Isolation and characterization of an AINTEGUMENTA-like gene in different coconut (Cocos nucifera L.) varieties from Sri Lanka

Development of an efficient tissue culture protocol in coconut is hampered by numerous technical constraints. Thus a greater understanding of the fundamental aspects of embryogenesis is essential. The role of AINTEGUMENTA-like genes in embryogenesis has been elucidated not only in model plants but also in economically important crops. A coconut gene, CnANT, that encodes two APETALA2 (AP2) domains and a conserved linker region similar to those of the BABY BOOM transcription factor was cloned, characterized, and its tissue specific expression was examined. The full-length cDNA of 1,780 bp contains a 1,425-bp open reading frame that encodes a putative peptide of 474 amino acids. The genomic DNA sequence includes 2,317 bp and consists of nine exons interrupted by eight introns. The exon/intron organization of CnANT is similar to that of homologous genes in other plant species. Analysis of differential tissue expression by real-time polymerase chain reaction indicated that CnANT is expressed more highly in in vitro grown tissues than in other vegetative tissues. Sequence comparison of the genomic sequence of CnANT in different coconut varieties revealed one single nucleotide polymorphism and one indel in the first exon and first intron, respectively, which differentiate the Tall group of trees from Dwarfs. The indel sequence, which can be considered a simple sequence repeats marker, was successfully used to distinguish the Tall and Dwarf groups as well as to develop a marker system, which may be of value in the identification of parental varieties that are used in coconut breeding programs in Sri Lanka.

Ovarian expression of insulin-like peptide 3 (INSL3) and its receptor (RXFP2) during development of bovine antral follicles and corpora lutea and measurement of circulating INSL3 levels during synchronized estrous cycles

Insulin-like peptide 3 (INSL3), a major product of testicular Leydig cells, is also expressed by the ovary but its functional role remains poorly understood. Here, we quantified expression of INSL3 and its receptor RXFP2 in theca interna (TIC) and granulosa (GC) compartments of developing bovine antral follicles and in corpora lutea (CL). INSL3 and RXFP2 mRNA levels were much higher in TIC than GC and increased progressively during follicle maturation with INSL3 peaking in large (11-18mm) estrogen-active follicles and RXFP2 peaking in 9-10mm follicles before declining in larger (11-18mm) follicles. Expression of both INSL3 and RXFP2 in CL was much lower than in TIC. In situ hybridization and immunohistochemistry confirmed abundant expression of INSL3 mRNA and protein in TIC. These observations indicate follicular TIC rather than CL as the primary site of both INSL3 production and action, implying a predominantly auto-/paracrine role in TIC. To corroborate the above findings, we showed that in vitro exposure of TIC to a luteinizing concentration of LH greatly attenuated expression of both INSL3 and its receptor while increasing progesterone secretion and expression of STAR and CYP11A1. Moreover, in vivo, a significant cyclic variation in plasma INSL3 was observed during synchronized estrous cycles. INSL3 and estradiol-17β followed a similar pattern, both increasing after luteolysis, before falling sharply after the LH surge. Thus, theca-derived INSL3, likely from the dominant pre-ovulatory follicle, is detectable in peripheral blood of cattle and expression is down-regulated during luteinisation induced by the pre-ovulatory LH surge. Collectively, these findings underscore the likely role of INSL3 as an important intrafollicular modulator of TIC function/steroidogenesis, whilst raising doubts about its potential contribution to CL function.

Transcriptome analysis for discovering candidate genes involve in embryogenesis in coconut (Cocos nucifera L.) through 454 pyrosequencing

Coconut, Cocos nucifera L. is a major plantation crop, which ensures income for millions of people in the tropical region. Detailed molecular studies on zygotic embryo development would provide valuable clues for the identification of molecular markers to improve somatic embryogenesis. Since there is no ongoing genome project for this species, coconut expressed sequence tags (EST) would be an interesting technique to identify important coconut embryo specific genes as well as other functional genes in different biochemical pathways. The goal of this study was to analyse the ESTs by examining the transcriptome data of the different embryo tissue types together with one somatic tissue. Here, four cDNA libraries from immature embryo, mature embryo, microspore derived embryo and mature leaves were constructed. cDNA was sequenced by the Roche-454 GS-FLX system and assembled into 32621 putative unigenes and 155017 singletons. Of these unigenes, 18651 had significant sequence similarities to non-redundant protein database, from which 16153 were assigned to one or more gene ontology categories. Homologue genes, which are responsible for embryo development such as chitinase, beta-1,3-glucanase, ATP synthase CF0 subunit, thaumatin-like protein and metallothionein-like protein were identified among the embryo EST collection. Of the unigenes, 6694 were mapped into 139 KEGG pathways including carbohydrate metabolism, energy metabolism, lipid metabolism, amino acid metabolism and nucleotide metabolism. This collection of 454-derived EST data generated from different tissue types provides a significant resource for genome wide studies and gene discovery of coconut, a non-model species.

