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1.
Sci Rep ; 11(1): 19313, 2021 Sep 29.
Artigo em Inglês | MEDLINE | ID: mdl-34588569

RESUMO

Shigellosis, caused by Shigella bacterial spp., is one of the leading causes of diarrheal morbidity and mortality. An increasing prevalence of multidrug-resistant Shigella species has revived the importance of bacteriophages as an alternative therapy to antibiotics. In this study, a novel bacteriophage, Sfk20, has been isolated from water bodies of a diarrheal outbreak area in Kolkata (India) with lytic activity against many Shigella spp. Phage Sfk20 showed a latent period of 20 min and a large burst size of 123 pfu per infected cell in a one-step growth analysis. Phage-host interaction and lytic activity confirmed by phage attachment, intracellular phage development, and bacterial cell burst using ultrathin sectioning and TEM analysis. The genomic analysis revealed that the double-stranded DNA genome of Sfk20 contains 164,878 bp with 35.62% G + C content and 241 ORFs. Results suggested phage Sfk20 to include as a member of the T4 myoviridae bacteriophage group. Phage Sfk20 has shown anti-biofilm potential against Shigella species. The results of this study imply that Sfk20 has good possibilities to be used as a biocontrol agent.

2.
FASEB J ; 35(9): e21814, 2021 09.
Artigo em Inglês | MEDLINE | ID: mdl-34369624

RESUMO

Alteration in glucose homeostasis during cancer metabolism is an important phenomenon. Though several important transcription factors have been well studied in the context of the regulation of metabolic gene expression, the role of epigenetic readers in this regard remains still elusive. Epigenetic reader protein transcription factor 19 (TCF19) has been recently identified as a novel glucose and insulin-responsive factor that modulates histone posttranslational modifications to regulate glucose homeostasis in hepatocytes. Here we report that TCF19 interacts with a non-histone, well-known tumor suppressor protein 53 (p53) and co-regulates a wide array of metabolic genes. Among these, the p53-responsive carbohydrate metabolic genes Tp53-induced glycolysis and apoptosis regulator (TIGAR) and Cytochrome C Oxidase assembly protein 2 (SCO2), which are the key regulators of glycolysis and oxidative phosphorylation respectively, are under direct regulation of TCF19. Remarkably, TCF19 can form different transcription activation/repression complexes which show substantial overlap with that of p53, depending on glucose-mediated variant stress situations as obtained from IP/MS studies. Interestingly, we observed that TCF19/p53 complexes either have CBP or HDAC1 to epigenetically program the expression of TIGAR and SCO2 genes depending on short-term high glucose or prolonged high glucose conditions. TCF19 or p53 knockdown significantly altered the cellular lactate production and led to increased extracellular acidification rate. Similarly, OCR and cellular ATP production were reduced and mitochondrial membrane potential was compromised upon depletion of TCF19 or p53. Subsequently, through RNA-Seq analysis from patients with hepatocellular carcinoma, we observed that TCF19/p53-mediated metabolic regulation is fundamental for sustenance of cancer cells. Together the study proposes that TCF19/p53 complexes can regulate metabolic gene expression programs responsible for mitochondrial energy homeostasis and stress adaptation.


Assuntos
Proteínas Reguladoras de Apoptose/genética , Carcinoma Hepatocelular/genética , Neoplasias Hepáticas/genética , Mitocôndrias/genética , Chaperonas Moleculares/genética , Monoéster Fosfórico Hidrolases/genética , Fatores de Transcrição/genética , Transcrição Genética/genética , Proteína Supressora de Tumor p53/genética , Adaptação Biológica/genética , Apoptose/genética , Linhagem Celular Tumoral , Metabolismo Energético/genética , Glucose/genética , Células Hep G2 , Homeostase/genética , Humanos , Potencial da Membrana Mitocondrial/genética , Estresse Fisiológico/genética , Ativação Transcricional/genética
3.
3 Biotech ; 11(1): 28, 2021 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-33442526

