RESUMEN
Photoacoustic transfection consists in the use of photoacoustic waves, generated in the thermoelastic expansion of a confined material absorbing a short pulse of a laser, to produce temporary mechanical deformations of the cell membrane and facilitate the delivery of plasmid DNA into cells. We show that high stress gradients, produced when picosecond laser pulses with a fluence of 100 mJ/cm2 are absorbed by piezophotonic materials, enable transfection of a plasmid DNA encoding Green Fluorescent Protein (gWizGFP, 3.74 MDa) in COS-7 monkey fibroblast cells with an efficiency of 5% at 20 °C, in 10 minutes. We did not observe significant cytotoxicity under these conditions. Photoacoustic transfection is scalable, affordable, enables nuclear localization and the dosage is easily controlled by the laser parameters.
Asunto(s)
Membrana Celular/metabolismo , Técnicas Fotoacústicas/métodos , Plásmidos/metabolismo , Transfección/métodos , Animales , Células COS/metabolismo , Chlorocebus aethiops , Proteínas Fluorescentes Verdes/genética , HumanosRESUMEN
Late endosome-resident interferon-induced transmembrane protein 3 (IFITM3) inhibits fusion of diverse viruses, including Influenza A virus (IAV), by a poorly understood mechanism. Despite the broad antiviral activity of IFITM3, viruses like Lassa virus (LASV), are fully resistant to its inhibitory effects. It is currently unclear whether resistance arises from a highly efficient fusion machinery that is capable of overcoming IFITM3 restriction or the ability to enter from cellular sites devoid of this factor. Here, we constructed and validated a functional IFITM3 tagged with EGFP or other fluorescent proteins. This breakthrough allowed live cell imaging of virus co-trafficking and fusion with endosomal compartments in cells expressing fluorescent IFITM3. Three-color single virus and endosome tracking revealed that sensitive (IAV), but not resistant (LASV), viruses become trapped within IFITM3-positive endosomes where they underwent hemifusion but failed to release their content into the cytoplasm. IAV fusion with IFITM3-containing compartments could be rescued by amphotericin B treatment, which has been previously shown to antagonize the antiviral activity of this protein. By comparison, virtually all LASV particles trafficked and fused with endosomes lacking detectable levels of fluorescent IFITM3, implying that this virus escapes restriction by utilizing endocytic pathways that are distinct from the IAV entry pathways. The importance of virus uptake and transport pathways is further reinforced by the observation that LASV glycoprotein-mediated cell-cell fusion is inhibited by IFITM3 and other members of the IFITM family expressed in target cells. Together, our results strongly support a model according to which IFITM3 accumulation at the sites of virus fusion is a prerequisite for its antiviral activity and that this protein traps viral fusion at a hemifusion stage by preventing the formation of fusion pores. We conclude that the ability to utilize alternative endocytic pathways for entry confers IFITM3-resistance to otherwise sensitive viruses.
Asunto(s)
Endosomas/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas de la Membrana/fisiología , Proteínas de Unión al ARN/metabolismo , Proteínas de Unión al ARN/fisiología , Células A549/metabolismo , Animales , Antivirales/metabolismo , Células COS/metabolismo , Chlorocebus aethiops , Endosomas/virología , Células HEK293/metabolismo , Interacciones Huésped-Patógeno , Humanos , Virus de la Influenza A/patogenicidad , Interferones/metabolismo , Virus Lassa/patogenicidad , Imagen Óptica/métodos , Transporte de Proteínas , Internalización del VirusRESUMEN
Pre-embedding nanogold silver and gold intensification methods involve immunoreactions with nanogold-labeled antibodies and intensification of the nanogold particles before embedding and ultrathin sectioning. These highly sensitive methods show good resolution and ultrastructural preservation. They also are useful for simultaneous observation of immunolabeled cells under light and electron microscopes, and for three-dimensional immunoelectron microscopic analyses. Silver intensification is usually superior for immunolabeling. On the other hand, ultrastructural preservation is better when gold intensification is used. In this chapter, we introduce pre-embedding nanogold silver and gold intensification procedures for use primarily with cultured cells.
