RESUMO
The Transient Receptor Potential Vanilloid 4 (TRPV4) channel has been shown to function in many physiological and pathophysiological processes. Despite abundant information on its importance in physiology, very few endogenous agonists for this channel have been described, and very few underlying mechanisms for its activation have been clarified. TRPV4 is expressed by several types of cells, such as vascular endothelial, and skin and lung epithelial cells, where it plays pivotal roles in their function. In the present study, we show that TRPV4 is activated by lysophosphatidic acid (LPA) in both endogenous and heterologous expression systems, pinpointing this molecule as one of the few known endogenous agonists for TRPV4. Importantly, LPA is a bioactive glycerophospholipid, relevant in several physiological conditions, including inflammation and vascular function, where TRPV4 has also been found to be essential. Here we also provide mechanistic details of the activation of TRPV4 by LPA and another glycerophospholipid, lysophosphatidylcholine (LPC), and show that LPA directly interacts with both the N- and C-terminal regions of TRPV4 to activate this channel. Moreover, we show that LPC activates TRPV4 by producing an open state with a different single-channel conductance to that observed with LPA. Our data suggest that the activation of TRPV4 can be finely tuned in response to different endogenous lipids, highlighting this phenomenon as a regulator of cell and organismal physiology. KEY POINTS: The Transient Receptor Potential Vaniloid (TRPV) 4 ion channel is a widely distributed protein with important roles in normal and disease physiology for which few endogenous ligands are known. TRPV4 is activated by a bioactive lipid, lysophosphatidic acid (LPA) 18:1, in a dose-dependent manner, in both a primary and a heterologous expression system. Activation of TRPV4 by LPA18:1 requires residues in the N- and C-termini of the ion channel. Single-channel recordings show that TRPV4 is activated with a decreased current amplitude (conductance) in the presence of lysophosphatidylcholine (LPC) 18:1, while LPA18:1 and GSK101 activate the channel with a larger single-channel amplitude. Distinct single-channel amplitudes produced by LPA18:1 and LPC18:1 could differentially modulate the responses of the cells expressing TRPV4 under different physiological conditions.
Assuntos
Canais de Potencial de Receptor Transitório , Canais de Cátion TRPV/metabolismo , Lisofosfatidilcolinas/farmacologia , Lisofosfolipídeos/farmacologiaRESUMO
Transient Receptor Potential Vanilloid 4 (TRPV4) ion channel is a sensor for multiple physical and chemical stimuli of ubiquitous expression that participates in various functions either in differentiated tissues or during differentiation. We recently demonstrated the nuclear localization of the full-length TRPV4 in the renal epithelial cells MDCK and its interaction with the transcriptional regulator ß-catenin. Here, we describe the presence of a functional nuclear localization signals (NLS) in the N-terminal domain of TRPV4. Simultaneous substitution R404Q, K405Q, and K407Q, produces a channel that fail to reach the nucleus, while K177Q, K178Q, and R179Q mutant channel reaches the nucleus but does not arrive to the plasma membrane (PM). Similar result was observed with the S824D phosphomimetic mutant and the K407E mutation associated with skeletal dysplasia. Structural analysis of these mutants showed important remodeling in their C-terminal domains. Our observations suggest that nucleus-PM trafficking of TRPV4 is important for its cellular functions and may help to explain some deleterious effect of mutations causing TRPV4 channelopathies.
Assuntos
Núcleo Celular/metabolismo , Canais de Cátion TRPV/química , Canais de Cátion TRPV/metabolismo , Sequência de Aminoácidos , Animais , Membrana Celular/metabolismo , Cães , Células Madin Darby de Rim Canino , Modelos Moleculares , Mutação/genética , Domínios Proteicos , Transporte Proteico , Relação Estrutura-Atividade , Canais de Cátion TRPV/genéticaRESUMO
TRPV4 is a nonselective cationic channel responsive to several physical and chemical stimuli. Defects in TRPV4 channel function result in human diseases, such as skeletal dysplasias, arthropathies, and peripheral neuropathies. Nonetheless, little is known about the role of TRPV4 in other cellular functions, such as nuclear Ca2+ homeostasis or Ca2+ -regulated transcription. Here, we confirmed the presence of the full-length TRPV4 channel in the nuclei of nonpolarized Madin-Darby canine kidney cells. Confocal Ca2+ imaging showed that activation of the channel increases cytoplasmic and nuclear Ca2+ leading to translocation of TRPV4 out of the nucleus together with ß-catenin, a transcriptional regulator in the Wnt signaling pathway fundamental in embryogenesis, organogenesis, and cellular homeostasis. TRPV4 inhibits ß-catenin transcriptional activity through a direct interaction dependent upon channel activity. This interaction also occurs in undifferentiated osteoblastoma and neuroblastoma cell models. Our results suggest a mechanism in which TRPV4 may regulate differentiation in several cellular contexts.
