RESUMEN
We have previously shown that mice challenged with a lethal dose of A/Puerto Rico/8/34-OVA(I) are protected by injection of 4-8 × 10(6) in vitro-generated Tc1 or Tc17 CD8(+) effectors. Viral load, lung damage, and loss of lung function are all reduced after transfer. Weight loss is reduced and survival increased. We sought in this study to define the mechanism of this protection. CD8(+) effectors exhibit multiple effector activities, perforin-, Fas ligand-, and TRAIL-mediated cytotoxicity, and secretion of multiple cytokines (IL-2, IL-4, IL-5, IL-9, IL-10, IL-17, IL-21, IL-22, IFN-γ, and TNF) and chemokines (CCL3, CCL4, CCL5, CXCL9, and CXCL10). Transfer of CD8(+) effectors into recipients, before infection, elicits enhanced recruitment of host neutrophils, NK cells, macrophages, and B cells. All of these events have the potential to protect against viral infections. Removal of any one, however, of these potential mechanisms was without effect on protection. Even the simultaneous removal of host T cells, host B cells, and host neutrophils combined with the elimination of perforin-mediated lytic mechanisms in the donor cells failed to reduce their ability to protect. We conclude that CD8(+) effector T cells can protect against the lethal effects of viral infection by means of a large number of redundant mechanisms.
Asunto(s)
Linfocitos T CD8-positivos/inmunología , Linfocitos T CD8-positivos/virología , Virus de la Influenza A/inmunología , Infecciones por Orthomyxoviridae/inmunología , Infecciones por Orthomyxoviridae/prevención & control , Animales , Linfocitos T CD8-positivos/trasplante , Diferenciación Celular/genética , Diferenciación Celular/inmunología , Células Cultivadas , Pruebas Inmunológicas de Citotoxicidad/métodos , Células de Riñón Canino Madin Darby , Ratones , Ratones Endogámicos BALB C , Ratones Endogámicos C57BL , Ratones Noqueados , Ratones Transgénicos , Infecciones por Orthomyxoviridae/virología , Linfocitos T Citotóxicos/inmunología , Linfocitos T Citotóxicos/trasplante , Linfocitos T Citotóxicos/virologíaRESUMEN
Hepatic gluconeogenesis is required for maintaining blood glucose homeostasis; yet, in diabetes mellitus, this process is unrestrained and is a major contributor to fasting hyperglycemia. To date, the impacts of chromatin modifying enzymes and chromatin landscape on gluconeogenesis are poorly understood. Through catalyzing the removal of methyl groups from specific lysine residues in the histone tail, histone demethylases modulate chromatin structure and, hence, gene expression. Here we perform an RNA interference screen against the known histone demethylases and identify a histone H3 lysine 36 (H3K36) demethylase, Jhdm1a, as a key negative regulator of gluconeogenic gene expression. In vivo, silencing of Jhdm1a promotes liver glucose synthesis, while its exogenous expression reduces blood glucose level. Importantly, the regulation of gluconeogenesis by Jhdm1a requires its demethylation activity. Mechanistically, we find that Jhdm1a regulates the expression of a major gluconeogenic regulator, C/EBPα. This is achieved, at least in part, by its USF1-dependent association with the C/EBPα promoter and its subsequent demethylation of dimethylated H3K36 on the C/EBPα locus. Our work provides compelling evidence that links histone demethylation to transcriptional regulation of gluconeogenesis and has important implications for the treatment of diabetes.
