Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: UNIPROT:P08758 (annexin V)
 9,383 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Endonexin is a 32kDa, calcium-dependent membrane-binding protein that is one of a group of proteins that binds to chromaffin granule membranes and may regulate membrane fusion events occurring during exocytosis. In this study an oligonucleotide probe that codes for a highly conserved, repeated sequence present in this and related proteins was used to isolate a 2,048 nucleotide cDNA encoding endonexin from a bovine liver cDNA library. The translated amino acid sequence of endonexin shows the four domain structure characteristic of proteins in this class. The nucleotide sequence is 55 to 61% identical to that of the related membrane-binding proteins lipocortin, calpactin, endonexin II and (half of) 68kDa calelectrin. Southern blot analysis of bovine genomic DNA suggests the presence of a single gene for this protein. A consensus nucleotide sequence (TCTGGGAACTTC) was identified in the 5' nontranslated portion of the endonexin mRNA that is also represented in the messages for calpactin and endonexin II.
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PMID:Cloning and characterization of a cDNA encoding bovine endonexin (chromobindin 4). 284 15

A 235-bp DNA coding for the leech blood coagulation inhibitor, hirudin, was chemically synthesized. The synthesis involved preparation of seven long oligodeoxyribonucleotide pairs which were assembled and cloned using a rapid and simple procedure. More than half of the transformed Escherichia coli cells expressed a biosynthetic polypeptide having biological properties which were very similar to authentic hirudin from the leech Hirudo medicinalis. To achieve efficient expression, we fused the hirudin DNA to a truncated C1 repressor gene of bacteriophage lambda to create a hybrid protein. An additional methionine at the fusion point allowed the active hirudin to be cleaved off by cyanogen bromide.
DNA 1986 Dec
PMID:Cloning and expression in Escherichia coli of a synthetic DNA for hirudin, the blood coagulation inhibitor in the leech. 354 21

In the early stages of apoptosis changes occur at the cell surface, which until now have remained difficult to recognize. One of these plasma membrane alterations is the translocation of phosphatidylserine (PS) from the inner side of the plasma membrane to the outer layer, by which PS becomes exposed at the external surface of the cell. Annexin V is a Ca2+ dependent phospholipid-binding protein with high affinity for PS. Hence this protein can be used as a sensitive probe for PS exposure upon the cell membrane. Translocation of PS to the external cell surface is not unique to apoptosis, but occurs also during cell necrosis. The difference between these two forms of cell death is that during the initial stages of apoptosis the cell membrane remains intact, while at the very moment that necrosis occurs the cell membrane looses its integrity and becomes leaky. Therefore the measurement of Annexin V binding to the cell surface as indicative for apoptosis has to be performed in conjunction with a dye exclusion test to establish integrity of the cell membrane. This paper describes the results of such an assay, as obtained in cultured HSB-2 cells, rendered apoptotic by irradiation and in human lymphocytes, following dexamethasone treatment. Untreated and treated cells were evaluated for apoptosis by light microscopy, by measuring the amount of hypo-diploid cells using of DNA flow cytometry (FCM) and by DNA electrophoresis to establish whether or not DNA fragmentation had occurred. Annexin V binding was assessed using bivariate FCM, and cell staining was evaluated with fluorescein isothiocyanate (FITC)-labelled Annexin V (green fluorescence), simultaneously with dye exclusion of propidium iodide (PI) (negative for red fluorescence). The test described, discriminates intact cells (FITC-/PI-), apoptotic cells (FITC+/PI-) and necrotic cells (FITC+/PI+). In comparison with existing traditional tests the Annexin V assay is sensitive and easy to perform. The Annexin V assay offers the possibility of detecting early phases of apoptosis before the loss of cell membrane integrity and permits measurements of the kinetics of apoptotic death in relation to the cell cycle. More extensive FCM will allow discrimination between different cell subpopulations, that may or may not be involved in the apoptotic process.
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PMID:A novel assay for apoptosis. Flow cytometric detection of phosphatidylserine expression on early apoptotic cells using fluorescein labelled Annexin V. 762 68

We have previously reported that neutrophilic granulocytes rapidly release part of their Fc gamma RIII from the plasma membrane upon in vitro activation, probably by proteolytic cleavage. In plasma and other body fluids, released or soluble Fc gamma RIII has been found in considerable amounts. In the present study, neutrophils were kept in maintenance culture for 18 to 24 hours. Forty percent of the neutrophils completely lost Fc gamma RIII, and the remainder of the cells showed a 60% decrease in Fc gamma RIII expression on their surface. Released Fc gamma RIII was detected in the culture supernatant. Nevertheless, more than 90% of the cells was viable as judged by hydrolysis of fluorescein diacetate. The presence of interferon gamma, granulocyte colony-stimulating factor, or granulocyte-macrophage colony-stimulating factor, but not interleukin-3 (IL-3), IL-6, or IL-8, in the culture medium increased the number of cells that still expressed Fc gamma RIII. We found that this loss of Fc gamma RIII was not the result of cell activation but correlated strongly with apoptosis. The Fc gamma RIII-negative subpopulation exhibited typical morphologic changes, such as nuclear condensation and DNA fragmentation. Furthermore, this subpopulation appeared to have acquired the property of binding Annexin V, a calcium-dependent, phospholipid-binding protein with high affinity for phosphatidylserine. The external exposure of this phospholipid by cells has been reported to occur during apoptosis. The property of Annexin V binding was not shared by the nonapoptotic, Fc gamma RIII-positive subpopulation. In this respect, we identified binding of Annexin V as an convenient marker for apoptotic cells. Our results indicate that soluble Fc gamma RIII in body fluids might be derived for a large part from neutrophils undergoing apoptosis in the tissues.
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PMID:Human neutrophils lose their surface Fc gamma RIII and acquire Annexin V binding sites during apoptosis in vitro. 781 8