Assuntos
Cálcio/metabolismo , Núcleo Celular/metabolismo , Células Epiteliais/metabolismo , Rim/citologia , Modelos Biológicos , Canais de Cátion TRPV/metabolismo , Transcrição Gênica , beta Catenina/genética , Animais , Sinalização do Cálcio , Diferenciação Celular , Linhagem Celular Tumoral , Cães , Humanos , Ativação do Canal Iônico , Células Madin Darby de Rim Canino , Neuroblastoma/patologia , Osteossarcoma/patologia , Ligação Proteica , Domínios Proteicos , Transporte Proteico , Canais de Cátion TRPV/química , beta Catenina/metabolismoRESUMO
The use of optimal cutting temperature (OCT) medium has served to improve the long-term preservation of surgical tissue specimens. Unfortunately, the presence of polymers in OCT has been found to generate signal interference in proteomic-based techniques. Indeed the presence of OCT medium in tissue lysates precludes the analysis of activity based proteomic profiles obtained from lung adenocarcinoma (LuAdCa) resection specimens. In order to probe this question further tissue lysates were prepared from 47 lung non-neoplastic and tumour, node, metastasis (TNM) stage 1 LuAdCa resection specimens embedded with or without OCT, and data of activity based multiplex profiles of protein tyrosine kinase peptide substrates were obtained. We found that changes in overall phosphorylation level coincided with the use of OCT and subsequently developed an OCT per peptide median correcting strategy by performing median centering on the values of each peptide. Application of this post-analytical strategy not only can identify changes in kinase activity but can also assist in identifying novel targets for therapeutic intervention against LuAdCa.
Assuntos
Adenocarcinoma de Pulmão/metabolismo , Neoplasias Pulmonares/metabolismo , Proteínas de Neoplasias/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteômica/métodos , Adenocarcinoma de Pulmão/patologia , Feminino , Humanos , Neoplasias Pulmonares/patologia , Masculino , FosforilaçãoRESUMO
TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca2+ imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Diferenciação Celular , Epitélio Corneano/citologia , Modelos Biológicos , Canais de Cátion TRPV/metabolismo , Junções Íntimas/metabolismo , Células 3T3 , Animais , Cálcio/metabolismo , Técnicas de Cultura de Células , Diferenciação Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Claudina-4/metabolismo , Impedância Elétrica , Fator de Crescimento Epidérmico/farmacologia , Ativação do Canal Iônico/efeitos dos fármacos , Camundongos , Transporte Proteico/efeitos dos fármacos , Coelhos , Frações Subcelulares/metabolismo , Junções Íntimas/efeitos dos fármacos , Fatores de TempoRESUMO
BACKGROUND: The neurotrophin Brain-Derived Neurotrophic Factor (BDNF) influences nigral dopaminergic neurons via autocrine and paracrine mechanisms. The reduction of BDNF expression in Parkinson's disease substantia nigra (SN) might contribute to the death of dopaminergic neurons because inhibiting BDNF expression in the SN causes parkinsonism in the rat. This study aimed to demonstrate that increasing BDNF expression in dopaminergic neurons of rats with one week of 6-hydroxydopamine lesion recovers from parkinsonism. The plasmids phDAT-BDNF-flag and phDAT-EGFP, coding for enhanced green fluorescent protein, were transfected using neurotensin (NTS)-polyplex, which enables delivery of genes into the dopaminergic neurons via neurotensin-receptor type 1 (NTSR1) internalization. RESULTS: Two weeks after transfections, RT-PCR and immunofluorescence techniques showed that the residual dopaminergic neurons retain NTSR1 expression and susceptibility to be transfected by the NTS-polyplex. phDAT-BDNF-flag transfection did not increase dopaminergic neurons, but caused 7-fold increase in dopamine fibers within the SN and 5-fold increase in innervation and dopamine levels in the striatum. These neurotrophic effects were accompanied by a significant improvement in motor behavior. CONCLUSIONS: NTS-polyplex-mediated BDNF overexpression in dopaminergic neurons has proven to be effective to remit hemiparkinsonism in the rat. This BDNF gene therapy might be helpful in the early stage of Parkinson's disease.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/biossíntese , Neurônios Dopaminérgicos , Neurotensina , Doença de Parkinson , Substância Negra , Transfecção/métodos , Animais , Fator Neurotrófico Derivado do Encéfalo/genética , Modelos Animais de Doenças , Neurônios Dopaminérgicos/metabolismo , Neurônios Dopaminérgicos/patologia , Terapia Genética/métodos , Masculino , Neurotensina/química , Neurotensina/farmacologia , Doença de Parkinson/genética , Doença de Parkinson/metabolismo , Doença de Parkinson/patologia , Doença de Parkinson/terapia , Ratos , Ratos Wistar , Receptores de Neurotensina/metabolismo , Substância Negra/metabolismo , Substância Negra/patologiaRESUMO
BACKGROUND/AIMS: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). METHODS: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. RESULTS: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. CONCLUSION: Ouabain 10 nM increases GJC in MDCK cells.
