Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
Navegar
Item Antileishmanial activity and mechanism of action from a purified fraction of Zingiber officinalis Roscoe against Leishmania amazonensis(Experimental Parasitology, 2016-03-22) Duarte, Mariana C.; Tavares, Grasiele S.V.; Valadares, Diogo G.; Lage, Daniela P.; Ribeiro, Tatiana G.; Lage, Letícia M.R.; Rodrigues, Marcella R.; Faraco, André A.G.; Soto, Manuel; Silva, Eduardo S. da; Fumagalli, Miguel A. Chávez; Tavares, Carlos A.P.; Leite, João Paulo V.; Oliveira, Jamil S.; Castilho, Rachel O.; Coelho, Eduardo A.F.In recent years, considerable attention has been given to identify new antileishmanial products derived from medicinal plants, although, to date, no new effective compound has been recently applied to treat leishmaniasis. In the present study, the antileishmanial activity of a water extract from Zingiber officinalis Roscoe (ginger) was investigated and a purified fraction, named F10, was identified as responsible by this biological activity. The chemical characterization performed for this fraction showed that it is mainly composed by flavonoids and saponins. The water extract and the F10 fraction presented IC50 values of 125.5 and 49.8 μg/mL, respectively. Their selectivity indexes (SI) were calculated and values were seven and 40 times higher, respectively, in relation to the value found for amphotericin B, which was used as a control. Additional studies were performed to evaluate the toxicity of these compounds in human red blood cells, besides of the production of nitrite, as an indicator of nitric oxide (NO), in treated and infected macrophages. The results showed that both F10 fraction and water extract were not toxic to human cells, and they were able to stimulate the nitrite production, with values of 13.6 and 5.4 μM, respectively, suggesting that their biological activity could be due to macrophages activation via NO production. In conclusion, the present study shows that a purified fraction from ginger could be evaluated in future works as a therapeutic alternative, on its own or in association with other drugs, to treat disease caused by L. amazonensis.Item Differential abundances of four forms of binder of SPerm 1 in the seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability(Theriogenology, 2016-02-27) Magalhães Jr, Marcos Jorge; Martins, Leonardo Franco; Senra, Renato Lima; Santos, Thaís Ferreira do; Okano, Denise Silva; Pereira, Paulo Roberto Gomes; Faria-Campos, Alessandra; Campos, Sérgio Vale Aguiar; Guimarães, José Domingos; Baracat-Pereira, Maria CristinaThe Binder of SPerm 1 (BSP1) protein is involved in the fertilization and semen cryopreservation processes and is described to be both beneficial and detrimental to sperm. Previously, the relationship of BSP1 with freezability events has not been completely understood. The objective of this work was to determine the differential abundance of the forms of the BSP1 protein in cryopreserved seminal plasma of Bos taurus indicus bulls with different patterns of semen freezability using proteomics. A wide cohort of adult bulls with high genetic value from an artificial insemination center was used as donors of high quality, fresh semen. Nine bulls presenting different patterns of semen freezability were selected. Two-dimensional gel electrophoresis showed differential abundance in a group of seven protein spots in the frozen/thawed seminal plasma from the bulls, ranging from 15 to 17 kDa, with pI values from 4.6 to 5.8. Four of these spots were confirmed to be BSP1 using mass spectrometry, proteomics, biochemical, and computational analysis (Tukey's test at P < 0.05). The protein spot weighing 15.52 ± 0.53 kDa with a pI value of 5.78 ± 0.12 is highlighted by its high abundance in bulls with low semen freezability and its absence in bulls presenting high semen freezability. This is the first report showing that more than two forms of BSP1 are found in the seminal plasma of Nelore adult bulls and not all animals have a similar abundance of each BSP1 form. Different BSP1 forms may be involved in different events of fertilization and the cryopreservation process.Item Evolutionary history of the cobalamin-independent methionine synthase gene family across the land plants(Molecular Phylogenetics and Evolution, 2017-12-06) Rody, Hugo Vianna Silva; Oliveira, Luiz Orlando dePlants are successful paleopolyploids. The wide diversity of land plants is driven strongly by their gene duplicates undergoing distinct evolutionary fates after duplication. We used genomic resources from 35 model plant species to unravel the evolutionary fate of gene copies (paralogs) of the cobalamin-independent methionine synthase (metE) gene family across the land plants. To explore genealogical relationships and characterize positive selection as a driving force in the evolution of metE paralogs within a single species, we carried out complementary analyses on genomic data of 32 genotypes of soybean. The size of the metE gene family remained small across the land plants; most of the studied species possessed 1–6 paralogs. Gene products were either cytosolic or chloroplastic; this dual subcellular distribution arose early during the divergence of the land plants and reached all extant lineages. Biased gene loss and gene retention events took place multiple times; recurrent evolution remodeled redundant metE paralogs to recover and maintain the dual subcellular distribution of MetE. Shared whole-genome duplication events gave rise to the metE paralogs of both soybean and Medicago truncatula. In soybean, the ancestral paralog pair GlymaPP2A encoded a cytosolic isoform of MetE, was under strong purifying selection, and retained high levels of expression across eight RNA-seq expression libraries. The daughters GlymaPP1 and GlymaPP2B showed accelerated rates of evolution, accumulated many sites predicted to be under positive selection, and possessed low levels of expression. Our results suggest that the metE paralogs of soybean follow Ohno’s