RESUMEN
The industrial yeast Pichia pastoris can utilize amino acids as the sole source of carbon. It possesses a post-transcriptional regulatory circuit that governs the synthesis of cytosolic glutamate dehydrogenase 2 (GDH2) and phosphoenolpyruvate carboxykinase (PEPCK), key enzymes of amino acid catabolism. Here, we demonstrate that the post-transcriptional regulatory circuit is activated during carbon starvation resulting in the translation of GDH2 and PEPCK mRNAs. GDH2 and PEPCK synthesis is abrogated in Δatg1 indicating a key role for autophagy or an autophagy-related process. Finally, carbon-starved Δgdh2 and Δpepck exhibit poor survival. This study demonstrates a key role for amino acid catabolism during carbon starvation, a phenomenon hitherto unreported in other yeast species.
Asunto(s)
Carbono/deficiencia , Proteínas Fúngicas/genética , Glutamato Deshidrogenasa (NADP+)/genética , Fosfoenolpiruvato Carboxiquinasa (ATP)/genética , ARN Mensajero/genética , Saccharomycetales/efectos de los fármacos , Aminoácidos/metabolismo , Autofagia/genética , Proteínas Relacionadas con la Autofagia , Carbono/farmacología , Proteínas Fúngicas/agonistas , Proteínas Fúngicas/biosíntesis , Regulación Fúngica de la Expresión Génica , Glutamato Deshidrogenasa (NADP+)/biosíntesis , Metabolismo/genética , Viabilidad Microbiana , Fosfoenolpiruvato Carboxiquinasa (ATP)/biosíntesis , Biosíntesis de Proteínas , ARN Mensajero/agonistas , ARN Mensajero/biosíntesis , Saccharomycetales/enzimología , Saccharomycetales/genética , Saccharomycetales/crecimiento & desarrolloRESUMEN
Thyroid cancer is the most frequent cancer of the endocrine system. Most patients are treated with thyroidectomy followed by radioiodine therapy. However, in part of the patients, a reduction of the sodium-iodide symporter (NIS) occurs, rendering radioiodine therapy ineffective. Moreover, epithelial-mesenchymal transition (EMT) may occur, leading to more aggressive and invasive features. Herein, we evaluated the effect of the flavonoid quercetin on EMT and NIS expression in BCPAP, a papillary thyroid carcinoma cell line. BCPAP was treated with 100 µM quercetin for 24 h and cell viability, apoptosis, EMT markers and NIS were evaluated. Quercetin decreased cell viability by enhancing apoptosis. The flavonoid also reduced matrix metalloproteinase 9 and increased E-cadherin mRNA levels, inhibiting BCPAP adhesion and migration. Additionally, quercetin increased NIS expression and function. Thus, our results suggest that quercetin could be useful as adjuvant in thyroid cancer therapy, inducing apoptosis, reducing invasion and increasing the efficacy of radioiodine therapy.
Asunto(s)
Antígenos CD/genética , Antineoplásicos Fitogénicos/farmacología , Cadherinas/genética , Quercetina/farmacología , ARN Mensajero/genética , Simportadores/genética , Glándula Tiroides/efectos de los fármacos , Antígenos CD/metabolismo , Apoptosis/efectos de los fármacos , Cadherinas/agonistas , Cadherinas/metabolismo , Adhesión Celular/efectos de los fármacos , Línea Celular Tumoral , Movimiento Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Transición Epitelial-Mesenquimal/efectos de los fármacos , Flavanonas/farmacología , Regulación Neoplásica de la Expresión Génica , Humanos , Metaloproteinasa 9 de la Matriz/genética , Metaloproteinasa 9 de la Matriz/metabolismo , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Rutina/farmacología , Transducción de Señal , Simportadores/agonistas , Simportadores/metabolismo , Glándula Tiroides/metabolismo , Glándula Tiroides/patologíaRESUMEN
Streptococcus pneumoniae causes pneumonia by infecting the alveolar epithelium via binding to host receptors, such as the platelet-activating factor receptor (PAFR). Although chronic periodontitis has been identified as a pneumonia risk factor, how periodontopathic bacteria cause pneumonia is not known. We found that S. pneumoniae adhered to PAFR expressed on A549 human alveolar epithelial cells stimulated by Porphyromonas gingivalis culture supernatant, and this was abrogated by a PAFR-specific inhibitor. Among the major virulence factors of P. gingivalis [lipopolysaccharide (LPS), fimbriae and gingipains (Rgps and Kgp)], PAFR expression and pneumococcal adhesion were executed in an Rgp-dependent manner. LPS and fimbriae did not induce PAFR expression. Hence, our findings suggest that P. gingivalis enhances pneumococcal adhesion to human alveoli by inducing PAFR expression and that gingipains are responsible for this.
