Histamine selectively interrupts VE-cadherin adhesion independently of capacitive calcium entry

Histamine is an important agent of innate immunity, transiently increasing the flux of immune-competent molecules from the vascular space to the tissues and then allowing rapid restoration of the integrity of the endothelial barrier. In previous work we found that histamine alters the endothelial barrier by disrupting cell-cell adhesion and identified VE-cadherin as an essential participant in this process. The previous work did not determine whether histamine directly interrupted VE-cadherin adhesion, whether the effects of histamine were selective for cadherin adhesion, or whether capacitive calcium flux across the cell membrane was necessary for the effects of histamine on cell-cell adhesion. In the current work we found that histamine directly interrupts adhesion of L cells expressing the type 1 histamine (H1) receptor and VE-cadherin to a VE-cadherin-Fc fusion protein. In contrast, integrin-mediated adhesion to fibronectin of the same L cells expressing the H1 receptor was not affected by histamine, demonstrating that the effects of histamine are selective for cadherin adhesion. Some of the effects of many edemagenic agonists on endothelium are dependent on the capacitive flux of calcium across the endothelial cell membrane. Blocking capacitive calcium flux with LaCl3 did not prevent histamine from interrupting VE-cadherin adhesion of transfected L cells, nor did it prevent histamine from interrupting cell-cell adhesion of human umbilical vein endothelial cells. These data support the contentions that histamine directly and selectively interrupts cadherin adhesion and this effect on cadherin adhesion is independent of capacitive calcium flux. endothelium; integrin; human histamine receptor H1; vascular endothelial cadherin ACUTE INFLAMMATORY EDEMA is an important component of innate immunity that facilitates the transfer of immune-competent molecules from the vascular space to the tissues. Histamine and serotonin are well-acknowledged physiological agonists of acute inflammatory edema that disrupt cell-cell apposition in postcapillary venules Vascular endothelial (VE)-cadherin is an endothelial unique cadherin that mediates cell-cell adhesion of endothelial cells (17). Function-interrupting antibody to VE-cadherin increases permeability of endothelium in vivo, indicating that integrity of VE-cadherin adhesion is essential to the integrity of the endothelial barrier (9, 16). It was of note that although antibody to VE-cadherin increased in vivo endothelial permeability within 1 h of its administration, no light microscopic changes were evident in the endothelium even at 2 h, demonstrating that no large gaps had formed between the cells. However, similar to the effects of histamine and serotonin, transmission electron micrographs detected very small gaps in lung microvessels exposed to antibody to VE-cadherin (9). ECV304 cells are a transformed bladder epithelial cell line that express the type 1 histamine receptor (H1) and P-and N-cadherin, but not VE-cadherin. When mock-transfected ECV304 cells were exposed to histamine, there was no change in the electrical resistance of the cell-cell barrier they created (24). However, when ECV304 cells transfected with VEcadherin were exposed to histamine, the electrical resistance of the cell-cell barrier fell, identifying an important role for VE-cadherin in the response to histamine of a monolayer of polarized cells (24). ECV304 cells express claudin 2 and develop tight junctions (5). This tight junction is a necessary component of the electrical resistance decreased by histamine. The observation that histamine decreased the integrity of the cell-cell barrier created by VE-cadherin transfected ECV304 cells did not discriminate between effects of histamine on an interaction of VE-cadherin with the tight junction versus a direct effect of histamine on VE-cadherin adhesion. Histamine and other agonists that increase endothelial permeability initiate signaling in endothelial cells that increases cell calcium (6, 7). Although some of the increase in cell calcium caused by these agonists reflects release of calcium from intracellular stores, capacitive flux of calcium across the cell membrane is also activated, and the capacitive calcium flux is necessary for some of the effects of these agonists on endothelial cells In these investigations we used a model of cadherin adhesion that lacks tight junctions and asked whether histamine directly affects VE-cadherin adhesion as opposed to affecting an interaction between VE-cadherin and the tight junction. Our results indicate that histamine directly affects VE-cadherin adhesion. Using a similar model of integrin-based adhesion, we foun

Testing and Diversity in Postsecondary Education: The Case of California

The past several years have seen numerous efforts to scale back or eliminate affirmative action in postsecondary admissions. In response, policymakers and postsecondary institutions in many states are searching for ways to maintain the diversity of student populations without resorting to a prohibited focus on race. In response to these changes, this study used data from California and a simplified model of the University of California admissions process to explore how various approaches to admissions affect the diversity of the admitted student population. "Race-neutral" admissions based solely on test scores and grades were compared with the results of actual admissions before and after the elimination of affirmative action. A final set of analyses explored the effects on diversity of alternative approaches that take into account factors other than grades and scores, but not race or ethnicity. Replacing the former admissions process that included preferences with a race-neutral model based solely on GPA and SAT-I scores substantially reduced minority representation at the two most selective UC campuses but had much smaller effects at the other six, less selective campuses. SAT-I scores contributed to but were not the sole cause of the underrepresentation of African American and Hispanic students. A race-neutral model based solely on GPA also produced an underrepresentation of minorities, albeit a less severe one. None of the alternative admissions models analyzed could replicate the composition of the student population that was in place before the termination of affirmative action in California. The only approach that substantially increased the representation of minority students was accepting most students on the basis of within-school rather than statewide rankings, and this approach caused a sizable drop in both the average SAT scores and the average GPA of admitted applicants, particularly among African American and Hispanic students. Although admissions systems differ, the basic findings of this study are likely to apply at a general level to many universities and underscore the difficulty of providing proportional representation for underserved minority students at highly selective institutions without explicit preferences

