RESUMEN
The aim of this study was to determine the effect of prolactin (PRL) on intracellular calcium (Ca2+) concentration and its neuroprotective role in a model of kainic acid (KA) excitotoxicity in primary cultures of hippocampal neurons. Cell viability and intracellular Ca2+ concentrations were determined by MTT and Fura-2 assays, respectively, either after induction by KA as an agonist or after treatment with NBQX antagonist alone or in combination with PRL administration. Expression of ionotropic glutamatergic receptors (iGluRs) subunits in neuronal cells was determined by RT-qPCR. Dose-response treatments with KA or glutamate (Glu), the latter used as endogenous agonist control, induced a significant increase in neuronal intracellular Ca2+ concentration followed by a significant decrease in hippocampal neuronal viability. Administration of PRL induced a significant increase in neuronal viability after treatment with KA. Furthermore, administration of PRL decreased intracellular Ca2+ concentrations induced by KA treatment. Independent administration of the AMPAR-KAR antagonist reversed cell death and reduced intracellular Ca2+ concentration in a similar manner as PRL. Additionally, mRNA expression of AMPAR, KAR and NMDAR subtypes were detected in hippocampal neurons; however, no significant changes in iGluRs subunit expression were observed due to excitotoxicity or PRL treatment. The results suggest that PRL inhibits the increase in intracellular Ca2+ concentration induced by KA, leading to neuroprotection.
Asunto(s)
Ácido Kaínico , Prolactina , Prolactina/farmacología , Ácido Kaínico/toxicidad , Neuroprotección , Hipocampo/metabolismo , Neuronas/metabolismoRESUMEN
The Entamoeba histolytica Ehcp112 and Ehadh112 genes that encode the EhCPADH complex are separated by a non-coding 188pb region. Their proximity suggests a coordinated expression regulation for both genes. Here, we studied the structure and function of 996 bp (p996CAT) upstream the ATG start codon of the Ehadh112 gene. The p996CAT plasmid drove CAT transcription with a 78% of the activity showed by actin promoter. Deletion of 330 bp at the 5' end of p966CAT to produce the p776CAT plasmid, decreased activity to 40% in relation to actin promoter and to 50% of p996CAT, suggesting the presence of a silencer in this region. Interestingly, deletion of other 297 bp to the p776CAT to generate the p469CAT plasmid, augmented activity in 2.5-fold compared with p776CAT construction, showing the presence of a proximal enhancer promoter. Transcription initiation sites (-69 and -150 bp), TATA like box, GAAC, and Inr elements, as well as putative DNA binding motifs, were mapped in the -1 to -469 bp core promoter region.
Asunto(s)
Entamoeba histolytica/genética , Lectinas/genética , Glicoproteínas de Membrana/genética , Regiones Promotoras Genéticas/fisiología , Proteínas Protozoarias/genética , Animales , Secuencia de Bases , Cloranfenicol O-Acetiltransferasa/genética , ADN Protozoario/química , Lectinas/fisiología , Glicoproteínas de Membrana/fisiología , Datos de Secuencia Molecular , Plásmidos , Regiones Promotoras Genéticas/genética , Proteínas Protozoarias/fisiología , TransfecciónRESUMEN
Ehcp112 encodes the Entamoeba histolytica EhCP112 cysteine protease that is part of the EhCPADH complex. By in silico analysis we identified putative transcription factor-binding sites along 837 bp upstream the Ehcp112 gene ATG codon. A TATA-like motif (TATATAAA) was located at -36 to -29 bp, a GAAC box (GAACC) was found at -10 to -14 bp and an Inr sequence (TTCAAC) at -8 to -2 bp. These tripartite promoter elements are in non-canonical positions, downstream the transcription initiation site (-280 bp). We cloned four Ehcp112 promoter fragments in pBSCAT-ACT plasmid to obtain pI (355 bp), pII (681 bp), pIII (833 bp), and pIV (554 bp) constructs. In transfected trophozoites, only pIII drove CAT activity with 44% efficiency in relation to actin promoter activity. Our results showed the presence of a distal and weak promoter in the Ehcp112 gene. The active DNA region is inside the open reading frame of the Ehrab B gene, suggesting that expression of both genes could be coordinately regulated.