RESUMEN
Many animal life histories entail changing feeding ecology, but the molecular bases for these transitions are poorly understood. The amphibian tadpole is typically a growth and dispersal life-history stage. Tadpoles are primarily herbivorous, and they capitalize on growth opportunities to reach a minimum body size to initiate metamorphosis. During metamorphic climax, feeding declines, at which time the gastrointestinal (GI) tract remodels to accommodate the carnivorous diet of the adult frog. Here we show that anorexigenic hypothalamic feeding controls are absent in the tadpole, but develop during metamorphosis concurrent with the production of the satiety signal leptin. Before metamorphosis there is a large increase in leptin mRNA in fat tissue. Leptin receptor mRNA increased during metamorphosis in the preoptic area/hypothalamus, the key brain region involved with the control of food intake and metabolism. This corresponded with an increase in functional leptin receptor, as evidenced by induction of socs3 mRNA and phosphorylated STAT3 immunoreactivity, and suppression of feeding behaviour after injection of recombinant frog leptin. Furthermore, we found that immunoneutralization of leptin in tadpoles at metamorphic climax caused them to resume feeding. The absence of negative regulation of food intake in the tadpole allows the animal to maximize growth prior to metamorphosis. Maturation of leptin-responsive neural circuits suppresses feeding during metamorphosis to facilitate remodelling of the GI tract.
Asunto(s)
Proteínas Anfibias/metabolismo , Ingestión de Alimentos , Conducta Alimentaria , Hipotálamo/metabolismo , Leptina/fisiología , Xenopus laevis/fisiología , Tejido Adiposo/metabolismo , Proteínas Anfibias/genética , Animales , Larva/genética , Larva/fisiología , Leptina/genética , Metamorfosis Biológica/genética , Metamorfosis Biológica/fisiología , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores de Leptina/fisiología , Proteínas Recombinantes/farmacología , Proteína 3 Supresora de la Señalización de Citocinas/genética , Proteína 3 Supresora de la Señalización de Citocinas/metabolismo , Proteínas de Xenopus/genética , Proteínas de Xenopus/metabolismo , Xenopus laevis/genética , Xenopus laevis/crecimiento & desarrolloRESUMEN
Ghrelin is a potent orexigenic peptide implicated in appetite regulation in rodents. However, except for teleost fish, the involvement of ghrelin in the regulation of feeding in non-mammalian vertebrates has not been well studied. Anuran amphibian larvae feed and grow during the pre- and prometamorphic stages, but, thereafter they stop feeding as the metamorphic climax approaches. Therefore, orexigenic factors seem to play important roles in growing larvae. In the present study, we examined the effect of intraperitoneal (IP) or intracerebroventricular (ICV) administration of synthetic bullfrog ghrelin (n-octanoylated 28-amino acid form) on food intake in larvae at the prometamorphic stages. Cumulative food intake was significantly increased by IP (8 and 16pmol/g body weight (BW)) or ICV (0.5 and 1pmol/g BW) administration of ghrelin during a 15-min observation period. The orexigenic action of ghrelin at 8pmol/g BW (IP) or at 0.5pmol/g BW (ICV) was blocked by treatment with a growth hormone secretagogue-receptor antagonist, [D-Lys(3)]GHRP-6 at 80pmol/g BW (IP) or at 5pmol/g BW (ICV). We then investigated the effect of feeding status on expression levels of the ghrelin transcript in the hypothalamus and gastrointestinal tract. Ghrelin mRNA levels in both were decreased 15 and 60min after feeding. These results indicate that ghrelin acts as an orexigenic factor in bullfrog larvae.
Asunto(s)
Proteínas Anfibias/administración & dosificación , Estimulantes del Apetito/administración & dosificación , Ingestión de Energía/efectos de los fármacos , Ghrelina/administración & dosificación , Proteínas Anfibias/genética , Proteínas Anfibias/metabolismo , Animales , Tracto Gastrointestinal/metabolismo , Expresión Génica , Ghrelina/metabolismo , Hipotálamo/metabolismo , Inyecciones Intraperitoneales , Inyecciones Intraventriculares , Larva/efectos de los fármacos , Larva/metabolismo , Rana catesbeianaRESUMEN
Solanum tuberosum plants were transformed with three genetic constructions expressing the Nicotiana tabacum AP24 osmotine, Phyllomedusa sauvagii dermaseptin and Gallus gallus lysozyme, and with a double-transgene construction expressing the AP24 and lysozyme sequences. Re-transformation of dermaseptin-transformed plants with the AP24/lysozyme construction allowed selection of plants simultaneously expressing the three transgenes. Potato lines expressing individual transgenes or double- and triple-transgene combinations were assayed for resistance to Erwinia carotovora using whole-plant and tuber infection assays. Resistance levels for both infection tests compared consistently for most potato lines and allowed selection of highly resistant phenotypes. Higher resistance levels were found in lines carrying the dermaseptin and lysozyme sequences, indicating that theses proteins are the major contributors to antibacterial activity. Similar results were obtained in tuber infection tests conducted with Streptomyces scabies. Plant lines showing the higher resistance to bacterial infections were challenged with Phytophthora infestans, Rhizoctonia solani and Fusarium solani. Considerable levels of resistance to each of these pathogens were evidenced employing semi-quantitative tests based in detached-leaf inoculation, fungal growth inhibition and in vitro plant inoculation. On the basis of these results, we propose that stacking of these transgenes is a promising approach to achieve resistance to both bacterial and fungal pathogens.
