RESUMEN
Citrus canker is a major disease caused by Xanthomonas citri pv. citri. Snakin-1 is an antimicrobial peptide, which was previously shown to be effective against different bacterial and fungal diseases in potato, wheat and lettuce when expressed in transgenic plants. We generated transgenic Citrange Troyer citrus rootstocks constitutively expressing this peptide and 5 different transgenic lines were challenged against virulent X. citri isolates. Challenge assays conducted in vitro using detached leaves and in planta by infiltration revealed a significant reduction of the number and size of canker lesions in some of the transgenic lines.
Asunto(s)
Antiinfecciosos , Citrus , Solanum tuberosum , Xanthomonas , Enfermedades de las Plantas , Solanum tuberosum/genética , Xanthomonas/genéticaRESUMEN
The incidence of chronic liver disease is constantly increasing, owing to the obesity epidemic. However, the causes and mechanisms of inflammation-mediated liver damage remain poorly understood. Endoplasmic reticulum (ER) stress is an initiator of cell death and inflammatory mechanisms. Although obesity induces ER stress, the interplay between hepatic ER stress, NLRP3 inflammasome activation and hepatocyte death signaling has not yet been explored during the etiology of chronic liver diseases. Steatosis is a common disorder affecting obese patients; moreover, 25% of these patients develop steatohepatitis with an inherent risk for progression to hepatocarcinoma. Increased plasma LPS levels have been detected in the serum of patients with steatohepatitis. We hypothesized that, as a consequence of increased plasma LPS, ER stress could be induced and lead to NLRP3 inflammasome activation and hepatocyte death associated with steatohepatitis progression. In livers from obese mice, administration of LPS or tunicamycin results in IRE1α and PERK activation, leading to the overexpression of CHOP. This, in turn, activates the NLRP3 inflammasome, subsequently initiating hepatocyte pyroptosis (caspase-1, -11, interleukin-1ß secretion) and apoptosis (caspase-3, BH3-only proteins). In contrast, the LPS challenge is blocked by the ER stress inhibitor TUDCA, resulting in: CHOP downregulation, reduced caspase-1, caspase-11, caspase-3 activities, lowered interleukin-1ß secretion and rescue from cell death. The central role of CHOP in mediating the activation of proinflammatory caspases and cell death was characterized by performing knockdown experiments in primary mouse hepatocytes. Finally, the analysis of human steatohepatitis liver biopsies showed a correlation between the upregulation of inflammasome and ER stress markers, as well as liver injury. We demonstrate here that ER stress leads to hepatic NLRP3 inflammasome pyroptotic death, thus contributing as a novel mechanism of inflammation-mediated liver injury in chronic liver diseases. Inhibition of ER-dependent inflammasome activation and cell death pathways may represent a potential therapeutic approach in chronic liver diseases.
Asunto(s)
Proteínas Portadoras/genética , Proteínas Portadoras/metabolismo , Estrés del Retículo Endoplásmico/genética , Hepatocitos/metabolismo , Inflamasomas/metabolismo , Lipopolisacáridos/metabolismo , Hepatopatías/genética , Obesidad/complicaciones , Animales , Muerte Celular , Enfermedad Crónica , Humanos , Hepatopatías/metabolismo , Ratones , Proteína con Dominio Pirina 3 de la Familia NLR , Transducción de SeñalRESUMEN
KEY MESSAGE: The novel sunflower gene HaGLP1 is the first germin-like protein characterized from the family Asteraceae. It alters the host redox status and confers protection against Sclerotinia sclerotiorum and Rhizoctonia solani. Germin-like proteins (GLPs) are a large, diverse and ubiquitous family of plant glycoproteins belonging to the Cupin super family. These proteins have been widely studied because of their diverse roles in important plant processes, including defence. The novel sunflower gene HaGLP1 encodes the first germin-like protein characterized from the family Asteraceae. To analyse whether constitutive in vivo expression of the HaGLP1 gene may lead to disease tolerance, we developed transgenic Arabidopsis plants that were molecularly characterized and biologically assessed after inoculation with Sclerotinia sclerotiorum or Rhizoctonia solani. HaGLP1 expression in Arabidopsis plants conferred tolerance to S. sclerotiorum at the first stages of disease and interfered with R. solani infection, thus giving rise to significant protection against the latter. Furthermore, HaGLP1 expression in Arabidopsis plants elevated endogenous ROS levels. HaGLP1-induced tolerance does not appear to be related to a constitutive induction of the plant defence or the ROS-related genes examined here. In conclusion, our data suggest that HaGLP1 is an interesting candidate for the engineering of plants with increased fungal tolerance and that this gene could also be useful for the selection of naturally overexpressing sunflower genotypes for conventional breeding purposes.
