Simultaneous polarized neutron reflectometry and anisotropic magnetoresistance measurements.

A novel experimental facility to carry out simultaneous polarized neutron reflectometry (PNR) and anisotropic magnetoresistance (AMR) measurements is presented. Performing both techniques at the same time increases their strength considerably. The proof of concept of this method is demonstrated on a CoO/Co bilayer exchange bias system. Although information on the same phenomena, such as the coercivity or the reversal mechanism, can be separately obtained from either of these techniques, the simultaneous application optimizes the consistency between both. In this way, possible differences in experimental conditions, such as applied magnetic field amplitude and orientation, sample temperature, magnetic history, etc., can be ruled out. Consequently, only differences in the fundamental sensitivities of the techniques can cause discrepancies in the interpretation between the two. The almost instantaneous information obtained from AMR can be used to reveal time-dependent effects during the PNR acquisition. Moreover, the information inferred from the AMR measurements can be used for optimizing the experimental conditions for the PNR measurements in a more efficient way than with the PNR measurements alone.

Differential regulation of epidermal function by VDR coactivators.

The transcriptional activity of the vitamin D receptor (VDR) is regulated by a number of coactivator and corepressor complexes, which bind to the VDR in a ligand (1,25(OH)2D3) dependent (coactivators) or inhibited (corepressors) process. In the keratinocyte the major coactivator complexes include the vitamin D interacting protein (DRIP) complex and the steroid receptor coactivator (SRC) complexes. These coactivator complexes are not interchangeable in their regulation of keratinocyte proliferation and differentiation. We found that the DRIP complex is the main complex binding to VDR in the proliferating keratinocyte, whereas SRC2 and 3 and their associated proteins are the major coactivators binding to VDR in the differentiated keratinocyte. Moreover, we have found a specific role for DRIP205 in the regulation of beta-catenin pathways regulating keratinocyte proliferation, whereas SRC3 uniquely regulates the ability of 1,25(OH)2D3 to induce more differentiated functions such as lipid synthesis and processing required for permeability barrier formation and the innate immune response triggered by disruption of the barrier. These findings provide a basis by which we can understand how one receptor (VDR) and one ligand (1,25(OH)2D3) can regulate a large number of genes in a sequential and differentiation specific fashion.

Sequential regulation of keratinocyte differentiation by 1,25(OH)2D3, VDR, and its coregulators.

Keratinocyte differentiation requires the sequential regulation of gene expression. We have explored the role of 1,25(OH)(2)D(3) and its receptor (VDR) in this process. VDR sequentially binds to coactivator complexes such as Vitamin D receptor interacting protein (DRIP) and steroid receptor coactivator (SRC) during differentiation. Different genes respond differently to the VDR/coactivator complexes as determined by knockdown studies. The binding of DRIP205 and SRC to VDR is ligand (i.e. 1,25(OH)(2)D(3)) dependent. LXXLL motifs in these coactivators are critical for this binding; however, the affinity for VDR of the different LXXLL motifs in these coactivators varies. Hairless is an inhibitor of 1,25(OH)(2)D(3) dependent gene transcription. A phiXXphiphi motif in hairless is crucial for hairless binding to VDR, and its binding is ligand independent. 1,25(OH)(2)D(3) displaces hairless and recruits the coactivators to VDREs. Hsp90 and p23 are chaperone proteins recruited to the DRIP/VDR complex, where they block the binding of the complex to VDREs and block 1,25(OH)(2)D(3) stimulated transcription. Thus four mechanisms explain the ability of 1,25(OH)(2)D(3) to sequentially regulate gene transcription during differentiation: changes in coregulator levels, their differential binding to VDR, differential gene responsiveness to the VDR/coregulator complexes, and chaperone proteins facilitating the cycling of VDR/coregulator complexes on and off the VDREs.

In vivo expression profile of an endothelial nitric oxide synthase promoter-reporter transgene.

Endothelium-derived nitric oxide (NO) is primarily attributable to constitutive expression of the endothelial nitric oxide synthase (eNOS) gene. Although a more comprehensive understanding of transcriptional regulation of eNOS is emerging with respect to in vitro regulatory pathways, their relevance in vivo warrants assessment. In this regard, promoter-reporter insertional transgenic murine lines were created containing 5,200 bp of the native murine eNOS promoter directing transcription of nuclear-localized beta-galactosidase. Examination of beta-galactosidase expression in heart, lung, kidney, liver, spleen, and brain of adult mice demonstrated robust signal in large and medium-sized blood vessels. Small arterioles, capillaries, and venules of the microvasculature were notably negative, with the exception of the vasa recta of the medullary circulation of the kidney, which was strongly positive. Only in the brain was the reporter expressed in non-endothelial cell types, such as the CA1 region of the hippocampus. Epithelial cells of the bronchi, bronchioles, and alveoli were scored as negative, as was renal tubular epithelium. Cardiac myocytes, skeletal muscle, and smooth muscle of both vascular and nonvascular sources failed to demonstrate beta-galactosidase staining. Expression was uniform across multiple founders and was not significantly affected by genomic integration site. These transgenic eNOS promoter-reporter lines will be a valuable resource for ongoing studies addressing the regulated expression of eNOS in vivo in both health and disease.

Characterization of the murine endothelial nitric oxide synthase promoter.

As our understanding of the contributory roles of NO in the blood vessel wall evolves, so does the need to firmly understand the basic principles governing the regulated expression of the endothelial nitric oxide synthase (eNOS) gene. Because a robust approach to dissecting the relative contribution of a given cardiovascular gene exploits the use of murine genetic models, P1 murine genomic clones were isolated, characterized and functionally assessed to gain further insight into the regulated expression of the eNOS gene in the mouse. Sequence analysis of 1.8 kb of 5' flanking regions revealed important regions of sequence conservation with human and bovine sequences. Functional promoter activity was confirmed using transient transfection analysis of cultured endothelial cells.