scholarly journals Localization of human heparan glucosaminyl N-deacetylase/N-sulphotransferase to the trans-Golgi network

1997 ◽  
Vol 325 (2) ◽  
pp. 351-357 ◽  
Author(s):  
Donald E. HUMPHRIES ◽  
Brandon M. SULLIVAN ◽  
M. Deize ALEIXO ◽  
Jennifer L. STOW
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Umbilical Vein ◽  
Intracellular Location ◽  
Trans Golgi Network ◽  
Liver And Pancreas ◽  
Umbilical Vein Endothelial Cells ◽  
Golgi Network ◽  
Tissues Expression ◽  
Rna Blot Analysis

In order to determine the intracellular location of heparan N-deacetylase/N-sulphotransferase, cDNAs encoding human heparan glucosaminyl N-deacetylase/N-sulphotransferase were cloned from human umbilical vein endothelial cells. The deduced amino acid sequence was identical to that of the human heparan N-sulphotransferase cloned previously [Dixon, Loftus, Gladwin, Scambler, Wasmuth and Dixon (1995) Genomics 26, 239–244]. RNA blot analysis indicated that two heparan N-sulphotransferase transcripts of approx. 8.5 and 4 kb were produced in all tissues. Expression was most abundant in heart, liver and pancreas. A cDNA encoding a Flag-tagged human heparan N-sulphotransferase (where Flag is an epitope with the sequence DYKDDDDK) was transfected into mouse LTA cells. Immunofluorescence detection using anti-Flag monoclonal antibodies demonstrated that the enzyme was localized to the trans-Golgi network. A truncated Flag-tagged heparan N-sulphotransferase was also retained in the Golgi, indicating that, as for many other Golgi enzymes, the N-terminal region of heparan N-sulphotransferase is sufficient for retention in the Golgi apparatus.

scholarly journals Envelopment of Human Cytomegalovirus Occurs by Budding into Golgi-Derived Vacuole Compartments Positive for gB, Rab 3, Trans-Golgi Network 46, and Mannosidase II

2003 ◽  
Vol 77 (5) ◽  
pp. 3191-3203 ◽  
Author(s):  
M. Homman-Loudiyi ◽  
K. Hultenby ◽  
W. Britt ◽  
C. Söderberg-Nauclér
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Human Cytomegalovirus ◽  
Virus Assembly ◽  
Umbilical Vein ◽  
Immunogold Labeling ◽  
Lung Fibroblasts ◽  
Trans Golgi Network ◽  
Immunofluorescence Labeling ◽  
Golgi Network

ABSTRACT Although considerable progress has been made towards characterizing virus assembly processes, assignment of the site of tegumentation and envelopment for human cytomegalovirus (HCMV) is still not clear. In this study, we examined the envelopment of HCMV particles in human lung fibroblasts (HF) HL 411 and HL 19, human umbilical vein endothelial cells, human pulmonary arterial endothelial cells, and arterial smooth muscle cells at different time points after infection by electron microscopy (EM), immunohistochemistry, and confocal microscopy analysis. Double-immunofluorescence labeling experiments demonstrated colocalization of the HCMV glycoprotein B (gB) with the Golgi resident enzyme mannosidase II, the Golgi marker TGN (trans-Golgi network) 46, and the secretory vacuole marker Rab 3 in all cell types investigated. Final envelopment of tegumented capsids was observed at 5 days postinfection by EM, when tegumented capsids budded into subcellular compartments located in the cytoplasm, in close proximity to the Golgi apparatus. Immunogold labeling and EM analysis confirmed staining of the budding compartment with HCMV gB, Rab 3, and mannosidase II in HL 411 cells. However, the markers Rab 1, Rab 2, Rab 7, Lamp 1 (late endosomes and lysosomes), and Lamp 2 (lysosomes) neither showed specific staining of the budding compartment in the immunogold labeling experiments nor colocalized with gB in the immunofluorescent colocalization experiments in any cell type studied. Together, these results suggest that the final envelopment of HCMV particles takes place mainly into a Golgi-derived secretory vacuole destined for the plasma membrane, which may release new infectious virus particles by fusion with the plasma membrane.