Cadherin-23, myosin VIIa and harmonin, encoded by Usher syndrome type I genes, form a ternary complex and interact with membrane phospholipids

Cadherin-23 is a component of early transient lateral links of the auditory sensory cells' hair bundle, the mechanoreceptive structure to sound. This protein also makes up the upper part of the tip links that control gating of the mechanoelectrical transduction channels. We addressed the issue of the molecular complex that anchors these links to the hair bundle F-actin core. By using surface plasmon resonance assays, we show that the cytoplasmic regions of the two cadherin-23 isoforms that do or do not contain the exon68-encoded peptide directly interact with harmonin, a submembrane PDZ (post-synaptic density, disc large, zonula occludens) domain-containing protein, with unusually high affinity. This interaction involves the harmonin Nter-PDZ1 supramodule, but not the C-terminal PDZ-binding motif of cadherin-23. We establish that cadherin-23 directly binds to the tail of myosin VIIa. Moreover, cadherin-23, harmonin and myosin VIIa can form a ternary complex, which suggests that myosin VIIa applies tension forces on hair bundle links. We also show that the cadherin-23 cytoplasmic region, harmonin and myosin VIIa interact with phospholipids on synthetic liposomes. Harmonin and the cytoplasmic region of cadherin-23, both independently and as a binary complex, can bind specifically to phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), which may account for the role of this phospholipid in the adaptation of mechanoelectrical transduction in the hair bundle. The distributions of cadherin-23, harmonin, myosin VIIa and PI(4,5)P2 in the growing and mature auditory hair bundles as well as the abnormal locations of harmonin and myosin VIIa in cadherin-23 null mutant mice strongly support the functional relevance of these interactions

Characterization of the Endothelial Cell Cytoskeleton following HLA Class I Ligation

Vascular endothelial cells (ECs) are a target of antibody-mediated allograft rejection. In vitro, when the HLA class I molecules on the surface of ECs are ligated by anti-HLA class I antibodies, cell proliferation and survival pathways are activated and this is thought to contribute to the development of antibody-mediated rejection. Crosslinking of HLA class I molecules by anti-HLA antibodies also triggers reorganization of the cytoskeleton, which induces the formation of F-actin stress fibers. HLA class I induced stress fiber formation is not well understood.The present study examines the protein composition of the cytoskeleton fraction of ECs treated with HLA class I antibodies and compares it to other agonists known to induce alterations of the cytoskeleton in endothelial cells. Analysis by tandem mass spectrometry revealed unique cytoskeleton proteomes for each treatment group. Using annotation tools a candidate list was created that revealed 12 proteins, which were unique to the HLA class I stimulated group. Eleven of the candidate proteins were phosphoproteins and exploration of their predicted kinases provided clues as to how these proteins may contribute to the understanding of HLA class I induced antibody-mediated rejection. Three of the candidates, eukaryotic initiation factor 4A1 (eIF4A1), Tropomyosin alpha 4-chain (TPM4) and DDX3X, were further characterized by Western blot and found to be associated with the cytoskeleton. Confocal microscopy analysis showed that class I ligation stimulated increased eIF4A1 co-localization with F-actin and paxillin.Colocalization of eIF4A1 with F-actin and paxillin following HLA class I ligation suggests that this candidate protein could be a target for understanding the mechanism(s) of class I mediated antibody-mediated rejection. This proteomic approach for analyzing the cytoskeleton of ECs can be applied to other agonists and various cells types as a method for uncovering novel regulators of cytoskeleton changes

Ste20-Related Proline/Alanine-Rich Kinase (SPAK) Regulated Transcriptionally by Hyperosmolarity Is Involved in Intestinal Barrier Function

The Ste20-related protein proline/alanine-rich kinase (SPAK) plays important roles in cellular functions such as cell differentiation and regulation of chloride transport, but its roles in pathogenesis of intestinal inflammation remain largely unknown. Here we report significantly increased SPAK expression levels in hyperosmotic environments, such as mucosal biopsy samples from patients with Crohn's disease, as well as colon tissues of C57BL/6 mice and Caco2-BBE cells treated with hyperosmotic medium. NF-κB and Sp1-binding sites in the SPAK TATA-less promoter are essential for SPAK mRNA transcription. Hyperosmolarity increases the ability of NF-κB and Sp1 to bind to their binding sites. Knock-down of either NF-κB or Sp1 by siRNA reduces the hyperosmolarity-induced SPAK expression levels. Furthermore, expression of NF-κB, but not Sp1, was upregulated by hyperosmolarity in vivo and in vitro. Nuclear run-on assays showed that hyperosmolarity increases SPAK expression levels at the transcriptional level, without affecting SPAK mRNA stability. Knockdown of SPAK expression by siRNA or overexpression of SPAK in cells and transgenic mice shows that SPAK is involved in intestinal permeability in vitro and in vivo. Together, our data suggest that SPAK, the transcription of which is regulated by hyperosmolarity, plays an important role in epithelial barrier function

G77-383 Marketing Your Timber

Marketing is the key to getting the most from your timber. Buying and selling trees is a business transaction. Marketing is the key to getting the most for your timber. Anyone can sell something if they are not concerned about the price received. When you market something, you are selling in a competitive market for the best possible price. A satisfactory sale occurs when the needs of both buyer and seller are met. The buyer must acquire material at current market prices in order to stay in business and still remain competitive. The timber owner should be paid the fair market value and have his land and remaining trees left in a productive condition for future sales. The woodland owner must receive a fair price to insure sustained yields and to protect the forest land base from conversion to other uses