Asunto(s)
Cisteína-Endopeptidasas Gingipaínas/farmacología , Glicoproteínas de Membrana Plaquetaria/genética , Porphyromonas gingivalis/metabolismo , ARN Mensajero/genética , Receptores Acoplados a Proteínas G/genética , Factores de Virulencia/farmacología , Células A549 , Adhesión Bacteriana/efectos de los fármacos , Técnicas de Cocultivo , Medios de Cultivo Condicionados/química , Medios de Cultivo Condicionados/farmacología , Fimbrias Bacterianas/química , Regulación de la Expresión Génica , Cisteína-Endopeptidasas Gingipaínas/deficiencia , Cisteína-Endopeptidasas Gingipaínas/genética , Interacciones Huésped-Patógeno/genética , Humanos , Lipopolisacáridos/farmacología , Modelos Biológicos , Glicoproteínas de Membrana Plaquetaria/agonistas , Glicoproteínas de Membrana Plaquetaria/metabolismo , Porphyromonas gingivalis/genética , Porphyromonas gingivalis/patogenicidad , Alveolos Pulmonares/microbiología , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/metabolismo , Factores de Virulencia/deficiencia , Factores de Virulencia/genéticaRESUMEN
BACKGROUND: Loss of skin integrity due to injury, burning, or illness makes the development of new treatment options necessary. Skin tissue engineering provides some solutions for these problems. OBJECTIVE: The potential of a biodegradable star-shaped copolymer [Poly(CLâCOâLA)-b-PEG] and penta-block copolymer hydrogel (PNIPAAm-PCL-PEG-PCL-PNIPAAm) was assessed for skin tissue engineering applications. METHODS: Two copolymers were synthesized for cellular culture scaffolds and their mechanical properties were compared. The resulting star-shaped copolymer and thermosensitive penta-block copolymer were characterized using Fourier transform infrared and nuclear magnetic resonance spectroscopy. The crystallizability of the two copolymers was analyzed using X-ray diffraction. The resulting thermosensitive penta-block copolymer was evaluated by differential thermal analysis, differential scanning calorimetry and thermogravimetric analysis. Scanning electron microscopy and in vitro degradation of the polymer network in phosphate buffer solutions (pH 7.4) at 37°C were also examined. The pore size of the gels was calculated with Image Analyzer software. Finally, the cytotoxic, morphological, and gene expression effects of copolymers on the skin fibroblast were evaluated. RESULTS: The experiments showed that the PNIPAAm-PCL-PEG-PCL-PNIPAAm polymer with the right composition and the expected molecular weight was achieved. The hydrogel had less crystallizability compared with its precursors. The resulting thermosensitive hydrogel had a three-dimensional structure with interconnected pores that mimicked the extracellular matrix. The control of the degradability rate can be possible by weight percent changes. The pore size correlated with the polymer concentration in aqueous solution and the pore sizes of the 20 wt% hydrogel were better for fibroblast cultivation than those of the 10 wt% hydrogel. Cell proliferation on the 20% gel was more than that of the 10% gel. The hydrogel not only preserved the viability and phenotypical morphology of the entrapped cells but also stimulated the initial cell-cell interactions and proliferation of fibroblasts. The hydrogel did not influence cell conformation and this property of the polymer underlined its safety. Cells seeded on this copolymer showed a normal and spear shape and formed a focal adhesion with the hydrogel surface. Notably, the hydrogel increased collagen I α1 and collagen III mRNAs expression. CONCLUSION: Due to the low molecular weight and poor mechanical strength of the star-shaped copolymer, it was not considered for fabrication of the scaffolds for wound healing. The biodegradable, biocompatible, injectable and thermosensitive PNIPAAm-PCL-PEG-PCL-PNIPAAm hydrogel in 20 wt% demonstrated a desirable potential for future application as a cell scaffold in skin tissue engineering and wound healing.
Asunto(s)
Implantes Absorbibles , Fibroblastos/efectos de los fármacos , Hidrogeles/síntesis química , Poliésteres/síntesis química , Polietilenglicoles/síntesis química , Resinas Acrílicas/química , Biomarcadores/metabolismo , Línea Celular , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Colágeno Tipo I/agonistas , Colágeno Tipo I/genética , Colágeno Tipo I/metabolismo , Cadena alfa 1 del Colágeno Tipo I , Colágeno Tipo III/agonistas , Colágeno Tipo III/genética , Colágeno Tipo III/metabolismo , Fibroblastos/citología , Fibroblastos/metabolismo , Expresión Génica/efectos de los fármacos , Humanos , Hidrogeles/farmacología , Poliésteres/farmacología , Polietilenglicoles/farmacología , Porosidad , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Piel/citología , Temperatura , Ingeniería de Tejidos/métodos , Cicatrización de Heridas/efectos de los fármacosRESUMEN
The therapeutic pathways that modulate transcription mechanisms currently include gene knockdown and splicing modulation. However, additional mechanisms may come into play as more understanding of molecular biology and disease etiology emerge. Building on advances in chemistry and delivery technology, oligonucleotide therapeutics is emerging as an established, validated class of drugs that can modulate a multitude of genetic targets. These targets include over 10,000 proteins in the human genome that have hitherto been considered undruggable by small molecules and protein therapeutics. The approval of five oligonucleotides within the last 2 years elicited unprecedented excitement in the field. However, there are remaining challenges to overcome and significant room for future innovation to fully realize the potential of oligonucleotide therapeutics. In this review, we focus on the translational strategies encompassing preclinical evaluation and clinical development in the context of approved oligonucleotide therapeutics. Translational approaches with respect to pharmacology, pharmacokinetics, cardiac safety evaluation, and dose selection that are specific to this class of drugs are reviewed with examples. The mechanism of action, chemical evolution, and intracellular delivery of oligonucleotide therapies are only briefly reviewed to provide a general background for this class of drugs.
