491 research outputs found
Temperature-Dependence of Weibel-Palade Body Exocytosis and Cell Surface Dispersal of von Willebrand Factor and Its Propolypeptide
No full textBackground: Weibel-Palade bodies (WPB) are endothelial cell (EC) specific secretory organelles containing Von Willebrand factor (VWF). The temperature-dependence of Ca2+-driven WPB exocytosis is not known, although indirect evidence suggests that WPB exocytosis may occur at very low temperatures. Here we quantitatively analyse the temperature-dependence of Ca2+-driven WPB exocytosis and release of secreted VWF from the cell surface of ECs using fluorescence microscopy of cultured human ECs containing fluorescent WPBs.
Principal Findings: Ca2+-driven WPB exocytosis occurred at all temperatures studied (7–37°C). The kinetics and extent of WPB exocytosis were strongly temperature-dependent: Delays in exocytosis increased from 0.92 s at 37°C to 134.2 s at 7°C, the maximum rate of WPB fusion decreased from 10.0±2.2 s−1 (37°C) to 0.80±0.14 s−1 (7°C) and the fractional extent of degranulation of WPBs in each cell from 67±3% (37°C) to 3.6±1.3% (7°C). A discrepancy was found between the reduction in Ca2+-driven VWF secretion and WPB exocytosis at reduced temperature; at 17°C VWF secretion was reduced by 95% but WPB exocytosis by 75–80%. This discrepancy arises because VWF dispersal from sites of WPB exocytosis is largely prevented at low temperature. In contrast VWF-propolypeptide (proregion) dispersal from WPBs, although slowed, was complete within 60–120 s. Novel antibodies to the cleaved and processed proregion were characterised and used to show that secreted proregion more accurately reports the secretion of WPBs at sub-physiological temperatures than assay of VWF itself.
Conclusions : We report the first quantitative analysis of the temperature-dependence of WPB exocytosis. We provide evidence; by comparison of biochemical data for VWF or proregion secretion with direct analysis of WPB exocytosis at reduced temperature, that proregion is a more reliable marker for WPB exocytosis at reduced temperature, where VWF-EC adhesion is increased
Differential cargo mobilisation within Weibel-Palade bodies after transient fusion with the plasma membrane.
Get PDFInflammatory chemokines can be selectively released from Weibel-Palade bodies (WPBs) during kiss-and-run exocytosis. Such selectivity may arise from molecular size filtering by the fusion pore, however differential intra-WPB cargo re-mobilisation following fusion-induced structural changes within the WPB may also contribute to this process. To determine whether WPB cargo molecules are differentially re-mobilised, we applied FRAP to residual post-fusion WPB structures formed after transient exocytosis in which some or all of the fluorescent cargo was retained. Transient fusion resulted in WPB collapse from a rod to a spheroid shape accompanied by substantial swelling (>2 times by surface area) and membrane mixing between the WPB and plasma membranes. Post-fusion WPBs supported cumulative WPB exocytosis. To quantify diffusion inside rounded organelles we developed a method of FRAP analysis based on image moments. FRAP analysis showed that von Willebrand factor-EGFP (VWF-EGFP) and the VWF-propolypeptide-EGFP (Pro-EGFP) were immobile in post-fusion WPBs. Because Eotaxin-3-EGFP and ssEGFP (small soluble cargo proteins) were largely depleted from post-fusion WPBs, we studied these molecules in cells preincubated in the weak base NH4Cl which caused WPB alkalinisation and rounding similar to that produced by plasma membrane fusion. In these cells we found a dramatic increase in mobilities of Eotaxin-3-EGFP and ssEGFP that exceeded the resolution of our method (∼ 2.4 µm2/s mean). In contrast, the membrane mobilities of EGFP-CD63 and EGFP-Rab27A in post-fusion WPBs were unchanged, while P-selectin-EGFP acquired mobility. Our data suggest that selective re-mobilisation of chemokines during transient fusion contributes to selective chemokine secretion during transient WPB exocytosis. Selective secretion provides a mechanism to regulate intravascular inflammatory processes with reduced risk of thrombosis
Traffic Problems and Their Solution in Animal Cells
No full text(This information was taken from the Distinguished Scientist Lecture Series Program 1987-1988).
Recording is distorted.
