Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997." Typescript. Vita. Includes bibliographical references (l. 176-196). Also available on the Internet.

Thesis (Ph. D.)--University of Missouri--Columbia, 1997. / "December 1997" Typescript. Vita. Includes bibliographical references (leaves: 176-196). Also available on the Internet.

No description available.

The cytokeratins from various human tissue were isolated using chromatographic techniques. The cytokeratins were first extracted from crude tissue using high and low salt buffers. It was necessary to use a denaturing agent such as urea to solubilize the resulting cytokeratin pellet. Imidazole also seemed to help solubilize the pellet and a reducing agent such as 2-Mercaptoethanol was not needed as previously believed. The acidic cytokeratins were separated from the neutral-basic cytokeratins using a DEAE ion-exchange column. The acidic cytokeratin fraction was further separated on a moderately polar reverse phase column with an acetonitrile gradient to eluted the proteins. Tetramethylammonium tetrafluoroborate was added to the mobile phase to react with any unreacted silanol groups on the stationary phase and trifluoroacetic acid was added to ion pair with the protein. The peaks were analyzed for purity using two dimensional electrophoresis and monoclonal antibodies that recognize the cytokeratins.

No description available.

No description available.

Integrins are major adhesion receptors for the extracellular matrix (ECM). This thesis focuses on the motifs and interactions within integrin cytoplasmic tails during integrin-mediated cell adhesion and spreading. The present study investigated the significance of the skelemin-αIIbβ3 interaction using Chinese Hamster Ovary (CHO) cells expressing wild-type or mutant αIIbβ3 receptors defective in skelemin binding. Most mutant cells displayed unimpaired adhesive capacity and spreading on immobilized fibrinogen at the early stages of cell spreading. In addition, they formed normal focal adhesions and stress fibers with no indication of impaired cell spreading. K716A and H722A mutant cells exhibited the greatest cell spreading, which was associated with enhanced p-Src activation. The K716 residue appeared to be the most important for skelemin binding in previous in vitro studies. Here, the protrusions of the leading edge of K716A cells showed strong colocalization of talin with αIIbβ3 which was associated with a loss in skelemin binding. These data suggest that the binding of skelemin to αIIbβ3 is not essential for normal cell spreading, but may act to exert contractile forces on cell spreading and coordinate the binding of talin to the membrane proximal region of integrin tails. The functional mode of peptides corresponding to the central motifs of the αIIb and αV tail, KRNRPPLEED (αIIb peptide) and KRVRPPQEEQ (αV peptide) was also investigated. Both peptides inhibited Mn2+-activated αIIbβ3 binding to soluble fibrinogen as well as the binding of αIIbβ3-expressing CHO cells to immobilized fibrinogen. Breast cancer progression has been linked to tumor cell interaction with ECM. Our αIIb and αV peptides also inhibited adhesion of two breast cancer cell lines (MDA-MB-435 and MCF7) to αV integrin ECM ligand vitronectin. Replacement of RPP with AAA significantly attenuated the inhibitory activity of the αIIb peptide. β-tubulin was identified as a potential αIIb peptide-binding partner, suggesting that microtubule cytoskeleton may participate in the regulation of integrin functions. These results provide insights into the mechanisms by which the central motifs of αIIb and αV tails regulate integrin activation and integrin-mediated cell adhesion

No description available.

Staphylococcus aureus (S. aureus) is a medically important bacterial pathogen associated with many diseases and infections of the respiratory system, wound sites, surgical incisions, and other portals of entry and exit. S. aureus is able to invade cells via mechanisms that have yet to be fully characterized. Vimentin, a protein filament of the animal cell cytoskeleton, and phosphoinositide 3-kinase (PI3K), a family of kinases responsible for initiating several cell signaling events, were found to be associated with S. aureus invasion. Confocal microscopy revealed that the vimentin network in human umbilical vein endothelial cells (HUVECs) undergoes dynamic rearrangement in steady state under control conditions. However, cells infected with S. aureus demonstrated peri-nuclear collapse of the vimentin network. Pre-treatment with LY294002, a drug that inhibits PI3K activity, decreased invasion of S. aureus and paralyzed the vimentin network. These data suggest that PI3K mediates S. aureus infection and vimentin rearrangement. / Department of Biology

Diss. (sammanfattning) Göteborg : Göteborgs universitet, 2007. / Härtill 3 uppsatser.