RESUMEN

A new pyridone alkaloid, (+)-N-deoxymilitarinone A (1), was isolated from Paecilomyces farinosus RCEF 0097 along with the related metabolites, militarinone D and militarinone B. The sterol 22E,4R-ergosta-7,22-diene-3beta,5alpha,6beta,9alpha-tetraol was also identified. The structures were established by spectroscopic methods, in particular with the aid of extensive NMR experiments. Compound 1 induced neurite sprouting in PC 12 cells when tested at 33 and 100 microM concentrations. A cytotoxic effect was observed in human neurons (IMR-32) at a concentration of 100 microM.

Asunto(s)

Alcaloides/aislamiento & purificación , Factores de Crecimiento Nervioso/aislamiento & purificación , Paecilomyces/química , Piridonas/aislamiento & purificación , Alcaloides/química , Alcaloides/farmacología , Animales , China , Humanos , Lepidópteros , Estructura Molecular , Factores de Crecimiento Nervioso/química , Factores de Crecimiento Nervioso/farmacología , Neuronas/efectos de los fármacos , Células PC12 , Piridonas/química , Piridonas/farmacología , Ratas

RESUMEN

Two new yellow pigments, farinosones A (1) and B (2), were isolated from the mycelial extract of the entomogenous fungal strain Paecilomyces farinosus RCEF 0101, together with farinosone C (3), a new metabolite derived from an early step of pyridone alkaloid biosynthesis. The structures were determined by spectroscopic means, in particular by extensive NMR experiments. Compounds 1 and 3 induced neurite outgrowth in the PC-12 cell line at concentrations of 50 microM, while compound 2 was inactive. No cytotoxicity was observed for compounds 1-3 in PC-12 cells when tested at 50 microM concentration in the MTT assay.

Asunto(s)

Factores de Crecimiento Nervioso/metabolismo , Paecilomyces/química , Fenoles/aislamiento & purificación , Pigmentos Biológicos/aislamiento & purificación , Piridonas/aislamiento & purificación , Animales , China , Ensayos de Selección de Medicamentos Antitumorales , Estructura Molecular , Resonancia Magnética Nuclear Biomolecular , Células PC12 , Fenoles/química , Fenoles/farmacología , Pigmentos Biológicos/química , Pigmentos Biológicos/farmacología , Piridonas/química , Piridonas/farmacología , Ratas

RESUMEN

The fungal metabolite militarinone A (MILI A) promotes neurite outgrowth in PC12 cells. This study was conducted to investigate the signaling pathways involved in the cellular differentiation processes induced by the compound, with a focus on cascades implicated with nerve growth factor (NGF)-mediated neuritogenesis. MILI A possessed pronounced amphiphilic properties. The compound rapidly accumulated in the cell membrane and was slowly released into the cytoplasma. In primed PC12 cells, an early activation of protein kinase B (Akt), representing a downstream target of phosphoinositol 3 (PI3) kinase, and a delayed phosphorylation of extracellular signal-regulated kinases 1 and 2 (ERK1/2), and of transcription factor cAMP responsive element binding protein (CREB) was found. The NGF-dependent activation of c-Jun amino terminal kinase (SAPK/JNK1) was potentiated. Morphological differentiation of cells and the phosphorylation of specific signal molecules were blocked by the MAP kinase (MEK1) inhibitor PD098059, the PI3-kinase (PI3K) inhibitor wortmannin and the adenylyl cyclase inhibitor 9-cyclopentyladenine.

Asunto(s)

Alcaloides/farmacología , Diferenciación Celular/efectos de los fármacos , Proteínas Quinasas Activadas por Mitógenos/metabolismo , Proteínas Serina-Treonina Quinasas/metabolismo , Proteínas Proto-Oncogénicas/metabolismo , Piridonas/farmacología , Transducción de Señal , Adenilil Ciclasas/efectos de los fármacos , Alcaloides/química , Androstadienos/farmacología , Animales , Western Blotting , Proteína de Unión a CREB , Membrana Celular/metabolismo , Citoplasma/metabolismo , Activación Enzimática , Inhibidores Enzimáticos/farmacología , Flavonoides/farmacología , Proteínas Quinasas JNK Activadas por Mitógenos/metabolismo , Cinética , Estructura Molecular , Factor de Crecimiento Nervioso/farmacología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Proteínas Nucleares/metabolismo , Células PC12 , Fosfatidilinositol 3-Quinasas/metabolismo , Fosforilación , Unión Proteica , Proteínas Proto-Oncogénicas c-akt , Piridonas/química , Ratas , Fracciones Subcelulares , Transactivadores/metabolismo , Wortmanina

