Dynamics of biochemical and morphophysiological changes during zygotic embryogenesis in Acca sellowiana (Berg.) Burr.

Acca sellowiana (Berg.) Burr. is a native Myrtaceae from southern Brazil and Uruguay, now the subject of a domestication and breeding program. Biotechnological tools have been used to assist in this program. The establishment of a reliable protocol of somatic embryogenesis has been pursued, with a view to capturing and fixing genetic gains. The rationale behind this work relies on the fact that deepening comprehension of the general metabolism of zygotic embryogenesis may certainly improve the protocol for somatic embryogenesis. Thus, in the present work we studied the accumulation of protein, total sugars, starch, amino acids, polyamines (PAs), IAA and ABA, in different stages of A. sellowiana zygotic embryogenesis. Starch is the predominant storage compound during zygotic embryo development. Increased synthesis of amino acids in the cotyledonary stage, mainly of asparagine, was observed throughout development. Total free PAs showed increased synthesis, whereas total conjugated PAs were mainly observed in the early developmental stages. IAA decreased and ABA increased with the progression from early to late embryogenesis. Besides providing basic information on the morphophysiological and biochemical changes of zygotic embryogenesis, the results here obtained may provide adequate strategies towards the modulation of somatic embryogenesis in this species as well as in other woody angiosperms.

Biochemical and biophysical properties of a highly active recombinant arginase from Leishmania (Leishmania) amazonensis and subcellular localization of native enzyme

Arginase (L-arginine amidinohydrolase, E.C. 3.5.3.1) is a metalloenzyme that catalyses the hydrolysis Of L-arginine to L-ornithine and urea. In Leishmania spp., the biological role of the enzyme may be involved in modulating NO production upon macrophage infection. Previously, we cloned and characterized the arginase gene from Leishmania (Leishmania) amazonensis. In the present work, we successfully expressed the recombinant enzyme in E. coli and performed biochemical and biophysical characterization of both the native and recombinant enzymes. We obtained K-M and V-max. values of 23.9(+/- 0.96) mM and 192.3 mu mol/min mg protein (+/- 14.3), respectively, for the native enzyme. For the recombinant counterpart, K-M was 21.5(+/- 0.90) mM and V-max was 144.9(+/- 8.9) mu mol/min mg. Antibody against the recombinant protein confirmed a glycosomal cellular localization of the enzyme in promastigotes. Data from light scattering and small angle X-ray scattering showed that a trimeric state is the active form of the protein. We determined empirically that a manganese wash at room temperature is the best condition to purify active enzyme. The interaction of the recombinant protein with the immobilized nickel also allowed us to confirm the structural disposition of histidine at positions 3 and 324. The determined structural parameters provide substantial data to facilitate the search for selective inhibitors of parasitic sources of arginase, which could subsequently point to a candidate for leishmaniasis therapy. (c) 2008 Elsevier B.V. All rights reserved.

Morphometrical, biochemical and molecular tools for assessing biodiversity. An example in Plebeia remota (Holmberg, 1903) (Apidae, Meliponini)

We see today many efforts to quantify biodiversity in different biomes. It is very important then to develop and to apply other methodologies that allow us to assess biodiversity. Here we present an example of application of three tools with this goal. We analyzed two populations of Plebeia remota from two distinct biomes that already showed several differences in morphology and behavior. Based on these differences, it has been suggested that the populations of Cunha and Prudentopolis do not represent a single species. In order to verify the existence or absence of gene flow between these two groups, we characterized the patterns of mtDNA through RFLP, the patterns of wing venation through geometric morphometry, and the cuticular hydrocarbons through gas chromatography-mass spectrometry. We used bees collected in these two locations and also from colonies which have being kept for around 9 years at Sao Paulo University. We found six different haplotypes in these specimens, of which three of them occurred exclusively in the population of Cunha and three only in the Prudentopolis population. The fact that the populations do not share haplotypes suggests no maternal gene flow between them. The two populations were differentiated by the pattern of the wing veins. They also had different mixtures of cuticle hydrocarbons. Furthermore it was shown that the colonies kept at the university did not hybridize. These two groups may constitute different species. We also show here the importance of using other methodologies than traditional taxonomy to assess and understand biodiversity, especially in bees.

