Biomedical subjects

Luis A López-Fernández

Publications and source records attributed to Luis A López-Fernández.

7 recordsLinked to original sources

PubMed2006

Human gene profiling in response to the active protein kinase, interferon-induced serine/threonine protein kinase (PKR), in infected cells. Involvement of the transcription factor ATF-3 IN PKR-induced apoptosis.

The interferon-induced serine/threonine protein kinase (PKR) has an essential role in cell survival and cell death after viral infection and under stress conditions, but the host genes involved in these processes are not well defined. We used human cDNA microarrays to identify, in infected cells, genes differentially expressed after PKR expression and analyzed the requirement of catalytic activity of the enzyme. To express PKR, we used vaccinia virus (VV) recombinants producing wild type PKR (VV-PKR) and the catalytically inactive mutant K296R (VV-PKR-K296R). Most regulated genes were classified according to biological function, including apoptosis, stress, defense, and immune response. Transcriptional changes detected by microarray analysis were confirmed for selected genes by quantitative real time reverse transcription PCR. A total of 111 genes were regulated specifically by PKR catalytic activity. Of these, 97 were up-regulated, and 14 were down-regulated. The ATF-3 transcription factor, involved in stress-induced beta-cell apoptosis, was up-regulated. Activation of endogenous PKR with a VV mutant lacking the viral protein E3L (VVDeltaE3L), a PKR inhibitor, triggered an increase in ATF-3 expression that was not observed in PKR(-/-) cells. Using null cells for ATF-3 and for the p65 subunit of NF-kappaB, we showed that induction of apoptosis by PKR at late times of infection was dependent on ATF-3 expression and regulated by NF-kappaB activation. Here, we identified human genes selectively induced by expression of active PKR in infected cells and linked ATF-3 to a novel mechanism used by PKR to induce apoptosis.

3T3 Cells↗

PubMed2006

Host response to the attenuated poxvirus vector NYVAC: upregulation of apoptotic genes and NF-kappaB-responsive genes in infected HeLa cells.

NYVAC has been engineered as a safe, attenuated vaccinia virus (VV) vector for use in vaccination against a broad spectrum of pathogens and tumors. Due to the interest in NYVAC-based vectors as vaccines and current phase I/II clinical trials with this vector, there is a need to analyze the human host response to NYVAC infection. Using high-density cDNA microarrays, we found 368 differentially regulated genes after NYVAC infection of HeLa cells. Clustering of the regulated genes identified six discrete gene clusters with altered expression patterns. Clusters 1 to 3 represented 47.5% of the regulated genes, with three patterns of gene activation kinetics, whereas clusters 4 to 6 showed distinct repression kinetics. Quantitative real-time reverse transcription-PCR analysis of selected genes validated the array data. Upregulated transcripts correlated with genes implicated in immune responses, including those encoding interleukin-1 receptor 2 (IL-1R2), IL-6, ISG-15, CD-80, and TNFSF7. NYVAC upregulated several intermediates of apoptotic cascades, including caspase-9, correlating with its ability to induce apoptosis. NYVAC infection also stimulated the expression of NF-kappaB1 and NF-kappaB2 as well as that of NF-kappaB target genes. Expression of the VV host range K1L gene during NYVAC infection prevented NF-kappaB activation, but not the induction of apoptosis. This study is the first overall analysis of the transcriptional response of human cells to NYVAC infection and provides a framework for future functional studies to evaluate this vector and its derivatives as human vaccines.

Activating Transcription Factors↗

PubMed2004

Microarray analysis reveals characteristic changes of host cell gene expression in response to attenuated modified vaccinia virus Ankara infection of human HeLa cells.

The potential use of the modified vaccinia virus Ankara (MVA) strain as a live recombinant vector to deliver antigens and elicit protective immune responses against infectious diseases demands a comprehensive understanding of the effect of MVA infection on human host gene expression. We used microarrays containing more than 15,000 human cDNAs to identify gene expression changes in human HeLa cell cultures at 2, 6, and 16 h postinfection. Clustering of the 410 differentially regulated genes identified 11 discrete gene clusters with altered expression patterns after MVA infection. Clusters 1 and 2 (accounting for 16.59% [68 of 410] of the genes) contained 68 transcripts showing a robust induction pattern that was maintained during the course of infection. Changes in cellular gene transcription detected by microarrays after MVA infection were confirmed for selected genes by Northern blot analysis and by real-time reverse transcription-PCR. Upregulated transcripts in clusters 1 and 2 included 20 genes implicated in immune responses, including interleukin 1A (IL-1A), IL-6, IL-7, IL-8, and IL-15 genes. MVA infection also stimulated the expression of NF-kappaB and components of the NF-kappaB signal transduction pathway, including p50 and TRAF-interacting protein. A marked increase in the expression of histone family members was also induced during MVA infection. Expression of the Wiskott-Aldrich syndrome family members WAS, WASF1, and the small GTP-binding protein RAC-1, which are involved in actin cytoskeleton reorganization, was enhanced after MVA infection. This study demonstrates that MVA infection triggered the induction of groups of genes, some of which may be involved in host resistance and immune modulation during virus infection.

