Expression Pattern and Promoter Analysis of a Eucalyptus grandis Germin-like Gene

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Systematic analysis of protein-detergent complexes applying dynamic light scattering to optimize solutions for crystallization trials

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Comparative analysis of three hyperthermophilic GH1 and GH3 family members with industrial potential

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Regulation of glycogen metabolism by the CRE-1, RCO-1 and RCM-1 proteins in Neurospora crassa. The role of CRE-1 as the central transcriptional regulator

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Influence of the Paracoccidioides brasiliensis 14-3-3 and gp43 proteins on the induction of apoptosis in A549 epithelial cells

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

A new heterologous fibrin sealant as scaffold to recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins for the repair of tibial bone defects

Tissue engineering has special interest in bone tissue aiming at future medical applications Studies have focused on recombinant human bone morphogenetic protein-2 (rhBMP-2) and natural latex proteins due to the osteogenic properties of rhBMP-2 and the angiogenic characteristic of fraction 1 protein (P-1) extracted from the rubber tree Hevea brasiliensis. Furthermore, heterologous fibrin sealant (FS) has been shown as a promising alternative in regenerative therapies. The aim of this study was to evaluate these substances for the repair of bone defects in rats. A bone defect measuring 3 mm in diameter was created in the proximal metaphysis of the left tibia of 60 rats and was implanted with rhBMP-2 or P-1 in combination with a new heterologous FS derived from snake venom. The animals were divided into six groups: control (unfilled bone defect), rhBMP-2 (defect filled with 5 mu g rhBMP-2), P-1 (defect filled with 5 mu g P-1), FS (defect filled with 8 mu g FS), FS/rhBMP-2 (defect filled with 8 mu g FS and 5 mu g rhBMP-2), FS/P-1 (defect filled with 8 mu g FS and 5 mu g P-1). The animals were sacrificed 2 and 6 weeks after surgery. The newly formed bone projected from the margins of the original bone and exhibited trabecular morphology and a disorganized arrangement of osteocyte lacunae. Immunohistochemical analysis showed intense expression of osteocalcin in all groups. Histometric analysis revealed a significant difference in all groups after 2 weeks (p < 0.05), except for the rhBMP-2 and FS/rhBMP-2 groups (p > 0.05). A statistically significant difference (p < 0.05) was observed in all groups after 6 weeks in relation to the volume of newly formed bone in the surgical area. In conclusion, the new heterologous fibrin sealant was found to be biocompatible and the combination with rhBMP-2 showed the highest osteogenic and osteoconductive capacity for bone healing. These findings suggest a promising application of this combination in the regeneration surgery.

Venomics of the Australian eastern brown snake (Pseudonaja textilis): detection of new venom proteins and splicing variants

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

The genome sequence of the plant pathogen Xylella fastidiosa: The Xylella fastidiosa consortium of the organization for nucleotide sequencing and analysis, Sao Paulo, Brazil

Xylella fastidiosa is a fastidious, xylem-limited bacterium that causes a range of economically important plant diseases. Here we report the complete genome sequence of X. fastidiosa clone 9a5c, which causes citrus variegated chlorosis - a serious disease of orange trees. The genome comprises a 52.7% GC-rich 2,679,305-base-pair (bp) circular chromosome and 'two plasmids of 51,158 bp and 1,285 bp. We can assign putative functions to47% of the 2,904 predicted coding regions. Efficient metabolic functions are predicted, with sugars as the principal energy and carbon source, supporting existence in the nutrient-poor xylem sap. The mechanisms associated with pathogenicity and virulence involve toxins, antibiotics and ion sequestration systems, as well as bacterium-bacterium and bacterium-host interactions mediated by a range of proteins. Orthologues of some of these proteins have only been identified in animal and human pathogens; their presence in X. fastidiosa indicates that the molecular basis for bacterial pathogenicity is both conserved and independent of host. At least 83 genes are bacteriophage-derived and include virulence-associated genes from other bacteria, providing direct evidence of phage-mediated horizontal gene transfer.

The homeostasis of iron, copper, and zinc in paracoccidioides brasiliensis, cryptococcus neoformans var. Grubii, and cryptococcus gattii: a comparative analysis