Asunto(s)
Glucemia , Gluconeogénesis , Histona Demetilasas con Dominio de Jumonji , Hígado/metabolismo , Animales , Glucemia/metabolismo , Proteínas Potenciadoras de Unión a CCAAT/genética , Proteínas Potenciadoras de Unión a CCAAT/metabolismo , Células Cultivadas , Regulación de la Expresión Génica , Gluconeogénesis/genética , Glucosa-6-Fosfatasa/metabolismo , Hepatocitos/metabolismo , Humanos , Histona Demetilasas con Dominio de Jumonji/genética , Histona Demetilasas con Dominio de Jumonji/metabolismo , Ratones , Ratones Endogámicos C57BL , Proteínas Serina-Treonina Quinasas/metabolismo , ARN Interferente Pequeño/genética , Ratas , Factores Estimuladores hacia 5'/metabolismoRESUMEN
Host cells use metabolic signaling through the LXRα nuclear receptor to defend against Listeria monocytogenes infection. 25-Hydroxycholesterol is a natural ligand of LXRs that is produced by the enzyme cholesterol 25-hydroxylase (CH25H). We found that expression of Ch25h is upregulated following L. monocytogenes infection in a beta interferon (IFN-ß)-dependent fashion. Moreover, increased Ch25h expression promotes survival of L. monocytogenes-infected cells and increases sensitivity of the host to infection. We determined that expression of Cd5l, a prosurvival gene, is controlled by CH25H. In addition, we found that CD5L inhibits activation of caspase-1, promoting survival of infected macrophages. Our results reveal a mechanism by which an intracellular pathogen can prolong survival of infected cells, thus providing itself with a protected environment in which to replicate.
Asunto(s)
Listeria monocytogenes/fisiología , Listeriosis/metabolismo , Macrófagos/metabolismo , Macrófagos/microbiología , Transducción de Señal/fisiología , Esteroide Hidroxilasas/biosíntesis , Animales , Proteínas Reguladoras de la Apoptosis/biosíntesis , Western Blotting , Femenino , Expresión Génica , Regulación de la Expresión Génica/genética , Etiquetado Corte-Fin in Situ , Listeriosis/inmunología , Ratones , Ratones Endogámicos C57BL , Receptores Inmunológicos/biosíntesis , Receptores Depuradores , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
Spinal Muscular Atrophy (SMA) is a neurodegenerative disease that is caused by deletion of the SMN (Survival of Motor Neuron) gene. The SMN protein is essential for cell survival and co-localized with TIA-1/R and G3BP, two characteristic markers of stress granules (SGs). To further study the SMN function in stress granules and in response to stress, we generated stable cell lines with SMN knockdown. Our data indicate that suppression of SMN drastically reduces cellular ability to form stress granules in response to stress treatment. In addition, we show that SMN deficiency sensitizes cells to sodium arsenite and H(2)O(2), two well-known stress inducers, leading to cell death at a much lower concentration of inducers in SMN knockdown cells than in control cells. Interestingly, the cell death is correlated with formation of stress granules, suggesting that involvement of SMN in formation of stress granules may play an important role in cell survival. Furthermore, rescue of SGs formation by overexpression of G3BP can reverse the defective formation of stress granules and results in partial abrogation of cell death against SMN deficiency. We deduce that modulation of stress response may be useful for potential SMN treatment.
Asunto(s)
Gránulos Citoplasmáticos/metabolismo , Estrés Fisiológico , Proteína 1 para la Supervivencia de la Neurona Motora/metabolismo , Animales , Arsenitos/farmacología , Proteínas Portadoras/metabolismo , Muerte Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Gránulos Citoplasmáticos/efectos de los fármacos , Citoprotección/efectos de los fármacos , Regulación hacia Abajo/efectos de los fármacos , Factor 2 Eucariótico de Iniciación/metabolismo , Técnicas de Silenciamiento del Gen , Células HEK293 , Humanos , Peróxido de Hidrógeno/farmacología , Ratones , Neuronas/citología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Células PC12 , Fosforilación/efectos de los fármacos , Interferencia de ARN/efectos de los fármacos , ARN Interferente Pequeño/metabolismo , Ratas , Compuestos de Sodio/farmacología , Estrés Fisiológico/efectos de los fármacos , TransfecciónRESUMEN
Tau aggregation is one of the major features in Alzheimer's disease and in several other tauopathies, including frontotemporal dementia with Parkinsonism linked to chromosome 17 (FTDP-17), and progressive supranuclear palsy (PSP). More than 35 mutations in the tau gene have been identified from FTDP-17 patients. A group of these mutations alters splicing of exon 10, resulting in an increase in exon 10 inclusion into tau mRNA. Abnormal splicing with inclusion of exon 10 into tau mRNA has also been observed in PSP and AD patients. These results indicate that abnormal splicing of exon 10, leading to the production of tau with exon 10, is probably one of the mechanisms by which tau accumulates and aggregates in tauopathic brains. Therefore, modulation of exon 10 splicing in the tau gene could potentially be targeted to prevent tauopathies. To identify small molecules or compounds that could potentially be developed into drugs to treat tauopathies, we established a cell-based high-throughput screening assay. In this review, we will discuss how realistic, specific biological molecules can be found to regulate exon 10 splicing in the tau gene for potential treatment of tauopathies.
