Vibrio parahaemolyticus ScrC modulates cyclic dimeric GMP regulation of gene expression relevant to growth on surfaces

In Vibrio parahaemolyticus, scrC participates in controlling the decision to be a highly mobile swarmer cell or a more adhesive, biofilm-proficient cell type. scrC mutants display decreased swarming motility over surfaces and enhanced capsular polysaccharide production. ScrC is a cytoplasmic membrane protein that contains both GGDEF and EAL conserved protein domains. These domains have been shown in many organisms to respectively control the formation and degradation of the small signaling nucleotide cyclic dimeric GMP (c-di-GMP). The scrC gene is part of the three-gene scrABC operon. Here we report that this operon influences the cellular nucleotide pool and that c-di-GMP levels inversely modulate lateral flagellar and capsular polysaccharide gene expression. High concentrations of this nucleotide prevent swarming and promote adhesiveness. Further, we demonstrate that ScrC has intrinsic diguanylate cyclase and phosphodiesterase activities, and these activities are controlled by ScrAB. Specifically, ScrC acts to form c-di-GMP in the absence of ScrA and ScrB; whereas ScrC acts to degrade c-di-GMP in the presence of ScrA and ScrB. The scrABC operon is specifically induced by growth on a surface, and the analysis of mutant phenotypes supports a model in which the phosphodiesterase activity of ScrC plays a dominant role during surface translocation and in biofilms.     

Brassinosteroid increases the cytokinin efficiency to induce direct somatic embryogenesis in leaf explants of <Emphasis Type="Italic">Coffea arabica</Emphasis> L. (Rubiaceae)

The combination of different plant growth regulators can result in beneficial effects in the induction of in vitro morphogenetic pathways. The present study reports the effect of 24-epibrassinolid (24-epiBR; brassinosteroid) when added alone and in association with N⁶-(2-isopentnyl) adenine (2-iP; cytokinin) in the induction of direct somatic embryogenesis in Coffea arabica. Leaf explants were cultivated in a modified Murashige and Skoog (MS) medium with 0 or 10 µM 2-iP and different concentrations (0.01, 0.10 or 1.0 µM) of 24-epiBR. Explants cultured on MS medium supplemented with 1.0 µM 24-epiBR in association with 2-iP produced 6.8 times more somatic embryos than the explants cultured with only 2-iP. Histological analyses also provided evidence that the supplementation of brassinosteroids in the culture medium could have influenced somatic embryogenesis differentiation. Somatic embryos obtained in the presence of brassinosteroid and cytokinin were better structured morpho-histologically as compared to those obtained in the medium with just cytokinin. This study opens new perspectives for the use of brassinosteroids in the somatic embryogenesis of C. arabica, so as to optimize the in vitro regeneration systems used in genetic improvement programs in C. arabica productive systems.     

Probing a polar cluster in the retinal binding pocket of bacteriorhodopsin by a chemical design approach

Bacteriorhodopsin has a polar cluster of amino acids surrounding the retinal molecule, which is responsible for light harvesting to fuel proton pumping. From our previous studies, we have shown that threonine 90 is the pivotal amino acid in this polar cluster, both functionally and structurally. In an attempt to perform a phenotype rescue, we have chemically designed a retinal analogue molecule to compensate the drastic effects of the T90A mutation in bacteriorhodopsin. This analogue substitutes the methyl group at position C(13) of the retinal hydrocarbon chain by and ethyl group (20-methyl retinal). We have analyzed the effect of reconstituting the wild-type and the T90A mutant apoproteins with all-trans-retinal and its 20-methyl derivative (hereafter, 13-ethyl retinal). Biophysical characterization indicates that recovering the steric interaction between the residue 90 and retinal, eases the accommodation of the chromophore, however it is not enough for a complete phenotype rescue. The characterization of these chemically engineered chromoproteins provides further insight into the role of the hydrogen bond network and the steric interactions involving the retinal binding pocket in bacteriorhodopsin and other microbial sensory rhodopsins.     

Recovery and purification of aprotinin from industrial insulin-processing effluent by immobilized chymotrypsin and negative IMAC chromatographies

Aprotinin, a bovine protease inhibitor currently also produced in recombinant bacteria, yeast, and corn, has valuable applications as a human therapeutic and in tissue culture. The objective of this work was to develop the basis of a large-scale aprotinin purification process centered on immobilized metal ion affinity chromatography (IMAC). This technique uses ligands—metal ions—of a lower cost and higher stability than those traditionally used in affinity chromatography. Since aprotinin does not interact with IMAC ligands, collection is from the nonretained fractions (negative chromatography). Stirred-tank batch IMAC adsorption experiments indicated that one-step aprotinin purification could not be successful. Immobilized chymotrypsin chromatography was then used as a prepurification step, yielding a suitable feed for IMAC (with purification factors as high as 476). IMAC column fed with these prepurified materials produced purified aprotinin in the nonretained fractions with purification factors as high as 952.     

Modeling human mitochondrial diseases in flies

Human mitochondrial diseases are associated with a wide range of clinical symptoms, and those that result from mutations in mitochondrial DNA affect at least 1 in 8500 individuals. The development of animal models that reproduce the variety of symptoms associated with this group of complex human disorders is a major focus of current research. Drosophila represents an attractive model, in large part because of its short life cycle, the availability of a number of powerful techniques to alter gene structure and regulation, and the presence of orthologs of many human disease genes. We describe here Drosophila models of mitochondrial DNA depletion, deafness, encephalopathy, Freidreich's ataxia, and diseases due to mitochondrial DNA mutations. We also describe several genetic approaches for gene manipulation in flies, including the recently developed method of targeted mutagenesis by recombinational knock-in.     

Flooding tolerance of Tabebuia cassinoides: Metabolic, morphological and growth responses

Tabebuia cassinoides (Lam.) DC (Bignoniaceae) is a tree species that occurs in swampy areas of the coastal “restinga” in SE Brazil (a coastal sandy plains scrub and forest formation). To elucidate possible adaptive strategies that enable this species to occupy areas subjected to seasonal or perennial waterlogging, metabolic, morphological and growth responses of plants under flooding conditions were studied. The root system of T. cassinoides plants presented elevated amounts of ethanol (10.6 μmol g⁻¹ fresh wt) only in the first 5 d of soil water saturation. The two-fold increase in ethanol production under flooding was corroborated by an increase in ADH activity in the same period. Lactic acid concentrations did not change significantly during four months of flooding treatment. The decrease of alcoholic fermentation under hypoxia was associated with the appearing of new roots. The induction of aerenchyma formation in roots developed under flooding conditions, allowed oxygen transport from the shoot to these organs, thus maintaining an aerobic respiration. We conclude that this characteristic and the capacity to oxidize the rhizosphere are probably responsible for the survival and growth of plants while flooded and for their success in an environment, which restricts the presence of the majority of competing tree species.