Purification, and Biochemical and Biophysical Characterization of Cellobiohydrolase I from Trichoderma harzianum IOC 3844

Because of its elevated cellulolytic activity, the filamentous fungus Trichoderma harzianum has a considerable potential in biomass hydrolysis applications. Trichoderma harzianum cellobiohydrolase I (ThCBHI), an exoglucanase, is an important enzyme in the process of cellulose degradation. Here, we report an easy single-step ion-exchange chromatographic method for purification of ThCBHI and its initial biophysical and biochemical characterization. The ThCBHI produced by induction with microcrystalline cellulose under submerged fermentation was purified on DEAE-Sephadex A-50 media and its identity was confirmed by mass spectrometry. The ThCBHI biochemical characterization showed that the protein has a molecular mass of 66 kDa and pi of 5.23. As confirmed by small-angle X-ray scattering (SAXS), both full-length ThCBHI and its catalytic core domain (CCD) obtained by digestion with papain are monomeric in solution. Secondary structure analysis of ThCBHI by circular dichroism revealed alpha-helices and beta-strands contents in the 28% and 38% range, respectively. The intrinsic fluorescence emission maximum of 337 nm was accounted for as different degrees of exposure of ThCBHI tryptophan residues to water. Moreover, ThCBHI displayed maximum activity at pH 5.0 and temperature of 50 degrees C with specific activities against Avicel and p-nitrophenyl-beta-D-cellobioside of 1.25 U/mg and 1.53 U/mg, respectively.

Skull Modularity in Neotropical Marsupials and Monkeys: Size Variation and Evolutionary Constraint and Flexibility

An organism is built through a series of contingent factors, yet it is determined by historical, physical, and developmental constraints. A constraint should not be understood as an absolute obstacle to evolution, as it may also generate new possibilities for evolutionary change. Modularity is, in this context, an important way of organizing biological information and has been recognized as a central concept in evolutionary biology bridging on developmental, genetics, morphological, biochemical, and physiological studies. In this article, we explore how modularity affects the evolution of a complex system in two mammalian lineages by analyzing correlation, variance/covariance, and residual matrices (without size variation). We use the multivariate response to selection equation to simulate the behavior of Eutheria and Metharia skulls in terms of their evolutionary flexibility and constraints. We relate these results to classical approaches based on morphological integration tests based on functional/developmental hypotheses. Eutherians (Neotropical primates) showed smaller magnitudes of integration compared with Metatheria (didelphids) and also skull modules more clearly delimited. Didelphids showed higher magnitudes of integration and their modularity is strongly influenced by within-groups size variation to a degree that evolutionary responses are basically aligned with size variation. Primates still have a good portion of the total variation based on size; however, their enhanced modularization allows a broader spectrum of responses, more similar to the selection gradients applied (enhanced flexibility). Without size variation, both groups become much more similar in terms of modularity patterns and magnitudes and, consequently, in their evolutionary flexibility. J. Exp. Zool. (Mol. Dev. Evol.) 314B:663-683, 2010. (C) 2010 Wiley-Liss, Inc.

Microcystins -LA, -YR, and -LR action on neutrophil migration

Microcystins (MCs) produced by some freshwater cyanobacterial species possess potent liver toxicity as evidenced by acute neutrophil infiltration. Here, we investigate the ability of three structurally distinct toxins (MC-LA, MC-LR, and MC-YR) to evoke neutrophil recruitment per se and their effects on migration pathways. Intravital Microscopic Studies showed that topical application of only MC-LR enhanced the numbers of rolling and adhered leukocytes in the endothelium of postcapillary mesenteric venules. The latter effects may be dependent upon induction of the synthesis and expression Of L-selectin and beta(2)-integrin in neutrophils, as assessed by flow cytometry and RT-PCR, respectively. Conversely, the three toxins promoted direct locomotion of neutrophils and enhanced their migration in response to NO, as measured by Boyden chamber assays, and increased intracellular calcium, a messenger in the chemotaxic process. In conclusion, our results show that MCs act on specific pathways of neutrophil recruitment, indicating their potential effect on neutrophils activation. (C) 2009 Elsevier Inc. All rights reserved.