Asunto(s)
Empalme Alternativo/genética , Tauopatías/genética , Proteínas tau/genética , Empalme Alternativo/efectos de los fármacos , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/genética , Enfermedad de Alzheimer/patología , Animales , Demencia/tratamiento farmacológico , Demencia/genética , Demencia/patología , Exones/genética , Humanos , Mutación , Oligorribonucleótidos Antisentido/genética , Oligorribonucleótidos Antisentido/uso terapéutico , Tauopatías/tratamiento farmacológico , Tauopatías/patologíaRESUMEN
Exon 2 of the Bcl-x gene undergoes alternative splicing in which the Bcl-xS splice variant promotes apoptosis in contrast to the anti-apoptotic splice variant Bcl-xL. In this study, the regulation of the alternative splicing of pre-mRNA of Bcl-x was examined in response to emetine. Treatment of different types of cancer cells with emetine dihydrochloride downregulated the level of Bcl-xL mRNA with a concomitant increase in the mRNA level of Bcl-xS in a dose- and time-dependent manner. Pretreatment with calyculin A, an inhibitor of protein phosphatase 1 (PP1) and protein phosphatase 2A (PP2A), blocked emetine-induced alternative splicing in contrast to okadaic acid, a specific inhibitor of PP2A in cells, demonstrating a PP1-mediated mechanism. Our finding on the regulation of RNA splicing of members of the Bcl-2 family in response to emetine presents a potential target for cancer treatment.
Asunto(s)
Empalme Alternativo/efectos de los fármacos , Emetina/farmacología , Proteína Fosfatasa 1/metabolismo , Proteína bcl-X/genética , Neoplasias de la Mama/genética , Neoplasias de la Mama/metabolismo , Neoplasias de la Mama/patología , Línea Celular Tumoral , Cicloheximida/farmacología , Regulación hacia Abajo/efectos de los fármacos , Regulación hacia Abajo/genética , Inhibidores Enzimáticos/farmacología , Femenino , Regulación Neoplásica de la Expresión Génica/efectos de los fármacos , Humanos , Neoplasias Pulmonares/genética , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patología , Masculino , Toxinas Marinas , Ácido Ocadaico/farmacología , Oxazoles/farmacología , Neoplasias de la Próstata/genética , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Proteína Fosfatasa 1/antagonistas & inhibidores , Proteína Fosfatasa 2/antagonistas & inhibidores , Proteína Fosfatasa 2/metabolismo , Regulación hacia Arriba/efectos de los fármacos , Regulación hacia Arriba/genética , Neoplasias del Cuello Uterino/genética , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/patologíaRESUMEN
BACKGROUND AND AIM: Tissue-type plasminogen activator (tPA) is one of the major components in the matrix proteolytic network whose role in the pathogenesis of liver fibrosis remains unknown. The aim of this study is to investigate the role of tPA in carbon tetrachloride (CCl(4))-induced liver fibrosis. METHODS: Wild-type and tPA knockout mice (8 mice per group) were injected interperitoneumly with 25% CCl(4) 2 ml/kg twice per week as CCl(4) administration groups and olive oil 2 ml/kg as controls. After 4 weeks, the livers of mice were removed under deep anesthesia and prepared for further studies such as histology, immunostaining, hydroxyproline assay, zymography and western blot analysis. RESULTS: Mice lacking tPA developed more severe morphological injury and displayed an increased deposition of collagen in the liver after CCl(4) administration compared with wild-type counterparts. Deficiency of tPA increased alpha-smooth muscle actin expression in the mice livers. On the other hand, the decrease of matrix metalloproteinase-2 (MMP-2), matrix metalloproteinase-9 (MMP-9) activities, metalloproteinase-13 (MMP-13) expression and a marked increase of tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) expression were found in the liver of CCl(4) administrated tPA(-/-) mice compared with wild-type counterparts. CONCLUSIONS: Deficiency of tPA aggravated liver fibrosis through promoting hepatic stellate cells (HSCs) activation and inhibiting ECM degradation by decreasing MMP-2, MMP-9 activities and disrupting the balance between MMP-13 and TIMP-1.