neofunctionalization model of gene duplicate evolution.Item Molecular evidence of hybrid zones of Cedrela (Meliaceae) in the Yungas of Northwestern Argentina(Molecular Phylogenetics and Evolution, 2016-05-19) Zelener, Noga; Tosto, Daniela; Oliveira, Luiz Orlando de; Soldati, María Cristina; Inza, María Virginia; Fornes, Luis FernandoIn the Yungas of Northwestern Argentina, three endangered species of Cedrela (C. angustifolia, C. saltensis, and C. balansae) follow altitudinal gradients of distribution with contact zones between them. We sampled 210 individuals from 20 populations that spanned most of Cedrela’s geographical range in the Yungas, and used Amplified Fragment Length Polymorphism (AFLP) markers and DNA sequences of the nuclear Internal Transcribed Spacer (ITS) to investigate hybrid zones. Data analyses employed an array of complementary methods, including principal coordinate analyses, Bayesian clustering analyses, maximum likelihood tree-building, and network techniques. Both nuclear molecular systems – AFLP and ITS – provided insights into the evolutionary history of Cedrela in the Yungas in a congruent manner. We uncovered strong support for the occurrence of natural hybridization between C. balansae and C. saltensis. Additionally, we identified hybrid zones in areas of sympatry (at both the Calilegua National Park and the San Andrés farm) and in transition zones from 820 to 1100 meters above sea level (localities of Pintascayo and Acambuco). There was no evidence for hybridization of either C. balansae or C. saltensis with C. angustifolia. The role of hybrid populations in conservation and use of genetic resources in the Yungas were discussed.Item Partial purification and characterization of digestive trypsin-like proteases from the velvet bean caterpillar, Anticarsia gemmatalis(Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 2004-10-31) Oliveira, M.G.A.; Simone, S.G. De; Xavier, L.P.; Guedes, R.N.C.Trypsin-like proteases from the midgut of Anticarsia gemmatalis Hübner (Lepidoptera: Noctuidae) were purified on an aprotinin-agarose column equilibrated with 0.01 M Tris–HCl containing 5 mM CaCl2 (pH 7.5). The yield was 66.7% with a purification factor of 107 and a final specific activity of 6.88 mM/min/mg protein with the substrate N-α-benzoyl-l-Arg-p-nitroanilide (l-BApNA). The purified fraction showed three bands with proteolytic activity and molecular weights of 66,000, 71,000 and 91,000 (sodium dodecyl sulphate (SDS)-polyacrylamide gel electrophoresis (PAGE)). Enzyme specificity assays were carried out using seven synthetic peptides containing 13 amino acid residues, but differing only on the 5th residue (K, R, Y, L, W or P). Peptide cleavage takes place only with amino acids K or R at the 5th position, which is typical of trypsin. The partially purified enzymes hydrolyzed casein and the synthetic trypsin substrates l-BApNA and N-α-p-tosyl-l-Arg methyl ester (l-TAME). Higher activity was observed at pH 8.5 and 35 °C when using l-BApNA as substrate and at pH 8.0 and 30 °C when using l-TAME. Maximum enzyme activity against l-BApNA was obtained with 20 mM CaCl2 in the reaction mixture. The partially purified enzymes showing trypsin activity were sensitive to inhibition by ethylenediaminetetraacetic acid (EDTA), phenylmethyl sulphonyl fluoride (PMSF), N-α-tosyl-l-lysine chloromethyl ketone (TLCK), benzamidine and aprotinin. Highest inhibition was obtained with TLCK and benzamidine. KM values obtained were 0.32 mM for l-BApNA and 52.5 μM for l-TAME.Item Vaccination with a CD4+ and CD8+ T-cell epitopes-based recombinant chimeric protein derived from Leishmania infantum proteins confers protective immunity against visceral leishmaniasis(Translational Research, 2018-10) Mendes, Tiago A. O.; Dias, Daniel S.; Ribeiro, Patrícia A. F.; Martins, Vívian T.; Lage, Daniela P.; Costa, Lourena E.; Chávez-Fumagalli, Miguel A.; Ramos, Fernanda F.; Santos, Thaís T. O.; Ludolf, Fernanda; Oliveira, Jamil S.; Silva, Eduardo S.; Galdino, Alexsandro S.; Duarte, Mariana C.; Roatt, Bruno M.; Menezes-Souza, Daniel; Teixeira, Antonio L.; et al.Vaccination seems to be the best approach to control visceral leishmaniasis (VL). Resistance against infection is based on the development of a Th1 immune response characterized by the production of interferons-γ (IFN-γ), interleukin-12 (IL-12), granulocyte-macrophage-colony-stimulating factor (GM-CSF), and tumor necrosis factor-α (TNF-α), among others. A number of antigens have been tested as potential targets against the disease; few of them are able to stimulate human immune cells. In the present study, 1 prediction of MHC class I and II molecules-specific epitopes in the amino acid sequences of 3 Leishmania proteins: 1 hypothetical, prohibitin, and small glutamine-rich tetratricopeptide repeat-containing proteins, was performed using bioinformatics tools, and a T-cell epitopes-based recombinant chimeric protein was constructed, synthetized and purified to be evaluated in invitro and in vivo experiments. The purified protein was tested regarding its immunogenicity in peripheral blood mononuclear cells (PBMCs) from healthy subjects and VL patients, as well as to its immunogenicity and protective efficacy in a murine model against Leishmania infantum infection. Results showed a Th1 response based on high IFN-γ and low IL-10 levels derived from in chimera-stimulated PBMCs in both healthy subjects and VL patients. In addition, chimera and/or saponin-immunized mice presented significantly lower parasite burden in distinct evaluated organs, when compared to the controls, besides higher levels of IFN-γ, IL-2, IL-12, and GM-CSF, and an IgG2a isotype-based humoral response. In addition, the CD4+ and CD8+ T-cell subtypes contributed to IFN-γ production in the protected animals. The results showed the immunogenicity in human cells and the protective efficacy against L. infantum in a murine model, and well indicate that this recombinant chimera can be considered as a promising strategy to be used against human disease.