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1.
Protein Pept Lett ; 28(10): 1138-1147, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34132177

RESUMO

BACKGROUND: Brucellosis is a zoonotic disease that causes serious economic losses due to factors, such as miscarriages and decreased milk yield in animals. Existing live vaccines have some disadvantages, so effective vaccines need to be developed with new technological approaches. OBJECTIVE: The primary objectives of this study were the expression and purification of recombinant Omp25 fusion protein from B. abortus, and the evaluation of the effect of the Omp25 protein on cell viability and inflammatory response. METHODS: The omp25 gene region was amplified by a polymerase chain reaction and cloned into a Pet102/D-TOPO expression vector. The protein expression was carried out using the prokaryotic expression system. The recombinant Omp25 protein was purified with affinity chromatography followed by GPC (Gel Permeation Chromatography). The MTS assay and cytokine-release measurements were carried out to evaluate cell viability and inflammatory response, respectively. RESULTS: It was determined that doses of the recombinant Omp25 protein greater than 0.1 µg/mL are toxic to RAW cells. Doses of 1 µg/mL and lower significantly increased inflammation due to Nitric Oxide (NO) levels. ELISA results showed that IFN-γ was produced in stimulated RAW 264.7 cells at a dose that did not affect the viability (0.05 µg/mL). However, IL-12, which is known to have a dual role in the activation of macrophages, did not show a statistically significant difference at the same dose. CONCLUSION: Studies on cell viability and Th1-related cytokine release suggest Omp25 protein to be a promising candidate molecule for vaccine development.


Assuntos
Brucella abortus/genética , Brucelose/tratamento farmacológico , Proteínas de Membrana/farmacologia , Proteínas Recombinantes de Fusão/farmacologia , Vacinas Sintéticas/farmacologia , Animais , Sobrevivência Celular/efeitos dos fármacos , Citocinas/metabolismo , Relação Dose-Resposta a Droga , Escherichia coli/química , Escherichia coli/genética , Humanos , Imunogenicidade da Vacina , Proteínas de Membrana/química , Proteínas de Membrana/genética , Camundongos , Óxido Nítrico/metabolismo , Células RAW 264.7 , Proteínas Recombinantes de Fusão/química , Proteínas Recombinantes de Fusão/genética , Desenvolvimento de Vacinas , Vacinas Sintéticas/química , Vacinas Sintéticas/genética
2.
J Biotechnol ; 316: 17-26, 2020 Jun 10.
Artigo em Inglês | MEDLINE | ID: mdl-32315688

RESUMO

In this study, juglone nanoparticles were prepared by single emulsion solvent evaporation method and their effect against Candida albicans biofilm was investigated in comparison with the free juglone and Fluconazole by performing XTT, crystal violet, standard plate count, confocal microscopy and membrane depolarization analyses. Juglone nanoparticles and free juglone were found to inhibit biofilm formation and pre-established biofilms (98-100%) at all doses tested, whereas Fluconazole did not cause a significant inhibition, even at the highest dose applied, especially against pre-established biofilms. Membrane depolarization analysis showed that free juglone and juglone loaded nanoparticles were effective on C. albicans membrane structure and have fluorescence quenching effect on DiSC3(5). It is extremely important that the antibiofilm activity of the juglone nanoparticles is similar to that of the juglone used at the same concentration, since similar effect is provided by using less active substance due to controlled release. Accordingly, it can easily be said that juglone loaded nanoparticles are much more effective in the formation and elimination of C. albicans biofilm than the free juglone and Fluconazole.


Assuntos
Antifúngicos/administração & dosagem , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Nanopartículas/administração & dosagem , Naftoquinonas/administração & dosagem , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/administração & dosagem , Antifúngicos/química , Candida albicans/fisiologia , Liberação Controlada de Fármacos , Nanopartículas/química , Naftoquinonas/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química
3.
J Cell Sci ; 130(21): 3631-3636, 2017 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-28923837

RESUMO

We characterized the tension response of clathrin-mediated endocytosis by using various cell manipulation methodologies. Elevated tension in a cell hinders clathrin-mediated endocytosis through inhibition of de novo coat initiation, elongation of clathrin coat lifetimes and reduction of high-magnitude growth rates. Actin machinery supplies an inward pulling force necessary for internalization of clathrin coats under high tension. These findings suggest that the physical cues cells receive from their microenvironment are major determinants of clathrin-mediated endocytic activity.


Assuntos
Actinas/metabolismo , Vesículas Revestidas por Clatrina/metabolismo , Clatrina/metabolismo , Endocitose/fisiologia , Células Epiteliais/metabolismo , Mecanotransdução Celular , Actinas/genética , Animais , Fenômenos Biomecânicos , Linhagem Celular , Linhagem Celular Tumoral , Tamanho Celular , Chlorocebus aethiops , Clatrina/genética , Vesículas Revestidas por Clatrina/ultraestrutura , Invaginações Revestidas da Membrana Celular/metabolismo , Invaginações Revestidas da Membrana Celular/ultraestrutura , Células Epiteliais/ultraestrutura , Proteínas de Ligação a Ácido Graxo/genética , Proteínas de Ligação a Ácido Graxo/metabolismo , Feminino , Expressão Gênica , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Humanos , Rim/citologia , Rim/metabolismo , Pressão Osmótica , Estresse Mecânico
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