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1.
Blood ; 137(14): 1895-1904, 2021 04 08.
Artigo em Inglês | MEDLINE | ID: mdl-33036024

RESUMO

Chronic lymphocytic leukemia (CLL) major stereotyped subset 2 (IGHV3-21/IGLV3-21, ∼2.5% of all cases of CLL) is an aggressive disease variant, irrespective of the somatic hypermutation (SHM) status of the clonotypic IGHV gene. Minor stereotyped subset 169 (IGHV3-48/IGLV3-21, ∼0.2% of all cases of CLL) is related to subset 2, as it displays a highly similar variable antigen-binding site. We further explored this relationship through next-generation sequencing and crystallographic analysis of the clonotypic B-cell receptor immunoglobulin. Branching evolution of the predominant clonotype through intraclonal diversification in the context of ongoing SHM was evident in both heavy and light chain genes of both subsets. Molecular similarities between the 2 subsets were highlighted by the finding of shared SHMs within both the heavy and light chain genes in all analyzed cases at either the clonal or subclonal level. Particularly noteworthy in this respect was a ubiquitous SHM at the linker region between the variable and the constant domain of the IGLV3-21 light chains, previously reported as critical for immunoglobulin homotypic interactions underlying cell-autonomous signaling capacity. Notably, crystallographic analysis revealed that the IGLV3-21-bearing CLL subset 169 immunoglobulin retains the same geometry and contact residues for the homotypic intermolecular interaction observed in subset 2, including the SHM at the linker region, and, from a molecular standpoint, belong to a common structural mode of autologous recognition. Collectively, our findings document that stereotyped subsets 2 and 169 are very closely related, displaying shared immunoglobulin features that can be explained only in the context of shared functional selection.


Assuntos
Genes de Cadeia Pesada de Imunoglobulina/genética , Leucemia Linfocítica Crônica de Células B/genética , Receptores de Antígenos de Linfócitos B/genética , Cristalografia por Raios X , Regulação Leucêmica da Expressão Gênica , Rearranjo Gênico , Células HEK293 , Humanos , Modelos Moleculares , Domínios Proteicos , Receptores de Antígenos de Linfócitos B/química , Hipermutação Somática de Imunoglobulina
2.
Oncoimmunology ; 9(1): 1794359, 2020 07 16.
Artigo em Inglês | MEDLINE | ID: mdl-32923157

RESUMO

Pancreatic ductal adenocarcinoma (PDAC) is characterized by a prominent stromal reaction that has been variably implicated in both tumor growth and tumor suppression. B-lymphocytes have been recently implicated in PDAC progression but their contribution to the characteristic stromal desmoplasia has never been assessed before. In the present work, we aimed to verify whether B-lymphocytes contribute to stromal cell activation in PDAC. CD19+ B-lymphocytes purified from peripheral blood of patients with PDAC were cultivated in the presence of human pancreatic fibroblasts and cancer-associated fibroblasts. Released pro-fibrotic soluble factors and collagen production were assessed by ELISA and Luminex assays. Quantitative RT-PCR was used to assess fibroblast activation in the presence of B cells. The expression of selected pro-fibrotic and inflammatory molecules was confirmed on PDAC tissue sections by multi-color immunofluorescence studies. We herein demonstrate that B-cells from PDAC patients (i) produce the pro-fibrotic molecule PDGF-B and stimulate collagen production by fibroblasts; (ii) express enzymes implicated in extracellular matrix remodeling including LOXL2; and (iii) produce the chemotactic factors CCL-4, CCL-5, and CCL-11. In addition we demonstrate that circulating plasmablasts are expanded in the peripheral blood of patients with PDAC, stimulate collagen production by fibroblasts, and infiltrate pancreatic lesions. Our results indicate that PDAC is characterized by perturbations of the B-cell compartment with expansion of B-lymphocyte subsets that directly contribute to the stromal reaction observed at disease site. These findings provide an additional rationale for modulating B-cell activity in patients with pancreatic cancer.


