RESUMEN
Regulation of eukaryotic cell cycle progression requires sequential activation and inactivation of cyclin-dependent kinases (CDKs). Activation of the cyclin B-cdc2 kinase complex is a pivotal step in mitotic initiation and the tyrosine kinase Wee1 is a key regulator of cell cycle sequence during G2/M transition and inhibits mitotic entry by phosphorylating the inhibitory tyrosine 15 on the cdc2 M-phase-inducing kinase. Wee1 degradation is essential for the exit from the G2 phase. In trypanosomatids, little is known about the genes that regulate cyclin B-cdc2 complexes at the G2/M transition of their cell cycle. Although canonical tyrosine kinases are absent in the genome of trypanosomatids, phosphorylation on protein tyrosine residues has been reported in Trypanosoma brucei. Here, we characterized a Wee1-like protein kinase gene from T. brucei. Expression of TbWee1 in a Schizosaccharomyces pombe strain null for Wee1 inhibited cell division and caused cell elongation. This demonstrates the lengthening of G2, which provided cells with extra time to grow before dividing. The Wee1-like protein kinase was expressed in the procyclic and bloodstream proliferative slender forms of T. brucei and the role of Wee1 in cell cycle progression was analyzed by generating RNA interference cell lines. In the procyclic form of T. brucei, the knock-down of TbWee1 expression by RNAi led to inhibition of parasite growth. Abnormal phenotypes showing an increase in the percentage of cells with 1N0K, 0N1K and 2N1K were observed in these RNAi cell lines. Using parasites with a synchronized cell cycle, we demonstrated that TbWee1 is linked to the G2/M phase. We also showed that TbWee1 is an essential gene necessary for proper cell cycle progression and parasite growth in T. brucei. Our results provide evidence for the existence of a functional Wee1 in T. brucei with a potential role in cell division at G2/M.
Asunto(s)
Proteínas Tirosina Quinasas/genética , Trypanosoma brucei brucei/citología , Trypanosoma brucei brucei/genética , Secuencia de Aminoácidos , División Celular , Bases de Datos Genéticas , Regulación hacia Abajo , Fase G2 , Genoma de Protozoos/genética , Datos de Secuencia Molecular , Fenotipo , Fosforilación , Proteínas Tirosina Quinasas/química , Proteínas Tirosina Quinasas/metabolismo , Schizosaccharomyces/genética , Homología de Secuencia de Ácido Nucleico , Análisis de Supervivencia , Trypanosoma brucei brucei/enzimología , Trypanosoma brucei brucei/crecimiento & desarrolloRESUMEN
It is expected that the next generation of biotech crops displaying enhanced quality traits with benefits to both farmers and consumers will have a better acceptance than first generation biotech crops and will improve public perception of genetic engineering. This will only be true if they are proven to be as safe as traditionally bred crops. In contrast with the first generation of biotech crops where only a single trait is modified, the next generation of biotech crops will add a new level of complexity inherent to the mechanisms underlying their output traits. In this study, a comprehensive evaluation of the comparative safety approach on a quality-improved biotech crop with metabolic modifications is presented. Three genetically engineered potato lines with silenced polyphenol oxidase (Ppo) transcripts and reduced tuber browning were characterized at both physiological and molecular levels and showed to be equivalent to wild-type (WT) plants when yield-associated traits and photosynthesis were evaluated. Analysis of the primary metabolism revealed several unintended metabolic modifications in the engineered tubers, providing evidence for potential compositional inequivalence between transgenic lines and WT controls. The silencing construct sequence was in silico analysed for potential allergenic cross-reactivity, and no similarities to known allergenic proteins were identified. Moreover, in vivo intake safety evaluation showed no adverse effects in physiological parameters. Taken together, these results provide the first evidence supporting that the safety of next generation biotech crops can be properly assessed following the current evaluation criterion, even if the transgenic and WT crops are not substantially equivalent.
