Purification of Human Plasma/Cellular Fibronectin and Fibronectin Fragments

A method is described for the purification of plasma fibronectins based on a combination of gelatin- and arginine-Sepharose chromatography steps. Cellular fibronectin can be purified from an osteosarcoma fibroblast cell line by affinity chromatography using a monoclonal antibody anti-fibronectin as ligand. Furthermore, we also provide a protocol for the purification of fibronectin domains obtained by fractionation of thermolysin-digested plasma fibronectin on ion-exchange/gel filtration chromatography columns. Assessment of the fibronectin purity is performed by SDS-PAGE, while the ligand binding activities of specific fibronectin domains are determined by ELISA

External validation suggests Integrin beta 3 as prognostic biomarker in serous ovarian adenocarcinomas

<p>Abstract</p> <p>Background</p> <p>The majority of women with ovarian cancer are diagnosed in late stages, and the mortality rate is high. The use of biomarkers as prognostic factors may improve the treatment and clinical outcome of these patients. We performed an external validation of the potential biomarkers CLU, ITGB3, CAPG, and PRAME to determine if the expression levels are relevant to use as prognostic factors.</p> <p>Methods</p> <p>We analysed the gene expression of CLU, ITGB3, CAPG, and PRAME in 30 advanced staged serous adenocarcinomas with quantitative real-time polymerase chain reaction (QPCR) and the protein levels were analysed in 98 serous adenocarcinomas with western blot for semiquantitative analysis. Statistical differences in mRNA and protein expressions between tumours from survivors and tumours from deceased patients were evaluated using the Mann-Whitney U test.</p> <p>Results</p> <p>The gene and protein ITGB3 (Integrin beta 3) were significantly more expressed in tumours from survivors compared to tumours from deceased patients, which is in concordance with our previous results. However, no significant differences were detected for the other three genes or proteins CLU, CAPG, and PRAME.</p> <p>Conclusion</p> <p>The loss of ITGB3 expression in tumours from deceased patients and high expression in tumours from survivors could be used as a biomarker for patients with advanced serous tumours.</p

The CS1 segment of fibronectin is involved in human OSCC pathogenesis by mediating OSCC cell spreading, migration, and invasion

<p>Abstract</p> <p>Background</p> <p>The alternatively spliced V region or type III connecting segment III (IIICS) of fibronectin is important in early development, wound healing, and tumorigenesis, however, its role in oral cancer has not been fully investigated. Thus, we investigated the role of CS-1, a key site within the CSIII region of fibronectin, in human oral squamous cell carcinoma (OSCC).</p> <p>Methods</p> <p>To determine the expression of CS-1 in human normal and oral SCC tissue specimens immunohistochemical analyses were performed. The expression of CS1 was then associated with clinicopathological factors. To investigate the role of CS-1 in regulating OSCC cell spreading, migration and invasion, OSCC cells were assayed for spreading and migration in the presence of a CS-1 peptide or a CS-1 blocking peptide, and for invasion using Matrigel supplemented with these peptides. In addition, integrin α4siRNA or a focal adhesion kinase (FAK) anti-sense oligonucleotide was transfected into OSCC cells to examine the mechanistic role of integrin α4 or FAK in CS1-mediated cell spreading and migration, respectively.</p> <p>Results</p> <p>CS-1 expression levels were significantly higher in OSCC tissues compared to normal tissues (p < 0.05). Also, although, high levels of CS-1 expression were present in all OSCC tissue samples, low-grade tumors stained more intensely than high grade tumors. OSCC cell lines also expressed higher levels of CS-1 protein compared to normal human primary oral keratinocytes. There was no significant difference in total fibronectin expression between normal and OSCC tissues and cells. Inclusion of CS-1 in the in vitro assays enhanced OSCC cell spreading, migration and invasion, whereas the CS1 blocking peptide inhibited these processes. Suppression of integrin α4 significantly inhibited the CS1-mediated cell spreading. Furthermore, this migration was mediated by focal adhesion kinase (FAK), since FAK suppression significantly blocked the CS1-induced cell migration.</p> <p>Conclusion</p> <p>These data indicate that the CS-1 site of fibronectin is involved in oral cancer pathogenesis and in regulating OSCC cell spreading, migration and invasion.</p

