Purification and characterization of a novel pectinase from Acrophialophora nainiana with emphasis on its physicochemical properties

An extracellular pectinase (PECI) was purified to apparent homogeneity from liquid state cultures of the thermophilic fungus Acrophialophora nainiana by ultrafiltration and a combination of gel filtration and ion-exchange chromatographic procedures. The molecular masses of PECI were 35,500 and 30,749 Da, as determined by SDS-PAGE and mass spectrometry, respectively. It was more active at 60 degrees C and pH 8.0 and showed high stability at 50 degrees C with half-life of 7 days. However at 60 and 70 degrees C, PECI was much less stable with half lives of approximately 20 and 3 min, respectively. The thermostability of purified PECI was also investigated by fluorescence and circular dichroism spectroscopy. Fluorescence revealed that the unfolding transition region was observed between 45 and 70 degrees C. A major decrease in the stability was found at 70 degrees C. Circular dichroism measurements at pH between 5.0 and 9.0 showed a transition temperature (T(m)) range of 50-55 degrees . The thermodynamic analysis of these results showed that EPGI is thermal stable protein exhibiting maximum stability (DeltaG(25)) of 22.65 and 19.19 kcal/mol at pH 8.0 and 9.0, respectively. The apparent K(m) value on pectin from citrus fruits was 4.22 mgml(-1). PECI exhibited no detectable activity of pectin methylesterase, endo-polygalacturonase, mannanase, xylanase and cellulase. However, it showed exo-polygalacturonase and pectin lyase activities. The presence of carbohydrate was detected in the pure PECI. It was activated by l-tryptophan, DEPC, DTT, DTNB, DTP, l-cystein and beta-mercaptoethanol and inhibited by NBS, Fe(2+), Cu(2+), Zn(2+), Mn(2+), Al(3+) and Ca(2+). The enzyme showed homology with a pectin lyases from Xanthomonas campestris and Bacillus licheniformis.     

Histological characterization of in vitro adventitious organogenesis in Citrus sinensis

The adventitious bud development was induced in epicotyl segments of Valencia sweet orange (Citrus sinensis L. Osbeck). Seeds were cultured in vitro for three weeks in the dark, followed by one week at a 16-h photoperiod. Epicotyl segments were cultured horizontally for the induction of organogenesis in Murashige and Tucker (1969, MT) culture medium supplemented with 1.0 mg dm⁻³ benzylaminopurine. Samples were observed by light and scanning electron microscopy from day zero to day 25, when buds were well grown. It was shown that the adventitious buds originated directly from the cambial region on the cut ends of the explants.     

Biochemical,physicochemical and molecular characterization of a genuine 2-Cys-peroxiredoxin purified from cowpea [Vigna unguiculata (L.) Walpers] leaves

Background

Peroxiredoxins have diverse functions in cellular defense-signaling pathways. 2-Cys-peroxiredoxins (2-Cys-Prx) reduce H₂O₂ and alkyl-hydroperoxide. This study describes the purification and characterization of a genuine 2-Cys-Prx from Vigna unguiculata (Vu-2-Cys-Prx).

Methods

Vu-2-Cys-Prx was purified from leaves by ammonium sulfate fractionation, chitin affinity and ion exchange chromatography.

Results

Vu-2-Cys-Prx reduces H₂O₂ using NADPH and DTT. Vu-2-Cys-Prx is a 44 kDa (SDS-PAGE)/46 kDa (exclusion chromatography) protein that appears as a 22 kDa molecule under reducing conditions, indicating that it is a homodimer linked intermolecularly by disulfide bonds and has a pI range of 4.56–4.72; its NH₂-terminal sequence was similar to 2-Cys-Prx from Phaseolus vulgaris (96%) and Populus tricocarpa (96%). Analysis by ESI-Q-TOF MS/MS showed a molecular mass/pI of 28.622 kDa/5.18. Vu-2-Cys-Prx has 8% α-helix, 39% β-sheet, 22% of turns and 31% of unordered forms. Vu-2-Cys-Prx was heat stable, has optimal activity at pH 7.0, and prevented plasmid DNA degradation. Atomic force microscopy shows that Vu-2-Cys-Prx oligomerized in decamers which might be associated with its molecular chaperone activity that prevented denaturation of insulin and citrate synthase. Its cDNA analysis showed that the redox-active Cys⁵² residue and the amino acids Pro⁴⁵, Thr⁴⁹ and Arg¹²⁸ are conserved as in other 2-Cys-Prx.

