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.

Cytogenetic Instability in Childhood Acute Lymphoblastic Leukemia Survivors

Contemporary anticancer therapies have largely improved the outcome for children with cancer, especially for Acute Lymphoblastic Leukemia (ALL). Actually, between 78% and 85% of patients achieve complete remission and are alive after 5 years of therapy completion. However, as cure rates increase, new concerns about the late effects of genotoxic treatment emerge, being the risk of developing secondary neoplasias, the most serious life-threatening rising problem. In the present paper, we describe and review the cytogenetic findings in peripheral lymphocytes from ALL survivors, and discuss aspects associated to the occurrence of increased chromosome rearrangements in this growing cohort.

Tetraploidization in Wilms tumor in an infant

Genetic instability is frequent in human cancer. Unscheduled tetraploidization can trigger cell transformation and tumorigenesis. We made a cytogenetic analysis by Giemsa-trypsin banding of a stage I, biphasic Wilms tumor diagnosed in a 10-month-old male. An evident karyotypic heterogeneity was found. Four different subclones of tumor cells were observed, with DNA content varying from diploid to near-tetraploid complements. The genetic events involved in the acquisition of aneuploidy in Wilms tumor remain unclear. We hypothesize that initial tetraploidization caused aberrant cell division, leading to abnormal chromosomal segregation, cell transformation and tumorigenesis.

PAMM: A Redox Regulatory Protein That Modulates Osteoclast Differentiation

The central role of reactive oxygen species (ROS) in osteoclast differentiation and in bone homeostasis prompted us to characterize the redox regulatory system of osteoclasts. In this report, we describe the expression and functional characterization of PAMM, a CXXC motif-containing peroxiredoxin 2-like protein expressed in bone marrow monocytes on stimulation with M-CSF and RANKL. Expression of wild-type (but not C to G mutants of the CXXC domain) PAMM in HEK293 cells results in an increased GSH/GSSG ratio, indicating a shift toward a more reduced environment. Expression of PAMM in RAW264.7 monocytes protected cells from hydrogen peroxide-induced oxidative stress, indicating that PAMM regulates cellular redox status. RANKL stimulation of RAW 264.7 cells caused a decrease in the GSH/GSSG ratio (reflecting a complementary increase in ROS). In addition, RANKL-induced osteoclast formation requires phosphorylation and translocation of NF-kappa B and c-Jun. In stably transfected RAW 264.7 cells, PAMM overexpression prevented the reduction of GSH/GSSG induced by RANKL. Concurrently, PAMM expression completely abolished RANKL-induced p100 NF-kappa B and c-Jun activation, as well as osteoclast formation. We conclude that PAMM is a redox regulatory protein that modulates osteoclast differentiation in vitro. PAMM expression may affect bone resorption in vivo and help to maintain bone mass. Antioxid. Redox Signal. 13, 27-37.

Karyotypic evolution trends in Rhamdia quelen (Siluriformes, Heptapteridae) with considerations about the origin and differentiation of its supernumerary chromosomes

Among catfish species of the genus Rhamdia reported for the Brazilian territory, R. quelen is the most widespread, being found in nearly all hydrographic basins of Brazil. Nowadays, R. quelen is a synonym for at least 47 other species in this genus, its taxonomic status still being controversial. The available cytogenetic reports show a wide variation in the karyotypic macrostructure, with the frequent presence of supernumerary chromosomes. The remarkable cytogenetic variability associated with taxonomic issues in this species indicates that R. quelen is actually a species complex. In order to carry out a wide comparative cytogenetic study in R. quelen from southern and southeastern Brazil and examine a species complex, we analyzed the chromosomes of 14 populations from the main hydrographic basins of these two regions. Using classic and molecular cytogenetic techniques, we found seven distinct karyotypic formulae, all bearing 2n = 58 chromosomes. Supernumerary chromosomes were present in most of the populations; their number, size and C-banding pattern allowed us to differentiate populations with similar karyotypic compositions. We examined patterns of chromosomal evolution as well as the probable mechanisms involved in the origin and morphological differentiation of their supernumerary chromosomes.

