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Biomedical subjects

Panos Ioannou

Publications and source records attributed to Panos Ioannou.

6 recordsLinked to original sources
PubMed2006

Chromatin-binding regions of EBNA1 protein facilitate the enhanced transfection of Epstein-Barr virus-based vectors.

Epstein-Barr virus (EBV)-based vectors can stably maintain large genomic fragments in mammalian cells, offering great potential for the treatment/correction of many acquired and inherited disorders. Numerous studies report marked increases in the transfection efficiency of EBV-based vectors after delivery into cell lines constitutively expressing Epstein-Barr nuclear antigen-1 (EBNA1), compared with cells not expressing EBNA1. We employ a novel strategy, involving the transfection of mRNA encoding EBNA1, to transiently express EBNA1 protein in human cells. Subsequently we show that the transfection efficiency of a 21-kb EBVbased vector is improved significantly when codelivered with mRNA encoding EBNA1. Similar increases in transfection efficiency were observed after delivery of the plasmid into cells constitutively expressing EBNA1. We also investigate the mechanism by which EBNA1 facilitates the transfection of EBV-based vectors, using mRNA encoding modified versions of the protein. Previous studies suggest that the EBNA1 DNA-binding domain (DBD), together with the nuclear localization signal (NLS), may enhance transfection of EBV plasmids by facilitating their nuclear transport. We demonstrate that an EBNA1 derivative comprising only the NLS and DBD does not facilitate transfection of EBV-based vectors. However, cells expressing an EBNA1 derivative devoid of a functional NLS but retaining the chromatin-binding regions, domains A and B, enhances plasmid transfection efficiency by up to 10-fold. Moreover, a variant of EBNA1 comprising two copies of domain A fused to the DBD enhances DNA transfection to an even greater extent than wild-type EBNA1. We therefore propose that EBNA1-mediated transfection of EBV-based vectors is dependent on the presence of chromatin- binding regions and the DBD, but not the NLS.

Active Transport, Cell Nucleus↗
PubMed2004

Friedreich's ataxia, no changes in mitochondrial labile iron in human lymphoblasts and fibroblasts: a decrease in antioxidative capacity?

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with (55)Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H(2)O(2) was significantly increased in patient cells, and H(2)O(2) toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2'-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA.

Adolescent↗
PubMed2003

Friedreich ataxia-update on pathogenesis and possible therapies.

Friedreich ataxia is the most-common inherited ataxia. Since the causative genetic basis was described in 1996, much has been learnt about the pathogenesis from human, animal, and yeast studies. This has led to the development of rational therapeutic approaches. In this review, the current state of knowledge regarding the pathogenesis of Friedreich ataxia is presented and possible therapeutic strategies based on this knowledge are discussed.

Animals↗
PubMed2002

Wnt-4 regulation by the Wilms' tumour suppressor gene, WT1.

The Wilms' tumour suppressor gene, WT1, encodes multiple nuclear protein isoforms, all containing four C-terminal zinc finger motifs. WT1 proteins can both activate and repress putative target genes in vitro, although the in vivo relevance of these putative target genes is often unverified. WT1 mutations can result in Wilms' tumour and the Denys-Drash Syndrome (DDS) of infantile nephropathy, XY pseudohermaphroditism and predisposition to Wilms' tumour. We have established stable transfectants of the mouse mesonephric cell line, M15, which express WT1 harbouring a common DDS point mutation (R394W). A comparison of the expression profiles of M15 and transfectant C2A was performed using Nylon-based arrays. Very few genes showed differential expression. However Wnt-4, a member of the Wnt gene family of secreted glycoproteins, was downregulated in C2A and other similar clones. Doxycycline induction of WT1-A or WT1-D expression in HEK293 stable transfectants also elicited an elevation in Wnt4 expression. Wnt4 is critical for the mesenchyme-to-epithelial transition during kidney development, making it an attractive putative WT1 target. We have mapped human Wnt-4 gene to chromosome 1p35-36, a region of frequent LOH in WT, have characterized the genomic structure of the human Wnt-4 gene and isolated 9 kb of immediate promoter. While several potential WT1 binding sites exist within this promoter, reporter analysis does not strongly support the direct regulation of Wnt4 by WT1. We propose that Wnt-4 regulation by WT1 occurs at a more distant promoter or enhancer site, or is indirect.

Amino Acid Substitution↗