New additions to the herpetofauna of the Dois Irmãos State Park, an urban Atlantic Rainforest fragment in northeastern Brazil

The Atlantic Rainforest is the second largest forest block in the Neotropics, which originally comprised more than 150 million ha in highly heterogeneous environmental conditions. In this study, we updated the list of amphibians and reptiles in the Dois Irmãos State Park (PEDI), a conservation unit that is an Atlantic Rainforest remnant in Pernambuco State, northeastern Brazil. Active and passive surveys were carried out weekly, from June 2008 to April 2009, respectively by searching different forest microenvironments and by using pitfall traps. We recorded 34 species of amphibians belonging to 10 families and 27 species of reptiles belonging to 17 families. This study adds three species of amphibians and 18 species of reptiles to previous surveys of the herpetofauna at the PEDI. The PEDI comprises a richness equivalent to 49% of the anurans and 32% of the reptiles recorded in the Atlantic Rainforest in Pernambuco State. This area makes up the group of urban protected areas in which the local herpetofauna has been inventoried, as well as isolated areas in the metropolitan region of the municipality of Recife. Regarding these urban areas, the PEDI Reserve comprises 38.7% of the local richness, being considered a well-maintained remnant in an urban area. Thus, we highlight the need to invest in the conservation of urban forest fragments, given the significant richness that PEDI displays in relation to other extremely impoverished Atlantic Rainforest areas.

New additions to the herpetofauna of the Dois Irmãos State Park, an urban Atlantic Rainforest fragment in northeastern Brazil

The Atlantic Rainforest is the second largest forest block in the Neotropics, which originally comprised more than 150 million ha in highly heterogeneous environmental conditions. In this study, we updated the list of amphibians and reptiles in the Dois Irmãos State Park (PEDI), a conservation unit that is an Atlantic Rainforest remnant in Pernambuco State, northeastern Brazil. Active and passive surveys were carried out weekly, from June 2008 to April 2009, respectively by searching different forest microenvironments and by using pitfall traps. We recorded 34 species of amphibians belonging to 10 families and 27 species of reptiles belonging to 17 families. This study adds three species of amphibians and 18 species of reptiles to previous surveys of the herpetofauna at the PEDI. The PEDI comprises a richness equivalent to 49% of the anurans and 32% of the reptiles recorded in the Atlantic Rainforest in Pernambuco State. This area makes up the group of urban protected areas in which the local herpetofauna has been inventoried, as well as isolated areas in the metropolitan region of the municipality of Recife. Regarding these urban areas, the PEDI Reserve comprises 38.7% of the local richness, being considered a well-maintained remnant in an urban area. Thus, we highlight the need to invest in the conservation of urban forest fragments, given the significant richness that PEDI displays in relation to other extremely impoverished Atlantic Rainforest areas.

A proteomic approach to guarana seed and pericarp maturation.

Paullinia cupana Kunth var. sorbilis (Mart.) Ducke, the cultivated guarana plant, is native to the Amazon and has been valued for its medicinal, stimulant and energetic properties for centuries. The seeds are the main commercial product of the plant and the source of high amounts of purine alkaloids (caffeine and theobromine) and polyphenols (flavonoids, catechins, and tannins). Proteins involved in the development and maturation of guarana fruits in its native habitat are interesting issues for proteomics. This study presents the proteomic profile of the seed and pericarp of healthy guarana in different maturation stages. Protein contents were higher in the mature seed compared to other stages due to the accumulation of storage proteins - 11S globulins. Proteins selected for identification by mass spectrometry are mostly related to stress responses and defense and this is not unexpected for fast growing and differentiating reproductive tissues.

Method for obtaining high-resolution proteomic analysis from pericarps of guarana.

