RESUMEN
UNLABELLED: In screening for natural products with antiparasitic activity, an endophytic fungus, strain F2611, isolated from above-ground tissue of the tropical grass Paspalum conjugatum (Poaceae) in Panama, was chosen for bioactive principle elucidation. Cultivation on malt extract agar (MEA) followed by bioassay-guided chromatographic fractionation of the extract led to the isolation of the new polyketide integrasone B (1) and two known mycotoxins, sterigmatocystin (2) and secosterigmatocystin (3). Sterigmatocystin (2) was found to be the main antiparasitic compound in the fermentation extract of this fungus, possessing potent and selective antiparasitic activity against Trypanosoma cruzi, the cause of Chagas disease, with an IC50 value of 0.13 µmol l(-1) . Compounds 2 and 3 showed high cytotoxicity against Vero cells (IC50 of 0.06 and 0.97 µmol l(-1) , respectively). The new natural product integrasone B (1), which was co-purified from the active fractions, constitutes the second report of a natural product possessing an epoxyquinone with a lactone ring and exhibited no significant biological activity. Strain F2611 represents a previously undescribed taxon within the Microthyriaceae (Dothideomycetes, Ascomycota). SIGNIFICANCE AND IMPACT OF THE STUDY: The present study attributes new antiparasitic and psychoactive biological activities to sterigmatocystin (2), and describes the structure elucidation of the new natural product integrasone B (1), which possesses a rare epoxyquinone with a lactone ring moiety. This is also the first report of sterigmatocystin (2) isolation in a fungal strain from this family, broadening the taxonomic range of sterigmatocystin-producing fungi. The study also presents taxonomic analyses indicating that strain F2611 is strongly supported as a member of the Microthyriaceae (Ascomycota), but is not a member of any previously known or sequenced genus.
Asunto(s)
Ascomicetos/química , Policétidos/farmacología , Esterigmatocistina/análogos & derivados , Esterigmatocistina/farmacología , Tripanocidas/farmacología , Animales , Ascomicetos/clasificación , Ascomicetos/genética , Supervivencia Celular/efectos de los fármacos , Chlorocebus aethiops , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , Endófitos/química , Concentración 50 Inhibidora , Datos de Secuencia Molecular , Tipificación Molecular , Técnicas de Tipificación Micológica , Poaceae/microbiología , Policétidos/aislamiento & purificación , Esterigmatocistina/aislamiento & purificación , Tripanocidas/aislamiento & purificación , Trypanosoma cruzi/efectos de los fármacos , Células VeroRESUMEN
Melanoma development is a multi-step process arising from a series of genetic and epigenetic events. Although the sequential stages involved in progression from melanocytes to malignant melanoma are clearly defined, our current understanding of the mechanisms leading to melanoma onset is still incomplete. Growing evidence show that the activation of endogenous retroviral sequences might be involved in transformation of melanocytes as well as in the increased ability of melanoma cells to escape immune surveillance. Here we show that human melanoma cells in vitro undergo a transition from adherent to a more malignant, non-adherent phenotype when exposed to stress conditions. Melanoma-derived non-adherent cells are characterized by an increased proliferative potential and a decreased expression of both HLA class I molecules and Melan-A/MART-1 antigen, similarly to highly malignant cells. These phenotypic and functional modifications are accompanied by the activation of human endogenous retrovirus K expression (HERV-K) and massive production of viral-like particles. Down-regulation of HERV-K expression by RNA interference prevents the transition from the adherent to the non-adherent growth phenotype in low serum. These results implicate HERV-K in at least some critical steps of melanoma progression.
