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Jesus M Aizpurua,
José Ignacio Ganboa,
Claudio Palomo,
Iraida Loinaz,
Joseba Oyarbide,
Xabier Fernandez,
Eva Balentová,
Raluca M Fratila,
Azucena Jiménez,
José Ignacio Miranda,
Antonio Laso,
Silvia Avila, José Luis Castrillo
ChemBioChem 02/2011; 12(3):401-5. · 3.94 Impact Factor
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ABSTRACT: Expression of POU1F1 gene, a member of the POU homeodomain family of transcription factors, is necessary for normal differentiation, development and survival of three anterior pituitary cell types (thyrotrophs, somatotrophs and lactotrophs) and for the proper expression of growth hormone (GH), prolactin (PRL), thyroid-stimulating hormone (TSH) genes and POU1F1 gene itself. Alternative splicing forms of this gene have been reported in different species, with few functional studies. Apart from the POU1F1-Wild-type with the expected length, in this work we isolated three additional splicing variants: POU1F1-beta, with a 78 bp insert in the trans-activation domain; POU1F1-gamma that lacks exon 3 and POU1F1-delta that lacks exons 3, 4 and 5. Four different protein isoforms were also detected by Western blot in the sheep pituitary tissue. Functional assays were performed to study the trans-activation of GH and PRL promoters by the splicing variants. Regarding the PRL promoter, the beta variant presented only 12% of the Wild-type trans-activation capacity. Variants gamma and delta showed no capacity to trans-activate PRL promoter. Both gamma and delta variants acted as repressors of Wt, reducing significantly the trans-activation made by Wt alone (p<0.05). Concerning the GH promoter, the beta variant presented a trans-activation capacity 10% higher than Wt. Wt and beta variants strongly interact in the activation of GH promoter doubling the trans-activation potential of Wt. Variants gamma and delta showed no capacity to trans-activate the GH promoter and both acted as repressors, reducing significantly (p<0.001) the trans-activation performed by Wt. This work presents, for the first time, the characterization of four splicing forms of Ovis aries POU1F1 gene.
Gene 11/2006; 382:12-9. · 2.34 Impact Factor
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Lucía Quintana,
Alberto Monasterio,
Kepa Escuredo,
Jokin del Amo,
Pilar Alfonso,
Felix Elortza,
Simon Santa Cruz,
Laureano Simón,
Antonio Martínez,
Pilar Giraldo,
Miguel Pocoví, José Luis Castrillo
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ABSTRACT: Chitotriosidase protein (ChT) is the most important biochemical marker described for Gaucher disease (GD). ChT activity is increased several hundred-fold in plasma of GD patients and shows a strong positive correlation with the severity of the disease. However, a recessively inherited enzyme deficiency, with an incidence of about 6% in the Caucasian population, means that not all patients with GD can be monitored by measuring ChT activity. Applying two-dimensional gel electrophoresis (2-DE) technology this study describes the localization and identification of five ChT isoforms in 2-DE images obtained from plasma of GD patients. All these isoforms were unequivocally identified using MALDI-TOF mass spectrometry (MS) and validated by western blot analysis. The features of each ChT isoform separated by 2-DE in plasma from GD patients homozygous for the wild-type ChT allele, carriers of one defective allele and patients homozygous for the mutant allele are presented. We also show the correlation between each ChT isoform and the plasma ChT enzymatic activity of the GD patients sampled in this study.
Biochimica et Biophysica Acta 08/2006; 1764(7):1292-8. · 4.66 Impact Factor
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ABSTRACT: POU1F1 (PIT-1/GHF-1) is a transcription factor with critical role in the transcriptional regulation of multiple genes in the pituitary and also important for the survival, differentiation and proliferation of three pituitary cell types. To understand the regulation of POU1F1 gene in Ovis aries we report its cloning, sequencing and characterization. The sequenced 5787 bp included six exons and two complete introns. Ovine POU1F1 gene has a high level of conservation with its bovine, human and rat counterparts showing 98.2%, 91.2% and 86.2% of similarity at the coding level, respectively. All six exons were analyzed for polymorphism detection in 100 animals of the Portuguese indigenous ovine breed 'Churra da Terra Quente'. One polymorphism was found at codon 58 in exon 2, in one allele of 4 animals leading to a change from cysteine to tyrosine (2% allelic frequency). In exon 3 two polymorphisms were detected: a G to A transition altering a glycine to an asparagine at codon 89 in one allele of one animal (0.5% allelic frequency) and another G to A transition at codon 105 converting an alanine into a threonine in one allele of 3 animals (1.5% allelic frequency). These polymorphisms might change the structure of the POU1F1 protein and modify gene-expression. In intron 4, an A to G transition was detected in one allele of six animals (3% allelic frequency). Exons 1, 4 and 6 showed no polymorphisms.
