RESUMO
Phaseic acid (PA) is a phytohormone regulating important physiological functions in higher plants. Here, we show the presence of naturally occurring (-)-PA in mouse and rat brains. (-)-PA is exclusively present in the choroid plexus and the cerebral vascular endothelial cells. Purified (-)-PA has no toxicity and protects cultured cortical neurons against glutamate toxicity through reversible inhibition of glutamate receptors. Focal occlusion of the middle cerebral artery elicited a significant induction in (-)-PA expression in the cerebrospinal fluid but not in the peripheral blood. Importantly, (-)-PA induction only occurred in the penumbra area, indicting a protective role of PA in the brain. Indeed, elevating the (-)-PA level in the brain reduced ischemic brain injury, whereas reducing the (-)-PA level using a monoclonal antibody against (-)-PA increased ischemic injury. Collectively, these studies showed for the first time that (-)-PA is an endogenous neuroprotective molecule capable of reversibly inhibiting glutamate receptors during ischemic brain injury.
Assuntos
Isquemia Encefálica/tratamento farmacológico , Encéfalo/efeitos dos fármacos , Ácido Glutâmico/metabolismo , Infarto da Artéria Cerebral Média/tratamento farmacológico , Fármacos Neuroprotetores/uso terapêutico , Receptores de Glutamato/química , Sesquiterpenos/uso terapêutico , Animais , Encéfalo/metabolismo , Encéfalo/patologia , Isquemia Encefálica/etiologia , Isquemia Encefálica/metabolismo , Células Cultivadas , Infarto da Artéria Cerebral Média/etiologia , Infarto da Artéria Cerebral Média/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Ratos , Ratos Sprague-Dawley , Receptores de Glutamato/metabolismoRESUMO
The phytohormone abscisic acid (ABA) regulates stress responses and controls numerous aspects of plant growth and development. Biosynthetic precursors and catabolites of ABA have been shown to trigger ABA responses in physiological assays, but it is not clear whether these are intrinsically active or whether they are converted into ABA in planta. In this study, we analyzed the effect of ABA precursors, conjugates, and catabolites on hormone signaling in Arabidopsis (Arabidopsis thaliana). The compounds were also tested in vitro for their ability to regulate the phosphatase moiety of ABA receptor complexes consisting of the protein phosphatase 2C ABI2 and the coreceptors RCAR1/PYL9, RCAR3/PYL8, and RCAR11/PYR1. Using mutants defective in ABA biosynthesis, we show that the physiological activity associated with ABA precursors derives predominantly from their bioconversion to ABA. The ABA glucose ester conjugate, which is the most widespread storage form of ABA, showed weak ABA-like activity in germination assays and in triggering ABA signaling in protoplasts. The ABA conjugate and precursors showed negligible activity as a regulatory ligand of the ABI2/RCAR receptor complexes. The majority of ABA catabolites were inactive in our assays. To analyze the chemically unstable 8'- and 9'-hydroxylated ABA catabolites, we used stable tetralone derivatives of these compounds, which did trigger selective ABA responses. ABA synthetic analogs exhibited differential activity as regulatory ligands of different ABA receptor complexes in vitro. The data show that ABA precursors, catabolites, and conjugates have limited intrinsic bioactivity and that both natural and synthetic ABA-related compounds can be used to probe the structural requirements of ABA ligand-receptor interactions.
Assuntos
Ácido Abscísico/farmacologia , Proteínas de Arabidopsis/metabolismo , Arabidopsis/efeitos dos fármacos , Arabidopsis/fisiologia , Reguladores de Crescimento de Plantas/farmacologia , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Regulação da Expressão Gênica de Plantas/efeitos dos fármacos , Genes Reporter , Germinação/efeitos dos fármacos , Germinação/genética , Germinação/fisiologia , Peptídeos e Proteínas de Sinalização Intracelular , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Mutação , Fosfoproteínas Fosfatases/genética , Fosfoproteínas Fosfatases/metabolismo , Reguladores de Crescimento de Plantas/química , Reguladores de Crescimento de Plantas/metabolismo , Folhas de Planta/efeitos dos fármacos , Folhas de Planta/genética , Folhas de Planta/fisiologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/genética , Raízes de Plantas/fisiologia , Estômatos de Plantas/efeitos dos fármacos , Estômatos de Plantas/genética , Estômatos de Plantas/fisiologia , Plantas Geneticamente Modificadas , Protoplastos , Proteínas Recombinantes de Fusão , Plântula/efeitos dos fármacos , Plântula/genética , Plântula/fisiologia , Sementes/efeitos dos fármacos , Sementes/genética , Sementes/fisiologia , Transdução de Sinais/efeitos dos fármacos , Tetralonas/química , Tetralonas/metabolismo , Tetralonas/farmacologiaRESUMO
Brain microglia are resident macrophage-like cells representing the first and main form of active immune response during brain injury. Microglia-mediated inflammatory events in the brain are known to be associated with chronic degenerative diseases such as Multiple Sclerosis, Parkinson's, or Alzheimer's disease. Therefore, identification of mechanisms activating microglia is not only important in the understanding of microglia-mediated brain pathologies, but may also lead to the development of new anti-inflammatory drugs for the treatment of chronic neurodegenerative diseases. Recently, abscisic acid (ABA), a phytohormone regulating important physiological functions in higher plants, has been proposed to activate murine microglial cell line N9 through increased intracellular calcium. In the present study, we determined the response to ABA and its analogues from murine primary microglia and immortalized murine microglial cell line BV-2 and N9 cells. A Fura-2-acetoxymethyl ester (Fura-2AM)-based ratiometric calcium imaging and measurement technique was used to determine the intracellular calcium changes in these cells when treated with (-)-ABA, (+)-ABA, (-)-trans-ABA and (+)-trans-ABA. Both primary microglia and microglial cell lines (BV-2 and N9 cells) showed significant increase in intracellular calcium ([Ca(2+)]i) in response to treatment with ATP and ionomycine. However, ABAs failed to evoke dose- and time-dependent [Ca(2+)]i changes in mouse primary microglia, BV-2 and N9 cells. Together, these surprising findings demonstrate that, contrary to that reported in N9 cells [3], ABAs do not evoke intracellular calcium changes in primary microglia and microglial cell lines. The broad conclusion that ABA evokes [Ca(2+)]i in microglia requires more evidence and further careful examination.
