KLEICY CAVALCANTE AMARAL

KLEICY CAVALCANTE AMARAL

Página do item completo

Resultados de Busca

Agora exibindo 1 - 7 de 7

Cytototic activity and chemical profile of methanolic extract obtained from Avelós stem (Euphorbia tirucalli L.) Euphorbiaceae
2022 - RUSSO, DANIELA C.; AMARAL, KLEICY C.; CALDAS, LHAIS A.; VIEIRA, DANIEL P.; SARTORELLI, PATRICIA; RIBEIRO FILHO, WALDEMAR A.
Avelós (Euphorbia tirucalli Linnaeus), a plant selected for this study, has been popularly used in the fight against tumors, arousing the interest of researchers in this area so that it can be used safely in the auxiliary treatment of different types of cancer.1 It belongs to the family Euphorbiaceae and to the genus Euphorbia, it is also the object of studies related to the treatment of a range of infectious and inflammatory diseases. Herbaceous much used by popular and traditional medicine, presents, a latex rich in molecules that confirm its high toxicity. The objective of this research was to verify the chemical profile of the methanolic extract obtained from the stem (modified leaves) of the plant in question and to determine the cytotoxicity of the crude extract by cytotoxic assay against the lineages of adenocarcinoma (MCF-7) and neoplastic cells of human melanoma (SK-MEL-37).2 For this, the plant was collected, and after drying and milling the material was extracted with methanol. Subsequently, the present compounds were separated by the thin-layer chromatography technique and the classes of substances found in the extract were identified by the technique of Nuclear Magnetic Resonance of Hydrogen and Carbon-13 (NMR).3 The combination of cyclohexane with Acetone and Hexane (5:3:2) provided a suitable polarity for the elution of the extract, which was revealed with ultraviolet detection and different reagents: sulfuric acid solutions; aluminum chloride; ferric chloride; 10% potassium hydroxide in ethanol; green bromocresol indicator solution; potassium permanganate, Dragendorff Reagent, vanillin and iodine vapors. The phytochemical study of the methanolic extract of Euphorbia tirucalli allowed to identify the presence of phenolic compounds, flavonoids and terpenes, a result confirmed by NMR spectra. The cytotoxic potential assays, although they are in low concentration thus altering the result, show that the methanolic extract of Euphorbia tirucalli shows activity against the tested cell lines. The observed activity may be related, according to information available in the literature, with the classes identified in the samples studied.
Synthesis of human bone morphogenetic protein-2 (hBMP-2) in E. coli periplasmic space
2021 - OLIVEIRA, JOAO E.; SUZUKI, MIRIAM F.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M.S.; MAGALHAES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; BARTOLINI, PAOLO
Human BMP-2, a homodimeric protein that belongs to the TGF- family, is a recognized osteoinductor due to its capacity of inducing bone regeneration and ectopic bone formation. The administration of its recombinant form is an alternative to autologous bone grafting. A variety of E. coli-derived hBMP-2 has been synthesized through refolding of cytoplasmic inclusion bodies. The present work reports the synthesis, purification, and characterization of periplasmic hBMP-2, obtained directly in its correctly folded and authentic form, i.e., without the initial methionine typical of the cytoplasmic product that can induce undesired immunoreactivity. A bacterial expression vector was constructed including the DsbA signal peptide and the cDNA of hBMP-2. The periplasmic fluid was extracted by osmotic shock and analyzed via SDS-PAGE, Western blotting, and reversed-phase high-performance liquid chromatography (RP-HPLC). The purification was carried out by heparin affinity chromatography, followed by high-performance size-exclusion chromatography (HPSEC). HPSEC was used for qualitative and quantitative analysis of the final product, which showed >95% purity. The classical in vitro bioassay based on the induction of alkaline phosphatase activity in myoblastic murine C2C12 cells and the in vivo bioassay consisting of treating calvarial critical-size defects in rats confirmed its bioactivity, which matched the analogous literature data for hBMP-2.
Cytotoxic activity and chemical profile of methanolic extract obtained from Avelós stem (Euphorbia tirucalli L.) Euphorbiaceae
2020 - RUSSO, DANIELA C.; AMARAL, KLEICY C.; CALDAS, LHAIS A.; VIEIRA, DANIEL P.; SARTORELLI, PATRICIA; RIBEIRO FILHO, WALDEMAR A.
