RESUMEN
CONTEXT: Podophyllotoxin, a pharmaceutically important bioactive compound of Podophyllum sps. (Berberidaceae), is in great demand worldwide as an anticancer and antivirus drug precursor. However, the source of podophyllotoxin is very limited due to the endangered status of the Podophyllum plant. OBJECTIVE: The aim of this study was to isolate podophyllotoxin-producing endophytic fungi from Sinopodophyllum hexandrum (Royle) Ying (1979) (Berberidaceae) plants of the Taibai Mountains of China in order to obtain bioactive compounds. MATERIALS AND METHODS: The strains producing kaempferol and podophyllotoxin were screened by thin-layer chromatography (TLC) analysis. The presence of kaempferol and podophyllotoxin in extracts of these strains was further confirmed by high-performance liquid chromatography (HPLC) and nuclear magnetic resonance (NMR) analyses. RESULTS: Among six endophytic fungi isolated from the rhizomes of S. hexandrum, one strain was able to produce kaempferol. Another strain, named TW5, was able to produce both kaempferol and podophyllotoxin simultaneously according to the TLC, HPLC, and NMR results. The podophyllotoxin yield of TW5 was calculated to be 49.3 µg/g of mycelial dry weight after 7-d fermentation. Strain TW5 was identified morphologically and phylogenetically to be Mucor fragilis Fresen. (Mucoraceae). These results suggest that the podophyllotoxin-synthesizing ability is obtained by uptaking genes involved in the podophyllotoxin synthesis from the host plant into endophytic fungal genomes. CONCLUSION: Our results showed, for the first time, that the endophytic fungus M. fragilis is able to produce simultaneously the same two bioactive metabolites, podophyllotoxin and kaempferol, as its host plant. Furthermore, the relatively high podophyllotoxin yield obtained may improve the industrial production of podophyllotoxin, which may help protect this endangered plant.
Asunto(s)
Quempferoles/análisis , Mucor , Podofilotoxina/análisis , Rizoma , Cromatografía en Capa Delgada/métodos , Quempferoles/química , Podofilotoxina/químicaRESUMEN
Limitation of damage after ischemia and reperfusion injury to the myocardium remains an elusive clinical goal. Previous studies have suggested that molecular chaperones, which include members of the heat shock protein (Hsp) family, may have cardioprotective effects, although the protective role of endogenous chaperones has not been well documented. CHIP (carboxyl terminus of Hsp70-interacting protein) is a cochaperone/ubiquitin ligase that integrates the response to stress at multiple levels. We tested the response of CHIP(-/-) mice to in vivo ischemia and reperfusion injury induced by left anterior descending coronary artery ligation. Compared with wild-type littermates, CHIP(-/-) mice had decreased survival and increased incidence of arrhythmias during reperfusion. The size of myocardial infarction, as assessed by the ratio of infarct area to area at risk, was 50% greater in CHIP(-/-) mice. Increased infarct size was accompanied by impaired upregulation of the chaperone Hsp70 after ischemia-reperfusion injury. In situ analysis also indicated that hearts of CHIP(-/-) mice were more prone to develop apoptosis in cardiomyocytes and especially endothelial cells of intramural vessels. Previous studies have found that CHIP plays a central role in maintaining protein quality control and coordinating the response to stress. The present data indicate that these functions of CHIP provide a critical cardioprotective effect in the setting of ischemia-reperfusion injury due in part to increased apoptosis in cardiac cells. Quality control mechanisms therefore may be underappreciated clinical targets for maximizing myocardial protection after injury.
Asunto(s)
Cardiotónicos/farmacología , Proteínas Portadoras/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Infarto del Miocardio/fisiopatología , Ubiquitina-Proteína Ligasas/fisiología , Proteínas Adaptadoras Transductoras de Señales , Animales , Arritmias Cardíacas/prevención & control , Proteínas Portadoras/genética , Ecocardiografía , Proteínas HSP70 de Choque Térmico/deficiencia , Proteínas HSP70 de Choque Térmico/genética , Frecuencia Cardíaca/fisiología , Ratones , Ratones Noqueados , Isquemia Miocárdica/prevención & control , Daño por Reperfusión Miocárdica/prevención & control , Ubiquitina-Proteína Ligasas/genética , Función Ventricular Izquierda/fisiologíaRESUMEN
A reproducible, transcriptionally diverse common reference RNA is required for accurate comparisons of data generated from most spotted microarray experiments in different experiments. Several methods have been proposed to make such a reference RNA, such as pooling RNAs isolated from multiple cell lines or tissues, amplifying pooled RNAs, or synthesizing RNAs or DNAs complementary to microarray features. We report an approach to prepare a large amount of mouse reference RNA from whole neonatal mice. This approach is simple, quick, reliable, reproducible, and inexpensive. The whole mouse reference RNA is highly representative when compared to two commercially available universal mouse reference RNAs isolated and pooled from multiple cell lines or organs.
Asunto(s)
Animales Recién Nacidos/genética , Perfilación de la Expresión Génica/métodos , ARN/análisis , Animales , Carbocianinas , ADN Complementario , Colorantes Fluorescentes , Perfilación de la Expresión Génica/normas , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Análisis de Secuencia por Matrices de Oligonucleótidos/normas , ARN/genética , ARN/normas , Reproducibilidad de los ResultadosRESUMEN
Microarray technology has greatly aided the identification of genes that are expressed differentially. Statistical analysis of such data by multiple comparisons procedures has been slow to develop, in part, because methods to cluster the results of such comparisons in biologically meaningful ways have not been available. We isolated and analyzed, by Northern blot and GeneChip, replicate liver RNA samples (n = 4/group) from rats fed with control diet or diet containing one of three chemopreventive compounds, selected because their pharmacological activities, including RNA expression response, are relatively well understood. We report on a classification tree, based on the results of nonparametric multiple comparisons, which results in the bipolar hierarchical clustering of genes in relation to their response to treatment. In addition to identifying treatment-responsive genes, application of this procedure to our test study identified the known pharmacological relationships among the treatment groups without supervision. Also, small treatment-specific subsets of genes were identified that may be indicative of additional pharmacophores present in the test compounds.