After much effort in surface chemistry development and optimization by several groups, fluorescent semiconductor nanocrystals probes, also known as quantum dots or qdots, are now entering the realm of biological applications with much to offer to biologists. The road to success has been paved with hurdles but from these efforts has stemmed a multitude of original surface chemistries that scientists in the biological fields can draw from for their specific biological applications. The ability to easily modulate the chemical nature of qdot surfaces by employing one or more of the recently developed qdot coatings, together with their exceptional photophysics have been key elements for qdots to acquire a status of revolutionary fluorescent bio-probes. Indeed, the unique properties of qdots not only give biologists the opportunity to explore advanced imaging techniques such as single molecule or lifetime imaging but also to revisit traditional fluorescence imaging methodologies and extract yet unobserved or inaccessible information in vitro or in vivo.

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106-6 and ROS 17/23 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear 'run-on' transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6-dichloro-l-~-~-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear posttranscriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position -2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions -906 to -931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated =IS-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated. Abbreviations. BSP, bone sialoprotein ; GRE, glucocorticoid response element; ERE, estrogen response element. Keywords. Mineralized tissuesNote. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X86100 for the R. nowegicus BSP gene. blast precursors [6, 71 and from an increase of bone...

No abstract

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106-6 and ROS 17/23 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear 'run-on' transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6-dichloro-l-~-~-ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear posttranscriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position -2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half-sites and a putative AP-1 site, located within positions -906 to -931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated =IS-fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated. Abbreviations. BSP, bone sialoprotein ; GRE, glucocorticoid response element; ERE, estrogen response element. Keywords. Mineralized tissuesNote. The novel nucleotide sequence data published here have been deposited with the EMBL sequence data bank and are available under the accession number X86100 for the R. nowegicus BSP gene. blast precursors [6, 71 and from an increase of bone...

No abstract