In Cellulo DNA Analysis: LMPCR Footprinting.

The in cellulo analysis of protein-DNA interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic sequencing technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate complex animal genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.

Chronic psychotropic drug use among frail elderly women receiving home care services.

Over the years, psychotropic drugs have been prescribed for symptoms of anxiety and/or insomnia. Elderly women are especially at risk of chronic use and ensuing side-effects. We examined psychosocial processes associated with long-term psychotropic drug use. We conducted in-depth interviews with 21 frail elderly women in a home care program and 14 of their primary caregivers. Results yielded a descriptive model of chronic use that takes into account antecedents of use, initial and subsequent prescription processes, individual contextual circumstances, the effect of the social context, and the women's cognitive strategies employed to make prolonged use coherent with their self-image.

In cellulo DNA analysis (LMPCR footprinting).

The in cellulo analysis of DNA protein interactions and chromatin structure is very important to better understand the mechanisms involved in the regulation of gene expression. The nuclease-hypersensitive sites and sequences bound by transcription factors often correspond to genetic regulatory elements. Using the Ligation-mediated polymerase chain reaction (LMPCR) technology, it is possible to precisely analyze these DNA sequences to demonstrate the existence of DNA-protein interactions or unusual DNA structures directly in living cells. Indeed, the ideal chromatin substrate is, of course, found inside intact cells. LMPCR, a genomic-sequencing, technique that map DNA single-strand breaks at the sequence level of resolution, is the method of choice for in cellulo footprinting and DNA structure studies because it can be used to investigate any complex genomes, including human. The detailed conventional and automated LMPCR protocols are presented in this chapter.

In vivo DNase I-mediated footprinting analysis along the human bradykinin B1 receptor (BDKRB1) gene promoter: evidence for cell-specific regulation.

By applying in vivo dimethyl sulphate and UV light type C-footprinting analysis, we previously showed that specific DNA sequences in the -1349/+42 core promoter region of the inducible human BDKRB1 (bradykinin B1 receptor) gene correlated with its transcriptional activity. In the present study we used the highly sensitive DNase I in vivo footprinting approach to delineate more precisely the functional domains of the BDKRB1 gene promoter in human SMCs (smooth muscle cells). Human lymphocytes that do not express a functional BDKRB1 were also studied as a reference using dimethyl sulphate, UV light type C and DNase I treatments. An obvious difference was found in the DNase I-footprinting patterns between cellular systems that express a functional BDKRB1 (SMCs) in comparison with human lymphocytes, where randomly distributed nucleosome-like footprinting patterns were found in the bulk of the core promoter region studied. Gel-shift assays and expression studies pointed to the implication of the YY1 and a TBP/TFIIB (TATA-box-binding protein/transcription factor IIB) transcription factor in the regulation of BDKRB1 gene expression in SMCs and possible YY1 involvement in the mechanisms of nuclear factor kappaB-mediated regulation of the receptor expression. No significant changes in the promoter foot-printing pattern were found after treatment with interleukin-1beta or serum (known BDKRB1 gene inducers), indicating that definite regulatory motifs could exist outside the BDKRB1 gene core promoter region studied.

Chromatin modification of the human imprinted NDN (necdin) gene detected by in vivo footprinting.

Allele-specific transcription is a characteristic feature of imprinted genes. Many imprinted genes are also transcribed in a tissue- or cell type-specific manner. Overlapping epigenetic signals must, therefore, modulate allele-specific and tissue-specific expression at imprinted loci. In addition, long-range interactions with an Imprinting Center (IC) may influence transcription, in an allele-specific or cell-type specific manner. The IC on human chromosome 15q11 controls parent-of-origin specific allelic identity of a set of genes located in cis configuration within 2 Mb. We have now examined the chromatin accessibility of the promoter region of one of the Imprinting Centre-controlled genes, NDN encoding necdin, using in vivo DNA footprinting to identify sites of DNA-protein interaction and altered chromatin configuration. We identified sites of modified chromatin that mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that long-lasting allele-specific marks and more labile tissue-specific marks layer epigenetic information that can be discriminated using DNA footprinting methodologies. Sites of modified chromatin mark the parental alleles in NDN-expressing cells, and in cells in which NDN is not expressed. Our results suggest that a layering of epigenetic information controls allele- and tissue-specific gene expression of this imprinted gene.

