Purification and characterization of a thermostable alpha-amylase produced by the fungus Paecilomyces variotii

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of alpha-amylase on Reagen (R) and Sigma (R) starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

Isolation and characterization of microsatellite markers from the stingless bee Nannotrigona testaceicornis

Conservation of natural populations and handling of breeding programs would benefit from the availability of molecular markers. Stingless bees are one of the most important pollinators in several ecosystems. Thus, seventeen microsatellite markers were developed from an enriched genomic library of Nannotrigona testaceicornis. They were characterized using 50 samples. The expected and observed heterozygosities ranged from 0.59 to 0.89 and from 0.39 to 0.79, respectively. These markers will contribute to advance researches on the genetic conservation, characterization and preservation of the Brazilian native bees.

Development and characterization of microsatellite loci in the marsh deer (Blastocerus dichotomus Cervidae)

Marsh deer (Blastocerus dichotomus) is one of the most exposed large mammals in South America. To aid in the conservation management of the species, nine polymorphic microsatellite loci were isolated and tested on up to 50 animals, showing 3-12 alleles and expected heterozygosity values varying from 0.69 to 0.89. These markers should be of considerable utility in future population and ecological genetics studies of this species. The marsh deer (Blastocerus dichotomus) is the biggest South American species of deer. Originally distributed across a large part of South America, stretching from the south bank of the Amazon river to northern Argentina, significant wild populations are now restricted to the Pantanal, swamplands that cover about 40% of southwest Brazil. The marsh deer is listed as Vulnerable on the Red List of the IUCN. Three populations of the species from three areas in the Parana River basin (between the states of Sao Paulo and Mato Grosso do Sul) were recently studied by observing protein polymorphism at 17 loci (Oliveira et al. 2005). Now we are presenting data about isolation of microsatellite markers to improve the results regarding population structure.

Transferability and characterization of nine microsatellite markers for the tropical tree species Tabebuia roseo-alba

Microsatellite loci that were previously developed in the tropical tree Tabebuia aurea were used for the genetic analysis of Tabebuia roseo-alba populations. Nine of 10 simple sequence repeat markers were amplified, and the polymorphism was assessed in 58 individuals sampled from two stands in southeastern Brazil. All loci were polymorphic with Mendelian inheritance. The allele numbers were high, ranging from 5 to 13 in population I and 3 to 7 in population II, with means of 8.9 and 5.5, respectively. We conclude that these markers can be efficiently used for parentage and gene-flow studies.

Resurrection of Two Species From Synonymy of Anopheles (Nyssorhynchus) strodei Root, and Characterization of a Distinct Morphological Form From the Strodei Complex (Diptera: Culicidae)

Anopheles albertoi Unti and Anopheles arthuri Unti are revived from the synonymy with Anopheles strodei Root, and a distinct morphological form (designated in this study as Anopheles CP Form) from the Strodei Complex of Anopheles (Nyssorhynchus) is characterized. The male genitalia of An. arthuri and An. albertoi are described and illustrated for the first time. An. strodei, An. arthuri, and An. albertoi were first distinguished based on scanning electron microphotos of the eggs, and then each egg type was associated with diagnostic characters of the male genitalia. Identification of Anopheles CP Form was based on morphological characters of the male genitalia, characterized and illustrated in this study. Molecular phylogenetic analysis was most clear when an outgroup was not included, in which case using the nuclear white gene, or the white gene in combination with the mitochondrial cytochrome c oxidase subunit I (COI) gene, clearly separated these four taxa. When Anopheles quadrimaculatus Say and Anopheles stephensi Liston were included as an outgroup, combined white and COI data resolved An. strodei and An. albertoi, whereas An. arthuri was not well resolved. The single sequence of Anopheles CP Form was recovered well separated from other groups in all analyses.

Pozzolanic behavior of bamboo leaf ash: Characterization and determination of the kinetic parameters

The paper presents a characterization and study of the pozzolanic behavior between calcium hydroxide (CH) and bamboo leaf ash (BLAsh), which was obtained by calcining bamboo leaves at 600 degrees C for 2 h in a laboratory electric furnace. To evaluate the pozzolanic behavior the conductometric method was used, which is based on the measurement of the electrical conductivity in a BLAsh/CH solution with the reaction time. Later, the kinetic parameters are quantified by applying a kinetic-diffusive model. The kinetic parameters that characterize the process (in particular, the reaction rate constant and free energy of activation) were determined with relative accuracy in the fitting process of the model. The pozzolanic activity is quantitatively evaluated according to the values obtained of the kinetic parameters. Other experimental techniques, such as X-ray diffraction (XRD) and scanning electron microscopy (SEM), were also employed. The results show that this kind of ash is formed by silica with a completely amorphous nature and a high pozzolanic activity. The correlation between the values of free energy of activation (Delta G(#)) and the reaction rate constants (K) are in correspondence with the theoretical studies about the rate processes reported in the literature. (C) 2010 Elsevier Ltd. All rights reserved.