Asunto(s)
Arabidopsis/metabolismo , Arabidopsis/microbiología , Ascomicetos/fisiología , Glicoproteínas/metabolismo , Helianthus/metabolismo , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente/microbiología , Especies Reactivas de Oxígeno/metabolismo , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas/genética , Regulación de la Expresión Génica de las Plantas/fisiología , Glicoproteínas/genética , Helianthus/genética , Enfermedades de las Plantas/genética , Enfermedades de las Plantas/microbiología , Proteínas de Plantas/genética , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/metabolismoRESUMEN
Melanin-concentrating hormone (MCH) is a cyclic peptide highly conserved in vertebrates and was originally identified as a skin-paling factor in Teleosts. In fishes, MCH also participates in the regulation of the stress-response and feeding behaviour. Mammalian MCH is a hypothalamic neuropeptide that displays multiple functions, mostly controlling feeding behaviour and energy homeostasis. Transgenic mouse models and pharmacological studies have shown the importance of the MCH system as a potential target in the treatment of appetite disorders and obesity as well as anxiety and psychiatric diseases. Two G-protein-coupled receptors (GPCRs) binding MCH have been characterized so far. The first, named MCH-R1 and also called SLC1, was identified through reverse pharmacology strategies by several groups as a cognate receptor of MCH. This receptor is expressed at high levels in many brain areas of rodents and primates and is also expressed in peripheral organs, albeit at a lower rate. A second receptor, designated MCH-R2, exhibited 38% identity to MCH-R1 and was identified by sequence analysis of the human genome. Interestingly, although MCH-R2 orthologues were also found in fishes, dogs, ferrets and non-human primates, this MCH receptor gene appeared either lacking or non-functional in rodents and lagomorphs. Both receptors are class I GPCRs, whose main roles are to mediate the actions of peptides and neurotransmitters in the central nervous system. However, examples of action of MCH on neuronal and non-neuronal cells are emerging that illustrate novel MCH functions. In particular, the functionality of endogenously expressed MCH-R1 has been explored in human neuroblastoma cells, SK-N-SH and SH-SY5Y cells, and in non-neuronal cell types such as the ependymocytes. Indeed, we have identified mitogen-activated protein kinase (MAPK)-dependent or calcium-dependent signalling cascades that ultimately contributed to neurite outgrowth in neuroblastoma cells or to modulation of ciliary beating in ependymal cells. The putative role of MCH on cellular shaping and plasticity on one side and volume transmission on the other must be now considered.
RESUMEN
Populations of Western countries are severely deficient in omega-3 intake, both in the form of alpha-linolenic acid (ALA) and the Long Chain derivatives (LC-n-3), Eicosa-Pentaenoic-Acid and Docosa-Hexaenoic-Acid. Omega-3 insufficiency is a risk factor for cardiovascular and cerebral diseases such as coronary heart disease and stroke. Stroke is a major cause of mortality and morbidity, and induces a significant socioeconomic cost and a marked increase in patient/family burden. To date, preventive treatments and neuroprotective drugs identified in preclinical studies failed in clinical trials, in part because of an inability to tolerate drugs at neuroprotective concentrations. Therefore testing alternative protective strategies, such as functional foods/nutraceuticals, are of considerable interest. We have previously demonstrated that a single injection of ALA reduced ischemic damage by limiting glutamate-mediated neuronal death, whereas repeated injections displayed additive protective benefits as a result of increased neurogenesis, synaptogenesis and neurotrophin expression. Because intravenous injections are not a suitable long-term strategy in humans, the present study investigated the effect of ALA supplementation by an experimental diet containing rapeseed oil (RSO, a rich source of ALA) as the only source of lipids for stroke prevention. We tested several experimental diets which included 5, 10, and 20% RSO-enriched diet and feeding paradigms (fresh diet was provided once or twice a week for 4 or 6 weeks). Our results showed that ALA supplemented diets are more sensitive to lipid peroxidation than a regular chow diet. Because the diet affected feeding behavior and animal growth, we defined concrete guidelines to investigate the effect of omega-3 supplementation on neuropathology. Among the different sets of experiments, animals fed with 10% and 20% RSO-enriched diet displayed a reduced mortality rate, infarct size and increased probability of spontaneous reperfusion in the post-ischemic period. In addition, a drastic reduction of lipid peroxidation levels was observed in the ischemic brain of RSO-fed animals. Overall, our findings provide new insights into the potential of employing rapeseed oil as a functional food/nutraceutical aiding in stroke prevention and protection.