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Anticuerpos/química , Oro Coloide/química , Inmunohistoquímica/métodos , Microscopía Inmunoelectrónica/métodos , Plata/química , Fijación del Tejido/métodos , Aldehído Deshidrogenasa/genética , Aldehído Deshidrogenasa/metabolismo , Animales , Células COS/metabolismo , Células COS/ultraestructura , Chlorocebus aethiops , Retículo Endoplásmico/metabolismo , Retículo Endoplásmico/ultraestructura , Resinas Epoxi/química , Fijadores/química , Formaldehído/química , Congelación , Expresión Génica , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas del Choque Térmico HSP47 , Ratones , Microtomía , Células 3T3 NIH , Polímeros/química , Coloración y Etiquetado/métodos , Adhesión del Tejido/métodosRESUMEN
To differentiate subtypes of microglia (MG), we developed a novel monoclonal antibody, 9F5, against one subtype (type 1) of rat primary MG. The 9F5 showed high selectivity for this cell type in Western blot and immunocytochemical analyses and no cross-reaction with rat peritoneal macrophages (Mφ). We identified the antigen molecule for 9F5: the 50- to 70-kDa fragments of rat glycoprotein nonmetastatic melanoma protein B (GPNMB)/osteoactivin, which started at Lys(170) . In addition, 9F5 immunoreactivity with GPNMB depended on the activity of furin-like protease(s). More important, rat type 1 MG expressed the GPNMB fragments, but type 2 MG and Mφ did not, although all these cells expressed mRNA and the full-length protein for GPNMB. These results suggest that 9F5 reactivity with MG depends greatly on cleavage of GPNMB and that type 1 MG, in contrast to type 2 MG and Mφ, may have furin-like protease(s) for GPNMB cleavage. In neonatal rat brain, amoeboid 9F5+ MG were observed in specific brain areas including forebrain subventricular zone, corpus callosum, and retina. Double-immunοstaining with 9F5 antibody and anti-Iba1 antibody, which reacts with MG throughout the CNS, revealed that 9F5+ MG were a portion of Iba1+ MG, suggesting that MG subtype(s) exist in vivo. We propose that 9F5 is a useful tool to discriminate between rat type 1 MG and other subtypes of MG/Mφ and to reveal the role of the GPNMB fragments during developing brain. GLIA 2016;64:1938-1961.
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Anticuerpos Monoclonales/metabolismo , Encéfalo/citología , Glicoproteínas de Membrana/inmunología , Glicoproteínas de Membrana/metabolismo , Microglía/enzimología , Microglía/inmunología , Animales , Animales Recién Nacidos , Antígenos/metabolismo , Antígenos CD/metabolismo , Células COS/efectos de los fármacos , Células COS/metabolismo , Proteínas de Unión al Calcio/metabolismo , Células Cultivadas , Chlorocebus aethiops , Ectodisplasinas/metabolismo , Embrión de Mamíferos , Ojo/embriología , Ojo/crecimiento & desarrollo , Ojo/metabolismo , Femenino , Furina/genética , Furina/farmacología , Regulación del Desarrollo de la Expresión Génica/efectos de los fármacos , Regulación del Desarrollo de la Expresión Génica/fisiología , Interleucina-12/farmacología , Glicoproteínas de Membrana/química , Glicoproteínas de Membrana/genética , Ratones , Ratones Endogámicos BALB C , Proteínas de Microfilamentos/metabolismo , Microglía/clasificación , Microglía/efectos de los fármacos , Proteoglicanos/metabolismo , Ratas , Ratas WistarRESUMEN
Research on drug metabolism and pharmacokinetics in large animal species including the horse is scarce because of the challenges in conducting in vivo studies. The metabolic reactions catalyzed by cytochrome P450s (CYPs) are central to drug pharmacokinetics. This study elucidated the characteristics of equine CYPs using diazepam (DZP) as a model compound as this drug is widely used as an anesthetic and sedative in horses, and is principally metabolized by CYPs. Diazepam metabolic activities were measured in vitro using horse and rat liver microsomes to clarify the species differences in enzyme kinetic parameters of each metabolite (temazepam [TMZ], nordiazepam [NDZ], p-hydroxydiazepam [p-OH-DZP], and oxazepam [OXZ]). In both species microsomes, TMZ was the major metabolite, but the formation rate of p-OH-DZP was significantly less in the horse. Inhibition assays with a CYP-specific inhibitors and antibody suggested that CYP3A was the main enzyme responsible for DZP metabolism in horse. Four recombinant equine CYP3A isoforms expressed in Cos-7 cells showed that CYP3A96, CYP3A94, and CYP3A89 were important for TMZ formation, whereas CYP3A97 exhibited more limited activity. Phylogenetic analysis suggested diversification of CYP3As in each mammalian order. Further study is needed to elucidate functional characteristics of each equine CYP3A isoform for effective use of diazepam in horses.
