RESUMEN
BACKGROUND AND AIMS: Light interception is closely related to canopy architecture. Few studies based on multi-view photography have been conducted in a field environment, particularly studies that link 3-D plant architecture with a radiation model to quantify the dynamic canopy light interception. In this study, we combined realistic 3-D plant architecture with a radiation model to quantify and evaluate the effect of differences in planting patterns and row orientations on canopy light interception. METHODS: The 3-D architectures of maize and soybean plants were reconstructed for sole crops and intercrops based on multi-view images obtained at five growth dates in the field. We evaluated the accuracy of the calculated leaf length, maximum leaf width, plant height and leaf area according to the measured data. The light distribution within the 3-D plant canopy was calculated with a 3-D radiation model. Finally, we evaluated canopy light interception in different row orientations. KEY RESULTS: There was good agreement between the measured and calculated phenotypic traits, with an R2 >0.97. The light distribution was more uniform for intercropped maize and more concentrated for sole maize. At the maize silking stage, 85 % of radiation was intercepted by approx. 55 % of the upper canopy region for maize and by approx. 33 % of the upper canopy region for soybean. There was no significant difference in daily light interception between the different row orientations for the entire intercropping and sole systems. However, for intercropped maize, near east-west orientations showed approx. 19 % higher daily light interception than near south-north orientations. For intercropped soybean, daily light interception showed the opposite trend. It was approx. 49 % higher for near south-north orientations than for near east-west orientations. CONCLUSIONS: The accurate reconstruction of 3-D plants grown in the field based on multi-view images provides the possibility for high-throughput 3-D phenotyping in the field and allows a better understanding of the relationship between canopy architecture and the light environment.
Asunto(s)
Productos Agrícolas , Hojas de la Planta , Estaciones del Año , Glycine max , Zea maysRESUMEN
MMP13 (matrix metallopeptidase 13) plays a key role in bone metabolism and cancer development, but has no known functions in Alzheimer's disease. In this study, we used high-throughput small molecule screening in SH-SY5Y cells that stably expressed a luciferase reporter gene driven by the BACE1 (ß-site amyloid precursor protein cleaving enzyme 1) promoter, which included a portion of the 5' untranslated region (5'UTR). We identified that CL82198, a selective inhibitor of MMP13, decreased BACE1 protein levels in cultured neuronal cells. This effect was dependent on PI3K (phosphatidylinositide 3-kinase) signalling, and was unrelated to BACE1 gene transcription and protein degradation. Further, we found that eukaryotic translation initiation factor 4B (eIF4B) played a key role, as the mutation of eIF4B at serine 422 (S422R) or deletion of the BACE1 5'UTR attenuated MMP13-mediated BACE1 regulation. In APPswe/PS1E9 mice, an animal model of Alzheimer's disease, hippocampal Mmp13 knockdown or intraperitoneal CL82198 administration reduced BACE1 protein levels and the related amyloid-ß precursor protein processing, amyloid-ß load and eIF4B phosphorylation, whereas spatial and associative learning and memory performances were improved. Collectively, MMP13 inhibition/CL82198 treatment exhibited therapeutic potential for Alzheimer's disease, via the translational regulation of BACE1.
Asunto(s)
Enfermedad de Alzheimer/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Ácido Aspártico Endopeptidasas/metabolismo , Benzofuranos/uso terapéutico , Disfunción Cognitiva/tratamiento farmacológico , Metaloproteinasa 13 de la Matriz/metabolismo , Inhibidores de la Metaloproteinasa de la Matriz/uso terapéutico , Morfolinas/uso terapéutico , Enfermedad de Alzheimer/genética , Precursor de Proteína beta-Amiloide/genética , Animales , Células Cultivadas , Factores Eucarióticos de Iniciación/genética , Técnicas de Silenciamiento del Gen , Hipocampo/metabolismo , Humanos , Ratones , Ratones Transgénicos , Mutación , Oligopéptidos/genética , Fosfatidilinositol 3-Quinasas/metabolismo , RatasRESUMEN
Furin is a proprotein convertase implicated in a variety of pathological processes including neurodegenerative diseases. However, the role of furin in neuronal plasticity and learning and memory remains to be elucidated. Here, we report that in brain-specific furin transgenic (Furin-Tg) mice, the dendritic spine density and proliferation of neural progenitor cells were significantly increased. These mice exhibited enhanced long-term potentiation (LTP) and spatial learning and memory performance, without alterations of miniature excitatory/inhibitory postsynaptic currents. In the cortex and hippocampus of Furin-Tg mice, the ratio of mature brain-derived neurotrophic factor (mBDNF) to pro-BDNF, and the activities of extracellular signal-related kinase (ERK) and cAMP response element-binding protein (CREB) were significantly elevated. We also found that hippocampal knockdown of CREB diminished the facilitation of LTP and cognitive function in Furin-Tg mice. Together, our results demonstrate that furin enhances dendritic morphogenesis and learning and memory in transgenic mice, which may be associated with BDNF-ERK-CREB signaling pathway.
