Mahogunin ring finger-1 (MGRN1) is a RING domain-containing ubiquitin ligase mutated in mahoganoid, a mouse mutation causing coat color darkening, congenital heart defects, high embryonic lethality, ...and spongiform neurodegeneration. The melanocortin hormones regulate pigmentation, cortisol production, food intake, and body weight by signaling through five G protein-coupled receptors positively coupled to the cAMP pathway (MC1R–MC5R). Genetic analysis has shown that mouse Mgrn1 is an accessory protein for melanocortin signaling that may inhibit MC1R and MC4R by unknown mechanisms. These melanocortin receptors (MCRs) regulate pigmentation and body weight, respectively. We show that human melanoma cells express 4 MGRN1 isoforms differing in the C-terminal exon 17 and in usage of exon 12. This exon contains nuclear localization signals. MGRN1 isoforms decreased MC1R and MC4R signaling to cAMP, without effect on β2-adrenergic receptor. Inhibition was independent on receptor plasma membrane expression, ubiquitylation, internalization, or stability and occurred upstream of Gαs binding to/activation of adenylyl cyclase. MGRN1 co-immunoprecipitated with MCRs, suggesting a physical interaction of the proteins. Significantly, overexpression of Gαs abolished the inhibitory effect of MGRN1 and decreased co-immunoprecipitation with MCRs, suggesting competition between MGRN1 and Gαs for binding to MCRs. Although all MGRN1s were located in the cytosol in the absence of MCRs, exon 12-containing isoforms accumulated in the nuclei upon co-expression with the receptors. Therefore, MGRN1 inhibits MCR signaling by a new mechanism involving displacement of Gαs, thus accounting for key features of the mahoganoid phenotype. Moreover, MGRN1 might provide a novel pathway for melanocortin signaling from the cell surface to the nucleus.
We assessed the role of age and disease activity as new factors contributing to establish the risk of progressive multifocal leucoencephalopathy in multiple sclerosis patients treated with ...natalizumab in 36 University Hospitals in Europe. We performed the study in 1,307 multiple sclerosis patients (70.8% anti-John Cunninghan virus positive antibodies) treated with natalizumab for a median time of 3.28 years. Epidemiological, clinical, and laboratory variables were collected. Lipid-specific IgM oligoclonal band status was available in 277 patients. Factors associated with progressive multifocal leucoencephalopathy onset were explored by uni- and multivariate logistic regression.
Thirty-five patients developed progressive multifocal leucoencephalopathy. The multivariate analysis identified anti-John Cunninghan virus antibody indices and relapse rate as the best predictors for the onset of this serious opportunistic infection in the whole cohort. They allowed to stratify progressive multifocal leucoencephalopathy risk before natalizumab initiation in individual patients area under the curve (AUC) = 0.85. The risk ranged from <1/3,300 in patients with anti-John Cunninghan virus antibody indices <0.9 and relapse rate >0.5, to 1/50 in the opposite case. In patients with lipid-specific IgM oligoclonal bands assessment, age at natalizumab onset, anti-John Cunninghan virus antibody indices, and lipid-specific IgM oligoclonal band status predicted progressive multifocal leucoencephalopathy risk (AUC = 0.92). The absence of lipid-specific IgM oligoclonal bands was the best individual predictor (OR = 40.94). The individual risk ranged from <1/10,000 in patients younger than 45 years at natalizumab initiation, who showed anti John Cunningham virus antibody indices <0.9 and lipid-specific IgM oligoclonal bands to 1/33 in the opposite case.
In a perspective of personalized medicine, disease activity, anti-lipid specific IgM oligoclonal bands, anti Jonh Cunninghan virus antibody levels, and age can help tailor natalizumab therapy in multiple sclerosis patients, as predictors of progressive multifocal leucoencephalopathy.
Several studies have revealed a correlation between chronic inflammation and nicotinamide adenine dinucleotide (NAD+) metabolism, but the precise mechanism involved is unknown. Here, we report that ...the genetic and pharmacological inhibition of nicotinamide phosphoribosyltransferase (Nampt), the rate-limiting enzyme in the salvage pathway of NAD+ biosynthesis, reduced oxidative stress, inflammation, and keratinocyte DNA damage, hyperproliferation, and cell death in zebrafish models of chronic skin inflammation, while all these effects were reversed by NAD+ supplementation. Similarly, genetic and pharmacological inhibition of poly(ADP-ribose) (PAR) polymerase 1 (Parp1), overexpression of PAR glycohydrolase, inhibition of apoptosis-inducing factor 1, inhibition of NADPH oxidases, and reactive oxygen species (ROS) scavenging all phenocopied the effects of Nampt inhibition. Pharmacological inhibition of NADPH oxidases/NAMPT/PARP/AIFM1 axis decreased the expression of pathology-associated genes in human organotypic 3D skin models of psoriasis. Consistently, an aberrant induction of NAMPT and PARP activity, together with AIFM1 nuclear translocation, was observed in lesional skin from psoriasis patients. In conclusion, hyperactivation of PARP1 in response to ROS-induced DNA damage, fueled by NAMPT-derived NAD+, mediates skin inflammation through parthanatos cell death.