Assuntos
Ácido Abscísico/análogos & derivados , Ácido Abscísico/farmacologia , Cálcio/metabolismo , Microglia/efeitos dos fármacos , Animais , Linhagem Celular Transformada , Camundongos , Microglia/metabolismoRESUMO
Conjugated 1,3,5-hexatrienes encased in bridged bicyclic skeletons are prepared by cross-coupling followed by half-reduction of the resulting dienynes. The trienes undergo 6pi electrocyclization at an ambient or elevated temperature to furnish complex, polycyclic cyclohexadienes. In all cases, complete selectivity in favor of cyclization from the exo face of the bridged bicyclic system was seen, in contrast to the corresponding 4pi Nazarov cyclizations.
RESUMO
Abscisic acid (ABA) is a phytohormone known to mediate numerous plant developmental processes and responses to environmental stress. In Arabidopsis thaliana, ABA acts, through a genetically redundant family of ABA receptors entitled Regulatory Component of ABA Receptor (RCAR)/Pyrabactin Resistant 1 (PYR1)/Pyrabactin Resistant-Like (PYL) receptors comprised of thirteen homologues acting in concert with a seven-member set of phosphatases. The individual contributions of A. thaliana RCARs and their binding partners with respect to specific physiological functions are as yet poorly understood. Towards developing efficacious plant growth regulators selective for specific ABA functions and tools for elucidating ABA perception, a panel of ABA analogs altered specifically on positions around the ABA ring was assembled. These analogs have been used to probe thirteen RCARs and four type 2C protein phosphatases (PP2Cs) and were also screened against representative physiological assays in the model plant Arabidopsis. The 1'-O methyl ether of (S)-ABA was identified as selective in that, at physiologically relevant levels, it regulates stomatal aperture and improves drought tolerance, but does not inhibit germination or root growth. Analogs with the 7'- and 8'-methyl groups of the ABA ring replaced with bulkier groups generally retained the activity and stereoselectivity of (S)- and (R)-ABA, while alteration of the 9'-methyl group afforded an analog that substituted for ABA in inhibiting germination but neither root growth nor stomatal closure. Further in vitro testing indicated differences in binding of analogs to individual RCARs, as well as differences in the enzyme activity resulting from specific PP2Cs bound to RCAR-analog complexes. Ultimately, these findings highlight the potential of a broader chemical genetics approach for dissection of the complex network mediating ABA-perception, signaling and functionality within a given species and modifications in the future design of ABA agonists.
Assuntos
Ácido Abscísico , Arabidopsis , Fosfoproteínas Fosfatases/metabolismo , Reguladores de Crescimento de Plantas/metabolismo , Ácido Abscísico/agonistas , Ácido Abscísico/análogos & derivados , Ácido Abscísico/química , Ácido Abscísico/metabolismo , Arabidopsis/química , Arabidopsis/genética , Arabidopsis/metabolismo , Estrutura Molecular , Transdução de Sinais , Relação Estrutura-AtividadeRESUMO
Readily available five- and six-membered lactones and N-sulfonyllactams undergo efficient addition of t-butyl propiolate, and the resulting adducts undergo cycloisomerization to six- and seven-membered cyclic ethers or amines in the presence of pyridinium acetate. The ring expansion process occurs in generally good yields and is proposed to involve a nucleophilic catalysis mechanism.
Assuntos
Alcinos/química , Carbono/química , Substâncias Intercalantes/química , Lactamas/química , Lactonas/química , Propionatos/química , Ciclização , IsomerismoRESUMO
Stille coupling under standard carbonylative conditions proceeds in poor yield when using hindered alkenylstannane and enol triflate partners. The inclusion of 35 mol % CuI or CuBr significantly improves the efficiency of the coupling, providing a variety of complex 1,4-dien-3-ones in good to excellent yield.