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.

Polyamines, IAA and ABA during germination in two recalcitrant seeds: Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm)

Background and Aims Plant growth regulators play an important role in seed germination. However, much of the current knowledge about their function during seed germination was obtained using orthodox seeds as model systems, and there is a paucity of information about the role of plant growth regulators during germination of recalcitrant seeds. In the present work, two endangered woody species with recalcitrant seeds, Araucaria angustifolia (Gymnosperm) and Ocotea odorifera (Angiosperm), native to the Atlantic Rain Forest, Brazil, were used to study the mobilization of polyamines (PAs), indole-acetic acid (IAA) and abscisic acid (ABA) during seed germination. Methods Data were sampled from embryos of O. odorifera and embryos and megagametophytes of A. angustifolia throughout the germination process. Biochemical analyses were carried out in HPLC. Key Results During seed germination, an increase in the (Spd + Spm) : Put ratio was recorded in embryos in both species. An increase in IAA and PA levels was also observed during seed germination in both embryos, while ABA levels showed a decrease in O. odorifera and an increase in A. angustifolia embryos throughout the period studied. Conclusions The (Spd + Spm) : Put ratio could be used as a marker for germination completion. The increase in IAA levels, prior to germination, could be associated with variations in PA content. The ABA mobilization observed in the embryos could represent a greater resistance to this hormone in recalcitrant seeds, in comparison to orthodox seeds, opening a new perspective for studies on the effects of this regulator in recalcitrant seeds. The gymnosperm seed, though without a connective tissue between megagametophyte and embryo, seems to be able to maintain communication between the tissues, based on the likely transport of plant growth regulators.

Chloroplast microsatellite markers for the Neotropical orchid genus Epidendrum, and cross-amplification in other Laeliinae species (Orchidaceae)

One of the most significant challenges confronting orchid researchers is the lack of specific molecular markers, mainly for species in the Neotropics. Here we report the first set of specific chloroplast microsatellite primers (cpSSR) developed for Neotropical orchids. In total, nine polymorphic cpSSR loci were isolated and characterized in four species occurring in the Brazilian Atlantic Rainforest: Epidendrum cinnabarinum, E. denticulatum, E. fulgens and E. puniceoluteum. Levels of intraspecific polymorphism were characterized using two populations for each species, with 13-20 individuals each. Allele numbers varied from two to three per locus, while the number of haplotypes ranged from three to six per species. Extensive differentiation among the taxa was detected. All markers were successfully cross-amplified in eight other different genera. These cpSSRs markers will enable novel insights into the evolution of this important Neotropical genus.

Effects of voluntary running on spatial memory and mature brain-derived neurotrophic factor expression in mice hippocampus after status epilepticus

Voluntary physical activity improves memory and learning ability in rodents, whereas status epilepticus has been associated with memory impairment. Physical activity and seizures have been associated with enhanced hippocampal expression of BDNF, indicating that this protein may have a dual role in epilepsy. The influence of voluntary physical activity on memory and BDNF expression has been poorly studied in experimental models of epilepsy. In this paper, we have investigated the effect of voluntary physical activity on memory and BDNF expression in mice with pilocarpine-incluced epilepsy. Male Swiss mice were assigned to four experimental groups: pilocarpine sedentary (PS), pilocarpine runners (PRs), saline sedentary (SS) and saline runners (SRs). Two days after pilocarpine-induced status epilepticus, the affected mice (PR) and their running controls (SR) were housed with access to a running wheel for 28 days. After that, the spatial memory and the expression of the precursor and mature forms of hippocampal BDNF were assessed. PR mice performed better than PS mice in the water maze test. In addition, PR mice had a higher amount of mature BDNF (14 kDa) relative to the total BDNF (14 kDa + 28 kDa + 32 kDa forms) content when compared with PS mice. These results show that voluntary physical activity improved the spatial memory and increased the hippocampal content of mature BDNF of mice with pilocarpine-induced status epilepticus. (C) 2009 Elsevier B.V. All rights reserved.