Asunto(s)
Grasas Insaturadas en la Dieta/administración & dosificación , Suplementos Dietéticos , Aceites de Plantas/administración & dosificación , Accidente Cerebrovascular/prevención & control , Ácido alfa-Linolénico/administración & dosificación , Animales , Ácidos Grasos Monoinsaturados , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Masculino , Ratones , Ratones Endogámicos C57BL , Aceite de Brassica napus , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/patologíaRESUMEN
Accumulating evidence show that chemokines can modulate the activity of neurons through various mechanisms. Recently, we demonstrated that CCR2, the main receptor for the chemokine CCL2, is constitutively expressed in dopamine neurons in the rat substantia nigra. Here we show that unilateral intranigral injections of CCL2 (50 ng) in freely moving rats increase extracellular concentrations of dopamine and its metabolites and decrease dopamine content in the ipsilateral dorsal striatum. Furthermore, these CCL2 injections are responsible for an increase in locomotor activity resulting in contralateral circling behavior. Using patch-clamp recordings of dopaminergic neurons in slices of the rat substantia nigra, we observed that a prolonged exposure (>8 min) to 10 nM CCL2 significantly increases the membrane resistance of dopaminergic neurons by closure of background channels mainly selective to potassium ions. This leads to an enhancement of dopaminergic neuron discharge in pacemaker or burst mode necessary for dopamine release. We provide here the first evidence that application of CCL2 on dopaminergic neurons increases their excitability, dopamine release and related locomotor activity.
Asunto(s)
Quimiocina CCL2/fisiología , Cuerpo Estriado/metabolismo , Dopamina/metabolismo , Sustancia Negra/metabolismo , Animales , Membrana Celular/fisiología , Quimiocina CCL2/farmacología , Cuerpo Estriado/efectos de los fármacos , Técnicas In Vitro , Activación del Canal Iónico , Masculino , Microdiálisis , Actividad Motora/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/fisiología , Técnicas de Placa-Clamp , Canales de Potasio/fisiología , Ratas , Ratas Wistar , Conducta Estereotipada/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Factores de TiempoRESUMEN
Dopaminergic neurons of the substantia nigra constitutively express the CXCR4 receptor for the chemokine stromal-cell-derived factor 1alpha (CXCL12) but, to date, no direct effect of CXCR4 activation by CXCL12 on membrane conductance of dopaminergic neurons has been demonstrated. We tested the effects of CXCL12 on whole-cell currents of dopaminergic neurons recorded in patch clamp in substantia nigra slices and showed that CXCL12 (0.01-10 nm) increased the amplitude of total high-voltage-activated (HVA) Ca currents through CXCR4 activation. This effect was reversibly reduced by varpi-conotoxin-GVIA, suggesting that CXCL12 acted on N-type Ca currents, known to be involved in dopamine (DA) release. We therefore investigated the effects of CXCL12 on DA release from cultured dopaminergic neurons from the rat mesencephalon. In basal conditions, CXCL12 alone had no effect on DA release. When neurons were depolarized with KCl (20 mm), and thus when HVA Ca currents were activated, low CXCL12 concentrations (1-50 nm) increased DA release via CXCR4 stimulation. These data strongly suggest that the chemokine CXCL12 can act directly as a neuromodulator of dopaminergic neuronal electrical activity through the modulation of HVA currents.