Assuntos
Carcinoma Ductal Pancreático , Neoplasias Pancreáticas , Linfócitos B , Humanos , Pâncreas , Células Estromais
3.
Cells ; 9(1)2020 01 10.
Artigo em Inglês | MEDLINE | ID: mdl-31936702

RESUMO

Eukaryotic initiation factor 6 (eIF6) is necessary for the nucleolar biogenesis of 60S ribosomes. However, most of eIF6 resides in the cytoplasm, where it acts as an initiation factor. eIF6 is necessary for maximal protein synthesis downstream of growth factor stimulation. eIF6 is an antiassociation factor that binds 60S subunits, in turn preventing premature 40S joining and thus the formation of inactive 80S subunits. It is widely thought that eIF6 antiassociation activity is critical for its function. Here, we exploited and improved our assay for eIF6 binding to ribosomes (iRIA) in order to screen for modulators of eIF6 binding to the 60S. Three compounds, eIFsixty-1 (clofazimine), eIFsixty-4, and eIFsixty-6 were identified and characterized. All three inhibit the binding of eIF6 to the 60S in the micromolar range. eIFsixty-4 robustly inhibits cell growth, whereas eIFsixty-1 and eIFsixty-6 might have dose- and cell-specific effects. Puromycin labeling shows that eIF6ixty-4 is a strong global translational inhibitor, whereas the other two are mild modulators. Polysome profiling and RT-qPCR show that all three inhibitors reduce the specific translation of well-known eIF6 targets. In contrast, none of them affect the nucleolar localization of eIF6. These data provide proof of principle that the generation of eIF6 translational modulators is feasible.


Assuntos
Fatores de Iniciação de Peptídeos/metabolismo , Biossíntese de Proteínas , Subunidades Ribossômicas Maiores de Eucariotos/metabolismo , Linhagem Celular , Nucléolo Celular/efeitos dos fármacos , Nucléolo Celular/metabolismo , Sobrevivência Celular , Ensaio de Imunoadsorção Enzimática , Humanos , Iniciação Traducional da Cadeia Peptídica/efeitos dos fármacos , Polirribossomos/efeitos dos fármacos , Polirribossomos/metabolismo , Ligação Proteica/efeitos dos fármacos , Puromicina/farmacologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Reprodutibilidade dos Testes
4.
J Struct Biol ; 210(1): 107462, 2020 04 01.
Artigo em Inglês | MEDLINE | ID: mdl-31962159

RESUMO

Methionine adenosyltransferases catalyse the biosynthesis of S-adenosylmethionine, the primary methyl group donor in biochemical reactions, through the condensation of methionine and ATP. Here, we report the structural analysis of the Pyrococcus furiosus methionine adenosyltransferase (PfMAT) captured in the unliganded, substrate- and product-bound states. The conformational changes taking place during the enzymatic catalytic cycle are allosterically propagated by amino acid residues conserved in the archaeal orthologues to induce an asymmetric dimer structure. The distinct occupancy of the active sites within a PfMAT dimer is consistent with a half-site reactivity that is mediated by a product-induced negative cooperativity. The structures of intermediate states of PfMAT reported here suggest a distinct molecular mechanism for S-adenosylmethionine synthesis in Archaea, likely consequence of the evolutionary pressure to achieve protein stability under extreme conditions.


Assuntos
Metionina Adenosiltransferase/metabolismo , Pyrococcus furiosus/enzimologia , Sítios de Ligação , Catálise , Cristalografia por Raios X/métodos , Metionina Adenosiltransferase/química , Conformação Proteica , Pyrococcus furiosus/metabolismo
5.
Sci Rep ; 9(1): 13530, 2019 09 19.
Artigo em Inglês | MEDLINE | ID: mdl-31537859

RESUMO

Several cellular processes depend on networks of proteins assembled at specific sites near the plasma membrane. Scaffold proteins assemble these networks by recruiting relevant molecules. The scaffold protein ERC1/ELKS and its partners promote cell migration and invasion, and assemble into dynamic networks at the protruding edge of cells. Here by electron microscopy and single molecule analysis we identify ERC1 as an extended flexible dimer. We found that ERC1 scaffolds form cytoplasmic condensates with a behavior that is consistent with liquid phases that are modulated by a predicted disordered region of ERC1. These condensates specifically host partners of a network relevant to cell motility, including liprin-α1, which was unnecessary for the formation of condensates, but influenced their dynamic behavior. Phase separation at specific sites of the cell periphery may represent an elegant mechanism to control the assembly and turnover of dynamic scaffolds needed for the spatial localization and processing of molecules.


