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Query: UNIPROT:P06889 (Mol)
 630,302 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The production of potentially targetable VSV-G-pseudotyped retrovirus vectors has been hampered by inadequate understanding of the structure-function relationships of the VSV-G protein. In these studies we demonstrate assembly and production of MLV-based and HIV-1-based vector particles using VSV-G proteins modified by the insertion of a peptide ligand into a site corresponding to amino acid position 24 of the native VSV-G molecule. The inserted ligand represents the decapeptide encoding the collagen-binding domain of von Willebrand factor. We have used deconvolution microscopy to demonstrate that the modified VSV-G molecules sequester in perinuclear structures and are unavailable for assembly of infectious virus particles at the cell surface under standard tissue culture conditions at 37 degrees C. In contrast, at a lower permissive temperature of 30 degrees C, the modified VSV-G protein traffics appropriately to the cell surface and participates in useful titers. Furthermore, VSV-G-pseudotyped MLV-based and HIV-1-based vectors displaying the collagen-binding domain demonstrate a statistically significant increased attachment to a collagen matrix as indicated by an ELISA-like cell binding assay and by a focus transduction assay.
Mol Ther 2004 Jan
PMID:Ligand-modified vesicular stomatitis virus glycoprotein displays a temperature-sensitive intracellular trafficking and virus assembly phenotype. 1474 80

Hematopoietic stem cells have a remarkable plastic capacity, which allows them to differentiate into various cells, such as immune cells, nervous cells, muscle cells, bone and cartilaginous cells. The aim of this study was to show the capacity of stem cells to differentiate into endothelial cells, in culture, after addition of endothelial cells growth supplement (ECGS). We also compared the behavior of these cells with that of endothelial cells obtained from human umbilical vein (HUVEC). CD34+ cells obtained by immunomagnetic separation from human umbilical cord and placental blood were used. After 12-15 days of culture in a medium containing ECGS, the cells showed morphological changes characteristic to endothelial cells and immunocytochemical analysis revealed the presence of CD31 surface antigen and von Willebrand factor. The flow-cytometric analysis of endothelial cells adhesion molecules (ECAM) showed that endothelial cells derived from CD34+ cells expressed CD54/ICAM-1 9.65+/-0.2% and CD106/VCAM 7.73+/-0.3%, values similar to those expressed by HUVECs. After TNF incubation, ECAM expression increased only in HUVECs. These data demonstrate that a fraction of circulating CD34+ cells may develop some endothelial cell characteristics when cultured with ECGS, but they are functionally different from HUVECs.
J Cell Mol Med
PMID:Endothelial cells from hematopoietic stem cells are functionally different from those of human umbilical vein. 1475 14

An angiogenesis assay based on gene transfer would be extremely useful for angiogenic gene therapy. A simple, reproducible, and quantitative assay to test angiogenic genes would provide more accurate predictions than conventional peptide-based assays. Here, we have developed a semiquantitative angiogenesis assay utilizing gene transfer into skeletal muscle, which is a target tissue for ischemic limb diseases. To facilitate quick and clean analysis, a naked plasmid DNA vector combined with an electroporation procedure was used for gene transfer. When the plasmid vector encoding vascular endothelial growth factor cDNA (pJDK-VEGF165) was injected into the tibialis anterior muscle of BALB/c mice, followed by in vivo electroporation and explant culture in growth factor-reduced Matrigel, the outward migration of sprouting cells was observed as early as day 2. The cells soon formed capillary networks, which peaked at day 7 and persisted until day 14. The capillary-like structures were positive for von Willebrand factor, platelet endothelial cell adhesion molecule, and vimentin, suggesting they were endothelial cells. There was little, if any, sprouting or formation of capillaries from the control vector (pJDK)-injected group. Consistent with the region of sprouting and network formation, the amount of secreted VEGF increased in the conditioned medium of explant cultures. The angiogenic potential of connective tissue growth factor (CTGF) was examined using the new assay. Whereas the CTGF gene alone induced weak sprouting activity, it appeared to inhibit the angiogenic activity of the VEGF165 gene during cotreatment. This attenuating activity of CTGF on VEGF was reproduced in vivo in a murine model of hindlimb ischemia. In a group of mice treated with both pJDK-CTGF and pJDK-VEGF165, the blood flow measured by laser Doppler imaging was significantly lower than that of the pJDK-VEGF165-treated group 10 days after femoral artery excision. These results are consistent with recent reports that suggest that CTGF inhibits VEGF. This confirms the usefulness of this novel ex vivo assay in assessing the angiogenic capacity of genes of interest. In summary, this new gene-based angiogenesis assay should be widely applicable in the study of angiogenic or antiangiogenic genes because it can readily predict the angiogenic potential of specific genes and their combinations.
Mol Ther 2004 Mar
PMID:A novel ex vivo angiogenesis assay based on electroporation-mediated delivery of naked plasmid DNA to skeletal muscle. 1500 15