Leishmania-Specific Surface Antigens Show Sub-Genus Sequence Variation and Immune Recognition

Background: A family of hydrophilic acylated surface (HASP) proteins, containing extensive and variant amino acid repeats, is expressed at the plasma membrane in infective extracellular (metacyclic) and intracellular (amastigote) stages of Old World Leishmania species. While HASPs are antigenic in the host and can induce protective immune responses, the biological functions of these Leishmania-specific proteins remain unresolved. Previous genome analysis has suggested that parasites of the sub-genus Leishmania (Viannia) have lost HASP genes from their genomes. Methods/Principal Findings: We have used molecular and cellular methods to analyse HASP expression in New World Leishmania mexicana complex species and show that, unlike in L. major, these proteins are expressed predominantly following differentiation into amastigotes within macrophages. Further genome analysis has revealed that the L. (Viannia) species, L. (V.) braziliensis, does express HASP-like proteins of low amino acid similarity but with similar biochemical characteristics, from genes present on a region of chromosome 23 that is syntenic with the HASP/SHERP locus in Old World Leishmania species and the L. (L.) mexicana complex. A related gene is also present in Leptomonas seymouri and this may represent the ancestral copy of these Leishmania-genus specific sequences. The L. braziliensis HASP-like proteins (named the orthologous (o) HASPs) are predominantly expressed on the plasma membrane in amastigotes and are recognised by immune sera taken from 4 out of 6 leishmaniasis patients tested in an endemic region of Brazil. Analysis of the repetitive domains of the oHASPs has shown considerable genetic variation in parasite isolates taken from the same patients, suggesting that antigenic change may play a role in immune recognition of this protein family. Conclusions/Significance: These findings confirm that antigenic hydrophilic acylated proteins are expressed from genes in the same chromosomal region in species across the genus Leishmania. These proteins are surface-exposed on amastigotes (although L. (L.) major parasites also express HASPB on the metacyclic plasma membrane). The central repetitive domains of the HASPs are highly variant in their amino acid sequences, both within and between species, consistent with a role in immune recognition in the host.

Retinoic Acid-Treated Pluripotent Stem Cells Undergoing Neurogenesis Present Increased Aneuploidy and Micronuclei Formation

The existence of loss and gain of chromosomes, known as aneuploidy, has been previously described within the central nervous system. During development, at least one-third of neural progenitor cells (NPCs) are aneuploid. Notably, aneuploid NPCs may survive and functionally integrate into the mature neural circuitry. Given the unanswered significance of this phenomenon, we tested the hypothesis that neural differentiation induced by all-trans retinoic acid (RA) in pluripotent stem cells is accompanied by increased levels of aneuploidy, as previously described for cortical NPCs in vivo. In this work we used embryonal carcinoma (EC) cells, embryonic stem (ES) cells and induced pluripotent stem (iPS) cells undergoing differentiation into NPCs. Ploidy analysis revealed a 2-fold increase in the rate of aneuploidy, with the prevalence of chromosome loss in RA primed stem cells when compared to naive cells. In an attempt to understand the basis of neurogenic aneuploidy, micronuclei formation and survivin expression was assessed in pluripotent stem cells exposed to RA. RA increased micronuclei occurrence by almost 2-fold while decreased survivin expression by 50%, indicating possible mechanisms by which stem cells lose their chromosomes during neural differentiation. DNA fragmentation analysis demonstrated no increase in apoptosis on embryoid bodies treated with RA, indicating that cell death is not the mandatory fate of aneuploid NPCs derived from pluripotent cells. In order to exclude that the increase in aneuploidy was a spurious consequence of RA treatment, not related to neurogenesis, mouse embryonic fibroblasts were treated with RA under the same conditions and no alterations in chromosome gain or loss were observed. These findings indicate a correlation amongst neural differentiation, aneuploidy, micronuclei formation and survivin downregulation in pluripotent stem cells exposed to RA, providing evidence that somatically generated chromosomal variation accompanies neurogenesis in vitro.