In vivo blockade of Ca(+2)-dependent nitric oxide synthases impairs expressions of L-selectin and PECAM-1

Interactions of leukocytes with endothelium play a role for the immune system modulated by endogenous agents, such as glucocorticoids and nitric oxide (NO). Glucocorticoids inhibit leukocyte-endothelial interactions whereas the role of NO is still controversial. In this study, the activity of Ca(+2)-dependent nitric oxide synthases was in vivo blocked in male Wistar rats by given L-NAME, 20 mg kg(-1) for 14 days dissolved in drinking water and expression of adhesion molecules involved in leukocyte-endothelial interactions was investigated. Expressions of L-selectin and PECAM-I in peripheral leukocytes and PECAM-1 in endothelial cells were reduced by L-NAME treatment. Only L-selectin expression was controlled at transcriptional levels. These effects were not dependent on endogenous glucocorticoids, as corticosterone levels were not altered in NAME-treated rats. Our results show that NO, produced at physiological levels, controls expression of constitutive adhesion molecules expressions in cell membranes by different mechanisms of action. Published by Elsevier Inc.

TAF15 and the leukemia-associated fusion protein TAF15-CIZ/NMP4 are cleaved by caspases-3 and-7

Caspases are central players in proteolytic pathways that regulate cellular processes Such as apoptosis and differentiation. To accelerate the discovery of novel caspase substrates we developed a method combining in silico screening and in vitro validation. With this approach, we identified TAH15 as a novel caspase Substrate in a trial Study. We find that TAF15 was specifically cleaved by caspases-3 and -7. Site-directed mutagenesis revealed the consensus sequence (106)DQPD/Y(110) as the only site recognized by these caspases. Surprisingly, TAF15 was cleaved at more than one site in staurosporine-treated Jurkat cells. In addition, we generated two oncogenic TAF15-CIZ/NMP4-fused proteins which have been found in acute myeloid leukemia and demonstrate that caspases-3 and -7 cleave the fusion proteins at one single site. Broad application of this combination approach should expedite identification of novel caspase-interacting proteins and provide new insights into the regulation of caspase pathways leading to cell death in normal and cancer cells. (C) 2009 Elsevier Inc. All rights reserved.

Long-term nitric oxide deficiency causes muscarinic supersensitivity and reduces beta(3)-adrenoceptor-mediated relaxation, causing rat detrusor overactivity

Background and purpose: Overactive bladder is a complex and widely prevalent condition, but little is known about its physiopathology. We have carried out morphological, biochemical and functional assays to investigate the effects of long-term nitric oxide (NO) deficiency on muscarinic receptor and beta-adrenoceptor modulation leading to overactivity of rat detrusor muscle. Experimental approach: Male Wistar rats received No-nitro-L-arginine methyl ester (L-NAME) in drinking water for 7-30 days. Functional responses to muscarinic and b-adrenoceptor agonists were measured in detrusor smooth muscle (DSM) strips in Krebs-Henseleit solution. Measurements of [H-3] inositol phosphate, NO synthase (NOS) activity, [H-3] quinuclidinyl benzilate ([H-3]QNB) binding and bladder morphology were also performed. Key results: Long-term L-NAME treatment significantly increased carbachol-induced DSM contractile responses after 15 and 30 days; relaxing responses to the beta(3)-adrenoceptor agonist BRL 37-344 were significantly reduced at 30 days. Constitutive NOS activity in bladder was reduced by 86% after 7 days and maintained up to 30 days of L-NAME treatment. Carbachol increased sixfold the [H-3] inositol phosphate in bladder tissue from rats treated with L-NAME. [H-3] QNB was bound with an apparent KD twofold higher in bladder membranes after L-NAME treatment compared with that in control. No morphological alterations in DSM were found. Conclusions and implications: Long-term NO deficiency increased rat DSM contractile responses to a muscarinic agonist, accompanied by significantly enhanced KD values for muscarinic receptors and [H-3] inositol phosphate accumulation in bladder. This supersensitivity for muscarinic agonists along with reductions of beta(3)-adrenoceptor-mediated relaxations indicated that overactive DSM resulted from chronic NO deficiency.