RESUMO

Reducible sugar solution has been produced from waste broken rice by a novel saccharification process using a combination of bio-enzyme (bakhar) and commercial enzyme (α-amylase). The reducible sugar solution thus produced is a promising raw material for the production of bioethanol using the fermentation process. Response surface methodology (RSM) and Artificial neural network-genetic algorithm (ANN-GA) have been used separately to optimize the multivariable process parameters for maximum yield of the total reducing sugar (TRS) in saccharification process. The maximum yield (0.704 g/g) of TRS is predicted by the ANN-GA model at a temperature of 93 °C, saccharification time of 250 min, 6.5 pH and 1.25 mL/kg of enzyme dosages, while the RSM predicts the maximum yield of 0.7025 g/g at a little different process conditions. The fresh experimental validation of the said model predictions by ANN-GA and RSM is found to be satisfactory with the relative mean error of 2.4% and 3.8% and coefficients of determination of 0.997 and 0.996.

4.
Cell Death Dis ; 11(12): 1073, 2020 12 15.
Artigo em Inglês | MEDLINE | ID: mdl-33323928

RESUMO

The major challenge in chemotherapy lies in the gain of therapeutic resistance properties of cancer cells. The relatively small fraction of chemo-resistant cancer cells outgrows and are responsible for tumor relapse, with acquired invasiveness and stemness. We demonstrate that zinc-finger MYND type-8 (ZMYND8), a putative chromatin reader, suppresses stemness, drug resistance, and tumor-promoting genes, which are hallmarks of cancer. Reinstating ZMYND8 suppresses chemotherapeutic drug doxorubicin-induced tumorigenic potential (at a sublethal dose) and drug resistance, thereby resetting the transcriptional program of cells to the epithelial state. The ability of ZMYND8 to chemo-sensitize doxorubicin-treated metastatic breast cancer cells by downregulating tumor-associated genes was further confirmed by transcriptome analysis. Interestingly, we observed that ZMYND8 overexpression in doxorubicin-treated cells stimulated those involved in a good prognosis in breast cancer. Consistently, sensitizing the cancer cells with ZMYND8 followed by doxorubicin treatment led to tumor regression in vivo and revert back the phenotypes associated with drug resistance and stemness. Intriguingly, ZMYND8 modulates the bivalent or poised oncogenes through its association with KDM5C and EZH2, thereby chemo-sensitizing the cells to chemotherapy for better disease-free survival. Collectively, our findings indicate that poised chromatin is instrumental for the acquisition of chemo-resistance by cancer cells and propose ZMYND8 as a suitable epigenetic tool that can re-sensitize the chemo-refractory breast carcinoma.


Assuntos
Oncogenes , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos/farmacologia , Neoplasias da Mama/tratamento farmacológico , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Carcinogênese/efeitos dos fármacos , Carcinogênese/genética , Carcinogênese/patologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Movimento Celular/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Doxorrubicina/farmacologia , Doxorrubicina/uso terapêutico , Resistencia a Medicamentos Antineoplásicos/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos/genética , Proteína Potenciadora do Homólogo 2 de Zeste/metabolismo , Epigênese Genética/efeitos dos fármacos , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Transição Epitelial-Mesenquimal/genética , Feminino , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Genoma Humano , Histona Desmetilases/metabolismo , Humanos , Camundongos Endogâmicos BALB C , Camundongos Nus , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Fenótipo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Resultado do Tratamento , Neoplasias de Mama Triplo Negativas/tratamento farmacológico , Neoplasias de Mama Triplo Negativas/genética , Neoplasias de Mama Triplo Negativas/patologia
5.
Biochem J ; 477(19): 3803-3818, 2020 10 16.
Artigo em Inglês | MEDLINE | ID: mdl-32926159

