RESUMEN
Trypsin Modulating Oostatic Factor (TMOF) receptor was solubilized from the guts of female Ae. Aegypti and cross linked to His6-TMOF and purified by Ni affinity chromatography. SDS PAGE identified two protein bands (45 and 61 kDa). The bands were cut digested and analyzed using MS/MS identifying a protein sequence (1306 amino acids) in the genome of Ae. aegypti. The mRNA of the receptor was extracted, the cDNA sequenced and cloned into pTAC-MAT-2. E. coli SbmA- was transformed with the recombinant plasmid and the receptor was expressed in the inner membrane of the bacterial cell. The binding kinetics of TMOF-FITC was then followed showing that the cloned receptor exhibits high affinity to TMOF (KD = 113.7 ± 18 nM ± SEM and Bmax = 28.7 ± 1.8 pmol ± SEM). Incubation of TMOF-FITC with E. coli cells that express the receptor show that the receptor binds TMOF and imports it into the bacterial cells, indicating that in mosquitoes the receptor imports TMOF into the gut epithelial cells. A 3D modeling of the receptor indicates that the receptor has ATP binding sites and TMOF transport into recombinant E. coli cells is inhibited with ATPase inhibitors Na Arsenate and Na Azide.
Asunto(s)
Aedes/genética , Clonación Molecular/métodos , Proteínas de Insectos/química , Proteínas de Insectos/genética , Receptores de Péptidos/química , Receptores de Péptidos/genética , Secuencia de Aminoácidos , Animales , Femenino , Tracto Gastrointestinal/fisiología , Estructura Secundaria de Proteína , Estructura Terciaria de ProteínaRESUMEN
Amyotrophic lateral sclerosis (ALS) is a devastating progressive neurodegenerative disease characterized by the selective death of motor neurons. Disease pathophysiology is complex and not yet fully understood. Higher gene expression of the inositol 1,4,5-trisphosphate receptor 2 gene (ITPR2), encoding the IP3 receptor 2 (IP3R2), was detected in sporadic ALS patients. Here, we demonstrate that IP3R2 gene expression was also increased in spinal cords of ALS mice. Moreover, an increase of IP3R2 expression was observed in other models of chronic and acute neurodegeneration. Upregulation of IP3R2 gene expression could be induced by lipopolysaccharide (LPS) in murine astrocytes, murine macrophages and human fibroblasts indicating that it may be a compensatory response to inflammation. Preventing this response by genetic deletion of ITPR2 from SOD1G93A mice had a dose-dependent effect on disease duration, resulting in a significantly shorter lifespan of these mice. In addition, the absence of IP3R2 led to increased innate immunity, which may contribute to the decreased survival of the SOD1G93A mice. Besides systemic inflammation, IP3R2 knockout mice also had increased IFNγ, IL-6 and IL1α expression. Altogether, our data indicate that IP3R2 protects against the negative effects of inflammation, suggesting that the increase in IP3R2 expression in ALS patients is a protective response.
Asunto(s)
Esclerosis Amiotrófica Lateral/genética , Inflamación/genética , Receptores de Inositol 1,4,5-Trifosfato/genética , Superóxido Dismutasa-1/genética , Esclerosis Amiotrófica Lateral/patología , Animales , Modelos Animales de Enfermedad , Humanos , Inflamación/patología , Interferón gamma/biosíntesis , Interleucina-1beta/biosíntesis , Interleucina-6/biosíntesis , Lipopolisacáridos , Masculino , Ratones , Ratones Noqueados , Neuronas Motoras/metabolismo , Neuronas Motoras/patología , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Two surveys of over 1,700 publications whose authors use quantitative real-time PCR (qPCR) reveal a lack of transparent and comprehensive reporting of essential technical information. Reporting standards are significantly improved in publications that cite the Minimum Information for Publication of Quantitative Real-Time PCR Experiments (MIQE) guidelines, although such publications are still vastly outnumbered by those that do not.