Global gene expression under nitrogen starvation in Xylella fastidiosa: contribution of the sigma(54) regulon

Background: Xylella fastidiosa, a Gram-negative fastidious bacterium, grows in the xylem of several plants causing diseases such as citrus variegated chlorosis. As the xylem sap contains low concentrations of amino acids and other compounds, X. fastidiosa needs to cope with nitrogen limitation in its natural habitat. Results: In this work, we performed a whole-genome microarray analysis of the X. fastidiosa nitrogen starvation response. A time course experiment (2, 8 and 12 hours) of cultures grown in defined medium under nitrogen starvation revealed many differentially expressed genes, such as those related to transport, nitrogen assimilation, amino acid biosynthesis, transcriptional regulation, and many genes encoding hypothetical proteins. In addition, a decrease in the expression levels of many genes involved in carbon metabolism and energy generation pathways was also observed. Comparison of gene expression profiles between the wild type strain and the rpoN null mutant allowed the identification of genes directly or indirectly induced by nitrogen starvation in a sigma(54)-dependent manner. A more complete picture of the sigma(54) regulon was achieved by combining the transcriptome data with an in silico search for potential sigma(54)-dependent promoters, using a position weight matrix approach. One of these sigma(54)-predicted binding sites, located upstream of the glnA gene (encoding glutamine synthetase), was validated by primer extension assays, confirming that this gene has a sigma(54)-dependent promoter. Conclusions: Together, these results show that nitrogen starvation causes intense changes in the X. fastidiosa transcriptome and some of these differentially expressed genes belong to the sigma(54) regulon.

An insight into the sialotranscriptome of the brown dog tick, Rhipicephalus sanguineus

Background: Rhipicephalus sanguineus, known as the brown dog tick, is a common ectoparasite of domestic dogs and can be found worldwide. R. sanguineus is recognized as the primary vector of the etiological agent of canine monocytic ehrlichiosis and canine babesiosis. Here we present the first description of a R. sanguineus salivary gland transcriptome by the production and analysis of 2,034 expressed sequence tags (EST) from two cDNA libraries, one consctructed using mRNA from dissected salivary glands from female ticks fed for 3-5 days (early to mid library, RsSGL1) and the another from ticks fed for 5 days (mid library, RsSGL2), identifying 1,024 clusters of related sequences. Results: Based on sequence similarities to nine different databases, we identified transcripts of genes that were further categorized according to function. The category of putative housekeeping genes contained similar to 56% of the sequences and had on average 2.49 ESTs per cluster, the secreted protein category contained 26.6% of the ESTs and had 2.47 EST's/clusters, while 15.3% of the ESTs, mostly singletons, were not classifiable, and were annotated as ""unknown function"". The secreted category included genes that coded for lipocalins, proteases inhibitors, disintegrins, metalloproteases, immunomodulatory and antiinflammatory proteins, as Evasins and Da-p36, as well as basic-tail and 18.3 kDa proteins, cement proteins, mucins, defensins and antimicrobial peptides. Comparison of the abundance of ESTs from similar contigs of the two salivary gland cDNA libraries allowed the identification of differentially expressed genes, such as genes coding for Evasins and a thrombin inhibitor, which were over expressed in the RsSGL1 (early to mid library) versus RsSGL2 (mid library), indicating their role in inhibition of inflammation at the tick feeding site from the very beginning of the blood meal. Conversely, sequences related to cement (64P), which function has been correlated with tick attachment, was largely expressed in the mid library. Conclusions: Our survey provided an insight into the R. sanguineus sialotranscriptome, which can assist the discovery of new targets for anti-tick vaccines, as well as help to identify pharmacologically active proteins.

Functional characterization of the sciarid BhC4-1 core promoter in transgenic Drosophila