Asunto(s)
Proteínas Anfibias/genética , Péptidos Catiónicos Antimicrobianos/genética , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Solanum tuberosum/genética , Proteínas Anfibias/metabolismo , Animales , Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/genética , Pollos/genética , Hongos/genética , Regulación de la Expresión Génica de las Plantas , Muramidasa/genética , Muramidasa/metabolismo , Pectobacterium carotovorum/patogenicidad , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/metabolismo , Solanum tuberosum/microbiología , Nicotiana/genéticaRESUMEN
Retinoic acid receptor beta 2 (RARß2) has been proposed as an important receptor mediating retinoid-induced axonal growth and regeneration in developing mammalian spinal cord and brain. In urodele amphibians, organisms capable of extensive central nervous system (CNS) regeneration as adults, this receptor had not been isolated, nor had its function been characterized. We have cloned a full-length RARß2 cDNA from adult newt CNS. This receptor, NvRARß2, is expressed in various adult organs capable of regeneration, including the spinal cord. Interestingly, both the NvRARß2 mRNA and protein are up-regulated during the first 2 weeks after amputation of the tail, primarily in the ependymoglial and meningeal tissues near the rostral cut surface of the cord. Treatment with LE135, a RARß-selective antagonist, caused a significant inhibition of ependymal outgrowth and a decrease in tail regenerate length. These data support an early role for this receptor in caudal spinal cord and tail regeneration in this amphibian.
Asunto(s)
Proteínas Anfibias/biosíntesis , Regulación de la Expresión Génica/fisiología , Receptores de Ácido Retinoico/biosíntesis , Regeneración/fisiología , Médula Espinal/fisiología , Cola (estructura animal)/fisiología , Proteínas Anfibias/antagonistas & inhibidores , Proteínas Anfibias/genética , Animales , Clonación Molecular , ADN Complementario/genética , ADN Complementario/metabolismo , Dibenzazepinas/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Notophthalmus viridescens , Especificidad de Órganos/efectos de los fármacos , Especificidad de Órganos/fisiología , Ratas , Receptores de Ácido Retinoico/antagonistas & inhibidores , Receptores de Ácido Retinoico/genética , Regeneración/efectos de los fármacos , Médula Espinal/patología , Traumatismos Vertebrales/genética , Traumatismos Vertebrales/metabolismo , Traumatismos Vertebrales/patología , Cola (estructura animal)/lesiones , Cola (estructura animal)/patologíaRESUMEN
In anurans, recognition of species-specific acoustic signals is essential to finding a mate. In many species, behavioral tests have elucidated which acoustic features contribute to species recognition, but the mechanisms by which the brain encodes these species-specific signal components are less well understood. The túngara frog produces a `whine' mating call that is characterized by a descending frequency sweep. However, much of the signal is unnecessary for recognition, as recognition behavior can be triggered by a descending two-tone step that mimics the frequency change in a portion of the whine. To identify the brain regions that contribute to species recognition in the túngara frog, we exposed females to a full-spectrum whine, a descending two-tone step that elicits recognition, the reversed two-tone step that does not elicit recognition, or no sound, and we measured expression of the neural activity-dependent gene egr-1 in the auditory brainstem and thalamus. We found that the behavioral relevance of the stimuli was the best predictor of egr-1 expression in the laminar nucleus of the torus semicircularis but not elsewhere. That is, the laminar nucleus responded more to the whine and the two-tone step that elicits recognition than to the reversed two-tone step. In contrast, in other brainstem and thalamic nuclei, whines induced egr-1 expression but tones did not. These data demonstrate that neural responses in the laminar nucleus correspond to behavioral responses of females and they suggest that the laminar nucleus may act as a feature detector for the descending frequencies characteristic of conspecific calls.
