Electrocoagulation as a clarifiation and pre-purification step in plant based bioprocesses

Electrocoagulation is a technique that has been applied to water and wastewater treatment. We propose here extension of this technique to the field of plant downstream processing (DSP), where plants are used as a bioreactors for recombinant protein production. The main problem in plant-based bioreactors is the presence of chlorophyll and phenolic compounds in plant extracts, which tend to precipitate and denature proteins. Their removal from the extracts is essential, since they have a large impact on the DSP performance. In the present work we studied the application of an electrocoagulation based technique to clarify tobacco leaf extracts by removing chlorophyll and phenolic compounds. By manipulating the pH of electrocoagulation, 90% of chlorophyll, 65% of phenolic compounds, and only 23% of total protein were removed from the extract. The process preferentially removes proteins with more acidic pI (below 6.03) and the pH of the process differentially effects removal of acidic and basic proteins.     

Hepatic stellate cells uptake of retinol associated with retinol-binding protein or with bovine serum albumin

Retinol is stored in liver, and the dynamic balance between its accumulation and mobilization is regulated by hepatic stellate cells (HSC). Representing less than 1% total liver protein, HSC can reach a very high intracellular retinoid (vitamin-A and its metabolites) concentration, which elicits their conversion from the myofibroblast to the fat-storing lipocyte phenotype. Circulating retinol is associated with plasma retinol-binding protein (RBP) or bovine serum albumin (BSA). Here we have used the in vitro model of GRX cells to compare incorporation and metabolism of BSA versus RBP associated [(3)H]retinol in HSC. We have found that lipocytes, but not myofibroblasts, expressed a high-affinity membrane receptor for RBP-retinol complex (KD = 4.93 nM), and both cell types expressed a low-affinity one (KD = 234 nM). The RBP-retinol complex, but not the BSA-delivered retinol, could be dislodged from membranes by treatments that specifically disturb protein-protein interactions (high RBP concentrations). Under both conditions, treatments that disturb the membrane lipid layer (detergent, cyclodextrin) released the membrane-bound retinol. RBP-delivered retinol was found in cytosol, microsomal fraction and, as retinyl esters, in lipid droplets, while albumin-delivered retinol was mainly associated with membranes. Disturbing the clathrin-mediated endocytosis did not interfere with retinol uptake. Retinol derived from the holo-RBP complex was differentially incorporated in lipocytes and preferentially reached esterification sites close to lipid droplets through a specific intracellular traffic route. This direct influx pathway facilitates the retinol uptake into HSC against the concentration gradients, and possibly protects cell membranes from undesirable and potentially noxious high retinol concentrations.     

Biochemical characterization of recombinant guaA-encoded guanosine monophosphate synthetase (EC 6.3.5.2) from Mycobacterium tuberculosis H37Rv strain

Administration of the current tuberculosis (TB) vaccine to newborns is not a reliable route for preventing TB in adults. The conversion of XMP to GMP is catalyzed by guaA-encoded GMP synthetase (GMPS), and deletions in the Shiguella flexneri guaBA operon led to an attenuated auxotrophic strain. Here we present the cloning, expression, and purification of recombinant guaA-encoded GMPS from Mycobacterium tuberculosis (MtGMPS). Mass spectrometry data, oligomeric state determination, steady-state kinetics, isothermal titration calorimetry (ITC), and multiple sequence alignment are also presented. The homodimeric MtGMPS catalyzes the conversion of XMP, MgATP, and glutamine into GMP, ADP, PP(i), and glutamate. XMP, NH(4)(+), and Mg(2+) displayed positive homotropic cooperativity, whereas ATP and glutamine displayed hyperbolic saturation curves. The activity of ATP pyrophosphatase domain is independent of glutamine amidotransferase domain, whereas the latter cannot catalyze hydrolysis of glutamine to NH(3) and glutamate in the absence of substrates. ITC data suggest random order of binding of substrates, and PP(i) is the last product released. Sequence comparison analysis showed conservation of both Cys-His-Glu catalytic triad of N-terminal Class I amidotransferase and of amino acid residues of the P-loop of the N-type ATP pyrophosphatase family.     