Cysteine proteases of Trypanosoma (Megatrypanum) theileri: Cathepsin L-like gene sequences as targets for phylogenetic analysis, genotyping diagnosis

Although Trypanosoma theileri and allied trypanosomes are the most widespread trypanosomes in bovids little is known about proteolytic enzymes in these species. We have characterized genes encoding for cathepsin L-like (CATL) cysteine proteases from isolates of cattle, water buffalo and deer that largely diverged from homologues of other trypanosome species. Analysis of 78 CATL catalytic domain sequences from 22 T. theileri trypanosomes disclosed 6 genotypes tightly clustered together into the T. theileri clade. The CATL genes in these trypanosomes are organized in tandem arrays of similar to 1.7 kb located in 2 chromosomal bands of 600-720 kb. A diagnostic PCR assay targeting CATL sequences detected T. theileri of all genotypes from cattle, buffaloes and cervids and also from tabanid vectors. Expression of T. theileri cysteine proteases was demonstrated by proteolytic activity in gelatin gels and hydrolysis of Z-Phe-Arg-AMC substrate. Results from this work agree with previous data using ribosomal and spliced leader genes demonstrating that CATL gene sequences are useful for diagnosis, population genotyping and evolutionary studies of T. theileri trypanosomes. (c) 2010 Elsevier Ireland Ltd. All rights reserved.

Genes of cathepsin L-like proteases in Trypanosoma rangeli isolates: Markers for diagnosis, genotyping and phylogenetic relationships

We have sequenced genes encoding cathepsin L-like (CatL-like) cysteine proteases from isolates of Trypanosoma rangeli from humans, wild mammals and Rhodnius species of Central and South America. Phylogenetic trees of sequences encoding mature CatL-like enzymes of T rangeli and homologous genes from other trypanosomes, Leishmania spp. and bodonids positioned sequences of T rangeli (rangelipain) closest to T cruzi (cruzipain). Phylogenetic tree of kinetoplastids based on sequences of CatL-like was totally congruent with those derived from SSU rRNA and gGAPDH genes. Analysis of sequences from the CatL-like catalytic domains of 17 isolates representative of the overall phylogenetic diversity and geographical range of T rangeli supported all the lineages (A-D) previously defined using ribosomal and spliced leader genes. Comparison of the proteolytic activities of T rangeli isolates revealed heterogeneous banding profiles of cysteine proteases in gelatin gels, with differences even among isolates of the same lineage. CatL-like sequences proved to be excellent targets for diagnosis and genotyping of T rangeli by PCR. Data from CatL-like encoding genes agreed with results from previous studies of kDNA markers, and ribosomal and spliced leader genes, thereby corroborating clonal evolution, independent transmission cycles and the divergence of T rangeli lineages associated with sympatric species of Rhodnius. (c) 2009 Elsevier B.V. All rights reserved.

Synthesis, Structure, and Phosphatase-Like Activity of a New Trinuclear Gd Complex with the Unsymmetrical Ligand H(3)L As a Model for Nucleases

The new trinuclear gadolinium complex [Gd(3)L(2)(NO(3))(2)(H(2)O)(4)]NO(3)center dot 8H(2)O (1) with the unsymmetrical ligand 2-[N-bis-(2-pyridylmethyl)aminomethyl]-4-methyl-6-[N-bis(2-hydroxy-2-oxoethyl)aminomethyl] phenol (H(3)L) was synthesized and characterized. The new ligand H(3)L was obtained in good yield. Complex I crystallizes in an orthorhombic cell, space group Pcab. Kinetic studies show that complex 1 is highly active in the hydrolysis of the substrate 2,4-bis(dinitrophenyl)phosphate (K(m) = 4.09 mM, V(max) = 2.68 x 10(-2) mM s(-1), and k(cat) = V(max)/[1] = 0.67 s(-1)). Through a potentiometric study and determination of the kinetic behavior of 1 in acetonitrile/water solution, the species present in solution could be identified, and a trinuclear monohydroxo species appears to be the most prominent catalyst under mild conditions. Complex 1 displays high efficiency in DNA hydrolytic cleavage, and complete kinetic studies were carried out (K(m) = 4.57 x 10(-4) M, K(cat)` = 3.42 h(-1), and k(cat)`/K(m) = 7.48 x 10(3) M(-1) h(-1)). Studies with a radical scavenger (dimethyl sulfoxide, DMSO) showed that it did not inhibit the activity, indicating the hydrolytic action of 1 in the cleavage of DNA, and studies on the incubation of distamycin with plasmid DNA suggest that 1 is regio-specific, interacting with the minor groove of DNA.