The lemurs of Madagascar are a unique radiation of primates that show an extraordinary diversity of lifestyles, morphologies and behaviours. However, very little is known about the relative antiquity of lemuriform clades due to the lack of terrestrial fossils for the Tertiary of Madagascar. Here, we employ a Bayesian method to estimate divergence dates within the lemuriform radiation using several unlinked gene loci and multiple fossil calibrations outside the lemuriform clade. Two mitochondrial genes (cytochrome oxidase II and cytochrome b), two nuclear introns (transthyretin intron 1 and von Willebrand factor gene intron 11) and one nuclear exon (interphotoreceptor retinoid binding protein, exon 1) are used in separate and combined analyses. The genes differ in taxon sampling and evolutionary characteristics but produce congruent date estimates. Credibility intervals narrow considerably in combined analyses relative to separate analyses due to the increased amount of data. We also test the relative effects of multiple vs. single calibration points, finding that, when only single calibration points are employed, divergence dates are systematically underestimated. For the mitochondrial DNA data set, we investigate the effects of sampling density within the mouse lemur radiation (genus Microcebus). When only two representative species are included, estimated dates throughout the phylogeny are more recent than with the complete-species sample, with basal nodes less affected than recent nodes. The difference appears to be due to the manner in which priors on node ages are constructed in the two analyses. In nearly all analyses, the age of the lemuriform clade is estimated to be approximately 62-65 Ma, with initial radiation of mouse lemurs and true lemurs (genus Eulemur) occurring approximately 8-12 Ma. The antiquity of the mouse lemur radiation is surprising given the near uniform morphology among species. Moreover, the observation that mouse lemurs and true lemurs are of similar ages suggests discrepancies in rates of morphological, behavioural and physiological evolution in the two clades, particularly with regard to characteristics of sexual signalling. These differences appear to correlate with the nocturnal vs. diurnal lifestyles, respectively, of these two primate groups.
Mol Ecol 2004 Apr
PMID:Divergence dates for Malagasy lemurs estimated from multiple gene loci: geological and evolutionary context. 1501 54

The Ku heterodimer (Ku70/Ku80) plays a central role in DNA double-strand breaks recognition and repair. However, Ku is expressed also on the surface of different types of cells along with its intracellular pool within the nucleus and the cytoplasm. Participation of membrane-associated Ku in cell-cell interaction has been reported recently. Here, we describe a novel function of cell-surface Ku as an adhesion receptor for fibronectin (Fn). The role of Ku in cell adhesion was investigated by comparing the Ku80 deficient Chinese hamster ovary (CHO) cell line, xrs-6, with clones transfected stably with either the hamster or human Ku80 cDNA. Ku expression in transfectant cells resulted in a significant increased adhesion on Fn and type IV collagen as compared to control cells. The observed increase in cell adhesion relied on Ku cell-surface expression, since antibodies directed against Ku70 or Ku80 subunit inhibited adhesion on Fn of Ku80, but not control vector, transfected xrs-6 cells. In addition, both Ku70 and Ku80 present a structural relationship with integrin I (or A) domains and the A1 and A3 domains of von Willebrand factor, domains known to be involved in Fn binding. Both Ku70 and Ku80 exhibit a complete set of residues compatible in their position and chemical nature with the formation of a metal ion-dependent adhesion (MIDAS) site implicated in ligand binding and integrin activation. Taken together, these functional and structural approaches support a new role for Ku as an adhesion receptor for Fn.
J Mol Biol 2004 Mar 26
PMID:The Membrane-associated form of the DNA repair protein Ku is involved in cell adhesion to fibronectin. 1501 72

The C-terminal fragment, Bb, of factor B combines with C3b to form the pivotal C3-convertase, C3bBb, of alternative complement pathway. Bb consists of a von Willebrand factor type A (vWFA) domain that is structurally similar to the I domains of integrins and a serine protease (SP) domain that is in inactive conformation. The structure of the C3bBb complex would be important in deciphering the activation mechanism of the SP domain. However, C3bBb is labile and not amenable to X-ray diffraction studies. We engineered a disulfide bond in the vWFA domain of Bb homologous to that shown to lock I domains in active conformation. The crystal structures of Bb(C428-C435) and its inhibitor complexes reveal that the adoption of the "active" conformation by the vWFA domain is not sufficient to activate the C3-convertase catalytic apparatus and also provide insights into the possible mode of C3-convertase activation.
Mol Cell 2004 Apr 09
PMID:Structural analysis of engineered Bb fragment of complement factor B: insights into the activation mechanism of the alternative pathway C3-convertase. 1506