Asunto(s)
Cirrosis Hepática/metabolismo , Hígado/metabolismo , Activador de Tejido Plasminógeno/metabolismo , Actinas/metabolismo , Animales , Tetracloruro de Carbono , Colágeno/metabolismo , Modelos Animales de Enfermedad , Hígado/enzimología , Hígado/patología , Cirrosis Hepática/inducido químicamente , Cirrosis Hepática/genética , Cirrosis Hepática/patología , Masculino , Metaloproteinasa 13 de la Matriz/metabolismo , Metaloproteinasa 2 de la Matriz/metabolismo , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Índice de Severidad de la Enfermedad , Factores de Tiempo , Inhibidor Tisular de Metaloproteinasa-1/metabolismo , Activador de Tejido Plasminógeno/deficiencia , Activador de Tejido Plasminógeno/genéticaRESUMEN
SUBJECTS: The aim of this study is to compare the efficacy and safety of en bloc bladder tumor-endoscopic submucosal dissection (BT-ESD) and conventional transurethral resection of BT (TURBT) in nonmuscle invasive bladder cancer (NMIBC) patients. METHODS: A retrospective cohort study was carried out in Shaanxi Provincial People's Hospital. A total of 193 eligible NMIBC (Ta/T1) patients were enrolled in this study (95 cases in BT-ESD group and 98 cases in TURBT group), between November 2013 and January 2017. The operation time, blood loss, postoperative bladder irrigation time, catheter indwelling time, hospital stay time, and complications were compared. Data were presented as median (range). Chi-squared or rank-sum test, two-way ANOVA, and Mantel-Cox (Log-Rank) test were performed using statistical software. A threshold of P < 0.05 was defined as statistically significant. RESULTS: The average operation time in the BT-ESD group was longer than that of in the TURBT group (40.0 [5.0, 100.0] min vs. 19.5 [3.0, 55.5] min); however, no significant longer operating time (P < 0.05) were observed in the smaller tumor (0 cm-3 cm). The postoperative bladder irrigation time, catheter indwelling time, and hospital stay in BT-ESD group were significantly shorter than that of in TURBT group (9.0 [5.0, 18.0] h, 2.5 [1.0, 4.0] d and 3.5 [2.0, 5.0] d for BT-ESD; 18.0 [12.0, 48.0] h, 3.5 [2.0, 7.0] d, and 4.5 [3.0, 8.0] d for TURBT). In addition, the BT-ESD group showed the decreased overall incidence of complications (2.1% vs. 9.2%). The univariate and multivariate analyses indicated an association between surgical option and tumor recurrence (hazard ratio = 5.624, odds ratio = 95% confidence interval = 1.582-19.991), Kaplan-Meir analysis showed significant difference in recurrence-free survival (RFS) (94.7% for ESD group vs. 78.4% for TURBT group) at 33 months. CONCLUSIONS: The application of the HybridKnife lead to a decrease in complications and RFS rate, which was a more safe and effective approach for NMIBC than conventional TURBT.
