Navegando por Palavras-chave "binding"

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    Fear conditioning performance and NMDA receptor subtypes: NR2A differential expression in the striatum
    (Elsevier B.V., 2006-04-28) Schenberg, E. E.; Ferreira, T. L.; Figueredo, L. Z.; Hipolide, D. C.; Nobrega, J. N.; Oliveira, MGM; Universidade Federal de São Paulo (UNIFESP); Ctr Addict & Mental Hlth
    While considerable evidence implicates NMDA receptors in the hippocampus in contextual fear conditioning, the role of other brain regions is less well understood. To further investigate this issue, rats were subjected to a contextual fear conditioning task and then classified as high or low responders according to performance. Density of NMDA receptors was evaluated using [H-3]MK-801 autoradiography in 52 brain areas and expression of NR2A and NR2B subunits was studied with in situ hybridization in the same brains. Results revealed no differences between high- and low-performance rats in NMDA receptor binding in any of the brain areas studied. Similarly, NR2B subunit expression was also not different between groups. However, NR2A expression was significantly higher in the caudate-putamen of low-performance rats. These results suggest that NMDA receptors in the caudate-putamen may also be involved in contextual fear conditioning performance. (c) 2006 Elsevier Inc. All rights reserved.
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    Functional assessment of angiotensin II and bradykinin analogues containing the paramagnetic amino acid TOAC
    (Elsevier B.V., 2008-02-01) Santos, Edson L. [UNIFESP]; Souza, Kely de Picoli [UNIFESP]; Sabatini, Regiane A. [UNIFESP]; Martin, Renan P. [UNIFESP]; Fernandes, Litiam [UNIFESP]; Nardi, Daniela T. [UNIFESP]; Malavolta, Luciana [UNIFESP]; Shimuta, Suma I. [UNIFESP]; Nakaie, Clovis R. [UNIFESP]; Pesquero, Joao B. [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    This study characterized pharmacologically the functional responses to agonists angiotensin II (AngII) and bradykinin (BK) derivatives containing the TOAC (2,2,6,6-tetramethylpiperidine-Noxyl-4-amino-4-carboxylic acid) spin [abet at the N-terminal (TOAC(1)-AngII and TOAC(0)-BK) and internal (TOAC(3)-AngII and TOAC(3)-BK) positions of these vasoactive peptides. Affinity constants of the ligands for AT(1) and B-2 receptors were evaluated in vitro by binding assays and biological effects by extracellular acidification rates and in vivo by blood pressure responses. in contrast to internally labeled analogues (TOAC(3)-AngII or TOAC(3)-BK), the TOAC(1)-AngII and TOAC(0)-BK derivatives dose-dependently increased the extracellular acidification rate in adherent cultured Chinese hamster ovary (CHO) cells expressing AT, or B2 receptors, respectively. in addition, TOAC(1)-AngII induced an increase in blood pressure when injected intravenously in awaken rats although with a potency four times smaller when compared to native AngII. Similarly to BK, TOAC(0)-BK dose-dependently decreased blood pressure when injected intra-arterially in rats with a lower potency when compared to the native peptide. On the contrary, TOAC(3)-AngII or TOAC(3)-BK did not provoke any alteration in blood pressure levels. in summary, our results confirmed that the insertion of TOAC-probe in the N-terminal region of peptides does not significantly modify the affinity or biological activity in vitro and in vivo conditions and could be an important toool to evaluate peptide-receptor interaction mechanism. Conversely, possibly due to the unique bend-inducing property of the cyclic TOAC probe, its insertion at position 3 in both AngII and BK structures seems to restrict the interaction and the activation of the AT, and B2 receptors. (c) 2007 Elsevier B.V. All rights reserved.