Iron, copper, and zinc are essential for all living organisms. Moreover, the homeostasis of these metals is vital to microorganisms during pathogenic interactions with a host. Most pathogens have developed specific mechanisms for the uptake of micronutrients from their hosts in order to counteract the low availability of essential ions in infected tissues. We report here an analysis of genes potentially involved in iron, copper, and zinc uptake and homeostasis in the fungal pathogens Paracoccidioides brasiliensis, Cryptococcus neoformans var. grubii, and Cryptococcus gattii. Although prior studies have identified certain aspects of metal regulation in Cryptococcus species, little is known regarding the regulation of these elements in P. brasiliensis. We also present amino acid sequences analyses of deduced proteins in order to examine possible conserved domains. The genomic data reveals, for the first time, genes associated to iron, copper, and zinc assimilation and homeostasis in P. brasiliensis. Furthermore, analyses of the three fungal species identified homologs to genes associated with high-affinity uptake systems, vacuolar and mitochondrial iron storage, copper uptake and reduction, and zinc assimilation. However, homologs to genes involved in siderophore production were only found in P. brasiliensis. Interestingly, in silico analysis of the genomes of P. brasiliensis Pb01, Pb03, and Pb18 revealed significant differences in the presence and/or number of genes involved in metal homeostasis, such as in genes related to iron reduction and oxidation. The broad analyses of the genomes of P. brasiliensis, C. neoformans var. grubii, and C. gattii for genes involved in metal homeostasis provide important groundwork for numerous interesting future areas of investigation that are required in order to validate and explore the function of the identified genes and gene pathways.

Cultivation and bromatological analysis of the medicinal mushroom Ganoderma lucidum (Curt.: Fr.) P. Karst cultivated in agricultural waste

The objective of the study was to evaluate the production of two strains of Ganoderma lucidum on agricultural waste and carry out bromatological analyses of the basidiomata obtained from the cultivation. The experiment was carried out at the Mushroom Module at the School of Agronomic Sciences of the São Paulo State University (FCA/UNESP - Botucatu, SP, Brazil) and two strains were used (GLM-09/01 and GLM-10/02) which were cultivated on waste, oat straw, bean straw, brachiaria grass straw, Tifton grass straw and eucalyptus sawdust under two situations: with (20%) and without (0%) supplementation with wheat bran. All the waste was taken from dumps of agricultural activities in Botucatu-SP. Both treatments were carried out in 10 repetitions, totaling 200 packages. The mushrooms cultivation took 90 days. Next, the biological efficiency of the treatments and the bromatological analysis of the basidiomata were evaluated. The biological efficiency (BE) values (%) varied from 0.0 to 6.7%. In the mushroom bromatological analyses, the results ranged from 8.7 to 13.7%, from 2.0 to 6.7%, from 0.83 to 1.79% and from 38.8 to 54.5%, for total protein, ethereal extract, ash and crude fiber, respectively. Thus, we conclude that the substrates which presented the greater yield were the brachiaria straw, 20% in both strains tested (GLM-09/01 and GLM-10/02) and the bean straw, 20% in the strain GLM-10/02. The mushrooms showed high levels of ethereal extract, fibers and ashes and a low level of proteins.

Assignment of Putative Functions to Membrane "Hypothetical Proteins" from the Trypanosoma cruzi Genome

Protozoan parasites cause thousands of deaths each year in developing countries. The genome projects of these parasites opened a new era in the identification of therapeutic targets. However, the putative function could be predicted for fewer than half of the protein-coding genes. In this work, all Trypanosoma cruzi proteins containing predicted transmembrane spans were processed through an automated computational routine and further analyzed in order to assign the most probable function. The analysis consisted of dissecting the whole predicted protein in different regions. More than 5,000 sequences were processed, and the predicted biological functions were grouped into 19 categories according to the hits obtained after analysis. One focus of interest, due to the scarce information available on trypanosomatids, is the proteins involved in signal-transduction processes. In the present work, we identified 54 proteins belonging to this group, which were individually analyzed. The results show that by means of a simple pipeline it was possible to attribute probable functions to sequences annotated as coding for "hypothetical proteins.'' Also, we successfully identified the majority of candidates participating in the signal-transduction pathways in T. cruzi.

Analysis of expressed sequence tags from the agarophyte Gracilaria tenuistipitata (Rhodophyta)

A total of 3,631 expressed sequence tags (ESTs) were established from two size-selected cDNA libraries made from the tetrasporophytic phase of the agarophytic red alga Gracilaria tenuistipitata. The average sizes of the inserts in the two libraries were 1,600 bp and 600 bp, with an average length of the edited sequences of 850 bp. Clustering gave 2,387 assembled sequences with a redundancy of 53%. Of the ESTs, 65% had significant matches to sequences deposited in public databases, 11% to proteins without known function, and 35% were novel. The most represented ESTs were a Na/K-transporting ATPase, a hedgehog-like protein, a glycine dehydrogenase and an actin. Most of the identified genes were involved in primary metabolism and housekeeping. The largest functional group was thus genes involved in metabolism with 14% of the ESTs; other large functional categories included energy, transcription, and protein synthesis and destination. The codon usage was examined using a subset of the data, and the codon bias was found to be limited with all codon combinations used.