Augmentation of eosinophil degranulation and LTC4secretion by integrin-mediated endothelial cell adhesion

1999 ◽  
Vol 277 (4) ◽  
pp. L802-L810 ◽  
Author(s):  
Nilda M. Muñoz ◽  
Kimm J. Hamann ◽  
Klaus F. Rabe ◽  
Hiroyuki Sano ◽  
Xiangdong Zhu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Priming Effect ◽  
Cytochalasin B ◽  
Umbilical Vein ◽  
Before And After ◽  
Interleukin 1Α ◽  
Umbilical Vein Endothelial Cells ◽  
Endothelial Cell Adhesion ◽  
Eosinophil Degranulation

We examined the effect of eosinophil ligation to cultured human umbilical vein endothelial cells (HUVECs) in augmenting the stimulated secretion of leukotriene (LT) C4and eosinophil peroxidase (EPO). The effects of adhesion were compared before and after specific blockade with monoclonal antibodies directed against eosinophil surface integrins or endothelial counterligands. Adhesion to HUVECs augmented EPO release caused by formyl-methionyl-leucyl-phenylalanine plus cytochalasin B from 403 ± 15.3 (BSA control) to 778 ± 225 ng/106cells for eosinophils exposed to interleukin-1α-treated HUVECs ( P < 0.05) and also caused a twofold increase in stimulated LTC4secretion ( P < 0.05). To determine whether augmented secretion resulted directly from adhesive ligation, studies were also performed with paraformaldehyde-treated HUVECs; stimulated secretion of LTC4from eosinophils was comparable to that for living HUVECs. Our study is the first demonstration that adhesion to HUVECs through ligation to α4- or β2-integrin on the eosinophil surface causes augmentation of stimulated secretion of both EPO and LTC4and that blockade of adhesion molecules on either eosinophils or HUVECs prevents the priming effect on eosinophil secretion.

Characterization of Endothelial Cell Differential Attachment to Fibrin and Fibrinogen and Its Inhibition by Arg-Gly-Asp-Containing Peptides

1995 ◽  
Vol 74 (02) ◽  
pp. 764-769 ◽  
Author(s):  
Mei-Chi Chang ◽  
Bih-Ru Wang ◽  
Tur-Fu Huang
Keyword(s):  
Endothelial Cells ◽  
Monoclonal Antibodies ◽  
Endothelial Cell ◽  
Cell Attachment ◽  
Umbilical Vein ◽  
Integrin Αvβ3 ◽  
Snake Venoms ◽  
Mediate Cell ◽  
Umbilical Vein Endothelial Cells

SummaryWe investigated the adhesion of human umbilical vein endothelial cells (HUVECs) to fibrin(ogen) molecule of varying structure for identifying sites that mediate cell attachment. Fibrin was prepared either with ancrod which liberates only FPA from fibrinogen, or with thrombin, which liberates both FPA and FPB. Both fibrin preparations equally supported HUVEC attachment. GRGDS, RGD-containing peptides of snake venoms, and monoclonal antibodies against αvβ3 (23C6 and 7E3) inhibited the attachment of HUVECs to fibrin by 65–75%. In contrast, the attachment of HUVECs to fibrinogen was less effective and was almost completely inhibited by both RGD-containing peptides and by antibodies against integrin αvβ3 (85-95% inhibition). The C-terminal dodecapeptide of fibrinogen γ chain (residues 400–411) inhibited minimally the attachment of HUVECs to fibrin. Additionally, the binding of RGD-containing snake venom peptides to HUVECs was both RGD- and divalent-cation-dependent. The IC50s for inhibition of HUVEC attachment to fibrin were 0.09 μM (rhodostomin), 1.54 μM (trigramin) and 1.64 μM (halysin).These results indicate that fibrin mediated support of cell attachment is independent of the cleavage of FPB from fibrinogen. HUVEC attachment to fibrinogen was almost completely inhibited by RGD-containing peptides and by antibodies against αvβ3. In contrast, the attachment to fibrin was partially resistant to RGD-containing peptides and to the monoclonal antibodies against integrin αvβ3. However, αvβ3 is the major receptor mediating HUVEC attachment to fibrin.