RESUMO

hTERT, the catalytic component of the human telomerase enzyme, is regulated by post-translational modifications, like phosphorylation and ubiquitination by multiple proteins which remarkably affects the overall activity of the enzyme. Here we report that hTERT gets SUMOylated by SUMO1 and polycomb protein CBX4 acts as the SUMO E3 ligase of hTERT. hTERT SUMOylation positively regulates its telomerase activity which can be inhibited by SENP3-mediated deSUMOylation. Interestingly, we have established a new role of hTERT SUMOylation in the repression of E-cadherin gene expression and consequent triggering on the epithelial-mesenchymal-transition (EMT) program in breast cancer cells. We also observed that catalytically active CBX4, leads to retention of hTERT/ZEB1 complex onto E-cadherin promoter leading to its repression through hTERT-SUMOylation. Further through wound healing and invasion assays in breast cancer cells, we showed the tumor promoting ability of hTERT was significantly compromised upon overexpression of SUMO-defective mutant of hTERT. Thus our findings establish a new post-translational modification of hTERT which on one hand is involved in telomerase activity maintenance and on the other hand plays a crucial role in the regulation of gene expression thereby promoting migration and invasion of breast cancer cells.


Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Movimento Celular , Ligases/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Telomerase/metabolismo , Transcrição Genética , Antígenos CD/genética , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Caderinas/genética , Feminino , Células HeLa , Humanos , Ligases/genética , Células MCF-7 , Invasividade Neoplásica , Proteínas de Neoplasias/genética , Proteínas do Grupo Polycomb/genética , Telomerase/genética
6.
J Phys Chem Lett ; 11(13): 5367-5372, 2020 Jul 02.
Artigo em Inglês | MEDLINE | ID: mdl-32522003

RESUMO

Copper doping in II-VI semiconductor nanocrystals (NCs) has sparked enormous debate regarding the oxidation state of Cu ions and their hugely differing consequences in optoelectronic applications. The identity of a magnetically active Cu2+ ion or a magnetically inactive d10 Cu+ ion has generally been probed using optical techniques, and confusion arises from the spatial clutter that is part of the technique. One major probe that could declutter the data obtained from ensemble emission is single-particle fluorescence spectroscopy. In this work, using this very technique along with X-ray absorption spectroscopy probing the local environment of dopant ions, we study Cu-doped II-VI semiconductor NCs to find conclusive evidence on the oxidation state of Cu dopants and hence the mechanism of their emission. Detailed analysis of blinking properties has been used to study the single-particle nature of the NCs.

7.
Carcinogenesis ; 41(12): 1767-1780, 2020 12 31.
Artigo em Inglês | MEDLINE | ID: mdl-32386317

RESUMO

Previously, our laboratory demonstrated that a deregulated E2F5/p38/SMAD3 axis was associated with uncontrolled cellular proliferation in prostate cancer (PCa). Here, we investigate the role of E2F5 in PCa in further details. RNAi-mediated E2F5 knockdown and pathway-focused gene expression profiling in PC3 cells identified TFPI2 as a downstream target of E2F5. Manipulation of E2F5 expression was also found to alter MMP-2 and MMP-9 levels as detected by Proteome Profiler array, western blot and reverse transcription coupled quantitative polymerase chain reaction Site-directed mutagenesis, dual-luciferase assays and chromatin immunoprecipitation with anti-E2F5-IgG coupled with qPCR confirmed recruitment of E2F5 on TFPI2, MMP-2 and MMP-9 promoters. RNAi-mediated knockdown of E2F5 expression in PC3 caused a significant alteration of cell migration while that of TFFI2 resulted in a modest change. Abrogation of E2F5 and TFPI2 expression was associated with significant changes in the gelatinolytic activity of active forms of MMP-2 and MMP-9. Moreover, E2F5, MMP-2 and MMP-9 levels were elevated in biopsies of PCa patients relative to that of benign hyperplasia, while TFPI2 expression was reduced. MMP-9 was coimmunoprecipitated with anti-TFPI2-IgG in PCa tissue samples suggesting a direct interaction between the proteins. Finally, artemisinin treatment in PC3 cells repressed E2F5 along with MMP-2/MMP-9 while triggering TFPI2 expression which alleviated PC3 aggressiveness possibly through inhibition of MMP activities. Together, our study reinstates an oncogenic role of E2F5 which operates as a dual-function transcription factor for its targets TFPI2, MMP-2 and MMP-9 and promotes cellular invasiveness. This study also indicates a therapeutic potential of artemisinin, a natural compound which acts by correcting dysfunctional E2F5/TFPI2/MMP axis in PCa.