Effects of gamma radiation on the growth of Alternaria alternata and on the production of alternariol and alternariol monomethyl ether in sunflower seeds

The objective of the present study was to evaluate the effects of different gamma radiation doses on the growth of Alternaria alternata and on the production of toxins alternariol (AOH), and alternariol monomethyl ether (AME) in sunflower seed samples. After irradiation with 2, 5 and 7 kGy, the spore mass was resuspended in sterile distilled water and the suspension was inoculated into sunflower seeds. The number of colony-forming units per gram (CFU/g) was determined after culture on Dichloran Rose Bengal Chloramphenicol and Dichloran Chloramphenicol Malt Extract Agar. The presence of AOH and AME was investigated by liquid chromatography coupled to mass spectrometry. The radiation doses used resulted in a reduction of the number of A. alternata CFU/g and of AOH and AME levels when compared to the nonirradiated control group. Maximum reduction of the fungus (98.5%) and toxins (99.9%) was observed at a dose of 7 and 5 kGy, respectively. Under the present conditions, gamma radiation was found to be an alternative for the control of A. alternata and, consequently, of AOH and AME production in sunflower seeds. (C) 2009 Elsevier Ltd. All rights reserved.

Characterisation, immunolocalisation and antifungal activity of a lipid transfer protein from chili pepper (Capsicum annuum) seeds with novel alpha-amylase inhibitory properties

Lipid transfer proteins (LTPs) were thus named because they facilitate the transfer of lipids between membranes in vitro. This study was triggered by the characterization of a 9-kDa LTP from Capsicum annuum seeds that we call Ca-LTP(1). Ca-LTP(1) was repurified, and in the last chromatographic purification step, propanol was used as the solvent in place of acetonitrile to maintain the protein`s biological activity. Bidimensional electrophoresis of the 9-kDa band, which corresponds to the purified Ca-LTP(1), showed the presence of three isoforms with isoelectric points (pIs) of 6.0, 8.5 and 9.5. Circular dichroism (CD) analysis suggested a predominance of alpha-helices, as expected for the structure of an LTP family member. LTPs immunorelated to Ca-LTP(1) from C. annuum were also detected by western blotting in exudates released from C. annuum seeds and also in other Capsicum species. The tissue and subcellular localization of Ca-LTP(1) indicated that it was mainly localized within dense vesicles. In addition, isolated Ca-LTP(1) exhibited antifungal activity against Colletotrichum lindemunthianum, and especially against Candida tropicalis, causing several morphological changes to the cells including the formation of pseudohyphae. Ca-LTP(1) also caused the yeast plasma membrane to be permeable to the dye SYTOX green, as verified by fluorescence microscopy. We also found that Ca-LTP(1) is able to inhibit mammalian alpha-amylase activity in vitro.

Isolation of a lectin and a galactoxyloglucan from Mucuna sloanei seeds

A lectin and a galactoxyloglucan were characterized from Mucuna sloanei seed cotyledons. The galactoxyloglucan, isolated by water extraction and ethanol precipitation, had Glc:Xyl:Gal proportions in a molar ratio of 1.8:1.7:1.0 and a molar mass (M(w)) of 1.6 x 10(6) g mol(-1). The lectin (sloanin), isolated from the same seed by affinity chromatography on cross-linked Adenanthera pavonina galactomannan, gave two protein bands by SDS-PAGE (36 and 34 kDa) and one peak by gel filtration (63.6 kDa). Its N-terminal sequence indicated similar to 69% identity with soybean agglutinin to leguminous lectins. Circular dichroism (CD) spectra established that sloanin predominantly contains beta-sheet structures. Sloanin has similar to 5.5% carbohydrate and displayed hemagglutinating activity against rabbit and enzyme treated human erythrocytes, inhibited only by D-Gal containing sugars. The interaction between sloanin and storage cell-wall galactoxyloglucan was tested by affinity chromatography and fluorescence spectroscopy. (C) 2009 Elsevier Ltd. All rights reserved.