Assuntos
Movimento Celular/fisiologia , Proteínas do Tecido Nervoso/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Células COS , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Chlorocebus aethiops , Citoplasma/metabolismo , Humanos , Proteínas do Tecido Nervoso/fisiologia , Proteínas Associadas à Matriz Nuclear/metabolismo , Proteínas Associadas à Matriz Nuclear/fisiologia , Proteínas rab de Ligação ao GTP/fisiologia
6.
ACS Chem Biol ; 14(8): 1845-1854, 2019 08 16.
Artigo em Inglês | MEDLINE | ID: mdl-31345020

RESUMO

Mutations in the NPHP1 gene, coding for human nephrocystin-1 (NPHP1), cause the autosomal recessive disease nephronophthisis, the most common cause of end-stage renal disease in children and adolescents. The function and structure of NPHP1 are still poorly characterized. NPHP1 presents a modular structure well in keeping with its role as an adaptor protein: it harbors an SH3 domain flanked by two glutamic acid-rich regions and a conserved C-terminal nephrocystin homology domain (NHD). Similar to other NPHP protein family members, its N-terminus contains a putative coiled-coil domain (NPHP1CC) that is supposed to play an important role in NPHP1 self-association and/or protein-protein interactions. Structural studies proving its structure and its oligomerization state are still lacking. Here we demonstrate that NPHP1CC is monomeric in solution and unexpectedly folds into an autonomous domain forming a three-stranded antiparallel coiled coil suitable for protein-protein interactions. Notably, we found that the NPHP1CC shares remarkable structural similarities with the three-stranded coiled coil of the BAG domain protein family, which is known to mediate the anti-apoptotic function of these proteins, suggesting a possible similar role for NPHP1CC. In agreement with this hypothesis, we show that in the context of the full-length protein the NPHP1CC is fundamental to regulate resistance to apoptotic stimuli.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas Reguladoras de Apoptose/metabolismo , Proteínas do Citoesqueleto/metabolismo , Sequência de Aminoácidos , Animais , Cães , Humanos , Células Madin Darby de Rim Canino , Ressonância Magnética Nuclear Biomolecular , Conformação Proteica em alfa-Hélice , Domínios Proteicos , Dobramento de Proteína , Alinhamento de Sequência
7.
PLoS Comput Biol ; 14(2): e1006021, 2018 02.
Artigo em Inglês | MEDLINE | ID: mdl-29489822

RESUMO

Hypoxia inducible factors (HIFs) are transcription factors belonging to the basic helix-loop-helix PER-ARNT-SIM (bHLH-PAS) protein family with a role in sensing oxygen levels in the cell. Under hypoxia, the HIF-α degradation pathway is blocked and dimerization with the aryl hydrocarbon receptor nuclear translocator (ARNT) makes HIF-α transcriptionally active. Due to the common hypoxic environment of tumors, inhibition of this mechanism by destabilization of HIF-α:ARNT dimerization has been proposed as a promising therapeutic strategy. Following the discovery of a druggable cavity within the PAS-B domain of HIF-2α, research efforts have been directed to identify artificial ligands that can impair heterodimerization. Although the crystallographic structures of the HIF-2α:ARNT complex have elucidated the dimer architecture and the 0X3-inhibitor placement within the HIF-2α PAS-B, unveiling the inhibition mechanism requires investigation of how ligand-induced perturbations could dynamically propagate through the structure and affect dimerization. To this end, we compared evolutionary features, intrinsic dynamics and energetic properties of the dimerization interfaces of HIF-2α:ARNT in both the apo and holo forms. Residue conservation analysis highlighted inter-domain connecting elements that have a role in dimerization. Analysis of domain contributions to the dimerization energy demonstrated the importance of bHLH and PAS-A of both partners and of HIF-2α PAS-B domain in dimer stabilization. Among quaternary structure oscillations revealed by Molecular Dynamics simulations, the hinge-bending motion of the ARNT PAS-B domain around the flexible PAS-A/PAS-B linker supports a general model for ARNT dimerization in different heterodimers. Comparison of the HIF-2α:ARNT dynamics in the apo and 0X3-bound forms indicated a model of inhibition where the HIF-2α-PAS-B interfaces are destabilised as a result of water-bridged ligand-protein interactions and these local effects allosterically propagate to perturb the correlated motions of the domains and inter-domain communication. These findings will guide the design of improved inhibitors to contrast cell survival in tumor masses.