Asunto(s)
Terapia Genética/métodos , ARN Mensajero/genética , Animales , Anticuerpos Monoclonales/administración & dosificación , Anticuerpos Monoclonales/farmacocinética , Ensayos Clínicos como Asunto , Aprobación de Drogas , Sistemas de Liberación de Medicamentos/métodos , Evaluación Preclínica de Medicamentos , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Oligorribonucleótidos Antisentido/administración & dosificación , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/farmacocinética , Interferencia de ARN , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/agonistas , ARN Mensajero/antagonistas & inhibidores , ARN Interferente Pequeño/administración & dosificación , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/farmacocinética , Transcripción Genética/efectos de los fármacosRESUMEN
BACKGROUND: Liver X receptors (LXRs) are involved in maintaining epidermal barrier and suppressing inflammatory responses in model systems. The LXR agonist VTP-38543 showed promising results in improving barrier function and inflammatory responses in model systems. OBJECTIVE: To assess the safety, tolerability, cellular and molecular changes, and clinical efficacy of the topical VTP-38543 in adults with mild to moderate atopic dermatitis (AD). METHODS: A total of 104 ambulatory patients with mild to moderate AD were enrolled in this randomized, double-blind, vehicle-controlled trial between December 2015 and September 2016. VTP-38543 cream in 3 concentrations (0.05%, 0.15%, and 1.0%) or placebo was applied twice daily for 28 days. Pretreatment and posttreatment skin biopsy specimens were obtained from a subset of 33 patients. Changes in SCORing of Atopic Dermatitis, Eczema Area and Severity Index, Investigator's Global Assessment, and tissue biomarkers (by real-time polymerase chain reaction and immunostaining) were evaluated. RESULTS: Topical VTP-38543 was safe and well tolerated. VTP-38543 significantly increased messenger RNA (mRNA) expression of epidermal barrier differentiation (loricrin and filaggrin, P = .02) and lipid (adenosine triphosphate-binding cassette subfamily G member 1 and sterol regulatory element binding protein 1c, P < .01) measures and reduced epidermal hyperplasia markers (thickness, keratin 16 mRNA). VTP-38543 nonsignificantly suppressed cellular infiltrates and down-regulated mRNA expression of several TH17/TH22-related (phosphatidylinositol 3, S100 calcium-binding protein A12) and innate immunity (interleukin 6) markers. CONCLUSION: Topical VTP-38543 is safe and well tolerated. Its application led to improvement in barrier differentiation and lipids. Longer-term studies are needed to clarify whether a barrier-based approach can induce meaningful suppression of immune abnormalities. TRIAL REGISTRATION: clinicaltrials.gov Identifier: NCT02655679.
Asunto(s)
Antiinflamatorios/uso terapéutico , Dermatitis Atópica/tratamiento farmacológico , Epidermis/efectos de los fármacos , Factores Inmunológicos/uso terapéutico , Receptores X del Hígado/agonistas , ARN Mensajero/agonistas , Transportadoras de Casetes de Unión a ATP/genética , Transportadoras de Casetes de Unión a ATP/inmunología , Administración Cutánea , Adulto , Transporte Biológico/efectos de los fármacos , Transporte Biológico/inmunología , Dermatitis Atópica/genética , Dermatitis Atópica/inmunología , Dermatitis Atópica/patología , Método Doble Ciego , Epidermis/inmunología , Epidermis/patología , Femenino , Proteínas Filagrina , Regulación de la Expresión Génica/inmunología , Humanos , Interleucina-6/genética , Interleucina-6/inmunología , Proteínas de Filamentos Intermediarios/genética , Proteínas de Filamentos Intermediarios/inmunología , Queratina-16/genética , Queratina-16/inmunología , Receptores X del Hígado/genética , Receptores X del Hígado/inmunología , Masculino , Proteínas de la Membrana/genética , Proteínas de la Membrana/inmunología , Persona de Mediana Edad , ARN Mensajero/genética , ARN Mensajero/inmunología , Proteína S100A12/genética , Proteína S100A12/inmunología , Índice de Severidad de la Enfermedad , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/genética , Proteína 1 de Unión a los Elementos Reguladores de Esteroles/inmunología , Resultado del TratamientoRESUMEN
HIV-1 latent reservoirs harbouring silenced but replication-competent proviruses are a major obstacle against viral eradication in infected patients. The "shock and kill" strategy aims to reactivate latent provirus with latency reversing agents (LRAs) in the presence of antiretroviral drugs, necessitating the development of effective and efficient LRAs. We screened a chemical library for potential LRAs and identified two dual Polo-like kinase (PLK)/bromodomain inhibitors, BI-2536 and BI-6727 (volasertib), which are currently undergoing clinical trials against various cancers. BI-2536 and BI-6727 significantly reactivated silenced HIV-1 provirus at both the mRNA and protein level in two latently infected model cell lines (ACH2 and U1). BI-2536 dramatically reactivated transcription of latent HIV-1 provirus in peripheral blood mononuclear cells derived from infected patients. Long terminal repeat activation by the inhibitors was associated with bromodomain rather than PLK inhibition. We also found that BI-2536 synergistically activates the latent provirus in combination with SAHA, a histone deacetylase inhibitor, or the non-tumour-promoting phorbol ester prostratin. Our findings strongly suggest that BI-2536 and BI-6727 are potent LRAs for the "shock and kill" HIV-1 eradication strategy.
Asunto(s)
Regulación Viral de la Expresión Génica/efectos de los fármacos , VIH-1/efectos de los fármacos , Pteridinas/farmacología , ARN Viral/genética , Activación Viral/efectos de los fármacos , Latencia del Virus/efectos de los fármacos , Línea Celular Tumoral , Combinación de Medicamentos , VIH-1/genética , VIH-1/crecimiento & desarrollo , Inhibidores de Histona Desacetilasas/farmacología , Interacciones Huésped-Patógeno/efectos de los fármacos , Interacciones Huésped-Patógeno/genética , Humanos , Concentración 50 Inhibidora , Leucocitos Mononucleares/efectos de los fármacos , Leucocitos Mononucleares/metabolismo , Leucocitos Mononucleares/virología , Linfocitos/efectos de los fármacos , Linfocitos/metabolismo , Linfocitos/virología , Monocitos/efectos de los fármacos , Monocitos/metabolismo , Monocitos/virología , Ésteres del Forbol/farmacología , Cultivo Primario de Células , Dominios Proteicos , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Viral/agonistas , ARN Viral/metabolismo , Células THP-1 , Transcripción Genética/efectos de los fármacos , Activación Viral/genética , Latencia del Virus/genética , Vorinostat/farmacologíaRESUMEN
Friedreich's Ataxia (FA) is an inherited neurologic disorder caused by an expanded GAA repeat within intron 1 of the frataxin (FXN) gene that reduces expression of FXN protein. Agents that increase expression of FXN have the potential to alleviate the disease. We previously reported that duplex RNAs (dsRNAs) and antisense oligonucleotides (ASOs) complementary to the GAA repeat could enhance expression of FXN protein. We now explore the potential of a diverse group of chemically modified dsRNAs and ASOs to define the breadth of repeat-targeted synthetic nucleic acids as a platform for therapeutic development for FA. ASOs and dsRNAs can activate FXN protein expression in FA patient-derived cell lines that possess varied numbers of GAA repeats. Increased FXN protein expression was achieved by ASOs incorporating diverse chemical modifications with low nanomolar potencies, suggesting substantial flexibility in choosing compounds for further chemical optimization and animal studies. Our data encourage further development of ASOs as agents to treat FA.
