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J Biomed Biotechnol ; 2012: 736472, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-23093860


A novel 68 kDa laccase was purified from the mycorrhizal fungus Agaricus placomyces by utilizing a procedure that comprised three successive steps of ion exchange chromatography and gel filtration as the final step. The monomeric enzyme exhibited the N-terminal amino acid sequence of DVIGPQAQVTLANQD, which showed only a low extent of homology to sequences of other fungal laccases. The optimal temperature for A. placomyces laccase was 30°C, and optimal pH values for laccase activity towards the substrates 2,7'-azinobis[3-ethylbenzothiazolone-6-sulfonic acid] diammonium salt (ABTS) and hydroquinone were 5.2 and 6.8, respectively. The laccase displayed, at 30°C and pH 5.2, K(m) values of 0.392 mM towards hydroquinone and 0.775 mM towards ABTS. It potently suppressed proliferation of MCF 7 human breast cancer cells and Hep G2 hepatoma cells and inhibited human immunodeficiency virus type 1 (HIV-1) reverse transcriptase (RT) activity with an IC(50) of 1.8 µM, 1.7 µM, and 1.25 µM, respectively, signifying that it is an antipathogenic protein.

Agaricus/enzimologia , Transcriptase Reversa do HIV/antagonistas & inibidores , Lacase/administração & dosagem , Lacase/metabolismo , Neoplasias Experimentais/tratamento farmacológico , Inibidores da Transcriptase Reversa/química , Proliferação de Células/efeitos dos fármacos , Proteínas Fúngicas/metabolismo , Proteínas Fúngicas/farmacologia , Células Hep G2 , Humanos , Células MCF-7 , Neoplasias Experimentais/patologia
PLoS One ; 7(8): e43181, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22905227


A novel Chinese chestnut (Castanea mollissima Bl.) mutant with extreme short catkins, here was named sck1 and has been characterized in the present study. This sck1 caused 6-fold shorter than wild-type catkins. Endogenous gibberellic acids markedly decreased in the mutant, and application of exogenous GA(3) could partially restore the sck1 phenotype to the wild-type phenotype. Paclobutrazol (PP(333)), an antagonist of GAs biosynthesis, could significantly inhibit the wild-type catkins growth, and lead to a short catkins phenotype similar to the sck1. In addition, compared to the wild-type catkins, the mRNA expression level of ent-kaurenoic acid oxidase (KAO), a gibberellin biosynthesis key gene, was significantly down-regulated (P<0.01) in the sck1. Importantly, transient over-expression of a normal CmKAO gene in short catkins also could partially restore the wild-type phenotype. Real-time PCR and semi-quantitative analysis showed that the mRNA expression level of KAO was significantly up-regulated. In addition, transient RNA interference of CmKAO in wild-type catkins led the mRNA expression level of KAO decrease significantly and inhibited the wild-type catkins elongation strongly. Taken together, our results suggest that the lower gibberellic acids content that is due to decreased CmKAO expression level may contribute to the generation of the extreme short male catkins, sck1.

Regulação da Expressão Gênica de Plantas , Oxigenases de Função Mista/genética , Nozes/genética , Clonagem Molecular , Giberelinas/genética , Mutação , Fenótipo , Folhas de Planta/metabolismo , Brotos de Planta/metabolismo , Reação em Cadeia da Polimerase/métodos , Regiões Promotoras Genéticas , RNA/metabolismo , Interferência de RNA , RNA Mensageiro/metabolismo