Asunto(s)
Dendritas/fisiología , Furina/metabolismo , Aprendizaje por Laberinto/fisiología , Memoria/fisiología , Animales , Factor Neurotrófico Derivado del Encéfalo/metabolismo , Corteza Cerebral/metabolismo , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Dendritas/genética , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Furina/genética , Hipocampo/metabolismo , Potenciación a Largo Plazo/genética , Potenciación a Largo Plazo/fisiología , Masculino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurogénesis/genética , Precursores de Proteínas/metabolismo , Interferencia de ARNRESUMEN
ADAM10 (a disintegrin and metalloproteinase domain-containing protein 10) is the α-secretase that is involved in APP (ß-amyloid precursor protein) processing. Enhancement of the nonamyloidogenic APP pathway by ADAM10 provides therapeutic potential for Alzheimer's disease (AD). By using high-throughput screening that targeted ADAM10, we determined that apicidin-an inhibitor of HDACs (histone deacetylases)-significantly increased mRNA and protein levels of ADAM10 in SH-SY5Y cells. A luciferase assay revealed that the nucleotides -444 to -300 in the ADAM10 promoter were sufficient to mediate this effect. In addition, knockdown of USF1 (upstream transcription factor 1) and HDAC2/3 prevented apicidin regulation of ADAM10. Moreover, USF1 acetylation was increased by apicidin, which enhanced the association of USF1 with HDAC2/3 and with the ADAM10 promoter. We further found that apicidin did not affect the phosphorylation of ERK or USF1; however, ERK inhibitor U0126 blocked the effect of apicidin on ADAM10. Finally, apicidin increased the level of α-site C-terminal fragment from APP and reduced the production of ß-amyloid peptide 1-42. Collectively, our study provides evidence that ADAM10 expression can be regulated by HDAC2/3 inhibitor apicidin via USF1-dependent mechanisms in which ERK signaling plays an important role. Thus, HDAC regulation of ADAM10 might shed new light on the understanding of AD pathology.-Hu, X.-T., Zhu, B.-L., Zhao, L.-G., Wang, J.-W., Liu, L., Lai, Y.-J., He, L., Deng, X.-J., Chen, G.-J. Histone deacetylase inhibitor apicidin increases expression of the α-secretase ADAM10 through transcription factor USF1-mediated mechanisms.
Asunto(s)
Proteína ADAM10/genética , Secretasas de la Proteína Precursora del Amiloide/genética , Inhibidores de Histona Desacetilasas/farmacología , Proteínas de la Membrana/genética , Péptidos Cíclicos/farmacología , Factores Estimuladores hacia 5'/metabolismo , Proteína ADAM10/metabolismo , Secretasas de la Proteína Precursora del Amiloide/metabolismo , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Animales , Línea Celular Tumoral , Humanos , Proteínas de la Membrana/metabolismo , Ratones , Regiones Promotoras Genéticas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Activación Transcripcional/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacos , Factores Estimuladores hacia 5'/genéticaRESUMEN
Background and Aims: Global agriculture is facing the challenge of a phenotyping bottleneck due to large-scale screening/breeding experiments with improved breeds. Phenotypic analysis with high-throughput, high-accuracy and low-cost technologies has therefore become urgent. Recent advances in image-based 3D reconstruction offer the opportunity of high-throughput phenotyping. The main aim of this study was to quantify and evaluate the canopy structure of plant populations in two and three dimensions based on the multi-view stereo (MVS) approach, and to monitor plant growth and development from seedling stage to fruiting stage. Methods: Multi-view images of flat-leaf cucumber, small-leaf pepper and curly-leaf eggplant were obtained by moving a camera around the plant canopy. Three-dimensional point clouds were reconstructed from images based on the MVS approach and were then converted into surfaces with triangular facets. Phenotypic parameters, including leaf length, leaf width, leaf area, plant height and maximum canopy width, were calculated from reconstructed surfaces. Accurate evaluation in 2D and 3D for individual leaves was performed by comparing reconstructed phenotypic parameters with referenced values and by calculating the Hausdorff distance, i.e. the mean distance between two surfaces. Key Results: Our analysis demonstrates that there were good agreements in leaf parameters between referenced and estimated values. A high level of overlap was also found between surfaces of image-based reconstructions and laser scanning. Accuracy of 3D reconstruction of curly-leaf plants was relatively lower than that of flat-leaf plants. Plant height of three plants and maximum canopy width of cucumber and pepper showed an increasing trend during the 70 d after transplanting. Maximum canopy width of eggplants reached its peak at the 40th day after transplanting. The larger leaf phenotypic parameters of cucumber were mostly found at the middle-upper leaf position. Conclusions: High-accuracy 3D evaluation of reconstruction quality indicated that dynamic capture of the 3D canopy based on the MVS approach can be potentially used in 3D phenotyping for applications in breeding and field management.