Asunto(s)
Proteínas de Unión a Hierro/genética , Oligonucleótidos Antisentido/genética , Oligonucleótidos/genética , ARN Bicatenario/genética , ARN Mensajero/genética , Expansión de Repetición de Trinucleótido , Adolescente , Adulto , Línea Celular , Niño , Femenino , Fibroblastos/metabolismo , Fibroblastos/patología , Ataxia de Friedreich/genética , Ataxia de Friedreich/metabolismo , Ataxia de Friedreich/patología , Ataxia de Friedreich/terapia , Regulación de la Expresión Génica , Terapia Genética/métodos , Humanos , Intrones , Proteínas de Unión a Hierro/agonistas , Proteínas de Unión a Hierro/metabolismo , Masculino , Oligonucleótidos/metabolismo , Oligonucleótidos Antisentido/metabolismo , Cultivo Primario de Células , ARN Bicatenario/metabolismo , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Triazoles/química , FrataxinaRESUMEN
The expression of VEGFR2 (Flk-1, according to immunohistochemistry) and of cyclin D2 mRNA (according to real-time PCR) in the myocardium of rats is studied in doxorubicin-induced cardiomyopathy and in response to betulonic acid amide. Doxorubicin alone and in combination with betulonic acid amide causes after 3 days a manifest reduction of cyclin D2 mRNA expression (by 38 and 63%, respectively), while injection of betulonic acid amide alone causes a 23-fold increase of cyclin D2 mRNA expression. An increase of cyclin D2 mRNA expression has been detected in all experimental groups after 14 days of experiment, the most pronounced in response to betulonic acid amide (63 times). The expression of Flk-1 in cardiomyocytes increases significantly in response to both chemical agents starting from day 3 of experiment. These results indicate that doxorubicin and betulonic acid amide induce cytoprotective reactions in the myocardium, first at the intracellular, then at the cellular levels.
Asunto(s)
Amidas/farmacología , Cardiomiopatías/tratamiento farmacológico , Cardiotónicos/farmacología , Ciclina D2/genética , Ácido Oleanólico/análogos & derivados , Tirosina Quinasa 3 Similar a fms/genética , Amidas/síntesis química , Animales , Cardiomiopatías/inducido químicamente , Cardiomiopatías/genética , Cardiomiopatías/patología , Cardiotónicos/síntesis química , Ciclina D2/agonistas , Ciclina D2/antagonistas & inhibidores , Ciclina D2/metabolismo , Doxorrubicina/antagonistas & inhibidores , Doxorrubicina/toxicidad , Regulación de la Expresión Génica , Masculino , Miocardio/metabolismo , Miocardio/patología , Miocitos Cardíacos/efectos de los fármacos , Miocitos Cardíacos/metabolismo , Miocitos Cardíacos/patología , Ácido Oleanólico/síntesis química , Ácido Oleanólico/farmacología , ARN Mensajero/agonistas , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Transducción de Señal , Tirosina Quinasa 3 Similar a fms/agonistas , Tirosina Quinasa 3 Similar a fms/antagonistas & inhibidores , Tirosina Quinasa 3 Similar a fms/metabolismoRESUMEN
The genome of the malaria parasite Plasmodium falciparum contains the surf gene family which encodes large transmembrane proteins of unknown function. While some surf alleles appear to be expressed in sexual stages, others occur in asexual blood stage forms and may be associated to virulence-associated processes and undergo transcriptional switching. We accessed the transcription of surf genes along multiple invasions by real time PCR. Based on the observation of persistent expression of gene surf4.1, we created a parasite line which expresses a conditionally destabilized SURFIN4.1 protein. Upon destabilization of the protein, no interference of parasite growth or morphological changes were detected. However, we observed a strong increase in the transcript quantities of surf4.1 and sometimes of other surf genes in knocked-down parasites. While this effect was reversible when SURFIN4.1 was stabilized again after a few days of destabilization, longer destabilization periods resulted in a transcriptional switch away from surf4.1. When we tested if a longer transcript half-life was responsible for increased transcript detection in SURFIN4.1 knocked-down parasites, no alteration was found compared to control parasite lines. This suggests a specific feedback of the expressed SURFIN protein to its transcript pointing to a novel type of regulation, inedited in Plasmodium.
Asunto(s)
Antígenos de Protozoos/genética , Retroalimentación Fisiológica , Estadios del Ciclo de Vida/genética , Plasmodium falciparum/genética , ARN Mensajero/genética , Alelos , Antígenos de Protozoos/metabolismo , Clonación de Organismos , Eritrocitos/parasitología , Regulación de la Expresión Génica , Técnicas de Silenciamiento del Gen , Semivida , Humanos , Morfolinas/farmacología , Plasmodium falciparum/crecimiento & desarrollo , Plasmodium falciparum/metabolismo , Isoformas de Proteínas/antagonistas & inhibidores , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Estabilidad del ARN , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , TransfecciónRESUMEN
()Epigallocatechin gallate (EGCG) and chlorogenic acid (CGA), major flavonoids in green tea, and coffee, respectively, are recognized as possessing potential benefits in a multitude of human health conditions, including bone disorders. We have previously demonstrated that prostaglandin F2α (PGF2α), a potent bone remodeling mediator, stimulates the synthesis of osteoprotegerin (OPG) through the activation of p44/p42 mitogenactivated protein kinase (MAPK), p38 MAPK and stress activated protein kinase/cJun Nterminal kinase (SAPK/JNK) in osteoblastlike MC3T3E1 cells. In the present study, the effects of EGCG and CGA on PGF2αstimulated OPG synthesis in MC3T3E1 cells were investigated. EGCG significantly upregulated PGF2αstimulated OPG release, whereas CGA did not affect OPG release. The PGF2αinduced expression level of OPG mRNA was enhanced by EGCG. Regarding the intracellular signaling underlying the effect of EGCG, EGCG failed to affect PGF2αstimulated phosphorylation of p44/p42 MAPK, p38 MAPK or SAPK/JNK. EGCG by itself markedly induced the phosphorylation of p44/p42 MAP kinase for up to 10 min and the status decreased subsequently, whereas EGCG did not significantly affect the phosphorylation status of p38 MAPK or SAPK/JNK within 60 min. These results indicated that EGCG, but not CGA amplifies the PGF2αstimulated OPG synthesis in osteoblasts.