The hypothesis of sympatric speciation as the dominant generator of endemism in a global hotspot of biodiversity

Allopatric or sympatric speciation influence the degree to which closely related species coexist in different manners, altering the patterns of phylogenetic structure and turnover among and between communities. The objective of this study was to examine whether phylogenetic community structure and turnover in the Brazilian Atlantic Forest permit conclusions about the dominant process for the formation of extant angiosperm richness of tree species. Therefore, we analyzed phylogenetic community structure (MPD, MNTD) as well as taxonomic (Jaccard similarity) and phylogenetic turnover (betaMPD, betaMNTD) among and between 49 tree communities distributed among three different habitat types. Mean annual precipitation and mean annual temperature in each survey area were estimated. Phylogenetic community structure does not differ between habitat types, although MPD reduces with mean annual temperature. Jaccard similarity decreases and betaMNTD increases with spatial distance and environmental differences between study sites. Spatial distance explains the largest portions of variance in the data, indicating dispersal limitation and the spatial aggregation of recently formed taxa, as betaMNTD is related to more recent evolutionary events. betaMPD, that is related to deep evolutionary splits, shows no spatial or environmental pattern, indicating that older clades are equally distributed across the Brazilian Atlantic Forest. While similarity pattern indicates dispersal limitations, the spatial turnover of betaMNTD is consistent with a high degree of sympatric speciation generating extant diversity and endemism in the Brazilian Atlantic Forest. More comprehensive approaches are necessary to reduce spatial sampling bias, uncertainties regarding angiosperm diversification patterns and confirm sympatric speciation as the dominant generator for the formation of extant species diversity in the Brazilian Atlantic Forest.     

Faunistic analysis of sharpshooters (Hemiptera: Auchenorrhyncha, Cicadellidae) in a 'Westin' sweet orange orchard

The purpose of this study was to analyze sharpshooter fauna in a five-year-old 'Westin' sweet orange orchard. Yellow sticky traps were placed on the edge of a forest, and on the periphery and inside the citrus stand. The traps were evaluated fortnightly, for three years. The most frequent species were Acrogonia citrina Marucci & Cavichioli, Bucephalogonia xanthophis (Berg), and Oncometopia facialis (Signoret). B. xanthophis occurred more in the forest edge, especially on spring and winter. A. citrina occurred most frequently in the forest edge, especially on spring. The highest incidence of O. facialis was inside the citrus stand, on spring and summer. Other cicadellids occurred more often in the forest edge, especially on summer. A. citrina, B. xanthophis, Dilobopterus costalimai Young, and O. facialis were predominant in all places studied. A. citrina, B. xanthophis and O. facialis were super dominant, super abundant, super frequent, and constant, except inside the stand, where B. xanthophis was dominant, very abundant, very frequent, and constant. D. costalimai and Homalodisca ignorata Melichar were dominant, very abundant, and very frequent in the forest edge and in the periphery of the stand, and D. costalimai was also predominant inside the stand. Scopogonalia subolivacea (St?l) was predominant in the forest edge and inside the stand, while Plesiommata corniculata Young was predominant in the periphery (both were dominant, very abundant, very frequent, and accessory).     