Revisiting cangitoxin, a sea anemone peptide: Purification and characterization of cangitoxins II and III from the venom of Bunodosoma cangicum

Sodium channel toxins from sea anemones are employed as tools for dissecting the biophysical properties of inactivation in voltage-gated sodium channels. Cangitoxin (CGTX) is a peptide containing 48 amino acid residues and was formerly purified from Bunodosoma cangicum. Nevertheless, previous works reporting, the isolation procedures for such peptide from B. cangicum secretions are controversial and may lead to incorrect information. In this paper, we report a simple and rapid procedure, consisting of two chromatographic steps, in order to obtain a CGTX analog directly from sea anemone venom. We also report a substitution of N16D in this peptide sample and the co-elution of an inseparable minor isoform presenting the R14H substitution. Peptides are named as CGTX-II and CGTX-III, and their effects over Nav1.1 channels in patch clamp experiments are demonstrated. (c) 2008 Elsevier Ltd. All rights reserved.

Isolation and characterization of 15 microsatellite loci in the stingless bee Plebeia remota (Apidae: Meliponini)

The destruction of Brazilian natural habitats has reduced bee populations and negative impacts of native flora pollination have been noticed. This work describes the isolation and characterization of microsatellite loci and evaluates them as molecular markers to study genetic variability of the stingless bee Plebeia remota. A microsatellite enriched genomic library was constructed and 15 primer pairs were designed for this species. The survey was conducted by analyzing 21 unrelated individuals. Genetic diversity indexes were calculated. The mean allelic richness was 6.3, the observed heterozygosity was 0.568, and the percentage of polymorphic loci was 93.33%. Also the primers were tested in cross-species amplification and showed promising results for P. droryana, P. emerina, P. lucii, P. meridionalis, P. pugnax, and P. saiqui. The microsatellite loci described here will be useful to evaluate genetic variability of stingless bees, and certainly will improve our knowledge about population dynamics especially in threatened environments.

Isolation and characterization of microsatellite loci in the Brazilian orchid Epidendrum fulgens

Epidendrum fulgens has a patchy distribution along the Atlantic Rainforest in the Brazilian coast, due to the destruction of its native habitat. Here, we report on both the development of nine new microsatellite markers isolated from this species and the characterization of their allele variability in two distant and unrelated populations. The number of alleles observed for each locus ranged from 2 to 17 with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying the effect of habitat fragmentation on the genetic structure of E. fulgens populations.

Isolation and characterization of microsatellite loci in Epidendrum puniceoluteum, an endemic orchid from the Atlantic Rainforest

Epidendrum puniceoluteum is an endemic orchid of Atlantic Rainforest, restricted to few populations only due to the destruction and fragmentation of its native habitat. Here, we report on the development of 10 microsatellite markers isolated from this orchid species. Genetic variability was characterized in two distant populations from Brazil coast. The number of alleles observed for each locus ranged from two to 12 and with an average of 6.4 alleles per locus. These microsatellites should be valuable tools for studying both fine-scale genetic structure of scattered E. puniceoluteum population and patterns will be useful genetic markers for other closely related taxa.

Notes on distribution, variation and characterization of Erythrolamprus pseudocorallus Roze, 1959 (Serpentes: Colubridae) with the first records from Colombia

The Maracaibo false coral snake Erythrolamprus pseudocorallus, previously known only from Venezuela, is recorded from five departments in Colombia. These new data include the westernmost and the southernmost records presently known for the species. Two specimens previously identified as E. aesculapii, from the localities of El Valle, Distrito Federal, Venezuela, and Yarumal, Antioquia, Colombia, are now attributed to E. pseudocorallus, the first one representing the northeasternmost record of the species. Morphological characterization of E. pseudocorallus is expanded based on the new specimens.

Immunocytochemical characterization of the pregeniculate nucleus and distribution of retinal and neuropeptide Y terminals in the suprachiasmatic nucleus of the Cebus monkey