Asunto(s)
Transformación Celular Viral , Retrovirus Endógenos/fisiología , Melanoma/virología , Activación Viral/fisiología , Células CACO-2 , Proliferación Celular , Transformación Celular Viral/genética , Células Cultivadas , Células Clonales/virología , Progresión de la Enfermedad , Retrovirus Endógenos/genética , Humanos , Células Jurkat , Células K562 , Melanocitos/patología , Melanocitos/ultraestructura , Melanocitos/virología , Melanoma/etiología , Melanoma/genética , Melanoma/patología , Modelos Biológicos , ARN Viral/aislamiento & purificación , Virión/crecimiento & desarrollo , Activación Viral/genéticaRESUMEN
We show here that a reverse transcriptase (RT) activity is present in murine epididymal spermatozoa. Sperm cells incubated with human poliovirus RNA can take up exogenous RNA molecules and internalize them in nuclei. Direct PCR amplification of DNA extracted from RNA-incubated spermatozoa indicate that poliovirus RNA is reverse-transcribed in cDNA fragments. PCR analysis of two-cell embryos shows that poliovirus RNA-challenged spermatozoa transfer retrotranscribed cDNA molecules into eggs during in vitro fertilization. Finally, RT molecules can be visualized on sperm nuclear scaffolds by immunogold electron microscopy. These results, therefore, reveal a novel metabolic function in spermatozoa, which may play a role during early embryonic development.
Asunto(s)
Núcleo Celular/enzimología , ARN Viral/metabolismo , ADN Polimerasa Dirigida por ARN/metabolismo , Espermatozoides/enzimología , Animales , Núcleo Celular/ultraestructura , Epidídimo , Femenino , Humanos , Cinética , Masculino , Ratones , Ratones Endogámicos C57BL , Ratones Endogámicos , Oocitos/fisiología , Poliovirus/genética , Reacción en Cadena de la Polimerasa , Motilidad Espermática , Espermatozoides/fisiología , Espermatozoides/ultraestructuraRESUMEN
Transformed cells express high levels of non-telomeric reverse-transcriptase (RT) activity of retrotransposon and endogenous retrovirus origin. We previously reported that RT inhibition, either pharmacological or through transient silencing of RT-encoding LINE-1 (L1) elements by RNA interference (RNAi), reduced proliferation, induced differentiation and reprogrammed gene expression in human tumorigenic cell lines. Moreover, the antiretroviral drug efavirenz antagonized tumor progression in animal models in vivo. To get insight into the role of retroelements in tumorigenesis, we have now produced two cell lines derived from A-375 melanoma, in which the expression of either L1 retrotransposon, or HERV-K endogenous retrovirus, was stably suppressed by RNAi. Compared to the parental A-375 cell line, cells with stably interfered L1 expression show a lower proliferation rate, a differentiated morphology and lower tumorigenicity when inoculated in nude mice. L1 silencing modulates expression of several genes and, unexpectedly, also downregulates HERV-K expression. In HERV-K interfered cells, instead, L1 expression was unaffected, and cell proliferation and differentiation remained unchanged compared to parental A-375 cells. In vivo, however, their tumorigenic potential was found to be reduced after inoculation in nude mice. These results suggest that L1 and HERV-K play specific and distinct roles in cell transformation and tumor progression.
Asunto(s)
Diferenciación Celular , Proliferación Celular , Retrovirus Endógenos/genética , Elementos de Nucleótido Esparcido Largo/fisiología , Melanoma Experimental/patología , Retroelementos , Animales , Diferenciación Celular/genética , Línea Celular Tumoral , Transformación Celular Neoplásica/genética , Progresión de la Enfermedad , Vectores Genéticos , Humanos , Elementos de Nucleótido Esparcido Largo/genética , Melanoma Experimental/genética , Melanoma Experimental/prevención & control , Melanoma Experimental/virología , Ratones , Ratones Desnudos , Trasplante de Neoplasias , ARN Interferente Pequeño/genética , Retroelementos/genéticaRESUMEN
Previous successful transplantation of human primary prostatic carcinomas into nude mice has been described as "close to zero." When injected in Matrigel instead of culture medium, 25,000-fold fewer cells of the PC-3 human prostatic carcinoma cell line were required for the growth of tumors in nude mice during a 3-month period of observation; similar enhancement was observed with two other human prostatic carcinoma cell lines. Six of ten primary human prostatic carcinomas were transplanted successfully into nude mice when Matrigel was used as the vehicle.
