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Now showing 1 - 10 of 32
  • Publication
    Interaction of the Gelsolin-Derived Antibacterial PBP 10 Peptide with Lipid Bilayers and Cell Membranes
    (2006-09-01) Bucki, Robert; Janmey, Paul; Bucki, Robert; Janmey, Paul
    PBP 10, an antibacterial, cell membrane-permeant rhodamine B-conjugated peptide derived from the polyphosphoinositide binding site of gelsolin, interacts selectively with both lipopolysaccharides (LPS) and lipoteichoic acid (LTA), the distinct components of gram-negative and gram-positive bacteria, respectively. Isolated LPS and LTA decrease the antimicrobial activities of PBP 10, as well as other antimicrobial peptides, such as cathelicidin-LL37 (LL37) and mellitin. In an effort to elucidate the mechanism of bacterial killing by PBP 10, we compared its effects on artificial lipid bilayers and eukaryotic cell membranes with the actions of the mellitin, magainin II, and LL37 peptides. This study reveals that pore formation is unlikely to be involved in PBP 10-mediated membrane destabilization. We also investigated the effects of these peptides on platelets and red blood cells (RBCs). Comparison of these antimicrobial peptides shows that only mellitin has a toxic effect on platelets and RBCs in a concentration range concomitant with its bactericidal activity. The hemolytic activities of the PBP 10 and LL37 peptides significantly increase when RBCs are osmotically swollen in hypotonic solution, indicating that these antibacterial peptides may take advantage of the more extended form of bacterial membranes in exerting their killing activities. Additionally, we found that LL37 hemolytic activity was much higher when RBCs were induced to expose phosphatidylserine to the external leaflet of their plasma membranes. This finding suggests that asymmetrical distribution of phospholipids in the external membranes of eukaryotic cells may represent an important factor in determining the specificity of antibacterial peptides for targeting bacteria rather than eukaryotic cells.
  • Publication
    Biopolymer Networks and Cellular Mechanosensing
    (2004-06-01) Georges, Penelope; Wagner, Oliver; Janmey, Paul; Janmey, Paul
    Cells and tissues are mechanical as well as biochemical machines, and cellular response to mechanical cues can have as large an influence on structure and function as chemical signals. The mechanical properties of cells are largely determined by networks of semiflexible polymers forming the cytoskeleton, which has viscoelastic properties that differ in important ways from the viscoelasticity of common synthetic materials. Two such features are the high resistance to deformation achieved by a remarkably low volume fraction of protein, and the increase in stiffness that occurs when the cytoskeletal network is deformed. The actin filaments, microtubules and intermediate filaments that comprise the cytoskeleton of most cell types are linear polymers with some important similarities but also some fundamental differences. The stiffness of the individual polymer types is vastly different, with persistence lengths ranging from 1 mm for the 24 nm diameter microtubules to a few 100 nm for the 10-14 nm diameter intermediate filaments. The material properties of these biopolymer networks are proposed to function as part of the mechanosensing mechanism in cells, and the stiffness of cytoskeletal networks is similar to that of common extracellular protein networks such as those formed by collagen and fibrin in which many cell types function. Examples of the morphologic differences in fibroblasts and astrocytes grown on chemically identical surfaces overlying gels with elastic moduli spanning the range from 50 to 12,000 Pa illustrate the large effect of stiffness differences on cell structure and function.
  • Publication
    Lamellar Phase of Stacked Two-Dimensional Rafts of Actin Filaments
    (2003-07-04) Wong, Gerard C.L.; Lin, Alison; Tang, Jay X.; Janmey, Paul; Janmey, Paul; Safinya, Cyrus R
    We examined liquid crystalline phases of the cytoskeletal polyelectrolyte filamentous (F-)actin in the presence of multivalent counterions. As a function of increasing ion concentration, the F-actin rods in either an isotropic or a nematic phase will transform into a new and unexpected lamellar phase of crosslinked rafts (LXR phase), before condensing into a bundled phase of parallel, close-packed rods. This behavior is generic for alkali earth divalent ions Mg2+, Ca2+, Sr2+, and Ba2+, and the structural transitions are achieved without any architecture-specific actin-binding linker proteins.
