Bioquímica e Biologia Molecular
URI permanente desta comunidadehttps://locus.ufv.br/handle/123456789/11837
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Item Antioxidant study indicative of antibacterial and antimutagenic activities of an ellagitannin-rich aqueous extract from the leaves of Miconia latecrenata(Journal of Ethnopharmacology, 2019-05-23) Diaz, Marisa Alves Nogueira; Fietto, Luciano Gomes; Leite, João Paulo Viana; Gontijo, Douglas Costa; Gontijo, Pablo Costa; Brandão, Geraldo Célio; Oliveira, Alaíde Braga deEthnopharmacological relevance; Several plant species of Miconia genus are commonly used in Brazilian folk medicine as anti-inflammatory agents and for the treatment of infectious diseases. Infusions and extracts of Miconia species are also reported as analgesic, antimicrobial, antimalarial, antioxidant, anti-inflammatory, antinociceptive, antimutagenic, and antitumoral. To determine the phytochemical composition of an aqueous extract of Miconia latecrenata leaves and to evaluate its antioxidant, antibacterial, antimutagenic and antigenotoxic activities. Materials and Methods; The following methods were used for the different effects: I) antioxidant - β-carotene/linoleic acid, lipid peroxidation, and DPPH• radical scavenging; II) antibacterial - agar well diffusion and MIC methods); III) antimutagenic assays - Ames Test; and IV) antigenotoxic - Plasmid cleavage test. The phytochemical analysis and phenolic quantification were carried out by UPLC-DAD-ESI-MS/MS and colorimetry, respectively. In addition, statistical correlation analysis was performed aiming to evaluate the Pearson correlation betweenphenolic compounds and biological assays. A high content of tannins was observed and the ellagitannin isomers of 1,2,3,5-tris-galloyl-4,6-HHDP-glucose were identified as the main constituents of the leaves aqueous extract. High antioxidant effect, in different tests, highantibacterial activity to gram-positive and negative strains, as well as high antimutagenic activity were observed. Statistical analysis showed a high Pearson correlation for the tannin content in relation to the results of the antioxidant and antibacterial tests. In general, the antioxidant action of the aqueous extract showed low correlation with the antimutagenic activity. The present results confirmed the expectations regarding the pharmacological profile of M. latecrenata supporting its therapeutic potential in relation to ROS/RNS related disorders. Furthermore, the phenolic compounds of M. latecrenata can act, in turn, minimizing or inhibiting the biological macromolecules damage, especially DNA.Item Biochemical properties of soybean leaf lipoxygenases: Presence of soluble and membrane-bound forms(Plant Physiology and Biochemistry, 2001-02) Baracat-Pereira, Maria Cristina; Oliveira, Maria Goreti de Almeida; Barros, Everaldo Gonçalves de; Moreira, Maurílio Alves; Santoro, Marcelo MatosLipoxygenases (EC 1.13.11.12, LOX) extracted from soybean leaves (Glycine max [L.] Merrill cv. IAC-100) at pH 6.5 showed low stability. Given the importance of correlating the biochemical roles with the physiological characteristics of each LOX isoenzyme, this work evaluates biochemical characteristics and stability conditions of these enzymes in order to plan a purification procedure. LOX activity (A234 at pH 6.0) increased four to five times when 0.25 % (v/v) Triton X-100, 1 % (w/v) polyvinylpyrrolidone, and 1 mM phenylmethylsulfonyl fluoride were added to leaf macerates. Fe2+ (1 mM) stabilised LOX (70.3 % of activity recovered after 48 h storage). Ammonium sulphate fractionation (35–65 % saturation) increased specific LOX activity five times and stabilised the enzymes. Two optimum LOX activities were observed at pH 6.0–6.5 and 4.0–5.0, and the greater storage stability was at pH 6.5 (after 24–28 h storage at different pH values). The results suggest the presence of at least two different forms of the enzyme. The forms of LOX that are active at acidic pH are more stable than the ones that are active at neutral pH. These stable forms were extracted in absence of detergents (soluble forms), while the forms of LOX that are active at pH 6.0–6.5 are unstable forms specially extracted in presence of Triton X-100, and possibly correspond to membrane-bound proteins.Item Evaluation of the effects of Quercetin and Kaempherol on the surface of MT-2 cells visualized by atomic force microscopy(Journal of Virological Methods, 2011-04-11) Almeida, Marcia R.; Reis, Jordana Grazziela A. Coelho dos; Gomes, Orlando Ab; Bortolini, Dener E.; Martins, Marina L.; Martins, Camila S.; Carvalho, Luciana D.; Souza, Jaqueline G.; Vilela, Jose Mario C.; Andrade, Margareth S.; Stancioli, Edel Figueiredo BarbosaThis study investigated the anti-viral effects of the polyphenolic compounds Quercetin and Kaempherol on the release of HTLV-1 from the surface of MT-2 cells. Atomic force microscopy (AFM) was used to scan the surface of the MT-2 cells. MT-2 cells were fixed with 100% methanol on round glass lamina or cleaved mica and dried under UV light and laminar flow. The images were captured on a Multimode equipment monitored by a NanoScope IIId controller from Veeco Instruments Inc operated in tapping mode and equipped with phase-imaging hardware. The images demonstrated viral budding structures 131 ± 57 nm in size, indicating profuse viral budding. Interestingly, cell-free viruses and budding structures visualized on the surface of cells were less common when MT-2 was incubated with Quercetin, and no particles were seen on the surface of cells incubated with Kaempherol. In summary, these data indicate that HTLV-1 is budding constantly from the MT-2 cell surface and that polyphenolic compounds were able to reduce this viral release. Biological samples were analyzed with crude cell preparations just after cultivation