Asunto(s)
Cucumis sativus/anatomía & histología , Procesamiento de Imagen Asistido por Computador/métodos , Piper/anatomía & histología , Solanum melongena/anatomía & histología , Agricultura , Cucumis sativus/crecimiento & desarrollo , Frutas/anatomía & histología , Frutas/crecimiento & desarrollo , Fenotipo , Piper/crecimiento & desarrollo , Fitomejoramiento , Hojas de la Planta/anatomía & histología , Hojas de la Planta/crecimiento & desarrollo , Plantones/anatomía & histología , Plantones/crecimiento & desarrollo , Solanum melongena/crecimiento & desarrolloRESUMEN
BACKGROUND: Nicotine is known to differentially regulate cortical interneuron and pyramidal neuron activities in the neocortex, while the underlying molecular mechanisms have not been well studied. In this study, RNA-sequencing was performed in acutely isolated cortical somatostatin (Sst)- positive interneurons and pyramidal neurons (Thy1) from mice treated with systemic nicotine for 14 days. We assessed the differentially expressed genes (DEGs) by nicotine in Sst- or Thy1- neurons, respectively, and then compared DEGs between Sst- and Thy1- neurons in the absence and presence of nicotine. RESULTS: In Sst-neurons, the DEGs by nicotine were associated with glycerophospholipid and nicotinate and nicotinamide metabolism; while in Thy1-neurons those related to immune response and purine and pyrimidine metabolisms were affected. Under basal condition, the DEGs between Sst- and Thy1- neurons were frequently associated with signal transduction, phosphorylation and potassium channel regulation. However, some new DEGs between Sst- and Thy1- neurons were found after nicotine, the majority of which belong to mitochondrial respiratory chain complex. CONCLUSIONS: Nicotine differentially affected subset of genes in Sst- and Thy1- neurons, which might contribute to the distinct effect of nicotine on interneuron and pyramidal neuron activities. Meanwhile, the altered transcripts associated with mitochondrial activity were found between interneurons and pyramidal neurons after chronic nicotine.
Asunto(s)
Encéfalo/citología , Perfilación de la Expresión Génica , Interneuronas/efectos de los fármacos , Interneuronas/metabolismo , Nicotina/farmacología , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Animales , Encéfalo/efectos de los fármacos , Ratones , Análisis de Secuencia de ARN , Factores de TiempoRESUMEN
The AMP-activated protein kinase (AMPK) is a key energy sensor. Its activator metformin could suppress epileptogenesis in the pentylenetetrazol (PTZ) kindling model. However, the effect of metformin on the acute and chronic seizures has not been studied. We first detected the expression of AMPK in the brain tissue of human and mice with chronic seizures, as well as in mice with acute seizures. Second, using behavioral assay and local filed potentials (LFPs) recording, we investigated the effect of chronic metformin treatment on seizures in a acute seizure model and a chronic seizure model. Our results showed that AMPK was expressed in neurons in the epileptic brain. The expression level was decreased in the brain tissue that experienced chronic and acute seizures. In PTZ-induced acute seizures model, behavioral assay showed that chronic metformin treatment decreased the mortality, and LFPs recording showed that chronic metformin treatment shortened the duration of generalized tonic-clonic seizures and prolonged the duration of postictal depression. Moreover, in kainic acid-induced chronic seizures model, LFPs recording showed that chronic metformin treatment shortened the duration of epileptic activity. Our study suggests that chronic metformin treatment could facilitate seizure termination.