  • Publication
    Impaired Notch Signaling Promotes De novo Squamous Cell Carcinoma Formation
    (2006-08-01) Lepore, John J.; Cheng, Lan; Seykora, John J; Proweller, Aaron; Millar, Sarah E; Tu, Lili; Pear, Warren S; Lepore, John J.; Parmacek, Michael S; Cheng, Lan; Lu, Min Min; Seykora, John J; Millar, Sarah E; Pear, Warren S; Parmacek, Michael S
    Signaling through Notch receptors in the skin has been implicated in the differentiation, proliferation, and survival of keratinocytes, as well as in the pathogenesis of basal cell carcinoma (BCC). To determine the composite function of Notch receptor–mediated signaling in the skin and overcome potential redundancies between receptors, conditional transgenic mice were generated that express the pan-Notch inhibitor, dominant-negative Mastermind Like 1 (DNMAML1), to repress all canonical [CBF-1/Suppressor of hairless/LAG-1 (CSL)–dependent] Notch signaling exclusively in the epidermis. Here, we report that DNMAML1 mice display hyperplastic epidermis and spontaneously develop cutaneous squamous cell carcinoma (SCC) as well as dysplastic precursor lesions, actinic keratoses. Mice expressing epidermal DNMAML1 display enhanced accumulation of nuclear ß-catenin and cyclin D1 in suprabasilar keratinocytes and in lesional cells from SCCs, which was also observed in human cutaneous SCC. These results suggest a model wherein CSL-dependent Notch signaling confers protection against cutaneous SCC. The demonstration that inhibition of canonical Notch signaling in mice leads to spontaneous formation of SCC and recapitulates the disease in humans yields fundamental insights into the pathogenesis of SCC and provides a unique in vivo animal model to examine the pathobiology of cutaneous SCC and for evaluating novel therapies.
  • Publication
    Kinetics of random aggregation-fragmentation processes with multiple components
    (2003-05-09) Diamond, Scott L; Diamond, Scott L
    A computationally efficient algorithm is presented for exact simulation of the stochastic time evolution of spatially homogeneous aggregation-fragmentation processes featuring multiple components or conservation laws. The algorithm can predict the average size and composition distributions of aggregating particles as well as their fluctuations, regardless of the functional form (e.g., composition dependence) of the aggregation or fragmentation kernels. Furthermore, it accurately predicts the complete time evolutions of all moments of the size and composition distributions, even for systems that exhibit gel transitions. We demonstrate the robustness and utility of the algorithm in case studies of linear and branched polymerization processes, the last of which is a two-component process. These simulation results provide the stochastic description of these processes and give new insights into their gel transitions, fluctuations, and long-time behavior when deterministic approaches to aggregation kinetics may not be reliable.
  • Publication
    Autocrine laminin-5 ligates {alpha}6{beta}4 integrin and activates RAC and NF{kappa}B to mediate anchorange-independent survival of mammary tumors
    (2003-12-22) Zahir, Nastaran; Lakins, Johnathon N; Chatterjee, Chandrima; Ming, WenYu; Chatterjee, Chandrima; Weaver, Valerie M.; Marinkovich, Matthew P; Weaver, Valerie M.
    Invasive carcinomas survive and evade apoptosis despite the absence of an exogenous basement membrane. How epithelial tumors acquire anchorage independence for survival remains poorly defined. Epithelial tumors often secrete abundant amounts of the extracellular matrix protein laminin 5 (LM-5) and frequently express α6β4 integrin. Here, we show that autocrine LM-5 mediates anchorage independent survival in breast tumors through ligation of a wild-type, but not a cytoplasmic tail–truncated α6β4 integrin. α6β4 integrin does not mediate tumor survival through activation of ERK or AKT. Instead, the cytoplasmic tail of β4 integrin is necessary for basal and epidermal growth factor–induced RAC activity, and RAC mediates tumor survival. Indeed, a constitutively active RAC sustains the viability of mammary tumors lacking functional β1 and β4 integrin through activation of NFκB, and overexpression of NFκB p65 mediates anchorage-independent survival of nonmalignant mammary epithelial cells. Therefore, epithelial tumors could survive in the absence of exogenous basement membrane through autocrine LM-5–α6β4 integrin–RAC–NFκB signaling.
  • Publication
    The convergence of haemodynamics, genomics, and endothelial structure in studies of the focal origin of atherosclerosis
    (2002-04-01) Davies, Peter F; Shi, Congzhu; Polacek, Denise C; Shi, Congzhu; Helmke, Brian P
    The completion of the Human Genome Project and ongoing sequencing of mouse, rat and other genomes has led to an explosion of genetics-related technologies that are finding their way into all areas of biological research; the field of biorheology is no exception. Here we outline how two disparate modern molecular techniques, microarray analyses of gene expression and real-time spatial imaging of living cell structures, are being utilized in studies of endothelial mechanotransduction associated with controlled shear stress in vitro and haemodynamics in vivo. We emphasize the value of such techniques as components of an integrated understanding of vascular rheology. In mechanotransduction, a systems approach is recommended that encompasses fluid dynamics, cell biomechanics, live cell imaging, and the biochemical, cell biology and molecular biology methods that now encompass genomics. Microarrays are a useful and powerful tool for such integration by identifying simultaneous changes in the expression of many genes associated with interconnecting mechanoresponsive cellular pathways.