Assuntos
Translocador Nuclear Receptor Aril Hidrocarboneto/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Simulação de Dinâmica Molecular , Domínios Proteicos , Animais , Cristalografia por Raios X , Ligantes , Camundongos , Mutação , Oscilometria , Oxigênio/química , Ligação Proteica , Multimerização Proteica , Termodinâmica , Transcrição Genética , Água/química
8.
Nat Commun ; 8: 15746, 2017 06 09.
Artigo em Inglês | MEDLINE | ID: mdl-28598442

RESUMO

Cell-autonomous B-cell receptor (BcR)-mediated signalling is a hallmark feature of the neoplastic B lymphocytes in chronic lymphocytic leukaemia (CLL). Here we elucidate the structural basis of autonomous activation of CLL B cells, showing that BcR immunoglobulins initiate intracellular signalling through homotypic interactions between epitopes that are specific for each subgroup of patients with homogeneous clinicobiological profiles. The molecular details of the BcR-BcR interactions apparently dictate the clinical course of disease, with stronger affinities and longer half-lives in indolent cases, and weaker, short-lived contacts mediating the aggressive ones. The diversity of homotypic BcR contacts leading to cell-autonomous signalling reconciles the existence of a shared pathogenic mechanism with the biological and clinical heterogeneity of CLL and offers opportunities for innovative treatment strategies.


Assuntos
Leucemia Linfocítica Crônica de Células B/metabolismo , Receptores de Antígenos de Linfócitos B/metabolismo , Linfócitos B/química , Linfócitos B/metabolismo , Linhagem Celular Tumoral , Dimerização , Humanos , Leucemia Linfocítica Crônica de Células B/genética , Modelos Moleculares , Ligação Proteica , Domínios Proteicos , Receptores de Antígenos de Linfócitos B/química , Receptores de Antígenos de Linfócitos B/genética , Transdução de Sinais
9.
Sci Rep ; 5: 15401, 2015 Oct 21.
Artigo em Inglês | MEDLINE | ID: mdl-26486184

RESUMO

Ribosomes function as platforms for binding of other molecules, but technologies for studying this process are lacking. Therefore we developed iRIA (in vitro Ribosomes Interaction Assay). In approach I, Artemia salina ribosomes spotted on solid phase are used for binding picomoles of analytes; in approach II, cellular extracts allow the measurement of ribosome activity in different conditions. We apply the method to analyze several features of eIF6 binding to 60S subunits. By approach I, we show that the off-rate of eIF6 from preribosomes is slower than from mature ribosomes and that its binding to mature 60S occurs in the nM affinity range. By approach II we show that eIF6 binding sites on 60S are increased with mild eIF6 depletion and decreased in cells that are devoid of SBDS, a ribosomal factor necessary for 60S maturation and involved in Swachman Diamond syndrome. We show binding conditions to immobilized ribosomes adaptable to HT and quantify free ribosomes in cell extracts. In conclusion, we suggest that iRIA will greatly facilitate the study of interactions on the ribosomal surface.


Assuntos
Fatores de Iniciação em Eucariotos/genética , Técnicas In Vitro/métodos , Ribossomos/genética , Animais , Artemia/genética , Artemia/metabolismo , Sítios de Ligação , Fatores de Iniciação em Eucariotos/metabolismo , Humanos , Ligação Proteica/genética , Ribossomos/metabolismo
10.
Biochim Biophys Acta ; 1844(3): 656-62, 2014 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-24473221