Assuntos
Comunicação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Junções Comunicantes/efeitos dos fármacos , Ouabaína/administração & dosagem , Animais , Cães , Células Epiteliais/efeitos dos fármacos , Células Madin Darby de Rim CaninoRESUMO
Polycystic kidney disease (PKD), a disease characterized by enlargement of the kidney through cystic growth is the fourth leading cause of end-stage kidney disease world-wide. TRPV4, a calcium-permeable TRP, channel participates in kidney cell physiology and since TRPV4 forms complexes with another channel whose malfunction is associated to PKD, TRPP2 (or PKD2), we sought to determine whether patients with PKD, exhibit previously unknown mutations in TRPV4. Here, we report the presence of mutations in the TRPV4 gene in patients diagnosed with PKD and determine that they produce gain-of-function (GOF). Mutations in the sequence of the TRPV4 gene have been associated to a broad spectrum of neuropathies and skeletal dysplasias but not PKD, and their biophysical effects on channel function have not been elucidated. We identified and examined the functional behavior of a novel E6K mutant and of the previously known S94L and A217S mutant TRVP4 channels. The A217S mutation has been associated to mixed neuropathy and/or skeletal dysplasia phenotypes, however, the PKD carriers of these variants had not been diagnosed with these reported clinical manifestations. The presence of certain mutations in TRPV4 may influence the progression and severity of PKD through GOF mechanisms. PKD patients carrying TRVP4 mutations are putatively more likely to require dialysis or renal transplant as compared to those without these mutations.
RESUMO
Cytokine adsorption can resolve different complications characteristic of transplantation medicine, such as cytokine storm activation and blood ABO and immune incompatibilities. Cytokine adsorption is also performed for the treatment of various life-threatening conditions, such as endotoxic septic shock, acute respiratory distress syndrome, and cardiogenic shock, all potentially leading to adverse clinical outcomes during transplantation. After surgery, dysmetabolism and stress response limit successful graft survival and can lead to primary or secondary graft dysfunction. In this clinical context, and given that a major problem in transplant medicine is that the demand for organs far exceeds the supply, a technological innovation such as a hemoadsorption system could greatly contribute to increasing the number of usable organ donors. The objectives of this review are to describe the specific advantages and disadvantages of the application of cytokine adsorption in the context of transplantation and examine, before and/or after organ transplantation, the benefits of the addition of a cytokine adsorption therapy protocol.
RESUMO
Some members of the transient receptor potential (TRP) family of cation channels mediate sensory responses to irritant substances. Although it is well known that TRPA1 channels are activated by pungent compounds found in garlic, onion, mustard and cinnamon extracts, activation of TRPV1 by these extracts remains controversial. Here we establish that TRPV1 is activated by pungent extracts from onion and garlic, as well as by allicin, the active compound in these preparations, and participates together with TRPA1 in the pain-related behavior induced by this compound. We found that in TRPV1 these agents act by covalent modification of cysteine residues. In contrast to TRPA1 channels, modification of a single cysteine located in the N-terminal region of TRPV1 was necessary and sufficient for all the effects we observed. Our findings point to a conserved mechanism of activation in TRP channels, which provides new insights into the molecular basis of noxious stimuli detection.