Asunto(s)
Carcinoma/patología , Colágeno/farmacología , Laminina/farmacología , Neoplasias de la Próstata/patología , Proteoglicanos/farmacología , Trasplante Heterólogo , Animales , Medios de Cultivo , Combinación de Medicamentos , Humanos , Masculino , Ratones , Ratones Desnudos , Trasplante de Neoplasias , Células Tumorales CultivadasRESUMEN
The chromatin structure of the ribosomal DNA in Xenopus laevis was studied by micrococcal nuclease digestions of blood, liver and embryonic cell nuclei. We have found that BglI-restricted DNA from micrococcal nuclease-digested blood cell nuclei has an increased electrophoretic mobility compared to the undigested control. Micrococcal nuclease digestion of liver cell nuclei causes a very slight shift in mobility, only in the region of the spacer containing the "Bam Islands". In contrast, the mobility of ribosomal DNA in chromatin of embryonic cells, under identical digestion conditions, remains unaffected by the nuclease activity. Denaturing gels or ligase action on the nuclease-treated DNA abolishes the differences in the electrophoretic mobility. Ionic strength and ethidium bromide influence the relative electrophoretic migration of the two DNA fragment populations, suggesting that secondary structure may play an important role in the observed phenomena. In addition, restriction analysis under native electrophoretic conditions of DNA prepared from blood, liver and embryonic cells shows that blood cell DNA restriction fragments always have a faster mobility than the corresponding fragments of liver and embryo cell DNA. We therefore propose that nicking activity by micrococcal nuclease modifies the electrophoretic mobility of an unusual DNA conformation, present in blood cell, and to a lesser extent, in liver cell ribosomal chromatin. A possible function for these structures is discussed. The differences of the ribosomal chromatin structures in adult and embryonic tissues may reflect the potential of the genes to be expressed.
Asunto(s)
Cromatina , ADN Ribosómico , Conformación de Ácido Nucleico , Animales , Desoxirribonucleasa I , Electroforesis en Gel de Agar , Embrión no Mamífero/análisis , Endodesoxirribonucleasas , Etidio , Hígado/análisis , Nucleasa Microcócica , Microscopía Electrónica , Hibridación de Ácido Nucleico , Concentración Osmolar , ARN Ribosómico , Xenopus laevis/genéticaRESUMEN
We have examined the murine genes encoding transcription factors E2F1, -3, -5 and -6 in gametes and early embryos. All genes are expressed as maternal transcripts and all are efficiently transcribed after the blastocyst stage. Between those two stages, each E2F mRNA is transcribed with a distinctive and unique pattern. E2F proteins are also differentially expressed and compartmentalized in pre-implantation embryos.
