In vitro culture and biochemical characterization of six trypanosome isolates from Peru and Brazil

Six trypanosomatids isolated from different geographical areas from South America (Peru and Brazil) and different vectors and reservoir hosts (the triatomine Panstrongylus chinai [TP1], Triatoma infestans [TP2], Rhodnius ecuadorensis [TP3], R. prolixus [TB1], Didelphys marsupialis [TB2]), and one from a human asymptomatic patient [TB3], were characterized using lectin agglutination, isoenzyme profile, in vitro culture final metabolite patterns, and compared with a reference strain (Trypanosoma cruzi, Maracay strain [TC]). The different isolates were cultured in vitro in Grace's medium supplemented with 10% inactivated bovine foetal serum. According to our results and the statistical study, the isolate obtained from R. ecuadorensis should be designed as a Trypanosoma rangeli sp., showing all other isolates strong similarities to T. cruzi. Between them, two clusters could be identified, strongly correlating with the geographical origin. Cluster I grouped isolates from Peru and T. cruzi reference strain, and cluster II grouped the three Brazilian isolates.     

Phylogenetic position of Mediterranean Astereae and character evolution of daisies (Bellis,Asteraceae) inferred from nrDNA ITS sequences

Phylogenetic analyses of nrITS sequences of Asteraceae revealed that the Bellis group is a natural assemblage comprising all the species of Bellis and Bellium, but not Rhynchospermum. In contrast, we propose to include the genera Bellis, Bellium, and Bellidastrum in the subtribe Bellidinae in the interest of circumscribing natural groups. Our results also suggest an early diversification in the western Mediterranean Basin of two monophyletic lineages, Bellis and Bellium. Three major groups can be distinguished within BELLIS: (1) the B. perennis group, containing five annual and perennial species with three ploidy levels (diploid, octoploid, and decaploid), which are distributed throughout the Mediterranean Basin despite lack of pappus; (2) the Bellis sylvestris group, with five annual and perennial species primarily from the western Mediterranean, in which there are five ploidy levels (diploid, tetraploid, hexaploid, octoploid, and decaploid); and (3) a basal grade consisting of three diploid, perennial species which displays remarkable diversification of morphologies. Striking characteristics, such as an annual life form, polyploidy, and loss of pappus, seem to have occurred in parallel several times and in different geographical areas during the early diversification of Bellis species in the western Mediterranean. Character evolution reconstructions allow us to describe a putative ancestor of the genus Bellis (proto-Bellis).     

Uptake Hydrogenase (Hup) in Common Bean (Phaseolus vulgaris) Symbioses

Strains of Rhizobium forming nitrogen-fixing symbioses with common bean were systematically examined for the presence of the uptake hydrogenase (hup) structural genes and expression of uptake hydrogenase (Hup) activity. DNA with homology to the hup structural genes of Bradyrhizobium japonicum was present in 100 of 248 strains examined. EcoRI fragments with molecular sizes of approximately 20.0 and 2.2 kb hybridized with an internal SacI fragment, which contains part of both bradyrhizobial hup structural genes. The DNA with homology to the hup genes was located on pSym of one of the bean rhizobia. Hup activity was observed in bean symbioses with 13 of 30 strains containing DNA homologous with the hup structural genes. However, the Hup activity was not sufficient to eliminate hydrogen evolution from the nodules. Varying the host plant with two of the Hup⁺ strains indicated that expression of Hup activity was host regulated, as has been reported with soybean, pea, and cowpea strains.     

Osmoprotectants in Halomonas elongata: high-affinity betaine transport system and choline-betaine pathway.

The osmoregulatory pathways of the moderately halophilic bacterium Halomonas elongata DSM 3043 have been investigated. This strain grew optimally at 1.5 to 2 M NaCl in M63 glucose-defined medium. It required at least 0.5 M NaCl for growth, which is a higher concentration than that exhibited by the H. elongata type strain ATCC 33173. Externally provided betaine, choline, or choline-O-sulfate (but not proline, ectoine, or proline betaine) enhanced the growth of H. elongata on 3 M NaCl-glucose-M63 plates, demonstrating the utilization of these compounds as osmoprotectants. Moreover, betaine and choline stimulated the growth of H. elongata DSM 3043 over the entire range of salinity, although betaine was more effective than choline at salinities below and above the optimum. We found that H. elongata DSM 3043 has at least one high-affinity transport system for betaine (K(m) = 3.06 microM and Vmax = 9.96 nmol of betaine min(-1) mg of protein(-1)). Competition assays demonstrated that proline betaine and ectoine, but not proline, choline, or choline-O-sulfate, are also transported by the betaine permease. Finally, thin-layer chromatography and 13C-nuclear magnetic resonance analysis showed that exogenous choline was taken up and transformed to betaine by H. elongata, demonstrating the existence of a choline-glycine betaine pathway in this moderately halophilic bacterium.     