Asunto(s)
Citocromo P-450 CYP3A/metabolismo , Diazepam/farmacocinética , Caballos/metabolismo , Hipnóticos y Sedantes/farmacocinética , Animales , Células COS/enzimología , Células COS/metabolismo , Chlorocebus aethiops , Citocromo P-450 CYP3A/genética , Diazepam/análogos & derivados , Masculino , Microsomas Hepáticos/enzimología , Microsomas Hepáticos/metabolismo , Nordazepam/farmacocinética , Oxazepam/farmacocinética , Filogenia , Isoformas de Proteínas/metabolismo , Ratas , Ratas Sprague-Dawley , Especificidad de la Especie , Temazepam/farmacocinéticaRESUMEN
Sodium-glucose cotransporter 2 (SGLT2) inhibitors showed a glucose lowering effect in type 2 diabetes patients through inducing renal glucose excretion. Detailed analysis of the mechanism of the glucosuric effect of SGLT2 inhibition, however, has been hampered by limitations of clinical study. Here, we investigated the mechanism of urinary glucose excretion using nonhuman primates with SGLT inhibitors tofogliflozin and phlorizin, both in vitro and in vivo. In cells overexpressing cynomolgus monkey SGLT2 (cSGLT2), both tofogliflozin and phlorizin competitively inhibited uptake of the substrate (α-methyl-d-glucopyranoside; AMG). Tofogliflozin was found to be a selective cSGLT2 inhibitor, inhibiting cSGLT2 more strongly than did phlorizin, with selectivity toward cSGLT2 1,000 times that toward cSGLT1; phlorizin was found to be a nonselective cSGLT1/2 inhibitor. In a glucose titration study in cynomolgus monkeys under conditions of controlled plasma drug concentration, both tofogliflozin and phlorizin increased fractional excretion of glucose (FEG) by up to 50% under hyperglycemic conditions. By fitting the titration curve using a newly introduced method that avoids variability in estimating the threshold of renal glucose excretion, we found that tofogliflozin and phlorizin lowered the threshold and extended the splay in a dose-dependent manner without significantly affecting the tubular transport maximum for glucose (TmG). Our results demonstrate the contribution of SGLT2 to renal glucose reabsorption (RGR) in cynomolgus monkeys and demonstrate that competitive inhibition of cSGLT2 exerts a glucosuric effect by mainly extending splay and lowering threshold without affecting TmG.
Asunto(s)
Compuestos de Bencidrilo/farmacología , Glucosa/metabolismo , Glucósidos/farmacología , Macaca fascicularis/orina , Florizina/farmacología , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Transportador 2 de Sodio-Glucosa/efectos de los fármacos , Animales , Células COS/metabolismo , Células COS/patología , Chlorocebus aethiops , ADN Complementario/genética , Relación Dosis-Respuesta a Droga , Técnicas In Vitro , Riñón/metabolismo , Riñón/patología , Masculino , Metilglucósidos/metabolismo , Modelos Animales , Transportador 1 de Sodio-Glucosa/antagonistas & inhibidores , Transportador 1 de Sodio-Glucosa/efectos de los fármacos , Transportador 1 de Sodio-Glucosa/genética , Transportador 2 de Sodio-Glucosa/genéticaRESUMEN
It has been shown that αA-mini-chaperone, a peptide representing the chaperone binding site in αA-crystallin, prevents destabilized protein aggregation. αA-Mini-chaperone has been shown to form amyloid fibrils. This study was undertaken to improve the stability of αA-mini-chaperone while preserving its anti-aggregation activity by fusing the flexible and solvent-exposed C-terminal 164-173 region of αA-crystallin to the mini-chaperone sequence DFVIFLDVKHFSPEDLT. The resulting chimeric chaperone peptide, DFVIFLDVKHFSPEDLTEEKPTSAPSS (designated CP1), was characterized. Circular dichroism studies showed that unlike αA-mini-chaperone with its ß-sheet structure, the CP1 peptide exhibited a random structure. Transmission electron microscopy (TEM) examination of the CP1 peptide incubated in a shaker at 37 °C for 72 h did not reveal amyloid fibrils, whereas αA-mini-chaperone showed distinct fibrils. Consistent with TEM observation, the thioflavin T binding assay showed an increased level of dye binding in the mini-chaperone incubated at 37 °C and subjected to shaking but not of the CP1 peptide incubated under similar conditions. The chaperone activity of the CP1 peptide was comparable to that of αA-mini-chaperone against denaturing alcohol dehydrogenase, citrate synthase, and α-lactalbumin. Transduction of both peptide chaperones to COS-7 cells showed no cytotoxic effects. The antioxidation assay involving the H2O2 treatment of COS-7 cells revealed that αA-mini-chaperone and the CP1 peptide have comparable cytoprotective properties against H2O2-induced oxidative damage in COS-7 cells. This study therefore shows that the addition of C-terminal sequence 164-173 of αA-crystallin to αA-mini-chaperone influences the conformation of αA-mini-chaperone without affecting its chaperone function or cytoprotective activity.