Avelós (Euphorbia tirucalli Linnaeus), a plant selected for this study, has been popularly used in the fight against tumors, arousing the interest of researchers in this area so that it can be used safely in the auxiliary treatment of different types of cancer.1 It belongs to the family Euphorbiaceae and to the genus Euphorbia, it is also the object of studies related to the treatment of a range of infectious and inflammatory diseases. Herbaceous much used by popular and traditional medicine, presents, a latex rich in molecules that confirm its high toxicity. The objective of this research was to verify the chemical profile of the metanolic extract obtained from the stem (modified leaves) of the plant in question and to determine the cytotoxicity of the crude extract by cytotoxic assay against the lineages of adenocarcinoma (MCF-7) and neoplastic cells of human melanoma (SK-MEL-37).2 For this, the plant was collected, and after drying and milling the material was extracted with methanol. Subsequently, the present compounds were separated by the thin-layer chromatography technique and the classes of substances found in the extract were identified by the technique of Nuclear Magnetic Resonance of Hydrogen and Carbon-13 (NMR).3 The combination of cyclohexane with Acetone and Hexane (5: 3: 2) provided a suitable polarity for the elution of the extract, which was revealed with ultraviolet detection and different reagents: sulfuric acid solutions; aluminum chloride; ferric chloride; 10% potassium hydroxide in ethanol; green bromocresol indicator solution; potassium permanganate, Dragendorff Reagent, vanillin and iodine vapors. The phytochemical study of the methanolic extract of Euphorbia tirucalli allowed to identify the presence of phenolic compounds, flavonoids and terpenes, a result that was confirmed by NMR spectra. The cytotoxic potential assays, although they are in low concentration thus altering the result, show that the methanolic extract of Euphorbia tirucalli shows activity against the tested cell lines. The observed activity may be related, according to information available in the literature, with the classes identified in the samples studied.
Human bone morphogenetic protein‑2 (hBMP‑2) characterization by physical–chemical, immunological and biological assays
2020 - SUZUKI, MIRIAM F.; OLIVEIRA, JOAO E.; DAMIANI, RENATA; LIMA, ELIANA R.; AMARAL, KLEICY C.; SANTOS, ANDERSON M. de S.; MAGALHÃES, GERALDO S.; FAVERANI, LEONARDO P.; PEREIRA, LUIS A.V.D.; SILVA, FABIANA M.; BARTOLINI, PAOLO
Commercially available preparations of methionyl-human BMP-2 and CHO-derived hBMP-2, which belongs to the transforming growth factor β (TGF-β) superfamily, were used for a complete characterization. This protein is an extremely efficient osteoinductor that plays an important role during bone regeneration and embryonic development. Characterization was carried out via SDS-PAGE and Western blotting, followed by reversed-phase HPLC, sizeexclusion HPLC and MALDI-TOF-MS. The classical in vitro bioassay, based on the induction of alkaline phosphatase activity in C2C12 cells, confirmed that hBMP-2 biological activity is mostly related to the dimeric form, being ~ 4-fold higher for the CHO-derived glycosylated form when compared with the E. coli counterpart. The E. coli-derived methBMP- 2 has shown, by MALDI-TOF-MS, a large presence of the bioactive dimer. A more complex molecular mass (MM) distribution was found for the CHO-derived product, whose exact MM has never been reported because of its variable glycosylation. A method based on RP-HPLC was set up, allowing a quantitative and qualitative hBMP-2 determination even directly on ongoing culture media. Considering that hBMP-2 is highly unstable, presenting moreover an extremely high aggregate value, we believe that these data pave the way to a necessary characterization of this important factor when synthesized by DNA recombinant techniques in different types of hosts.
Human bone morphogenetic protein (hBMP)-2 characterization by physical chemical, immunological and biological assays
2019 - SUZUKI, M.F.; OLIVEIRA, J.E.; DAMIANI, R.; LIMA, E.R.; AMARAL, K.C.; SILVA, F.M.; BARTOLINI, P.