[Psychological distress and psychotropic drugs as perceived by elderly women and their aides during loss of autonomy: qualitative and quantitative perspective]. / Détresse psychologique et psychotropes, perception de femmes âgées en perte d'autonomie et leurs aidantes: une perspective qualitative et quantitative.

Using qualitative data and quantitative measures of psychological distress, this research examines attitudes towards psychotropic drug use among 14 home care recipients and their caregivers. It relates these attitudes to the type of family support provided and the women's level of mental health (both self-reported and attributed to the aged drug user by the caregiver). Four categorical themes--"it's a habit", "it's useful and under control", "it keeps her under control", and "what drug use?"--describe the types of attitudes of caregivers towards psychotropic drug use by the elderly women they care for. These themes are associated with the level of congruence between self-reported and caregiver-ascribed scores of mental health. The results, although exploratory, indicate that a large divergence between self-reported and caregiver-ascribed scores of psychological distress was associated with caregivers' attitudes of indifference or resignation towards drug use. The drug is perceived as necessary not only by the user but also by the overworked caregiver, who sees it as a way of lightening her burden. From a clinical perspective, our findings indicate that the influence of caregivers needs to be taken into account in any effort to reduce or stop chronic psychotropic drug use by elderly users.

Characterization of the survival motor neuron (SMN) promoter provides evidence for complex combinatorial regulation in undifferentiated and differentiated P19 cells.

There exist two SMN (survival motor neuron) genes in humans, the result of a 500 kb duplication in chromosome 5q13. Deletions/mutations in the SMN1 gene are responsible for childhood spinal muscular atrophy, an autosomal recessive neurodegenerative disorder. While the SMN1 and SMN2 genes are not functionally equivalent, up-regulation of the SMN2 gene represents an important therapeutic target. Consequently, we exploited in silico, in vitro and in vivo approaches to characterize the core human and mouse promoters in undifferentiated and differentiated P19 cells. Phylogenetic comparison revealed four highly conserved regions that contained a number of cis-elements, only some of which were shown to activate/repress SMN promoter activity. Interestingly, the effect of two Sp1 cis-elements varied depending on the state of P19 cells and was only observed in combination with a neighbouring Ets cis-element. Electrophoretic mobility-shift assay and in vivo DNA footprinting provided evidence for DNA-protein interactions involving Sp, NF-IL6 and Ets cis-elements, whereas transient transfection experiments revealed complex interactions involving these recognition sites. SMN promoter activity was strongly regulated by an NF-IL6 response element and this regulation was potentiated by a downstream Ets element. In vivo results suggested that the NF-IL6 response must function either via a protein-tethered transactivation mechanism or a transcription factor binding an upstream element. Our results provide strong evidence for complex combinatorial regulation and suggest that the composition or state of the basal transcription complex binding to the SMN promoter is different between undifferentiated and differentiated P19 cells.

Nerve regeneration in a collagen-chitosan tissue-engineered skin transplanted on nude mice.

A reconstructed skin made of a collagen-chitosan sponge seeded with human fibroblasts and keratinocytes and grown in vitro for 31 days was developed for the treatment of deep and extensive burns. The aim of this study was to assess whether this tissue-engineered skin could promote nerve regeneration in vivo, since recovery of sensation is a major concern for burnt patients. The human reconstructed skin was transplanted on the back of nude mice and the growth of nerve fibres within it was assessed 40, 60, 90 and 120 days after graft. Nerve growth was monitored by confocal microscopy using immunohistochemical staining of PGP 9.5 and 150 kD neurofilament, while Schwann cell migration was observed using protein S100 expression and laminin deposition. Nerve growth was first detected 60 days after transplantation and was more abundant 90 and 120 days after graft. Linear arrangements of Schwann cells were observed in the graft as early as 40 days after graft. Nerve growth was observed along these Schwann cell extensions 60 days after transplantation. We conclude that the three-dimensional architecture of the collagen-chitosan tissue-engineered skin sponge encourages nerve growth. This result provides new perspectives to increase nerve regeneration within the tissue-engineered skin by linkage of neurotrophic factors in the sponge before transplantation.