Near-infrared (NIR) fluorochromes have become important reporter molecules for many biomedical applications, including FACS sorting, confocal microscopy, and more recently in vivo imaging. While the structures of several stable 800 nm fluorochromes have been published, their synthesis is often complex and there are difficulties in rapidly purifying these compounds in large quantities. Here we report on the synthesis of NIR820, ex/em = 790/820, with excellent physicochemical properties. Importantly, NIR820 is conveniently synthesized in a three-step reaction and can be purified by flash column chromatography rather than by HPLC. NIR820 is chemically stable and can be directly coupled to peptides during the solid-phase synthesis. In addition, NIR820 is also suitable for conjugation to proteins and other affinity molecules in aqueous buffer.
Background: Tissues undergoing a chronic inflammatory process, such as the synovium in rheumatoid arthritis, are characterized by the infiltration of lymphocytes of different subsets and activation of monocyte/macrophages. Interleukin-1 (IL-1), a monocyte/ macrophage product that stimulates synovial fibroblasts to produce matrix metalloproteinases (MMPs), prostaglandins, and other cytokines, also has profound effects on the synthesis of extracellular matrix components such as type I collagen. In previous studies, we have shown that synovial fibroblasts and chondrocytes isolated from human joint tissues are particularly sensitive to prostaglandins, which modulate the effects of IL-1 on collagen gene expression in an autocrine manner. Materials and Methods: BALBc/3T3 fibroblasts were treated with IL-1 and prostaglandins in the absence and presence of indomethacin to inhibit endogenous prostaglandin biosynthesis. Collagen synthesis was analyzed by SDS-PAGE as [ 3 H]prolinelabeled, secreted proteins, and prostaglandin production and cyclic adenosine 3',5'-cyclic monophosphate (camp) content were assayed. The expression of type I collagen gene (Col1a1) promoter-reporter gene constructs was examined in transient transfection experiments, and the binding of nuclear factors to the Col1a1 promoter region spanning Ϫ222 bpրϩ116 bp was analyzed by DNase I footprinting and electrophoretic mobility shift (EMSA) assays. Results: IL-1 increased the synthesis of type I and type III collagens in BALBc/3T3 fibroblasts; greater increases were observed when IL-1-stimulated synthesis of PGE 2 was blocked by indomethacin. Transient transfection experiments demonstrated dose-dependent inhibition of theϪ222 bp Col1a1 promoter by exogenously added prostaglandins with the order of potency of PGF 2 ␣ Ͼ PGE 2 Ͼ PGE 1. DNase I footprinting showed increased protection, which extended from the region immediately upstream of the TATA box, owing to the binding of nuclear factors from PGE 2-or PGE 1-treated BALBc/3T3 cells. EMSA analysis showed zinc-dependent differences in the binding of nuclear factors from untreated and prostaglandin-treated cells to the Ϫ84 bpրϪ29 bp region of the Col1a1 promoter. Conclusions: These results show that the inhibition of Col1a1 expression by IL-1 in fibroblasts is mediated by prostaglandins at the transcriptional level and suggest that PGE-responsive factors may interact directly or indirectly with basal regulatory elements in the proximal promoter region of the Col1a1 gene.
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