Dr. Palade, a Nobel laureate, is senior research Scientist and special advisor to the dean at Yale University School of Medicine. Born in Moldavia, Rumania, Dr. Palade earned the M.D. degree at the University of Bucharest while he was intern and resident at the Association of the Civil Hospital of Bucharest. He came to he United States in 1946 to be visiting investigator in the Department of Biology at New York Univerisity, and has since held positions at the Rockefeller Institute and Yale University. He is a member of the American Academy of Arts and Sciences, the . National Academy of Sciences, the Royal Society, and several other scientific societies. Honors for Dr. Palade\u27s work have included the National Medal of Science, the Henry Gray Award, the Schleiden Medaille, the Brown-Hazen Award, the Dickson Prize, and the Horwitz Prize, along others. In 1974 he received the Nobel Prize Physiology or Medicine for his discoveries, with A. Claude and C. DeDuve, on the structural and functional organization of the cell. He has also recieved a number of honorary degrees from both American and European universities. Dr. Palade is the author of many articles on issues in cell biology in the Journal of Biological Chemistry, Annals of Biochemistry, Journal of Cell Biology and other scientific journals, as well as in several anthologies and proceedings. He has been editor of the Journal of Cell Biology and Journal of Molecular Biology and is presently editor of the editor of Membrance Biology and Annual Review of Cell Biology.
His Work: Dr. Palade\u27s research has concentrated on cell fractionation and electron microscopy. A series of he that he made on the cytoplasmic membrane systems led to his important work on protein synthesis, especially in the pancreatic cell.
His Lecture: February 13, 1988: Traffic Problems and Their Solution in Animal Cellshttps://digitalcommons.bard.edu/dsls_1987_1988/1005/thumbnail.jp
Re-establishment of VWF-dependent Weibel-Palade bodies in VWD endothelial cells
Get PDFType 3 von Willebrand disease (VWD) is a severe hemorrhagic defect in humans. We now identify the homozygous mutation in the Chapel Hill strain of canine type 3 VWD that results in premature termination of von Willebrand factor (VWF) protein synthesis. We cultured endothelium from VWD and normal dogs to study intracellular VWF trafficking and Weibel-Palade body formation. Weibel-Palade bodies could not be identified in the canine VWD aortic endothelial cells (VWD-AECs) by P-selectin, VWFpp, or VWF immunostaining and confocal microscopy. We demonstrate the reestablishment of Weibel-Palade bodies that recruit endogenous P-selectin by expressing wild-type VWF in VWD-AECs. Expression of mutant VWF proteins confirmed that VWF multimerization is not necessary for Weibel-Palade body creation. Although the VWF propeptide is required for the formation of Weibel-Palade bodies, it cannot independently induce the formation of the granule. These VWF-null endothelial cells provide a unique opportunity to examine the biogenesis of Weibel-Palade bodies in endothelium from a canine model of type 3 VWD
Pharmacologic differentiation between inositol-1,4,5-trisphosphate-induced Ca2+ release and Ca2+- or caffeine-induced Ca2+ release from intracellular membrane systems.
No full textVarious known Ca2+ channel blockers and intracellular Ca2+ antagonists have been tested for effects of inositol-1,4,5-trisphosphate (IP3)-induced Ca2+ release from isolated canine brain microsomes. In agreement with previous reports, heparin, p-chloromercuribenzoic acid, W-7, cinnarizine, flunarizine, certain local anesthetics, La3+, and Ca2+ inhibit the release of Ca2+ induced by addition of IP3. In addition, we report here pronounced inhibition of IP3-induced Ca2+ release by low levels of Cd2+, by relatively high concentrations of TMB-8, and by phytic acid. In contrast, a number of blockers of other Ca2+ channels (nifedipine, verapamil, dantrolene, dithiothreitol, and ruthenium red) have relatively little or no effect on IP3-induced Ca2+ release from brain microsomes. The relative ineffectiveness of substances that inhibit Ca2+- or caffeine-induced Ca2+ release from skeletal muscle sarcoplasmic reticulum suggests that release of Ca2+ from caffeine- and IP3-sensitive neuronal Ca2+ stores is likely to be mediated by different channels. Further evidence that different channels are involved is presented by way of demonstration of the lack of Ca2+-induced Ca2+ release from these brain microsomes and the lack of effect on sarcoplasmic reticulum caffeine-induced Ca2+ release of certain inhibitors of IP3-induced Ca2+ release used here. Among IP3-induced Ca2+ release blockers, La3+ appeared to be exceptional in its ability to stimulate microsomal Ca2+ uptake sufficiently to attenuate release of Ca2+ induced by IP3. Most blockers of IP3-induced Ca2+ release appear not to function by way of inhibiting K+ counter-ion movements (valinomycin does not reverse the inhibition) but rather by way of direct interaction with the IP3 receptor or the Ca2+ channel that mediates the IP3-induced Ca2+ release. Inhibition of [3H]IP3 binding to the microsomes by phytic acid, heparin, pyrophosphate, p-chloromercuribenzoic acid, and Ca2+ could be demonstrated but not by the other substances tested
Direct inhibition of inositol-1,4,5-trisphosphate-induced Ca2+ release from brain microsomes by K+ channel blockers.
