Inhibition of extracellular nucleotides hydrolysis intensifies the allergic bronchospasm. A novel protective role of ectonucleotidases.

BACKGROUND: Nucleotides released to the extracellular space stimulate purinergic receptors, and their effects are modulated by ectonucleotidases. The role of ATP in the allergic bronchospasm has been scantly studied. METHODS: We used several techniques (plethysmography, organ baths, confocal microscopy, RT-PCR, ATP measurement) to explore the role of nucleotides and ectonucleotidases in the allergic bronchospasm in guinea pigs. RESULTS: While allergenic challenge with a low-dose ovalbumin (OVA) only produced a small bronchospasm (~2-fold the basal lung resistance), previous inhibition of ectonucleotidases by ARL-67156 greatly intensified this response (~11-fold the basal lung resistance, with 44% mortality). Bronchoalveolar lavage fluid obtained during this bronchospasm contained increased ATP concentration. This potentiation was abolished by antagonism of purinergic receptors (suramin+RB2) or TXA2 receptor (SQ29548), or by intratracheal apyrase. In tracheal rings and lung parenchyma strips, OVA caused a concentration-dependent contraction. Suramin+RB2 or levamisole produced a significant rightward displacement of this response, and ARL-67156 did not modify it. Platelets stimulated with OVA released ATP. Confocal images of nonsensitized tracheas showed slight fluorescence for P2Y6 receptors in epithelium and none for P2Y4 . Sensitized animals showed strong fluorescence to both receptors and to alkaline phosphatase in the airway epithelium. This correlated with a large increment in mRNA for P2Y4 and P2Y6 receptors in sensitized animals. CONCLUSIONS: Nucleotides greatly potentiate the allergic bronchospasm when ectonucleotidases activity is diminished, and this effect is probably favored by the upregulation of P2Y4 and P2Y6 receptors in airway epithelium during sensitization. These results prompt for further research on these mechanisms in human asthma.

Prolactin reduces the kainic acid-induced increase in intracellular Ca2+ concentration, leading to neuroprotection of hippocampal neurons.

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.

Improved molecular karyotype of Entamoeba histolytica.

Different electrophoretic conditions were used to improve the molecular karyotype of Entamoeba histolytica clone L6 derived from the heterogeneous strain HM1:IMSS. Eleven to 17 bands ranging between 0.3 and over 3 megabases (Mb) were resolved by transverse alternating field electrophoresis (TAFE). Amebic chromosomes presented similar pattern when they were TAFE separated at 72, 90 or 120 h running time, but resolution of the bands was increased at 90 h, and the best electrophoretic pattern was obtained at 120 h. Bal 31 digestion of DNA in the plugs suggested that most of the bands are lineal molecules and that pMD, an amebic DNA fragment, hybridizes with both lineal and nonlineal DNA molecules.

Entamoeba histolytica: structural and functional analysis of the Ehadh112 gene promoter.

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.

The Entamoeba histolytica Ehcp112 gene has a distal and weak promoter.

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.