The basis for the endothelial cell-restricted expression of endothelial nitric-oxide synthase (eNOS) is not known. While transgenic promoter/reporter mice demonstrated endothelium cell-specific eNOS expression, we found robust expression of episomal eNOS promoter/reporter constructs in cell types that do not express the native eNOS transcript. To explore the mechanism underlying this differential activity pattern of chromatin-versus episomebased eNOS promoters, we examined the methylation status of 5-regulatory sequences of the human eNOS gene. DNA methylation differed dramatically between endothelial and nonendothelial cell types, including vascular smooth muscle cells. This same cell type-specific methylation pattern was observed in vivo in endothelial and vascular smooth muscle cells of the mouse aorta at the native murine eNOS promoter. We addressed the functional consequences of methylation on eNOS transcription using transient transfection of in vitro methylated promoter/reporter constructs and found that methylated constructs exhibited a marked decrease in the synergistic action of Sp1, Sp3, and Ets1 on eNOS promoter activity. The addition of methyl-CpG-binding protein 2 further reduced the transcriptional activity of methylated eNOS constructs. Importantly, chromatin immunoprecipitation demonstrated the presence of Sp1, Sp3, and Ets1 at the native eNOS promoter in endothelial cells but not in vascular smooth muscle cells. Finally, robust expression of eNOS mRNA was induced in nonendothelial cell types following inhibition of DNA methyltransferase activity with 5-azacytidine, demonstrating the importance of DNA methylation-mediated repression. This report is the first to show that promoter DNA methylation plays an important role in the cell-specific expression of a constitutively expressed gene in the vascular endothelium.
The ability to create updated images as surgery progresses introduces the concept of 'near-real-time' CT guidance for head and neck surgery. We found that the use of CBCT increased surgical confidence in accessing the frontal recess, resolved ambiguities with anatomical variations, and provided valuable teaching information to surgeons in training in both preoperative planning and correlation between tri-planar CT scans and intraoperative endoscopic findings.
Rhinosinusitis is a common medical problem that interferes with patient quality of life and loss of work productivity. Because of the heterogeneity that underlies its pathology, no one treatment regimen exists for the management of rhinosinusitis.
It is well known that glucagon-like peptide-1 (7-36 amide) (tGLP-1) is a potent insulinotropic hormone with powerful antidiabetogenic effects. In the present study we sought to determine the precise regions of the intracellular domains of the tGLP-1 receptor that are required for its efficient coupling to adenylyl cyclase because cAMP is the primary candidate second messenger coupling tGLP-1 to insulin secretion. Recently, we identified an amino acid within the third intracellular loop, K334, that was required for efficient coupling of tGLP-1 receptor to adenylyl cyclase. A similar mutagenesis-based strategy was employed here to examine the first and second intracellular loops and to further define sequences in the third loop required for the efficient coupling of the receptor to its second messengers. Receptor mutants were expressed in COS-7 cells and examined for tGLP-1 binding and cAMP stimulation. Three alanine substitution mutations, V327A, 1328A, and V331A, resulted in significantly lower tGLP-1-stimulated cAMP production without reductions in receptor expression. Analysis of the first and second intracellular loops revealed only one mutation contained within the first loop, R176A, where a significant reduction in cAMP activation was observed with normal receptor expression. These studies suggest that specific determinants of coupling for tGLP-1 receptor are primarily localized to the predicted junction of the fifth transmembrane helix and the third intracellular loop. We predict that V327, 1328, and V331 form part of a hydrophobic face that directly contacts the G protein.
Long-term endoscopic confirmation of frontal ostium patency demonstrates that endoscopic frontal sinusotomy can yield high quality, durable results. There was no significant difference in patency results between ECRS and non-ECRS patients. Laryngoscope, 2009.
Dizziness is a common medical condition that impacts significantly on patients' activities of daily living. This review outlines the clinical approach to dizziness to facilitate timely diagnosis and management of this complex symptom.
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