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1.
Eur J Pharm Sci ; 55: 46-57, 2014 May 13.
Artigo em Inglês | MEDLINE | ID: mdl-24486483

RESUMO

Diacylglycerol (DAG) is a central mediator of signaling pathways that regulate cell proliferation, survival and apoptosis. Therefore, C1 domain, the DAG binding site within protein kinase C (PKC) and other DAG effector proteins, is considered a potential cancer drug target. Derivatives of 5-(hydroxymethyl)isophthalic acid are a novel group of C1 domain ligands with antiproliferative and differentiation-inducing effects. Our previous work showed that these isophthalate derivatives exhibit antiproliferative and elongation-inducing effects in HeLa human cervical cancer cells. In this study we further characterized the effects of bis(3-trifluoromethylbenzyl) 5-(hydroxymethyl)isophthalate (HMI-1a3) on HeLa cell proliferation and morphology. HMI-1a3-induced cell elongation was accompanied with loss of focal adhesions and actin stress fibers, and exposure to HMI-1a3 induced a prominent relocation of cofilin-1 into the nucleus regardless of cell phenotype. The antiproliferative and morphological responses to HMI-1a3 were not modified by pharmacological inhibition or activation of PKC, or by RNAi knock-down of specific PKC isoforms, suggesting that the effects of HMI-1a3 were not mediated by PKC. Genome-wide gene expression microarray and gene set enrichment analysis suggested that, among others, HMI-1a3 induces changes in small GTPase-mediated signaling pathways. Our experiments revealed that the isophthalates bind also to the C1 domains of ß2-chimaerin, protein kinase D (PKD) and myotonic dystrophy kinase-related Cdc42-binding kinase (MRCK), which are potential mediators of small GTPase signaling and cytoskeletal reorganization. Pharmacological inhibition of MRCK, but not that of PKD attenuated HMI-1a3-induced cell elongation, suggesting that MRCK participates in mediating the effects of HMI-1a3 on HeLa cell morphology.


Assuntos
Forma Celular/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Proteínas Serina-Treonina Quinases/metabolismo , Transdução de Sinais/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Cofilina 1/metabolismo , Relação Dose-Resposta a Droga , Adesões Focais/efeitos dos fármacos , Adesões Focais/enzimologia , Células HeLa , Humanos , Miotonina Proteína Quinase , Fenótipo , Fosforilação , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/genética , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/genética , Interferência de RNA , Fibras de Estresse/efeitos dos fármacos , Fibras de Estresse/enzimologia , Fatores de Tempo , Transfecção
2.
Pharmacol Res ; 73: 44-54, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23643828

RESUMO

Protein kinase C (PKC) is a family of serine/threonine phosphotransferases ubiquitously expressed and involved in multiple cellular functions, such as proliferation, apoptosis and differentiation. The C1 domain of PKC represents an attractive drug target, especially for developing PKC activators. Dialkyl 5-(hydroxymethyl)isophthalates are a novel group of synthetic C1 domain ligands that exhibit antiproliferative effect in HeLa cervical carcinoma cells. Here we selected two isophthalates, HMI-1a3 and HMI-1b11, and characterized their effects in the human neuroblastoma cell line SH-SY5Y. Both of the active isophthalates exhibited significant antiproliferative and differentiation-inducing effects. Since HMI-1b11 did not impair cell survival even at the highest concentration tested (20µM), and supported neurite growth and differentiation of SH-SY5Y cells, we focused on studying its downstream signaling cascades and effects on gene expression. Consistently, genome-wide gene expression microarray and gene set enrichment analysis indicated that HMI-1b11 (10µM) induced changes in genes mainly related to cell differentiation. In particular, further studies revealed that HMI-1b11 exposure induced up-regulation of GAP-43, a marker for neurite sprouting and neuronal differentiation. These effects were induced by a 7-min HMI-1b11 treatment and specifically depended on PKCα activation, since pretreatment with the selective inhibitor Gö6976 abolished the up-regulation of GAP-43 protein observed at 12h. In parallel, we found that a 7-min exposure to HMI-1b11 induced PKCα accumulation to the cytoskeleton, an effect that was again prevented by pretreatment with Gö6976. Despite similar binding affinities to PKC, the isophthalates had different effects on PKC-dependent ERK1/2 signaling: HMI-1a3-induced ERK1/2 phosphorylation was transient, while HMI-1b11 induced a rapid but prolonged ERK1/2 phosphorylation. Overall our data are in accordance with previous studies showing that activation of the PKCα and ERK1/2 pathways participate in regulating neuronal differentiation. Furthermore, since PKC has been classified as one of the cognitive kinases, and activation of PKC is considered a potential therapeutic strategy for the treatment of cognitive disorders, our findings suggest that HMI-1b11 represents a promising lead compound in research aimed to prevent or counteract memory impairment.