Diapocynin versus apocynin as pretranscriptional inhibitors of NADPH oxidase and cytokine production by peripheral blood mononuclear cells

Apocynin has been extensively used as an inhibitor of NADPH oxidase (NOX) in many experimental models using phagocytic and non-phagocytic cells. Currently, there is some controversy about the efficacy of apocynin in non-phagocytic cells, but in phagocytes the reported results are consistent, which could be due to the presence of myeloperoxidase in these cells. This enzyme has been proposed as responsible for activating apocynin by generating its dimer, diapocynin, which is supposed to be the active compound that prevents NADPH oxidase complex assembly and activation. Here, we synthesized diapocynin and studied its effect on inhibition of gp91(phox) RNA expression. We found that diapocynin strongly inhibited the expression of gp91(phox)mRNA in peripheral blood mononuclear cells (PBMC). Only at a higher concentration, apocynin was able to exert the same effect. We also compared the apocynin and diapocynin efficacy as inhibitors of tumor necrosis factor-alpha (TNF-alpha) and interleukin-10 (IL-10) production in response to lipopolysaccharide (LPS)-activated PBMC. Although apocynin did inhibit TNF-alpha production, diapocynin had a much more pronounced effect, on both TNF-alpha and IL-10 production. In conclusion, these findings suggest that the bioconversion of apocynin to diapocynin is an important issue not limited to enzymatic activity inhibition, but also for other biological effects as gp91(phox) mRNA expression and cytokine production. Hence, as diapocynin can be easily prepared from apocynin, a one-step synthesis, we recommend its use in studies where the biological effects of apocynin are searched. (C) 2010 Elsevier Inc. All rights reserved.

Different mechanisms underlie the effects of acute and long-term inhibition of nitric oxide synthases in antigen-induced pulmonary eosinophil recruitment in BALB/C mice

Nitric oxide synthase (NOS) inhibitors are largely used to evaluate the NO contribution to pulmonary allergy, but contrasting data have been reported. In this study, pharmacological, biochemical and pharmacokinetic assays were performed to compare the effects of acute and long-term treatment of BALB/C mice with the non-selective NOS inhibitor L-NAME in ovalbumin (OVA)-challenged mice. Acute L-NAME treatment (50 mg/kg, gavage) significantly reduced the eosinophil number in bronchoalveolar lavage fluid (BALF). The inducible NOS (iNOS) inhibitor aminoguanidine (20 mg/kg/day in the drinking water) also significantly reduced the eosinophil number in BALF In contrast, 3-week L-NAME treatment (50 and 150 mg/kg/day in the drinking water) significantly increased the pulmonary eosinophil influx. The constitutive NOS (cNOS) activity in brain and lungs was reduced by both acute and 3-week L-NAME treatments. The pulmonary iNOS activity was reduced by acute L-NAME (or aminoguanidine), but unaffected by 3-week L-NAME treatment. Acute L-NAME (or aminoguanidine) treatment was more efficient to reduce the NO(x) levels compared with 3-week L-NAME treatment. The pharmacokinetic study revealed that L-NAME is not bioavailable when given orally. After acute L-NAME intake, serum concentrations of the metabolite N(omega)-nitro-L-arginine decreased from 30 min to 24 h. In the 3-week L-NAME treatment, the N(omega)-nitro-L-arginine concentration was close to the detection limit. In conclusion, 3-week treatment with L-NAME yields low serum N(omega)-nitro-L-arginine concentrations, causing preferential inhibition of cNOS activity. Therefore, eosinophil influx potentiation by 3-week L-NAME treatment may reflect removal of protective cNOS-derived NO, with no interference on the ongoing inflammation due to iNOS-derived NO. (c) 2008 Elsevier Ltd. All rights reserved.

Toxicokinetics and toxicological effects of single oral dose of fumonisin B1 containing Fusarium verticillioides culture material in weaned piglets

Toxicokinetics and the toxicological effects of culture material containing fumonisin B(1) (FB(1)) were studied in male weaned piglets by clinical, pathological, biochemical and sphingolipid analyses. The animals received a single oral dose of 5 mg FB(1)/kg of body weight. obtained from Fusarium verticillioides culture material. FB(1) was detected by H PLC in plasma collected at 1-h intervals up to 6 h and at 12-h intervals up to 96 h. FB(1) eliminated in feces and urine was quantified over a 96-h period and in liver samples collected 96 h post-intoxication. Blood samples were obtained at the beginning and end of the experiment to determine serum enzyme activity, total bilirubin, cholesterol, sphinganine (Sa), sphingosine (So) and the Sa/So ratio. FB(1) was detected in plasma between 30 min and 36 h after administration. The highest concentration of FB(1) was observed after 2 h, with a mean concentration of 282 mu g/ml. Only 0.93% of the total FB(1) was detected in urine between 75 min and 41 h after administration, the highest mean concentration (561 mu g/ml) was observed during the interval after 8 at 24 h. Approximately 76.5% of FB(1) was detected in feces eliminated between 8 and 84 h after administration, with the highest levels observed between 8 and 24 h. Considering the biochemical parameters, a significant increase only occurred in cholesterol, alkaline phosphatase and aspartate aminotransferase activities. In plasma and urine, the highest Sa and Sa/So ratios were obtained at 12 and 48 h, respectively. (C) 2010 Elsevier Ireland Ltd. All rights reserved.

