RESUMEN
OBJECTIVE: Vesicular acetylcholine transporter (VAChT) is a rate-limiting factor for synaptic acetylcholine transport. Our study focused on whether [18 F] VAT, a novel positron emission tomography (PET) tracer, could be used in detecting cognitive deficits in epilepsy. METHODS: Morris water maze test was used to evaluate learning and memory deficits in pilocarpine-induced chronic epilepsy rats 12 weeks after status epilepticus. Interictal [18 F] VAT PET was performed 13 weeks after status epilepticus to evaluate the level of VAChT in cholinergic pathways compared with [18 F] fluorodeoxyglucose PET. The association between VAChT levels and memory measures was analyzed. Neuropathological tests were performed. RESULTS: Epileptic rats exhibited significant memory deficits in Morris water maze test. [18 F] VAT uptake decreased in septum, hippocampus, thalamus, and basal forebrain, and correlated to memory function. Of note, the level of VAChT in basal forebrain significantly decreased, yet no glucose hypometabolism was detected. Immunofluorescence and Western blot demonstrated decreased expression of VAChT in hippocampus and basal forebrain in the epilepsy group, but no change of expression of acetyltransferase or activity of acetylcholinesterase was detected. SIGNIFICANCE: [18 F] VAT PET is a promising method to test the level of VAChT as a valuable biomarker for memory deficits in pilocarpine-induced chronic epileptic rats.
Asunto(s)
Encéfalo/diagnóstico por imagen , Epilepsia/complicaciones , Trastornos de la Memoria/diagnóstico por imagen , Trastornos de la Memoria/etiología , Proteínas de Transporte Vesicular de Acetilcolina/metabolismo , Acholeplasmataceae/metabolismo , Animales , Encéfalo/efectos de los fármacos , Enfermedad Crónica , Modelos Animales de Enfermedad , Epilepsia/inducido químicamente , Epilepsia/diagnóstico por imagen , Fluorodesoxiglucosa F18/farmacocinética , Masculino , Aprendizaje por Laberinto/efectos de los fármacos , Agonistas Muscarínicos/toxicidad , Naftoles/farmacocinética , Pilocarpina/toxicidad , Piperidinas/farmacocinética , Tomografía de Emisión de Positrones , Ratas , Ratas Sprague-DawleyRESUMEN
Genetic relatedness of phytoplasmas is commonly defined on the basis of differences in the highly conserved 16S rRNA gene, which may not resolve closely related phytoplasmas. An example of this is the closely related tomato big bud (TBB) and sweet potato little leaf strain V4 (SPLL-V4) phytoplasmas, which cannot easily be differentiated by their 16S rRNA gene sequences. This study aimed to identify genes on the TBB phytoplasma chromosome which could be used to examine genetic variation between these two closely related phytoplasmas. Random clones generated from TBB phytoplasma genomic DNA were sequenced and characterized by database analysis. Twenty-three genes were identified within 19 random clones, which contained approximately 18.0 kbp of TBB phytoplasma genomic DNA. Half of the TBB phytoplasma genes identified were involved in DNA replication, transcription and translation. The remaining TBB phytoplasma genes were involved in protein secretion, cellular processes and energy metabolism. Phylogenetic analysis of representative genes showed that the TBB phytoplasma grouped with the mycoplasmas with the exception of the TBB phytoplasma secA gene, which grouped with the onion yellows phytoplasma. PCR primers were designed based on the new genes and tested on isolates of the TBB and SPLL-V4 phytoplasmas. Most primers amplified a product from TBB and SPLL-V4 phytoplasma samples. When amplified products were subjected to RFLP analysis, the restriction patterns were the same as the respective original clones. This result confirmed that the same sequence had been amplified by PCR and showed that these isolates were indistinguishable using the new genes. This study showed that in fact the TBB and SPLL-V4 phytoplasmas are closely related even with the analysis of new genes. These new genes have, however, provided insight into the biology of the TBB and SPLL-V4 phytoplasmas.
Asunto(s)
Acholeplasmataceae/genética , Genes Bacterianos , Proteasas ATP-Dependientes , Acholeplasmataceae/metabolismo , Adenosina Trifosfatasas/genética , Proteínas Bacterianas/genética , Secuencia de Bases , ADN Bacteriano/genética , Metabolismo Energético , Proteínas de Escherichia coli/genética , Genoma Bacteriano , Ipomoea batatas/microbiología , Solanum lycopersicum/microbiología , Proteínas de la Membrana/genética , Proteínas de Transporte de Membrana/genética , Datos de Secuencia Molecular , Filogenia , Canales de Translocación SEC , Proteína SecA , Especificidad de la EspecieRESUMEN
Genes encoding SecA and SecY proteins, essential components of the Sec protein translocation system, were cloned from onion yellows phytoplasma, an unculturable plant pathogenic bacterium. The secA gene consists of 2,505 nucleotides encoding an 835 amino acid protein (95.7 kDa) and shows the highest similarity with SecA of Bacillus subtilis. Anti-SecA rabbit antibody was prepared from a purified partial SecA protein, with a histidine tag expressed in Escherichia coli. Western blot analysis confirmed that SecA protein (approximately 96 kDa) is produced in phytoplasma-infected plants. Immunohistochemical thin sections observed by optical microscopy showed that SecA is characteristically present in plant phloem tissues infected with phytoplasma. The secY gene consists of 1,239 nucleotides encoding a 413 amino acid protein (45.9 kDa) and shows the highest similarity with SecY of B. subtilis. These results suggest the presence of a functional Sec system in phytoplasmas. Because phytoplasmas are endocellular bacteria lacking cell walls, this system might secrete bacterial proteins directly into the host cytoplasm. This study is what we believe to be the first report of the sequence and expression analysis of phytoplasma genes encoding membrane proteins with a predicted function.