Assuntos
Proteína GAP-43/metabolismo , Neuritos/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Proteína Quinase C-alfa/metabolismo , Diferenciação Celular , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Proteína GAP-43/genética , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Neuritos/fisiologia , Neuroblastoma/metabolismo , Ácidos Ftálicos/química , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/química , Inibidores de Proteínas Quinases/farmacologia , Estrutura Terciária de Proteína
3.
PLoS One ; 7(4): e35630, 2012.
Artigo em Inglês | MEDLINE | ID: mdl-22536418

RESUMO

Dysregulation of PKCε is involved in several serious diseases such as cancer, type II diabetes and Alzheimer's disease. Therefore, specific activators and inhibitors of PKCε hold promise as future therapeutics, in addition to being useful in research into PKCε regulated pathways. We have previously described llama single chain antibodies (VHHs) that specifically activate (A10, C1 and D1) or inhibit (E6 and G8) human recombinant PKCε. Here we report a thorough kinetic analysis of these VHHs. The inhibiting VHHs act as non-competitive inhibitors of PKCε activity, whereas the activating VHHs have several different modes of action, either increasing V(max) and/or decreasing K(m) values. We also show that the binding of the VHHs to PKCε is conformation-dependent, rendering the determination of affinities difficult. Apparent affinities are in the micromolar range based on surface plasmon resonance studies. Furthermore, the VHHs have no effect on the activity of rat PKCε nor can they bind the rat form of the protein in immunoprecipitation studies despite the 98% identity between the human and rat PKCε proteins. Finally, we show for the first time that the VHHs can influence PKCε function also in cells, since an activating VHH increases the rate of PKCε translocation in response to PMA in HeLa cells, whereas an inhibiting VHH slows down the translocation. These results give insight into the mechanisms of PKCε activity modulation and highlight the importance of protein conformation on VHH binding.


Assuntos
Ativadores de Enzimas/farmacologia , Proteína Quinase C-épsilon/metabolismo , Anticorpos de Cadeia Única/farmacologia , Animais , Encéfalo/enzimologia , Ativação Enzimática , Proteínas de Fluorescência Verde/metabolismo , Células HeLa , Humanos , Cinética , Ligação Proteica , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/química , Transporte Proteico , Ratos , Proteínas Recombinantes de Fusão/metabolismo , Anticorpos de Cadeia Única/biossíntese , Anticorpos de Cadeia Única/química , Especificidade da Espécie , Ressonância de Plasmônio de Superfície , Acetato de Tetradecanoilforbol/farmacologia
4.
PLoS One ; 6(5): e20053, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21629792