14-3-3 Mediates Histone Cross-Talk during Transcription Elongation in Drosophila

Post-translational modifications of histone proteins modulate the binding of transcription regulators to chromatin. Studies in Drosophila have shown that the phosphorylation of histone H3 at Ser10 (H3S10ph) by JIL-1 is required specifically during early transcription elongation. 14-3-3 proteins bind H3 only when phosphorylated, providing mechanistic insights into the role of H3S10ph in transcription. Findings presented here show that 14-3-3 functions downstream of H3S10ph during transcription elongation. 14-3-3 proteins localize to active genes in a JIL-1–dependent manner. In the absence of 14-3-3, levels of actively elongating RNA polymerase II are severely diminished. 14-3-3 proteins interact with Elongator protein 3 (Elp3), an acetyltransferase that functions during transcription elongation. JIL-1 and 14-3-3 are required for Elp3 binding to chromatin, and in the absence of either protein, levels of H3K9 acetylation are significantly reduced. These results suggest that 14-3-3 proteins mediate cross-talk between histone phosphorylation and acetylation at a critical step in transcription elongation

S-Phase Favours Notch Cell Responsiveness in the Drosophila Bristle Lineage

We have studied cell sensitivity to Notch pathway signalling throughout the cell cycle. As model system, we used the Drosophila bristle lineage where at each division N plays a crucial role in fate determination. Using in vivo imaging, we followed this lineage and activated the N-pathway at different moments of the secondary precursor cell cycle. We show that cells are more susceptible to respond to N-signalling during the S-phase. Thus, the period of heightened sensitivity coincided with the period of the S-phase. More importantly, modifications of S-phase temporality induced corresponding changes in the period of the cell's reactivity to N-activation. Moreover, S-phase abolition was correlated with a decrease in the expression of tramtrack, a downstream N-target gene. Finally, N cell responsiveness was modified after changes in chromatin packaging. We suggest that high-order chromatin structures associated with the S-phase create favourable conditions that increase the efficiency of the transcriptional machinery with respect to N-target genes

Malleable Machines in Transcription Regulation: The Mediator Complex

The Mediator complex provides an interface between gene-specific regulatory proteins and the general transcription machinery including RNA polymerase II (RNAP II). The complex has a modular architecture (Head, Middle, and Tail) and cryoelectron microscopy analysis suggested that it undergoes dramatic conformational changes upon interactions with activators and RNAP II. These rearrangements have been proposed to play a role in the assembly of the preinitiation complex and also to contribute to the regulatory mechanism of Mediator. In analogy to many regulatory and transcriptional proteins, we reasoned that Mediator might also utilize intrinsically disordered regions (IDRs) to facilitate structural transitions and transmit transcriptional signals. Indeed, a high prevalence of IDRs was found in various subunits of Mediator from both Saccharomyces cerevisiae and Homo sapiens, especially in the Tail and the Middle modules. The level of disorder increases from yeast to man, although in both organisms it significantly exceeds that of multiprotein complexes of a similar size. IDRs can contribute to Mediator's function in three different ways: they can individually serve as target sites for multiple partners having distinctive structures; they can act as malleable linkers connecting globular domains that impart modular functionality on the complex; and they can also facilitate assembly and disassembly of complexes in response to regulatory signals. Short segments of IDRs, termed molecular recognition features (MoRFs) distinguished by a high protein–protein interaction propensity, were identified in 16 and 19 subunits of the yeast and human Mediator, respectively. In Saccharomyces cerevisiae, the functional roles of 11 MoRFs have been experimentally verified, and those in the Med8/Med18/Med20 and Med7/Med21 complexes were structurally confirmed. Although the Saccharomyces cerevisiae and Homo sapiens Mediator sequences are only weakly conserved, the arrangements of the disordered regions and their embedded interaction sites are quite similar in the two organisms. All of these data suggest an integral role for intrinsic disorder in Mediator's function

Integrins as therapeutic targets: lessons and opportunities.

The integrins are a large family of cell adhesion molecules that are essential for the regulation of cell growth and function. The identification of key roles for integrins in a diverse range of diseases, including cancer, infection, thrombosis and autoimmune disorders, has revealed their substantial potential as therapeutic targets. However, so far, pharmacological inhibitors for only three integrins have received marketing approval. This article discusses the structure and function of integrins, their roles in disease and the chequered history of the approved integrin antagonists. Recent advances in the understanding of integrin function, ligand interaction and signalling pathways suggest novel strategies for inhibiting integrin function that could help harness their full potential as therapeutic targets

Multiple Roles for ISWI in Transcription, Chromosome Organization and DNA Replication

ISWI functions as the ATPase subunit of multiple chromatin-remodeling complexes. These complexes use the energy of ATP hydrolysis to slide nucleosomes and increase chromatin fluidity, thereby modulating the access of transcription factors and other regulatory proteins to DNA. Here we discuss recent progress toward understanding the biological functions of ISWI, with an emphasis on its roles in transcription, chromosome organization and DNA replication