Asunto(s)
Chaperonas Moleculares/metabolismo , Cadena A de alfa-Cristalina/metabolismo , Secuencia de Aminoácidos , Animales , Benzotiazoles , Células COS/efectos de los fármacos , Células COS/metabolismo , Chlorocebus aethiops , Dicroismo Circular , Peróxido de Hidrógeno/farmacología , Microscopía Electrónica de Transmisión , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Datos de Secuencia Molecular , Fragmentos de Péptidos/química , Fragmentos de Péptidos/genética , Fragmentos de Péptidos/metabolismo , Conformación Proteica , Ingeniería de Proteínas , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Tiazoles/metabolismo , Cadena A de alfa-Cristalina/química , Cadena A de alfa-Cristalina/genéticaRESUMEN
Reduced production of nitric oxide (NO) is one of the first indications of endothelial dysfunction and precedes overt cardiovascular disease. Increased expression of Arginase has been proposed as a mechanism to account for diminished NO production. Arginases consume l-arginine, the substrate for endothelial nitric oxide synthase (eNOS), and l-arginine depletion is thought to competitively reduce eNOS-derived NO. However, this simple relationship is complicated by the paradox that l-arginine concentrations in endothelial cells remain sufficiently high to support NO synthesis. One mechanism proposed to explain this is compartmentalization of intracellular l-arginine into distinct, poorly interchangeable pools. In the current study, we investigated this concept by targeting eNOS and Arginase to different intracellular locations within COS-7 cells and also BAEC. We found that supplemental l-arginine and l-citrulline dose-dependently increased NO production in a manner independent of the intracellular location of eNOS. Cytosolic arginase I and mitochondrial arginase II reduced eNOS activity equally regardless of where in the cell eNOS was expressed. Similarly, targeting arginase I to disparate regions of the cell did not differentially modify eNOS activity. Arginase-dependent suppression of eNOS activity was reversed by pharmacological inhibitors and absent in a catalytically inactive mutant. Arginase did not directly interact with eNOS, and the metabolic products of arginase or downstream enzymes did not contribute to eNOS inhibition. Cells expressing arginase had significantly lower levels of intracellular l-arginine and higher levels of ornithine. These results suggest that arginases inhibit eNOS activity by depletion of substrate and that the compartmentalization of l-arginine does not play a major role.
Asunto(s)
Arginasa/metabolismo , Arginina/metabolismo , Células COS/metabolismo , Endotelio Vascular/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Animales , Aorta/citología , Aorta/metabolismo , Arginina/farmacología , Células COS/citología , Bovinos , Línea Celular , Células Cultivadas , Chlorocebus aethiops , Citrulina/farmacología , Endotelio Vascular/citología , Endotelio Vascular/efectos de los fármacos , Modelos Animales , Óxido Nítrico/metabolismo , Ornitina/metabolismoRESUMEN
Cellular processes are tightly controlled through well-coordinated signaling networks that respond to conflicting cues, such as reactive oxygen species (ROS), endoplasmic reticulum (ER) stress signals, and survival factors to ensure proper cell function. We report here a direct interaction between inhibitor of κB kinase (IKK) and apoptosis signal-regulating kinase 1 (ASK1), unveiling a critical node at the junction of survival, inflammation, and stress signaling networks. IKK can be activated by growth factor stimulation or tumor necrosis factor alpha engagement. IKK forms a complex with and phosphorylates ASK1 at a sensor site, Ser967, leading to the recruitment of 14-3-3, counteracts stress signal-triggered ASK1 activation, and suppresses ASK1-mediated functions. An inhibitory role of IKK in JNK signaling has been previously reported to depend on NF-κB-mediated gene expression. Our data suggest that IKK has a dual role: a transcription-dependent and a transcription-independent action in controlling the ASK1-JNK axis, coupling IKK to ROS and ER stress response. Direct phosphorylation of ASK1 by IKK also defines a novel IKK phosphorylation motif. Because of the intimate involvement of ASK1 in diverse diseases, the IKK/ASK1 interface offers a promising target for therapeutic development.
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Quinasa I-kappa B/metabolismo , MAP Quinasa Quinasa Quinasa 5/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Animales , Apoptosis/fisiología , Células COS/efectos de los fármacos , Células COS/metabolismo , Chlorocebus aethiops , Cromonas/farmacología , Peróxido de Hidrógeno/farmacología , Quinasa I-kappa B/genética , MAP Quinasa Quinasa Quinasa 5/genética , Ratones , Morfolinas/farmacología , Neuritas/fisiología , Células PC12 , Fosforilación , Proteínas Proto-Oncogénicas c-akt/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-akt/genética , Ratas , Serina/metabolismo , Transducción de SeñalRESUMEN
Campylobacter jejuni is the major cause of bacterial food-borne illness in the USA and Europe. An important virulence attribute of this bacterial pathogen is its ability to enter and survive within host cells. Here we show through a quantitative proteomic analysis that upon entry into host cells, C. jejuni undergoes a significant metabolic downshift. Furthermore, our results indicate that intracellular C. jejuni reprograms its respiration, favoring the respiration of fumarate. These results explain the poor ability of C. jejuni obtained from infected cells to grow under standard laboratory conditions and provide the bases for the development of novel anti microbial strategies that would target relevant metabolic pathways.
