RESUMO
(2'S)-2'-Deoxy-2'-C-methyluridine and (2'R)-2'-deoxy-2'-C-methyluridine were incorporated in the 3'-overhang region of the sense and antisense strands and in positions 2 and 5 of the seed region of siRNA duplexes directed against Renilla luciferase, whereas (2'S)-2'-deoxy-2'-C-methylcytidine was incorporated in the 6-position of the seed region of the same constructions. A dual luciferase reporter assay in transfected HeLa cells was used as a model system to measure the IC50 values of 24 different modified duplexes. The best results were obtained by the substitution of one thymidine unit in the antisense 3'-overhang region by (2'S)- or (2'R)-2'-deoxy-2'-C-methyluridine, reducing IC50 to half of the value observed for the natural control. The selectivity of the modified siRNA was measured, it being found that modifications in positions 5 and 6 of the seed region had a positive effect on the ON/OFF activity.
Assuntos
RNA Interferente Pequeno/química , Uridina/análogos & derivados , Animais , Ensaios Enzimáticos , Células HeLa , Humanos , Concentração Inibidora 50 , Luciferases de Renilla/genética , Estabilidade de RNA , RNA Interferente Pequeno/síntese química , RNA Interferente Pequeno/genética , Renilla/enzimologia , Estereoisomerismo , Temperatura , Uridina/químicaRESUMO
The corticotropin-releasing factor (CRF)-producing neurons of the amygdala have been implicated in behavioral and physiological responses associated with fear, anxiety, stress, food intake and reward. To overcome the difficulties in identifying CRF neurons within the amygdala, a novel transgenic mouse line, in which the humanized recombinant Renilla reniformis green fluorescent protein (hrGFP) is under the control of the CRF promoter (CRF-hrGFP mice), was developed. First, the CRF-hrGFP mouse model was validated and the localization of CRF neurons within the amygdala was systematically mapped. Amygdalar hrGFP-expressing neurons were located primarily in the interstitial nucleus of the posterior limb of the anterior commissure, but also present in the central amygdala. Secondly, the marker of neuronal activation c-Fos was used to explore the response of amygdalar CRF neurons in CRF-hrGFP mice under different experimental paradigms. C-Fos induction was observed in CRF neurons of CRF-hrGFP mice exposed to an acute social defeat stress event, a fasting/refeeding paradigm or lipopolysaccharide (LPS) administration. In contrast, no c-Fos induction was detected in CRF neurons of CRF-hrGFP mice exposed to restraint stress, forced swimming test, 48-h fasting, acute high-fat diet (HFD) consumption, intermittent HFD consumption, ad libitum HFD consumption, HFD withdrawal, conditioned HFD aversion, ghrelin administration or melanocortin 4 receptor agonist administration. Thus, this study fully characterizes the distribution of amygdala CRF neurons in mice and suggests that they are involved in some, but not all, stress or food intake-related behaviors recruiting the amygdala.
Assuntos
Tonsila do Cerebelo/citologia , Tonsila do Cerebelo/fisiologia , Hormônio Liberador da Corticotropina/metabolismo , Neurônios/citologia , Neurônios/fisiologia , Proteínas de Anfíbios/genética , Proteínas de Anfíbios/metabolismo , Tonsila do Cerebelo/efeitos dos fármacos , Tonsila do Cerebelo/fisiopatologia , Animais , Dieta Hiperlipídica , Dominação-Subordinação , Ingestão de Alimentos/fisiologia , Jejum/fisiologia , Grelina/administração & dosagem , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Lipopolissacarídeos , Masculino , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Neurônios/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-fos/metabolismo , Receptor Tipo 4 de Melanocortina/antagonistas & inibidores , Receptor Tipo 4 de Melanocortina/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Renilla , Restrição Física , Estresse Psicológico/fisiopatologia , Natação/fisiologiaRESUMO
Calcified sclerites are common in many benthic marine invertebrates, and despite their widespread occurrence, little is known about their ecological roles. Previous studies suggested that the sclerite composition of coral colonies may be altered in response to environmental cues such as predation and water motion. Furthermore, larger sclerites are thought to be more effective than small ones in deterring predators, while small sclerites may provide greater stiffness and resistance to deformation. The present study compared the length of the sclerites of the sea pansy Renilla muelleri from three depths in Guanabara Bay in southeastern Brazil. Our results show that sclerites are larger in deep-water specimens than in those from shallow water. Field assays were conducted in which sclerites from sea pansies at three depths were incorporated into artificial foods and offered to a natural assemblage of fish. These assays demonstrate that sclerites from R. muelleri from all three depths significantly reduced consumption by generalist carnivorous fishes. We conclude that R. muelleri uses skeletal elements not only to give the body its form but also as a defense against biotic threats.