RESUMO

Diacylglycerol (DAG)-mediated signaling pathways, such as those mediated by protein kinase C (PKC), are central in regulating cell proliferation and apoptosis. DAG-responsive C1 domains are therefore considered attractive drug targets. Our group has designed a novel class of compounds targeted to the DAG binding site within the C1 domain of PKC. We have previously shown that these 5-(hydroxymethyl)isophthalates modulate PKC activation in living cells. In this study we investigated their effects on HeLa human cervical cancer cell viability and proliferation by using standard cytotoxicity tests and an automated imaging platform with machine vision technology. Cellular effects and their mechanisms were further characterized with the most potent compound, HMI-1a3. Isophthalate derivatives with high affinity to the PKC C1 domain exhibited antiproliferative and non-necrotic cytotoxic effects on HeLa cells. The anti-proliferative effect was irreversible and accompanied by cell elongation. HMI-1a3 induced down-regulation of retinoblastoma protein and cyclins A, B1, D1, and E. Effects of isophthalates on cell morphology, cell proliferation and expression of cell cycle-related proteins were different from those induced by phorbol 12-myristate-13-acetate (PMA) or bryostatin 1, but correlated closely to binding affinities. Therefore, the results strongly indicate that the effect is C1 domain-mediated.


Assuntos
Ciclo Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Ácidos Ftálicos/farmacologia , Proteína Quinase C/metabolismo , Western Blotting , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Ciclina A/metabolismo , Ciclina B1/metabolismo , Ciclina D1/metabolismo , Diglicerídeos/metabolismo , Células HeLa , Humanos , Ácidos Ftálicos/química , Proteína Quinase C/química , Estrutura Terciária de Proteína , Proteína do Retinoblastoma/metabolismo
5.
Curr Top Med Chem ; 11(11): 1370-92, 2011.
Artigo em Inglês | MEDLINE | ID: mdl-21513495

RESUMO

The second messenger diacylglycerol (DAG) plays a central role in the signal transduction of G-protein coupled receptors and receptor tyrosine kinases by binding to C1 domain of effector proteins. C1 domain was first identified in protein kinase C (PKC) which comprises a family of ten isoforms that play roles in diverse cellular processes such as proliferation, apoptosis and differentiation. Aberrant signaling through PKC isoforms and other C1 domain-containing proteins has been implicated in several pathological disorders. Drug discovery concerning C1 domains has exploited both natural products and rationally designed compounds. Currently, molecules from several classes of C1 domain-binding compounds are in clinical trials; however, still more have the potential to enter the drug development pipeline. This review gives a summary of the recent developments in C1 domain-binding compounds.


Assuntos
Fatores Biológicos/farmacologia , Descoberta de Drogas , Proteína Quinase C/antagonistas & inibidores , Proteína Quinase C/química , Inibidores de Proteínas Quinases/farmacologia , Animais , Fatores Biológicos/síntese química , Fatores Biológicos/química , Humanos , Ligantes , Proteína Quinase C/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/química , Estrutura Terciária de Proteína , Relação Estrutura-Atividade
6.
Eur J Pharm Sci ; 42(4): 332-9, 2011 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-21220006

RESUMO

The 10 isozymes of the protein kinase C (PKC) family can have different roles on the same biological process, making isozyme specific analysis of function crucial. Currently, only few pharmacological compounds with moderate isozyme specific effects exist thus hampering research into individual PKC isozymes. The antigen binding regions of camelid single chain antibodies (VHHs) could provide a solution for obtaining PKC isozyme specific modulators. In the present study, we have successfully selected and characterized PKCɛ specific VHH antibodies from two immune VHH libraries using phage display. The VHHs were shown to exclusively bind to PKCɛ in ELISA and immunoprecipitation studies. Strikingly, five of the VHHs had an effect on PKCɛ kinase activity in vitro. VHHs A10, C1 and D1 increased PKCɛ kinase activity in a concentration-dependent manner (EC(50) values: 212-310nM), whereas E6 and G8 inhibited PKCɛ activity (IC(50) values: 103-233nM). None of these VHHs had an effect on the activity of the other novel PKC isozymes PKCδ and PKCθ. To our knowledge, these antibodies are the first described VHH activators and inhibitors for a protein kinase. Furthermore, the development of PKCɛ specific modulators is an important contribution to PKC research.