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Proteínas Bacterianas/metabolismo , Células COS/microbiología , Campylobacter jejuni/metabolismo , Metabolismo Energético , Metaboloma , Proteómica , Animales , Fenómenos Fisiológicos Bacterianos , Proteínas Bacterianas/genética , Células COS/metabolismo , Campylobacter jejuni/genética , Campylobacter jejuni/patogenicidad , Reprogramación Celular , Chlorocebus aethiops , Contaminación de Alimentos , Fumaratos/metabolismo , Regulación de la Expresión Génica , Interacciones Huésped-PatógenoRESUMEN
Niacin raises high-density lipoprotein and lowers low-density lipoprotein through the activation of the ß-hydroxybutyrate receptor hydroxycarboxylic acid 2 (HCA2) (aka GPR109a) but with an unwanted side effect of cutaneous flushing caused by vascular dilation because of the stimulation of HCA2 receptors in Langerhans cells in skin. HCA1 (aka GPR81), predominantly expressed in adipocytes, was recently identified as a receptor for lactate. Activation of HCA1 in adipocytes by lactate results in the inhibition of lipolysis, suggesting that agonists for HCA1 may be useful for the treatment of dyslipidemia. Lactate is a metabolite of glucose, suggesting that HCA1 may also be involved in the regulation of glucose metabolism. The low potency of lactate to activate HCA1, coupled with its fast turnover rate in vivo, render it an inadequate tool for studying the biological role of lactate/HCA1 in vivo. In this article, we demonstrate the identification of 3-hydroxybenzoic acid (3-HBA) as an agonist for both HCA2 and HCA1, whereas 3,5-dihydroxybenzoic acid (3,5-DHBA) is a specific agonist for only HCA1 (EC(50) â¼150 µM). 3,5-DHBA inhibits lipolysis in wild-type mouse adipocytes but not in HCA1-deficient adipocytes. Therefore, 3,5-DHBA is a useful tool for the in vivo study of HCA1 function and offers a base for further HCA1 agonist design. Because 3-HBA and 3,5-DHBA are polyphenolic acids found in many natural products, such as fruits, berries, and coffee, it is intriguing to speculate that other heretofore undiscovered natural substances may have therapeutic benefits.
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Adipocitos/efectos de los fármacos , Hidroxibenzoatos/farmacología , Lipólisis/efectos de los fármacos , Receptores Acoplados a Proteínas G/agonistas , Animales , Células COS/metabolismo , Chlorocebus aethiops , AMP Cíclico/metabolismo , Expresión Génica , Humanos , Ácido Láctico/metabolismo , Células de Langerhans/metabolismo , Lipoproteínas HDL/metabolismo , Lipoproteínas LDL/metabolismo , Masculino , Ratones , Niacina/metabolismo , Receptores Acoplados a Proteínas G/genética , Receptores Nicotínicos/genética , Resorcinoles , TransfecciónRESUMEN
We previously demonstrated that endogenous hNUDC and Mpl co-localized in the perinuclear and cytoplasmic regions of megakaryocyte cells by indirect immunofluorescence. We further reported that hNUDC accumulated in the Golgi when NIH 3T3 cells were transfected with an hNUDC expression vector alone. However, co-transfection with hNUDC and Mpl expression vectors caused both proteins to co-localize predominantly in the cytosol. These observations led us to hypothesize that a complex containing hNUDC and Mpl may alter hNUDC subcellular location and induce its secretion. In the present study, we test this hypothesis by employing bimolecular fluorescence complementation (BiFC) to detect and visualize the complex formation of hNUDC/Mpl in living cells. We further examined in detail the subcellular locations of the hNUDC/Mpl complex by co-transfection of BiFC chimeras with known subcellular markers. The distribution of hNUDC/Mpl in the endoplasmic reticulum (ER), Golgi and cell surface was determined. Furthermore, the N-terminal 159 amino acids of hNUDC, but not C-terminal half, bound to Mpl in vivo and exhibited a similar localization pattern to that of full-length hNUDC in Cos-1 cells. Adenovirus-mediated overexpression of hNUDC or its N-terminal 159 residues in a human megakaryocyte cell line (Dami) resulted in increased levels of hNUDC or hNUDC(1-159) secretion. In contrast, depletion of Mpl by transfecting Dami cells with adenovirus bearing Mpl-targeting siRNA significantly blocked hNUDC secretion. Thus, we provide the first evidence that the N-terminal region of hNUDC contains all of the necessary information to complex with Mpl and traffic through the secretory pathway.
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Proteínas de Ciclo Celular/metabolismo , Membrana Celular/metabolismo , Retículo Endoplásmico/metabolismo , Aparato de Golgi/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Trombopoyetina/metabolismo , Adenoviridae/genética , Animales , Western Blotting , Células COS/metabolismo , Células COS/ultraestructura , Proteínas de Ciclo Celular/genética , Chlorocebus aethiops , Medios de Cultivo Condicionados/farmacología , Citometría de Flujo , Transferencia Resonante de Energía de Fluorescencia , Humanos , Microscopía Confocal , Proteínas Nucleares/genética , ARN Interferente Pequeño/genética , Receptores de Trombopoyetina/antagonistas & inhibidores , Receptores de Trombopoyetina/genética , Fracciones SubcelularesRESUMEN
Inflammation is believed to be integral to the pathogenesis of Alzheimer's disease (AD). Arachidonic acid (AA) is the most important omega-6 fatty acid and a mediator of inflammatory pathways. High-sensitivity enzyme linked immunosorbent assay shows that AA and its various metabolites; prostaglandins, thromboxanes, and leukotriene B4 resulted in significantly higher secretion of both Abeta40 and 42 peptides. A combination of identical number of alternate cis and trans double bonds either at positions Δ5 or 7Z,13 or 15E (such as PGE(2), PGF(2α), THXB2 and PGF(2α)EA) or at positions Δ6Z,8E,10E,14Z (such as LB4) built in the 3-dimensional structure of 20-carbon fatty acyl chains believed to be responsible for their detrimental action. CP 24,879 and sesamin, 2 inhibitors of the AA pathway suppressed the production of amyloid-beta (Aß) peptides. Immunoblotting experiments and use of SP-C99 transfected COS-7 cells suggested that AA and its metabolites-driven altered production of Aß is mediated through gamma-secretase cleavage of amyloid precursor protein (APP). An early-onset AD transgenic mouse model expressing the double-mutant form of human amyloid precursor protein, Swedish (K670N/M671L) and Indiana (V717F), corroborated our in vitro findings by showing higher levels of Abeta and amyloid plaques in the brains, when they were fed chow supplemented with 2% AA. Our work not only supports that AA and its metabolites are involved in the production of Aß and in the pathogenesis of AD but also contributes to clarify aspects of structure-activity relationship helpful for future nonsteroidal anti-inflammatory drugs (NSAIDs) research.