Genetica 04/2006; 126(3):303-14. · 2.15 Impact Factor
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ABSTRACT: Here we report the identification and expression analysis of two novel human genes--DIPLA1 (Differentially expressed in placenta 1) and DIPAS (DIPLA1 Antisense). These genes are located at chromosomal region 9q33.1, in opposite orientations, and are flanked by the pregnancy-associated plasma protein-A (PAPP-A) and astrotactin 2 (ASTN2) genes. The mRNA sequences of both genes contain several upstream AUGs (uAUG) and various potential open reading frames (ORFs). DIPLA1 mRNA is 1.8 kb long and contains a 285 nt ORF coding for a polypeptide designated as replicative senescence up-regulated (RSU) protein. Antisense DIPAS mRNA is 2.7 kb long and contains a 309 nt ORF coding for a protein with partial similitude to the gamma isoform variant of the human Ca(2+)/calmodulin (CaM)-dependent protein kinase II. Both genes are conserved in placental-species and are presumably transcribed from initiator (Inr) promoter elements located at opposite strands. In 20 human normal tissues tested, DIPLA1 mRNA expression was placenta-specific, whereas DIPAS mRNA expression was higher in placenta, brain, kidney and testis. In addition, DIPAS mRNA hybridizes with the 3'UTR region from PAPP-A mRNA, which spans over 4 kb more than previously reported, forming a potential sense-antisense double stranded RNA (dsRNA) duplex. Our results are of interest for placenta gene expression regulation and for the identification of novel genes in the human genome.
Gene 02/2005; 344:241-50. · 2.34 Impact Factor
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ABSTRACT: In the past, human endometrial receptivity has been investigated by chasing specific molecules throughout the menstrual cycle. Now the genomic approach allows us to investigate the hierarchical contribution of a high number of genes to a specific function. In this study, we analyzed differentially the gene expression pattern of 375 human cytokines, chemokines, and related factors, plus that of their receptors, in endometrial receptivity. To do this, we used a combined approach of human endometrium and cell lines. We have compared the gene expression pattern in receptive vs. prereceptive human endometria and contrasted the results with gene expression in the highly adhesive cell line (to JAR cells and mouse blastocysts) RL95-2 vs. HEC-1A, a cell line with markedly less adhesiveness. IGF-binding protein-related protein 1 (IGFBP-rP1), also known as IGFBP-7/mac 25, was the second most up-regulated gene in both of the investigated models. These results were corroborated by performing RT-PCR on the same RNA samples and validated by quantitative fluorescent RT-PCR and in situ hybridization in endometrium throughout the menstrual cycle. Interestingly, a 35-fold increase in expression during the receptive phase was compared with the prereceptive phase followed by a sharp increase in the late luteal. Further quantitative fluorescent RT-PCR experiments using the epithelial and stromal endometrial fraction throughout the menstrual cycle confirmed that IGFBP-rP1 expression was localized in the epithelial and stromal compartments and up-regulated mainly in the latter. In situ experiments confirmed the endometrial localization and regulation of IGFBP-rP1 mRNA. At the protein level, IGFBP-rP1 was localized by immunohistochemistry at the apical part of the luminal and glandular epithelium, stromal, and endothelial cells. In conclusion, using a genomic approach with a combined experimental design of receptivity in vivo and in vitro, we have discovered the implication of IGFBP-rP1 in endometrial physiology, which seems related to endometrial receptivity.
Journal of Clinical Endocrinology & Metabolism 05/2003; 88(4):1849-57. · 6.50 Impact Factor
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ABSTRACT: Endometrial receptivity is a self-limited period in which the endometrial epithelium (EE) acquires a functional and transient ovarian steroid-dependent status that allows blastocyst adhesion. Termed as "the window of implantation", this specific period opens 4-5 days after progesterone production or administration and closes after 9-10 days. Scientific knowledge on the endometrial receptivity process is fundamental for the understanding of human reproduction, but so far none of the proposed biochemical markers for endometrial receptivity has been proven to be clinically useful. In this work, we present strategies of cDNA analysis technologies that aim to clarify the fragmented information in this field. Specifically, the objective is the differential identification, cloning and sequencing of genes linked to endometrial receptivity in humans, combining differential display PCR and cDNA microarray analysis of endometrial epithelial-derived cell lines and endometrial samples obtained in the same patient 2 and 7 days after the luteinizing hormone (LH) surge (day LH+2) and (day LH+7), respectively.
Journal of Reproductive Immunology 55(1-2):131-9. · 2.97 Impact Factor
