Pérdida de las neuronas dopaminérgicas esencefálicas por la inactivación del gen Nurr1

Nurr 1 es un factor de transcripción que se expresa predominantemente en el cerebro. En el presente trabajo se clonó y caracterizó el gen murino nurr1 y se determinó mediante hibridación in situ su patrón de expresión durante el desarrollo embrionario y en el cerebro del ratón adulto. Además, utilizando la tecnología de inactivación de genes mediante recombinación homóloga, se generó un modelo animal murino carente de nurr1. La inactivación de este gen es letal y resulta en la degeneración de las neuronas dopaminérgicas en la región ventral mesencefálica, y como onsecuencia se pierde la síntesis del neurotransmisor dopamina

Binding of hnRNP H and U2AF65 to Respective G-codes and a Poly-Uridine Tract Collaborate in the N50-5'ss Selection of the REST N Exon in H69 Cells

The splicing of the N exon in the pre-mRNA coding for the RE1-silencing transcription factor (REST) results in a truncated protein that modifies the expression pattern of some of its target genes. A weak 3’ss, three alternative 5’ss (N4-, N50-, and N62-5’ss) and a variety of putative target sites for splicing regulatory proteins are found around the N exon; two GGGG codes (G2-G3) and a poly-Uridine tract (N-PU) are found in front of the N50-5’ss. In this work we analyzed some of the regulatory factors and elements involved in the preferred selection of the N50-5’ss (N50 activation) in the small cell lung cancer cell line H69. Wild type and mutant N exon/b-globin minigenes recapitulated N50 exon splicing in H69 cells, and showed that the N-PU and the G2-G3 elements are required for N50 exon splicing. Biochemical and knockdown experiments identified these elements as U2AF65 and hnRNP H targets, respectively, and that they are also required for N50 exon activation. Compared to normal MRC5 cells, and in keeping with N50 exon activation, U2AF65, hnRNP H and other splicing factors were highly expressed in H69 cells. CLIP experiments revealed that hnRNP H RNA-binding occurs first and is a prerequisite for U2AF65 RNA binding, and EMSA and CLIP experiments suggest that U2AF65-RNA recognition displaces hnRNP H and helps to recruit other splicing factors (at least U1 70K) to the N50-5’ss. Our results evidenced novel hnRNP H and U2AF65 functions: respectively, U2AF65-recruiting to a 5’ss in humans and the hnRNP H-displacing function from two juxtaposed GGGG codes

Production of biologically active human lymphotactin (XCL1) by Lactococcus lactis

Lymphotactin-XCL1 is a chemokine produced mainly by activated CD8? T-cells and directs migration of CD4? and CD8? lymphocytes and natural killer (NK) cells. We expressed human lymphotactin (LTN) by the lactic-acid bacterium Lactococcus lactis. Biological activity of LTN was confirmed by chemo-attraction of human T-cells by chemotaxis demonstrating, for the first time, how this chemokine secreted by a food-grade prokaryote retains biological activity and chemoattracts T lymphocytes. This strain thus represents a feasible well-tolerated vector to deliver active LTN at a mucosal level

Efficient secretion of a modified E7 protein from human papilloma virus type-16 by Lactococcus lactis

Aims: To create and provide a strain of the food-grade bacterium Lactococcus lactis able to efficiently secrete a modified form of the E7 protein from the human papilloma virus (HPV) type-16. Methods and Results: We cloned the coding sequence of a modified E7 (E7m)from the HPV-16 in a plasmid regulated by the strong expression promoter p59. Secretion of the E7m was made by the signal peptide of the usp45 gene.The E7m was detected by Western blot in the cell-free-medium fraction, showing no degradation or aberrant forms. Conclusions: We constructed a strain of L. lactis able to secrete efficiently aHPV-16 E7 modified protein with diminished transforming activity. Significance and Impact of the Study: Human papilloma virus infection is associated with more than 99% of cervical cancers. Immunotherapy targeting E7 to treat HPV-associated cervical malignancies has been demonstrated to be highly efficient. However, native E7 maintains transforming activity. We present this new strain of a food-grade bacterium able to efficiently secrete a HPV-16 E7-modified protein with diminished transforming activity. This new strain could be used as a live vaccine to deliver E7 at a mucosal level and generate antitumour immune responses against HPV-associated tumours

DNA vaccine encoding human papillomavirus antigens flanked by a signal peptide and a KDEL sequence induces a potent therapeutic antitumor effect

Abstract. Cellular immune responses play a critical role in the eradication of intracellular infections and malignant cells through the recognition and subsequent removal of the infection or malignant cells. Effective antigen presentation is crucial for stimulating the immune system against malignant cells. Calreticulin (CRT) has been used to improve antigen presentation. However, CRT overexpression has been previously associated with the development of pancreatic and breast cancer. The import and retention signals of CRT in the endoplasmic reticulum (ER) can be used to overcome CRT overexpression. The present study describes the potent antitumor effect of a DNA vaccine encoding human papillomavirus type 16 E6 and E7 antigens flanked by ER import and retention signals (SP-E6E7m-KDEL). The effect of this vaccine was compared with that of E6 and E7 antigens fused to human full-length CRT (hCRT-E6E7m). In the present study, the effectiveness of SP-E6E7m-KDEL for inducing an interferon-γ antigen‑specific, response and its therapeutic effect against tumors was demonstrated, which was as effective as immunization against those antigens fused to CRT. This simplified strategy, using ER import and retention signal peptides to direct antigens to this organelle, provides an efficient alternative to traditional vaccines and, more importantly, a safe and potent system to induce a therapeutic antitumor response