General significance

The biochemical and molecular features of Vu-2-Cys-Prx are similar to other members of 2-Cys-Prx family. To date, only one publication reported on the purification of native 2-Cys-Prx from leaves and the subsequent analysis by N-terminal Edman sequencing, which is crucial for construction of stromal recombinant 2-Cys-Prx proteins.     

A new species of <Emphasis Type="Italic">Symplocos</Emphasis> (Symplocaceae) from the Itatiaia Plateau of Brazil

Symplocos minima, a new species of Symplocos section Hopea from the Itatiaia Plateau in the Atlantic Rain Forest biome of southeastern Brazil, is described and illustrated. This species is distinguished by its densely compact shrubby habit, ascending leaves, fasciculate inflorescences with several persistent bracts, corolla with five to six erect lobes, pistillate flowers with the disc not thickened along the margin, fruiting calyx lobes obscuring the disc, and seeds sub-orbicular in cross section. The new species is morphologically related to S. itatiaiae and S. pentandra, but can be differenciated from them mainly due to the tree habit and fruiting calyx lobes not obscuring the disc in S. itatiaiae and the pistillate flowers with a disc that is thickened along the margin in S. pentandra.     

Exploring patterns of interspecific variation in quantitative traits using sequential phylogenetic eigenvector regressions

A number of metrics have been developed for estimating phylogenetic signal in data and to evaluate correlated evolution, inferring broad-scale evolutionary and ecological processes. Here, we proposed an approach called phylogenetic signal-representation (PSR) curve, built upon phylogenetic eigenvector regression (PVR). In PVR, selected eigenvectors extracted from a phylogenetic distance matrix are used to model interspecific variation. In the PSR curve, sequential PVR models are fitted after successively increasing the number of eigenvectors and plotting their R(2) against the accumulated eigenvalues. We used simulations to show that a linear PSR curve is expected under Brownian motion and that its shape changes under alternative evolutionary models. The PSR area, expressing deviations from Brownian motion, is strongly correlated (r= 0.873; P < 0.01) with Blomberg's K-statistics, so nonlinear PSR curves reveal if traits are evolving at a slower or higher rate than expected by Brownian motion. The PSR area is also correlated with phylogenetic half-life under an Ornstein-Uhlenbeck process, suggesting how both methods describe the shape of the relationship between interspecific variation and time since divergence among species. The PSR curve provides an elegant exploratory method to understand deviations from Brownian motion, in terms of acceleration or deceleration of evolutionary rates occurring at large or small phylogenetic distances.     

Heavy Metals in Hair of Wild Canids from the Brazilian Cerrado

In this study, we aimed to assess whether free-ranging wild canids are exposed to heavy metals in one of the most developed and populated regions of Brazil. Hair of 26 wild canids (maned wolves Chrysocyon brachyurus, crab-eating foxes Cerdocyon thous, and hoary foxes Lycalopex vetulus) from the Cerrado biome in Southeast Brazil were analyzed by spectrophotometry to detect cadmium, chromium, and lead, and also the essential copper, iron, manganese, and zinc traces. All samples showed traces of copper, iron, manganese, and zinc. Non-essential lead was detected in 57% (2.35 ± 0.99 mg/kg), and chromium in 88% (2.98 ± 1.56 mg/kg) of samples. Cadmium traces (detection limit 0.8 mg/kg) were not found. Crab-eating foxes had more copper, iron, and manganese in hair than maned wolves. Correlations among element levels differed between maned wolves and crab-eating foxes. Concentrations of chromium and lead were outstandingly higher than in wild canids from other areas. Addressing the causes of such levels and the impacts of the heavy metal pollution in Neotropical ecosystems is urgent for animal health and conservation purposes. We argue that heavy metal pollution should be considered as dangerous threats to wildlife health in Brazil and recommend hair sampling as a biomonitoring tool for heavy metals in Neotropical terrestrial mammals.     

Regeneration and characterization of somatic hybrids combining sweet orange and mandarin/mandarin hybrid cultivars for citrus scion improvement