Circadian rhythms generated by the suprachiasmatic nucleus (SCN) are modulated by photic and non-photic stimuli. In rodents, direct photic stimuli reach the SCN mainly through the retinohypothalamic tract (RHT), whereas indirect photic stimuli are mainly conveyed by the geniculohypothalamic tract (GHT). In rodents, retinal cells form a pathway that reaches the intergeniculate leaflet (IGL) where they establish synapses with neurons that express neuropeptide Y (NPY), hence forming the GHT projecting to the SCN. In contrast to the RHT, which has been well described in primates, data regarding the presence or absence of the IGL and GHT in primates are contradictory. Some studies have suggested that an area of the pregeniculate nucleus (PGN) of primates might be homologous to the IGL of rodents, but additional anatomical and functional studies on primate species are necessary to confirm this hypothesis. Therefore, this study investigated the main histochemical characteristics of the PGN and the possible existence of the GHT in the SCN of the primate Cebus, comparing the distribution of NPY immunoreactivity, serotonin (5-HT) immunoreactivity and retinal terminal fibers in these two structures. The results show that a collection of cell bodies containing NPY and serotonergic immunoreactivity and retinal innervations are present within a zone that might be homologous to the IGL of rodents. The SCN also receives dense retinal innervations and we observed an atypical distribution of NPY- and 5-HT-immunoreactive fibers without regionalization in the ventral part of the nucleus as described for other species. These data may reflect morphological differences in the structures involved in the regulation of circadian rhythms among species and support the hypothesis that the GHT is present in some higher primates (diurnal animals). (C) 2009 Elsevier B.V. All rights reserved.

Identification and characterization of antigenic proteins potentially expressed during the infectious process of Paracoccidioides brasiliensis

Paracoccidioides brasiliensis causes paracoccidioidomycosis (PCM), a systemic mycosis presenting clinical manifestations ranging from mild to severe forms. A P. brasiliensis cDNA expression library was produced and screened with pooled sera from PCM patients adsorbed against antigens derived from in vitro-grown P. brasiliensis yeast cells. Sequencing DNA inserts from clones reactive with PCM patients sera indicated 35 open reading frames presenting homology to genes involved in metabolic pathways, transport, among other predicted functions. The complete cDNAs encoding aromatic-L-amino-acid decarboxylase (Pbddc), lumazine synthase (Pbls) and a homologue of the high affinity copper transporter (Pbctr3) were obtained. Recombinant proteins PbDDC and PbLS were obtained; a peptide was synthesized for PbCTR3. The proteins and the synthetic peptide were recognized by sera of patients with confirmed PCM and not by sera of healthy patients. Using the in vivo-induced antigen technology (IVIAT), we identified immunogenic proteins expressed at high levels during infection. Quantitative real time RTPCR demonstrated high transcript levels of Pbddc, Pbls and Pbctr3 in yeast cells infecting macrophages. Transcripts in yeast cells derived from spleen and liver of infected mice were also measured by qRT-PCR. Our results suggest a putative role for the immunogenic proteins in the infectious process of P. brasiliensis. (C) 2009 Elsevier Masson SAS. All rights reserved.

EPR in the Characterization of the Shade Effect on Translucence, Remaining Free Radicals, and Polymerization Depth of Commercially Available Resin Composites

The aim of this study was to assess the relation between the number of free radicals generated and the polymerization depth in two different commercial brands of resin composites with different colors and translucence. Electron paramagnetic resonance quantified the radical populations through relative intensity (I (r)) of free radicals generated, and radical decay was monitored. Sample translucence and the classical polymerization depth were measured. The analysis indicated that resin with more color pigments (MA4, I (r) = 0.73 a.u) or more opacity components (ODA2, I (r) = 0.84 a.u) generated smaller populations of free radicals and have the lower polymerization depth than clearer (M, I (r) = 1.20 a.u and MA2, I (r) = 1.02) or more translucent (OEA2, I (r) = 1.00 a.u) composites for the same light-curing time. It seems that irradiation doses have to be adequate to more colored and less translucent resins.

Characterization of the optical properties of atmospheric aerosols in Amazonia from long-term AERONET monitoring (1993-1995 and 1999-2006)

We present a new climatology of atmospheric aerosols (primarily pyrogenic and biogenic) for the Brazilian tropics on the basis of a high-quality data set of spectral aerosol optical depth and directional sky radiance measurements from Aerosol Robotic Network (AERONET) Cimel Sun-sky radiometers at more than 15 sites distributed across the Amazon basin and adjacent Cerrado region. This network is the only long-term project (with a record including observations from more than 11 years at some locations) ever to have provided ground-based remotely-sensed column aerosol properties for this critical region. Distinctive features of the Amazonian area aerosol are presented by partitioning the region into three aerosol regimes: southern Amazonian forest, Cerrado, and northern Amazonian forest. The monitoring sites generally include measurements from the interval 1999-2006, but some sites have measurement records that date back to the initial days of the AERONET program in 1993. Seasonal time series of aerosol optical depth (AOD), angstrom ngstrom exponent, and columnar-averaged microphysical properties of the aerosol derived from sky radiance inversion techniques (single-scattering albedo, volume size distribution, fine mode fraction of AOD, etc.) are described and contrasted for the defined regions. During the wet season, occurrences of mineral dust penetrating deep into the interior were observed.