Asunto(s)
Anuros/fisiología , Conducta Sexual Animal , Vocalización Animal , Estimulación Acústica , Proteínas Anfibias/genética , Animales , Anuros/metabolismo , Tronco Encefálico/metabolismo , Proteína 1 de la Respuesta de Crecimiento Precoz/genética , Femenino , Expresión Génica , Especificidad de la Especie , Tálamo/metabolismoRESUMEN
LAR-like receptor protein tyrosine phosphatases (RPTPs), which are abundantly expressed in the nervous systems of most if not all bilaterian animals thus far examined, have been implicated in regulating a variety of critical neuronal processes. These include neuronal pathfinding, adhesion and synaptogenesis during development and, in adult mammals, neuronal regeneration. Here we explored a possible role of a LAR-like RPTP (HmLAR1) in response to mechanical trauma in the adult nervous system of the medicinal leech. In situ hybridization and QPCR analyses of HmLAR1 expression in individual segmental ganglia revealed a significant up-regulation in receptor expression following CNS injury, both in situ and following a period in vitro. Furthermore, we observed up-regulation in the expression of the leech homologue of the Abelson tyrosine kinase, a putative signaling partner to LAR receptors, but not among other tyrosine kinases. The effects on neuronal regeneration were assayed by comparing growth across a nerve crush by projections of individual dorsal P neurons (P(D)) following single-cell injection of interfering RNAs against the receptor or control RNAs. Receptor RNAi led to a significant reduction in HmLAR1 expression by the injected cells and resulted in a significant decrease in sprouting and regenerative growth at the crush site relative to controls. These studies extend the role of the HmLARs from leech neuronal development to adult neuronal regeneration and provide a platform to investigate neuronal regeneration and gene regulation at the single cell level.
Asunto(s)
Proteínas Anfibias/metabolismo , Sistema Nervioso Central/metabolismo , Sanguijuelas/metabolismo , Regeneración Nerviosa/fisiología , Neuronas/metabolismo , Proteínas Tirosina Fosfatasas/metabolismo , Proteínas Anfibias/genética , Animales , Sistema Nervioso Central/lesiones , Expresión Génica , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Hibridación Fluorescente in Situ , Compresión Nerviosa , Proteínas Tirosina Fosfatasas/genética , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Dermaseptin B1 is a potent cationic antimicrobial peptide found in skin secretions of the arboreal frog Phyllomedusa bicolor. A synthetic derivative of dermaseptin B1, MsrA2 (N-Met-dermaseptin B1), elicited strong antimicrobial activities against various phytopathogenic fungi and bacteria in vitro. To assess its potential for plant protection, MsrA2 was expressed at low levels (1-5 microg/g of fresh tissue) in the transgenic potato (Solanum tuberosum L.) cv. Desiree. Stringent challenges of these transgenic potato plants with a variety of highly virulent fungal phytopathogens--Alternaria, Cercospora, Fusarium, Phytophthora, Pythium, Rhizoctonia and Verticillium species--and with the bacterial pathogen Erwinia carotovora demonstrated that the plants had an unusually broad-spectrum and powerful resistance to infection. MsrA2 profoundly protected both plants and tubers from diseases such as late blight, dry rot and pink rot and markedly extended the storage life of tubers. Due to these properties in planta, MsrA2 is proposed as an ideal antimicrobial peptide candidate to significantly increase resistance to phytopathogens and improve quality in a variety of crops worldwide with the potential to obviate fungicides and facilitate storage under difficult conditions.
Asunto(s)
Proteínas Anfibias/metabolismo , Péptidos Catiónicos Antimicrobianos/metabolismo , Inmunidad Innata/genética , Enfermedades de las Plantas/microbiología , Solanum tuberosum/genética , Solanum tuberosum/metabolismo , Proteínas Anfibias/genética , Animales , Péptidos Catiónicos Antimicrobianos/genética , Anuros , Northern Blotting , Clonación Molecular , Hongos/fisiología , Técnicas de Transferencia de Gen , Oligonucleótidos , Plantas Modificadas GenéticamenteRESUMEN
Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) was purified from two amphibian species, Xenopus laevis and Pleurodeles waltl. Comparative studies revealed that the two proteins differ by their subunit molecular masses, pI values and V8 digested peptide maps. The effect of zinc, cadmium and copper ions on GAPDH enzymatic activity has been examined in vitro. A time, metal concentration and metal type dependent inhibition was observed for both enzymes. X. laevis and P. waltl GAPDHs exhibit a much greater sensitivity to copper than to cadmium or zinc ions. Different half-lives and differential sensitivity to various metals was observed between the two enzymes with P. waltl GAPDH being remarkably tolerant to cadmium ions compared to the X. laevis enzyme. In order to understand the differential sensitivity of the two enzymes to metals, we produced 3D models of both X. laevis and P. waltl GAPDH structures based upon known 3D structures of GAPDHs from other species. This necessitated, in a first step, to clone a 900 bp cDNA fragment encoding the nearly full-length P. waltl GAPDH. Spatial motif searches on the homology models indicated potential metal binding sites involving cysteine and histidine residues outside the catalytic sites, existing only in either the X. laevis or the P. waltl GAPDH sequences.