Protoplast fusion between sweet orange and mandarin/mandarin hybrids scion cultivars was performed following the model ??diploid embryogenic callus protoplast?+?diploid mesophyll-derived protoplast??. Protoplasts were isolated from embryogenic calli of ??Pera?? and ??Westin?? sweet orange cultivars (Citrus sinensis) and from young leaves of ??Fremont??, Nules??, and ??Thomas?? mandarins (C. reticulata), and ??Nova?? tangelo [C. reticulata?×?(C. paradisi?×?C. reticulata)]. The regenerated plants were characterized based on their leaf morphology (thickness), ploidy level, and simple sequence repeat (SSR) molecular markers. Plants were successfully generated only when ??Pera?? sweet orange was used as the embryogenic parent. Fifteen plants were regenerated being 7 tetraploid and 8 diploid. Based on SSR molecular markers analyses all 7 tetraploid regenerated plants revealed to be allotetraploids (somatic hybrids), including 2 from the combination of ??Pera?? sweet orange?+???Fremont?? mandarin, 3 ??Pera?? sweet orange?+???Nules?? mandarin, and 2 ??Pera?? sweet orange?+???Nova?? tangelo, and all the diploid regenerated plants showed the ??Pera?? sweet orange marker profile. Somatic hybrids were inoculated with Alternaria alternata and no disease symptoms were detected 96?h post-inoculation. This hybrid material has the potential to be used as a tetraploid parent in interploid crosses for citrus scion breeding.     

Hepatitis C virus quantification in serum and saliva of HCV-infected patients

The hepatitis C virus (HCV) can be detected in blood and other bodily fluids, such as saliva, semen and gastric juices. The aim of this study was to compare the HCV viral loads in the serum and saliva of infected patients. Twenty-nine patients with detectable HCV RNA in their serum and saliva were included in this study. The HCV viral loads were determined through quantitative real-time polymerase chain reactions. The median viral RNA levels were 5.78 log10 copies in the serum and 3.32 log10 copies in the saliva. We observed that the salivary HCV viral load was significantly lower than the viral load in the serum. Further studies are required to understand the role of saliva in the diagnosis, management and potential transmission of HCV.     

On the selection of phylogenetic eigenvectors for ecological analyses

Among the statistical methods available to control for phylogenetic autocorrelation in ecological data, those based on eigenfunction analysis of the phylogenetic distance matrix among the species are becoming increasingly important tools. Here, we evaluate a range of criteria to select eigenvectors extracted from a phylogenetic distance matrix (using phylogenetic eigenvector regression, PVR) that can be used to measure the level of phylogenetic signal in ecological data and to study correlated evolution. We used a principal coordinate analysis to represent the phylogenetic relationships among 209 species of Carnivora by a series of eigenvectors, which were then used to model log‐transformed body size. We first conducted a series of PVRs in which we increased the number of eigenvectors from 1 to 70, following the sequence of their associated eigenvalues. Second, we also investigated three non‐sequential approaches based on the selection of 1) eigenvectors significantly correlated with body size, 2) eigenvectors selected by a standard stepwise algorithm, and 3) the combination of eigenvectors that minimizes the residual phylogenetic autocorrelation. We mapped the mean specific component of body size to evaluate how these selection criteria affect the interpretation of non‐phylogenetic signal in Bergmann's rule. For comparison, the same patterns were analyzed using autoregressive model (ARM) and phylogenetic generalized least‐squares (PGLS). Despite the robustness of PVR to the specific approaches used to select eigenvectors, using a relatively small number of eigenvectors may be insufficient to control phylogenetic autocorrelation, leading to flawed conclusions about patterns and processes. The method that minimizes residual autocorrelation seems to be the best choice according to different criteria. Thus, our analyses show that, when the best criterion is used to control phylogenetic structure, PVR can be a valuable tool for testing hypotheses related to heritability at the species level, phylogenetic niche conservatism and correlated evolution between ecological traits.     

Effects of (-) mammea A/BB isolated from Calophyllum brasiliense leaves and derivatives on mitochondrial membrane of Leishmania amazonensis

We have previously demonstrated antileishmanial activity on Leishmania amazonensis of the natural (1-2), synthetic (7) and derivatives of coumarin (-) mammea A/BB (3-6) isolated from the dichloromethane extract of Calophyllum brasiliense leaves. The aim of the present study was to evaluate morphological and ultrastructural alterations in Leishmania amazonensis induced by these compounds. In promastigote forms, all seven compounds produced significant morphological and ultrastructural alterations, as revealed by scanning and transmission electron microscopy. The compound 5,7-dihydroxy-8-(2-methylbutanoyl)-6-(3-methylbutyl)-4-phenyl-chroman-2-one (3), the most active antileishmanial with LD?? of 0.9 μM), induced cell shrinkage and a rounded appearance of the cells. Parasites incubated in the presence of compound (3) showed ultrastructural changes, such as the appearance of mitochondrial swelling with a reduction in the density of the mitochondrial matrix and the presence of vesicles inside the mitochondrion, indicating damage and significant change in this organelle; abnormal chromatin condensation, alterations in the nuclear envelope, intense atypical cytoplasmic vacuolization, and the appearance of autophagic vacuoles were also observed. In addition, the compound (3) may be acting to depolarize the mitochondrial membrane potential of the cells, leading to death of the parasite.