A Component of the Xanthomonadaceae Type IV Secretion System Combines a VirB7 Motif with a N0 Domain Found in Outer Membrane Transport Proteins

Type IV secretion systems (T4SS) are used by Gram-negative bacteria to translocate protein and DNA substrates across the cell envelope and into target cells. Translocation across the outer membrane is achieved via a ringed tetradecameric outer membrane complex made up of a small VirB7 lipoprotein (normally 30 to 45 residues in the mature form) and the C-terminal domains of the VirB9 and VirB10 subunits. Several species from the genera of Xanthomonas phytopathogens possess an uncharacterized type IV secretion system with some distinguishing features, one of which is an unusually large VirB7 subunit (118 residues in the mature form). Here, we report the NMR and 1.0 angstrom X-ray structures of the VirB7 subunit from Xanthomonas citri subsp. citri (VirB7(XAC2622)) and its interaction with VirB9. NMR solution studies show that residues 27-41 of the disordered flexible N-terminal region of VirB7(XAC2622) interact specifically with the VirB9 C-terminal domain, resulting in a significant reduction in the conformational freedom of both regions. VirB7(XAC2622) has a unique C-terminal domain whose topology is strikingly similar to that of N0 domains found in proteins from different systems involved in transport across the bacterial outer membrane. We show that VirB7(XAC2622) oligomerizes through interactions involving conserved residues in the N0 domain and residues 42-49 within the flexible N-terminal region and that these homotropic interactions can persist in the presence of heterotropic interactions with VirB9. Finally, we propose that VirB(7XAC2622) oligomerization is compatible with the core complex structure in a manner such that the N0 domains form an extra layer on the perimeter of the tetradecameric ring.

Chemical changes in bromeliad leaves at different vegetative stages

Studies concerning the accumulating capacity of native epiphytic bromeliads are of utmost relevance, due to the continuous incorporation of chemical elements provided by these organisms in the ecosystems. Bromeliad species from diverse So Paulo State conservation units, Brazil, were sampled for young, mature and old leaves using a sustainable sampling method. By applying INAA, the accumulation of ten chemical elements, i.e. Br, Ca, Co, Fe, K, Na, Rb, Sc, Sr and Zn, was investigated in different leaf vegetative stages. The bromeliads showed divergent chemical element distribution patterns, demonstrating a real complexity in the accumulation and translocation mechanisms utilized by these plants.

Disparity between Multilocus Enzyme Electrophoresis, Microsatellite Markers and Pulsed-Field Gel Electrophoresis in epidemiological tracking of Candida albicans

Various molecular systems are available for epidemiological, genetic, evolutionary, taxonomic and systematic studies of innumerable fungal infections, especially those caused by the opportunistic pathogen C. albicans. A total of 75 independent oral isolates were selected in order to compare Multilocus Enzyme Electrophoresis (MLEE), Electrophoretic Karyotyping (EK) and Microsatellite Markers (Simple Sequence Repeats - SSRs), in their abilities to differentiate and group C. albicans isolates (discriminatory power), and also, to evaluate the concordance and similarity of the groups of strains determined by cluster analysis for each fingerprinting method. Isoenzyme typing was performed using eleven enzyme systems: Adh, Sdh, M1p, Mdh, Idh, Gdh, G6pdh, Asd, Cat, Po, and Lap (data previously published). The EK method consisted of chromosomal DNA separation by pulsed-field gel electrophoresis using a CHEF system. The microsatellite markers were investigated by PCR using three polymorphic loci: EF3, CDC3, and HIS3. Dendrograms were generated by the SAHN method and UPGMA algorithm based on similarity matrices (S(SM)). The discriminatory power of the three methods was over 95%, however a paired analysis among them showed a parity of 19.7-22.4% in the identification of strains. Weak correlation was also observed among the genetic similarity matrices (S(SM)(MLEE) x S(SM)(EK) x S(SM)(SSRs)). Clustering analyses showed a mean of 9 +/- 12.4 isolates per cluster (3.8 +/- 8 isolates/taxon) for MLEE, 6.2 +/- 4.9 isolates per cluster (4 +/- 4.5 isolates/taxon) for SSRs, and 4.1 +/- 2.3 isolates per cluster (2.6 +/- 2.3 isolates/taxon) for EK. A total of 45 (13%), 39(11.2%), 5 (1.4%) and 3 (0.9%) clusters pairs from 347 showed similarity (Si) of 0.1-10%, 10.1-20%, 20.1-30% and 30.1-40%, respectively. Clinical and molecular epidemiological correlation involving the opportunistic pathogen C. albicans may be attributed dependently of each method of genotyping (i.e., MLEE, EK, and SSRs) supplemented with similarity and grouping analysis. Therefore, the use of genotyping systems that give results which offer minimum disparity, or the combination of the results of these systems, can provide greater security and consistency in the determination of strains and their genetic relationships. (C) 2010 Elsevier B.V. All rights reserved.