Poster Sessions AP01: Gene Expression and Regulation. Regulation of gene expression during the development of cortico‐thalamic connection in rat

The formation of Cortico‐Thalamic projections requires the precise spatial and temporal expression of proteins that are involved in the different stages of synaptogenesis. We reasoned that the underlying molecular mechanism of this process is the differential expression of genes that code for stage specific proteins. Our research objective was to identify the differential expressed mRNAs during the main stages of synapses formation, which starts at embryonic day 12 (E12) and finishes on the first postnatal days in the rat. We approach this problem using Differential Display technique on three distinct ages of rat cerebral cortex that were: E13, E18 and postnatal day 0 (P0). We found 80 differential bands using 54 random primers and 18 of them were cloned and sequenced. The sequence analysis showed among others, a cDNA fragment highly homologous with the human A Kinase Anchoring Protein 450/350 also called CG‐NAP. We found that this cDNA fragment homologous to AKAP was up regulated at E15 when cortical cells are undergoing active axogenesis. The expression pattern of this cDNA was confirmed by Real Time PCR. Our findings suggest a possible function for AKAP 450 in the regulation of the state of phosphorylation of centrosomal components during the initial stages of synapses formation during the establishment of Cortico‐Thalamic connection.     

Genetic composition,population structure and phylogeography of the loggerhead sea turtle: colonization hypothesis for the Brazilian rookeries

The loggerhead sea turtle, Caretta caretta, is the most common species of sea turtle nesting in Brazil and is listed as endangered by the IUCN. Our study characterizes the genetic structure of loggerheads in Brazil based on mitochondrial DNA control region variability and presents a hypothesis for the colonization of Brazilian rookeries. We analyzed 329 samples from Brazilian rookeries and an oceanic foraging ground, and we compared our results with previously published data for other loggerhead populations. Brazilian rookeries had four haplotypes, none of which have been reported for rookeries outside Brazil. Six haplotypes were found in the foraging aggregation. The presence of the CC-A4 haplotype at all sampled sites and the low nucleotide diversity suggest a common origin for all rookeries, with CC-A4 being the ancestral haplotype of the Brazilian populations. The occurrence of three haplotypes in the foraging aggregation that are known only from rookeries outside of Brazil is consistent with the transoceanic migratory behavior of loggerheads. Our results indicated that the colonization of Brazilian rookeries probably occurred from the southern USA stock. This recent colonization most likely followed a north to south route along the Brazilian coastline, influenced by the Brazilian warm current. Our results further suggest the existence of two genetic population units of loggerheads in Brazil and corroborate natal homing behavior in loggerheads.     

Efficacy of a Vaccine Formula against Tuberculosis in Cattle

“Test-and-slaughter” has been successful in industrialized countries to control and eradicate tuberculosis from cattle; however, this strategy is too expensive for developing nations, where the prevalence is especially high. Vaccination with the Calmette-Guérin (BCG) strain has been shown to protect against the development of lesions in vaccinated animals: mouse, cattle and wildlife species. In this study, the immune response and the pathology of vaccinated (BCG-prime and BCG prime-CFP-boosted) and unvaccinated (controls) calves were evaluated under experimental settings. A 10⁶ CFU dose of the BCG strain was inoculated subcutaneously on the neck to two groups of ten animas each. Thirty days after vaccination, one of the vaccinated groups was boosted with an M. bovis culture filtrate protein (CFP). Three months after vaccination, the three groups of animals were challenged with 5×10⁵ CFU via intranasal by aerosol with a field strain of M. bovis. The immune response was monitored throughout the study. Protection was assessed based on immune response (IFN-g release) prechallenge, presence of visible lesions in lymph nodes and lungs at slaughter, and presence of bacilli in lymph nodes and lung samples in histological analysis. Vaccinated cattle, either with the BCG alone or with BCG and boosted with CFP showed higher IFN-g response, fewer lesions, and fewer bacilli per lesion than unvaccinated controls after challenge. Animals with low levels of IFN-g postvaccine-prechallenge showed more lesions than animals with high levels. Results from this study support the argument that vaccination could be incorporated into control programs to reduce the incidence of TB in cattle in countries with high prevalence.     