  • Publication
    Hypoxia-inducible Factor Regulates αvß3 Integrin Cell Surface Expression
    (2005-04-01) Weaver, Valerie M.; Simon, M. Celeste; Weaver, Valerie M.; Simon, M. Celeste
    Hypoxia-inducible factor (HIF)-deficient placentas exhibit a number of defects, including changes in cell fate adoption, lack of fetal angiogenesis, hypocellularity, and poor invasion into maternal tissue. HIF is a heterodimeric transcription factor consisting of α and ß aryl hydrocarbon receptor nuclear translocator or ARNT) subunits. We used undifferentiated trophoblast stem (TS) cells to characterize HIF-dependent adhesion, migration, and invasion. Arnt-/- and Hifα-/- TS cells exhibit reduced adhesion and migration toward vitronectin compared with wild-type cells. Furthermore, this defect is associated with decreased cell surface expression of integrin αvß3 and significantly decreased expression of this integrin in focal adhesions. Because of the importance of adhesion and migration in tumor progression (in addition to placental development), we examined the affect of culturing B16F0 melanoma cells in 1.5% oxygen (O2). Culturing B16F0 melanoma cells at 1.5% O2 resulted in increased αvß3 integrin surface expression and increased adhesion to and migration toward vitronectin. Together, these data suggest that HIF and O2 tension influence placental invasion and tumor migration by increasing cell surface expression of αvß3 integrin.
  • Publication
    Role of lateral cell–cell border location and extracellular/transmembrane domains in PECAM/CD31 mechanosensation
    (2004-08-06) Kaufman, David A.; Albelda, Steven M.; Davies, Peter F.; Davies, Peter F.
    Phosphorylation of tyrosine residues on platelet–endothelial cell adhesion molecule-1 (PECAM-1), followed by signal trans- 13 duction events, has been described in endothelial cells following exposure to hyperosmotic and fluid shear stress. However, it is 14 unclear whether PECAM-1 functions as a primary mechanosensor in this process. Utilizing a PECAM-1–null EC-like cell line, we 15 examined the importance of cellular localization and the extracellular and transmembrane domains in PECAM-1 phosphorylation 16 responses to mechanical stress. Tyrosine phosphorylation of PECAM-1 was stimulated in response to mechanical stress in null cells 17 transfected either with full length PECAM-1 or with PECAM-1 mutants that do not localize to the lateral cell–cell adhesion site and 18 that do not support homophilic binding between PECAM-1 molecules. Furthermore, null cells transfected with a construct that 19 contains the intact cytoplasmic domain of PECAM-1 fused to the extracellular and transmembrane domains of the interleukin-2 20 receptor also underwent mechanical stress-induced PECAM-1 tyrosine phosphorylation. These findings suggest that mechano- 21 sensitive PECAM-1 may lie downstream of a primary mechanosensor that activates a tyrosine kinase.
  • Publication
    The requirement for Notch signaling at the β-selection checkpoint in vivo is absolute and independent of the pre–T cell receptor
    (2006-10-02) Sambandam, Arivazhagan; Maillard, Ivan; Tu, LiLi; Shestova, Olga; Yashiro-Ohtani, Yumi; Bhandoola, Avinash; Pear, Warren S; Shestova, Olga; Xu, Lanwei; Bhandoola, Avinash; Pear, Warren S
    Genetic inactivation of Notch signaling in CD4−CD8− double-negative (DN) thymocytes was previously shown to impair T cell receptor (TCR) gene rearrangement and to cause a partial block in CD4+CD8+ double-positive (DP) thymocyte development in mice. In contrast, in vitro cultures suggested that Notch was absolutely required for the generation of DP thymocytes independent of pre-TCR expression and activity. To resolve the respective role of Notch and the pre-TCR, we inhibited Notch-mediated transcriptional activation in vivo with a green fluorescent protein–tagged dominant-negative Mastermind-like 1 (DNMAML) that allowed us to track single cells incapable of Notch signaling. DNMAML expression in DN cells led to decreased production of DP thymocytes but only to a modest decrease in intracellular TCRβ expression. DNMAML attenuated the pre-TCR–associated increase in cell size and CD27 expression. TCRα or TCRαβ transgenes failed to rescue DNMAML-related defects. Intrathymic injections of DNMAML− or DNMAML+ DN thymocytes revealed a complete DN/DP transition block, with production of DNMAML+ DP thymocytes only from cells undergoing late Notch inactivation. These findings indicate that the Notch requirement during the β-selection checkpoint in vivo is absolute and independent of the pre-TCR, and it depends on transcriptional activation by Notch via the CSL/RBP-J–MAML complex.