Search

Background Excessive extracellular matrix (ECM) deposition is a hallmark feature in fibrosis and tissue remodelling diseases. Typically, mesenchymal cells will produce collagens under standard 2D cell culture conditions, however these do not assemble into fibrils. Existing assays for measuring ECM production are often low throughput and not disease relevant. Here we describe a robust, high content, pseudo-3D phenotypic assay to quantify mature fibrillar collagen deposition which is both physiologically relevant and amenable to high throughput compound screening. Using pulmonary fibroblasts derived from patients with idiopathic pulmonary fibrosis (IPF), we developed the ‘scar-in-a-jar’ assay into a medium-throughput phenotypic assay to robustly quantify collagen type I deposition and other extracellular matrix (ECM) proteins over 72 h. Results This assay utilises macromolecular crowding to induce an excluded volume effect and enhance enzyme activity, which in combination with TGF-β1 stimulation significantly accelerates ECM production. Collagen type I is upregulated approximately 5-fold with a negligible effect on cell number. We demonstrate the robustness of the assay achieving a Z prime of approximately 0.5, and % coefficient of variance (CV) of

One of the key control points in the trafficking of the T cell effector subsets, Th1 and Th2, to sites of inflammation is their migration out of the bloodstream. The mechanism by which the cells initially adhere to the endothelium is dependent on the selectin family of adhesion molecules. Only polarised Th1 cells are capable of binding P-selectin despite both Th1 and Th2 cells expressing PSGL-1, the P-selectin ligand. This may be due to a secondary modification of PSGL-1 that is present on Th1 but not Th2 cells. One key modification of PSGL-1 is the alpha3 fucosylation of the O-glycans. To address whether the binding of Th1 and Th2 cells may be regulated by fucosylation, we have studied the expression of the alpha3 fucosyltransferases, FucT-IV and VII, using in vitro differentiated mouse T cells. Messenger RNA levels for both FucT-IV and VII were found to be higher in Th1 than Th2 cells. alpha3 fucosyltransferase enzyme activities were also elevated in Th1 cells. The increased expression of the alpha3 fucosyltransferases in Th1 cells correlated with the ability of Th1, but not Th2, cells to bind to P-selectin. Thus, the regulation of the binding of effector T cells to the endothelium, and subsequent trafficking to inflammatory sites, may be controlled by the fucosylation state of PSGL-1 mediated by the selective expression of the alpha3 fucosyltransferases.

The POU domain transcription factor Oct-6 (SCIP/Tst-1) is likely to control important stages of Schwann cell development, including the initiation of myelination around birth. Here, we use immunocytochemical and reverse transcriptase-polymerase chain reaction techniques to examine Oct-6 earlier in nerve development, to test the idea that Oct-6 has an additional role in Schwann cell precursors or early embryonic Schwann cells, a possibility raised by previous studies on transgenic mice. Consistent with this, we find low but unambiguous levels of Oct-6 mRNA and protein in Schwann cell precursors of mouse and rat (nerves from 12- and 14-day-old embryos, respectively), with expression levels gradually increasing during early Schwann cell development and towards birth. Unexpectedly, Oct-6 immunoreactivity is clearly present in nuclei of most myelinating cells at least as late as postnatal day 12. Furthermore, many nonmyelinating Schwann cells express Oct-6 in adult life. A comparison of Oct-6 mRNA with other Schwann cell transcription factors-namely, Oct-1, Krox-20, and Pax-3-reveals that each factor exhibits strong developmental regulation and a unique expression pattern in embryonic nerves. Therefore, they are likely to play distinct regulatory roles in early development of the Schwann cell lineage.

We have determined the complete sequence of chick brain alpha-actinin (892 amino acids; 107,644 Da). The sequence differs from that of smooth muscle alpha-actinin only in the region of the first EF-hand calcium-binding motif, where 27 residues in brain alpha-actinin are replaced by just 22 residues in the smooth muscle isoform. This probably accounts for the different calcium sensitivities of the two isoforms with respect to actin binding. Analysis of the gene structure showed that this region of sequence divergence is encoded by two separate exons whose incorporation is mutually exclusive. We have determined the proportion of the two transcripts in various tissues and cell lines using poly(A)+ RNA and a quantitative assay based on the polymerase chain reaction. MRC-5 fibroblasts and HeLa cells express mRNAs encoding both isoforms, whereas Namalwa lymphoblastoid cells, which lack actin stress fibers, express only the non-muscle mRNA. Both isoforms of alpha-actinin became incorporated into stress fibers and cell-matrix junctions when full-length chick alpha-actinin cDNAs were expressed in monkey COS cells. The levels of chick alpha-actinin mRNAs were found to be serum-inducible, suggesting that alpha-actinin may be an early response gene.