Isolation and characterization of four type 2 ribosome inactivating pulchellin isoforms from Abrus pulchellus seeds

Abrus pulchellus seeds contain at least seven closely related and highly toxic type 2 ribosome-inactivating pulchellins, each consisting of a toxic A-chain linked to a sugar binding B-chain. In the present study, four pulchellin isoforms (termed P I, P II, P III and P IV) were isolated by affinity, ion exchange and chromatofocusing chromatographies, and investigated with respect to toxicity and sugar binding specificity. Half maximal inhibitory concentration and median lethal dose values indicate that P I and P II have similar toxicities and that both are more toxic to cultured HeLa cells and mice than P III and P IV. Interestingly, the secondary structural characteristics and sugar binding properties of the respective pairs of isoforms correlate well with the two toxicity levels, in that P I/P II and P III/P IV form two specific subgroups. From the deduced amino acids sequences of the four isoforms, it is clear that the highest similarity within each subgroup is found to occur within domain 2 of the B-chains, suggesting that the disparity in toxicity levels might be attributed to subtle differences in B-chain-mediated cell surface interactions that precede and determine toxin uptake pathways.

Potassium Current in Mature Bovine Spermatozoa

Ion channels have been assigned a pivotal importance in various sperm functions and are therefore promising targets for contraceptive development. The lack of data on channel functionality and pharmacology has hampered this goal. This is a consequence of technical problems of applying electrophysiological techniques to spermatozoa due to their small size and form. By using a laminin coating to increase adherence of spermatozoa and nystatin in the patch pipette for pore formation, we have adapted the whole-cell recording technique to study currents in mature uncapacitated bovine spermatozoa. Employing these conditions, in the head region, patched spermatozoa could be transferred into the whole-cell configuration. For the first time we document an outward rectifying current in mature bovine spermatozoa was blocked by tetraethyl ammonium (TEA) chloride. The observation of a shift in the reversal potential as a response to changes in the extracellular concentration of K+ ions allowed us to identify this current as K+ selective. This result shows that K+ channels in the head region of mature uncapacitated bovine spermatozoa can be suitably investigated using the whole-cell recording patch-clamp technique.

Water-soluble Cu, Fe, Mn and Zn species in nuts and seeds

An approach was developed to estimate molecular weight distribution of water-soluble Cu, Fe, Mn and Zn species in Brazil nut, cupuassu seed and coconut pulp by size exclusion chromatography (SEC) coupled on-line to ultra-violet (UV) and off-line to graphite furnace atomic absorption spectrometry (GF-AAS) detectors and matrix-assisted laser desorption ionisation time-of-flight mass spectrometry (MALDI-TOF-MS). SEC-UV analytical signals showed the prevalence of high molecular weight (HMW) species (79-1.7 kDa for Brazil nut, 50-1.7 kDa for coconut pulp, and 34-1.7 kDa for cupuassu seeds). The Brazil nut SEC-UV, GF-AAS and MALDI-TOF mass spectra gave confirmation of the association of the elements with water-soluble compounds. The elemental profiles were associated with fractions of compounds of molecular weight 1.2-16 kDa for Brazil nut, 1.7-13 kDa for coconut pulp, and 1.2-7.6 kDa for cupuassu seeds. (C) 2009 Elsevier Inc. All rights reserved.

Elemental analysis of nuts and seeds by axially viewed ICP OES

A method for the multi-elemental determination of metals (Al, Ba, Ca, Cu, Fe, K, Mg, Mn, Sr and Zn), metalloids (B and Si), and non-metals (Cl, P and 5) in the babassu nut and mesocarp, sapucaia nut, coconut pulp, cupuassu pulp and seed, and cashew nut by axially viewed inductively coupled plasma optical emission spectrometry is presented. A diluted oxidant mixture (2 ml HNO(3) + 1 ml H(2)O(2) + 3 ml H(2)O) was used to achieve the complete decomposition of the organic matrix in a closed-vessel microwave oven. The accuracy of the entire proposed method was confirmed by standard reference material analysis (peach leaves-NIST SRM1547). The certified values showed a good agreement at a 95% confidence limit (Student`s t-test). The average RSD for repeatability of calibration solutions measurements were in the range of 1.1-6.7%. Limits of quantification (LOQ = 10 x LOD) were in the level of 0.00072-0.0532 mg/l. The macro and micronutrient ranges in the different nuts and seeds did not exceed the dietary reference intake (DRI), except for Mn in the babassu nut. (C) 2010 Published by Elsevier Ltd.