Commercial preparations of human-met-BMP-2 (GenScript) and of CHO-derived hBMP-2 (Infuse-Medtronic) provided a complete characterization of this protein, which belongs to the “transforming growth factors β” superfamily, via SDS-PAGE, Western blotting, reversed-phase HPLC, high-performance size-exclusion chromatography and MALDI-TOF-MS. E.coli-derived met-hBMP-2 has shown a large presence of dimer (MM= 26,054 Da), versus a theoretic value of 26,072 Da. More complex was the distribution of the CHO-derived product, whose exact MM has never been reported due to variable glycosylation: via MALDI-TOF-MS a dimer (28,732 Da) and a large amount of monomer (14,377 Da) were found. A novel method based on RP-HPLC was also validated for hBMP-2 qualitative and quantitative analysis directly in ongoing culture media. The classical “in vitro” bioassay, via alkaline phosphatase induction in murine myoblastic cells C2C12, confirmed that hBMP-2 bioactivity is mostly related to the dimer, being ∼6-fold higher for the CHO-derived glycosylated form. Considering that hBMP-2 is a highly effective osteoinductors, plays an important role during bone regeneration and repair, as well as during embryonic development, and presents an extremely high aggregate value, we believe that these data pave the way to the characterization of this important factor when obtained by DNA recombinant techniques in different host cells.
Effects of an inhibitor of nitric oxide production on cell cycle and micronucleus frequency in irradiated human breast adenocarcinoma cells
2019 - AMARAL, KLEICY C.; CARVALHO, LUMA R. de; ALBIERO, ANA LIGIA; LAUBE, RAQUEL; NASCIMENTO, ANA C.G.; BONFIM, LETICIA; VIEIRA, DANIEL P.
Breast adenocarcinomas ar e the most frequent malignant tumor (about 25% of cases), and its malignant outcome causes about 15% of all deaths of women with cancer. The production of nitric oxide (NO) by isoforms of nitric ox ide synthases (NOS’s) are related to increased malignancy a nd stimuli to metastatic progression of breast adenocarcinomas, but its presence in irradiated cells can lead to higher frequencies of DNA damage. The work used aminoguanidine, an inhibitor of isof orm 2 of NOS (NOS 2) to treat human breast cancer cells (MCF 7) in non toxic concentrations (1 and 2mM) before exposure to gamma irradiation 60 Co) to assess if reduction of intracellular NO can protect from, or induce radio induced cell damage, cell cycle disruption or death. Cells were treated and irradiated at 0, 0.5, 1, 2, 4 or 8Gy . A dministration of aminoguanidine arrest ed the cell cycle in the synthesis (S) phase, altering the DNA repair capacity of cells. T he higher concentration (2mM) led to less genotoxic damage (about 50%) in cells irradiated at 8Gy as obse rved using micronucleus scoring by flow cytometry. Alternatively, 1mM of aminoguanidin e increased genotoxic damage and induced a less significant increase of S phase cells. Despite the findings, no significant alterations in cell proliferation rates were o bserved. Finding s showed that aminogua ni dine can modulate radio induced ef fects on cells.
Expressão, purificação e caracterização físico-química da rhBMP-2 (recombinante humana BMP-2)
2019 - AMARAL, KLEICY C.
As proteínas morfogenéticas ósseas (BMPs) são um grupo proteico pertencente à superfamília dos fatores transformadores de crescimento beta (TGF-β). Dentre estas, a BMP-2 humana recombinante (rhBMP-2) tem sido amplamente estudada, devido suas propriedades osteoindutoras. Já existe a comercialização desta proteína produzida por tecnologia de DNA recombinante em células de ovário de hamster chinês (CHO) e para fins de pesquisa, a expressa no citoplasma de bactérias Escherichia coli (E. coli). A rhBMP-2 na sua forma homodimérica possui alta atividade biológica, induzindo a mineralização óssea em ossos endocondrais e a diferenciação de células mesenquimais em osteoblastos e osteoclastos. O objetivo desse trabalho consistiu em obter a rhBMP-2 através da produção em bactérias E. coli, altamente purificada através de técnicas cromatográficas e com atividade biológica. As cepas utilizadas para produção foram a W3110 e a BL21(DE), transformadas com plasmídeos contendo o cDNA da hBMP-2 cultivadas em três temperaturas de expressão: 25 °C, 37 °C e 42 °C. Para purificação dessa proteína foram utilizadas duas técnicas cromatográficas: afinidade a heparina e exclusão molecular por cromatografia líquida de alto desempenho (HPSEC). As coletas destas etapas de purificação levaram a separação de oito produtos que foram caracterizados por eletroforese em gel de poliacrilamida (SDS-PAGE) e Western Blotting (WB). A atividade biológica foi averiguada por produção de fosfatase alcalina em células mioblásticas de camundongo (C2C12). A avaliação dos produtos comerciais também foi feita através dessas técnicas. Os resultados indicaram que dois dos oito produtos purificados, um obtido a partir da produção na cepa BL21(DE) à 25 °C e outro da produção na cepa W3110 à 42 °C, apresentaram relevante grau de pureza e atividade biológica.