Asunto(s)
Catequina/análogos & derivados , Dinoprost/farmacología , Osteoblastos/efectos de los fármacos , Osteoprotegerina/genética , ARN Mensajero/genética , Animales , Catequina/farmacología , Línea Celular , Ácido Clorogénico/farmacología , Sinergismo Farmacológico , Regulación de la Expresión Génica , Ratones , Proteína Quinasa 1 Activada por Mitógenos/genética , Proteína Quinasa 1 Activada por Mitógenos/metabolismo , Proteína Quinasa 8 Activada por Mitógenos/genética , Proteína Quinasa 8 Activada por Mitógenos/metabolismo , Osteoblastos/citología , Osteoblastos/metabolismo , Osteoprotegerina/agonistas , Osteoprotegerina/metabolismo , Fosforilación/efectos de los fármacos , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Transducción de Señal , Proteínas Quinasas p38 Activadas por Mitógenos/genética , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
Methylprednisolone sodium succinate (MPSS) has been suggested as a treatment for spinal cord injury (SCI), but its use has been limited due to its adverse effects. Erythropoietin (EPO) has been suggested as a promising candidate for limiting SCI in mammals. The aim of the present study was to investigate the effects of EPO in combination with MPSS on astrocytes following ischemic injury in vitro. Astrocytes were isolated from the cerebral cortex of postnatal day 3 SpragueDawley rats and cultured in vitro. Astrocyte ischemic injury was induced by oxygen and glucose deprivation for 4 h, and reperfusion was simulated by subsequent culture under normoxic conditions. The effects of EPO and MPSS on the expression of aquaporin4 (AQP4) were investigated. Ischemic astrocytes were treated with EPO (10 U/ml), MPSS (10 µg/ml), or EPO (10 U/ml) in combination with MPSS (10 µg/ml) during reperfusion. The cell viability of astrocytes was assessed using an MTT assay. The mRNA and protein expression levels of AQP4 were determined using reverse transcriptionquantitative polymerase chain reaction and western blot analysis, respectively. The role of the protein kinase C (PKC) signaling pathway in the molecular mechanisms underlying the effects of EPO and MPSS was also investigated. The present results demonstrated that following treatment with EPO and MPSS, the mRNA expression levels of AQP4 were upregulated and cell viability was enhanced. EPO and MPSS effectively inhibited the oxygen and glucose deprivationmediated downregulation of AQP4 following reperfusion. In addition, the combined treatment with EPO and MPSS exhibited higher AQP4 expression levels and cell viability compared with each treatment alone. Finally, the effects of EPO and MPSS on AQP4 expression were partially reversed by pretreatment with the PKC inhibitor Ro 318220. The present study indicated that EPO and MPSS had a synergistic effect on AQP4 expression following reperfusion, and suggest that they may be combined in the treatment of SCI.
Asunto(s)
Acuaporina 4/genética , Astrocitos/efectos de los fármacos , Eritropoyetina/farmacología , Hemisuccinato de Metilprednisolona/farmacología , ARN Mensajero/genética , Animales , Animales Recién Nacidos , Acuaporina 4/agonistas , Acuaporina 4/metabolismo , Astrocitos/citología , Astrocitos/metabolismo , Hipoxia de la Célula/genética , Supervivencia Celular/efectos de los fármacos , Femenino , Regulación de la Expresión Génica , Glucosa/deficiencia , Glucosa/farmacología , Hipotálamo/citología , Hipotálamo/efectos de los fármacos , Hipotálamo/metabolismo , Indoles/farmacología , Fármacos Neuroprotectores/farmacología , Oxígeno/farmacología , Cultivo Primario de Células , Proteína Quinasa C/antagonistas & inhibidores , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Inhibidores de Proteínas Quinasas/farmacología , ARN Mensajero/agonistas , ARN Mensajero/metabolismo , Ratas , Ratas Sprague-Dawley , Daño por Reperfusión/genética , Daño por Reperfusión/metabolismo , Transducción de SeñalRESUMEN
In-feed antibiotics have been commonly used to promote the growth performance of piglets. The antibiotics can increase protein utilization, but the underlying mechanism is largely unknown. The present study investigated the effects of in-feed antibiotics on intestinal AA transporters and receptors to test the hypothesis that the alteration of circulating AA profiles may be concomitant with the change of intestinal AA transporters and receptors. Sixteen litters of piglets at day 7 started to receive creep feed with (Antibiotic) or without (Control) antibiotic. Piglets were weaned at day 23 after birth, and fed the same diets until day 42. In-feed antibiotics did not affect the BW of 23-day-old (P = 0.248), or 42-day-old piglets (P = 0.089), but increased the weight gain to feed ratio from day 23 to 42 (P = 0.020). At day 42 after birth, antibiotic treatment increased the concentrations of most AAs in serum (P < 0.05), and decreased the concentrations of most AAs in jejunal and ileal digesta. Antibiotics upregulated (P < 0.05) the mRNA expression levels for jejunal AAs transporters (CAT1, EAAC1, ASCT2, y+LAT1), peptide transporters (PepT1), and Na+-K+-ATPase (ATP1A1), and ileal AA transporters (ASCT2, y+LAT1, b0,+AT, and B0AT1), and ATP1A1. The antibiotics also upregulated the mRNA expression of jejunal AAs receptors T1R3 and CaSR, and ileal T1R3. Protein expression levels for jejunal AA transporters (EAAC1, b0,+AT, and ASCT2) and PepT1 were also upregulated. Correlation analysis revealed that the alterations of AA profiles in serum after the in-feed antibiotics were correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. In conclusion, the in-feed antibiotics increased serum level of most AAs and decreased most AAs in the small intestine. These changes correlated with the upregulations of mRNA expression levels for key AA transporters and receptors in the small intestine. The findings provide further insights into the mechanism of in-feed antibiotics, which may provide new framework for designing alternatives to antibiotics in animal feed in the future.