Asunto(s)
Inocuidad de los Alimentos , Ingeniería Genética , Solanum tuberosum/genética , Alérgenos/análisis , Animales , Catecol Oxidasa/genética , Biología Computacional , Femenino , Silenciador del Gen , Ratones , Ratones Endogámicos BALB C , FotosíntesisRESUMEN
Dione juno and Agraulis vanillae are very common butterflies in natural gardens in South America, and also bred worldwide. In addition, larvae of these butterflies are considered as pests in crops of Passiflora spp. For these reasons, it is important to identify and describe pathogens of these species, both for preservation purposes and for use in pest control. Baculoviridae is a family of insect viruses that predominantly infect species of Lepidoptera and are used as bioinsecticides. Larvae of D. juno and A. vanillae exhibiting symptoms of baculovirus infection were examined for the presence of baculoviruses by PCR and transmission electron microscopy. Degenerate primers were designed and used to amplify partial sequences from the baculovirus p74, cathepsin, and chitinase genes, along with previously designed primers for amplification of lef-8, lef-9, and polh. Sequence data from these six loci, along with ultrastructural observations on occlusion bodies isolated from the larvae, confirmed that the larvae were infected with nucleopolyhedroviruses from genus Alphabaculovirus. The NPVs from the two different larval hosts appear to be variants of the same, previously undescribed baculovirus species. Phylogenetic analysis of the sequence data placed these NPVs in Alphabaculovirus group I/clade 1b.
Asunto(s)
Mariposas Diurnas/virología , Nucleopoliedrovirus/aislamiento & purificación , Animales , ADN Viral/genética , Cuerpos de Inclusión/virología , Larva/virología , Nucleopoliedrovirus/genética , Control Biológico de Vectores , América del SurRESUMEN
Sensory analysis studies are critical in the development of quality enhanced crops, and may be an important component in the public acceptance of genetically modified foods. It has recently been established that odor preferences are shared between humans and mice, suggesting that odor exploration behavior in mice may be used to predict the effect of odors in humans. We have previously found that mice fed diets supplemented with engineered nonbrowning potatoes (-PPO) consumed more potato than mice fed diets supplemented with wild-type potatoes (WT). This prompted us to explore a possible role of potato odor in mice preference for nonbrowning potatoes. Taking advantage of two well established neuroscience paradigms, the "open field test" and the "nose-poking preference test", we performed experiments where mice exploration behavior was monitored in preference assays on the basis of olfaction alone. No obvious preference was observed towards -PPO or WT lines when fresh potato samples were tested. However, when oxidized samples were tested, mice consistently investigated -PPO potatoes more times and for longer periods than WT potatoes. Congruently, humans discriminated WT from -PPO samples with a considerably better performance when oxidized samples were tested than when fresh samples were tested in blind olfactory experiments. Notably, even though participants ranked all samples with an intermediate level of pleasantness, there was a general consensus that the -PPO samples had a more intense odor and also evoked the sense-impression of a familiar vegetable more often than the WT samples. Taken together, these findings suggest that our previous observations might be influenced, at least in part, by differential odors that are accentuated among the lines once oxidative deterioration takes place. Additionally, our results suggest that nonbrowning potatoes, in addition to their extended shelf life, maintain their odor quality for longer periods of time than WT potatoes. To our knowledge this is the first report on the use of an animal model applied to the sensory analysis of a transgenic crop.
Asunto(s)
Odorantes/análisis , Tubérculos de la Planta/metabolismo , Plantas Modificadas Genéticamente/metabolismo , Solanum tuberosum/metabolismo , Análisis de Varianza , Animales , Catecol Oxidasa/genética , Catecol Oxidasa/metabolismo , Color , Conducta Exploratoria/fisiología , Femenino , Análisis de los Alimentos , Preferencias Alimentarias/fisiología , Humanos , Ratones , Ratones Endogámicos BALB C , Percepción Olfatoria/fisiología , Oxidación-Reducción , Tubérculos de la Planta/química , Tubérculos de la Planta/genética , Plantas Modificadas Genéticamente/genética , Olfato/fisiología , Solanum tuberosum/genéticaRESUMEN
BACKGROUND: Anticarsia gemmatalis is a pest in South America's soybean crops, which could be controlled by the Multinucleopolyhedrovirus of A. gemmatalis (AgMNPV). Currently, its commercial production is based on infected larvae. However, the possibility of using modified baculoviruses in Integrated Pest Management programs has stimulated an interest to develop alternative multiplication processes. This study evaluated the AgMNPV production in UFL-Ag-286 cells previously deprived Fetal Bovine Serum. RESULTS: Culture media containing 1% FBS during the previous 48 hours achieved a synchronized condition where 90% of cells were found in G0/G1 stage, showing the presence of non-filamentous actin. All characteristics were estimated from cellular viability tests, cell actin detection trials and flow cytometer cell cycle analysis. AgMNPV production was tested by transcript studies and budded viruses (BVs) and occlusion bodies (OBs) yield quantitation. Results showed that the productivity in FBS deprived cells was 9.8 times more in BVs and 3.8 times more in OBs with respect to non-treated cells. CONCLUSIONS: UFL-Ag-286 cells previously deprived in FBS shown to be a better host for AgMNPV propagation, increasing the useful for both in vitro bioinsecticide production and applications such as recombinant protein expression or gene delivery.