Assuntos
Allium/química , Dor/induzido quimicamente , Dor/metabolismo , Extratos Vegetais/farmacologia , Canais de Cátion TRPV/efeitos dos fármacos , Canais de Cátion TRPV/metabolismo , Sequência de Aminoácidos/fisiologia , Animais , Linhagem Celular , Sequência Conservada , Cisteína/química , Dissulfetos , Evolução Molecular , Feminino , Alho/química , Humanos , Masculino , Potenciais da Membrana/efeitos dos fármacos , Potenciais da Membrana/genética , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Nociceptores/efeitos dos fármacos , Nociceptores/metabolismo , Cebolas/química , Estrutura Terciária de Proteína , Ácidos Sulfínicos/farmacologia , Canais de Cátion TRPV/químicaRESUMO
Pax-6 is a regulatory gene with a major role during visual system development, but its association with corneal epithelial differentiation is not clearly established. Using the RCE1-(5T5) cell line, which mimics corneal epithelial differentiation, we analyzed Pax-6 biological role. Immunostaining of proliferating colonies and confluent sheets showed that Pax-6-positive cells were also K3 keratin-positive, suggesting that Pax-6 is expressed in differentiating cells. Pax-6 mRNA was barely expressed in early cell cultures; but after confluence, its levels raised up to fivefold as demonstrated by Northern blot and RT-qPCR. The raise in Pax-6 expression preceded for 9 h the increase in LDH-H and LDH-M mRNAs, previously shown as early markers of corneal epithelial cell differentiation. The full-length mRNAs encoding for the two major Pax-6 isoforms were found at very low levels in proliferating cells, and abundantly expressed in the confluent stratified epithelia; Pax-6 mRNA was 2- to 2.5-fold more abundant than Pax-6(5a) mRNA. The ectopic expression of Pax-6 or Pax-6(5a) decreased proliferative ability leading to the formation of abortive, non-proliferative colonies. In contrast, culture conditions that delay or block corneal epithelial cell differentiation reduced or inhibited the expression of Pax-6. Collectively, results show that Pax-6 is the earlier differentiation marker expressed by corneal epithelial cells, and open the possibility for a major role of Pax-6 as the main driver of the differentiation of corneal epithelial cells.
Assuntos
Diferenciação Celular/fisiologia , Córnea , Células Epiteliais/fisiologia , Proteínas do Olho/metabolismo , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Box Pareados/metabolismo , Proteínas Repressoras/metabolismo , Animais , Biomarcadores/metabolismo , Cadaverina/metabolismo , Linhagem Celular , Córnea/citologia , Córnea/fisiologia , Células Epiteliais/citologia , Proteínas do Olho/genética , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Queratina-3/genética , Queratina-3/metabolismo , Lactato Desidrogenases/genética , Lactato Desidrogenases/metabolismo , Fator de Transcrição PAX6 , Fatores de Transcrição Box Pareados/genética , Fenótipo , Coelhos , Proteínas Repressoras/genéticaRESUMO
Na(x) is a sodium channel, thought to be a descendant of the voltage-gated sodium channel family. Nevertheless, Na(x) is not activated by voltage but rather by augmentation of extracellular sodium over 150 mM. In the brain, it is localized to the circumventricular organs, important regions for salt and water homeostasis in mammals, where it operates as a sodium-level sensor of body fluid. Na(x) channel is expressed in lung, uterus, and heart, and it is also found in trigeminal and dorsal root ganglia and in nonmyelinating Schwann cells, where its physiological role remains unclarified. Here we identified the promoter and transcription start sites of Na(x) sodium channel in dorsal root ganglia neurons from mouse. We report a characterization of the basal TATA-less promoter and the sequence requirements for promoter activity in Neuro 2A neuroblastoma cells and in dorsal root ganglia neurons, where basal promoter activity seems to require NGFI-C and Ebox DNA elements. Finally, we provide evidence that a repression mechanism that inhibits Na(x) expression may be present in certain tissues. These findings provide the basis with which to understand tissue-specific regulation of Na(x) sodium channel gene (Scn7a) expression.