Background: Core promoters are cis-regulatory modules to which bind the basal transcriptional machinery and which participate in the regulation of transcription initiation. Although core promoters have not been extensively investigated through functional assays in a chromosomal context, the available data suggested that the response of a given core promoter might vary depending on the promoter context. Previous studies suggest that a (-57/+40) fragment constitutes the core promoter of the BhC4-1 gene which is located in DNA puff C4 of the sciarid fly Bradysia hygida. Here we tested this (-57/+40) fragment in distinct regulatory contexts in order to verify if promoter context affects its core promoter activity. Results: Consistent with the activity of a core promoter, we showed that in the absence of upstream regulatory sequences the (-57/+40) fragment drives low levels of reporter gene mRNA expression throughout development in transgenic Drosophila. By assaying the (-57/+40) fragment in two distinct regulatory contexts, either downstream of the previously characterized Fbp1 enhancer or downstream of the UAS element, we showed that the BhC4-1 core promoter drives regulated transcription in both the germline and in various tissues throughout development. Furthermore, the use of the BhC4-1 core promoter in a UAS construct significantly reduced salivary gland ectopic expression in third instar larvae, which was previously described to occur in the context of the GAL4/UAS system. Conclusions: Our results from functional analysis in transgenic Drosophila show that the BhC4-1 core promoter drives gene expression regardless of the promoter context that was assayed. New insights into the functioning of the GAL4/UAS system in Drosophila were obtained, indicating that the presence of the SV40 sequence in the 3' UTR of a UAS construct does not preclude expression in the germline. Furthermore, our analysis indicated that ectopic salivary gland expression in the GAL4/UAS system does not depend only on sequences present in the GAL4 construct, but can also be affected by the core promoter sequences in the UAS construct. In this context, we propose that the sciarid BhC4-1 core promoter constitutes a valuable core promoter which can be employed in functional assays in insects.

Genome Survey Sequence Analysis and Identification of Homologs of Major Surface Protease (gp63) Genes in Trypanosoma rangeli

In this study, 222 genome survey sequences were generated for Trypanosoma rangeli strain P07 isolated from an opossum (Didelphis albiventris) in Minas Gerais State, Brazil. T. rangeli sequences were compared by BLASTX (Basic Local Alignment Search Tool X) analysis with the assembled contigs of Leishmania braziliensis, Leishmania infantum, Leishmania major, Trypanosoma brucei, and Trypanosoma cruzi. Results revealed that 82% (182/222) of the sequences were associated with predicted proteins described, whereas 18% (40/222) of the sequences did not show significant identity with sequences deposited in databases, suggesting that they may represent T. rangeli-specific sequences. Among the 182 predicted sequences, 179 (80.6%) had the highest similarity with T. cruzi, 2 (0.9%) with T. brucei, and 1 (0.5%) with L. braziliensis. Computer analysis permitted the identification of members of various gene families described for trypanosomatids in the genome of T. rangeli, such as trans-sialidases, mucin-associated surface proteins, and major surface proteases (MSP or gp63). This is the first report identifying sequences of the MSP family in T. rangeli. Multiple sequence alignments showed that the predicted MSP of T. rangeli presented the typical characteristics of metalloproteases, such as the presence of the HEXXH motif, which corresponds to a region previously associated with the catalytic site of the enzyme, and various cysteine and proline residues, which are conserved among MSPs of different trypanosomatid species. Reverse transcriptase-polymerase chain reaction analysis revealed the presence of MSP transcripts in epimastigote forms of T. rangeli.

Hantavirus Pulmonary Syndrome, Central Plateau, Southeastern, and Southern Brazil

Hantavirus pulmonary syndrome (HPS) is an increasing health problem in Brazil because of encroachment of sprawling urban, agricultural, and cattle-raising areas into habitats of subfamily Sigmodontinae rodents, which serve as hantavirus reservoirs. From 1993 through June 2007, a total of 884 cases of HIPS were reported in Brazil (case-fatality rate 39%). To better understand this emerging disease, we collected 89 human serum samples and 68 rodent lung samples containing antibodies to hantavirus from a 2,500-km-wide area in Brazil. RNA was isolated from human samples and rodent lung tissues and subjected to reverse transcription-PCR. Partial sequences of nucleocapsid protein and glycoprotein genes from 22 human and 16 rodent sources indicated only Araraquara virus and Juquitiba virus lineages. The case-fatality rate of HPS was higher in the area with Araraquara virus. This virus, which may be the most virulent hantavirus in Brazil, was associated with areas that have had greater anthropogenic changes.