Asunto(s)
Señalización del Calcio/fisiología , Quimiocina CXCL12/metabolismo , Dopamina/metabolismo , Neuronas/metabolismo , Sustancia Negra/metabolismo , Animales , Canales de Calcio Tipo N/efectos de los fármacos , Canales de Calcio Tipo N/metabolismo , Señalización del Calcio/efectos de los fármacos , Células Cultivadas , Quimiocina CXCL12/farmacología , Conotoxinas/farmacología , Relación Dosis-Respuesta a Droga , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Ratas , Ratas Wistar , Sustancia Negra/citología , Sustancia Negra/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiologíaRESUMEN
We recently demonstrated that dopaminergic (DA) neurons of the rat substantia nigra constitutively expressed CXCR4, receptor for the chemokine stromal cell-derived factor-1 (SDF-1)/CXCL12 (SDF-1). To check the physiological relevance of such anatomical observation, in vitro and in vivo approaches were used. Patch clamp recording of DA neurons in rat substantia nigra slices revealed that SDF-1 (10 nmol/L) induced: (i) a depolarization and increased action potential frequency; and (ii) switched the firing pattern of depolarized DA neurons from a tonic to a burst firing mode. This suggests that SDF-1 could increase DA release from neurons. Consistent with this hypothesis, unilateral intranigral injection of SDF-1 (50 ng) in freely moving rat decreased DA content and increased extracellular concentrations of DA and metabolites in the ipsilateral dorsal striatum, as shown using microdialysis. Furthermore, intranigral SDF-1 injection induced a contralateral circling behavior. These effects of SDF-1 were mediated via CXCR4 as they were abrogated by administration of a selective CXCR4 antagonist. Altogether, these data demonstrate that SDF-1, via CXCR4, activates nigrostriatal DA transmission. They show that the central functions of chemokines are not restricted, as originally thought, to neuroinflammation, but extend to neuromodulatory actions on well-defined neuronal circuits in non-pathological conditions.
Asunto(s)
Quimiocinas CXC/farmacología , Cuerpo Estriado/efectos de los fármacos , Dopamina/metabolismo , Sustancia Negra/efectos de los fármacos , Potenciales de Acción/efectos de los fármacos , Animales , Conducta Animal/efectos de los fármacos , Química Encefálica/efectos de los fármacos , Quimiocina CXCL12 , Relación Dosis-Respuesta a Droga , Lateralidad Funcional , Masculino , Microdiálisis/métodos , Actividad Motora/efectos de los fármacos , Ratas , Ratas Wistar , Receptores CCR4 , Receptores de Quimiocina/metabolismo , Tirosina 3-Monooxigenasa/metabolismoRESUMEN
In rat substantia nigra (SN), Chemokine (CXC motif) receptor 4 (CXCR4) for the chemokine stromal cell-derived factor (SDF)-1alpha is expressed on dopaminergic (DA) neurones, but also on non-DA cells, suggesting presynaptic actions. Using whole-cell patch-clamp recordings in DA neurones of rat SN slices at a holding potential of -60 mV, we showed here that SDF-1alpha exerts multiple presynaptic effects. First, SDF-1alpha (10 nm) induced an increase in the frequency of spontaneous and miniature GABA(A) postsynaptic currents by presynaptic mechanisms, consistent with the presence of CXCR4 on GABAergic neurones of the SN, as revealed by immunocytochemistry. Second, SDF-1alpha (0.1-1 nm) induced a glutamatergic inward current resistant to tetrodotoxin (TTX), most probably the result of glutamate release from non-neuronal cells. This inward current was not blocked by the CXCR4 antagonist AMD 3100 (1 microm), consistent with the lack of CXCR4 on astrocytes as shown by immunocytochemistry under basal conditions. Finally, SDF-1alpha (10 nm) induced, via CXCR4, an outward G protein-activated inward rectifier (GIRK) current, which was TTX sensitive and prevented by application of the GABA(B) antagonist CGP55845A, suggesting GABA spillover on to GABA(B) receptors. Our results show that SDF-1alpha induces, via presynaptic mechanisms, alterations in the excitability of DA neurones as confirmed by current-clamp experiments.