Assuntos
Biomarcadores Tumorais/metabolismo , Fator de Transcrição E2F5/metabolismo , Regulação Neoplásica da Expressão Gênica , Glicoproteínas/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Metaloproteinase 9 da Matriz/metabolismo , Neoplasias da Próstata/patologia , Apoptose , Biomarcadores Tumorais/genética , Movimento Celular , Proliferação de Células , Fator de Transcrição E2F5/genética , Glicoproteínas/genética , Humanos , Masculino , Metaloproteinase 2 da Matriz/genética , Metaloproteinase 9 da Matriz/genética , Invasividade Neoplásica , Prognóstico , Neoplasias da Próstata/genética , Neoplasias da Próstata/metabolismo , Células Tumorais Cultivadas
8.
Biochemistry ; 59(4): 389-399, 2020 02 04.
Artigo em Inglês | MEDLINE | ID: mdl-31746185

RESUMO

Transcription factor 19 (TCF19) plays critical roles in type 1 diabetes and the maintenance of pancreatic ß cells. Recent studies have also implicated TCF19 in cell proliferation of hepatic carcinoma and non-small cell lung carcinoma; however, the mechanism underlying this regulation remains elusive. At the molecular level, TCF19 contains two modules, the plant homeodomain (PHD) finger and the forkhead-associated (FHA) domain, of unclear function. Here, we show that TCF19 mediates hepatocellular carcinoma HepG2 cell proliferation through its PHD finger that recognizes trimethylated lysine 4 of histone 3 (H3K4me3). W316 of the PHD finger of TCF19 is one of the critical residues eliciting this function. Whole genome microarray analysis and orthogonal cell-based assays identified a large subset of genes involved in cell survival and proliferation that depend on TCF19. Our data suggest that TCF19 acts as a pro-oncogene in hepatocellular carcinoma cells and that its functional PHD finger is critical in cell proliferation.


Assuntos
Histonas/metabolismo , Fatores de Transcrição/metabolismo , Carcinoma Hepatocelular/metabolismo , Linhagem Celular Tumoral , Proliferação de Células/fisiologia , Proteínas de Ligação a DNA/metabolismo , Células Hep G2 , Código das Histonas , Histonas/genética , Humanos , Neoplasias Hepáticas/metabolismo , Lisina/metabolismo , Metilação , Modelos Moleculares , Dedos de Zinco PHD/fisiologia , Ligação Proteica , Fatores de Transcrição/fisiologia
9.
ACS Synth Biol ; 8(11): 2585-2592, 2019 11 15.
Artigo em Inglês | MEDLINE | ID: mdl-31600062

RESUMO

Non-neuronal optogenetic approaches empower precise regulation of protein dynamics in live cells but often require target-specific protein engineering. To address this challenge, we developed a generalizable light-modulated protein stabilization system (GLIMPSe) to control the intracellular protein level independent of its functionality. We applied GLIMPSe to control two distinct classes of proteins: mitogen-activated protein kinase phosphatase 3 (MKP3), a negative regulator of the extracellular signal-regulated kinase (ERK) pathway, and a constitutively active form of MEK (CA MEK), a positive regulator of the same pathway. Kinetics study showed that light-induced protein stabilization could be achieved within 30 min of blue light stimulation. GLIMPSe enables target-independent optogenetic control of protein activities and therefore minimizes the systematic variation embedded within different photoactivatable proteins. Overall, GLIMPSe promises to achieve light-mediated post-translational stabilization of a wide array of target proteins in live cells.