Role of Ngamma-acetyldiaminobutyrate as an enzyme stabilizer and an intermediate in the biosynthesis of hydroxyectoine.

Strain CHR63 is a salt-sensitive mutant of the moderately halophilic wild-type strain Halomonas elongata DSM 3043 that is affected in the ectoine synthase gene (ectC). This strain accumulates large amounts of Ngamma-acetyldiaminobutyrate (NADA), the precursor of ectoine (D. Cánovas, C. Vargas, F. Iglesias-Guerra, L. N. Csonka, D. Rhodes, A. Ventosa, and J. J. Nieto, J. Biol. Chem. 272:25794-25801, 1997). Hydroxyectoine, ectoine, and glucosylglycerate were also identified by nuclear magnetic resonance (NMR) as cytoplasmic organic solutes in this mutant. Accumulation of NADA, hydroxyectoine, and ectoine was osmoregulated, whereas the levels of glucosylglycerate decreased at higher salinities. The effect of the growth stage on the accumulation of solutes was also investigated. NADA was purified from strain CHR63 and was shown to protect the thermolabile enzyme rabbit muscle lactate dehydrogenase against thermal inactivation. The stabilizing effect of NADA was greater than the stabilizing effect of ectoine or potassium diaminobutyrate. A (1)H NMR analysis of the solutes accumulated by the wild-type strain and mutants CHR62 (ectA::Tn1732) and CHR63 (ectC::Tn1732) indicated that H. elongata can synthesize hydroxyectoine by two different pathways-directly from ectoine or via an alternative pathway that converts NADA into hydroxyectoine without the involvement of ectoine.     

Molecular data and ecological niche modelling reveal the evolutionary history of the common and Iberian moles (Talpidae) in Europe

According to mitochondrial data, the common mole, Talpa europaea, is paraphyletic. This could be explained by either an ancient introgression of mtDNA from the Iberian blind mole T. occidentalis to T. europaea, or the existence of a differentiated taxonomic entity in northern Spain that needs to be described. In this study, we combined mitochondrial (Cytb) and nuclear (HDAC2) data to investigate these two alternative hypotheses. Based on both mitochondrial and nuclear data and an extensive geographical sampling (399 sequenced individuals), we show that the populations of T. europaea from Spain and south‐western France (south of the Loire River) are phylogenetically closer to T. occidentalis than to T. europaea. The Spanish–French lineage has some morphological characters resembling more to T. occidentalis (e.g. eyes) and others resembling more to T. europaea (external measurements, mesostyle of the first upper molar). It also seems to have several distinctive dental characters, suggesting that it should be recognized as a new species. Within the three lineages, we found a marked phylogeographical pattern, with several allopatric or parapatric lineages, dating from the Pleistocene. Our genetic data combined with species distribution models support the presence of several putative glacial refugia during glacial maxima for each species.     

Genetic polymorphism of serotonin transporter 5-HTTLPR: involvement in smoking behaviour

Data suggest that the serotonin (5-hydroxytryptamine, 5-HT) system is implicated in the pathogenesis of multiple neuropsychiatric disorders and may also be involved in smoking behaviour since nicotine increases brain serotonin secretion. It is known that smoking behaviour is influenced by both genetic and environmental factors. The present review examines the role of the serotonin transporter gene (5-HTT) in smoking behaviour and investigating studies that showed association of 5-HTT gene with smoking. This study discusses a polymorphism which has been investigated by many researchers, as the bi-allelic insertion/deletion polymorphism in the 5^′- flanking promoter region (5-HTTLPR). This gene has received considerable attention in attempts to understand the molecular determinants of smoking. Therefore, in the present study, the relationship between genetic polymorphism of serotonin transporter in smoking behaviour is reviewed considering the interactive effect of genetic factors.