RESUMEN

An assay for the HPLC-based search for monoamine oxidase-A (MAO-A) inhibitors in plant extracts was established. It combines human recombinant MAO-A, expressed as GST-fusion protein in yeast, with a kinetic measurement of the conversion of kynuramine to 4-hydroxyquinoline. Substrate selectivity and kinetic parameters of the GST-fusion protein were comparable to the wild-type enzyme. The applicability of the assay to HPLC-based activity profiling was tested with plant extracts spiked with small amounts of known MAO inhibitors.

Asunto(s)

Inhibidores de la Monoaminooxidasa/farmacología , Monoaminooxidasa/metabolismo , Extractos Vegetales/química , Cromatografía Líquida de Alta Presión , Humanos , Cinética , Extractos Vegetales/farmacología , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Saccharomyces cerevisiae/metabolismo , Sensibilidad y Especificidad , Especificidad por Sustrato

RESUMEN

Three yellow pigments were isolated from a mycelial extract of the entomopathogenic fungus Paecilomyces militaris. With the aid of spectroscopic means, one compound was identified as a new pyridone alkaloid, militarinone D (1). The two other metabolites were characterized as two novel 3-acyl tetramic acids, militarinones B (2) and C (3). In contrast to the structurally related pyridone militarinone A (4), compounds 1-3 showed only negliable neuritogenic activity in PC-12 cells, whereas militarinone D (1) exhibited cytotoxicity. On the basis of a co-occurrence of 3-acyl tetramic acids and biogenetically related pyridone alkaloids in P. militaris, a revised biosynthetic pathway for pyridone alkaloids is proposed.

Asunto(s)

Alcaloides/aislamiento & purificación , Antineoplásicos/aislamiento & purificación , Paecilomyces/química , Pigmentos Biológicos/aislamiento & purificación , Piridonas/aislamiento & purificación , Pirrolidinonas/aislamiento & purificación , Alcaloides/biosíntesis , Alcaloides/química , Alcaloides/farmacología , Animales , Antineoplásicos/química , Antineoplásicos/farmacología , China , Cromatografía en Capa Delgada , Insectos/patogenicidad , L-Lactato Deshidrogenasa/análisis , L-Lactato Deshidrogenasa/metabolismo , Estructura Molecular , Factores de Crecimiento Nervioso/metabolismo , Células PC12/efectos de los fármacos , Células PC12/enzimología , Pigmentos Biológicos/química , Pigmentos Biológicos/farmacología , Piridonas/química , Piridonas/farmacología , Pirrolidinonas/química , Pirrolidinonas/farmacología , Ratas

RESUMEN

The molecular mechanisms underlying the regulation of interleukin (IL)-10 transcription in monocytic cells by various stimuli during inflammation and the stress reaction are not fully understood. Recently, we provided evidence that stress-induced IL-10 promoter activation in monocytic cells is mediated by catecholamines via a cAMP-dependent signaling pathway including CREB/ATF (cAMP-responsive element binding protein/activating transcription factor) binding to two CRE motifs. However, the mutation of these sites diminished cAMP responsiveness by only 50%, suggesting a role for additional transcription factors and elements in the cAMP-dependent regulation of the human IL-10 promoter. Here, we analyze the functional role of one such factor, C/EBP, in two cell lines of myelomonocytic origin, THP-1 and HL-60, which are known to differ in their differentiation status and C/EBP protein content. We show that the level of basal as well as cAMP-stimulated IL-10 transcription depends on the expression of C/EBP alpha and beta and their binding to three motifs in the promoter/enhancer region. The C/EBP5 motif, which is located between the TATA-box and the translation start point, is essential for the C/EBP-mediated constitutive and most of the cAMP-stimulated expression as its mutation nearly abolished IL-10 promoter activity. Our results suggest a dominant role of C/EBP transcription factors relative to CREB/ATF in tissue-specific and differentiation-dependent IL-10 transcription.