Asunto(s)
Resección Endoscópica de la Mucosa , Neoplasias de la Vejiga Urinaria/diagnóstico por imagen , Neoplasias de la Vejiga Urinaria/patología , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Niño , Preescolar , Terapia Combinada , Resección Endoscópica de la Mucosa/métodos , Femenino , Estudios de Seguimiento , Humanos , Estimación de Kaplan-Meier , Masculino , Persona de Mediana Edad , Imagen Multimodal/métodos , Clasificación del Tumor , Invasividad Neoplásica , Estadificación de Neoplasias , Recurrencia , Carga Tumoral , Neoplasias de la Vejiga Urinaria/mortalidad , Adulto JovenRESUMEN
Deletion or mutation of the survival of motor neuron (SMN1) gene causes Spinal Muscular Atrophy (SMA), a motor neuron degenerative disease. To study the SMN function, we co-transfected mouse NSC34 cells with SMN and mutant superoxide dismutase 1 (SOD1) constructs. We demonstrated that SMN protected NSC34 cells against cell death induced by mutant SOD1 under oxidative stress. Further studies indicated that over-expression of wild-type SMN up-regulated chaperone activity. In contrast, chaperone activity was decreased in cells expressing SMN mutant Y272C or in cells with SMN suppressed by shRNA. In vitro assays using bacteria lysates expressing GST-SMN or purified GST-SMN protein showed that the GST-SMN reduced catalase aggregation, indicating that SMN may possess chaperone activity. We conclude that SMN plays a protective role in motor neurons by its chaperone activity. Our results provide support for the potential development of therapy for SMA and amyotrophic lateral sclerosis (ALS).
Asunto(s)
Apoptosis , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Chaperonas Moleculares/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Neuroblastoma/metabolismo , Proteínas de Unión al ARN/metabolismo , Superóxido Dismutasa/metabolismo , Animales , Línea Celular , Supervivencia Celular , Citoprotección , Ratones , Proteínas del Complejo SMN , Superóxido Dismutasa-1 , Proteína 1 para la Supervivencia de la Neurona MotoraRESUMEN
Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in the nervous system. We show that exogenous tPA or tPA/plasminogen (plg) promotes axonal regeneration, remyelination, and functional recovery after sciatic nerve injury in the mouse. Local application of tPA or tPA/plg 7 days after sciatic nerve crush significantly increased the total number of axons and myelinated axons, which is accompanied by enhanced expression of neurofilament. Treatment with tPA or tPA/plg reduced the deposition of fibrin(ogen) after nerve injury. Moreover, tPA or tPA/plg increased the number of macrophages and induced MMP-9 expression at the injury site, coincident with reduced collagen scar formation and accelerated clearance of myelin and lipid debris after treatment. Consequently, tPA or tPA/plg treatment protected muscles from atrophy after nerve injury, indicating better functional recovery. These results suggest that administration of exogenous tPA or tPA/plg promotes axonal regeneration and remyelination through removal of fibrin deposition and activation of MMP-9-positive macrophages, which may be responsible for myelin debris clearance and preventing collagen scar formation. Therefore, tPA may be useful for treatment of peripheral nerve injury.