Chemical analysis of the essential oil extracted from the seeds of Licaria puchury-major

The essential oil from seeds of Licaria puchury-major was isolated by hydrodistillation. The chemical composition of the oil was analyzed by GC and GUMS. Sixteen compounds were identified, representing 91.4% of the total oil. The major components were safrole (58.4%), dodecanoic acid (13.7%) and alpha-terpineol (8.4%). Oxygenated monoterpenoids were the main group of compounds.

Cultivares de batata para sistemas orgânicos de produção

Informações a respeito de cultivares adaptadas ao sistema de cultivo orgânico são escassas. O objetivo deste estudo foi avaliar, sob sistema de cultivo orgânico, genótipos nacionais e estrangeiros desenvolvidos para o cultivo convencional, quanto ao potencial produtivo, em condições de campo. O experimento foi conduzido em 2008, no Pólo APTA Leste Paulista, em Monte Alegre do Sul-SP. O delineamento experimental foi em blocos ao acaso, com 18 tratamentos e quatro repetições. Cada parcela foi constituída por 80 batatas-semente, dispostas em quatro linhas de 5 m de comprimento, espaçadas de 80 cm, com 25 cm entre tubérculos. Os genótipos avaliados foram Agata, Asterix, Caesar, Cupido, Éden, Melody, Novella e Vivaldi, de origem estrangeira; e Apuã, Aracy, Catucha, IAC Aracy Ruiva, Itararé, Monte Alegre 172, IAC 6090, APTA 16.5, APTA 15.20 e APTA 21.54, nacionais. Foram avaliadas as características de produtividade total e comercial de tubérculos, massa média total e comercial de tubérculos, teor de matéria seca e severidade da pinta-preta (Alternaria solani). Os clones APTA 16.5, APTA 21.54 e IAC 6090, e as cultivares Cupido, Apuã, Itararé e Monte Alegre 172 foram os mais produtivos. 'APTA 21.54' superou os demais em relação a produtividade comercial (18,07 t ha-1), sendo que 'APTA 16.5', 'Cupido', 'IAC 6090' e 'Itararé' formaram o segundo grupo. As maiores massas médias de tubérculos foram apresentadas pelas cultivares Itararé e Cupido. O clone IAC 6090 e as cultivares Aracy e Aracy Ruiva foram as que apresentaram maiores teores de matéria seca, com valor médio de 22,91%. 'APTA 16.5', 'Apuã', 'Aracy', 'Aracy Ruiva', 'Éden', 'Ibituaçú' e 'Monte Alegre 172' apresentaram alto nível de resistência à pinta-preta. As cultivares Itararé, Apuã e Cupido são adaptadas ao cultivo orgânico, e os clones avançados APTA 16.5, APTA 21.54 e IAC 6090 apresentam potencial de cultivo no sistema orgânico.

Two-dimensional gel electrophoretic protein profile analysis during seed development of Ocotea catharinensis: a recalcitrant seed species

The aim of the present work was to characterize changes in the protein profile throughout seed development in O. catharinensis, a recalcitrant species, by two-dimensional gel electrophoresis. Protein extraction was undertaken by using a thiourea/urea buffer, followed by a precipitation step with 10% TCA. Comparative analysis during seed development showed that a large number of proteins were exclusively detected in each developmental stage. The cotyledonary stage, which represents the transition phase between embryogenesis and the beginning of metabolism related to maturation, presents the highest number of stage-specific spots. Protein identification, through MS/MS analysis, resulted in the identification of proteins mainly related to oxidative metabolism and storage synthesis. These findings contribute to a better understanding of protein metabolism during seed development in recalcitrant seeds, besides providing information on established markers that could be useful in defining and improving somatic embryogenesis protocols, besides monitoring the development of somatic embryos in this species.