Artigos
URI permanente para esta coleçãohttps://locus.ufv.br/handle/123456789/11847
Navegar
Item Evidence for the association of the human regulatory protein Ki-1/57 with the translational machinery(FEBS Letters, 2011-08-19) Gonçalves, Kaliandra de Almeida; Bressan, Gustavo Costa; Saito, Ângela; Morello, Luis Gustavo; Zanchin, Nilson Ivo T.; Kobarg, JörgKi-1/57 is a cytoplasmic and nuclear protein of 57 kDa first identified in malignant cells from Hodgkin’s lymphoma. Based on yeast-two hybrid protein interaction we found out that Ki-1/57 interacts with adaptor protein RACK1 (receptor of activated kinase 1), CIRP (cold-inducible RNA-binding protein), RPL38 (ribosomal protein L38) and FXR1 (fragile X mental retardation-related protein 1). Since these proteins are involved in the regulation of translation we suspected that Ki-1/57 may have a role in it. We show by immunoprecipitation the association of Ki-1/57 with FMRP. Confocal microscopy revealed that Ki-1/57 colocalizes with FMRP/FXR1/2 to stress granules. Furthermore Ki-1/57 cosediments with free ribosomal particles and enhances translation, when tethered to a reporter mRNA, suggesting that Ki-1/57 may be involved in translational regulation.Item Functional analysis of the naturally recombinant DNA-A of the bipartite begomovirus tomato chlorotic mottle virus(Virus Research, 2007-03-26) Fontenelle, Mariana R.; Luz, Dirce F.; Gomes, Ana Paula S.; Florentino, Lilian H.; Zerbini, Francisco M.; Fontes, Elizabeth P.B.All geminiviruses found in Brazil belong to the Begomovirus genus with a bipartite genome that is split between two genomic components, DNA-A and DNA-B. The DNA-A of the bipartite begomovirus ToCMoV-[MG-Bt] (Tomato chlorotic mottle virus), however, possesses as a peculiar characteristic the capacity to systemically infect Nicotiana benthamiana. Here we further characterize this variant DNA-A and show that it also infects Solanum lycopersicum and other host plants, in the absence of DNA-B. The ToCMoV-[MG-Bt]-DNA-A encodes an additional ORF, designated AC5, but otherwise its genome organization is similar to other DNA-A from Western Hemisphere begomoviruses. We showed that this AC5 putative ORF is not essential for infection, as disruption of its coding capacity caused no effect on ToCMoV-[MG-Bt]-DNA-A-mediated infection process. Likewise, the ToCMoV-[MG-Bt]-DNA-A ac4 mutant was indistinguishable from its wild type counterpart in all hosts tested. In contrast, an av1 (coat protein) mutant was unable to infect systemically N. benthamiana and Chenopodium quinoa in the absence of DNA-B. However, inclusion of DNA-B in the infection assay fully rescued the movement defect of the ToCMoV-[MG-Bt]-DNA-A av1 mutant. These results suggest that at suboptimal conditions for infection the coat protein is required for ToCMoV-[MG-Bt] systemic movement.Item Hydrolysis of oligosaccharides in soybean flour by soybean α-galactosidase(Food Chemistry, 2015-12) Guimarães, Valéria Monteze; José, Inês Chamel; Oliveira, Maria Goreti de Almeida e; Oliveira, Maria Goreti de Almeida e; Barros, Everaldo Gonçalves de; Moreira, Maurílio Alves; Viana, Simone de Fátima; Rezende, Sebastião Tavares deRaffinose oligosaccharides (ROs) make up a substantial part (40%) of the soluble sugars found in soybean seeds and are responsible for flatulence after the ingestion of soybean and other legumes. Consequently, soy-based foods would find a broader approval if the ROs were removed from soybean products or hydrolysed by α-galactosidases. During soybean seed germination, of content the ROs decrease substantially, while the α-galactosidase activity increases. α-Galactosidase was partially purified from germinating seeds by partition in an aqueous two-phase system and ion-exchange chromatography. The enzyme preparation presented maximal activities against ρ-nitrophenyl-α-d-galactopyranoside (ρNPGal) at 60 °C and a pH of 5.0 and the KM app values for ρNPGal, melibiose, and raffinose of the enzyme preparation were 0.33, 0.42, and 6.01 mM, respectively. The enzyme was highly inhibited by SDS, copper, and galactose. Hydrolysis of soybean flour ROs by enzyme preparation reduced the stachyose and raffinose contents by 72.3% and 89.2%, respectively, after incubation for 6 h at 40 °C.Item Impact of the removal of N-terminal non-structured amino acids on activity and stability of xylanases from Orpinomyces sp. PC-2(International Journal of Biological Macromolecules, 2017-08-03) Ventorim, Rafaela Zandonade; Mendes, Tiago Antônio de Oliveira; Trevizano, Larissa Mattos; Camargos, Ana Maria dos Santos; Guimarães, Valéria MontezeXylanases catalyze the random hydrolysis of xylan backbone from plant biomass and thus, they have application in the production of biofuels, Kraft pulps biobleaching and feed industry. Here, xylanases derived from Orpinomyces sp. PC-2 were engineered guided by molecular dynamics methods to obtain more thermostable enzymes. Based on these models, 27 amino acid residues from the N-terminal were predicted to reduce protein stability and the impact