Palythine-threonine, a major novel mycosporine-like amino acid (MAA) isolated from the hermatypic coral Pocillopora capitata

Using a high-resolution reverse-phase liquid chromatography method we found that the tissues of the hermatypic coral Pocillopora capitato (collected in Santiago Bay, Mexico) contain a high diversity of primary and secondary mycosporine-like amino acids (MAAs) typical of some reef-building coral species: mycosporine-glycine, shinorine, porphyra-334, mycosporine-methylamine-serine, mycosporine-methylamine-threonine, palythine-serine, palythine and one additional novel predominant MAA, with an absorbance maximum of 320 nm. Here we document the isolation and characterization of this novel MAA from the coral A capitata. Using low multi-stage mass analyses of deuterated and non deuterated compounds, high-resolution mass analyses (Time of Flight, TOF) and other techniques, this novel compound was characterized as palythine-threonine. Palythine-threonine was also present in high concentrations in the corals Pocillopora eydouxi and Stylophora pistillata indicating a wider distribution of this MAA among reef-building corals. From structural considerations we suggest that palythine-threonine is formed by decarboxylation of porphyra-334 followed by demethylation of mycosporine-methylamine-threonine. (C) 2008 Elsevier B.V. All rights reserved.

Chemical composition and fumigant effect of essentialoil of Lippia sidoides Cham. and monoterpenes against Tenebrio molitor (L.) (coleoptera: tenebrionidae)

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Effect of chickpea (Cicer arietinum L.) germination on the major globulin content and in vitro digestibility

A germinação das sementes de grão-de-bico foi acompanhada por um período de 6 dias, no qual pequenas variações nos teores de nitrogênio e globulina total foram registradas. A globulina majoritária (tipo 11 S) apresentou maiores variações após o quarto dia de germinação. A natureza e distribuição da fração globulina majoritária isolada na cromatografia em Sepharose CL-6B mostrou pequenas modificações ao final do período de germinação. A eletroforese em gel de poliacrilamida com dodecilssulfato de sódio do pico eluído na cromatografia em Sepharose CL-6B demonstra modificações nas bandas de proteínas entre os pesos moleculares de 20 e 30 kDa e acima de 60 kDa, indicando degradação protéica durante o período. Atividade proteolítica foi detectada na fração albumina da semente que aumentou até o quarto dia, seguido de queda até o sexto dia de germinação, quando da utilização de globulina total isolada da semente e caseína como substratos. Farinha de grão-de-bico, frações albumina e globulina total isoladas não apresentaram aumento na digestibilidade in vitro; entretanto, a fração globulina majoritária isolada foi mais suscetível à hidrólise após germinação.

Plasmodium vivax circumsporozoite genotypes: a limited variation or new subspecies with major biological consequences?

Background: Plasmodium vivax circumsporozoite variants have been identified in several geographical areas. The real implication of the genetic variation in this region of the P. vivax genome has been questioned for a long time. Although previous studies have observed significant association between VK210 and the Duffy blood group, we present here that evidences of this variation are limited to the CSP central portion.Methods: The phylogenetic analyses were accomplished starting from the amplification of conserved domains of 18 SSU RNAr and Cyt B. The antibodies responses against the CSP peptides, MSP-1, AMA-1 and DBP were detected by ELISA, in plasma samples of individuals infected with two P. vivax CS genotypes: VK210 and P. vivax-like.Results: These analyses of the two markers demonstrate high similarity among the P. vivax CS genotypes and surprisingly showed diversity equal to zero between VK210 and P. vivax-like, positioning these CS genotypes in the same clade. A high frequency IgG antibody against the N- and C-terminal regions of the P. vivax CSP was found as compared to the immune response to the R- and V-repetitive regions (p = 0.0005, Fisher's Exact test). This difference was more pronounced when the P. vivax-like variant was present in the infection (p = 0.003, Fisher's Exact test). A high frequency of antibody response against MSP-1 and AMA-1 peptides was observed for all P. vivax CS genotypes in comparison to the same frequency for DBP.Conclusions: This results target that the differences among the P. vivax CS variants are restrict to the central repeated region of the protein, mostly nucleotide variation with important serological consequences.