Assuntos
Gânglios Espinais/metabolismo , Neurônios/metabolismo , Regiões Promotoras Genéticas , Canais de Sódio/genética , Canais de Sódio/metabolismo , Animais , Linhagem Celular Tumoral , Células Cultivadas , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Inibidores de Histona Desacetilases , Ácidos Hidroxâmicos/farmacologia , Masculino , Camundongos , Camundongos Endogâmicos BALB C , Mutação , Neurônios/efeitos dos fármacos , RNA Mensageiro/metabolismo , Transcrição Gênica , Canais de Sódio Disparados por VoltagemRESUMO
Previously we improved the neurotensin (NT)-polyplex by the coupling of HA2 fusogenic peptide (FP) and Vp1 SV40 karyophilic peptide (KP). We now report the proportion of [(125)I]-NT, [(3)H]-FP, and poly-l-lysine (PLL) in the NT-polyplex, and some of its biophysical properties. We concluded that the most efficient NT-polyplex comprised 1 NT, 4 FP, and 2 PLL molecules. Electrophoresis revealed that high acidity is detrimental for NT-polyplex stability. Electron microscopy and electrophoresis studies showed that 6 muM KP and 1% serum condensed the plasmid DNA (pDNA) before the appearance of toroid structures. Four plasmids were used to evaluate the transfection efficiency. In vitro, maximum expression was produced at molar ratios (pDNA : [(125)I]-NT-[(3)H]-FP-PLL conjugate) of 1:34 for pEGFP-N1 and 1:27 for pECFP-Nuc. Cotransfection of those plasmids was attained at their optimum molar ratios. In vivo, maximum expression of the pDAT-BDNF-flag in dopamine neurons was produced at a 1:45 molar ratio, whereas that of pDAT-EGFP was at 1:20. The NT-polyplex in the presence of 1 muM SR-48692, an NT-receptor specific antagonist, and untargeted polyplex did not cause transfection in vivo demonstrating the specificity of gene transfer via NT-receptor endocytosis. This information is essential for synthesizing an efficient NT-polyplex that can provide a useful tool for specific gene transfection.
Assuntos
Técnicas Genéticas , Neurotensina/química , Animais , Biofísica/métodos , Linhagem Celular Tumoral , Dopamina/metabolismo , Humanos , Concentração de Íons de Hidrogênio , Técnicas In Vitro , Masculino , Neurônios/metabolismo , Sinais de Localização Nuclear , Pirazóis/química , Quinolinas/química , Ratos , Ratos Wistar , TransfecçãoRESUMO
Despite constant improvement in existing therapeutic efforts, the overall survival rate of lung cancer patients remains low. Enzyme activities may identify new therapeutically targetable biomarkers and overcome the marked lack of correlation between cellular abundance of translated proteins and corresponding mRNA expression levels. We analysed tyrosine kinase activities to classify lung adenocarcinoma (LuAdCa) resection specimens based on their underlying changes in cellular processes and pathways that are agents of or result from malignant transformation. We characterised 71 same-patient pairs of early-stage LuAdCa and non-neoplastic LuAdCa resection specimen lysates in the presence or absence of a tyrosine kinase inhibitor. We performed ex vivo multiplex tyrosine phosphorylation assays using 144 selected microarrayed kinase substrates. The obtained 76 selected phosphotyrosine signature peptides were subsequently analysed in terms of follow-up treatments and outcomes recorded in the patient files. For tumour, node, metastasis (TNM) stage 1 LuAdCa patients, we noticed a larger tyrosine kinase inhibitor-induced decrease in tyrosine phosphorylation for long-term as opposed to short-term disease survivors, for which 26 of 76 selected peptides were significantly (p < 0.01, FDR < 3%) more inhibited in the long-term survivors. Using statistical class prediction analysis, we obtained a 'prognostic-signature' for long- versus short-term disease survivors and correctly predicted the survival status of 73% of our patients. Our translational approach may assist clinical disease management after surgical resection and may help to direct patients for an optimal treatment strategy.
RESUMO
Dp71 dystrophin is the main DMD gene product expressed in the central nervous system. Experiments using PC12 cells as a neuronal model have shown that Dp71 isoforms are involved in differentiation, adhesion, cell division, and nuclear architecture. To contribute to the knowledge of Dp71 domains function, we previously reported the isolation and partial characterization of the dystrophin Dp71[INCREMENT]78-79 (a mutant that lacks exons 71, 78, and 79), which stimulates the neuronal differentiation of PC12-C11 clone. In this article, we generated a doxycycline (Dox)-inducible expression system in PC12 Tet-On cells (B10 cells) to overexpress and control the transcription of Dp71[INCREMENT]78-79. Western blotting and confocal microscopy showed an increase in the amount of Dp71[INCREMENT]78-79 (217±75-fold) with the addition of Dox to growth medium. Cell proliferation assays and morphometric analyses demonstrated that Dp71[INCREMENT]78-79 increases the growth rate of B10 cells and reduces the nerve growth factor-neuronal differentiation. Western blotting analysis revealed an upregulation in the expression of proliferating cell nuclear antigen, focal adhesion kinase, and ß-dystroglycan in B10 cells compared with control cells. Our results show that the inducible expression of Dp71[INCREMENT]78-79 increases the growth rate of PC12 Tet-On cells, suggesting a role of this protein in cell proliferation.
