The radius of gyration of human plasma fibronectin was determined by light scattering both under conditions in which the molecule is in an extended conformation (ionic strength 1.01 M, pH 8) and close to its native, more compact conformation (ionic strength 0.16 M, pH 8). These values were found to be 17.5 +/‐ 0.8 nm and 10.7 +/‐ 0.9 nm respectively, for a constant mol. wt of 533,000 +/‐ 8000, in excellent agreement with the value of 520,000 deduced from its known composition. A set of models, each made of two identical, end‐to‐end joined chains of 28 beads, was then constructed, and their calculated physico‐chemical parameters were compared with those available for the whole fibronectin molecule and for some of its proteolytic fragments in both conformations. Two possible models for the circulating form are presented here: in both, the fibronectin molecule is in a compact, tangled conformation, with the amino‐terminal end of one chain folded over to the carboxy end of itself or of the other chain either in a hairpin or in a circular fashion. With the exception of the carboxy‐terminal fibrin(ogen)‐binding domains, all the domains appear to be well exposed to the solvent, and thus free to interact with potential ligands.
Abstract. Fibronectin molecules are dimers composed of subunits whose primary structures may differ. This is due to alternative splicing in at least two regions (ED and IIICS) of the pre-mRNA.Using two monoclonal antibodies specific for two different epitopes of domain 5 (high affinity for heparin), we have quantitatively analyzed the expression of the IIICS sequence in human fibronectins from different sources. The results demonstrated that the percentage of fibronectin subunits containing the IIICS is higher in fibronectins from tumor-derived or simian virus 40-transformed human cells than in fibronectins from human plasma or normal human fibroblasts.Furthermore, we observed that 45-65 % of fibronectin subunits from transformed cells or normal embryonic fibroblasts are sialylated on the heparinbinding domain 5, whereas this occurs in only 24-28% of fibronectin subunits from normal adult fibroblasts. On the contrary, no sialylation was observed on domain 5 in fibronectin from human plasma.F IBRONECTINS (FNs) ~ are high molecular weight adhesive glycoproteins present in soluble form in plasma and other body fluids and in insoluble form in the extracellular matrices and basement membranes. FN molecules act as bridges between the cell surface and extracellular material. In fact, the FN molecules contain a cell-binding site and binding sites for collagen, heparin, ganglioside, and fibrin. Due to their multiple interactions FNs play an important role in diverse biological phenomena including cell adhesion, cell migration, hemostasis and thrombosis, wound healing, and the ability to induce a more normal phenotype in transformed cells (for reviews on distribution, structure, and biological functions see references 1, 3, 4, 13, and 16).Recently it has been demonstrated that the FN polymorphism may be due to alternative splicing schemes since many different mRNAs may originate from the primary transcript of a single gene (5, 7-10, 17, 18, 22) localized on chromosome two (6, 27).Here, using the domain-specific monoclonal antibodies IST-7 and IST-2 we have studied the presence of the IIICS sequence in FN from plasma and from the conditioned media of normal and tumor-derived human cells. Materials and Methods Cell Lines and Monoclonal AntibodiesCultured normal human fibroblast cell lines (LZ, from adult human skin; GM-3651-C, from adult human skin; GM-5386, from embryonic human 1. Abbreviations used in this paper: FN, fibronectin; RIA, radioimmunoassay; SV-40, simian virus 40.skin; WI-38, from embryonic human lung) and transformed cell lines (HT-1080, from a human fbrosarcoma; RD, from an embryonic human rhabdomyosarcoma; IgR3, from a human melanoma; and WI-38VA13, simian virus 40 lSV-40]-transformed WI-38 cells) were grown in Eagle's minimum essential medium supplemented with 10% fetal calf serum (Flow Laboratories, Irvine, Scotland) that had been depleted of bovine FN by passage through a large capacity gelatin-Sepharose column.Monoclonal antibodies to human plasma FN were prepared as previously described (25). Partial ...
N-(4-hydroxyphenyl)retinamide (HPR) is a synthetic retinoid with anti-cancer properties and lower toxicity than all-trans retinoic acid (RA). Neuroblastoma cells treated with HPR and observed by fluorescence microscopy showed clear signs of apoptosis, such as chromatin condensation and margination, nuclear fragmentation and the presence of "apoptotic bodies". Moreover, measurements on a cell-by-cell basis by the flow-cytometric DNA-content in situ-terminal-deoxinucleotidyl-transferase(TDT) assay showed that apoptosis induced by HPR was dose- and time-dependent and that the fraction of apoptotic cells increased from approximately 15% at 1.25 microM at 2 days after treatment up to approximately 90% at 5 microM and 8 days of continuous treatment. Additionally, we found that cells were induced into apoptosis independently from the cell-cycle phase. In contrast, equimolar or higher doses of RA, from 5 microM to 80 microM, were able to inhibit growth by differentiation, but failed to induce apoptosis. We conclude that the functional effects of HPR and RA in LA-N-5 neuroblastoma cells are mediated by apoptosis and differentiation respectively, suggesting a potential clinical use of HPR in the management of neuroblastoma patients.
The aim of the study was to test the hypothesis that a human mutated K-ras protein induces abnormalities in mitosis and development of sub-clones characterized by changes in DNA ploidy and proliferation. For this purpose, we used control and NIH-3T3 mouse cells transfected with the human codon 12 G-C-mutated K-ras oncogene. We found that abnormal mitoses, mainly characterized by lagging chromosomes in prometaphase or anaphase, had a significantly higher frequency in transfected cells than in control cells. The generation of sub-clones was screened by limiting-dilution experiments followed by cell expansion. Cloning efficiency was much higher for the K-ras transfected cells with 858/2112 (41%) successful sub-clones than for control, which provided 564/2592 (22%) sub-clones. DNA flow cytometry of 4.6-diamidino-2-phenilindole-2-hydrochloride-stained nuclei from randomly selected sub-clones was performed in order to evaluate DNA index and S-phase fraction values. We found 9 out of 100 DNA aneuploid sub-clones generated by the K-ras-transfected cells vs. 1 out of 100 for the controls. Overall, our data indicate that high expression of the mutationally activated human K-ras product in NIH-3T3 cells was associated with abnormal mitoses, increase of cloning efficiency and DNA aneuploidization.
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