Asunto(s)
Regeneración Nerviosa/efectos de los fármacos , Recuperación de la Función/efectos de los fármacos , Neuropatía Ciática/tratamiento farmacológico , Activador de Tejido Plasminógeno/uso terapéutico , Animales , Antígenos de Diferenciación/metabolismo , Compuestos Azo , Western Blotting/métodos , Recuento de Células/métodos , Modelos Animales de Enfermedad , Interacciones Farmacológicas , Inmunoglobulina G/metabolismo , Inmunohistoquímica/métodos , Masculino , Metaloproteinasa 9 de la Matriz/metabolismo , Ratones , Ratones Endogámicos C57BL , Atrofia Muscular/tratamiento farmacológico , Atrofia Muscular/etiología , Atrofia Muscular/patología , Vaina de Mielina/metabolismo , Fibras Nerviosas Mielínicas/efectos de los fármacos , Fibras Nerviosas Mielínicas/metabolismo , Fibras Nerviosas Mielínicas/patología , Plasminógeno/administración & dosificación , Neuropatía Ciática/complicaciones , Neuropatía Ciática/metabolismoRESUMEN
Progression from brown preadipocytes to adipocytes engages two transcriptional programs: the expression of adipogenic genes common to both brown fat (BAT) and white fat (WAT), and the expression of BAT-selective genes. However, the dynamics of chromatin states and epigenetic enzymes involved remain poorly understood. Here we show that BAT development is selectively marked and guided by repressive H3K27me3 and is executed by its demethylase Jmjd3. We find that a significant subset of BAT-selective genes, but not common fat genes or WAT-selective genes, are demarcated by H3K27me3 in both brown and white preadipocytes. Jmjd3-catalyzed removal of H3K27me3, in part through Rreb1-mediated recruitment, is required for expression of BAT-selective genes and for development of beige adipocytes both in vitro and in vivo. Moreover, gain- and loss-of-function Jmjd3 transgenic mice show age-dependent body weight reduction and cold intolerance, respectively. Together, we identify an epigenetic mechanism governing BAT fate determination and WAT plasticity.
Asunto(s)
Tejido Adiposo Pardo/embriología , Tejido Adiposo Blanco/embriología , Regulación del Desarrollo de la Expresión Génica , Histona Demetilasas con Dominio de Jumonji/metabolismo , Adipocitos/citología , Adipocitos/metabolismo , Animales , Proteínas Reguladoras de la Apoptosis/metabolismo , Peso Corporal , Proteínas de Unión al ADN/metabolismo , Canales Iónicos/metabolismo , Masculino , Ratones , Ratones Endogámicos C57BL , Proteínas Mitocondriales/metabolismo , Mutación Puntual , Regiones Promotoras Genéticas , Análisis de Secuencia de ARN , Termogénesis/genética , Factores de Transcripción/metabolismo , Transgenes , Proteína Desacopladora 1RESUMEN
We conducted an analysis of the Kallmann syndrome 1 (KAL-1) genotype in 17 patients with Kallmann syndrome (KS), 9 patients with normosmic idiopathic hypogonadotropic hypogonadism (nIHH) and 20 age-matched normal men in Northwestern China. To do this, we used multiplex PCR analysis with exon-flanking primers and automated sequencing techniques with peripheral blood DNA samples. Intragenic deletions were found at the KAL-1 locus in two KS patients. One case with an atrial septal defect exhibited an intragenic deletion of exon 6. Another KS patient with cryptorchidism had intragenic deletions of exons 5 and 6. For the nIHH patients, no abnormalities were observed in the exonic and flanking sequences of KAL-1. This report describes two intragenic deletions of KAL-1 in two KS patients and suggests that KAL-1 deletion might be more prevalent in KS patients with other congenital organ abnormalities than those described previously in other series from Northwestern China.
Asunto(s)
Proteínas de la Matriz Extracelular/genética , Hipogonadismo/genética , Síndrome de Kallmann/genética , Proteínas del Tejido Nervioso/genética , Adolescente , Adulto , China , Eliminación de Gen , Humanos , MasculinoRESUMEN
Tissue plasminogen activator (tPA) is an essential component of the proteolytic cascade that lyses blood clots. Various studies also suggest that tPA plays important roles in peripheral nerve regeneration. Here we show that disruption of tPA gene reduces macrophage migration after sciatic nerve injury in mice. Moreover, lack of tPA activity attenuates migrating ability of macrophages and affects MMP-9 expression and activity in macrophages in vitro. Addition of ethylenediaminetetraacetic acid (EDTA), which inhibits MMPs, abolished the differences of migration ability of macrophages between tPA(+/+) and tPA(-/-) mice. Axonal regeneration is correlated with the increase of macrophage migration, suggesting that tPA may help create a beneficial environment for axonal regeneration through promoting macrophage infiltration. This study shows that tPA may play a role in nerve regeneration through regulating the migration ability of macrophages. This function of tPA may depend on, at least in part, upregulating MMP-9 expression and activity in macrophages.