Asunto(s)

Bucladesina/farmacología , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , AMP Cíclico/fisiología , Regulación de la Expresión Génica/inmunología , Interleucina-10/genética , Monocitos/citología , Regiones Promotoras Genéticas , Transcripción Genética/inmunología , Animales , Secuencia de Bases , Diferenciación Celular , Codón Iniciador , Regulación de la Expresión Génica/efectos de los fármacos , Células HL-60 , Humanos , Ratones , Datos de Secuencia Molecular , Monocitos/inmunología , Muridae , Oligodesoxirribonucleótidos , Biosíntesis de Proteínas , Proteínas Recombinantes/inmunología , TATA Box , Transfección

RESUMEN

Cold-pressed, non-raffinated evening primrose oil was found to contain lipophilic radical scavengers. A highly enriched fraction of these compounds could be obtained from the oil by extraction with aqueous ethanol and subsequent liquid-liquid partitioning with petroleum. LC-DAD-MS analysis revealed that the fraction contained three aromatic compounds with identical UV and ESI-MS spectra. The compounds were isolated by RP-HPLC and their structures established by chemical and spectroscopic means as 3-O-trans-caffeoyl derivatives of betulinic, morolic, and oleanolic acid. The morolic acid derivative was a new compound. The three esters exhibited pronounced radical scavenging activity against the stable 2,2-diphenyl-1-picrylhydrazyl radical and were potent inhibitors of neutrophil elastase and cyclooxygenase-1 and -2 in vitro. Commercial samples of evening primrose oils contained only traces of these lipophilic antioxidants.

Asunto(s)

Inhibidores de la Ciclooxigenasa/farmacología , Inhibidores Enzimáticos/farmacología , Ácidos Grasos Esenciales/química , Ácidos Grasos Esenciales/farmacología , Depuradores de Radicales Libres/farmacología , Elastasa de Leucocito/antagonistas & inhibidores , Antioxidantes/análisis , Cromatografía Líquida de Alta Presión , Cromatografía Liquida , Inhibidores de la Ciclooxigenasa/análisis , Inhibidores Enzimáticos/análisis , Ésteres/farmacología , Depuradores de Radicales Libres/análisis , Ácidos Linoleicos , Espectrometría de Masas/métodos , Oenothera biennis , Ácido Oleanólico/análisis , Triterpenos Pentacíclicos , Aceites de Plantas , Triterpenos/química , Ácido gammalinolénico , Ácido Betulínico

RESUMEN

To evaluate the invasive potential of early tumorous lesions of the breast, especially DCIS, we have analyzed semiquantitative telomerase activity in well defined microdissected tissue areas from malignant or benign breast lesions from 145 patients. In order to prove the relationship to cell cycle defects, p53 and p21 proteins were analyzed in corresponding cryostat sections by immunohistochemistry. Telomerase activity was detected in 3 (33.3%) out of 9 benign breast lesions and 109 (80.1%) of 136 malignant tumors. Our study failed to demonstrate an association between telomerase activity, p21, established prognostic factors, such as lymph node status and metastasis, and clinical outcome. We found a high rate of cases (42.2%) with intratumoral heterogeneity concerning telomerase activity status. Telomerase heterogeneity was more obvious in samples with mixed tissue types, although we could not assign specifically the telomerase activity to lobular, ductal and intraductal tissue areas. Telomerase activity was detected in 81.8% of ductal carcinoma in situ (DCIS) which indicates telomerase reactivation as an early event in carcinogenesis. Fifteen (19.5%) out of 77 cases were positive for p53 protein in immunohistochemistry and found to be significantly more frequent in the subgroup with poor outcome. There was only a trend to an association of p53 with high level of telomerase. The prognostic relevance of telomerase activity for patients with benign breast lesions must be further investigated.

Asunto(s)

Neoplasias de la Mama/enzimología , Neoplasias de la Mama/patología , Proteínas Proto-Oncogénicas p21(ras)/metabolismo , Telomerasa/metabolismo , Proteína p53 Supresora de Tumor/metabolismo , Adulto , Anciano , Anciano de 80 o más Años , Carcinoma Ductal de Mama/enzimología , Carcinoma Ductal de Mama/patología , Ciclo Celular , Femenino , Humanos , Inmunohistoquímica , Persona de Mediana Edad , Análisis Multivariante , Metástasis de la Neoplasia , Pronóstico , Resultado del Tratamiento

RESUMEN

Unusual, highly symmetrical cubes are formed by the dodecameric cationic phosphoraneiminato complexes of copper(I) and silver(I) [M12 (NPEt3 )8 ]4+ , in which the metal atoms occupy the edges and the N atoms of NPEt 3-3 groups the corners of the cube (see figure). The structures can be understood as molecular sections of the Cu3 N structure, which is inverse to the ReO3 -type structure.