Asunto(s)
Quimiocinas CXC/metabolismo , Dopamina/metabolismo , Inhibición Neural/fisiología , Terminales Presinápticos/metabolismo , Sustancia Negra/metabolismo , Transmisión Sináptica/fisiología , Animales , Bencilaminas , Quimiocina CXCL12 , Quimiocinas CXC/farmacología , Ciclamas , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/efectos de los fármacos , Canales de Potasio Rectificados Internamente Asociados a la Proteína G/metabolismo , Antagonistas del GABA/farmacología , Ácido Glutámico/metabolismo , Compuestos Heterocíclicos/farmacología , Canales Iónicos/efectos de los fármacos , Canales Iónicos/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Inhibición Neural/efectos de los fármacos , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Ratas , Ratas Wistar , Receptores CXCR4/efectos de los fármacos , Receptores CXCR4/metabolismo , Receptores de GABA-A/efectos de los fármacos , Receptores de GABA-A/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Sustancia Negra/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/fisiologíaRESUMEN
Stromal cell-derived factor-1alpha (SDF-1alpha) is a chemokine whose receptor, CXCR4, is distributed in specific brain areas including hypothalamus. SDF-1alpha has recently been found to play important roles in neurons, although direct modulation of voltage-gated ionic channels has never been shown. In order to clarify this issue, we performed patch-clamp experiments in fetal mouse hypothalamic neurons in culture. SDF-1alpha (10 nm) decreased the peak and rising slope of the action potentials and spike discharge frequency in 22% of hypothalamic neurons tested. This effect was blocked by the CXCR4 antagonist AMD 3100 (1 microm) but not by the metabotropic glutamate receptor antagonist MCPG (500 microm), indicating a direct action of SDF-1alpha on its cognate receptor. This effect involved a depression of both inward and outward voltage-dependent currents of the action potential. We confirmed these effects in the human neuroblastoma cell line SH-SY5Y, which endogenously expresses CXCR4. Voltage-clamp experiments revealed that SDF-1alpha induced a 20% decrease in the peak of the tetrodotoxin-sensitive sodium current and tetraethylammonium-sensitive delayed rectifier potassium current, respectively. Both effects were concentration dependent, and blocked by AMD 3100 (200 nm). This dual effect was reduced or blocked by 0.4 mm GTPgammaS G-protein pre-activation or by pre-treatment with the G-protein inhibitor pertussis toxin (200 ng/mL), suggesting that it is mediated via activation of a G(i/o) protein. This study extends the functions of SDF-1alpha to a direct modulation of voltage-dependent membrane currents of neuronal cells.
Asunto(s)
Potenciales de Acción/efectos de los fármacos , Quimiocinas CXC/farmacología , Glicina/análogos & derivados , Neuronas/efectos de los fármacos , Porinas/metabolismo , Animales , Bencilaminas , Cloruro de Cadmio/farmacología , Células Cultivadas , Quimiocina CXCL12 , Ciclamas , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Regulación de la Expresión Génica/efectos de los fármacos , Glicina/farmacología , Guanosina 5'-O-(3-Tiotrifosfato)/farmacología , Compuestos Heterocíclicos/farmacología , Humanos , Hipotálamo/citología , Inmunohistoquímica/métodos , Ratones , Neuroblastoma , Neuronas/metabolismo , Técnicas de Placa-Clamp/métodos , Porinas/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , ARN Mensajero/biosíntesis , Receptores CXCR4/antagonistas & inhibidores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Bloqueadores de los Canales de Sodio/farmacología , Tetraetilamonio/farmacología , Tetrodotoxina/farmacología , Canales Aniónicos Dependientes del VoltajeRESUMEN
Stromal cell-derived factor 1alpha (SDF-1alpha), a chemoattractant for leucocytes and neurons, and its receptor, CXCR4 are expressed in subsets of neurons of specific brain areas. In rat lateral hypothalamic area (LHA) we show, using immunocytochemistry, that CXCR4 is localized within melanin-concentrating hormone (MCH)-expressing neurons, mainly involved in feeding behaviour regulation. We investigated whether SDF-1alpha may control MCH neuronal activity. Patch-clamp recordings in rat LHA slices revealed multiple effects of SDF-1alpha on the membrane potential of MCH neurons, indirect through glutamate/GABA release and direct through GIRK current activation. Moreover, SDF-1alpha at 0.1-1 nM decreased peak and discharge frequency of action potential evoked by current pulses. These effects were further confirmed in voltage-clamp experiments, SDF-1alpha depressing both potassium and sodium currents. At 10 nM, however, SDF-1alpha increased peak and discharge frequency of action potential evoked by current pulses. Using a specific CXCR4 antagonist, we demonstrated that only the depressing effect on AP discharge was mediated through CXCR4 while the opposite effect was indirect. Together, our studies reveal for the first time a direct effect of SDF-1alpha on voltage-dependent membrane currents of neurons in brain slices and suggest that this chemokine may regulate MCH neuron activity.