Asunto(s)
Aminoácidos/sangre , Antibacterianos/farmacología , Regulación de la Expresión Génica/efectos de los fármacos , Aumento de Peso/efectos de los fármacos , Sistema de Transporte de Aminoácidos ASC/agonistas , Sistema de Transporte de Aminoácidos ASC/genética , Sistema de Transporte de Aminoácidos ASC/metabolismo , Alimentación Animal/análisis , Animales , Animales Recién Nacidos , Transporte Biológico/efectos de los fármacos , Transportador 3 de Aminoácidos Excitadores/agonistas , Transportador 3 de Aminoácidos Excitadores/genética , Transportador 3 de Aminoácidos Excitadores/metabolismo , Kitasamicina/farmacología , Transportador de Aminoácidos Neutros Grandes 1/genética , Transportador de Aminoácidos Neutros Grandes 1/metabolismo , Oxitetraciclina/farmacología , Transportador de Péptidos 1/agonistas , Transportador de Péptidos 1/genética , Transportador de Péptidos 1/metabolismo , Quinoxalinas/farmacología , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Receptores Sensibles al Calcio/agonistas , Receptores Sensibles al Calcio/genética , Receptores Sensibles al Calcio/metabolismo , Receptores Acoplados a Proteínas G/agonistas , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/genética , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Porcinos , Canales Catiónicos TRPV/agonistas , Canales Catiónicos TRPV/genética , Canales Catiónicos TRPV/metabolismo , DesteteRESUMEN
Tamoxifen-resistant (TAMR) estrogen receptor-positive (ER+) breast cancer is characterized by elevated Erb-B2 receptor tyrosine kinase 2 (ERBB2) expression. However, the underlying mechanisms responsible for the increased ERBB2 expression in the TAMR cells remain poorly understood. Herein, we reported that the ERBB2 expression is regulated at the post-transcriptional level by miR26a/b and the RNA-binding protein human antigen R (HuR), both of which associate with the 3'-UTR of the ERBB2 transcripts. We demonstrated that miR26a/b inhibits the translation of ERBB2 mRNA, whereas HuR enhances the stability of the ERBB2 mRNA. In TAMR ER+ breast cancer cells with elevated ERBB2 expression, we observed a decrease in the level of miR26a/b and an increase in the level of HuR. The forced expression of miR26a/b or the depletion of HuR decreased ERBB2 expression in the TAMR cells, resulting in the reversal of tamoxifen resistance. In contrast, the inactivation of miR26a/b or forced expression of HuR decreased tamoxifen responsiveness of the parental ER+ breast cancer cells. We further showed that the increase in HuR expression in the TAMR ER+ breast cancer cells is attributable to an increase in the HuR mRNA isoform with shortened 3'-UTR, which exhibits increased translational activity. This shortening of the HuR mRNA 3'-UTR via alternative polyadenylation (APA) was observed to be dependent on cleavage stimulation factor subunit 2 (CSTF2/CstF-64), which is up-regulated in the TAMR breast cancer cells. Taken together, we have characterized a model in which the interplay between miR26a/b and HuR post-transcriptionally up-regulates ERBB2 expression in TAMR ER+ breast cancer cells.
Asunto(s)
Regiones no Traducidas 3'/efectos de los fármacos , Neoplasias de la Mama/tratamiento farmacológico , Resistencia a Antineoplásicos , Proteína 1 Similar a ELAV/metabolismo , MicroARNs/metabolismo , Receptor ErbB-2/metabolismo , Tamoxifeno/farmacología , Antineoplásicos Hormonales/farmacología , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Proliferación Celular/efectos de los fármacos , Factor de Estimulación del Desdoblamiento , Femenino , Humanos , MicroARNs/antagonistas & inhibidores , Mutación , Proteínas de Neoplasias/agonistas , Proteínas de Neoplasias/antagonistas & inhibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Poliadenilación/efectos de los fármacos , Interferencia de ARN , Estabilidad del ARN/efectos de los fármacos , ARN Mensajero/agonistas , ARN Mensajero/antagonistas & inhibidores , ARN Mensajero/química , ARN Mensajero/metabolismo , ARN Neoplásico/agonistas , ARN Neoplásico/antagonistas & inhibidores , ARN Neoplásico/química , ARN Neoplásico/metabolismo , Proteínas de Unión al ARN/agonistas , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Receptor ErbB-2/agonistas , Receptor ErbB-2/antagonistas & inhibidores , Receptor ErbB-2/genética , Elementos de Respuesta/efectos de los fármacos , Regulación hacia Arriba/efectos de los fármacosRESUMEN
Oxidative stress contributes to inflammatory skin diseases, including psoriasis. Monomethylfumarate (MMF) is an antipsoriatic agent with a poorly understood mechanism of action. In other cell types MMF increases the expression of nuclear factor erythroid-derived 2-like 2 (Nrf2), a transcription factor that regulates cellular antioxidant responses, to reduce oxidative stress like that observed in inflammatory disorders such as multiple sclerosis. We tested the hypothesis that MMF enhances Nrf2 activity in keratinocytes, thereby improving their capacity to counteract environmental stresses. We used Western analysis, immunofluorescence, and real-time quantitative reverse-transcription polymerase chain reaction to examine the effect of MMF on the expression of Nrf2 and its targets. We also measured intracellular reactive oxygen species (ROS) levels following MMF treatment. Our data show that MMF increased total and nuclear Nrf2 levels in primary mouse keratinocytes and enhanced mRNA expression of several Nrf2-downstream effectors, including heme oxygenase-1 and peroxiredoxin-6. Moreover, MMF treatment attenuated the generation of ROS following hydrogen peroxide treatment. On the other hand, the expression and membranous localization of aquaporin-3 (AQP3), a glycerol channel implicated in keratinocyte differentiation, was stimulated by MMF, which also enhanced keratinocyte glycerol uptake. The Nrf2 activator sulforaphane also increased AQP3 levels, suggesting that AQP3 expression may be regulated by Nrf2. We show for the first time that MMF stimulates Nrf2 and AQP3 expression and function/activity in keratinocytes. This effect may account, in part, for the previously observed ability of MMF to inhibit proliferation and inflammatory mediator production and promote differentiation in keratinocytes and to treat psoriasis.