RESUMO

A non-specific nucleoside hydrolase from Escherichia coli (RihC) has been cloned, overexpressed, and purified to greater than 95% homogeneity. Size exclusion chromatography and sodium dodecyl sulfate polyacrylamide gel electrophoresis show that the protein exists as a homodimer. The enzyme showed significant activity against the standard ribonucleosides with uridine, xanthosine, and inosine having the greatest activity. The Michaelis constants were relatively constant for uridine, cytidine, inosine, adenosine, xanthosine, and ribothymidine at approximately 480µM. No activity was exhibited against 2'-OH and 3'-OH deoxynucleosides. Nucleosides in which additional groups have been added to the exocyclic N6 amino group also exhibited no activity. Nucleosides lacking the 5'-OH group or with the 2'-OH group in the arabino configuration exhibited greatly reduced activity. Purine nucleosides and pyrimidine nucleosides in which the N7 or N3 nitrogens respectively were replaced with carbon also had no activity.


Assuntos
Escherichia coli/enzimologia , N-Glicosil Hidrolases/química , Catálise , Cromatografia em Gel , Eletroforese em Gel de Poliacrilamida , Concentração de Íons de Hidrogênio , Peso Molecular , N-Glicosil Hidrolases/isolamento & purificação , Solventes/química , Especificidade por Substrato
11.
Biochemistry ; 51(22): 4590-9, 2012 Jun 05.
Artigo em Inglês | MEDLINE | ID: mdl-22551416

RESUMO

The purine- and pyrimidine-specific nucleoside hydrolases (NHs) from the archaeon Sulfolobus solfataricus participate in the fundamental pathway of nucleotide catabolism and function to maintain adequate levels of free nitrogenous bases for cellular function. The two highly homologous isozymes display distinct specificities toward nucleoside substrates, and both lack the amino acids employed for activation of the leaving group in the hydrolytic reaction by the NHs characterized thus far. We determined the high-resolution crystal structures of the purine- and pyrimidine-specific NHs from S. solfataricus to reveal that both enzymes belong to NH structural homology group I, despite the different substrate specificities. A Na(+) ion is bound at the active site of the pyrimidine-specific NH instead of the prototypical Ca(2+), delineating a role of the metals in the catalytic mechanism of NHs in the substrate binding rather than nucleophile activation. A conserved His residue, which regulates product release in other homologous NHs, provides crucial interactions for leaving group activation in the archaeal isozymes. Modeling of the enzyme-substrate interactions suggests that steric exclusion and catalytic selection underlie the orthogonal base specificity of the two isozymes.


Assuntos
N-Glicosil Hidrolases/química , N-Glicosil Hidrolases/metabolismo , Sulfolobus solfataricus/enzimologia , Domínio Catalítico , Cristalografia por Raios X , Isoenzimas/química , Isoenzimas/metabolismo , Modelos Moleculares , Nucleosídeos/metabolismo , Conformação Proteica , Purinas/metabolismo , Pirimidinas/metabolismo , Sódio/metabolismo , Especificidade por Substrato , Sulfolobus solfataricus/química
12.
Biochemistry ; 49(41): 8999-9010, 2010 Oct 19.
Artigo em Inglês | MEDLINE | ID: mdl-20825170

RESUMO

Trypanosomes are purine-auxotrophic parasites that depend upon nucleoside hydrolase (NH) activity to salvage nitrogenous bases necessary for nucleic acid and cofactor synthesis. Nonspecific and purine-specific NHs have been widely studied, yet little is known about the 6-oxopurine-specific isozymes, although they are thought to play a primary role in the catabolism of exogenously derived nucleosides. Here, we report the first functional and structural characterization of the inosine-guanosine-specific NH from Trypanosoma brucei brucei. The enzyme shows near diffusion-limited efficiency coupled with a clear specificity for 6-oxopurine nucleosides achieved through a catalytic selection of these substrates. Pre-steady-state kinetic analysis reveals ordered product release, and a rate-limiting structural rearrangement that is associated with the release of the product, ribose. The crystal structure of this trypanosomal NH determined to 2.5 Å resolution reveals distinctive features compared to those of both purine- and pyrimidine-specific isozymes in the framework of the conserved and versatile NH fold. Nanomolar iminoribitol-based inhibitors identified in this study represent important lead compounds for the development of novel therapeutic strategies against trypanosomal diseases.