Associations among genotype, clinical phenotype, and intracellular localization of trafficking proteins in ARC syndrome

Arthrogryposisrenal dysfunctioncholestasis (ARC) syndrome is a rare autosomal recessive multisystem disorder caused by mutations in vacuolar protein sorting 33 homologue B (VPS33B) and VPS33B interacting protein, apicalbasolateral polarity regulator (VIPAR). Cardinal features of ARC include congenital joint contractures, renal tubular dysfunction, cholestasis, severe failure to thrive, ichthyosis, and a defect in platelet alpha-granule biogenesis. Most patients with ARC do not survive past the first year of life. We report two patients presenting with a mild ARC phenotype, now 5.5 and 3.5 years old. Both patients were compound heterozygotes with the novel VPS33B donor splice-site mutation c.1225+5G>C in common. Immunoblotting and complementary DNA analysis suggest expression of a shorter VPS33B transcript, and cell-based assays show that c.1225+5G>C VPS33B mutant retains some ability to interact with VIPAR (and thus partial wild-type function). This study provides the first evidence of genotypephenotype correlation in ARC and suggests that VPS33B c.1225+5G>C mutation predicts a mild ARC phenotype. We have established an interactive online database for ARC (https://grenada.lumc.nl/LOVD2/ARC) comprising all known variants in VPS33B and VIPAR. Also included in the database are 15 novel pathogenic variants in VPS33B and five in VIPAR. Hum Mutat 33:16561664, 2012. (c) 2012 Wiley Periodicals, Inc.

Quantitative proteomic analysis and functional studies reveal that nucleophosmin is involved in cell death in glioblastoma cell line transfected with siRNA

Previously, we reported that nucleophosmin (NPM) was increased in glioblastoma multiforme (GBM). NPM is a phosphoprotein related to apoptosis, ribosome biogenesis, mitosis, and DNA repair, but details about its function remain unclear. We treated U87MG and A172 cells with small interference RNA (siRNA) and obtained a reduction of 80% in NPM1 expression. Knockdown at the protein level was evident after the 4th day and was maintained until the 7th day of transfection that was investigated by quantitative proteomic analysis using isobaric tags. The comparison of proteomic analysis of NPM1-siRNA against controls allowed the identification of 14 proteins, two proteins showed increase and 12 presented a reduction of expression levels. Gene ontology assigned most of the hypoexpressed proteins to apoptosis regulation, including GRP78. NPM1 silencing did not impair cell proliferation until the 7th day after transfection, but sensitized U87MG cells to temozolomide (TMZ), culminating with an increase in cell death and provoking at a later period a reduction of colony formation. In a large data set of GBM patients, both GRP78 and NPM1 genes were upregulated and presented a tendency to shorter overall survival time. In conclusion, NPM proved to participate in the apoptotic process, sensitizing TMZ-treated U87MG and A172 cells to cell death, and in association with upregulation of GRP78 may be helpful as a predictive factor of poor prognosis in GBM patients.

Proteomic Analysis of Chloroplast-to-Chromoplast Transition in Tomato Reveals Metabolic Shifts Coupled with Disrupted Thylakoid Biogenesis Machinery and Elevated Energy-Production Components

A comparative proteomic approach was performed to identify differentially expressed proteins in plastids at three stages of tomato (Solanum lycopersicum) fruit ripening (mature-green, breaker, red). Stringent curation and processing of the data from three independent replicates identified 1,932 proteins among which 1,529 were quantified by spectral counting. The quantification procedures have been subsequently validated by immunoblot analysis of six proteins representative of distinct metabolic or regulatory pathways. Among the main features of the chloroplast-to-chromoplast transition revealed by the study, chromoplastogenesis appears to be associated with major metabolic shifts: (1) strong decrease in abundance of proteins of light reactions (photosynthesis, Calvin cycle, photorespiration) and carbohydrate metabolism (starch synthesis/degradation), mostly between breaker and red stages and (2) increase in terpenoid biosynthesis (including carotenoids) and stress-response proteins (ascorbate-glutathione cycle, abiotic stress, redox, heat shock). These metabolic shifts are preceded by the accumulation of plastid-encoded acetyl Coenzyme A carboxylase D proteins accounting for the generation of a storage matrix that will accumulate carotenoids. Of particular note is the high abundance of proteins involved in providing energy and in metabolites import. Structural differentiation of the chromoplast is characterized by a sharp and continuous decrease of thylakoid proteins whereas envelope and stroma proteins remain remarkably stable. This is coincident with the disruption of the machinery for thylakoids and photosystem biogenesis (vesicular trafficking, provision of material for thylakoid biosynthesis, photosystems assembly) and the loss of the plastid division machinery. Altogether, the data provide new insights on the chromoplast differentiation process while enriching our knowledge of the plant plastid proteome.