Asunto(s)
Acuaporina 3/biosíntesis , Fumaratos/farmacología , Maleatos/farmacología , Factor 2 Relacionado con NF-E2/biosíntesis , Psoriasis , ARN Mensajero/biosíntesis , Animales , Animales Recién Nacidos , Acuaporina 3/agonistas , Acuaporina 3/genética , Secuencia de Bases , Células Cultivadas , Expresión Génica , Queratinocitos/efectos de los fármacos , Queratinocitos/metabolismo , Ratones , Factor 2 Relacionado con NF-E2/agonistas , ARN Mensajero/agonistas , ARN Mensajero/genéticaRESUMEN
The regulation of fat metabolism is important for maintaining functional and structural tissue homeostasis in biological systems. Reducing excessive lipids has been an important concern due to the concomitant health risks caused by metabolic disorders such as obesity, adiposity and dyslipidemia. A recent study revealed that unlike conventional care regimens (e.g., diet or medicine), low-energy visible radiation (VR) regulates lipid levels via autophagy-dependent hormone-sensitive lipase (HSL) phosphorylation in differentiated human adipose-derived stem cells. To clarify the underlying cellular and molecular mechanisms, we first verified the photoreceptor and photoreceptor-dependent signal cascade in nonvisual 3T3-L1 adipocytes. For a better understanding of the concomitant phenomena that result from VR exposure, mature 3T3-L1 adipocytes were exposed to four different wavelengths of VR (410, 505, 590 and 660nm) in this study. The results confirmed that specific VR wavelengths, especially 505nm than 590nm, increase intracellular cyclic adenosine monophosphate (cAMP) levels and decrease lipid droplets. Interestingly, the mRNA and protein levels of the Opn2 (rhodopsin) photoreceptor increased after VR exposure in mature 3T3-L1 adipocytes. Subsequent treatment of mature 3T3-L1 adipocytes at a specific VR wavelength induced rhodopsin- and ß3-adrenergic receptor (AR)-dependent lipolytic responses that consequently led to increases in intracellular cAMP and phosphorylated HSL protein levels. Our study indicates that photoreceptors are expressed and exert individual functions in nonvisual cells, such as adipocytes. We suggest that the VR-induced photoreceptor system could be a potential therapeutic target for the regulation of lipid homeostasis in a non-invasive manner.
Asunto(s)
Adipocitos/efectos de la radiación , Lipólisis/efectos de la radiación , ARN Mensajero/agonistas , Receptores Adrenérgicos beta 3/genética , Rodopsina/agonistas , Esterol Esterasa/genética , Células 3T3-L1 , Adipocitos/citología , Adipocitos/metabolismo , Animales , Diferenciación Celular , AMP Cíclico/agonistas , AMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de AMP Cíclico/genética , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Regulación de la Expresión Génica , Humanos , Luz , Fototransducción , Gotas Lipídicas/metabolismo , Gotas Lipídicas/efectos de la radiación , Lipólisis/genética , Ratones , Fosforilación/efectos de la radiación , ARN Mensajero/genética , ARN Mensajero/metabolismo , ARN Interferente Pequeño/genética , ARN Interferente Pequeño/metabolismo , Receptores Adrenérgicos beta 3/metabolismo , Rodopsina/antagonistas & inhibidores , Rodopsina/genética , Rodopsina/metabolismo , Esterol Esterasa/metabolismoRESUMEN
BACKGROUND: The novel compound XH601 is a synthesized derivative of formononetin. The present study was to investigate the hypolipidemia effect and potential mechanism of XH601. METHODS: Male Golden Syrian hamsters were induced by high-fat diet (HFD) for eight weeks and the hyperlipidemic model was established successfully. After XH601 treatment, serum and hepatic biochemistry parameters of hamsters were detected and the effect of XH601 on adipose tissue was also analyzed. Furthermore, 3 T3-L1 cell differentiation by Oil-Red-O staining was observed and the mRNA and protein expression of peroxisome proliferator-activated receptors (PPARs) were measured by qRT-PCR and Western-blot in mature adipocytes. RESULTS: The in vivo results suggest that XH601 significantly decreased the adipose weight and levels of serum triglyceride (TG), total cholesterol (TC), low-density lipoprotein (LDL-C), apolipoprotein B (Apo-B), apolipoprotein E (Apo-E), while increased serum high-density lipoprotein (HDL-C). The in vitro results implied that XH601 up-regulated the mRNA and protein expression of both PPARα and PPARß/δ in a dose-dependent manner. CONCLUSIONS: The study suggests that XH601 exhibited strong ability to improve the dyslipidemia in hamsters fed with high-fat diet. The potential mechanism of XH601 was associated with the up-regulation of PPARα and PPARß/δ mRNA and protein expression.