Central nitrergic system regulation of neuroendocrine secretion, fluid intake and blood pressure induced by angiotensin-II

Background: Nitric oxide (NO) synthesis has been described in several circumventricular and hypothalamic structures in the central nervous system that are implicated in mediating central angiotensin-II (ANG-II) actions during water deprivation and hypovolemia. Neuroendocrine and cardiovascular responses, drinking behavior, and urinary excretions were examined following central angiotensinergic stimulation in awake freely-moving rats pretreated with intracerebroventricular injections of N omega-nitro-L-arginine methyl ester (L-NAME, 40 mu g), an inhibitor of NO synthase, and L-arginine (20 ug), a precursor of NO. Results: Injections of L-NAME or ANG-II produced an increase in plasma vasopressin (VP), oxytocin (OT) and atrial natriuretic peptide (ANP) levels, an increase in water and sodium intake, mean arterial blood pressure and sodium excretion, and a reduction of urinary volume. L-NAME pretreatment enhanced the ANG-II response, while L-arginine attenuated VP and OT release, thirst, appetite for sodium, antidiuresis, and natriuresis, as well as pressor responses induced by ANG-II. Discussion and conclusion: Thus, the central nitrergic system participates in the angiotensinergic responses evoked by water deprivation and hypovolemia to refrain neurohypophysial secretion, hydromineral balance, and blood pressure homeostasis.

Concurrent sequence variation of TP53 and TP73 genes in anaplastic astrocytoma

Disruption or loss of tumor suppressor gene TP53 is implicated in the development or progression of almost all different types of human malignancies. Other members of the p53 family have been identified. One member, p73, not only shares a high degree of similarity with p53 in its primary sequence, but also has similar functions. Like p53, p73 can bind to DNA and activate transcription. Using PCR-SSCP and gene sequencing, we analyzed the TP53 and TP73 genes in a case of a grade III anaplastic astrocytoma that progressed to glioblastoma. We found a deletion of AAG at position 595-597 of TP53 (exon 6), resulting in the deletion of Glu 199 in the protein and a genomic polymorphism of TP73, identified as an A-to-G change, at position E8/+15 at intron 8 (IVS8-15A>G). The mutation found at exon 6 of the gene TP53 could be associated with the rapid tumoral progression found in this case, since the mutated p53 may inactivate the wild-type p53 and the p73 alpha protein, which was conserved here, leading to an increase in cellular instability.

First Report of the Isolation and Molecular Characterization of Rickettsia amblyommii and Rickettsia felis in Central America

During 2010, 15 adult ticks, identified as Amblyomma cajennense, were collected from horses in Cahuita and Turrialba districts, whereas 7 fleas, identified as Ctenocephalides felis, were collected from a dog in San Jose city, Costa Rica. In the laboratory, three A. cajennense specimens, two from Cahuita and one from Turrialba, were individually processed for rickettsial isolation in cell culture, as was a pool of seven fleas. Rickettsiae were successfully isolated and established in Vero cell culture from the three ticks and from a pool of seven fleas in C6/36 cell culture. The three tick isolates were genotypically identified as Rickettsia amblyommii, and the flea isolate was identified as Rickettsia felis through DNA sequencing of portions of the rickettsial genes gltA, ompA, and ompB of each isolate. In addition, other seven ticks were shown to contain rickettsial DNA. Polymerase chain reaction products of at least two of these ticks were sequenced and also showed to correspond to R. amblyommii. Overall, 66.7% (10/15) of the A. cajennense adult ticks were found to be infected with rickettsiae. This is the first report of a successful isolation in cell culture of R. amblyommii and R. felis from Central America.

Determination and Molecular Analysis of the Complete Genome Sequence of Two Wild-Type Rabies Viruses Isolated from a Haematophagous Bat and a Frugivorous Bat in Brazil

The complete genome sequences of two Brazilian wild-type rabies viruses (RABV), a BR-DR1 isolate from a haematophagous bat (Desmodus rotundus) and a BR-AL1 isolate from a frugivorous bat (Artibeus lituratus), were determined. The genomes of the BR-DR1 and RR-AL1 had 11,923 and 11,922 nt, respectively, and both encoded the five standard genes of rhabdoviruses. The complete nucleotide sequence identity between the BR-DR1 and BR-AL1 isolates was 97%. The BR-DR1 and BR-AL1 isolates had some conserved functional sites revealed by the fixed isolates, whereas both isolates had unique amino acid substitutions in the antigenic region IV of the nucleocapsid gene. Therefore, it is speculated that both isolates were nearly identical in virologic character. According to our phylogenetic analysis based on the complete genomes, both isolates belonged to genotype 1, and to the previously defined ""vampire bat-related RABV lineage"" which consisted of mainly D. rotundus- and A. lituratus- isolates; however, a branch pattern with high bootstrap values suggested that BR-DR1 was more closely related to the 9001FRA isolate, which was collected from a dog bitten by a bat in French Guiana, than to BR-AL1. This result suggests that the vampire bat-related RABV lineage includes Brazilian vampire bat and Brazilian frugivorous bat RABV and is further divided into Brazilian vampire bat and Brazilian frugivorous bat RABV sub-lineages. The phylogenetic analysis based on the complete genomes was valuable in discriminating among very closely related isolates.