The deubiquitylating enzyme OTUB1 is present in all tissues and targets many substrates, in both the cytosol and nucleus. We found that casein kinase 2 (CK2) phosphorylated OTUB1 at Ser(16) to ...promote its nuclear accumulation in cells. Pharmacological inhibition or genetic ablation of CK2 blocked the phosphorylation of OTUB1 at Ser(16), causing its nuclear exclusion in various cell types. Whereas we detected unphosphorylated OTUB1 mainly in the cytosol, we detected Ser(16)-phosphorylated OTUB1 only in the nucleus. In vitro, Ser(16)-phosphorylated OTUB1 and nonphosphorylated OTUB1 exhibited similar catalytic activity, bound K63-linked ubiquitin chains, and interacted with the E2 enzyme UBE2N. CK2-mediated phosphorylation and subsequent nuclear localization of OTUB1 promoted the formation of 53BP1 (p53-binding protein 1) DNA repair foci in the nucleus of osteosarcoma cells exposed to ionizing radiation. Our findings indicate that the activity of CK2 is necessary for the nuclear translocation and subsequent function of OTUB1 in DNA damage repair.
Prostaglandin E
(PGE
) plays an important role in immune activities in teleost fish, including seabream. However, receptors involved in PGE
signaling, as well as the pathways activated downstream, ...are largely unknown. In this study, one ortholog of mammalian PTGER1, PTGER3 and PTGER4, and two of PTGER2 (Ptger2a and Ptger2b) were identified and characterized in gilthead seabream. In silico analysis showed that all these receptors possessed the organization domain of G protein-coupled receptors, with the exception of Ptger2b. The corresponding in vivo studies revealed that they were expressed in all the tissues examined, the highest mRNA levels of ptger1 and ptger3 being observed in the spleen and of ptger2a and ptger4 in the blood. Bacterial infection induced higher mRNA levels of ptger2a, ptger3 and ptger4 in peritoneal exudate (the site of bacterial injection). In addition, head kidney acidophilic granulocytes and macrophages displayed different ptger1, ptger2a, ptger3 and ptger4 expression profiles. Furthermore, in macrophages the expression of the receptors was weakly affected by stimulation with bacterial DNA or with PGE
, while in acidophilic granulocytes stimulation resulted in the upregulation of ptger2a and ptger4. Taken together, these results suggest different roles for seabream PGE
receptors in the regulation of the immune responses.
Introducción: el dengue es considerada la enfermedad viral transmitida por artrópodos de mayor importancia en el humano a nivel global, con un estimado de 100 millones de infecciones anuales y más de ...20 000 muertes. La patogénesis de la enfermedad grave por dengue no está totalmente esclarecida. Sin embargo, existen estudios que la asocian con infecciones secuenciales por diferentes serotipos virales, activación de células T de memoria e hiperproducción de citocinas. Objetivos: determinar la expresión cualitativa de genes de las citoquinas IFNγ y TNFα en tejidos de bazo e hígado de casos fatales por dengue 3 o dengue 4 mediante la detección de ARNm y comprobar su papel en la patogénesis de la enfermedad. Métodos: El estudio se realizó a través de un método de retro-transcripción y reacción en cadena de la polimerasa (RT-PCR) con el uso de cebadores específicos. Resultados: la expresión de IFNγ predominó a la de TNFα, y fue más evidente en los tejidos de bazo que del hígado. Conclusiones: los resultados obtenidos apoyan el papel patogénico del patrón de respuesta Th1 en el dengue grave y constituye el primer estudio realizado de este tipo en la infección por dengue.
Introducción: durante años se ha especulado sobre el posible papel de los mastocitos en la patogenia de la infección por dengue, debido a su extensa ubicación en los tejidos. Sin embargo, su papel en ...relación con esta infección no ha sido bien explorado. Objetivo: evaluar el comportamiento de las plaquetas en el ratón Balb/c, deficiente de mastocitos peritoneales, en el cual se reproduce la infección secundaria. Métodos: se evaluó el comportamiento de las plaquetas en un modelo animal en ratón Balb/c en presencia o ausencia de mastocitos peritoneales. Resultados: se constató una disminución significativa de las plaquetas asociada a la presencia de los mastocitos (p= 0,0155). Conclusiones: los resultados sugieren que los mastocitos pudieran tener un papel patogénico en la infección por dengue.
Drug products used for treating tuberculosis are one of the most widely reported medicines to be classified as falsified or substandard in low- and middle-income countries, representing a major ...hazard to health. The aim of this study was, firstly, to develop an ultra-performance liquid chromatography (UPLC) method which is able to analyze fixed combination tablets with up to four active pharmaceutical ingredients, including isoniazid, pyrazinamide, rifampicin, and ethambutol. Secondly, we aimed to optimize it through the design of experiments and multi-linear regression based on a central composite design and to validate it according to the guidelines of the International Conference on Harmonization. The application of this tools enabled the identification of the influential factors (flow rate, formic acid, and temperature) and their effects on the studied responses (retention factor and percentage for each drug) as part of the quality by design approach. The method proved to be to be linear in the range from 5.0 to 15 µg/mL for isoniazid, pyrazinamide, and rifampicin, being precise (<1%) and accurate (97−101%). In addition, the method validated for ethambutol proved to be linear from 1.4 to 4.2 µg/mL, as well as precise (0.54%) and accurate (97.3%). The method was stability indicated for all the active pharmaceutical ingredients studied and was able to detect two substandard formulations sampled on the African market.
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