The structure of a native L-amino acid oxidase, the major component of the Vipera ammodytes ammodytes venomic, reveals dynamic active site and quaternary structure stabilization by divalent ions

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)

Citrus aurantium L. essential oil exhibits anxiolytic-like activity mediated by 5-HT1A-receptors and reduces cholesterol after repeated oral treatment

Background: The current treatments for anxiety disorders and depression have multiple adverse effects in addition to a delayed onset of action, which has prompted efforts to find new substances with potential activity in these disorders. Citrus aurantium was chosen based on ethnopharmacological data because traditional medicine refers to the Citrus genus as useful in diminishing the symptoms of anxiety or insomnia, and C. aurantium has more recently been proposed as an adjuvant for antidepressants. In the present work, we investigated the biological activity underlying the anxiolytic and antidepressant effects of C. aurantium essential oil (EO), the putative mechanism of the anxiolytic-like effect, and the neurochemical changes in specific brain structures of mice after acute treatment. We also monitored the mice for possible signs of toxicity after a 14-day treatment.Methods: The anxiolytic-like activity of the EO was investigated in a light/dark box, and the antidepressant activity was investigated in a forced swim test. Flumazenil, a competitive antagonist of benzodiazepine binding, and the selective 5-HT1A receptor antagonist WAY100635 were used in the experimental procedures to determine the mechanism of action of the EO. To exclude false positive results due to motor impairment, the mice were submitted to the rotarod test.Results: The data suggest that the anxiolytic-like activity observed in the light/dark box procedure after acute (5 mg/kg) or 14-day repeated (1 mg/kg/day) dosing was mediated by the serotonergic system (5-HT1A receptors). Acute treatment with the EO showed no activity in the forced swim test, which is sensitive to antidepressants. A neurochemical evaluation showed no alterations in neurotransmitter levels in the cortex, the striatum, the pons, and the hypothalamus. Furthermore, no locomotor impairment or signs of toxicity or biochemical changes, except a reduction in cholesterol levels, were observed after treatment with the EO.Conclusion: This work contributes to a better understanding of the biological activity of C. aurantium EO by characterizing the mechanism of action underlying its anxiolytic-like activity. © 2013 Costa et al; licensee BioMed Central Ltd.

Isolamento e caracterização de um gene que codifica uma isofalvona redutase like de café(Coffea arábica L.) e análise de sua região promotora

Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)

Papel dos receptores Toll-Like 2 e 4 do estado nutricional em pacientes com leishmaniose visceral

Pós-graduação em Doenças Tropicais - FMB

Photomorphogenic modulation of water stress in tomato (Solanum lycopersicum L.): the role of phytochromes A, B1, and B2

Phytochromes are red/far-red light photoreceptors that mediate a variety of photomorphogenic processes in plants, from germination to flowering. In addition, there is evidence that phytochromes are also part of the stress signalling response, especially in response to water deficit stress, which is the major abiotic factor limiting plant growth and crop productivity worldwide. In this study, we used the phyA (far red-insensitive; fri), phyB1 (temporary red-insensitive; tri) and phyB2 mutants of tomato (Solanum lycopersicum L.) to study the roles of these three phytochromes in drought stress responses. Compared to wild type (WT) plants grown under water-deficit stress conditions, the fri, tri, and phyB2 mutants did not exhibit altered dry weights, leaf areas, stomatal densities, or stomatal opening. The stomatal conductance of all three mutants was severely reduced under both fully-hydrated and water-deficit conditions. Although relative water contents did change after drought stress in each mutant, the most significant reduction in water potential during water stress was observed in the fri mutant. However, this mutant returned its water status to WT levels during rehydration. Although the phyB2 mutant lost more water from detached leaves during abscisic acid (ABA) treatment, phyB2 behaved like WT plants, indicating that this mutant was not insensitive to ABA. Overall, these results indicate that the phytochromes phyA, phyB1, and phyB2 modulate drought stress responses in tomato.