Assuntos
Isoenzimas/imunologia , Proteína Quinase C-épsilon , Anticorpos de Cadeia Única/imunologia , Anticorpos de Cadeia Única/isolamento & purificação , Sequência de Aminoácidos , Animais , Camelídeos Americanos/imunologia , Regiões Determinantes de Complementaridade , Humanos , Cadeias Pesadas de Imunoglobulinas , Concentração Inibidora 50 , Isoenzimas/antagonistas & inibidores , Biblioteca de Peptídeos , Ligação Proteica , Proteína Quinase C-épsilon/antagonistas & inibidores , Proteína Quinase C-épsilon/imunologia
7.
Eur J Pharm Sci ; 40(5): 404-11, 2010 Aug 11.
Artigo em Inglês | MEDLINE | ID: mdl-20433920

RESUMO

HIV-1 Tat-peptide is widely used as a vector for cargo delivery into intact cells. As a cationic, arginine-rich peptide it can readily penetrate the plasma membrane and facilitate the penetration of impermeable bioactive molecules such as proteins, peptides, nucleic acids and drugs. Although at first considered as an inert vector, recent studies have however shown that it might have effects on its own on various cellular processes. In the present study we have investigated the effects of the Tat-peptide(48-60) on two basic serine/threonine kinases, protein kinase C and A, since earlier studies have shown that certain arginine-rich peptides or proteins might have a modulatory effect on their activity. In in vitro studies, Tat-peptide inhibited PKC alpha in a concentration-dependent manner with an IC(50)-value of 22nM and PKA with an IC(50)-value of 1.2 microM. The mode of inhibition was studied in the presence of increasing concentrations of a substrate peptide or ATP. Tat-peptide competed with the kinase substrates, however it did not compete with ATP. In a panel of 70 kinases Tat-peptide showed inhibitory activity at least towards other AGC-family kinases (PKB, SGK1, S6K1, MSK1), CAMK-family kinases (CAMK1 and MELK) and a STE family kinase (MKK1). In HeLa cells Tat-peptide inhibited the phorbol ester-evoked ERK1/2 phosphorylation suggesting that Tat inhibited PKC also in intact cells. In thyroid cells Tat-peptide attenuated sphingosylphosphorylcholine-evoked Ca(2+)-fluxes, which have earlier been shown to be dependent on PKC. Taken together, these results indicate that the Tat-peptide(48-60) is a potent inhibitor which binds to the substrate binding site of the basophilic kinase domain.


Assuntos
Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Fragmentos de Peptídeos/farmacologia , Proteína Quinase C/antagonistas & inibidores , Produtos do Gene tat do Vírus da Imunodeficiência Humana/farmacologia , Trifosfato de Adenosina/metabolismo , Cálcio/metabolismo , Linhagem Celular Tumoral , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Células HeLa , Humanos , Cinética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fragmentos de Peptídeos/metabolismo , Peptídeos/metabolismo , Fosforilação , Proteína Quinase C/metabolismo , Glândula Tireoide/metabolismo , Produtos do Gene tat do Vírus da Imunodeficiência Humana/metabolismo
8.
J Med Chem ; 52(13): 3969-81, 2009 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-19438240

RESUMO

Protein kinase C (PKC) is a widely studied molecular target for the treatment of cancer and other diseases. We have approached the issue of modifying PKC function by targeting the C1 domain in the regulatory region of the enzyme. Using the X-ray crystal structure of the PKC delta C1b domain, we have discovered conveniently synthesizable derivatives of dialkyl 5-(hydroxymethyl)isophthalate that can act as potential C1 domain ligands. Structure-activity studies confirmed that the important functional groups predicted by modeling were indispensable for binding to the C1 domain and that the modifications of these groups diminished binding. The most promising compounds were able to displace radiolabeled phorbol ester ([(3)H]PDBu) from PKC alpha and delta at K(i) values in the range of 200-900 nM. Furthermore, the active isophthalate derivatives could modify PKC activation in living cells either by inducing PKC-dependent ERK phosphorylation or by inhibiting phorbol-induced ERK phosphorylation. In conclusion, we report here, for the first time, that derivatives of isophthalic acid represent an attractive novel group of C1 domain ligands that can be used as research tools or further modified for potential drug development.