Guarana has great agricultural potential and is largely used therapeutically and in the production of non-alcoholic energy drinks. Genomic and proteomic studies are crucial to identify proteins that play central roles in the maintenance and viability of fruits, as well as to identify proteins related to the main metabolic pathways. However, the success of any protein analysis starts with the protein extract preparation, which needs to offer an extract that is free of contaminants. This study aimed to evaluate different extraction methods to obtain high-quantity and high-quality extracts that are compatible with analysis by 2-dimensional electrophoresis and tandem mass spectrometry protein identification. Three different methods were tested: trichloroacetic acid (TCA)/acetone, sodium dodecyl sulfate (SDS)/phenol, and polyvinylpolypyrrolidone (PVPP)/SDS/phenol. The extract obtained from the TCA/acetone precipitation presented low solubility and contamination with lipids and carbohydrates. On the other hand, the quality of the extract gradually improved after using phenol and PVPP/phenol, enabling a yield up to 2 mg/g macerated tissues and the detection of 457 spots by 2-dimensional electrophoresis. The effectiveness of the procedure used was validated by identification of 10 randomly selected proteins by mass spectrometry. The procedure described here can be a starting point for applications using tissues of other organs of guarana or tissues of species that are similar to guarana.

Electrophoresis and spectrometric analyses of adaptation-related proteins in thermally stressed Chromobacterium violaceum.

Chromobacterium violaceum is a Gram-negative proteobacteria found in water and soil; it is widely distributed in tropical and subtropical regions, such as the Amazon rainforest. We examined protein expression changes that occur in C. violaceum at different growth temperatures using electrophoresis and mass spectrometry. The total number of spots detected was 1985; the number ranged from 99 to 380 in each assay. The proteins that were identified spectrometrically were categorized as chaperones, proteins expressed exclusively under heat stress, enzymes involved in the respiratory and fermentation cycles, ribosomal proteins, and proteins related to transport and secretion. Controlling inverted repeat of chaperone expression and inverted repeat DNA binding sequences, as well as regions recognized by sigma factor 32, elements involved in the genetic regulation of the bacterial stress response, were identified in the promoter regions of several of the genes coding proteins, involved in the C. violaceum stress response. We found that 30 °C is the optimal growth temperature for C. violaceum, whereas 25, 35, and 40 °C are stressful temperatures that trigger the expression of chaperones, superoxide dismutase, a probable small heat shock protein, a probable phasing, ferrichrome-iron receptor protein, elongation factor P, and an ornithine carbamoyltransferase catabolite. This information improves our comprehension of the mechanisms involved in stress adaptation by C. violaceum.

Transcriptome characterization of the dimorphic and pathogenic fungus Paracoccidioides brasiliensis by EST analysis.

Paracoccidioides brasiliensis is a pathogenic fungus that undergoes a temperature-dependent cell morphology change from mycelium (22 degrees C) to yeast (36 degrees C). It is assumed that this morphological transition correlates with the infection of the human host. Our goal was to identify genes expressed in the mycelium (M) and yeast (Y) forms by EST sequencing in order to generate a partial map of the fungus transcriptome. Individual EST sequences were clustered by the CAP3 program and annotated using Blastx similarity analysis and InterPro Scan. Three different databases, GenBank nr, COG (clusters of orthologous groups) and GO (gene ontology) were used for annotation. A total of 3,938 (Y = 1,654 and M = 2,274) ESTs were sequenced and clustered into 597 contigs and 1,563 singlets, making up a total of 2,160 genes, which possibly represent one-quarter of the complete gene repertoire in P. brasiliensis. From this total, 1,040 were successfully annotated and 894 could be classified in 18 functional COG categories as follows: cellular metabolism (44%); information storage and processing (25%); cellular processes-cell division, posttranslational modifications, among others (19%); and genes of unknown functions (12%). Computer analysis enabled us to identify some genes potentially involved in the dimorphic transition and drug resistance. Furthermore, computer subtraction analysis revealed several genes possibly expressed in stage-specific forms of P. brasiliensis. Further analysis of these genes may provide new insights into the pathology and differentiation of P. brasiliensis.

Single-chain Fv with Fc fragment of the human IgG1 tag: construction, Pichia pastoris expression and antigen binding characterization.

We report two expression vectors in Pichia pastoris that direct the synthesis of recombinant single chain antibody variable region (scFv), derived from anti-Z-DNA monoclonal antibody Z22. The first vector codes for a scFv fused to the Ig binding domain of staphylococcal Protein A. The second vector codes for the scFv fused to the Fc fragment of the human IgG1. The fusion partner simplified the detection and purification of the secreted protein. These constructs yielded high level expression of an scFv with specific binding activity toward a Z form of DNA, with binding activity comparable to that of the scFv molecule produced in an Escherichia coli expression system and the original monoclonal antibody.