Proteins and Peptidases from Conidia and Mycelia of Scedosporium apiospermum Strain HLPB

The conidia-mycelia transformation is an essential step during the life cycle of the fungal human pathogens of the Pseudallescheria boydii complex. In the present study, we have analyzed the protein and peptidase profiles in two distinct morphological stages, conidia and mycelia, of Scedosporium apiospermum sensu stricto. Proteins synthesized by the mycelia, migrating at the ranges of 62-48 and 22-18 kDa, were not detected from the conidial extract. Conidia produced a single cellular peptidase of 28 kDa able to digest copolymerized albumin, while mycelia yielded 6 distinct peptidases ranging from 90 to 28 kDa. All proteolytic enzymes were active at acidic pH and fully inhibited by 1,10-phenanthroline, characterizing these activities as metallo-type peptidases. Quantitative peptidase assay, using soluble albumin, showed a high metallopeptidase production in mycelial cells in comparison with conidia. The regulated expression of proteins and peptidases in different morphological stages of S. apiospermum represents a potential target for isolation of stage-specific markers for biochemical and immunological analysis.

Mode of operation and low-resolution structure of a multi-domain and hyperthermophilic endo-beta-1,3-glucanase from Thermotoga petrophila

1,3-beta-Glucan depolymerizing enzymes have considerable biotechnological applications including biofuel production, feedstock-chemicals and pharmaceuticals. Here we describe a comprehensive functional characterization and low-resolution structure of a hyperthermophilic laminarinase from Thermotoga petrophila (TpLam). We determine TpLam enzymatic mode of operation, which specifically cleaves internal beta-1,3-glucosidic bonds. The enzyme most frequently attacks the bond between the 3rd and 4th residue from the non-reducing end, producing glucose, laminaribiose and laminaritriose as major products. Far-UV circular dichroism demonstrates that TpLam is formed mainly by beta structural elements, and the secondary structure is maintained after incubation at 90 degrees C. The structure resolved by small angle X-ray scattering, reveals a multi-domain structural architecture of a V-shape envelope with a catalytic domain flanked by two carbohydrate-binding modules. Crown Copyright (C) 2011 Published by Elsevier Inc. All rights reserved.

alpha-Hydroxynitrile lyase protein from Xylella fastidiosa: Cloning, expression, and characterization

Xylella fastidiosa is a xylem-restricted plant pathogen that causes a range of diseases in several and important crops. Through comparative genomic sequence analysis many genes were identified and, among them, several potentially involved in plant-pathogen interaction. The experimental determination of the primary sequence of some markedly expressed proteins for X fastidiosa and the comparison with the nucleic acids sequence of genome identified one of them as being SCJ21.16 (XFa0032) gene product. The comparative analysis of this protein against SWISSPROT database, in special, resulted in similarity with a-hydroxynitrile lyase enzyme (HNL) from Arabidopsis thaliana, causing interest for being one of the most abundant proteins both in the whole cell extract as well as in the extracellular protein fraction. It is known that HNL enzyme are involved in a process termed ""cyanogenesis"", which catalyzes the dissociation of alpha-hydroxinitrile into carbonyle and HCN when plant tissue is damaged. Although the complete genome sequences of X.fastidiosa are available and the cyanogenesis process is well known, the biological role of this protein in this organism is not yet functionally characterized. In this study we presented the cloning, expression, characterization of recombinant HNL from X fastidiosa, and its probable function in the cellular metabolism. The successful cloning and heterologous expression in Escherichia coli resulted in a satisfactory amount of the recombinant HNL expressed in a soluble, and active form giving convenient access to pure enzyme for biochemical and structural studies. Finally, our results confirmed that the product of the gene XFa0032 can be positively assigned as FAD-independent HNLs. (C) 2009 Elsevier Ltd. All rights reserved.