Assuntos
Ácidos Ftálicos/síntese química , Proteína Quinase C/antagonistas & inibidores , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Desenho de Fármacos , Ativação Enzimática , Células HeLa , Humanos , Insetos , Fosforilação , Ácidos Ftálicos/farmacologia , Proteína Quinase C/metabolismo , Proteína Quinase C-alfa/antagonistas & inibidores , Proteína Quinase C-alfa/metabolismo , Proteína Quinase C-delta/antagonistas & inibidores , Proteína Quinase C-delta/metabolismo , Inibidores de Proteínas Quinases/síntese química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade
9.
Eur J Pharmacol ; 544(1-3): 21-30, 2006 Aug 21.
Artigo em Inglês | MEDLINE | ID: mdl-16846598

RESUMO

Chronic nicotine exposure induces upregulation of nicotinic receptors, but the mechanisms underlying this phenomenon are not well understood. The aim of this study was to examine the role of different second messenger systems in the nicotine-induced upregulation of alpha7-nicotinic receptors in SH-EP1-halpha7 human epithelial cells. We show here that chronic exposure to nicotine results in accumulation of cAMP. Furthermore, an enhanced cAMP signalling potentiates nicotine-induced upregulation of alpha7-nicotinic receptors measured by [3H]methyllycaconitine ([3H]MLA) binding suggesting that cAMP is involved in the alpha7-nicotinic receptor upregulation. Down-regulation of protein kinase C (PKC) with a phorbol ester abolishes the nicotine-induced upregulation of alpha7-nicotinic receptors. Furthermore, overexpression of PKCalpha in SH-EP1-halpha7 cells results in potentiation of nicotine-evoked upregulation indicating that PKC has a role in regulation of alpha7-nicotinic receptor number. The Ca2+-calmodulin kinase II (CaMKII) and extracellular signal regulated kinase 1/2 (ERK1/2) appear not to participate in alpha7-nicotinic receptor upregulation since the specific inhibitors of these kinases did not have an effect on the nicotine-induced upregulation. Taken together this study provides evidence that nicotine induces accumulation of cAMP and that the upregulation mechanisms of alpha7-nicotinic receptors are potentiated both by cAMP and PKC. As nicotine-evoked upregulation of heteromeric nicotinic receptors in SH-SY5Y cells was unaffected by the treatment with drugs affecting cAMP signalling or PKC activity, our results suggest that the upregulation mechanisms of homomeric alpha7-nicotinic receptors and heteromeric nicotinic receptors differ from each other.


Assuntos
AMP Cíclico/metabolismo , Células Epiteliais/metabolismo , Nicotina/metabolismo , Proteína Quinase C/metabolismo , Receptores Nicotínicos/biossíntese , Regulação para Cima , Cálcio/metabolismo , Calmodulina/metabolismo , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Humanos , Ligantes , Nicotina/farmacologia , Agonistas Nicotínicos/farmacologia , Proteína Quinase C-alfa/metabolismo , Receptor Nicotínico de Acetilcolina alfa7
10.
Cell Signal ; 18(10): 1671-8, 2006 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-16490345