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Enfermedad de Alzheimer/dietoterapia , Ácido Araquidónico/administración & dosificación , Ácido Araquidónico/metabolismo , Suplementos Dietéticos , Enfermedad de Alzheimer/genética , Péptidos beta-Amiloides/metabolismo , Precursor de Proteína beta-Amiloide/genética , Animales , Ácido Araquidónico/química , Vías Biosintéticas/efectos de los fármacos , Biotinilación , Células COS/efectos de los fármacos , Células COS/metabolismo , Moduladores de Receptores de Cannabinoides/farmacología , Chlorocebus aethiops , Cricetinae , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Inmunoprecipitación , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Mutación/genética , Fragmentos de Péptidos/metabolismo , Placa Amiloide/patología , Prostaglandinas/farmacología , Tromboxanos/farmacología , TransfecciónRESUMEN
INTRODUCTION: Arrhythmogenic right ventricular cardiomyopathy (ARVC) is a genetic disorder caused by mutations in desmosomal genes. It is often associated with life-threatening arrhythmias. Some affected individuals develop progressive heart failure and may require cardiac transplantation. METHODS: The explanted heart of a young adult with end-stage heart failure due to a null allele in desmoglein-2 was studied at macroscopic, microscopic, and molecular level. Myocardial samples were probed for junctional localization of desmosomal components and the gap junction protein connexin43 by immunohistochemical staining. In addition, the protein content of desmosomal and adherens junction markers as well as connexin43 was assessed by Western blotting. RESULTS: Histological analysis confirmed ARVC. Despite the loss of specific immunoreactive signal for desmosomal components at the cardiac intercalated disks (shown for plakoglobin, desmoplakin, and plakophilin-2), these proteins could be detected by Western blotting. Only for desmoglein-2, desmocollin-2, and plakoglobin were reduced protein levels observed. Adherens junction proteins were not affected. Lower phosphorylation levels were observed for connexin43; however, localization of the gap junction protein displayed regional differences. At the molecular level, disease progression was more severe in the right ventricle compared to the left ventricle. CONCLUSION: Our data suggest that, in the ARVC heart, plakoglobin is mainly redistributed from the junctions to other cellular pools and that protein degradation only plays a secondary role. Homogenous changes in the phosphorylation status of connexin43 were observed in multiple ARVC samples, suggesting that this might be a general feature of the disease.
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Displasia Ventricular Derecha Arritmogénica/genética , Desmogleína 2/genética , Silenciador del Gen , Insuficiencia Cardíaca/genética , Ventrículos Cardíacos/metabolismo , Adolescente , Animales , Displasia Ventricular Derecha Arritmogénica/patología , Displasia Ventricular Derecha Arritmogénica/cirugía , Western Blotting , Células COS/metabolismo , Chlorocebus aethiops , Conexina 43/genética , Conexina 43/metabolismo , Desmogleína 2/metabolismo , Insuficiencia Cardíaca/patología , Insuficiencia Cardíaca/cirugía , Trasplante de Corazón , Ventrículos Cardíacos/patología , Heterocigoto , Humanos , Masculino , Miocardio/metabolismo , Miocardio/patología , Fosforilación , Transfección , gamma Catenina/genética , gamma Catenina/metabolismoRESUMEN
Some protein transduction methods have already been developed for regenerative medicine application. These methods can be applied to soluble proteins but not to insoluble proteins, such as those that originate from inclusion bodies, for example, Escherichia coli. We have developed a method that allows the in vitro solubilization of denatured proteins without refolding and their efficient cellular internalization through conjugation to the peptide, octa-arginine (R8), via disulfide bonds with cysteine residues. Ovalbumin (OVA), denatured in urea solution containing dithiothreitol, was used as a model protein. The R8 peptide was conjugated with OVA in urea solution. Denatured OVA was recovered in the insoluble fraction after dialysis against phosphate-buffered saline. However, almost all the R8-conjugated OVA was recovered in the soluble fraction and used for translocation experiments in HeLa, Chinese hamster ovary-K1, Cos-7, and matured dendritic cells, where efficient internalization of the protein conjugate was observed. Furthermore, we formulated R8-conjugated ß-galactosidase and R8-conjugated luciferase using a similar procedure, and investigated how the conjugated proteins are processed after cell internalization. We also observed that only a small fraction of these proteins refolded and almost all underwent intracellular degradation. These results suggest that this method is suitable for the transduction of antigen-presenting cells and will benefit research and innovation in vaccine design and discovery.