Asunto(s)
Proteínas Portadoras , Proteínas de Ciclo Celular , Proteínas de Unión al ADN , Células Germinativas/metabolismo , Factores de Transcripción/genética , Células 3T3 , Animales , Factores de Transcripción E2F , Factor de Transcripción E2F1 , Factor de Transcripción E2F3 , Factor de Transcripción E2F5 , Factor de Transcripción E2F6 , Desarrollo Embrionario y Fetal , Femenino , Regulación del Desarrollo de la Expresión Génica , Masculino , Ratones , Ratones Endogámicos C57BL , Proteína 1 de Unión a Retinoblastoma , Factor de Transcripción DP1RESUMEN
Endogenous, non-telomeric Reverse Transcriptase (RT) is encoded by two classes of repeated genomic elements, retrotransposons and endogenous retroviruses, and is an essential component of the retrotransposition machinery of both types of elements. Expression of RT-coding genes is generally repressed in non-pathological, terminally differentiated cells, but is active in early embryos, germ cells, embryo and tumor tissues, all of which have a high proliferative potential. To clarify whether reverse transcription is functionally implicated in control of cell growth, differentiation and in embryogenesis, recent experiments have been undertaken to inactivate the endogenous RT activity. RT was inhibited in normal and transformed cell lines by exposure to nevirapine, a non-nucleosidic RT inhibitor. The endogenous RT was also blocked in murine embryos by microinjection of an anti-RT antibody. Both experimental approaches yielded a dramatic inhibition of proliferation. Murine embryos arrested at pre-implantation stages. Transformed cell lines underwent a significant reduction in the rate of cell growth, concomitant with the induction of differentiation. In addition, RT inhibition induced an extensive reprogramming of the gene expression profile both in cultured cell lines and in preimplantation embryos. From these studies, endogenous RT begins to emerge as a key function with a driving role in normal and pathological developmental processes.
Asunto(s)
Diferenciación Celular/fisiología , Proliferación Celular , ADN Polimerasa Dirigida por ARN/fisiología , Animales , Desarrollo Embrionario , RatonesRESUMEN
Foreign DNA is spontaneously taken up by mouse epididymal sperm cells and is further internalized into nuclei. The interaction and/or internalization of the exogenous DNA triggers the activation of sperm endogenous nucleases which mediate rearrangements of the internalized DNA. Foreign DNA sequences are found to be tightly bound to the sperm nuclear scaffold, and to undergo a recombination process with the sperm chromosomal DNA. Sequence analysis of randomly selected clones from a library of sperm genomic DNA transformed with pSV2CAT plasmid showed that foreign sequences were integrated in a unique site of the sperm genome. Preliminary results suggest that the integration process is mediated by a retrotranscription step.
Asunto(s)
Espermatozoides , Transformación Genética , Animales , Núcleo Celular , Humanos , Masculino , RatonesRESUMEN
Mouse epididymal sperm cells have the spontaneous ability to take up exogenous DNA. A proportion of the sperm-bound DNA is further internalized into sperm nuclei. In this work, we have followed up the fate of the foreign DNA upon internalization into nuclei. We have found that the internalized plasmid DNA becomes tightly associated with the nuclear scaffold, is extensively rearranged, and undergoes recombination with the sperm genomic DNA. Sequence analysis of two randomly selected clones independently recovered by plasmid rescue from pSV2CAT plasmid-challenged sperm cells shows that DNA fragments from the plasmid are integrated into the mouse sperm genome. The sites of integration are identical in both clones, suggesting that these events do not occur randomly, but take place at preferential sites. A topoisomerase II consensus sequence is found adjacent to one end of the integration site, suggesting a possible role of this enzyme in the process of nonhomologous recombination.
Asunto(s)
ADN/genética , Genoma , Plásmidos/genética , Transfección , Animales , Masculino , Ratones , Datos de Secuencia Molecular , Análisis de Secuencia de ADN , EspermatozoidesRESUMEN
Mature sperm cells, either of epididymal origin or ejaculated and depleted of seminal fluid, are spontaneously able to bind exogenous DNA molecules which are subsequently internalized into sperm nuclei. Southern blot analysis showed that the internalized DNA was specifically cleaved by sperm endonucleases and showed typical fragmentation patterns of localized hypersensitivity. Nucleases were activated in response to the internalization of exogenous DNA by sperm cells and their activity increased with the DNA concentration. Nuclease activation was efficient in epididymal sperm cells, while being drastically reduced in ejaculated washed spermatozoa. Nucleases were Ca++ dependent, and were, respectively, inhibited and activated by preincubating sperm cells with Aurintricarboxylic Acid (ATA) and Ca++ Ionophore A23187, which are known to, respectively, inhibit and activate apoptosis in somatic cells. Moreover, nuclease activation also caused a partial degradation of the sperm endogenous chromosomal DNA; cleaved DNA fragments were released from the sperm cells to the medium. Taken together, these results suggest that a metabolically active process similar to apoptosis is triggered in the nuclei of mature sperm cells upon interaction with exogenous DNA.