Rep-PCR of tropical rhizobia for strain fingerprinting, biodiversity appraisal and as a taxonomic and phylogenetic tool

With more than 30 million doses of rhizobial inoculants marketed per year, it is probable that Brazilian agriculture benefits more than any other country from symbiotic N₂ fixation. As a result of strain-selection programs, 142 strains of rhizobia are officially recommended for use in commercial inoculants for ninety-six leguminous crops. In this study, sixty-eight of these elite strains were characterized by rep-PCR with the BOX-primer. Reproducibility of the DNA profiles was confirmed, suggesting efficacy of BOX-PCR both for control of quality of inoculants and for preliminary characterization of rhizobial culture collections. Strains of different species never showed similarity higher than 70% in the BOX-PCR analysis, however, some strains of the same species fit into more than one cluster, and correlation between BOX-PCR products and l6S rRNA sequences was low (7.6%). On the other hand, a polyphasic approach — 20%∶80% of BOX-PCR:16S rRNA which correlated well with the l6S rRNA analysis (95%), and provided higher definition of the genotypes, resulting in clearer indications of the taxonomic groups — might expedite rhizobial diversity studies.     

Molecular mechanisms beyond glucose transport in diabetes-related male infertility

Diabetes mellitus (DM) is one of the greatest public health threats in modern societies. Although during a few years it was suggested that DM had no significant effect in male reproductive function, this view has been challenged in recent years. The increasing incidence of DM worldwide will inevitably result in a higher prevalence of this pathology in men of reproductive age and subfertility or infertility associated with DM is expected to dramatically rise in upcoming years. From a clinical perspective, the evaluation of semen parameters, as well as spermatozoa deoxyribonucleic acid (DNA) integrity, are often studied due to their direct implications in natural and assisted conception. Nevertheless, recent studies based on the molecular mechanisms beyond glucose transport in testicular cells provide new insights in DM-induced alterations in male reproductive health. Testicular cells have their own glucose sensing machinery that react to hormonal fluctuations and have several mechanisms to counteract hyper- and hypoglycemic events. Moreover, the metabolic cooperation between testicular cells is crucial for normal spermatogenesis. Sertoli cells (SCs), which are the main components of blood–testis barrier, are not only responsible for the physical support of germ cells but also for lactate production that is then metabolized by the developing germ cells. Any alteration in this tied metabolic cooperation may have a dramatic consequence in male fertility potential. Therefore, we present an overview of the clinical significance of DM in the male reproductive health with emphasis on the molecular mechanisms beyond glucose fluctuation and transport in testicular cells.     

<Emphasis Type=Italic>Lockhartia viruensis</Emphasis> (Orchidaceae-Oncidiinae), a new species from Roraima state,Brazilian Amazonia region

A new species, Lockhartia viruensis, is described and illustrated from the state of Roraima, in the Brazilian Amazon, and its affinities are discussed. The new species is similar to L. imbricata, but differs by having an entire lip with an obtuse to rounded apex, a callus on the disk of the lip with a longitudinal row of papillae with a rounded apex, and the margin of the column wings entire.     

Secondary succession impairment in restored mangroves

In this work it was hypothesized that secondary succession on sites that have been managed by single planting of mangrove species is compromised by residual stressors, which could reduce the ecosystem??s structural development and lower its functions. Forest structure and environmental characteristics of three planted mangrove stands are compared with reference sites. Structural attributes showed significant differences in the comparison of planted and reference stands. Avicennia schaueriana was the dominant species within both natural regeneration and old-growth stands in terms of basal area (99.2 and 99.4?%, 69.6 and 84.5?%, and 59.0 and 87.1?% for Itacorubi, Saco Grande, and Ratones, respectively). Restoration stands were dominated by Laguncularia racemosa (80.6 and 94.2?% for Saco Grande and Ratones, respectively), except at one site (Itacorubi), where A. schaueriana prevailed (99.7?%). Even though restoration and regeneration stands at Itacorubi showed similar species composition and dominance, cohort sorting revealed an inferior regeneration potential in the restoration stand. Multiple correlation analysis indicated that variables related to elevation disruptions (p _w ?=?0.521) were the environmental drivers responsible for the differences observed in forest structure. At restoration sites an impaired pattern of secondary succession was observed, indicating that single species plantings may be ineffective if characteristics of the site, as well as of the area surrounding it, are not considered. The inadequate management of restoration sites can therefore have implications for both immediate and long-term large-scale ecosystem services.