Blotting, Northern↗

PubMed2004

[Professional practice and gender in primary care].

In this paper we analyse the relations between sex of patients and professionals, social class and health insurance scheme, with the type of medical practice and level of satisfaction with care received by the patients. Following recent literature it can be said that medical practices based on the psychosocial paradigm obtain better results in terms of health and satisfaction. In the primary health care sector, there is evidence that women doctors follow a communication pattern grounded on the psychosocial model. In parallel, women patients frequently show their preferences for women doctors when their complaints are related to psychosocial issues. Sex and social class of patients and professionals are not consistently related to the level of satisfaction reached, although to date there is still a lack of research to support this point. It is necessary to incorporate in our research these analytical categories. Not only in data collection but also in the analysis. It would not be possible to fully understand the complexity of clinical activities without considering that gender and class shape our individual and social identity and therefore condition the diverse forms of interaction in patient-doctor relations.

Female↗

PubMed2003

Cellular gene expression survey of vaccinia virus infection of human HeLa cells.

Vaccinia virus (VV) is a cytocidal virus that causes major changes in host cell machinery shortly after infecting cells. To define the consequences of virus infection on host gene expression, we used microarrays of approximately 15,000 human cDNAs to examine expression levels of mRNAs isolated at 2, 6, and 16 h postinfection from cultures of infected HeLa cells. The majority of profiling changes during VV infection corresponded to downregulation of genes at 16 h postinfection. Differentially expressed genes were clustered into seven groups to identify common regulatory pathways, with most of them (90%) belonging to clusters 6 and 7, which represent genes whose expression was repressed after infection. Cluster 1, however, contained 37 transcripts (2.81%) showing a robust pattern of induction that was maintained during the course of infection. Genes in cluster 1 included those for Wiskott-Aldrich syndrome protein (WASP) family member WASF1, thymosine, adenosine A2a receptor, glutamate decarboxylase 2, CD-80 antigen, KIAA0888 protein, selenophosphate synthetase, pericentrin, and attractin as well as several expressed sequence tags. We analyzed in more detail the fate of WASP protein in VV-infected cells, because a related family member, N-WASP, is involved in viral motility. WASP protein accumulated in the course of infection; its increase required viral DNA replication and de novo protein synthesis, and it localized in cytoplasmic structures distinct from uninfected cells. This study is the first quantitative analysis of host gene expression following VV infection of cultured human cells, demonstrating global changes in the expression profile, and identifies upregulated genes with potential roles in the virus replication cycle.

Blotting, Western↗

PubMed2003

Developmental regulation of expression of Ran/M1 and Ran/M2 isoforms of Ran-GTPase in mouse testis.

Two isoforms of Ran-GTPase have been described: Ran/M1 and Ran/M2. Ran/M2 is testis specific, whereas the Ran/M1 isoform is also expressed in somatic tissues. Here we show that both mRNAs, differing in 35 of the 648 nucleotides included in the ORFs, are developmentally regulated during spermatogenesis. Real-time RT-PCR experiments demonstrated that the expression of Ran/M1 and Ran/M2 increased in pachytene spermatocytes with progressive transcript accumulation until they reached the round spermatid stage, in the seminiferous epithelium of adults. In the testis, the expression of both isoforms was found to be restricted to germ cells. An expression window from early pachytene spermatocytes to late round spermatids was detected by in situ hybridization.

Animals↗

PubMed2002

Ran GTPase expression during early development of the mouse embryo.

Ran is a small GTP-binding protein involved in several essential roles for cell viability. This observation implies Ran might be ubiquitously expressed during development. However, Ran shows a differentiated expression pattern that is restricted to specific tissues from embryo to adult. At early embryonic stages of mouse development we found persistent Ran expression in proliferating neural tissue, neural crest derived dorsal root ganglions and sensory pits. We also showed an accumulation of Ran transcripts in main embryonic haematopoietic tissues: blood islands first and then hepatic bud. In advanced stages of development Ran is also expressed in other tissues showing a high cell turnover.

Animals↗