Asunto(s)
Metformina/farmacología , Convulsiones/prevención & control , Proteínas Quinasas Activadas por AMP/metabolismo , Animales , Masculino , Ratones , Ratones Endogámicos BALB C , Pentilenotetrazol/toxicidad , Convulsiones/inducido químicamenteRESUMEN
HMGCS2 (mitochondrial 3-hydroxy-3-methylglutaryl-COA synthase 2) is a control enzyme in ketogenesis. The mitochondrial localization and interaction with APP (ß-amyloid precursor protein) suggest that HMGCS2 may play a role in the pathophysiology of AD (Alzheimer's disease). Here we report that overexpression of HMGCS2 decreased levels of APP and related CTFs (carboxy-terminal fragments), which was largely prevented by an autophagic inhibitor chloroquine. In addition, HMGCS2 enhancement of autophagic marker LC3II was diminished by rapamycin, an inhibitor of mechanistic target of rapamycin. Moreover, deprivation of EBSS (Earle's Balanced Salt Solution) significantly augmented the effect of HMGCS2 on LC3II, while acetoacetate reversed the reduction of LC3II, APP and CTFs which was induced by HMGCS2 knockdown. In the presence of acetoacetate, rapamycin failed to induce further increase of LC3II, which mimicked the effect of HMGCS2 overexpression. Finally, HMGCS2 enhanced the antioxidant response. Collectively, HMGCS2 shares with ketone bodies common features in autophagic clearance of APP and CTFs, suggesting that ketone bodies play an important role in HMGCS2 regulation of the autophagy.
Asunto(s)
Precursor de Proteína beta-Amiloide/metabolismo , Autofagia/genética , Hidroximetilglutaril-CoA Sintasa/genética , Cuerpos Cetónicos/metabolismo , Proteínas Asociadas a Microtúbulos/genética , Serina-Treonina Quinasas TOR/genética , Acetoacetatos/farmacología , Animales , Línea Celular , Cloroquina/farmacología , Regulación de la Expresión Génica , Células HEK293 , Hipocampo/citología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Humanos , Hidroximetilglutaril-CoA Sintasa/antagonistas & inhibidores , Hidroximetilglutaril-CoA Sintasa/metabolismo , Ratones , Proteínas Asociadas a Microtúbulos/metabolismo , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Proteolisis/efectos de los fármacos , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de Señal , Sirolimus/farmacología , Serina-Treonina Quinasas TOR/antagonistas & inhibidores , Serina-Treonina Quinasas TOR/metabolismo , TransgenesRESUMEN
Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1 (Pin1) is a unique PPIase belonging to the parvulin family, and it isomerizes peptide bond between phospho-(Ser/Thr) and Pro. Pin1 has been linked to the pathogenesis of various human diseases; however, its exact biological functions remain unclear. The aim of the present study is to explore the expression pattern of Pin1 in patients with refractory epilepsy and in a chronic pilocarpine-induced epileptic mouse model. Using Western blot, immunofluorescence and immunoprecipitation analysis, we found that Pin1 protein was mainly distributed in neurons, demonstrated by colocalization with the dendritic marker, MAP2. However, the expression of Pin1 decreased remarkably in epileptic patients and experimental mice. Furthermore, the reciprocal coimmunoprecipitation analysis showed that Pin1 interacted with NR2A and NR2B-containing NMDA receptors not AMPA receptors in epileptic mouse models. Our results are the first to indicate that the expression of Pin1 in epileptic brain tissue could play important roles in epilepsy.
Asunto(s)
Modelos Animales de Enfermedad , Regulación hacia Abajo/fisiología , Epilepsia del Lóbulo Temporal/metabolismo , Peptidilprolil Isomerasa de Interacción con NIMA/metabolismo , Adolescente , Adulto , Animales , Niño , Regulación hacia Abajo/efectos de los fármacos , Epilepsia del Lóbulo Temporal/inducido químicamente , Epilepsia del Lóbulo Temporal/patología , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Hipocampo/patología , Humanos , Masculino , Ratones , Ratones Endogámicos C57BL , Persona de Mediana Edad , Neocórtex/efectos de los fármacos , Neocórtex/metabolismo , Neocórtex/patología , Pilocarpina/toxicidad , Adulto JovenRESUMEN
Glypican-4 (Gpc4) has been found to play an important role in enhancing miniature excitatory postsynaptic currents (mEPSCs). But, the relationship between Gpc4 and epilepsy is still a mystery. In this study, we investigated the expression patterns of Gpc4 in patients with epilepsy and in a pilocarpine-induced rat model of epilepsy. We also determined if altered Gpc4 expression resulted in increased susceptibility to seizures. Western blotting and immunofluorescent methods were utilized. Gpc4 was significantly increased in patients and epileptic rats induced by pilocarpine injection. According to behavioral studies, downregulation of Gpc4 by Gpc4 siRNA decreased spontaneous seizure frequency, while upregulation of Gpc4 by recombinant Gpc4 overexpression led to a converse result. These findings support the hypothesis that increased expression of Gpc4 in the brain is associated with epileptic seizures.