Asunto(s)
ADN/metabolismo , Endodesoxirribonucleasas/metabolismo , Espermatozoides/enzimología , Animales , Apoptosis , Ácido Aurintricarboxílico/farmacología , Secuencia de Bases , Calcimicina/farmacología , Calcio/fisiología , Bovinos , Activación Enzimática , Ionóforos/farmacología , Lactoglobulinas/genética , Masculino , Ratones , Datos de Secuencia Molecular , Plásmidos/genética , Plásmidos/metabolismo , PorcinosRESUMEN
Neutralizing antibodies against West Nile (WNV) and Usutu (USUV) viruses were measured in 6000 samples collected, between 1 September 2010 and 30 June 2011, from blood donors living in different districts of Emilia-Romagna, northeastern Italy. On the basis of the microneutralization assay (MNTA), 47 (0.78%) subjects were positive for WNV and 14 (0.23%) for USUV. These results were compared with those obtained 2 years ago and suggest an increased circulation of USUV among humans in Emilia-Romagna.
Asunto(s)
Anticuerpos Antivirales/sangre , Donantes de Sangre/estadística & datos numéricos , Infecciones por Flavivirus/sangre , Infecciones por Flavivirus/epidemiología , Flavivirus/inmunología , Fiebre del Nilo Occidental/sangre , Virus del Nilo Occidental/inmunología , Adolescente , Adulto , Femenino , Flavivirus/aislamiento & purificación , Infecciones por Flavivirus/inmunología , Humanos , Italia/epidemiología , Masculino , Persona de Mediana Edad , Estudios Seroepidemiológicos , Fiebre del Nilo Occidental/epidemiología , Fiebre del Nilo Occidental/inmunología , Virus del Nilo Occidental/aislamiento & purificación , Adulto JovenAsunto(s)
Aminoacil-ARNt Sintetasas/metabolismo , ARN de Transferencia/metabolismo , Erizos de Mar/crecimiento & desarrollo , Envejecimiento , Animales , Arginina , Isótopos de Carbono , Cromatografía DEAE-Celulosa , Electroforesis Discontinua , Embrión no Mamífero , Femenino , Fertilización , Leucina , Lisina , Óvulo , Saccharomyces cerevisiae/enzimología , Erizos de Mar/enzimología , Erizos de Mar/metabolismo , Serina , Espectrofotometría Ultravioleta , Factores de Tiempo , TritioRESUMEN
Sperm cells from a variety of species share the spontaneous ability to take up foreign DNA. That feature has been exploited to generate genetically modified animals with variable efficiency in different species. An unexpectedly large set of factors appears to modulate the interaction of sperm cells with exogeneous DNA. The binding is mediated by specific DNA-binding proteins and is antagonized by an inhibitory factor in the seminal fluid. A portion of sperm-bound DNA is internalized in nuclei, a process mediated by CD4 molecules. Sperm interaction with foreign DNA triggers endogenous nuclease(s) that cleaves both the exogenous and the genomic DNA, eventually leading to a cell death process which resembles apoptosis. Internalized foreign DNA sequences reach the nuclear matrix and undergo recombination with chromosomal DNA. From these studies, a surprising network of metabolic functions is beginning to emerge in mature spermatozoa, which are normally repressed and are specifically activated upon exposure to appropriate stimuli.