Assuntos
Proliferação de Células , Distrofina/genética , Distrofina/metabolismo , Animais , Western Blotting , Éxons , Imunofluorescência , Microscopia Confocal , Mutação , Neurogênese/fisiologia , Células PC12 , Ratos , TransfecçãoRESUMO
We report an analysis in vivo of the RNA degradosome assembly of Escherichia coli. Employing fluorescence microscopy imaging and fluorescence energy transfer (FRET) measurements, we present evidence for in vivo pairwise interactions between RNase E-PNPase (polynucleotide phosphorylase), and RNase E-Enolase. These interactions are absent in a mutant strain with genomically encoded RNase E that lacks the C-terminal half, supporting the role of the carboxy-end domain as the scaffold for the degradosome. We also present evidence for in vivo proximity of Enolase-PNPase and Enolase-RhlB. The data support a model for the RNA degradosome (RNAD), in which the RNase E carboxy-end is proximal to PNPase, more distant to Enolase, and more than 10 nm from RhlB helicase. Our measurements were made in strains with mono-copy chromosomal fusions of the RNAD enzymes with fluorescent proteins, allowing measurement of the expression of the different proteins under different growth and stress conditions.
Assuntos
Endorribonucleases/metabolismo , Escherichia coli/enzimologia , Complexos Multienzimáticos/metabolismo , Polirribonucleotídeo Nucleotidiltransferase/metabolismo , RNA Helicases/metabolismo , RNA Helicases DEAD-box/metabolismo , Endorribonucleases/química , Endorribonucleases/genética , Proteínas de Escherichia coli/metabolismo , Transferência Ressonante de Energia de Fluorescência , Microscopia de Fluorescência , Complexos Multienzimáticos/química , Complexos Multienzimáticos/genética , Plasmídeos/genética , Polirribonucleotídeo Nucleotidiltransferase/química , Polirribonucleotídeo Nucleotidiltransferase/genética , Mapas de Interação de Proteínas/genética , RNA Helicases/química , RNA Helicases/genéticaRESUMO
The polarized distribution of K(+) channels in MDCK cells is lost upon harvesting and restored upon re-seeding. Using semi-quantitative PCR, in the present work we find that (i) Cells do not "wait" for the normal recycling of membrane proteins to restore their lost channels, but trigger their replacement, suggesting that the membrane has a way of engaging the nucleus. (ii) Replacement channels do not come from an internal reservoir, as it is the case with Na(+), K(+)-ATPase, but requires a de novo synthesis. (iii) Replacement is not an all-or-none response, since mRNA for MaxiK channels increases by 8-fold after re-seeding, but those for Kv1.6 and Kv1.7 are not affected by harvesting/re-seeding. (iv) TEA, charybdotoxin and iberiotoxin fail to trigger the replacement response in mature monolayers, suggesting that replacement is not due to suppression of channel function. (v) MDCK cells have a typical transporting epithelial phenotype (TEP) consisting of tight junctions (TJs) plus polarity. Although the polarized distribution of K-channels is a prominent attribute of TEP, blocking their function does not perturb the development of TEP, as gauged through the development of TJs, nor level of expression (Western blot) and distribution (confocal microscopy) of occludin, and claudins 1, 3 and 7.