No full textDihydropyridine receptor and ryanodine receptor gene expression in long-term denervated rat muscles
No full textFollowing disruption of the nerve supply, extensor digitorum longus (EDL) and soleus (SOL) muscles in rats are known to exhibit alterations in excitation-contraction coupling. After total RNA isolation from the denervated and the contralateral control muscles performed at 25 and 50 days following denervation, RNase protection assays were carried out with four cDNA probes specific for the skeletal and cardiac isoforms of both the DHPR alpha 1-subunit and the RyR. Longterm denervation increased the expression of the mRNA for skeletal DHPR and skeletal RyR in SOL muscle, but it also significantly increased the expression of the mRNA for the cardiac isoform of the DHPR alpha 1 subunit in EDL muscle
Ant-Based System Analysis on the Traveling Salesman Problem Under Real-World Settings
No full textMany optimization problems have huge solution spaces, deep restrictions, highly correlated parameters, and operate with uncertain or inconsistent data. Such problems sometimes elude the usual solving methods we are familiar with, forcing us to continuously improve these methods or to even completely reconsider the solving methodologies. When decision makers need faster and better results to more difficult problems, the quality of a decision support system is crucial. To estimate the quality of a decision support system when approaching difficult problems is not easy, but is very important when designing and conducting vital industrial processes or logistic operations. This paper studies the resilience of a solving method, initially designed for the static and deterministic TSP (Traveling Salesman Problem) variant, when applied to an uncertain and dynamic TSP version. This investigation shows how a supplementary level of complexity can be successfully handled. The traditional ant-based system under investigation is infused with a technique which allows the evaluation of its performances when uncertain input data are present. Like the real ant colonies do, the system rapidly adapts to repeated environmental changes. A comparison with the performance of another heuristic optimization method is also done
Storage of Tissue-Type Plasminogen Activator in Weibel-Palade Bodies of Human Endothelial Cells
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Abstract
—Tissue-type plasminogen activator (t-PA) is acutely released by endothelial cells. Although its endothelial storage compartment is still not well defined, t-PA release is often accompanied by release of von Willebrand factor (vWf), a protein stored in Weibel-Palade bodies. We investigated, therefore, whether t-PA is stored in these secretory organelles. Under basal culture conditions, a minority of human umbilical vein endothelial cells (HUVEC) exhibited immunofluorescent staining for t-PA, which was observed only in Weibel-Palade bodies. To increase t-PA expression, HUVEC were infected with a t-PA recombinant adenovirus (AdCMVt-PA). Overexpressed t-PA was detected in Weibel-Palade bodies and acutely released together with endogenous vWf by thrombin or calcium ionophore stimulation. In contrast, plasminogen activator inhibitor type 1 and urokinase were not detected in Weibel-Palade bodies after adenovirus-mediated overexpression. Infection of HUVEC with proinsulin recombinant adenovirus resulted in the storage of insulin in Weibel-Palade bodies, indicating that these organelles can also store nonendothelial proteins that show regulated secretion. Infection of AtT-20 pituitary cells, a cell type with regulated secretion, with AdCMVt-PA resulted in the localization of t-PA in adrenocorticotropic hormone–containing granules, indicating that t-PA can be diverted to secretory granules independently of vWf. Coinfection of AtT-20 cells with AdCMVt-PA and proinsulin recombinant adenovirus resulted in the colocalization of t-PA and insulin in the same granules. Taken together, these results suggest that HUVEC have protein sorting mechanisms similar to those of other regulated secretory cells. Although the results did not exclude an alternative storage site for t-PA in HUVEC, they established that t-PA can be stored in Weibel-Palade bodies. This finding may explain the acute coordinate secretion of t-PA and vWf.
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On the Effect of Adding Nodes to TSP Instances: An Empirical Analysis
No full textOur human society is experiencing complex problems nowadays, which require large amounts of computing resources, fast algorithms and efficient implementations. These real-world problems generate new instances for the classical, academic problems as well as new data collections that can be used for assessing the available solving packages. This paper focuses on the Traveling Salesman Problem, which is one of the most studied combinatorial optimization problems, with many variants and broad applications. In order to allow a smooth integration with the current Geographic Information Systems (GIS) technologies, the instances described in this work are specified by geographic coordinates, and they use the orthodromic distance. A sequence of similar instances is defined, and the characteristics of the state-of-the-art exact solver results on these instances are presented and discussed
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