Assuntos
Fosfatase 6 de Especificidade Dupla/metabolismo , MAP Quinase Quinase Quinases/metabolismo , Sistema de Sinalização das MAP Quinases , Optogenética/métodos , Engenharia de Proteínas/métodos , Processamento de Proteína Pós-Traducional/efeitos da radiação , Proteólise/efeitos da radiação , Animais , Células HEK293 , Humanos , Cinética , Luz , Luciferases de Vaga-Lume/genética , Luciferases de Vaga-Lume/metabolismo , Células PC12 , Estabilidade Proteica/efeitos da radiação , Ratos , Transfecção
10.
EBioMedicine ; 37: 442-452, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30337251

RESUMO

BACKGROUND: Plasmodium falciparum and Plasmodium vivax are two major parasites responsible for malaria which remains a threat to almost 50% of world's population despite decade-long eradication program. One possible reason behind this conundrum is that the bases of clinical variability in malaria caused by either species are complex and poorly understood. METHODS: Whole-genome transcriptome was analyzed to identify the active and predominant pathways in the PBMC of P. falciparum and P. vivax infected malaria patients. Deregulated genes were identified and annotated using R Bioconductor and DAVID/KEGG respectively. Genetic and functional regulation of CD14, a prioritized candidate, were established by quantitative RT-PCR, genotyping using RFLP and resequencing, mapping of transcription factor binding using CONSITE and TFBIND, dual luciferase assay, western blot analysis, RNAi- mediated gene knockdown and chromatin-immunoprecipation. FINDINGS: The study highlighted that deregulation of host immune and inflammatory genes particularly CD14 as a key event in P. falciparum malaria. An abundance of allele-C of rs5744454, located in CD14 promoter, in severe malaria motivated us to establish an allele-specific regulation of CD14 by SP1. An enhancement of SP1 and CD14 expression was observed in artemisinin treated human monocyte cell line. INTERPRETATION: Our data not only reinstates that CD14 of TLR pathway plays a predominant role in P. falciparum malaria, it establishes a functional basis for genetic association of rs5744454 with P. falciparum severe malaria by demonstrating a cis-regulatory role of this promoter polymorphism. Moreover, the study points towards a novel pharmacogenetic aspect of artemisinin-based anti-malarial therapy. FUND: DST-SERB, Govt. of India, SR/SO/HS-0056/2013.


Assuntos
Regulação da Expressão Gênica , Leucócitos Mononucleares/metabolismo , Receptores de Lipopolissacarídeos/metabolismo , Malária Falciparum/metabolismo , Plasmodium falciparum , Plasmodium vivax , Fator de Transcrição Sp1/metabolismo , Transcriptoma , Adulto , Feminino , Humanos , Leucócitos Mononucleares/parasitologia , Leucócitos Mononucleares/patologia , Malária Vivax , Masculino , Pessoa de Meia-Idade
11.
J Mol Biol ; 429(20): 2999-3017, 2017 10 13.
Artigo em Inglês | MEDLINE | ID: mdl-28882542

RESUMO

Temporal kinetics and spatial coordination of signal transduction in cells are vital for cell fate determination. Tools that allow for precise modulation of spatiotemporal regulation of intracellular signaling in intact cells and multicellular organisms remain limited. The emerging optobiological approaches use light to control protein-protein interaction in live cells and multicellular organisms. Optobiology empowers light-mediated control of diverse cellular and organismal functions such as neuronal activity, intracellular signaling, gene expression, cell proliferation, differentiation, migration, and apoptosis. In this review, we highlight recent developments in optobiology, focusing on new features of second-generation optobiological tools. We cover applications of optobiological approaches in the study of cellular and organismal functions, discuss current challenges, and present our outlook. Taking advantage of the high spatial and temporal resolution of light control, optobiology promises to provide new insights into the coordination of signaling circuits in intact cells and multicellular organisms.


Assuntos
Técnicas Citológicas/métodos , Regulação da Expressão Gênica , Luz , Transdução de Sinais , Análise Espaço-Temporal
12.
J Vis Exp ; (124)2017 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-28654043

