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1.
Planta Med ; 86(8): 565-570, 2020 May.
Artigo em Inglês | MEDLINE | ID: mdl-32325509

RESUMO

Bacopa monnieri is an Ayurvedic plant with rising interest in the pharmacological effects of its extract and constituents, including flavonoids, saponins, and triterpenes such as cucurbitacins, betulinic acid, and bacosine. The latter two compounds are isomeric 3-hydroxy lupenoic acids, which vary only in the arrangement of the carboxylic acid group and the methyl group at C-27 and C-28 and the orientation of the hydroxy group at C-3. In this study, we have reinvestigated the contents of betulinic acid and bacosine, respectively, in extracts from various commercially available B. monnieri powders and food supplements. To our surprise, HPLC-ion trap time-of-flight analyses identified only betulinic acid, but not bacosine, in all extracts under study, which was verified by GC-MS, HPLC-ELSD, 1D NMR (1H,13C), and 2D NMR (1H,1H COSY, 1H,13C HMBC, 1H,13C HSQC, 1H,1H NOESY) experiments. Moreover, it turned out that commercially available reference samples of bacosine were structurally identical with betulinic acid.


Assuntos
Bacopa , Saponinas , Triterpenos , Cromatografia Líquida de Alta Pressão , Extratos Vegetais
2.
J Am Chem Soc ; 134(26): 10773-6, 2012 Jul 04.
Artigo em Inglês | MEDLINE | ID: mdl-22712572

RESUMO

Alkenes and arenes represent two classes of feedstock compounds whose union has fundamental importance to synthetic organic chemistry. We report a new approach to alkene arylation using diaryliodonium salts and Cu catalysis. Using a range of simple alkenes, we have shown that the product outcomes differ significantly from those commonly obtained by the Heck reaction. We have used these insights to develop a number of new tandem and cascade reactions that transform readily available alkenes into complex arylated products that may have broad applications in chemical synthesis.


Assuntos
Alcenos/química , Derivados de Benzeno/química , Cobre/química , Oniocompostos/química , Catálise
3.
J Comp Neurol ; 520(3): 570-89, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-21858817

RESUMO

The adenosine A(2A) receptor (A(2A) R) is a potential drug target for the treatment of Parkinson's disease and other neurological disorders. In rodents, the therapeutic efficacy of A(2A) R modulation is improved by concomitant modulation of the metabotropic glutamate receptor 5 (mGluR5). To elucidate the anatomical substrate(s) through which these therapeutic benefits could be mediated, pre-embedding electron microscopy immunohistochemistry was used to conduct a detailed, quantitative ultrastructural analysis of A(2A) R localization in the primate basal ganglia and to assess the degree of A(2A) R/mGluR5 colocalization in the striatum. A(2A) R immunoreactivity was found at the highest levels in the striatum and external globus pallidus (GPe). However, the monkey, but not the rat, substantia nigra pars reticulata (SNr) also harbored a significant level of neuropil A(2A) R immunoreactivity. At the electron microscopic level, striatal A(2A) R labeling was most commonly localized in postsynaptic elements (58% ± 3% of labeled elements), whereas, in the GPe and SNr, the labeling was mainly presynaptic (71% ± 5%) or glial (27% ± 6%). In both striatal and pallidal structures, putative inhibitory and excitatory terminals displayed A(2A) R immunoreactivity. Striatal A(2A) R/mGluR5 colocalization was commonly found; 60-70% of A(2A) R-immunoreactive dendrites or spines in the monkey striatum coexpress mGluR5. These findings provide the first detailed account of the ultrastructural localization of A(2A) R in the primate basal ganglia and demonstrate that A(2A) R and mGluR5 are located to interact functionally in dendrites and spines of striatal neurons. Together, these data foster a deeper understanding of the substrates through which A(2A) R could regulate primate basal ganglia function and potentially mediate its therapeutic effects in parkinsonism.