Asunto(s)
Quimiocinas CXC/farmacología , Hormonas Hipotalámicas/fisiología , Melaninas/fisiología , Neuronas/fisiología , Hormonas Hipofisarias/fisiología , Animales , Quimiocina CXCL12 , Relación Dosis-Respuesta a Droga , Masculino , Potenciales de la Membrana/fisiología , Ratas , Ratas WistarRESUMEN
We report an asymptomatic case of diffuse intestinal diverticulosis simultaneously affecting duodenum, jejunum and colon. The patient presented signs of progressive systemic sclerosis, which supports the hypothesis in the literature of an aetiological link.
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Divertículo/diagnóstico , Enfermedades Intestinales/diagnóstico , Anciano de 80 o más Años , Colecistectomía , Divertículo/complicaciones , Divertículo/cirugía , Fiebre/complicaciones , Humanos , Hiperbilirrubinemia/complicaciones , Enfermedades Intestinales/complicaciones , Enfermedades Intestinales/cirugía , Masculino , Manometría , Enfermedad de Raynaud/complicacionesRESUMEN
Genetically engineered expression of replicase encoding sequences has been proposed as an efficient system to confer protection against virus diseases by eliciting protection mechanisms in the plant. Potato leaf-roll was one of the first diseases for which this kind of protection was engineered in potato plants. However, details of the protecting mechanism were not reported, so far. The ORF2b of an Argentinean strain of PLRV was cloned and sequenced finding 94% and 97% of homology with Australian and Dutch strains, respectively. To elucidate the mechanism of protection against PLRV infection, three versions of ORF2b (non-translatable sense, translatable sense with an engineered ATG and antisense) were constructed under the control of the 35S CaMV promoter and the nos terminator and introduced in potato plants (cv. Kennebec) by Agrobacterium tumefaciens-mediated transformation. Grafting infection experiments showed that resistant transgenic plants could be obtained with any of the constructs, suggesting that the mechanism of protection is independent of the expression of protein and is RNA mediated. Field trial infection confirmed that resistant transgenic events were obtained. Biolistic transient transformation experiments of leaves derived from transgenic plants using a gene coding for the fusion protein GUS-ORF2b, followed by scoring of the number of GUS expressing leaf spots, supported that the protection is mediated by a post-transcriptional gene silencing mechanism.
Asunto(s)
Silenciador del Gen , Luteovirus/genética , Plantas Modificadas Genéticamente/virología , ARN Polimerasa Dependiente del ARN/genética , Solanum tuberosum/virología , Transformación Genética , Clonación Molecular , Luteovirus/enzimología , Datos de Secuencia Molecular , Sistemas de Lectura Abierta , Homología de Secuencia de Ácido Nucleico , Solanum tuberosum/genéticaRESUMEN
A general strategy is presented for the dominant negative reduction in the levels of heterodimeric soluble proteins within the secretory pathway through fusion of one of its partners C-terminal to the lysosomal enzyme cathepsin B (CB). Stable transfectants of CB-7B2 chimeras in AT20 cells result in a drastic reduction of the endogenous levels of its partner, the proprotein convertase PC2. This dominant negative suppressive effect requires active CB. It was partially reversed by NH(4)Cl, the cell-permeable CB inhibitor CA-074Me, but not by the proteasome inhibitor Lactacystin, suggesting the potential participation of the lysosomal/endosomal degradative pathway in this process.
