RESUMO
The crude aqueous ethanol extract of the cones of Alnus glutinosa (L.) Gaertn. (Betulaceae; black alder, European alder) was obtained and further partitioned between water and various organic phases. The active water and butanol phases were subjected to assay-guided (DPPH) fractionation using repetitive RP HPLC until individual compounds were isolated. Their antioxidative activities, measured as SC50 values, were evaluated. The chemical structures of the isolated compounds were elucidated with the help of mass spectroscopy, (1)H NMR technique, UV spectroscopy, and chemical approaches. One novel ellagitannin, glutinoin (2), along with two known compounds, pedunculagin (1) and praecoxin D (3), were isolated and found to contribute to antioxidative activity of the A. glutinosa cones extract. The activities (SC50) of 1-3 were evaluated as 0.95 (1), 1.00 (2) and 1.01 µg mL⻹ (3). The scavenging effects of glutinoin (2) and praecoxin D (3) were reported for the first time.
Assuntos
Alnus/química , Antioxidantes/química , Antioxidantes/farmacologia , Taninos Hidrolisáveis/química , Taninos Hidrolisáveis/farmacologia , Antioxidantes/isolamento & purificação , Cromatografia Líquida de Alta Pressão , Avaliação Pré-Clínica de Medicamentos , Taninos Hidrolisáveis/isolamento & purificação , Espectroscopia de Ressonância Magnética , Espectrometria de Massas , Estrutura Molecular , Extratos Vegetais/química , Plantas Medicinais/química , Espectrofotometria UltravioletaRESUMO
An aqueous ethanol extract of Pyrola rotundifolia L. induced AMP-activated protein kinase (AMPK) phosphorylation in C2C12 myotubes. The bioassay-guided fractionation of the extract led to the isolation a 2-methyl-7-hydroxymethyl-1,4-naphthoquinone, or a 7'-hydroxy-chimaphilin, which showed concentration-dependent AMPK phosphorylation activity at 2.5-20 µg/ml. At a concentration of 10 µg/ml (50 µM), an approximately four-fold increase in the AMPKα(Thr¹7²) phosphorylation level was observed. The stimulatory effect of naphthoquinone on AMPK activity was comparable to that of known compounds found in natural sources that activate the AMPK signaling pathway.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Ativadores de Enzimas/farmacologia , Fibras Musculares Esqueléticas/efeitos dos fármacos , Naftoquinonas/farmacologia , Extratos Vegetais/farmacologia , Pyrola/química , Animais , Linhagem Celular , Relação Dose-Resposta a Droga , Ativadores de Enzimas/isolamento & purificação , Camundongos , Estrutura Molecular , Fibras Musculares Esqueléticas/metabolismo , Naftoquinonas/isolamento & purificação , Fosforilação , Extratos Vegetais/químicaRESUMO
An aqueous ethanol extract of Bergenia crassifolia rhizomes strongly inhibited human pancreatic lipase activity and increased scavenging of DPPH free radicals in vitro. Chromatographic separation of this extract led to isolation of the hydrolysable tannins (+)-catechin 3,5-di-O-gallate (1) and (+)-catechin 3-O-gallate (2). This is the first report of the isolation of compound 1 from plant material. This compound strongly inhibited human pancreatic lipase (with an IC(50) value of 0.42 µg/ml) and exhibited a remarkable free radical-scavenging ability (with an SC(50) value of 1.04 µg/ml). The chemical structures of 1 and 2 were elucidated using MS, NMR and chemical approaches.
Assuntos
Antioxidantes/farmacologia , Catequina/farmacologia , Inibidores Enzimáticos/farmacologia , Pancrelipase/antagonistas & inibidores , Extratos Vegetais/farmacologia , Saxifragaceae/química , Antioxidantes/isolamento & purificação , Compostos de Bifenilo/metabolismo , Catequina/análogos & derivados , Catequina/isolamento & purificação , Inibidores Enzimáticos/isolamento & purificação , Humanos , Concentração Inibidora 50 , Pâncreas , Picratos/metabolismo , Extratos Vegetais/química , RizomaRESUMO
Overexpression of the yeaS gene encoding a protein belonging to the RhtB transporter family conferred upon cells resistance to glycyl-l-leucine, leucine analogues, several amino acids and their analogues. yeaS overexpression promoted leucine and, to a lesser extent, methionine and histidine accumulation by the respective producing strains. Our results indicate that yeaS encodes an exporter of leucine and some other structurally unrelated amino acids. The expression of yeaS (renamed leuE for "leucine export") was induced by leucine, l-alpha-amino-n-butyric acid and, to a lesser extent, by several other amino acids. The global regulator Lrp mediated this induction.