It has been demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the role of CD34-positive endothelial cells in liver samples from patients with hepatitis B virus (HBV)-associated chronic liver diseases. Tissue sections were obtained by liver biopsy from 25 patients with HBV-associated chronic liver diseases and were examined by immunohistochemistry using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34-positive, but vWF-negative endothelial cells were observed, particularly in the sinusoids and vascular endothelial cells. We counted these cells and expressed the results as a CD34-labeling index (LI). The CD34 LI did not correlate with VEGF expression and the CD34 LI of patients who progressed to hepatocellular carcinoma (HCC) tended to increase compared to those that did not progress to HCC. CD34 LI was an independent risk factor for development of HCC (relative risk, 35.689; P = 0.033). We conclude that CD34-positive endothelial cells in patients with HBV-associated chronic liver diseases might play a role in hepatocarcinogenesis.
Int J Mol Med 2004 Aug
PMID:Expression of CD34-positive sinusoidal endothelial cells in patients with HBV-associated chronic liver diseases. 1525 62

A homology model for the A2 domain of von Willebrand factor (VWF) is presented. A large number of target-template alignments were combined into a consensus alignment and used for constructing the model from the structures of six template proteins. Molecular dynamics simulation was used to study the structural and dynamic effects of eight mutations introduced into the model, all associated with type 2A von Willebrand disease. It was found that the group I mutations G1505R, L1540P and S1506L cause significant deviations over multiple regions of the protein, coupled to significant thermal fluctuations for G1505R and L1540P. This suggests that protein instability may be responsible for their intracellular retention. The group II mutations R1597W, E1638K and G1505E caused single loop displacements near the physiologic VWF proteolysis site between Y1605-M1606. These modest structural changes may affect interactions between VWF and the ADAMTS13 protease. The group II mutations I1628T and L1503Q caused no significant structural change in the protein, suggesting that inclusion of the protease in this model is necessary for understanding their effect. [Figure: see text]. Homology model of the von Willebrand factor A2 domain
J Mol Model 2004 Aug
PMID:Molecular modeling of the von Willebrand factor A2 Domain and the effects of associated type 2A von Willebrand disease mutations. 1532 48

Bone marrow and peripheral blood of adults contain a special sub-type of progenitor cells which are able to differentiate into mature endothelial cells, thus contributing to re-endothelialization and neo-vascularization. These angiogenic cells have properties of embryonal angioblasts and were termed endothelial progenitor cells (EPCs). In general, three surface markers (CD133, CD34 and the vascular endothelial growth factor receptor-2) characterize the early functional angioblast, located predominantly in the bone marrow. Later, when migrating to the systemic circulation EPCs gradually lose their progenitor properties and start to express endothelial marker like VE-cadherin, endothelial nitric oxide synthase and von Willebrand factor. The number of circulating EPCs in healthy subjects is rather low and a variety of conditions or factors may further influence this number. In the context of possible therapeutic application of EPCs recent clinical studies employing these cells for neo-vascularization of ischemic organs have just been published. However, the specificity of the observed positive clinical effects, the mechanisms regulating the differentiation of EPCs and their homing to sites of injured tissue remain partially unknown at present.
J Cell Mol Med
PMID:Endothelial progenitor cells: characterization, pathophysiology, and possible clinical relevance. 1560 78

N-Ethyl-maleimide-sensitive factor (NSF) plays a critical role in the regulation of exocytosis. NSF regulates exocytosis by interacting with a complex containing soluble NSF attachment protein receptor (SNARE) molecules, hydrolyzing ATP, and disassembling the SNARE complex. We hypothesized that peptide inhibitors of NSF would decrease exocytosis. We now report the development of a novel set of peptides that block exocytosis by inhibiting NSF activity. These NSF inhibitors are fusion polypeptides composed of an 11 amino acid human immunodeficiency virus transactivating regulatory protein (TAT) domain fused to a 22 amino acid NSF domain. These TAT-NSF fusion polypeptides cross endothelial cell membranes, inhibit NSF hydrolysis of ATP, decrease NSF disassembly of SNARE molecules, and block exocytosis of von Willebrand factor. Control peptides have no effect on exocytosis. TAT-NSF inhibitors administered to mice prolong the bleeding time. Blood concentrations of these TAT-NSF peptides rapidly decrease within 5 min after injection and then remain constant from 10 to 60 min after injection. These TAT-NSF compounds may be useful in the treatment of a variety of diseases in which exocytosis plays a prominent role, including myocardial infarction, stroke, thrombosis, and autoimmune disorders.
Mol Pharmacol 2005 Apr
PMID:A novel class of fusion polypeptides inhibits exocytosis. 1567


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