Asunto(s)
Medios de Cultivo/química , Nucleopoliedrovirus/fisiología , Cultivo de Virus/métodos , Animales , Bovinos , Línea Celular , Medio de Cultivo Libre de Suero , Mariposas Nocturnas/virología , Suero/químicaRESUMEN
PURPOSE: We developed and characterized an orthotopic invasive bladder tumor model. MATERIAL AND METHODS: The MB49-I invasive bladder tumor cell line was obtained after 13 consecutive in vivo passages of primary tumor obtained by subcutaneous inoculation of MB49 bladder tumor cells in C57Bl/6J male mice. RESULTS: MB49-I tumor local invasiveness, tumor weight and spontaneous metastatic capacity were higher than in MB49 tumors. In MB49-I bladder tumors increased vimentin was observed, suggesting epithelial mesenchymal transition. In vitro the MB49-I cell line showed higher invasive properties associated with an increase in cathepsin B, metalloproteinase 9 and urokinase-type plasminogen activator proteolytic activities. Orthotopic bladder tumors induced by electrocautery of the bladder wall and subsequent instillation of MB49 and MB49-I bladder cancer cells generated superficial and invasive bladder tumors, respectively. CONCLUSIONS: The new murine bladder model described resembles human bladder disease, making it a useful tool for studying the molecular mechanisms of tumor progression and metastasis, and assaying antimetastatic and anti-invasive agents.
Asunto(s)
Catepsina B/fisiología , Modelos Animales de Enfermedad , Neoplasias de la Vejiga Urinaria/patología , Animales , Línea Celular Tumoral , Masculino , Ratones , Ratones Endogámicos C57BL , Invasividad NeoplásicaRESUMEN
Currently, to our knowledge, there are no continuous cell lines derived from estrogen dependent, tamoxifen sensitive spontaneous mouse mammary carcinomas. We describe here the establishment and characterization of a cell line derived from the M05 mouse mammary tumor, LM05-Mix, composed of both an epithelial and a fibroblastic component. From it the respective epithelial LM05-E and fibroblastic LM05-F cell lines were generated by limiting dilution. Immunofluorescence studies confirmed that the epithelial cells were positive for E-cadherin, cytokeratins and vimentin whereas the fibroblastic cells were negative for the epithelial markers and positive for alpha-smooth muscle actin and vimentin. Both cell types expressed estrogen and progesterone receptors, although only the epithelial LM05-E cells were stimulated by estradiol and inhibited by tamoxifen. In the bicellular LM05-Mix cell line estradiol proved to stimulate cell proliferation whereas the response to tamoxifen was dependent on confluency and the degree of epithelial-fibroblastic interactions. The presence of membrane estrogen receptors in both cell types was suggested by the achievement of non-genomic responses to short treatments with estradiol, leading to the phosphorylation of ERK1/2. Finally, cytogenetic studies suggest that these two cell types represent independent cell populations within the tumor and would not be the result of an epithelial-mesenchymal transition. This model presents itself as a valuable alternative for the study of estrogen responsiveness and tamoxifen resistance in the context of epithelial-stromal interactions.