Asunto(s)
Catepsina B/metabolismo , Lisosomas/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Hormonas Hipofisarias/metabolismo , Subtilisinas/metabolismo , Cloruro de Amonio/farmacología , Animales , Catepsina B/antagonistas & inhibidores , Células Cultivadas , Inhibidores de Cisteína Proteinasa/farmacología , Dipéptidos/farmacología , Lisosomas/efectos de los fármacos , Lisosomas/enzimología , Ratones , Proteínas del Tejido Nervioso/genética , Proteína 7B2 Secretora Neuroendocrina , Hormonas Hipofisarias/genética , Proproteína Convertasa 2 , Proteínas Recombinantes de Fusión/metabolismo , Subtilisinas/genéticaRESUMEN
PC1 and PC2 are two major enzymes involved in the processing of protein precursors directed to the regulated secretory pathway. Whereas transcripts encoding both enzymes are widely distributed in the central nervous system, information regarding the localization of proteins themselves is still lacking. In an attempt to gain insight into the neurobiologic roles of PC1 and PC2, both enzymes were immunolocalized in the rat brain by using C-terminally directed antibodies, which respectively recognize the 87-kDa PC1 and the 75 and 68-kDa PC2 forms. Adjacent sections immunoreacted with PC1 or PC2 antibodies exhibited selective patterns of immunostaining in regions well characterized with respect to their biosynthesis of multiple neuropeptides such as the cerebral cortex, hippocampus, and hypothalamus. PC1 signal intensity was generally weaker than that of PC2, although both enzymes displayed extensive overlapping patterns of expression. As assessed by double-labeling experiments at the cellular level, PC1 and PC2 immunoreactive signals were localized within the trans-Golgi network and nerve terminals, in keeping with the biosynthetic pathways of neuropeptides. Immunoreactive fibers were detected in many areas throughout the brain but were particularly densely distributed in the hypothalamus and the brainstem. Both enzymes were also localized within dendrites of numerous neurons, supporting the hypothesis that dendritic neuropeptide maturation and release may occur in a large number of brain regions. Taken together, our results provide new evidence that both convertases are efficiently targeted to the neuronal regulated secretory pathway and are well poised to process protein precursors in biologically active end-products within the mammalian brain.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Encéfalo/enzimología , Neuronas/enzimología , Sistemas Neurosecretores/enzimología , Ratas/metabolismo , Subtilisinas/metabolismo , Animales , Encéfalo/citología , Masculino , Neuronas/citología , Sistemas Neurosecretores/citología , Hipófisis/citología , Hipófisis/enzimología , Proproteína Convertasa 2 , Proproteína Convertasas , Ratas/anatomía & histología , Ratas Sprague-DawleyRESUMEN
Cellular trafficking of subtilisin/kexin-like precursor convertases (PCs) may be regulated by a number of motifs, some of which are present within the P-domain and in the C-terminal sequence. Six of the seven known PCs contain a conserved RGD sequence within the P domain. In order to investigate the functional importance of this motif, we generated mutants of PC1 that contain a Myc tag epitope inserted between the prosegment and the catalytic subunit. Cellular expression of vaccinia virus recombinants revealed that this tag did not seem to influence the autocatalytic conversion of proPC1 into PC1 or its bioactivity. The two PC1 variants produced possess either the wild type RGD sequence or its RGE mutant. Stable transfectants of these variants in AtT20 cells revealed that similar to the wild type enzyme, PC1-RGD-Myc is sorted to secretory granules. In contrast, PC1-RGE-Myc exits the cell via the constitutive secretory pathway. In vitro, a 14-mer peptide spanning the RGD sequence of PC1, but not its RGE mutant, binds to cell surface vitronectin-binding integrins of Chinese hamster ovary cells. However, within the endoplasmic reticulum and in an RGD-independent fashion, integrin alpha5beta1 associates primarily with the zymogens proPC1, proPC1-DeltaC (missing the C-terminal 137 residues), as well as proPC2. Thus, the observed discrimination between the secretion routes of PC1-RGD and PC1-RGE does not implicate integrins such as alpha5beta1.
Asunto(s)
Ácido Aspártico Endopeptidasas/metabolismo , Oligopéptidos/metabolismo , Receptores de Fibronectina/metabolismo , Secuencia de Aminoácidos , Animales , Ácido Aspártico Endopeptidasas/química , Secuencia de Bases , Transporte Biológico , Células CHO , Cricetinae , Cartilla de ADN , Activación Enzimática , Inmunohistoquímica , Proproteína Convertasas , Unión Proteica , Proteínas Proto-Oncogénicas c-myc/química , TransfecciónRESUMEN
Among the members of the proprotein convertase (PC) family, PC1 and PC2 have well established roles as prohormone convertases. Another good candidate for this role is PC5-A that has been shown to be present in the regulated secretory pathway of certain neuroendocrine tissues, but evidence that it can process prohormones is lacking. To determine whether PC5-A could function as a prohormone convertase and to compare its cleavage specificity with that of PC1 and PC2, we stably transfected the rat pheochromocytoma PC12 cell line with PC5-A and analyzed the biosynthesis and subcellular localization of the enzyme, as well as its ability to process pro-neurotensin/neuromedin N (pro-NT/NN) into active peptides. Our data showed that in transfected PC12 cells, PC5-A was converted from its 126-kDa precursor form into a 117-kDa mature form and, to a lesser extent, into a C-terminally truncated 65-kDa form of the 117-kDa product. Metabolic and immunochemical studies showed that PC5-A was sorted to early compartments of the regulated secretory pathway where it colocalized with immunoreactive NT. Furthermore, pro-NT/NN was processed in these compartments according to a pattern that differed from that previously described in PC1- and PC2-transfected PC12 cells. This pattern resembled that previously reported for pro-NT/NN processing in the adrenal medulla, a tissue known to express high levels of PC5-A. Altogether, these data demonstrate for the first time the ability of PC5-A to function as a prohormone convertase in the regulated secretory pathway and suggest a role for this enzyme in the physiological processing of pro-NT/NN.