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    Interaction between Shiga Toxin and Monoclonal Antibodies: Binding Characteristics and in Vitro Neutralizing Abilities
    (Mdpi Ag, 2012-09-01) Rocha, Leticia B.; Luz, Daniela E.; Moraes, Claudia T. P.; Caravelli, Andressa; Fernandes, Irene; Guth, Beatriz Ernestina Cabilio [UNIFESP]; Horton, Denise S. P. Q.; Piazza, Roxane M. F.; Butantan Inst; Universidade Federal de São Paulo (UNIFESP)
    Monoclonal antibodies (MAbs) have been employed either for diagnosis or treatment of infections caused by different pathogens. Specifically for Shiga toxin-producing Escherichia coli (STEC), numerous immunoassays have been developed for STEC diagnosis, showing variability in sensitivity and specificity when evaluated by reference laboratories, and no therapy or vaccines are currently approved. Thus, the aim of this work was the characterization of the interaction between MAbs against Stx1 and Stx2 toxins and their neutralizing abilities to enable their use as tools for diagnosis and therapy. the selected clones designated 3E2 (anti-Stx1) and 2E11 (anti-Stx2) were classified as IgG1. 3E2 recognized the B subunit of Stx1 with an affinity constant of 2.5 x 10(-10) M, detected as little as 6.2 ng of Stx1 and was stable up to 50 degrees C. in contrast, 2E11 recognized the A subunit of Stx2, was stable up to 70 degrees C, had a high dissociation constant of 6.1 x 10(-10) M, and detected as little as 12.5 ng of Stx2. Neutralization tests showed that 160 ng of 3E2 MAb inhibited 80% of Stx1 activity and 500 mu g 2E11 MAb were required for 60% inhibition of Stx2 activity. These MAb amounts reversed 25 to 80% of the cytotoxicity triggered by different STEC isolates. in conclusion, these MAbs show suitable characteristics for their use in STEC diagnosis and encourage future studies to investigate their protective efficacy.
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    Rapid eye movement sleep deprivation-induced down-regulation of beta-adrenergic receptors in the rat brainstem and hippocampus
    (Elsevier B.V., 2004-09-01) Pedrazzoli, M.; Venditti, Marco Antonio Campana [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    Rapid eye movement (REM) sleep deprivation induces a cortical down-regulation of beta-adrenergic receptors. Down-regulation of cortical beta-adrenergic receptors is consistently observed after a number of different chronic antidepressant treatments (drugs and electroconvulsive shock). REM sleep deprivation has an antidepressant effect in humans, and in rats, it decreases immobility in the behavioral despair test, an effect also produced by antidepressant treatments. To verify whether REM sleep deprivation also affects hippocampal beta-adrenergic receptors, we carried out the binding of [H-3]-dihydroalprenolol ([H-3]-DHA) to hippocampal membranes from rats deprived of REM sleep for 96 h. We also determined the binding of [H-3]-DHA to brainstem membranes, a brain region where noradrenergic nuclei are located. Rats were deprived of REM sleep using a water tank with multiple small platforms. [H-3-DHA] saturation conditions (concentrations ranging from 0.15 to 6 nM) were obtained in a crude hippocampus and brainstem membrane preparation. Nonspecific binding was determined using DL-propranolol in hippocampus homogenates. in the brainstem homogenates, nonspecific binding was determined in the presence of DL-propranolol or L-isoproterenol. the results obtained showed statistically significant down-regulation of beta-adrenergic receptors in both the hippocampus and the brainstem after REM sleep deprivation. in the hippocampus, there was also a significant decrease in the dissociation constant (K-D). in the brainstem, a significant decrease in K-D was observed when DL-propranolol was used to determine nonspecific binding. the down-regulation of beta-adrenergic receptors in the hippocampus and brainstem suggests the involvement of these brain areas in the antidepressant effect of REM sleep deprivation. (C) 2004 Elsevier Inc. All rights reserved.