Single-Chain and Two-Chain Tissue-Type Plasminogen Activator (t-PA) Bind Differently to Cultured Human Endothelial Cells

1989 ◽  
Vol 62 (02) ◽  
pp. 699-703 ◽  
Author(s):  
Rob J Aerts ◽  
Karin Gillis ◽  
Hans Pannekoek
Keyword(s):  
Endothelial Cells ◽  
Plasminogen Activator ◽  
Binding Sites ◽  
Umbilical Vein ◽  
Tissue Type ◽  
Single Chain ◽  
Human Endothelial Cells ◽  
Tissue Type Plasminogen Activator ◽  
Type Plasminogen Activator ◽  
Umbilical Vein Endothelial Cells

SummaryIt has recently been shown that the fibrinolytic components plasminogen and tissue-type plasminogen activator (t-PA) both bind to cultured human umbilical vein endothelial cells (HUVEC). After cleavage of t-PA by plasmin, “single-chain” t-PA (sct-PA) is converted into “two-chain” t-PA (tct-PA), which differs from the former in a number of respects. We compared binding of sct-PA and tct-PA to the surface of HUVEC. Removal of t-PA bound to HUVEC by a mild treatment with acid and a subsequent quantification of eluted t-PA both by activity- and immunoradiometric assays revealed that, at concentrations between 10 and 500 nM, HUVEC bind about 3-4 times more sct-PA than tct-PA. At these concentrations, both sct-PA and tct-PA remain active when bound to HUVEC. Mutual competition experiments showed that sct-PA and tct-PA can virtually fully inhibit binding of each other to HUVEC, but that an about twofold higher concentration of tct-PA is required to prevent halfmaximal binding of sct-PA than visa versa. These results demonstrate that sct-PA and tct-PA bind with different affinities to the same binding sites on HUVEC.

Studies of the Factor Xa-Dependent Inhibitor of Factor VIIa/Tissue Factor (Extrinsic Pathway Inhibitor) from Cell Supernates of Cultured Human Umbilical Vein Endothelial Cells

1989 ◽  
Vol 61 (01) ◽  
pp. 101-105 ◽  
Author(s):  
Bonnie J Warn-Cramer ◽  
Fanny E Almus ◽  
Samuel I Rapaport
Keyword(s):  
Molecular Weight ◽  
Endothelial Cells ◽  
Tissue Factor ◽  
Phorbol Ester ◽  
Umbilical Vein ◽  
Primary Cultures ◽  
Factor Xa ◽  
Extrinsic Pathway ◽  
Human Umbilical Vein ◽  
Umbilical Vein Endothelial Cells

SummaryCultured human umbilical vein endothelial cells (HUVEC) have been reported to produce extrinsic pathway inhibitor (EPI), the factor Xa-dependent inhibitor of factor VHa/tissue factor (TF). We examined the release of this inhibitor from HUVEC as a function of their growth state and in response to the induction of endothelial cell TF activity. HUVEC constitutively produced significant amounts of EPI at all stages of their growth in culture including the post-confluent state. Rate of release varied over a 3-fold range for primary cultures from 12 different batches of pooled umbilical cord cells. Constitutive EPI release was unaltered during a 6 hour period of induction of TF activity with thrombin or phorbol ester but slowed during longer incubation of the cells with phorbol ester. Whereas plasma contains two molecular weight forms of EPI, only the higher of these two molecular weight forms was demonstrable by Western analysis of HUVEC supernatants with 125I-factor Xa as the ligand.

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