Asunto(s)
Línea Celular Tumoral/citología , Células Epiteliales/citología , Fibroblastos/citología , Neoplasias Mamarias Experimentales/metabolismo , Ratones , Animales , Apoptosis/fisiología , Western Blotting , Línea Celular Tumoral/metabolismo , Resistencia a Antineoplásicos/fisiología , Células Epiteliales/metabolismo , Estradiol/farmacología , Estrógenos/farmacología , Femenino , Fibroblastos/metabolismo , Técnica del Anticuerpo Fluorescente , Hibridación Fluorescente in Situ , Etiquetado Corte-Fin in Situ , Técnicas In Vitro , Neoplasias Mamarias Experimentales/genética , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismo , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Moduladores Selectivos de los Receptores de Estrógeno/farmacología , Tamoxifeno/farmacología , Translocación GenéticaRESUMEN
Currently, an in vivo spontaneous model of estrogen dependent, tamoxifen sensitive breast cancer does not exist. We show here the characterization of the M05 mammary tumor that appeared spontaneously in a 1-year-old virgin female BALB/c mouse in our animal facility. The M05 tumor is a semi-differentiated adenocarcinoma that expresses estrogen and progesterone receptors. When it was transplanted to either male or ovariectomized female mice it did not grow. Moreover, ovariectomy or treatment with tamoxifen of tumor bearing mice led to a halt in tumor growth. Treatment of ovariectomized mice that had been inoculated with the M05 tumor showed that only estradiol, but not progesterone, promoted the re-growth of the tumor. Finally, after passage nine, tumor growth was achieved in male and ovariectomized female mice suggesting that the tumor had progressed to hormone independence. However, like often found in the clinic, expression of estrogen and progesterone receptors was maintained. This model mimics the biology of estrogen receptor positive breast cancer in humans and presents itself as an invaluable tool for the study of endocrine resistance in a physiologically relevant setting.
Asunto(s)
Neoplasias Mamarias Experimentales/tratamiento farmacológico , Tamoxifeno/uso terapéutico , Adenocarcinoma/tratamiento farmacológico , Adenocarcinoma/inmunología , Adenocarcinoma/patología , Animales , División Celular , Femenino , Inmunocompetencia , Masculino , Neoplasias Mamarias Experimentales/inmunología , Neoplasias Mamarias Experimentales/patología , Ratones , Ratones Endogámicos BALB C , Trasplante de Neoplasias/métodos , Trasplante de Neoplasias/patología , Ovariectomía , Receptores de Estrógenos/metabolismo , Receptores de Progesterona/metabolismoRESUMEN
P74 is a protein encoded in the genome of baculoviruses, associated with the envelopes of occluded virus. Its presence proved to be essential for per os infection. In first place, in this work we designed two universal primers to amplify a sequence region of the p74 ORF in baculoviruses from different classification groups. Then, by the use of these amplicons we obtained the complete sequence of the p74 locus from two isolates of AgMNPV, 2D (Brazil) and SF (Argentina). In the flanking regions we determined the complete sequence of p10 gene and a portion of p26 gene. Comparing both p74 sequence data (ORFs of 1935 bp) we found fifteen nucleotide changes that result in six amino acid changes. Comparisons of AgMNPV p74s with other baculovirus homologous genes indicate a close relationship with other group I Nucleopolyhedrovirus, in particular CfDEFNPV. These results were based on ORF sequence, amino acid sequence and gene order. The predictive studies about secondary structure and hydrophobic index point at six regions potentially associated to its function or native conformation. Finally, the detection of p74 mRNA after virus DNA replication confirms a late expression pattern.
Asunto(s)
Genes Virales , Nucleopoliedrovirus/genética , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Clonación Molecular , ADN Viral/genética , Genoma Viral , Lepidópteros/virología , Datos de Secuencia Molecular , Nucleopoliedrovirus/clasificación , Nucleopoliedrovirus/aislamiento & purificación , Filogenia , Reacción en Cadena de la Polimerasa , Transcripción Genética , Proteínas del Envoltorio Viral/química , Proteínas del Envoltorio Viral/genéticaRESUMEN
Mice bearing LP07 lung adenocarcinoma show some characteristics that are similar to those present in patients with NSCLC. LP07 tumor-bearing mice develop the paraneoplastic syndromes of cachexia, leukocytosis and hypercalcemia. These symptoms may be partly due to a systemic inflammatory response. Our aim was to determine if treatment with NSAIDs would lower tumor and metastasis growth and their accompanying syndromes. The nonselective COX inhibitor indomethacin and the selective COX-2 inhibitor celecoxib reduced tumor growth and metastasis outcome in s.c. LP07 tumor-bearing mice. Both drugs also inhibited the development of leukocytosis and the weight loss associated with LP07 progression. Serum levels of the inflammatory cytokines IL-1beta and IL-6, mediators of cachexia, were modulated by NSAIDs. Inhibition of in vitro migration and invasion and reduction in angiogenesis were attained when cells were treated with either indomethacin or celecoxib. MMP-9 activity was also reduced in conditioned media from LP07 cells treated with celecoxib. These data suggest that several processes implicated in tumor progression can be modulated with NSAID treatment. Improvement in performance status through modulation of cachexia may offer a possibility for combining anti-inflammatory treatments with more aggressive therapies.