Assuntos
Sistemas de Transporte de Aminoácidos Neutros/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Regulação Bacteriana da Expressão Gênica , Leucina/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Sistemas de Transporte de Aminoácidos Neutros/genética , Sequência de Bases , Proteínas de Ligação a DNA , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Histidina/metabolismo , Leucina/química , Proteína Reguladora de Resposta a Leucina , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Membrana Transportadoras/genética , Metionina/metabolismo , Fatores de Transcrição , Sítio de Iniciação de TranscriçãoRESUMO
The yicM gene of Escherichia coli was found by selection for resistance to 6-mercaptopurine. Translation and transcription initiation sites of yicM were determined. Overexpression of yicM increased resistance of sensitive cells to inosine and guanosine, decreased E. coli growth rate in medium containing these ribonucleosides as the sole carbon source, led to inosine accumulation by the E. coli strain deficient in purine nucleoside phosphorylase and enhanced the rate of inosine excretion by an inosine-producing strain. These results suggest that yicM encodes a purine ribonucleoside exporter and we have accordingly renamed it nepI (for 'nucleoside efflux permease-inosine').
Assuntos
Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Proteínas de Transporte de Nucleotídeos/genética , Proteínas de Transporte de Nucleotídeos/metabolismo , Nucleosídeos de Purina/metabolismo , Ribonucleosídeos/metabolismo , Sequência de Bases , Transporte Biológico Ativo , Clonagem Molecular , Meios de Cultura , DNA Bacteriano/genética , Farmacorresistência Bacteriana/genética , Escherichia coli/efeitos dos fármacos , Escherichia coli/crescimento & desenvolvimento , Expressão Gênica , Inosina/metabolismo , Mercaptopurina/farmacologia , Dados de Sequência Molecular , Biossíntese de Proteínas , Nucleosídeos de Purina/farmacologia , Purina-Núcleosídeo Fosforilase/metabolismo , Ribonucleosídeos/farmacologia , Transcrição GênicaRESUMO
Gamma-aminobutyraldehyde dehydrogenase (ABALDH) from wild-type E. coli K12 was purified to apparent homogeneity and identified as YdcW by MS-analysis. YdcW exists as a tetramer of 202+/-29 kDa in the native state, a molecular mass of one subunit was determined as 51+/-3 kDa. Km parameters of YdcW for gamma-aminobutyraldehyde, NAD+ and NADP+ were 41+/-7, 54+/-10 and 484+/-72 microM, respectively. YdcW is the unique ABALDH in E. coli K12. A coupling action of E. coli YgjG putrescine transaminase and YdcW dehydrogenase in vitro resulted in conversion of putrescine into gamma-aminobutyric acid.
Assuntos
Aldeído Oxirredutases/metabolismo , Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Aldeído Oxirredutases/química , Aldeído Oxirredutases/isolamento & purificação , Sequência de Aminoácidos , Cromatografia Líquida de Alta Pressão , Escherichia coli/enzimologia , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/isolamento & purificação , Dados de Sequência Molecular , Homologia de Sequência de AminoácidosRESUMO
Structural and functional properties of recombinant IL-4delta2, a naturally occurring splice variant of human IL-4 with a deletion of the loop region 22-37, have been analyzed. IL-4delta2 has alpha-helical structure and most likely preserves the "up-up-down-down" topology typical of the four-helix-bundle cytokines. IL-4delta2 interacts specifically with the alpha chain of IL-4R and competes effectively with IL-4 for the common binding sites. Thus, IL-4delta2 may act as a regulator of the cytokine net, being the natural antagonist of IL-4.
Assuntos
Processamento Alternativo , Interleucina-4/genética , Isoformas de Proteínas/genética , Divisão Celular/fisiologia , Dicroísmo Circular , Clonagem Molecular , Cistina/metabolismo , Interleucina-4/metabolismo , Ligantes , Isoformas de Proteínas/metabolismo , Estrutura Terciária de Proteína , Espectroscopia de Infravermelho com Transformada de Fourier , Timo/metabolismoRESUMO
BACKGROUND: Putrescine is the intermediate product of arginine decarboxylase pathway in Escherichia coli which can be used as an alternative nitrogen source. Transaminase and dehydrogenase enzymes seem to be implicated in the degradative pathway of putrescine, in which this compound is converted into gamma-aminobutyrate. But genes coding for these enzymes have not been identified so far. RESULTS: The 1.8-kbp DNA fragment containing E. coli K12 ygjG gene with aer-ygjG intergenic region was examined. It was found that the fragment contains sigma54-depended open reading frame (ORF) of 1,380 nucleotides encoding a 459-amino acid polypeptide of approximately 49.6 kDa. The cytidine (C) residue localized 10 bp downstream of the sigma54 promoter sequence was identified as the first mRNA base. The UUG translation initiation codon is situated 36 nucleotides downstream of the mRNA start. The YgjG was expressed as a his6-tag fused protein and purified to homogeneity. The protein catalyzed putrescine:2-oxoglutaric acid (2-OG) aminotransferase reaction (PATase, EC 2.6.1.29). The Km values for putrescine and 2-OG were found to be 9.2 mM and 19.0 mM, respectively. The recombinant enzyme also was able to transaminate cadaverine and, in lower extent, spermidine, and gave maximum activity at pH 9.0. CONCLUSION: Expression of E. coli K12 ygjG coding region revealed sigma54-depended ORF which encodes a 459-amino acid protein with putrescine:2-OG aminotransferase activity. The enzyme also was able to transaminate cadaverine and, in lower extent, spermidine.