Assuntos
Gânglios da Base/metabolismo , Gânglios da Base/ultraestrutura , Corpo Estriado/metabolismo , Corpo Estriado/ultraestrutura , Receptor A2A de Adenosina/ultraestrutura , Receptores de Glutamato Metabotrópico/ultraestrutura , Sequência de Aminoácidos , Animais , Gânglios da Base/química , Corpo Estriado/química , Dendritos/genética , Dendritos/metabolismo , Dendritos/ultraestrutura , Feminino , Globo Pálido/química , Globo Pálido/metabolismo , Globo Pálido/ultraestrutura , Células HEK293 , Haplorrinos , Humanos , Macaca mulatta , Masculino , Dados de Sequência Molecular , Neurônios/química , Neurônios/metabolismo , Neurônios/ultraestrutura , Ratos , Ratos Sprague-Dawley , Receptor A2A de Adenosina/genética , Receptor A2A de Adenosina/metabolismo , Receptor de Glutamato Metabotrópico 5 , Receptores de Glutamato Metabotrópico/genética , Receptores de Glutamato Metabotrópico/metabolismo
4.
Methods Mol Biol ; 756: 345-56, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21870238

RESUMO

Many transmembrane receptors are regulated by associations with scaffold proteins containing PDZ domains, which interact with receptor carboxyl-termini to control receptor signaling, trafficking, and localization. We describe here approaches for detecting and characterizing interactions between receptors and PDZ scaffolds. These approaches include the construction and screening of proteomic arrays, blot overlays using fusion proteins, and co-immunoprecipitation studies to assess interactions in cells.


Assuntos
Domínios PDZ , Análise Serial de Proteínas/métodos , Proteômica/métodos , Receptores Acoplados a Proteínas G/metabolismo , Animais , Linhagem Celular , Humanos , Imunoprecipitação/métodos , Proteínas Recombinantes de Fusão/metabolismo
5.
J Biol Chem ; 286(33): 28914-28921, 2011 Aug 19.
Artigo em Inglês | MEDLINE | ID: mdl-21708946

RESUMO

GPR56 is an adhesion G protein-coupled receptor that plays a key role in cortical development. Mutations to GPR56 in humans cause malformations of the cerebral cortex, but little is known about the normal function of the receptor. We found that the large N terminus (NT) of GPR56 is cleaved from the rest of the receptor during processing but remains non-covalently associated with the seven-transmembrane region of the receptor, as indicated by coimmunoprecipitation of the two GPR56 fragments from both transfected cells and native tissue. We also found that truncation of the GPR56 NT results in constitutive activation of receptor signaling, as revealed by increased GPR56-stimulated signaling upon transfection of HEK-293 cells with truncated GPR56, greatly enhanced binding of ß-arrestins by truncated GPR56 relative to the full-length receptor, extensive ubiquitination of truncated GPR56, and cytotoxicity induced by truncated GPR56 that could be rescued by cotransfection of cells with ß-arrestin 2. Furthermore, we found that the GPR56 NT is capable of homophilic trans-trans interactions that enhance receptor signaling activity. On the basis of these findings, we suggest a model of receptor activation in which the large N terminus of GPR56 constrains receptor activity but N-terminal interactions (GPR56 NT with an extracellular ligand and/or GPR56 NT homophilic trans-trans associations) can remove this inhibitory influence of the N terminus to activate receptor signaling.


Assuntos
Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais/fisiologia , Arrestinas/genética , Arrestinas/metabolismo , Córtex Cerebral/anormalidades , Córtex Cerebral/metabolismo , Células HEK293 , Humanos , Mutação , Estrutura Terciária de Proteína , Receptores Acoplados a Proteínas G/genética , Ubiquitinação/genética , beta-Arrestina 2 , beta-Arrestinas
6.
Neurosci Lett ; 487(1): 3-7, 2011 Jan 03.
Artigo em Inglês | MEDLINE | ID: mdl-20430067