Asunto(s)
Potenciales de Acción , Encéfalo/fisiopatología , Epilepsia/fisiopatología , Glipicanos/metabolismo , Adolescente , Adulto , Animales , Células Cultivadas , Niño , Preescolar , Femenino , Humanos , Masculino , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Distribución Tisular , Adulto JovenRESUMEN
It is long been suggested that one-carbon metabolism (OCM) is associated with Alzheimer's disease (AD), whereas the potential mechanisms remain poorly understood. Taking advantage of chemical biology, that mitochondrial serine hydroxymethyltransferase (SHMT2) directly regulated the translation of ADAM metallopeptidase domain 10 (ADAM10), a therapeutic target for AD is reported. That the small-molecule kenpaullone (KEN) promoted ADAM10 translation via the 5' untranslated region (5'UTR) and improved cognitive functions in APP/PS1 mice is found. SHMT2, which is identified as a target gene of KEN and the 5'UTR-interacting RNA binding protein (RBP), mediated KEN-induced ADAM10 translation in vitro and in vivo. SHMT2 controls AD signaling pathways through binding to a large number of RNAs and enhances the 5'UTR activity of ADAM10 by direct interaction with GAGGG motif, whereas this motif affected ribosomal scanning of eukaryotic initiation factor 2 (eIF2) in the 5'UTR. Together, KEN exhibits therapeutic potential for AD by linking OCM with RNA processing, in which the metabolic enzyme SHMT2 "moonlighted" as RBP by binding to GAGGG motif and promoting the 5'UTR-dependent ADAM10 translation initiation.
Asunto(s)
Enfermedad de Alzheimer , Glicina Hidroximetiltransferasa , Animales , Ratones , Regiones no Traducidas 5' , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/metabolismo , Glicina Hidroximetiltransferasa/genética , ARN Mensajero/genéticaRESUMEN
Porcine circovirus type 2 (PCV2) uses autophagy machinery to enhance its replication in PK-15 cells. However, the underlying mechanisms are unknown. By the use of specific inhibitors, RNA interference, and coimmunoprecipitation, we show that PCV2 induces autophagy in PK-15 cells through a pathway involving the kinases AMP-activated protein kinase (AMPK) and extracellular signal-regulated kinase 1/2 (ERK1/2), the tumor suppressor protein TSC2, and the mammalian target of rapamycin (mTOR). AMPK and ERK1/2 positively regulate autophagy through negative control of the mTOR pathway by phosphorylating TSC2 in PCV2-infected PK-15 cells. Thus, PCV2 might induce autophagy via the AMPK/ERK/TSC2/mTOR signaling pathway in the host cells, representing a pivotal mechanism for PCV2 pathogenesis.
Asunto(s)
Proteínas Quinasas Activadas por AMP/metabolismo , Autofagia , Infecciones por Circoviridae/metabolismo , Circovirus/metabolismo , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Serina-Treonina Quinasas TOR/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Animales , Línea Celular , Regulación Viral de la Expresión Génica , Modelos Biológicos , Fosforilación , ARN Interferente Pequeño/metabolismo , Transducción de Señal , Porcinos , Proteína 2 del Complejo de la Esclerosis TuberosaRESUMEN
The direct conversion of human skin fibroblasts to neurons has a low efficiency and unclear mechanism. Here, we show that the knockdown of PTBP2 significantly enhanced the transdifferentiation induced by ASCL1, MIR9/9∗-124, and p53 shRNA (AMp) to generate mostly GABAergic neurons. Longitudinal RNA sequencing analyses identified the continuous induction of many RNA splicing regulators. Among these, the knockdown of RBFOX3 (NeuN), significantly abrogated the transdifferentiation. Overexpression of RBFOX3 significantly enhanced the conversion induced by AMp; the enhancement was occluded by PTBP2 knockdown. We found that PTBP2 attenuation significantly favored neuron-specific alternative splicing (AS) of many genes involved in synaptic transmission, signal transduction, and axon formation. RBFOX3 knockdown significantly reversed the effect, while RBFOX3 overexpression occluded the enhancement. The study reveals the critical role of neuron-specific AS in the direct conversion of human skin fibroblasts to neurons by showing that PTBP2 attenuation enhances this mechanism in concert with RBFOX3.