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    Relação estrutura-atividade entre os receptores AT1 e AT2 da angiotensina II à luz da estrutura do receptor CXCR4 da quimiocina
    (Universidade Federal de São Paulo (UNIFESP), 2014) Martin, Renan Paulo [UNIFESP]; Shimuta, Suma Imura [UNIFESP]; Universidade Federal de São Paulo (UNIFESP)
    A angiotensina II (AngII) e um potente agente do sistema renina-angiotensina participando da regulacao da pressao arterial e na homeostase hidromineral. Seu efeitos fisiologicos sao mediados pelos receptores tipo 1 (AT1) e tipo 2 (AT2) que possuem respostas contrarias nas suas vias de sinalizacao. Alguns modelos de interacao agonista-receptor vem sendo propostos onde e possivel destacar alguns residuos importantes para interacao do agonista ao receptor como Asp1, Arg2 e Tyr4 alem das extremidades N e C-terminais. Numa etapa inicial foi proposta para avaliar a possiblidade da substituicao do metodo de estudo de ligacao tradicional baseado em uso de radioligantes por um nao radioativo (fluoroforo). Foi entao utilizado o lantanideo Europio (Eu3+) como marcador do ligante nos ensaios de competicao pelo sitio de ligacao comparando ao uso do 3H. Depois disso, o projeto foi dividido em duas etapas, sendo a primeira, uma abordagem pratica, onde diferentes ligantes foram testados e as afinidades de ligacao de cada um comparada com aquelas dos receptores AT1 e AT2. Posteriormente foram obtidas 3 mutacoes sitio dirigidas no receptor AT2 onde a primeira causou o rompimento da segunda ponte SS (C35S), a segunda a substituicao das Asn127 por Gly no meio da helice III (N127G) e a terceira a substituicao da Asp297 por Ala (D297A). Alem disso foi proposto um metodo teorico de estudo usando modelagem molecular dos receptores AT1 e AT2 por homologia ao CXCR4. Os testes de ligacao usando os dois metodos propostos mostraram resultados equivalentes, razao pela qual se adotou o metodo fluorimetrico para o prosseguimento dos experimentos. E interessante destacar que a substituicao da Tyr4 por Ile provocou uma queda de afinidade de cerca de 100 vezes enquanto o uso da Phe na mesma posicao quase nao afetou a afinidade do ligante ao receptor AT1. Por outro lado o mesmo experimento conduzido com o receptor AT2 mostrou nao haver diferenca significativa. Esses resultados estao de acordo com os da literatura, uma vez que o residuo Asn111 do AT1 parece funcionar como botao que controla as mudancas do estado nao ativado para um estado propicio a ativacao que e um estado essencial para a correta acomodacao do ligante no receptor. Entretanto o receptor AT2 parece ja estar nesse estado independentemente da presenca desse residuo. Alem disso, o receptor AT2 possui um sitio de interacao mais flexivel do que o do AT1 que parece ser mais critico as mudancas no ligante. Na analise mutacional abordada, tanto o mutante C35S quanto o N127G nao apresentaram mudancas significativas no perfil de resposta aos ligantes estudados. Por outro lado o receptor AT2 carregando a mutacao D297A sofreu uma queda drastica na capacidade de ligacao com todos os analogos testados, indicando assim que esse residuo e crucial para a interacao com o segmento N-terminal da AngII. Os resultados de dinamica corroboram com os dados experimentais reforcando a melhor acomodacao da molecula de AngII ao receptor AT2 quando comparada ao receptor AT1
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    Up-regulation of Ca2+ channels in vas deferens after chronic treatment of newborn rats with nifedipine
    (Elsevier B.V., 2002-05-17) Verde, L. F.; Lafayette, SSL; Caricati-Neto, A.; Jurkiewicz, N. H.; Jurkiewicz, A.; Universidade Federal de São Paulo (UNIFESP)
    Radioligand binding and contraction techniques were used to verify if L-type Ca2+ charnels are modified in rat vas deferens after treatment with the blocker nifedipine (15 mug), injected at 7, 14, 21 and 28 days after birth. Vas deferens tissue was used 10, 30 and 90 days after the last injection, to verify if modifications are persistent. Binding studies with cell membranes, using [H-3]isradipine, showed an increase of the density (B-max) of Ca2+ channels by more than 60%, after 10 and 30 days, without changes of affinity (K-d). Maximal contractions (E-max) of KCl, were increased by 106% and 37%, respectively, after 10 and 30 days, without changes of apparent affinity (pD(2)). After 90 days, the values of B-max, K-d, E-max and pD(2) were not different from the controls. Differences were also not found for rats injected when adult. It is concluded that treatment of newborn, but not of adult, rats with nifedipine produced a long-lasting, though reversible, upregulation of L-type Ca2+ channels. (C) 2002 Elsevier Science B.V. All rights reserved.