RESUMO

The astrocytic glutamate transporter GLAST (also known as EAAT1) is a key regulator of extracellular glutamate levels in many regions of vertebrate brains. To identify novel interacting partners that might regulate the localization and function of GLAST in astrocytes, we screened the transporter's C-terminus (GLAST-CT) against a proteomic array of 96 different PDZ domains. The GLAST-CT robustly and specifically interacted with PDZ domains from two related scaffolding proteins, the Na(+)/H(+) exchanger regulatory factors 1 and 2 (NHERF-1 and NHERF-2). Studies on cultured rat cortical astrocytes revealed that these cells are highly enriched in NHERF-2 relative to NHERF-1. Endogenous GLAST and NHERF-2 from cultured astrocytes were found to robustly co-immunoprecipitate, and further co-immunoprecipitation studies on mutant versions of GLAST expressed in transfected cells revealed the GLAST/NHERF-2 interaction to be dependent on the last amino acid of the GLAST-CT. Knockdown of endogenous NHERF-2 in astrocytes via siRNA treatment resulted in a significant reduction in GLAST activity, which corresponded to significantly reduced total expression of GLAST protein and reduced half-life of GLAST, as assessed in pulse-chase metabolic labeling studies. These findings reveal that NHERF-2 can interact with GLAST in astrocytes to enhance GLAST stability and activity.


Assuntos
Transportador 1 de Aminoácido Excitatório/metabolismo , Domínios PDZ/fisiologia , Fosfoproteínas/metabolismo , Trocadores de Sódio-Hidrogênio/metabolismo , Animais , Ácido Aspártico/farmacocinética , Astrócitos , Células Cultivadas , Embrião de Mamíferos , Transportador 1 de Aminoácido Excitatório/genética , Regulação da Expressão Gênica/genética , Humanos , Imunoprecipitação/métodos , Mutação de Sentido Incorreto/genética , Neocórtex/citologia , Domínios PDZ/efeitos dos fármacos , Fosfoproteínas/genética , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/genética , Estrutura Terciária de Proteína , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Trocadores de Sódio-Hidrogênio/genética , Transfecção/métodos , Trítio/farmacocinética
7.
Gastroenterology ; 140(3): 924-34, 2011 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-21134377

RESUMO

BACKGROUND & AIMS: Lysophosphatidic acid (LPA) is a potent inducer of colon cancer and LPA receptor type 2 (LPA(2)) is overexpressed in colon tumors. LPA(2) interacts with membrane-associated guanylate kinase with inverted orientation-3 (MAGI-3) and the Na+/H+ exchanger regulatory factor 2 (NHERF-2), but the biological effects of these interactions are unknown. We investigated the roles of MAGI-3 and NHERF-2 in LPA(2)-mediated signaling in human colon cancer cells. METHODS: We overexpressed or knocked down MAGI-3 in HCT116 and SW480 cells. The effects of MAGI-3 and NHERF-2 in LPA-induced cell migration, invasion, inositol phosphate generation, and nuclear factor-κB activation were determined. Expression of MAGI-3 and NHERF-2 in human colon tumor tissues was analyzed using tissue microarray analysis. RESULTS: NHERF-2 promoted migration and invasion of colon cancer cells, whereas MAGI-3 inhibited these processes. MAGI-3 competed with NHERF-2 for binding to LPA(2) and phospholipase C-ß3. However, NHERF-2 and MAGI-3 reciprocally regulated LPA(2)-induced phospholipase C activity. MAGI-3 increased the interaction of LPA(2) with Gα(12), whereas NHERF-2 preferentially promoted interaction between LPA(2) and Gα(q). MAGI-3 decreased the tumorigenic capacity of LPA(2) by attenuating the activities of nuclear factor-κB and c-Jun N-terminal kinase. MAGI-3 and NHERF-2 were expressed differentially in colon adenocarcinomas, consistent with their opposing effects. CONCLUSIONS: LPA(2) is dynamically regulated by 2 distinct PDZ proteins via modulation of G-protein coupling and receptor signaling. MAGI-3 is a negative regulator of LPA(2) signaling.