Assuntos
Células Epiteliais/metabolismo , Canais de Potássio/metabolismo , Animais , Sequência de Bases , Cálcio/farmacologia , Linhagem Celular , Sequência Conservada , Cães , Células Epiteliais/efeitos dos fármacos , Amplificação de Genes/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Dados de Sequência Molecular , Fenótipo , Bloqueadores dos Canais de Potássio/farmacologia , Canais de Potássio/química , Canais de Potássio/genética , RNA Mensageiro/genética , Alinhamento de Sequência , Transcrição Gênica/genéticaRESUMO
The transient receptor potential vanilloid 1 (TRPV1) channel is a nonselective cation channel activated by capsaicin and responsible for thermosensation. To date, little is known about the gating characteristics of these channels. Here we used tetrabutylammonium (TBA) to determine whether this molecule behaves as an ion conduction blocker in TRPV1 channels and to gain insight into the nature of the activation gate of this protein. TBA belongs to a family of classic potassium channel blockers that have been widely used as tools for determining the localization of the activation gate and the properties of the pore of several ion channels. We found TBA to be a voltage-dependent pore blocker and that the properties of block are consistent with an open-state blocker, with the TBA molecule binding to multiple open states, each with different blocker affinities. Kinetics of channel closure and burst-length analysis in the presence of blocker are consistent with a state-dependent blocking mechanism, with TBA interfering with closing of an activation gate. This activation gate may be located cytoplasmically with respect to the binding site of TBA ions, similar to what has been observed in potassium channels. We propose an allosteric model for TRPV1 activation and block by TBA, which explains our experimental data.
Assuntos
Detergentes/farmacologia , Compostos de Amônio Quaternário/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Linhagem Celular , Humanos , Estrutura Terciária de ProteínaRESUMO
Recently we showed that the neurotensin polyplex is a nanoparticle carrier system that targets reporter genes in nigral dopamine neurons in vivo. Herein, we report its first practical application in experimental parkinsonism, which consisted of transfecting dopamine neurons with the gene coding for human glial cell line-derived neurotrophic factor (hGDNF). Hemiparkinsonism was induced in rats by a single dose of 6-hydroxydopamine (30 microg) into the ventrolateral part of the striatum. We showed that transfection of the hGDNF gene into the substantia nigra of rats 1 week after the neurotoxin injection produced biochemical, anatomical, and functional recovery from hemiparkinsonism. RT-PCR analysis showed mRNA expression of exogenous hGDNF in the transfected substantia nigra. Western blot analysis verified transgene expression by recognizing the flag epitope added at the C-terminus of the hGDNF polypeptide, which was found mainly in dopamine neurons by double immunofluorescence techniques. These data indicate that the neurotensin polyplex holds great promise for the neuroprotective therapy of Parkinson disease.
Assuntos
Fator Neurotrófico Derivado de Linhagem de Célula Glial/genética , Neurônios/metabolismo , Neurotensina/química , Doença de Parkinson Secundária/terapia , Animais , Apomorfina/farmacologia , Corpo Estriado/efeitos dos fármacos , Corpo Estriado/metabolismo , Corpo Estriado/patologia , Dopamina/metabolismo , Terapia Genética/métodos , Vetores Genéticos/genética , Fator Neurotrófico Derivado de Linhagem de Célula Glial/química , Fator Neurotrófico Derivado de Linhagem de Célula Glial/fisiologia , Humanos , Imuno-Histoquímica , Metanfetamina/farmacologia , Nanopartículas/química , Oxidopamina/administração & dosagem , Oxidopamina/toxicidade , Doença de Parkinson Secundária/induzido quimicamente , Doença de Parkinson Secundária/genética , Ratos , Substância Negra/efeitos dos fármacos , Substância Negra/metabolismo , Substância Negra/patologia , Fatores de Tempo , Transfecção/métodosRESUMO
Lactate dehydrogenase (LDH) and glucose-6-phosphate dehydrogenase (G-6-PDH) activities were studied during corneal epithelial growth and differentiation in cell culture. LDH and G-6-PDH activities increased up to 60 and 150-fold, respectively, when corneal epithelial cells constituted a differentiated four to five layered epithelium; these increases showed a similar time-course to the expression of K3 keratin. Immunostaining experiments showed that in growing colonies, LDH staining is stronger in those cells that are K3 positive; in contrast, in confluent four to five layered epithelia LDH and K3 were located in all cell layers, similar to the pattern found in frozen sections from rabbit central cornea. During growth and differentiation, the LDH isoenzyme set from corneal epithelial cells did not change; and it was different from those observed in cultured conjunctival, esophageal and epidermal cells. The augment in LDH activity was due to a 25-fold increase in the LDH-H mRNA and a 12-fold augment in LDH-M mRNA. A computer-assisted search led to identify AP2 and Sp1 binding sites in the LDH and G-6-PDH promoters, suggesting that their expression might share common regulatory mechanisms with the regulation of the differentiation-linked keratins. It is proposed that LDH may be an early marker of corneal epithelial differentiation, and its isozyme pattern could be distinctive from other epithelial cell lineages.