RESUMO

Several sphingolipid derivatives, including sphingosylphosphorylcholine (SPC), regulate a multitude of biological processes. In the present study we show that both human thyroid cancer cells (FRO cells) and normal human thyroid cells express G protein-coupled receptor 4 (GPR4) and ovarian cancer G protein-coupled receptor 1 (OGR1), putative SPC-specific receptors. In FRO cells SPC evoked a concentration-dependent increase in intracellular free calcium concentration ([Ca2+]i) in a calcium containing, but not in a calcium-free buffer. Sphingosine 1-phosphate (S1P) evoked an increase in [Ca2+]i in both a calcium containing and a calcium-free buffer. The phospholipase C (PLC) inhibitor U 73122 potently attenuated the effect of SPC, suggesting that effects of SPC were mediated by a G protein coupled receptor. Overnight pretreatment of the cells with pertussis toxin did not affect the SPC-evoked response. Interestingly, SPC did not evoke an increase in inositol phosphates, although S1P did so. Furthermore, in cells pretreated with thapsigargin to deplete intracellular calcium stores, SPC still evoked an increase in [Ca2+]i, suggesting that SPC mainly evoked entry of extracellular calcium. When the cells were pretreated with the protein kinase C (PKC) inhibitor GF 109203X, or when the cells were pretreated with PMA for 24 h, the SPC-evoked calcium entry was attenuated. Thus, the SPC-evoked calcium entry was apparently dependent on PKC. In sharp contrast, the increase in [Ca2+]i evoked by S1P was not sensitive to GF 109203X. Furthermore, the calcium entry evoked by the diacylglycerol analog 1-oleoyl-2-acetyl-sn-glycerol was not inhibited by GF 109203X. In addition, SPC decreased the incorporation of 3H-thymidine in a concentration-dependent manner in FRO cells. Taken together, SPC may be an important factor regulating thyroid cancer cell function.


Assuntos
Sinalização do Cálcio/efeitos dos fármacos , Cálcio/metabolismo , Fosforilcolina/análogos & derivados , Proteína Quinase C/metabolismo , Esfingosina/análogos & derivados , Glândula Tireoide/citologia , Glândula Tireoide/efeitos dos fármacos , Compostos de Boro/farmacologia , Células Cultivadas , Gadolínio/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Humanos , Lisofosfolipídeos/farmacologia , Fosforilcolina/farmacologia , Isoformas de Proteínas/antagonistas & inibidores , Isoformas de Proteínas/metabolismo , Proteína Quinase C/antagonistas & inibidores , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Receptores Acoplados a Proteínas G/genética , Esfingosina/farmacologia , Tapsigargina/farmacologia , Timidina/metabolismo , Glândula Tireoide/enzimologia , Glândula Tireoide/metabolismo , Trítio
11.
Cell Signal ; 16(12): 1417-24, 2004 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-15381257

RESUMO

Tumor necrosis factor alpha (TNFalpha) alters the electrophysiological properties of many cell types. In thyroid cells however, the effects have not yet been elucidated. Here, we report the effect of TNFalpha and its second messenger ceramide on the resting membrane potential (RMP) of thyroid FRTL-5 cells. In patch-clamp experiments, we showed that TNFalpha and ceramide depolarise the RMP by inhibiting an acid-sensitive inwardly rectifying potassium current. This depolarisation depended on the activation of protein kinase Czeta (PKCzeta), because it can be blocked by calphostin C, a PKC-inhibitory peptide and a specific inhibitor peptide for PKCzeta. The activation of PKCzeta was confirmed by Western blotting, in which a stimulation with TNFalpha led to the translocation of PKCzeta to the particulate fraction. We conclude that TNFalpha and ceramide depolarise the RMP of thyroid FRTL-5 cells by attenuating a Ba(2+)- and acid-sensitive potassium conductance via activation of PKCzeta.


Assuntos
Ceramidas/farmacologia , Potenciais da Membrana/efeitos dos fármacos , Canais de Potássio/metabolismo , Proteína Quinase C/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Animais , Bário/farmacologia , Western Blotting , Linhagem Celular , Proliferação de Células , Ceramidas/química , Eletrofisiologia , Ativação Enzimática , Concentração de Íons de Hidrogênio , Íons , Técnicas de Patch-Clamp , Peptídeos/química , Transporte Proteico , Ratos , Fatores de Tempo
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