Platelet-derived growth factor (PDGF) has been directly implicated in developmental and physiological processes, as well as in human cancer, fibrotic diseases and arteriosclerosis. The PDGF family ...currently consists of at least three gene products, PDGF-A, PDGF-B and PDGF-C, which selectively signal through two PDGF receptors (PDGFRs) to regulate diverse cellular functions. After two decades of searching, PDGF-A and B were the only ligands identified for PDGFRs. Recently, however, database mining has resulted in the discovery of a third member of the PDGF family, PDGF-C, a functional analogue of PDGF-A that requires proteolytic activation. PDGF-A and PDGF-C selectively activate PDGFR-α, whereas PDGF-B activates both PDGFR-α and PDGFR-β. Here we identify and characterize a new member of the PDGF family, PDGF D, which also requires proteolytic activation. Recombinant, purified PDGF-D induces DNA synthesis and growth in cells expressing PDGFRs. In cells expressing individual PDGFRs, PDGF-D binds to and activates PDGFR-β but not PDGFR-α. However, in cells expressing both PDGFRs, PDGF-D activates both receptors. This indicates that PDGFR-α activation may result from PDGFR-α/β heterodimerization.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Background & Aims: We recently identified a novel member of the human fibroblast growth factor (FGF) family of signaling molecules, designated FGF-20. In the present study, we examined the activity ...of this protein in 2 animal models of acute intestinal inflammation and in mechanistic studies in vitro. Methods: In vivo experiments consisted of a murine dextran sulfate sodium (DSS) model of colitis and a rat indomethacin model of small intestinal ulceration/inflammation. Cell growth, restitution, gene expression (cyclooxygenase-2 COX-2 and intestinal trefoil factor ITF), and prostaglandin E2 (PGE2) levels were examined in vitro. Results: In the DSS-colitis model, prophylactic administration of FGF-20 significantly reduced the severity and extent of mucosal damage as indicated by a 55%–93% reduction in luminal blood loss, distal colonic edema, histologic inflammation, and epithelial cell loss relative to animals administered vehicle control. No toxicity was noted during administration of FGF-20 to normal controls. In addition, therapeutic administration of FGF-20 enhanced survival in this model. In the indomethacin–small bowel ulceration/inflammation model, administration of FGF-20 reduced small intestinal weight gain, necrosis, inflammation, and weight loss (36%–53% relative to vehicle control). In vitro studies demonstrated that FGF-20 stimulates growth, restitution, mRNA expression of COX-2 and ITF, and PGE2 levels in human intestinal epithelial cells and enhances the growth of human intestinal fibroblasts. Conclusions: FGF-20, having demonstrated therapeutic activity in 2 experimental models of intestinal inflammation, represents a promising new candidate for the treatment of human inflammatory bowel disease.
GASTROENTEROLOGY 2002;123:1151-1162
A previously undescribed human member of the cystatin superfamily called cystatin F has been identified by expressed sequence tag sequencing in human cDNA libraries. A full-length cDNA clone was ...obtained from a library made from mRNA of CD34-depleted cord blood cells. The sequence of the cDNA contained an open reading frame encoding a putative 19-residue signal peptide and a mature protein of 126 amino acids with two disulfide bridges and enzyme-binding motifs homologous to those of Family 2 cystatins. Unlike other human cystatins, cystatin F has 2 additional Cys residues, indicating the presence of an extra disulfide bridge stabilizing the N-terminal region of the molecule. Recombinant cystatin F was produced in a baculovirus expression system and characterized. The mature recombinant protein processed by insect cells had an N-terminal segment 7 residues longer than that of cystatin C and displayed reversible inhibition of papain and cathepsin L (Ki = 1.1 and 0.31 nm, respectively), but not cathepsin B. Like cystatin E/M, cystatin F is a glycoprotein, carrying two N-linked carbohydrate chains at positions 36 and 88. An immunoassay for quantification of cystatin F showed that blood contains low levels of the inhibitor (0.9 ng/ml). Six B cell lines in culture secreted barely detectable amounts of cystatin F, but several T cell lines and especially one myeloid cell line secreted significant amounts of the inhibitor. Northern blot analysis revealed that the cystatin F gene is primarily expressed in peripheral blood cells and spleen. Tissue expression clearly different from that of the ubiquitous inhibitor, cystatin C, was also indicated by a high incidence of cystatin F clones in cDNA libraries from dendritic and T cells, but no clones identified by expressed sequence tag sequencing in several B cell libraries and in >600 libraries from other human tissues and cells.
The angiopoietins comprise a family of proteins that have pro or antiangiogenic activities. Through a proprietary technology designed to identify transcripts of all expressed genes, we isolated a ...cDNA encoding an angiopoietin-related protein that we designate angioarrestin. The mRNA expression profile of angioarrestin was striking in that it was down-regulated in many tumor tissues when compared with adjacent nontumor tissue, suggesting a role for this protein in tumor inhibition. To test this hypothesis, we ectopically expressed angioarrestin in HT1080 tumor cells and measured pulmonary tumor nodule formation in nude mice. HT1080 cells expressing angioarrestin showed a marked reduction in the number and size of tumor nodules. In vitro, the recombinant protein was systematically tested in a number of endothelial cell assays and found to block critical processes involved in the angiogenic cascade, such as vascular endothelial growth factor/basic fibroblast growth factor-mediated endothelial cell proliferation, migration, tubular network formation, and adhesion to extracellular matrix proteins. These findings reveal a novel function for angioarrestin as an angiogenesis inhibitor and indicate that the molecule may be a potential cancer therapeutic.
The influence of various culture parameters on infection and replication of recombinant vaccinia virus in HeLa cells was examined during various phases of viral replication. A modified form of the ...model of Valentine and Allison (Biochim. Biophys. Acta 1960, 40, 393–399) model was used to predict successfully the viral adsorption rates in cell suspensions. An experimentally determined aggregation factor, ϵ, was included in the model to account for deviations of the observed adsorption rates from those predicted by the earlier model. It was also shown that the ionic strength, ionic species, and serum proteins present in the medium significantly altered the adsorption kinetics of the virus. The lysosomotropic base chloroquine was found to enhance viral infection more than 2‐fold during the penetration step of viral infection. It was also demonstrated that cells infected during the exponential growth phase gave higher viral yields than those infected during the lag or stationary growth phases and the initial viral MOI did not significantly alter viral yields. Finally, it was demonstrated that viral infection of HeLa cells grown in 4‐L bioreactor batch cultures resulted in increased death and glucose uptake rates and significantly lower growth rates.