Assuntos
N-Glicosil Hidrolases/química , Nucleosídeos/química , Proteínas de Protozoários/química , Purinonas/química , Trypanosoma brucei brucei/enzimologia , Animais , Cristalografia por Raios X , Cinética , N-Glicosil Hidrolases/metabolismo , Nucleosídeos/metabolismo , Proteínas de Protozoários/metabolismo , Purinonas/metabolismo , Relação Estrutura-Atividade
13.
Biochemistry ; 47(15): 4418-26, 2008 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-18361502

RESUMO

Enzymes with nucleoside hydrolase activity (NHs) belonging to homology group I either are markedly specific for pyrimidine nucleoside substrates or hydrolyze with comparable efficiencies the N-glycosidic bond in all common nucleosides. The biochemical and structural basis for these differences in substrate specificity is still unknown. Here we characterize the binding interactions between the slowly hydrolyzed substrate inosine and the Escherichia coli pyrimidine-specific NH YeiK using cryotrapping and X-ray crystallography. Guided by the structural features of the Michaelis complex, we show the synergic effect of two specific point mutations in YeiK that increase the catalytic efficiency toward purine nucleosides to values comparable to those of natural nonspecific NHs. We demonstrate that the integrity of an active-site catalytic triad comprised of two hydroxylated amino acids and one histidine residue is a requirement for the highly efficient hydrolysis of inosine by group I NHs. Instead, cleavage of the YeiK-preferred substrate uridine is not affected by mutations at the same locations, suggesting a different fine chemical mechanism for the hydrolysis of the two nucleoside substrates. Our study provides for the first time direct evidence that distinct subsets of amino acid residues are involved in the hydrolysis of purine or pyrimidine nucleosides in group I NHs.


Assuntos
Proteínas de Escherichia coli/química , N-Glicosil Hidrolases/química , Sequência de Aminoácidos , Cristalografia por Raios X , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Inosina/química , Cinética , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , N-Glicosil Hidrolases/genética , N-Glicosil Hidrolases/metabolismo , Alinhamento de Sequência , Especificidade por Substrato , Uridina/química
14.
Nutr Metab Cardiovasc Dis ; 16(5): 322-9, 2006 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-16829340

RESUMO

BACKGROUND AND AIM: Based on the reported cardioprotective effects of resveratrol, a polyphenolic antioxidant abundant in grapes that binds to estrogen receptors, and the well-characterized anti-inflammatory properties of 17beta-estradiol, the effects of resveratrol on the functional expression of inflammatory enzymes were assessed in vascular smooth muscle cells (SMC) from normoglycaemic and streptozotocin-diabetic rats. METHODS AND RESULTS: SMC were isolated from the aorta four weeks after treating rats with streptozotocin or its vehicle. In SMC exposed to a cytokine mixture for 24h, unexpectedly, treatment with resveratrol (0.1-100microM) as well as the structurally related isoflavone genistein (1nM-1microM) enhanced expression of inducible NO synthase (iNOS). Genistein failed to mimic the elevated iNOS activity induced by resveratrol. Inhibition of estrogen receptors by the pure antiestrogen ICI 182,780 reversed the action of resveratrol on iNOS. In addition, resveratrol failed to alter cyclooxygenase-2 protein levels but reduced the accumulation of prostaglandin E(2) in the culture medium of SMC from normoglycaemic, but not diabetic rats. CONCLUSIONS: These results indicate that resveratrol, at concentrations approaching putative peak plasma levels in vivo, exhibited no anti-inflammatory properties in vascular SMC from normal and diabetic rats. By contrast, resveratrol displayed a potential pro-inflammatory activity in settings of vascular inflammation.


Assuntos
Anti-Inflamatórios não Esteroides/farmacologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/enzimologia , Óxido Nítrico Sintase Tipo II/metabolismo , Estilbenos/farmacologia , Animais , Aorta/enzimologia , Aorta/metabolismo , Diabetes Mellitus Experimental , Modelos Animais de Doenças , Relação Dose-Resposta a Droga , Estradiol/farmacologia , Genisteína/farmacologia , Masculino , Ratos , Ratos Sprague-Dawley , Receptores de Estrogênio/metabolismo , Resveratrol , Técnicas de Cultura de Tecidos
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