Asunto(s)
Empalme Alternativo , Neuronas , Humanos , Empalme Alternativo/genética , Neuronas/metabolismo , Empalme del ARN , Axones/metabolismo , Fibroblastos/metabolismo , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Proteína de Unión al Tracto de Polipirimidina/genética , Proteína de Unión al Tracto de Polipirimidina/metabolismoRESUMEN
Analysis of the genome sequence of Vibrio parahaemolyticus reveals two IcmF family genes in putative type VI secretion system (vpT6SS) clusters in chromosomes 1 (icmF1) and 2 (icmF2). The icmF1 gene is present in majority of clinical isolates (87.5 %), but has a low fraction (25.0 %) in environmental isolates. However, icmF2 is contained in all strains of both clinical and environmental sources. Deletion of either icmF1 or hcp1 significantly reduced bacterial adhesion to Caco-2 cells or HeLa monolayers. However, the ΔicmF2 and Δhcp2 mutants showed decreased adhesion only to HeLa monolayers. Western blot analysis showed that Hcp2 was present both in the supernatant and pellet samples in the wild-type strain, but only in the pellet of the ΔicmF2 mutant, indicating that Hcp2 is a translocon of T6SS2. Although vpT6SS1 might be functional in cellular adhesion, the putative translocon Hcp1 was not detectable. Quantitative PCR revealed 10-fold and 17-fold less transcripts of hcp1 and icmF1 mRNA than those of hcp2 and icmF2 accordingly. Thus, we postulate that the putative vpT6SS systems contribute to adhesion of V. parahaemolyticus to host cells.
Asunto(s)
Adhesión Bacteriana/fisiología , Vibrio parahaemolyticus/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Células CACO-2 , Células Cultivadas , Regulación Bacteriana de la Expresión Génica , Células HeLa , Humanos , Vibrio parahaemolyticus/genética , Vibrio parahaemolyticus/patogenicidad , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Porcine circovirus type 2 (PCV2) is believed to be the primary causative agent of postweaning multisystemic wasting syndrome (PMWS). It is supposed that capsid protein of PCV may contribute to replication control via interaction between Cap and Rep in the nucleoplasm. In this study, we described the construction and in vitro characterization of NLS-exchanged PCV DNA clones based on a PMWS-associated PCV2b isolate from China to determine the role of ORF2 NLS in PCV replication. RESULTS: The PCV1, PCV2, PCV2-NLS1 and PCV1-NLS2 DNA clone were generated by ligating a copy of respective genome in tandem with a partial duplication. The PCV2-NLS1 and PCV1-NLS2 DNA clone contained a chimeric genome in which the ORF2 NLS was exchanged. The four DNA clones were all confirmed to be infectious in vitro when transfected into PK-15 cells, as PCV capsid protein were expressed in approximately 10-20% of the transfected cells. The in vitro growth characteristics of the DNA clones were then determined and compared. All the recovered progeny viruses gave rise to increasing infectious titers during passages and were genetically stable by genomic sequencing. The chimeric PCV1-NLS2 and PCV2-NLS1 viruses had the final titers of about 104.2 and 103.8 TCID50/ml, which were significantly lower than that of PCV1 and PCV2 (105.6 and 105.0 TCID50/ml, respectively). When the ORF2 NLS exchanged, the mutant PCV2 (PCV2-NLS1) still replicated less efficiently and showed lower infectious titer than did PCV1 mutant (PCV1-NLS2), which was consistent with the distinction between wild type PCV1 and PCV2. CONCLUSIONS: Recovery of the chimeiric PCV1-NLS2 and PCV2-NLS1 progeny viruses indicate that the nuclear localization signal sequence of capsid protein are functionally exchangeable between PCV1 and PCV2 with respect to the role of nuclear importing and propagation. The findings also reveal that ORF2 NLS play an accessory role in the replication of PCV. However, we found that ORF2 NLS was not responsible for the distinction of in vitro growth characteristic between PCV1 and PCV2. Further studies are required to determine the in vivo viral replication and pathogenicity of the NLS chimeric DNA clones.