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Hiperlipidemias/tratamiento farmacológico , Hipolipemiantes/farmacología , Isoflavonas/farmacología , PPAR alfa/agonistas , PPAR delta/agonistas , PPAR-beta/agonistas , Células 3T3-L1 , Tejido Adiposo/efectos de los fármacos , Tejido Adiposo/metabolismo , Tejido Adiposo/patología , Animales , Apolipoproteínas B/sangre , Apolipoproteínas E/sangre , Diferenciación Celular , HDL-Colesterol/sangre , LDL-Colesterol/sangre , Cricetinae , Dieta Alta en Grasa/efectos adversos , Regulación de la Expresión Génica , Hiperlipidemias/etiología , Hiperlipidemias/metabolismo , Hiperlipidemias/patología , Masculino , Mesocricetus , Ratones , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR delta/genética , PPAR delta/metabolismo , PPAR-beta/genética , PPAR-beta/metabolismo , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Triglicéridos/sangreRESUMEN
Acute and habitual dietary habits contribute to the onset and progression of many forms of cardiovascular disease. Circulating peripheral blood monocytes have been a target of pre-clinical research related to the risk of atherosclerosis. Specifically, when monocytes migrate into the subendothelial space and endocytosize modified LDL (i.e. acLDL or oxLDL) they phenotypically transform into foam cells. The endocytosis of modified LDL is mediated by the scavenger receptor CD36, whose expression is in tern regulated by the transcription factor PPAR-γ. In this report, we describe a novel technique for the simultaneous measurement of intracellular PPAR-γ mRNA and protein in peripheral blood monocytes collected from human subjects in fasted state or 3 and 5-h after consuming a high-calorie (65% of daily calorie needs), high-fat meal. Intracellular detection and co-localization of PPAR-γ was made possible using a combination of image-based flow cytometry (MilliporeSigma FlowSight) and an amplified mRNA FISH staining technique (Affymetrix/eBioscience PrimeFlow). Consumption of a high-calorie, high-fat meal increased the percentage of co-localization at both 3 and 5-h post prandial compared to pre-meal. No obvious difference in co-localization was observed when cells were treated by acLDL in vitro. More research is needed to determine how to best use this method to study pre-clinical risk of atherosclerosis.
Asunto(s)
Grasas de la Dieta/administración & dosificación , Citometría de Flujo/métodos , Citometría de Imagen/métodos , Lipoproteínas LDL/farmacología , Monocitos/metabolismo , PPAR gamma/genética , Anticuerpos/química , Antígenos CD36/genética , Antígenos CD36/metabolismo , Dieta Alta en Grasa/efectos adversos , Conducta Alimentaria , Expresión Génica , Humanos , Hibridación Fluorescente in Situ/métodos , Monocitos/efectos de los fármacos , Monocitos/patología , Monocitos/ultraestructura , PPAR gamma/agonistas , PPAR gamma/metabolismo , Cultivo Primario de Células , ARN Mensajero/agonistas , ARN Mensajero/genética , ARN Mensajero/metabolismo , Coloración y Etiquetado/métodos , Regulación hacia ArribaRESUMEN
Diabetes patients associated with liver disease carry a significant risk of morbidity and mortality. Cinnamon has been reported to reduce fructose-induced oxidative stress in the rat liver. However, the mechanism by which cinnamon protects the liver in a high-saccharide environment remains to be investigated. HepG2 cells were cultured with 30 mM D-ribose to mimic the high-oxidative-stress environment, typical of a liver in a diabetic patient. Three different chemical types of C. osmophloeum ethanol extracts (CEEs) were added in HepG2 culture media and the administration of all three CEEs protected HepG2 cells from D-ribose damage and increased cell survival by approximately 20 percent. Exclusively, the transcript variant 1 of the ghrelin gene, but not variant 3, was 2-3 times induced by the addition of these CEEs. Moreover, the mRNAs of ghrelin processing enzyme, furin, and mboat4 were detected in HepG2 cells. The ghrelin hormones in the culture media were increased 4-9 times by the addition of CEEs. The protective effects of ghrelin on HepG2 cells in D-ribose environment were further confirmed by recombinant ghrelin transfection. We conclude that the CEEs induce ghrelin gene expression and protect HepG2 cells from D-ribose-induced oxidative damage through ghrelin signalling.
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Antioxidantes/farmacología , Cinnamomum/química , Ghrelina/agonistas , Extractos Vegetales/farmacología , ARN Mensajero/agonistas , Aciltransferasas/genética , Aciltransferasas/metabolismo , Antioxidantes/química , Supervivencia Celular/efectos de los fármacos , Etanol/química , Furina/genética , Furina/metabolismo , Regulación de la Expresión Génica , Ghrelina/genética , Ghrelina/metabolismo , Células Hep G2 , Humanos , Estrés Oxidativo/efectos de los fármacos , Extractos Vegetales/química , Isoformas de Proteínas/agonistas , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , ARN Mensajero/genética , ARN Mensajero/metabolismo , Ribosa/antagonistas & inhibidores , Ribosa/farmacología , Transducción de Señal , Solventes/químicaRESUMEN
OBJECTIVE: We investigated α-7 nAchR expression in human peritoneal macrophages and examined whether activation of nAchR might be a new therapy for endometriosis. MATERIALS AND METHODS: Human peritoneal fluid mononuclear cells (PFMC) were stimulated with lipopolysaccharide (LPS) in the presence of α-7 nAChR agonists. In a murine endometriosis model, α-7 nAChR modulators were administered. RESULTS: Human PFMC expressed α-7 nAChR at the mRNA and protein levels. Activation of α-7 nAChR with its agonists led to significant (P<.01) suppression of LPS-induced interleukin (IL) -1ß expression. In a murine endometriosis model, one week after inoculation of endometrium to the peritoneal cavity, α-7 nAChR agonist significantly suppressed the expression of IL-1ß mRNA (P<.01), which was negated when α-7 nAChR antagonist was administered simultaneously. α-7 nAChR agonist significantly suppressed the formation of endometriotic lesions, which was reversed with α-7 nAChR antagonist. CONCLUSION: Activation of nAChR might be a new candidate for treatment of endometriosis.