Tooth Movement Activates the Central Amygdala and the Lateral Hypothalamus by the Magnitude of the Force Applied

Objective: To determine if the magnitude of the force used to induce incisor tooth movement promotes distinct activation in cells in the central amygdala (CEA) and lateral hypothalamus (LH) of rats. Also, the effect of morphine on Fos immunoreactivity (Fos-IR) was investigated in these nuclei. Materials and Methods: Adult male rats were anesthetized and divided into six groups: only anesthetized (control), without orthodontic appliance (OA), OA but without force, OA activated with 30g or 70g, OA with 70g in animals pretreated with morphine (2 mg/kg, intraperitoneal). Three hours after the onset of the experiment the rats were reanesthetized and perfused with 4% paraformaldehyde. The brains were removed and fixed, and sections containing CEA and LH were processed for Fos protein immunohistochemistry. Results: The results show that in the control group, the intramuscular injection of a ketamine/xylazine mixture did not induce Fos-IR cells in the CEA or in the LH. Again, the without force group showed a little Fos-IR. However, in the 70g group the Fos-IR was the biggest observed (P < .05, Tukey) in the CEA and LH compared with the other groups. In the 30g group, the Fos-IR did not differ from the control group, the without OA group, and the without force group. Furthermore, pretreatment with morphine in the 70g group reduced Fos-IR in these regions. Conclusions: Tooth movement promotes Fos-IR in the CEA and LH according to the magnitude of the force applied. (Angle Orthod. 2010;80:111-115.)

Mosquitoes infected with dengue viruses in Brazil

Dengue epidemics have been reported in Brazil since 1985. The scenery has worsened in the last decade because several serotypes are circulating and producing a hyper-endemic situation, with an increase of DHF/DSS cases as well as the number of fatalities. Herein, we report dengue virus surveillance in mosquitoes using a Flavivirus genus-specific RT-Hemi-Nested-PCR assay. The mosquitoes (Culicidae, n = 1700) collected in the Northeast, Southeast and South of Brazil, between 1999 and 2005, were grouped into 154 pools. Putative genomes of DENV-1, -2 and -3 were detected in 6 mosquito pools (3.8%). One amplicon of putative DENV-1 was detected in a pool of Haemagogus leucocelaenus suggesting that this virus could be involved in a sylvatic cycle. DENV-3 was found infecting 3 pools of larvae of Aedes albopictus and the nucleotide sequence of one of these viruses was identified as DENV-3 of genotype III, phylogenetically related to other DENV-3 isolated in Brazil. This is the first report of a nucleotide sequence of DENV-3 from larvae of Aedes albopictus.

Chorioallantoic placentation in Galea spixii (Rodentia, Caviomorpha, Caviidae)

Background: Placentas of guinea pig-related rodents are appropriate animal models for human placentation because of their striking similarities to those of humans. To optimize the pool of potential models in this context, it is essential to identify the occurrence of characters in close relatives. Methods: In this study we first analyzed chorioallantoic placentation in the prea, Galea spixii, as one of the guinea pig's closest relatives. Material was collected from a breeding group at the University of Mossoro, Brazil, including 18 individuals covering an ontogenetic sequence from initial pregnancy to term. Placentas were investigated by means of histology, electron microscopy, immunohistochemistry (vimentin, alpha-smooth muscle actin, cytokeration) and proliferation activity (PCNA). Results: Placentation in Galea is primarily characterized by an apparent regionalization into labyrinth, trophospongium and subplacenta. It also has associated growing processes with clusters of proliferating trophoblast cells at the placental margin, internally directed projections and a second centre of proliferation in the labyrinth. Finally, the subplacenta, which is temporarily supplied in parallel by the maternal and fetal blood systems, served as the center of origin for trophoblast invasion. Conclusion: Placentation in Galea reveals major parallels to the guinea pig and other caviomorphs with respect to the regionalization of the placenta, the associated growing processes, as well as trophoblast invasion. A principal difference compared to the guinea pig occurred in the blood supply of the subplacenta. Characteristics of the invasion and expanding processes indicate that Galea may serve as an additional animal model that is much smaller than the guinea pig and where the subplacenta partly has access to both maternal and fetal blood systems.