Asunto(s)
Neurotensina/metabolismo , Precursores de Proteínas/metabolismo , Procesamiento Proteico-Postraduccional , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Sustitución de Aminoácidos , Animales , Datos de Secuencia Molecular , Células PC12 , Proproteína Convertasa 5 , Ratas , Serina Endopeptidasas/química , Serina Endopeptidasas/genética , TransfecciónRESUMEN
The Authors report a particular case and make a careful international literature regarding etiopathogenesis and surgical treatment of oesophageal achalasia. First they consider the infection mechanisms as the cause of underlying motor disturbances of the oesophagus. Afterwards holding as parameters of reference for the evaluation of effectiveness of the therapy both the relief of dysphagia and the appearance of postoperative RGE, the various surgical options are then examined: pneumatic dilatation as opposed to surgery; the approach to thoracic and abdominal surgery and the possible assembly of a reflux protection mechanism. Finally, they propose the discussion on the challenge that the new technological systems have opened to the international surgical scene: the minimal-access surgery.
Asunto(s)
Acalasia del Esófago/etiología , Acalasia del Esófago/cirugía , Adulto , Humanos , MasculinoRESUMEN
Asr is a family of genes that maps to chromosome 4 of tomato. Asr2, a recently reported member of this family, is believed to be regulated by abscisic acid (ABA), stress and ripening. A genomic Asr2 clone has been fully sequenced, and candidate upstream regulatory elements have been identified. To prove that the promoter region is functional in vivo, we fused it upstream of the beta-glucuronidase (GUS) reporter gene. The resulting chimeric gene fusion was used for transient expression assays in papaya embryogenic calli and leaves. In addition, the same construct was used to produce transgenic tomato, papaya, tobacco, and potato plants. Asr2 upstream sequences showed promoter function in all of these systems. Under the experimental conditions tested, ABA stimulated GUS expression in papaya and tobacco, but not in tomato and potato systems.
Asunto(s)
Ácido Abscísico/farmacología , Regulación de la Expresión Génica , Proteínas de Plantas/genética , Solanum lycopersicum/genética , Glucuronidasa/genética , Plantas Modificadas Genéticamente , Regiones Promotoras Genéticas , Proteínas Recombinantes de Fusión/biosíntesisRESUMEN
The regulatory peptide neurotensin NT has been proposed to exert an autocrine trophic effect on human colon cancers. In the present study, pro-neurotensin/neuromedin N (proNT/NN) expression and processing were investigated in 13 human colon cancer cell lines using a combination of radioimmunoassay and HPLC techniques. All 13 cell lines displayed low to moderate levels of proNT/NN ranging from 10 to 250 fmol/mg protein. However, only 6 (HCT8, LoVo, HT29, C119A, LS174T, and coloDM320) processed the precursor. Three of the latter (HCT8, LS174T, and coloDM320) were analysed in detail with regard to proNT/NN processing pattern and were found to produce NT and large precursor fragments ending with the NT or NN sequence. They had no detectable level of NN. Such a processing pattern resembles that generated by the prohormone convertase PC5. Northern and Western blot analysis of prohormone convertase expression in the 3 cell lines revealed that they were devoid of PC1 and PC2, whereas they all expressed PC5. These data indicate that proNT/NN is a good marker of human colon cancer cell lines while NT is found in only about half of the cell lines. They also suggest that, in addition to NT, several proNT/NN-derived products, possibly generated by PC5, might exert an autocrine positive effect on human colon cancer growth.