Assuntos
Adenocarcinoma/metabolismo , Neoplasias do Colo/metabolismo , Lisofosfolipídeos/metabolismo , Proteínas de Membrana/metabolismo , Fosfoproteínas/metabolismo , Receptores de Ácidos Lisofosfatídicos/metabolismo , Transdução de Sinais , Trocadores de Sódio-Hidrogênio/metabolismo , Adenocarcinoma/genética , Adenocarcinoma/patologia , Polipose Adenomatosa do Colo/genética , Polipose Adenomatosa do Colo/metabolismo , Animais , Movimento Celular , Neoplasias do Colo/genética , Neoplasias do Colo/patologia , Modelos Animais de Doenças , Subunidades alfa G12-G13 de Proteínas de Ligação ao GTP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Genes APC , Células HCT116 , Humanos , Fosfatos de Inositol/metabolismo , Proteínas Quinases JNK Ativadas por Mitógeno/metabolismo , Proteínas de Membrana/genética , Camundongos , NF-kappa B/metabolismo , Invasividade Neoplásica , Fosfolipase C beta/metabolismo , Fosfoproteínas/genética , Interferência de RNA , Receptores de Ácidos Lisofosfatídicos/genética , Trocadores de Sódio-Hidrogênio/genética , Fatores de Tempo , Análise Serial de Tecidos , Transfecção
8.
Nat Rev Mol Cell Biol ; 10(12): 819-30, 2009 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-19935667

RESUMO

G protein-coupled receptors (GPCRs) mediate physiological responses to various ligands, such as hormones, neurotransmitters and sensory stimuli. The signalling and trafficking properties of GPCRs are often highly malleable depending on the cellular context. Such fine-tuning of GPCR function can be attributed in many cases to receptor-interacting proteins that are differentially expressed in distinct cell types. In some cases these GPCR-interacting partners directly mediate receptor signalling, whereas in other cases they act mainly as scaffolds to modulate G protein-mediated signalling. Furthermore, GPCR-interacting proteins can have a big impact on the regulation of GPCR trafficking, localization and/or pharmacological properties.


Assuntos
Proteína Serina-Treonina Quinases de Interação com Receptores/metabolismo , Receptores Acoplados a Proteínas G/metabolismo , Animais , Humanos , Ligantes , Transporte Proteico , Transdução de Sinais
9.
J Am Chem Soc ; 131(6): 2056-7, 2009 Feb 18.
Artigo em Inglês | MEDLINE | ID: mdl-19161306

RESUMO

An efficient method for the asymmetric gold(I)-catalyzed preparation of medium sized rings has been developed. The method provides 7- to 9-membered rings in excellent yield. High enantioselectivities can be achieved for 7- and 8-membered ring products employing chiral gold(I) complexes. The results provide insight into the mechanism, showing the fluxional nature of gold(I)-stabilized vinyl carbenoid intermediates.


Assuntos
Alcinos/química , Cicloeptanos/síntese química , Ciclo-Octanos/síntese química , Ciclopropanos/síntese química , Catálise , Ciclização , Ouro/química
10.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 9): o2150, 2009 Aug 15.
Artigo em Inglês | MEDLINE | ID: mdl-21577559

RESUMO

The title compound, C(18)H(20)O(4), was prepared in high yield from 3,5-dimethoxy-styrene via a Ru-catalysed homo-olefin metathesis. Exclusive formation of the E-configurated isomer was observed. Inter-estingly, one symmetric unit contains two mol-ecules adopting an s-syn-anti and and an all-s-anti conformation.

11.
Acta Crystallogr Sect E Struct Rep Online ; 65(Pt 9): o2229, 2009 Aug 26.
Artigo em Inglês | MEDLINE | ID: mdl-21577628

RESUMO

The centrosymmetric title compound, C(18)H(26)O(4), was prepared in high yield from 4-acetoxy-styrene via Ru-catalysed homo-olefin metathesis. Exclusive formation of the E-configurated isomer was observed. In the crystal, a strong C-H⋯π inter-molecular inter-action links the mol-ecules together.

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