Distinguishing post-AGB impostors in a sample of pre-main sequence stars

Context. A sample of 27 sources, cataloged as pre-main sequence stars by the Pico dos Dias Survey (PDS), is analyzed to investigate a possible contamination by post-AGB stars. The far-infrared excess due to dust present in the circumstellar envelope is typical of both categories: young stars and objects that have already left the main sequence and are suffering severe mass loss. Aims. The two known post-AGB stars in our sample inspired us to seek for other very likely or possible post-AGB objects among PDS sources previously suggested to be Herbig Ae/Be stars, by revisiting the observational database of this sample. Methods. In a comparative study with well known post-AGBs, several characteristics were evaluated: (i) parameters related to the circumstellar emission; (ii) spatial distribution to verify the background contribution from dark clouds; (iii) spectral features; and (iv) optical and infrared colors. Results. These characteristics suggest that seven objects of the studied sample are very likely post-AGBs, five are possible post-AGBs, eight are unlikely post-AGBs, and the nature of seven objects remains unclear.

Fast ray-tracing algorithm for circumstellar structures (FRACS) II. Disc parameters of the B[e] supergiant CPD-57 degrees 2874 from VLTI/MIDI data

Context. B[e] supergiants are luminous, massive post-main sequence stars exhibiting non-spherical winds, forbidden lines, and hot dust in a disc-like structure. The physical properties of their rich and complex circumstellar environment (CSE) are not well understood, partly because these CSE cannot be easily resolved at the large distances found for B[e] supergiants (typically greater than or similar to 1 kpc). Aims. From mid-IR spectro-interferometric observations obtained with VLTI/MIDI we seek to resolve and study the CSE of the Galactic B[e] supergiant CPD-57 degrees 2874. Methods. For a physical interpretation of the observables (visibilities and spectrum) we use our ray-tracing radiative transfer code (FRACS), which is optimised for thermal spectro-interferometric observations. Results. Thanks to the short computing time required by FRACS (<10 s per monochromatic model), best-fit parameters and uncertainties for several physical quantities of CPD-57 degrees 2874 were obtained, such as inner dust radius, relative flux contribution of the central source and of the dusty CSE, dust temperature profile, and disc inclination. Conclusions. The analysis of VLTI/MIDI data with FRACS allowed one of the first direct determinations of physical parameters of the dusty CSE of a B[e] supergiant based on interferometric data and using a full model-fitting approach. In a larger context, the study of B[e] supergiants is important for a deeper understanding of the complex structure and evolution of hot, massive stars.

Age distribution of the central stars of galactic disk planetary nebulae

Context. Determination of the ages of central stars of planetary nebulae (CSPN) is a complex problem, and there is presently no single method that can be generally applied. We have developed several methods of estimating the ages of CSPN, based on both the observed nebular properties and some properties of the stars themselves. Aims. Our aim is to estimate the ages and the age distribution of CSPN and to compare the derived results with mass and age determinations of CSPN and white dwarfs based on empirical determinations of these quantities. Methods. We considered a sample of planetary nebulae in the galactic disk, most of which (similar to 69%) are located in the solar neighbourhood, within 3 kpc from the Sun. We discuss several methods of deriving the age distribution of CSPN, namely; (i) the use of an age-metallicity relation that also depends on the galactocentric distance; (ii) the use of an age-metallicity relation obtained for the galactic disk; and (iii) the determination of ages from the central star masses obtained from the observed nitrogen abundances. Results. We estimated the age distribution of CSPN with average uncertainties of 1-2 Gyr, and compared our results with the expected distribution based both on the observed mass distribution of white dwarfs and on the age distribution derived from available mass distributions of CSPN. Based on our derived age distributions, we conclude that most CSPN in the galactic disk have ages under 6 Gyr, and that the age distribution is peaked around 2-4 Gyr.