RESUMEN
We characterized, identified, and cloned a major protein which comprised 16% of the total proteins from Cytophaga sp. cell lysate. After French pressing, the fraction of cell envelope was treated with 0.2% Triton X-100 to remove cell membranes. Subsequent SDS-PAGE analysis of the Triton X-100-insoluble cell wall revealed a protein of 120 kDa with a pI of 5.4, which was identified by gold immunostaining as the surface (S)-layer protein of this soil bacterium. The nucleotide sequence of the cloned S-layer protein gene (slp) encoding this protein consisted of 3144 nucleotides with an ORF for 1047 amino acids, which included a typical 32-amino acid leader peptide sequence. Amino acid sequence alignment revealed 29-48% similarity between this protein and the S-layer proteins from other prokaryotic organisms. The 120-kDa protein from the Cytophaga sp. cell lysate has been characterized as a member of the S-layer proteins, and the slp gene was cloned and expressed in Escherichia coli. E. coli harboring the plasmid containing the 600- or 800-bp DNA fragment upstream of the initiation codon of the slp gene, in the presence of the reporter gene rsda (raw starch digesting amylase), showed amylase activity in starch containing plate. The putative promoter region of slp located 600 bp upstream of the initiation codon might be used for foreign gene expression.
Asunto(s)
Clonación Molecular , Cytophaga/genética , Glicoproteínas de Membrana/química , Cytophaga/química , Cytophaga/metabolismo , Cytophaga/ultraestructura , Electroforesis en Gel de Poliacrilamida , Expresión Génica , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/aislamiento & purificación , Glicoproteínas de Membrana/metabolismo , Datos de Secuencia Molecular , Peso Molecular , Sistemas de Lectura Abierta , Regiones Promotoras Genéticas , Análisis de Secuencia de ADNRESUMEN
The antitumor activity of Anoectochilus formosanus Hayata (Orchidaceae), a popularly used folk medicine in the treatment of cancers in Asia, was investigated in MCF-7 human mammary carcinoma cells. Plant extracts of A. formosanus were observed to induce apoptosis of MCF-7 cells as evidenced by cell-morphological changes, an early redistribution of plasma membrane phosphatidylserine, and DNA content distribution studies. Bioactivity-guided fractionation of A. formosanus extracts produced a specific ethyl acetate (EA)-partitioned fraction in which apoptotic activity was enriched. The chemical profile and candidate index compounds of the active EA fraction were obtained using HPLC and various spectral analyses. Western blot analysis showed that upon treatment of MCF-7 cells with the EA fraction, cleavage of pro-caspases-8, -9, and -7, and poly(ADP-ribose) polymerase as well as significant release of mitochondrial cytochrome c into the cytosol were readily observed. Flow cytometry showed that the Fas ligand protein was overexpressed in EA-treated MCF-7 cells. Functional genomic studies indicated that specific genes related to cytoskeleton rearrangement, apoptotic signal transduction, and various transcription factors were differentially regulated in EA-treated MCF-7 cells. Putative apoptotic signaling pathways of MCF-7 cells in response to the EA extract of A. formosanus are proposed.
Asunto(s)
Antineoplásicos Fitogénicos/farmacología , Apoptosis , Neoplasias de la Mama/tratamiento farmacológico , Neoplasias de la Mama/patología , Orchidaceae/metabolismo , Extractos Vegetales/farmacología , Proteínas de Plantas/química , Western Blotting , Caspasas/metabolismo , Línea Celular Tumoral , Membrana Celular/metabolismo , Proliferación Celular , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Colorantes/farmacología , Biología Computacional , ADN Complementario/metabolismo , Activación Enzimática , Proteína Ligando Fas , Citometría de Flujo , Regulación Neoplásica de la Expresión Génica , Humanos , Glicoproteínas de Membrana/metabolismo , Hibridación de Ácido Nucleico , Análisis de Secuencia por Matrices de Oligonucleótidos , Fosfatidilserinas/metabolismo , Sales de Tetrazolio/farmacología , Tiazoles/farmacología , Factores de TiempoRESUMEN
Phytochemical characteristics and antioxidant activities of the crude and fractionated plant extracts of Anoectochilus formosanus were evaluated using five different assay systems. An acid-treatment (2 N HCl in 95% ethanol) was employed to treat a butanol fraction (BuOH), creating an acid-hydrolyzed BuOH fraction. The IC(50) values for DPPH radicals in the BuOH and acid-hydrolyzed BuOH fractions were 0.521 and 0.021 mg/mL, respectively. The acid-hydrolyzed BuOH exhibited approximately 5-fold higher activity in scavenging superoxide anion than catechin. The acid-hydrolyzed BuOH fraction also effectively protected phi x174 supercoiled DNA against strand cleavage induced by H(2)O(2) and reduced oxidative stress in HL-60 cells. Metabolite profiling showed that the aglycones of flavonoid glycosides in BuOH were produced after acid hydrolytic treatment, and this resulted in a significant increase in antioxidant activities of acid-hydrolyzed BuOH. One new diarylpentanoid, kinsenone, and three known flavonoid glycosides and their derivatives were identified for the first time from A. formosanus, with strong antioxidant properties.