in the presence of Quercetin and Kaempherol, showing that the AFM technique is a rapid and powerful tool for analysis of antiviral activity of new biological compounds.Item Evolutionary history of Manihot carthagenensis (Euphorbiaceae) and allied species in eastern South America(Molecular Phylogenetics and Evolution, 2019-03) Silveira, Thamyres Cardoso da; Martins, Márcio Lacerda Lopes; Rody, Hugo Vianna Silvapt-BR; Oliveira, Luiz Orlando deIntermittent episodes of climate changes, such as those that occurred during the Pleistocene, likely shaped the diversification of the young genus Manihot Mill. (Euphorbiacheae). One of such recently-derived congeners ─ M. carthagenensis ─ exhibits a widely disjunct distribution across dry environments in Eastern South America. Herein, we used molecular data from four nuclear gene regions (sts, ch_metE, g3pdh, and nia-i3) and seven nuclear microsatellite loci for reconstructing the phylogenetic relationships among M. carthagenensis and allied species and exploring likely phylogeographic scenarios that shaped the diversification and the distribution of gene pools of M. carthagenensis across the Caatinga and Chaco. Our data suggest that M. carthagenensis is not a monophyletic clade, as presently circumscribed. Morphological differences, genealogical relationships, and vegetation associations support three well-differentiated lineages, each of which merits the species rank: M. carthagenensis, M. glaziovii, and M. hahnii. Microsatellite data suggest that the newly circumscribed M. carthagenensis consists of at least three distinct gene pools, which are partly structured according to geography. The three gene pools likely evolved in allopatry, but remained interfertile. Population expansions after climate amelioration contributed to structuring hybrid zones. Moreover, we described two new single-copy gene regions (sts and ch_metE) as sources of molecular variation; they can facilitate the fine-scale probing of other parts of the phylogeny across Manihot.Item High-yield secretion of multiple client proteins in Aspergillus(Enzyme and Microbial Technology, 2012-07-15) Gonçalves, Thiago A.; Segato, Fernando; Damásio, André R. L.; Lucas, Rosymar C. de; Squina, Fabio M.; Decker, Stephen R.; Prade, Rolf A.Production of pure and high-yield client proteins is an important technology that addresses the need for industrial applications of enzymes as well as scientific experiments in protein chemistry and crystallization. Fungi are utilized in industrial protein production because of their ability to secrete large quantities of proteins. In this study, we engineered a high-expression-secretion vector, pEXPYR that directs proteins towards the extracellular medium in two Aspergillii host strains, examine the effect of maltose-induced over-expression and protein secretion as well as time and pH-dependent protein stability in the medium. We describe five client proteins representing a core set of hemicellulose degrading enzymes that accumulated up to 50–100 mg/L of protein. Using a recyclable genetic marker that allows serial insertion of multiple genes, simultaneous hyper-secretion of three client proteins in a single host strain was accomplished.Item Phenylcoumarins from Kielmeyera elata(Phytochemistry, 1999-06) Gramacho, R.da S; Oliveira, Tânia Toledo de; Queiroz, Maria Eliana L.R de; Neves, Antonio A.; Nagem, Tanus J.; Saddi, NagibFriedelin, the 4-phenylcoumarins mammeisin, 4-phenyl-5-hydroxy-6-(3-methyl-1-oxobutyl)-2′-(1-hydroxy-1-methylethyl)-3′,4′-dihydro(5′,4′:7,8) furancoumarin, and mammeigin have been purified from the stem of Kielmeyera elata. These compounds have previously been reported for the same species. In addition, a new derivative of mammeigin has been isolated and identified as 4-phenyl-5-hydroxy-6-(3-hydroxymethyl-1-oxo-butyl)-2′,2′-dimethyl(6′,5′:7,8)pyrancoumarin.Item A WW domain-containing protein forms immune nuclear bodies against begomoviruses(Molecular Plant, 2018-12-03) Calil, Iara P.; Quadros, Iana P. S.; Araújo, Thais C.; Duarte, Christiane E. M.; Gouveia-Mageste, Bianca C.; Silva, José Cleydson F.; Brustolini, Otávio J. B.; Teixeira, Ruan M.; Oliveira, Cauê N.; Milagres, Rafael W. M. M.; Martins, Gilberto S.; Reis, Pedro A. B.; Machado, Joao Paulo B.; Fontes, Elizabeth P. B.; Chory, JoanneThe bipartite begomoviruses (Geminiviridae family), which are DNA viruses that replicate in the nucleus of infected cells, encode the nuclear shuttle protein (NSP) to facilitate the translocation of viral DNA from the nucleus to the cytoplasm via nuclear pores. This intracellular trafficking of NSP–DNA complexes is accessorized by the NSP-interacting guanosine triphosphatase (NIG) at the cytosolic side. Here, we report the nuclear redistribution of NIG by AtWWP1, a WW domain-containing protein that forms immune nuclear bodies (NBs) against begomoviruses. We demonstrated that AtWWP1 relocates NIG from the cytoplasm to the nucleus where it is confined to AtWWP1-NBs, suggesting that the NIG-AtWWP1 interaction may interfere with the NIG pro-viral function associated with its cytosolic localization. Consistent with this assumption, loss of AtWWP1 function cuased plants more susceptible to begomovirus infection, whereas overexpression of AtWWP1 enhanced plant resistance to begomovirus. Furthermore, we found that a mutant version of AtWWP1 defective for NB formation was no longer capable of interacting with and relocating NIG to the nucleus and lost its immune function against begomovirus. The antiviral function of AtWWP1-NBs, however, could be antagonized by viral infection that induced either the disruption or a decrease in the number of AtWWP1-NBs. Collectively, these results led us to propose that AtWWP1 organizes nuclear structures into nuclear foci, which provide intrinsic immunity against begomovirus infection.