RESUMO

Kinase activity is crucial for a plethora of cellular functions, including cell proliferation, differentiation, migration, and apoptosis. During early embryonic development, kinase activity is highly dynamic and widespread across the embryo. Pharmacological and genetic approaches are commonly used to probe kinase activities. Unfortunately, it is challenging to achieve superior spatial and temporal resolution using these strategies. Furthermore, it is not feasible to control the kinase activity in a reversible fashion in live cells and multicellular organisms. Such a limitation remains a bottleneck for achieving a quantitative understanding of kinase activity during development and differentiation. This work presents an optogenetic strategy that takes advantage of a bicistronic system containing photoactivatable proteins Arabidopsis thaliana cryptochrome 2 (CRY2) and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). Reversible activation of the mitogen-activated protein kinase (MAPK) signaling pathway is achieved through light-mediated protein translocation in live cells. This approach can be applied to mammalian cell cultures and live vertebrate embryos. This bicistronic system can be generalized to control the activity of other kinases with similar activation mechanisms and can be applied to other model systems.


Assuntos
Diferenciação Celular/fisiologia , Embrião não Mamífero/enzimologia , Desenvolvimento Embrionário/fisiologia , Luz , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Optogenética/métodos , Animais , Proteínas de Arabidopsis/metabolismo , Diferenciação Celular/genética , Cricetinae , Desenvolvimento Embrionário/genética , Proteínas Quinases Ativadas por Mitógeno/genética , Transporte Proteico , Transdução de Sinais , Xenopus/embriologia
13.
Front Mol Neurosci ; 10: 4, 2017.
Artigo em Inglês | MEDLINE | ID: mdl-28163671

RESUMO

Synaptic transmission is a fundamental molecular process underlying learning and memory. Successful synaptic transmission involves coupled interaction between electrical signals (action potentials) and chemical signals (neurotransmitters). Defective synaptic transmission has been reported in a variety of neurological disorders such as Autism and Alzheimer's disease. A large variety of macromolecules and organelles are enriched near functional synapses. Although a portion of macromolecules can be produced locally at the synapse, a large number of synaptic components especially the membrane-bound receptors and peptide neurotransmitters require active transport machinery to reach their sites of action. This spatial relocation is mediated by energy-consuming, motor protein-driven cargo trafficking. Properly regulated cargo trafficking is of fundamental importance to neuronal functions, including synaptic transmission. In this review, we discuss the molecular machinery of cargo trafficking with emphasis on new experimental strategies that enable direct modulation of cargo trafficking in live cells. These strategies promise to provide insights into a quantitative understanding of cargo trafficking, which could lead to new intervention strategies for the treatment of neurological diseases.

14.
Development ; 143(21): 4085-4094, 2016 11 01.
Artigo em Inglês | MEDLINE | ID: mdl-27697903

RESUMO

A limited number of signaling pathways are repeatedly used to regulate a wide variety of processes during development and differentiation. The lack of tools to manipulate signaling pathways dynamically in space and time has been a major technical challenge for biologists. Optogenetic techniques, which utilize light to control protein functions in a reversible fashion, hold promise for modulating intracellular signaling networks with high spatial and temporal resolution. Applications of optogenetics in multicellular organisms, however, have not been widely reported. Here, we create an optimized bicistronic optogenetic system using Arabidopsis thaliana cryptochrome 2 (CRY2) protein and the N-terminal domain of cryptochrome-interacting basic-helix-loop-helix (CIBN). In a proof-of-principle study, we develop an optogenetic Raf kinase that allows reversible light-controlled activation of the Raf/MEK/ERK signaling cascade. In PC12 cells, this system significantly improves light-induced cell differentiation compared with co-transfection. When applied to Xenopus embryos, this system enables blue light-dependent reversible Raf activation at any desired developmental stage in specific cell lineages. Our system offers a powerful optogenetic tool suitable for manipulation of signaling pathways with high spatial and temporal resolution in a wide range of experimental settings.


Assuntos
Diferenciação Celular/genética , Desenvolvimento Embrionário/genética , Optogenética/métodos , Fosfotransferases/metabolismo , Animais , Arabidopsis , Proteínas de Arabidopsis/química , Proteínas de Arabidopsis/genética , Criptocromos/química , Criptocromos/genética , Luz , Sistema de Sinalização das MAP Quinases , Células PC12 , Fosforilação , Fosfotransferases/genética , Ratos , Transdução de Sinais , Transgenes , Xenopus , Quinases raf/metabolismo
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