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Disulfuros/química , Oligopéptidos/química , Oligopéptidos/metabolismo , Desnaturalización Proteica , Ingeniería de Proteínas/métodos , Proteínas Recombinantes/metabolismo , Animales , Células COS/metabolismo , Cricetinae , Cricetulus , Células Dendríticas/metabolismo , Diálisis , Células HeLa/metabolismo , Humanos , Luciferasas/química , Ratones , Ovalbúmina/química , Transporte de Proteínas , Proteínas Recombinantes/química , Solubilidad , Urea/química , beta-Galactosidasa/químicaRESUMEN
BACKGROUND: Fabry disease is a rare disorder caused by a large variety of mutations in the gene encoding lysosomal alpha-galactosidase. Many of these mutations are unique to individual families. Fabry disease can be treated with enzyme replacement therapy, but a promising novel strategy relies on small molecules, so called "pharmacological chaperones", which can be administered orally. Unfortunately only 42% of genotypes respond to pharmacological chaperones. RESULTS: A procedure to predict which genotypes responsive to pharmacological chaperones in Fabry disease has been recently proposed. The method uses a position-specific substitution matrix to score the mutations. Using this method, we have screened public databases for predictable responsive cases and selected nine representative mutations as yet untested with pharmacological chaperones. Mutant lysosomal alpha galactosidases were produced by site directed mutagenesis and expressed in mammalian cells. Seven out of nine mutations responded to pharmacological chaperones. Nineteen other mutations that were tested with pharmacological chaperones, but were not included in the training of the predictive method, were gathered from literature and analyzed in silico. In this set all five mutations predicted to be positive were responsive to pharmacological chaperones, bringing the percentage of responsive mutations among those predicted to be positive and not used to train the classifier to 86% (12/14). This figure differs significantly from the percentage of responsive cases observed among all the Fabry mutants tested so far. CONCLUSIONS: In this paper we provide experimental support to an "in silico" method designed to predict missense mutations in the gene encoding lysosomal alpha galactosidase responsive to pharmacological chaperones. We demonstrated that responsive mutations can be predicted with a low percentage of false positive cases. Most of the mutations tested to validate the method were described in the literature as associated to classic or mild classic phenotype. The analysis can provide a guideline for the therapy with pharmacological chaperones supported by experimental results obtained in vitro. We are aware that our results were obtained in vitro and cannot be translated straightforwardly into benefit for patients, but need to be validated by clinical trials.
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1-Desoxinojirimicina/farmacología , Enfermedad de Fabry/tratamiento farmacológico , Chaperonas Moleculares/farmacología , Mutación Missense , alfa-Galactosidasa/efectos de los fármacos , alfa-Galactosidasa/genética , 1-Desoxinojirimicina/uso terapéutico , Animales , Secuencia de Bases , Células COS/metabolismo , Dominio Catalítico , Chlorocebus aethiops , Enfermedad de Fabry/enzimología , Enfermedad de Fabry/genética , Femenino , Humanos , Masculino , Modelos Moleculares , Chaperonas Moleculares/uso terapéutico , Datos de Secuencia Molecular , Muramidasa , Mutagénesis Sitio-Dirigida , Valor Predictivo de las Pruebas , alfa-Galactosidasa/metabolismoRESUMEN
The HIV/SIV accessory protein Nef is known to down-modulate cell surface receptors that are required for virus entry such as CD4, CCR5 and CXCR4 to block lethal viral superinfection of the infected cell. The chemokine receptor CXCR4 also plays an important role in promoting cell proliferation, metastasis and tumor angiogenesis. Therefore it was of interest to evaluate if Nef can down-regulate CXCR4 in tumor cells since this could affect these critical prognostic parameters. The CXCR4-expressing cell line ACC3 that was derived from a salivary gland adenoid cystic carcinoma (ACC) of the head and neck was transfected with Nef from SIV(mac239) and cell surface expression of the receptor was monitored by FACS analysis. Real time proliferation of cells was measured with the xCELLigence system (Roche, Mannheim, Germany). Cell migration was detected by an in vitro scratch assay. Similarly, COS-7 cells were co-transfected with CXCR4 and Nef and were treated as described for ACC3. In vitro tube formation was deployed to assess the effect of Nef on angiogenesis. siRNA was used for CXCR4 knockdown. Cell surface down-modulation of endogenous CXCR4 could be observed in ACC3 cells after Nef-transfection as well as in COS-7 cells after co-transfection of CXCR4 and Nef. Proliferation as well as migration of Nef-transfected ACC3 tumor cells appeared significantly reduced. In vitro tube formation was significantly lowered after Nef-transfection or CXCR4 knockdown with siRNA. SIV-Nef could serve as an interesting tool to study the biologic behavior of CXCR4-expressing tumors such as ACC. Deploying SIV-Nef thereby could help in the discovery of new therapeutic approaches for the treatment of ACC and other CXCR4-expressing tumors.