of this removal was validated to two enzyme con- structs: small xylanase Wild-Type (SWT) obtained from Wild-Type xylanase (WT) and small xylanase Mutant (SM2) generated from M2 mutant xylanase (V135A, A226T). The tail removal promoted increase in specific activity of purified SWT and SM2, which achieved 5,801.7 and 5,106.8 U mg^−1 of protein, respec- tively, while the WT activity was 444.1 U mg^−1 of protein. WT, SWT and SM2 showed half-life values at 50 ◦ C of 0.8, 2.3 and 29.5 h, respectively. Overall, in view of the results, we propose that the presence of non-structured amino acid in the N-terminal leads to destabilization of the xylanases and may promote less access of the substrate to the active site. Therefore, its removal may promote increased stability and enzymatic activity, interesting properties that make them suitable for biotechnological applications.Item In vitro ruminal degradation of ricin and its effect on microbial growth(Animal Feed Science and Technology, 2010-04-21) Oliveira, M. R. C.; Oliveira, A. S. de; Campos, J. M. S.; Lana, R. P.; Machado, O. L. T.; Retamal, C. A.; Detmann, E.; Valadares Filho, S. C.Ricin is a toxic protein found in castorseed (Ricinus communis L.). We hypothesized that ruminal microbiota are capable of degrading ricin, and that the toxin inhibits ruminal microbial growth. Therefore, first we evaluated the in vitro ruminal degradation of ricin from solvent castorseed meal (SCM) by SDS-PAGE and densitometry analysis of culture medium (Experiment 1). Culture medium (three replicates) were collected after 0, 3, 6, 12, 24 and 48 h of incubation content initially 0, 61, 122 and 244 μg of ricin/mL or 122 μg of ricin/mL (without ruminal inoculum). No protein compounds were detected by SDS-PAGE in the culture medium without ricin, indicating an absence of interference from the ruminal inoculum. Ricin chains remained intact in the absence of rumen inoculum, but they were degraded at rates of 0.2725, 0.1504 and 0.0648 h^−1 with ruminal inoculum, at initial ricin concentrations of 61, 122 and 244 μg/mL. Next, the effect of ricin denaturation on rumen microbial specific growth rate (SGR) (OD-600 nm) and the average ammonia concentration at the same time of incubation were investigated (Experiment 2). This experiment had a completely randomized design in a 3 × 3 factorial (three replicates) arrangement, with three sources of protein (trypticase-control; crude extract of soluble protein at pH 3.8 buffer of solvent castorseed meal (CEP) intact, containing 1.46 mg of ricin/mL; and denatured CEP with calcium oxide, containing 0.04 mg of ricin/mL) and three protein levels (0.42, 0.84, and 1.68 mg/mL). There was interaction (P=0.021) between protein level and protein source for SGR. A linear increase (P<0.001) of SGR was observed with increase of trypticase level, but there was a quadratic effect (P=0.023) with increase of intact CEP level, with a minimum value of SGR of −0.004 h^−1 at a protein level of 1.45 mg/mL (210 μg of ricin/mL) of intact CEP. There was no effect (P=0.099) of denatured CEP level, but SGR increased (P<0.001) 3.2 times with denaturation of intact CEP. Ruminal microbial growth was inhibited by 50% with 89 μg of ricin/mL. Ammonia concentration was 91% lower (P<0.001) for the CEP source when compared to trypticase, but the denaturation of intact CEP had no effect (P=0.9560) on the ammonia concentration. Although ruminal microbiota was able to degrade ricin in in vitro conditions, the toxin inhibits ruminal microbial growth. Therefore, complete detoxification of CSM before using it to feed ruminants is recommended. The denatured CEP presents potential of use as modifier of rumen microbial fermentation.Item Label-free assay based on immobilized capillary enzyme reactor of Leishmania infantum nucleoside triphosphate diphosphohydrolase (LicNTPDase-2-ICER-LC/UV)(Journal of Chromatography B, 2016-01-01) Vasconcellos, Raphael de Souza; Magalhães, Luana; Oliveira, Arthur Henrique Cavalcante de; Mariotini-Moura, Christiane; Firmino, Rafaela de Cássia; Fietto, Juliana Lopes Rangel; Cardoso, Carmen LúciaNucleoside triphosphate diphosphohydrolase (NTPDase) is an enzyme belonging to the apyrase family that participates in the hydrolysis of the nucleosides di- and triphosphate to the corresponding nucleoside monophosphate. This enzyme underlies the virulence of parasites such as Leishmania. Recently, an NTPDase from Leishmania infantum (LicNTPDase-2) was cloned and expressed and has been considered as a new drug target for the treatment of leishmaniasis. With the intent of developing label-free online screening methodologies, LicNTPDase-2 was covalently immobilized onto a fused silica capillary tube in the present study to create an immobilized capillary enzyme reactor (ICER) based on LicNTPDase-2 (LicNTPDase-2-ICER). To perform the activity assays, a multidimensional chromatographic method was developed employing the LicNTPDase-2-ICER in the first dimension, and an analytical Ascentis C8 column was used in the second dimension to provide