Asunto(s)
Proteínas de la Cápside/genética , Circovirus/genética , Señales de Localización Nuclear , Recombinación Genética , Animales , Línea Celular , China , Prueba de Complementación Genética , Síndrome Multisistémico de Emaciación Posdestete Porcino/virología , Porcinos , Carga ViralRESUMEN
Myeloid differentiation factor 88 (MyD88) is one of the key adaptor proteins to signal transduction that triggers downstream cascades involved in innate immunity. In this study, the MyD88 gene from Chinese soft-shelled turtle (Trionyx sinensis) (tMyD88) was identified, representing the fist example from reptile species. The tMyD88 has a 894-bp ORF and encodes a polypeptide of 297 amino acids including a typical death domain (DD) at the N-terminus and a conservative Toll/IL-1R (TIR) domain at the C-terminus. It was expressed at high levels in spleen, blood, lungs and liver, but marginal in kidneys and intestines of turtles challenged with live cells of Aeromonas hydrophila, as determined by real-time PCR. RAW 264.7 cells transfected with pcDNA-tMyD88 showed higher NF-κB activity than the vector control (673.78 vs 410.72, P < 0.05). Expression of proinflammatory cytokines IL-1ß and TNF-α was also significantly higher in RAW 264.7 cells transfected with pcDNA-tMyD88 than those having pcDNA3.1 control vector (P < 0.01). These results indicate that tMyD88 might possess an important role in defense against microbial infection in Chinese soft-shelled turtles similar to that in mammals.
Asunto(s)
Factor 88 de Diferenciación Mieloide/genética , Factor 88 de Diferenciación Mieloide/metabolismo , Tortugas/metabolismo , Secuencia de Aminoácidos , Animales , Secuencia de Bases , Regulación de la Expresión Génica/fisiología , Datos de Secuencia Molecular , FilogeniaRESUMEN
An RT-nested PCR (RT-nPCR)-based restriction fragment length polymorphism (RFLP) analyses of the E2 gene were developed for genetic subtyping and differentiation of vaccinated and infected classical swine fever virus (CSFV) strains. RT-nPCR identified 96 CSFV-positive samples from 321 clinical specimens from southeastern China during 2003-2008. The PCR products of positive samples were further differentiated using MspI digestion, 23 were identified as the C-strain, 62 as field strains, and 11 as mixture of the vaccine strain and field ones. RFLP with BglI, DdeI, DraI, and PstI were then used for subtyping of the field CSFV isolates. Thirty-eight field isolates phylogenetically classified as subgroup 2.1 based on E2 were divided into 11 subtypes by this RFLP scheme. Both RFLP profiling and sequence-based phylogenetic analysis revealed genetic diversity of CSFV in the field. Three novel substitutions at amino acid positions 17, 93, and 286 were identified in the predominant subtype VI strains isolated in 2008 as compared to other strains including historical subtype VI strains. These results suggest that CSFV in China experienced gradual variations and evolutionary accumulation progress. Thus, the RFLP methods targeting on the CSFV E2 gene are suitable for epidemiological survey in endemic area where the C-strain is applied for vaccination. Combination of the RFLP schemes with sequence-based phylogenetic analysis could provide more detailed information on transmission of CSFV in the region or even its evolution.
Asunto(s)
Virus de la Fiebre Porcina Clásica/clasificación , Peste Porcina Clásica/virología , Reacción en Cadena de la Polimerasa/métodos , Polimorfismo de Longitud del Fragmento de Restricción , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa/métodos , Proteínas del Envoltorio Viral/genética , Virología/métodos , Sustitución de Aminoácidos/genética , Animales , China , Virus de la Fiebre Porcina Clásica/genética , Virus de la Fiebre Porcina Clásica/aislamiento & purificación , Análisis por Conglomerados , Dermatoglifia del ADN , Genotipo , Epidemiología Molecular , Datos de Secuencia Molecular , Filogenia , ARN Viral/genética , Análisis de Secuencia de ADNRESUMEN
The etiology of Alzheimer's disease (AD) has been intensively studied. However, little is known about the molecular alterations in early-stage and late-stage AD. Hence, we performed RNA sequencing and assessed differentially expressed genes (DEGs) in the hippocampus of 18-month and 7-month-old APP/PS1 mice. Moreover, the DEGs induced by treatment with nicotine, the nicotinic acetylcholine receptor agonist that is known to improve cognition in AD, were also analyzed in old and young APP/PS1 mice. When comparing old APP/PS1 mice with their younger littermates, we found an upregulation in genes associated with calcium overload, immune response, cancer, and synaptic function; the transcripts of 14 calcium ion channel subtypes were significantly increased in aged mice. In contrast, the downregulated genes in aged mice were associated with ribosomal components, mitochondrial respiratory chain complex, and metabolism. Through comparison with DEGs in normal aging from previous reports, we found that changes in calcium channel genes remained one of the prominent features in aged APP/PS1 mice. Nicotine treatment also induced changes in gene expression. Indeed, nicotine augmented glycerolipid metabolism, but inhibited PI3K and MAPK signaling in young mice. In contrast, nicotine affected genes associated with cell senescence and death in old mice. Our study suggests a potential network connection between calcium overload and cellular signaling, in which additional nicotinic activation might not be beneficial in late-stage AD.