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Ácido Aspártico Endopeptidasas/farmacología , Carcinoma Adenoide Quístico/metabolismo , Receptores CXCR4/metabolismo , Neoplasias de las Glándulas Salivales/metabolismo , Proteínas Reguladoras y Accesorias Virales/fisiología , Animales , Células COS/metabolismo , Línea Celular Tumoral , Movimiento Celular , Proliferación Celular , Chlorocebus aethiops , Regulación hacia Abajo , Humanos , Técnicas In Vitro , Neovascularización Patológica , ARN Interferente Pequeño , TransfecciónAsunto(s)
Dermatitis Atópica/genética , Calicreínas/genética , Calicreínas/metabolismo , ARN Mensajero/metabolismo , Regiones no Traducidas 3'/genética , Alelos , Animales , Células COS/metabolismo , Células Cultivadas , Chlorocebus aethiops , Expresión Génica , Células HeLa/metabolismo , Humanos , Mutagénesis Insercional , Polimorfismo de Nucleótido SimpleRESUMEN
AIMS: Much evidence suggests that vascular remodelling in the lung plays a crucial role in the development of pulmonary fibrosis. Therefore, anti-angiogenesis therapy may be a promising treatment for pulmonary fibrosis. Recently, a new inhibitor called vasohibin has been discovered to operate as an intrinsic and highly specific feedback inhibitor in the process of angiogenesis. However, to date, the effect of vasohibin on anti-angiogenesis of pulmonary fibrosis has not been examined. METHODS: In this study, we utilised vasohibin to test the potential of pulmonary fibrosis therapy. We examined the role of vasohibin in the pathophysiology of bleomycin-induced pneumopathy in mice by transfection of the vasohibin gene. RESULTS: The results demonstrated that transfection of the vasohibin gene could attenuate pulmonary fibrosis via inhibition of angiogenesis, which markedly decreased lymphocyte infiltration, cytokine secretion and fibroblast proliferation. CONCLUSIONS: This method may be beneficial for treating lung fibrosis and may provide a novel strategy for clinical application in the future.
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Proteínas de Ciclo Celular/genética , Terapia Genética/métodos , Neovascularización Patológica/prevención & control , Fibrosis Pulmonar/terapia , Animales , Bleomicina/toxicidad , Líquido del Lavado Bronquioalveolar/química , Líquido del Lavado Bronquioalveolar/citología , Células COS/efectos de los fármacos , Células COS/metabolismo , Proliferación Celular/efectos de los fármacos , Chlorocebus aethiops , Modelos Animales de Enfermedad , Fibroblastos/efectos de los fármacos , Fibroblastos/metabolismo , Fibroblastos/patología , Pulmón/efectos de los fármacos , Pulmón/metabolismo , Pulmón/patología , Linfocitos/metabolismo , Linfocitos/patología , Ratones , Ratones Endogámicos C57BL , Neovascularización Patológica/genética , Neovascularización Patológica/patología , Fibrosis Pulmonar/inducido químicamente , Fibrosis Pulmonar/patología , TransfecciónRESUMEN
PURPOSE: gammaD-Crystallin (CRYGD) is a major structural lens crystallin and its mutations result in congenital cataract formation. In this study, we attempted to correct the altered protein features of G165fsX8 CRYGD protein with small chemical molecules. METHODS: Recombinant FLAG-tagged mutants (R15C, R15S, P24T, R61C, and G165fsX8) of CRYGD were expressed in COS-7 cells and treated with small chemical molecules with reported protein chaperoning properties (sodium 4-phenylbutyrate [4-PBA], trimethylamine N-oxide [TMAO], and glycerol and DMSO [DMSO]). Protein solubility in 0.5% Triton X-100 and subcellular distribution was examined by western blotting and immunofluorescence, respectively. Apoptosis was assayed as the percentage of fragmented nuclei in transfected cells. Expression of heat-shock proteins (Hsp70 and Hsp90) was examined by reverse transcription-polymerase chain reaction analysis. RESULTS: Unlike WT and most mutants (R15C, R15S, P24T, and R61C) of CRYGD, G165fsX8 CRYGD was significantly insoluble in 0.5% Triton X-100. This insolubility was alleviated by dose-dependent 4-PBA treatment. The treatment relieved the mislocalization of G165fsX8 CRYGD from the nuclear envelope. Also, 4-PBA treatment reduced cell apoptosis and caused an upregulation of Hsp70. CONCLUSIONS: 4-PBA treatment reduced the defective phenotype of mutant G165fsX8 CRYGD and rescued the affected cells from apoptosis. This could be a potential treatment for lens structural protein and prevent lens opacity in cataract formation.