Barium uptake by maize plants as affected by sewage sludge in a long-term field study

Along-term experiment was carried out under field conditions in Jaboticabal, SP, Brazil, with the objective of evaluating the concentration of Ba in soil and in maize plants grown in a soil treated with sewage sludge for nine consecutive years. During 2005/2006, maize was used as test plants and the experimental design was in randomized complete blocks with four treatments and five replicates. Treatments consisted of: 0.0, 45.0, 90.0 and 127.5 t ha(-1) sewage sludge (dry basis). Sewage sludge application increased soil Ba concentration. Barium accumulated in the parts of maize plants were generally affected by the successive applications of sewage sludge to the soil. However, the concentration of Ba in maize grain did not exceed the critical levels of Ba for human consumption. Sewage sludge applied to soil for a long time did not affect dry matter and grain production, nevertheless had the similar effect of mineral fertilization. Published by Elsevier B.V.

Kallikrein-Kinin System Mediated Inflammation in Alzheimer`s Disease In Vivo

The Kallikrein-Kinin System (KKS) has been associated to inflammatory and immunogenic responses in the peripheral and central nervous system by the activation of two receptors, namely B1 receptor and B2 receptor. The B1 receptor is absent or under-expressed in physiological conditions, being up-regulated during tissue injury or in the presence of cytokines. The B2 receptor is constitutive and mediates most of the biological effects of kinins. Some authors suggest a link between the KKS and the neuroinflammation in Alzheimer`s disease (AD). We have recently described an increase in bradykinin (BK) in the cerebrospinal fluid and in densities of B1 and B2 receptors in brain areas related to memory, after chronic infusion of amyloid-beta (A beta) peptide in rats, which was accompanied by memory disruption and neuronal loss. Mice lacking B1 or B2 receptors presented reduced cognitive deficits related to the learning process, after acute intracerebroventricular (i.c.v). administration of A. Nevertheless, our group showed an early disruption of cognitive function by i.c.v. chronic infusion of A beta after a learned task, in the knock-out B2 mice. This suggests a neuroprotective role for B2 receptors. In knock-out B1 mice the memory disruption was absent, implying the participation of this receptor in neurodegenerative processes. The acute or chronic infusion of A beta can lead to different responses of the brain tissue. In this way, the proper involvement of KKS on neuroinflammation in AD probably depends on the amount of A beta injected. Though, BK applied to neurons can exert inflammatory effects, whereas in glial cells, BK can have a potential protective role for neurons, by inhibiting proinflammatory cytokines. This review discusses this duality concerning the KKS and neuroinflammation in AD in vivo.

Ischaemic preconditioning improves proteasomal activity and increases the degradation of delta PKC during reperfusion

The response of the myocardium to an ischaemic insult is regulated by two highly homologous protein kinase C (PKC) isozymes, delta and epsilon PKC. Here, we determined the spatial and temporal relationships between these two isozymes in the context of ischaemia/reperfusion (I/R) and ischaemic preconditioning (IPC) to better understand their roles in cardioprotection. Using an ex vivo rat model of myocardial infarction, we found that short bouts of ischaemia and reperfusion prior to the prolonged ischaemic event (IPC) diminished delta PKC translocation by 3.8-fold and increased epsilon PKC accumulation at mitochondria by 16-fold during reperfusion. In addition, total cellular levels of delta PKC decreased by 60 +/- 2.7% in response to IPC, whereas the levels of epsilon PKC did not significantly change. Prolonged ischaemia induced a 48 +/- 11% decline in the ATP-dependent proteasomal activity and increased the accumulation of misfolded proteins during reperfusion by 192 +/- 32%; both of these events were completely prevented by IPC. Pharmacological inhibition of the proteasome or selective inhibition of epsilon PKC during IPC restored delta PKC levels at the mitochondria while decreasing epsilon PKC levels, resulting in a loss of IPC-induced protection from I/R. Importantly, increased myocardial injury was the result, in part, of restoring a delta PKC-mediated I/R pro-apoptotic phenotype by decreasing pro-survival signalling and increasing cytochrome c release into the cytosol. Taken together, our findings indicate that IPC prevents I/R injury at reperfusion by protecting ATP-dependent 26S proteasomal function. This decreases the accumulation of the pro-apoptotic kinase, delta PKC, at cardiac mitochondria, resulting in the accumulation of the pro-survival kinase, epsilon PKC.

