisons revealed that reduction in genome size implied loss of redundant metabolic pathways, with maintenance of alternative routes in different species. Horizontal gene transfer was consistently observed between M. synoviae and Mycoplasma gallisepticum. Our analyses indicated a likely transfer event of hemagglutinin-coding DNA sequences from M. gallisepticum to M. synoviae.     

Histological analysis reveals the formation of shoots rather than embryos in regenerating cultures of <Emphasis Type="Italic">Eucalyptus globulus</Emphasis>

Eucalyptus globulus is an important species in international forestry in regions with Mediterranean climates and comprises 65?% of the plantation hardwood in Australia. Propagation by somatic embryogenesis would offer many advantages and its development has received much attention. Structures regenerating on explants from hypocotyls of mature zygotic embryos of E. globulus cultured on medium with NAA, reported previously to be effective for embryogenic regeneration, were analyzed morphologically and histologically to clarify their pathway of development. Analysis of series of sections revealed organogenic, rather than embryogenic, pathways of regeneration in this system. We show that protocols for propagation of E. globulus must be carefully evaluated by microscopic examination of adequate numbers of serial sections.     

When invasion may not be harmful: niche relations in a lizard assemblage

Some studies have suggested that non‐native species invasions may threaten local diversity by creating homogenized environments. However, many studies have been based on limited or anecdotal data, and/or have failed to consider the influence of habitat modification together with possible influences of non‐native species on native ones. Hemidactylus mabouia (Squamata, Gekkonidae) likely invaded natural environments in Brazil hundreds of years ago. Yet, little is known about whether it affects native lizard fauna. We tested whether H. mabouia negatively influences native lizard species richness and abundance on a regional scale and locally through niche overlap. We analyzed species abundance and richness of nine lizard assemblages, in five of which H. mabouia occurred. We evaluated niche overlap of species in a lizard assemblage with high H. mabouia abundance through null models. Niche axes included spatial use, temporal activity and diet. Although species abundance did not differ among sites with and without the invasive species, the presence of H. mabouia seems constrained to the richer assemblages sampled. We observed significantly higher niche overlap in spatial (?_obs = 0.63; ?_exp = 0.37; P_obs ≥ P_exp = 0.0002) and trophic axes (?_obs = 0.46; ?_exp = 0.17; P_obs ≥ P_exp < 0.001), but not in activity. When we considered all axes (three‐dimensional niche), there was no overlapping among the lizard species. Our findings did not support the hypothesis that this non‐native species negatively influences other sympatric lizard species.     

Changes in γ-aminobutyric acid concentration,gas exchange,and leaf anatomy in <Emphasis Type="Italic">Eucalyptus</Emphasis> clones under drought stress and rewatering

Drought stress promotes biochemical and physiological alterations in plant metabolism that limit growth and yield. This study investigated the accumulation of γ-aminobutyric acid (GABA) in plant tissue, the stomatal conductance (gs) and changes in leaf anatomy in Eucalyptus following drought stress situation. In this study, eight Eucalyptus clones were evaluated under normal water supply (control) and drought stress conditions (stress). For the control treatment, plants were irrigated every day with an automated system until the soil was saturated, and for the stress treatment, drought stress was imposed by non-irrigation of plants, and pots were covered using plastic sheeting to avoid rainfall and humidity. This study has shown that: (1) all clones decreased gs with increasing vapor pressure deficit (D) in both treatments. All plastics and drought-tolerant clones (except GG) presented lower stomatal sensitivity to D under stress conditions than drought-sensitive clones; (2) GABA concentrations increased fast after drought stress, but we could not find correlation with these changes and resistance to water stress; and (3) all clones increased the number of stomata and reduced leaf thickness after water stress. The finding is that GABA is a fast stress-signaling molecule in Eucalyptus, but the response of gs to D is a best physiological variable to differentiate drought-tolerant and drought-sensitive Eucalyptus clones.     

Nuclear markers reveal a complex introgression pattern among marine turtle species on the Brazilian coast

Surprisingly, a high frequency of interspecific sea turtle hybrids has been previously recorded in a nesting site along a short stretch of the Brazilian coast. Mitochondrial DNA data indicated that as much as 43% of the females identified as Eretmochelys imbricata are hybrids in this area (Bahia State of Brazil). It is a remarkable find, because most of the nesting sites surveyed worldwide, including some in northern Brazil, presents no hybrids, and rare Caribbean sites present no more than 2% of hybrids. Thus, a detailed understanding of the hybridization process is needed to evaluate natural or anthropogenic causes of this regional phenomenon in Brazil, which could be an important factor affecting the conservation of this population. We analysed a set of 12 nuclear markers to investigate the pattern of hybridization involving three species of sea turtles: hawksbill (E. imbricata), loggerhead (Caretta caretta) and olive ridley (Lepidochelys olivacea). Our data indicate that most of the individuals in the crossings L. olivacea × E. imbricata and L. olivacea × C. caretta are F1 hybrids, whereas C. caretta × E. imbricata crossings present F1 and backcrosses with both parental species. In addition, the C. caretta × E. imbricata hybridization seems to be gender and species biased, and we also found one individual with evidence of multispecies hybridization among C. caretta × E. imbricata × Chelonia mydas. The overall results also indicate that hybridization in this area is a recent phenomenon, spanning at least two generations or ～40 years.     

Plasmid-based controls to detect rpoB mutations in Mycobacterium tuberculosis by quantitative polymerase chain reaction-high-resolution melting

Quantitative polymerase chain reaction-high-resolution melting (qPCR-HRM) analysis was used to screen for mutations related to drug resistance in Mycobacterium tuberculosis. We detected the C526T and C531T mutations in the rifampicin resistance-determining region (RRDR) of the rpoB gene with qPCR-HRM using plasmid-based controls. A segment of the RRDR region from M. tuberculosis H37Rv and from strains carrying C531T or C526T mutations in the rpoB were cloned into pGEM-T vector and these vectors were used as controls in the qPCR-HRM analysis of 54 M. tuberculosis strains. The results were confirmed by DNA sequencing and showed that recombinant plasmids can replace genomic DNA as controls in the qPCR-HRM assay. Plasmids can be handled outside of biosafety level 3 facilities, reducing the risk of contamination and the cost of the assay. Plasmids have a high stability, are normally maintained in Escherichia coli and can be extracted in large amounts.