Asunto(s)
Proteínas de Unión al ADN/metabolismo , ADN/metabolismo , Técnicas de Transferencia de Gen , Espermatozoides/fisiología , Animales , Apoptosis , Núcleo Celular/metabolismo , ADN/química , Desoxirribonucleasas/metabolismo , Genoma , Humanos , Masculino , Espermatozoides/citologíaRESUMEN
Micrococcal nuclease digestion was used as a tool to study the organization of the ribosomal chromatin in liver, blood and embryo cells of X. laevis. It was found that in liver and blood cells, ribosomal DNA is efficiently protected from nuclease attack in comparison to bulk chromatin. Although ribosomal chromatin is fragmented in a typical nucleosomal pattern, a considerable portion of ribosomal DNA retains a high molecular weight even after extensive digestion. A greater accessibility of the coding region in comparison to the non-coding spacer was found. In embryos, when ribosomal DNA is fully transcribed, these genes are even more highly protected than in adult tissues: in fact, the nucleosomal ladder can hardly be detected and rDNA is preserved in high molecular weight. Treatment of chromatin with 0.8 M NaCl abolishes the specific resistance of the ribosomal chromatin to digestion. The ribosomal chromatin, particularly in its active state, seems to be therefore tightly complexed with chromosomal proteins which protect its DNA from nuclease degradation.
Asunto(s)
Cromatina/metabolismo , ADN Recombinante/metabolismo , Ribosomas/metabolismo , Animales , Núcleo Celular/metabolismo , Cromatina/ultraestructura , Clonación Molecular , ADN/genética , ADN Ribosómico , Embrión no Mamífero , Femenino , Hígado/metabolismo , Nucleasa Microcócica , Peso Molecular , Hibridación de Ácido Nucleico , Óvulo/metabolismo , Transcripción Genética , XenopusRESUMEN
The four histones H2A, H2B, H3 and H4 from calf thymus, CHO and sea urchin gastrula cells were associated by stepwise dialysis from 2 M NaCl with SV40 DNA Form I. The in vitro-assembled chromatins were visualized by electron microscopy and the size of the DNA fragments generated by digestion with DNase II was determined. Irrespective of the origin of the histones, the size of the smallest DNA band generated at early times of digestion was about 190 base pairs, whereas oligomeric DNA bands were multiples of 140 bp. These results support our previous proposal that the four histones H2A, H2B, H3 and H4 are able to organize more than 140 bp of DNA, but do not provide any evidence that the variability of histones H2A and H2B plays a role in the variability of the DNA repeat length of native chromatins.
Asunto(s)
Cromatina/ultraestructura , ADN Viral/metabolismo , Histonas/metabolismo , Animales , Sitios de Unión , Bovinos , Sistema Libre de Células , Peso Molecular , Unión Proteica , Erizos de Mar , Virus 40 de los Simios , Especificidad de la EspecieRESUMEN
Native rat liver chromatin fragments exposed to 600 mM NaCl at 37 degrees C for 45 min exhibit substantial modification of their original (approximately 200 base pairs) repeating subunit structure: a new repeat of 140 base pairs, superimposed on a high background, is observed after micrococcal nuclease digestion. The same material appears, in the electron microscope, as clusters of tightly packed beads connected by stretches of 'free' DNA. These modifications are not observed when the native chromatin is incubated at 37 degrees C at NaCl concentrations up to 400 mM. When native rat liver chromatin depleted of histone H1 by tRNA extraction is exposed to ionic strengths up to 600 mM NaCl at 4 degrees C, almost no modifications of the original native repeating structure are observed. However, when the incubation is carried out at 37 degrees C in 150, 300 or 400 mM NaCl, rearrangements of the native structure occur as indicated by micrococcal nuclease digestion and electron microscopic studies. Incubation of H1-depleted chromatin at 600 mM NaCl for 45 min at 37 degrees C induces, as for the native chromatin, a complete rearrangement characterized by the appearance of a 140-base-pair repeat superimposed on a high background upon digestion by micrococcal nuclease. It is suggested that these rearrangements are mediated by hydrophobic interactions between the histone cores and are prevented at ionic strengths lower than 500 mM by the presence of histone H1.