The effect of 5 days of aspartate and asparagine supplementation on glucose transport activity in rat muscle

The consumption of protein supplements containing amino acids is increasing around the world Aspartate (Asp) and asparagine (Asn) are amino acids metabolized by skeletal muscle. This metabolism involves biochemical pathways that are involved in increasing Krebs cycle activity via anaplerotic reactions. resulting in higher glutamine concentrations. A connection between amino acid supplementation, glycogen concentration, and glucose uptake has been previously demonstrated. The purpose of this study was to evaluate the effect of asp and Asn Supplementation on glucose uptake in rats using three different glycogen concentrations The results indicate that Asp and Asn supplementation in rats with high glycogen concentrations (fed state) further increased the glycogen concentration in the muscle, and decreased in vitro 2-deoxyglucose (a glucose analog.) uptake by the muscle at maximal insulin concentrations When animals had a medium glycogen concentration (consumed lard for 3 days). glucose uptake was higher in the supplemented group at sub-maximal insulin concentrations. We conclude that supplementation of Asp and Asn reduced glucose transport in rat muscle only at higher levels of glycogen. The ingestion of lard for 3 days changed the responsiveness and sensitivity to insulin, and that group had higher levels of insulin sensivity with Asp and Asn supplementation. Copyright (C) 2009 John Wiley & Sons, Ltd.

Cardiac anti-remodelling effect of aerobic training is associated with a reduction in the calcineurin/NFAT signalling pathway in heart failure mice

Cardiomyocyte hypertrophy occurs in response to a variety of physiological and pathological stimuli. While pathological hypertrophy in heart failure is usually coupled with depressed contractile function, physiological hypertrophy associates with increased contractility. In the present study, we explored whether 8 weeks of moderate intensity exercise training would lead to a cardiac anti-remodelling effect in an experimental model of heart failure associated with a deactivation of a pathological (calcineurin/NFAT, CaMKII/HDAC) or activation of a physiological (Akt-mTOR) hypertrophy signalling pathway. The cardiac dysfunction, exercise intolerance, left ventricle dilatation, increased heart weight and cardiomyocyte hypertrophy from mice lacking alpha(2A) and alpha(2C) adrenoceptors (alpha(2A)/alpha(2C)ARKO mice) were associated with sympathetic hyperactivity induced heart failure. The relative contribution of Ca(2+)-calmodulin high-affinity (calcineurin/NFAT) and low-affinity (CaMKII/HDAC) targets to pathological hypertrophy of alpha(2A)/alpha(2C)ARKO mice was verified. While nuclear calcineurin B, NFATc3 and GATA-4 translocation were significantly increased in alpha(2A)/alpha(2C)ARKO mice, no changes were observed in CaMKII/HDAC activation. As expected, cyclosporine treatment decreased nuclear translocation of calcineurin/NFAT in alpha(2A)/alpha(2C)ARKO mice, which was associated with improved ventricular function and a pronounced anti-remodelling effect. The Akt/mTOR signalling pathway was not activated in alpha(2A)/alpha(2C)ARKO mice. Exercise training improved cardiac function and exercise capacity in alpha(2A)/alpha(2C)ARKO mice and decreased heart weight and cardiomyocyte width paralleled by diminished nuclear NFATc3 and GATA-4 translocation as well as GATA-4 expression levels. When combined, these findings support the notion that deactivation of calcineurin/NFAT pathway-induced pathological hypertrophy is a preferential mechanism by which exercise training leads to the cardiac anti-remodelling effect in heart failure.