analytical separation of the substrates and products. The validated LicNTPDase-2-ICER method provided the following kinetic parameters of the immobilized enzyme: KM of 2.2 and 1.8 mmol L^−1 for the ADP and ATP substrates, respectively. Suramin (1 mmol L^−1) was also shown to inhibit 32.9% of the enzymatic activity. The developed method is applicable to kinetic studies and enables the recognition of the ligands. Furthermore, a comparison of the values of LicNTPDase-2-ICER with those obtained with an LC method using free enzyme in solution showed that LicNTPDase-2-ICER-LC/UV was an accurate and reproducible method that enabled automated measurements for the rapid screening of ligands.Item New antineoplastic agent based on a dibenzoylmethane derivative: Cytotoxic effect and direct interaction with DNA(Biophysical Chemistry, 2018-08) Nascimento, Fernanda R.; Moura, Tiago A.; Baeta, Jefferson V.P.B.; Publio, Bruno C.; Ferreira, Pollyanna M.F.; Santos, Anésia A.; França, Andressa A.P.; Rocha, Marcio S.; Diaz-Muñoz, Gaspar; Diaz, Marisa A.N.Melanoma accounts for only 4% of all skin cancers but is among the most lethal cutaneous neoplasms. Dacarbazine is the drug of choice for the treatment of melanoma in Brazil through the public health system mainly because of its low cost. However, it is an alkylating agent of low specificity and elicits a therapeutic response in only 20% of cases. Other drugs available for the treatment of melanoma are expensive, and tumor cells commonly develop resistance to these drugs. The fight against melanoma demands novel, more specific drugs that are effective in killing drug-resistant tumor cells. Dibenzoylmethane (1,3-diphenylpropane-1,3-dione) derivatives are promising antitumor agents. In this study, we investigated the cytotoxic effect of 1,3-diphenyl-2-benzyl-1,3-propanedione (DPBP) on B16F10 melanoma cells as well as its direct interaction with the DNA molecule using optical tweezers. DPBP showed promising results against tumor cells and had a selectivity index of 41.94. Also, we demonstrated the ability of DPBP to interact directly with the DNA molecule. The fact that DPBP can interact with DNA in vitro allows us to hypothesize that such an interaction may also occur in vivo and, therefore, that DPBP may be an alternative to treat patients with drug-resistant melanomas. These findings can guide the development of new and more effective drugs.Item Spectroscopic and thermodynamic properties of Debaryomyces hansenii UFV-1 α-galactosidases(International Journal of Biological Macromolecules, 2010-04-01) Rezende, Sebastião T.; Viana, Pollyanna A.; Meza, Andreia N.; Gomide, Felipe T.F.; Nagem, Ronaldo A.P.; Santos, Alexandre M.C.; Santoro, Marcelo M.; Guimarães, Valéria M.Spectroscopic and thermodynamic properties were determined for Debaryomyces hansenii UFV-1 extracellular and intracellular α-galactosidases. α-Galactosidases showed similar secondary structure compositions (α-helix, β-sheet parallel and β-turn). Effects of pH and temperature on the structure of α-galactosidases were investigated using circular dichroism spectroscopy. It was more pronounced at low pH. Microcalorimetry was employed for the determination of thermodynamic parameters. Immediate thermal denaturation reversibility was not observed for α-galactosidases; it occurred as a thermodynamically driven process. Extracellular α-galactosidase, at pH 5.5, showed lower Tm when compared to the intracellular enzyme. The CD and DSC data suggest that D. hansenii α-galactosidases have different behaviors although they possess some similar secondary structures.Item Thermostability improvement of Orpinomyces sp. xylanase by directed evolution(Journal of Molecular Catalysis B: Enzymatic, 2012-09) Trevizano, Larissa Mattos; Ventorim, Rafaela Zandonade; Rezende, Sebastião Tavares de; Silva Junior, Floriano Paes; Guimarães, Valéria MontezeThe methodology of directed evolution, using the mutagenic technique of error-prone PCR has been used to improve the thermostability of enzymes. This method was applied to the endo-β-1,4-xylanase from Orpinomyces strain PC-2. The constructed library of xylanase (xynA) mutants was subjected to several screening cycles in plates with azo-xylan-agarose as substrate and four thermostable mutants (M1–M4) were selected. Homology models for these thermostable mutants were constructed to identify the location of the residues changed by error-prone PCR and to investigate the effect of these mutations on the xylanase properties. Xylanase activities of the mutants and wild type were maximal at 60 °C and in the pH range of 5–7. The mutants displayed higher thermostability than the wild type XynA, where the wild type showed a half-life at 60 °C of 7.92 min, while half-life values for M1, M2, M3 and M4 were 209, 33.2, 401 and 15.3 min, respectively. Additionally, M3 and M4 presented a good performance in more extreme pH conditions. The mutants retained their ability to hydrolyze birchwood and oat spelt xylans, which are substrates presenting different degrees of branching.