Asunto(s)
Envejecimiento/metabolismo , Enfermedad de Alzheimer/genética , Hipocampo/metabolismo , Nicotina/farmacología , Transcriptoma , Envejecimiento/genética , Enfermedad de Alzheimer/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Canales de Calcio/genética , Canales de Calcio/metabolismo , Respiración de la Célula , Senescencia Celular , Hipocampo/efectos de los fármacos , Hipocampo/crecimiento & desarrollo , Sistema de Señalización de MAP Quinasas , Masculino , Ratones , Ratones Endogámicos C57BL , Fosfatidilinositol 3-Quinasas/genética , Fosfatidilinositol 3-Quinasas/metabolismo , Presenilina-1/genéticaRESUMEN
Methionine sulfoxide reductase B2 (MSRB2) is a mitochondrial protein that protects cell from oxidative stress. The antioxidant activity suggests that MSRB2 may play a role in the pathophysiology of Alzheimer's disease (AD). Here, we report that in APP/PS1 mice, an animal model of AD, MSRB2 protein levels were decreased in the hippocampus at both young (6 mon) and old (18 mon) age, and in the cortex only at an old age, respectively. In HEK293 cells that stably express human full-length ß-amyloid precursor protein (APP, HEK/APP), MSRB2 reduced the protein and mRNA levels of APP and ß-amyloid converting enzyme 1 (BACE1), and the consequent amyloid beta peptide (Aß) 1-40 and Aß1-42 levels. MSRB2 overexpression or knockdown also oppositely affected Tau phosphorylation at selective sites, with the concomitant alteration of the phosphorylated extracellular signal regulated kinase (p-ERK) and AMP-activated protein kinase (p-AMPK) levels. Moreover, in cells treated with long-term (24â¯h) hydrogen peroxide, the alterations of APP processing and Tau phosphorylation were reversed by MSRB2 overexpression. We further found that MSRB2-mediated regulation of APP transcription involved JNK and ERK signaling, as MSRB2 also reduced the levels of phosphorylated JNK (p-JNK), and JNK or ERK inhibitor attenuated the effect of MSRB2 on APP proteins and transcripts. Finally, MSRB2 reduced apoptosis-related proteins Bax and caspase3 and enhanced the anti-apoptotic protein Bcl2. These results indicated that the role for MSRB2 in AD-like pathology was closely associated with its antioxidant activity. By attenuating both amyloidogenesis and Tau phosphorylation, MSRB2 may serve as a potential therapeutic target for AD.
Asunto(s)
Enfermedad de Alzheimer/enzimología , Enfermedad de Alzheimer/patología , Metionina Sulfóxido Reductasas/metabolismo , Mitocondrias/enzimología , Estrés Oxidativo/fisiología , Animales , Células HEK293 , Humanos , Ratones , Ratones TransgénicosRESUMEN
Cav1.2 is the pore-forming subunit of L-type voltage-gated calcium channel (LTCC) that plays an important role in calcium overload and cell death in Alzheimer's disease. LTCC activity can be regulated by estrogen, a sex steroid hormone that is neuroprotective. Here, we investigated the potential mechanisms in estrogen-mediated regulation of Cav1.2 protein. We found that in cultured primary neurons, 17ß-estradiol (E2) reduced Cav1.2 protein through estrogen receptor α (ERα). This effect was offset by a proteasomal inhibitor MG132, indicating that ubiquitin-proteasome system was involved. Consistently, the ubiquitin (UB) mutant at lysine 29 (K29R) or the K29-deubiquitinating enzyme TRAF-binding protein domain (TRABID) attenuated the effect of ERα on Cav1.2. We further identified that the E3 ligase Mdm2 (double minute 2 protein) and the PEST sequence in Cav1.2 protein played a role, as Mdm2 overexpression and the membrane-permeable PEST peptides prevented ERα-mediated Cav1.2 reduction, and Mdm2 overexpression led to the reduced Cav1.2 protein and the increased colocalization of Cav1.2 with ubiquitin in cortical neurons in vivo. In ovariectomized (OVX) APP/PS1 mice, administration of ERα agonist PPT reduced cerebral Cav1.2 protein, increased Cav1.2 ubiquitination, and improved cognitive performances. Taken together, ERα-induced Cav1.2 degradation involved K29-linked UB chains and the E3 ligase Mdm2, which might play a role in cognitive improvement in OVX APP/PS1 mice.