We characterized the region of human chromosome 4q26-q28 that contains the gene encoding annexin V (placental anticoagulant protein I), a member of a family of calcium-dependent phospholipid binding proteins. A total of 14.5 kb, containing 9 introns, could be directly amplified from genomic DNA; the remainder was characterized from genomic clones in phage lambda and a yeast artificial chromosome. The gene was mapped with restriction enzymes BamHI, EcoRI, HindIII, SacI, StuI, and XbaI; the transcribed region spans 28 kb and contains 13 exons (44 tp 530 bp in size) and 12 introns (0.23 to 8.8 kb in size). Several putative transcription factor binding sites are present in the 5'-region, but the promoter has no recognizable TATA box. This study will facilitate further analysis of the functions of annexin V and its role in disease.
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PMID:Organization of the human annexin V (ANX5) gene. 803 19

Apoptosis, or programmed cell death, is a general mechanism for removal of unwanted cells from the immune system. It is characterized by chromatin condensation, a reduction in cell volume, and endonuclease cleavage of DNA into oligonucleosomal length fragments. Apoptosis is also accompanied by a loss of membrane phospholipid asymmetry, resulting in the exposure of phosphatidylserine at the surface of the cell. Expression of phosphatidylserine at the cell surface plays an important role in the recognition and removal of apoptotic cells by macrophages. Here we describe a new method for the detection of apoptotic cells by flow cytometry, using the binding of fluorescein isothiocyanate-labeled annexin V to phosphatidylserine. When Burkitt lymphoma cell lines and freshly isolated germinal center B cells are cultured under apoptosis inducing conditions, all cells showing chromatin condensation strongly stain with annexin V, whereas normal cells are annexin V negative. Moreover, DNA fragmentation is only found in the annexin V-positive cells. The nonvital dye ethidium bromide was found to stain a subpopulation of the annexin V-positive apoptotic cells, increasing with time. Our results indicate that the phase in apoptosis that is characterized by chromatin condensation coincides with phosphatidylserine exposure. Importantly, it precedes membrane damage that might lead to release from the cells of enzymes that are harmful to the surrounding tissues. Annexin V may prove important in further unravelling the regulation of apoptosis.
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PMID:Annexin V for flow cytometric detection of phosphatidylserine expression on B cells undergoing apoptosis. 806 38

Oxidized cholesterol compounds or oxysterols are thought to be potent membrane-destabilizing agents. Anionic phospholipids, chiefly phosphatidylserine, have a procoagulant potential due to their ability to favour the membrane assembly of the characteristic clotting enzyme complexes including the tissue factor-dependent initiating complex. However, in resting cells, phosphatidylserine is sequestered in the inner leaflet of the plasma membrane. When THP-1 monocytic cells were cultured in the presence of 7beta-hydroxycholesterol (7beta-OH) or 25-hydroxycholesterol (25-OH), prothrombinase, which reflects anionic phospholipid exposure and tissue factor (TF) procoagulant activities, increased in a time- and dose-dependent manner. 7beta-OH appeared 1.5- to 2-fold more potent than 25-OH. Interestingly, no effect of cholesterol itself could be detected on procoagulant activities. Nevertheless, no difference in TF activity could be detected between oxysterol-treated and control cells after disruption. TF antigen expression was the same in oxysterol-treated and control cells as shown by flow cytometry. In contrast, the use of labelled annexin V, a protein probe of anionic phospholipids, revealed an elevated number of cells with exposed phosphatidylserine. Because the latter also constitutes a signal for phagocyte recognition of apoptotic cells and fragments, and a proportion of cells displayed altered morphology with condensed chromatin and membrane blebs, analysis of DNA was performed and indicated apoptosis in oxysterol-treated cells. Hence, oxysterol-induced phosphatidylserine exposure and enhanced TF activity may results from apoptosis. These results suggest relationships between oxysterol and the amplification of coagulation reactions by monocytic cells resulting from induced phosphatidylserine exposure.
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PMID:Oyxsterols induce membrane procoagulant activity in monocytic THP-1 cells. 861 54

Plasma membrane binding of annexin V was used to detect and quantitate apoptotic cells induced by cytotoxic drug treatment in epithelial cell cultures. Chinese hamster ovary (CHO) cells were incubated for 2 h with the ID90 concentration of Cisplatin (20 microM), and 24, 48, 72, and 96 h later the unfixed cells were stained with fluorescein isothiocyanate (FITC)-conjugated annexin V. The fluorescence signal was quantitated by flow cytometry (FCM). During the early phase of the apoptotic response, the annexin V-binding frequency histograms showed two separate cell populations, a dimly and a brightly fluorescent one. At t = 96 h after drug incubation, when the process of apoptosis was completed, only the brightly fluorescent population was present. A dose-effect relationship could be established between the Cisplatin concentration used in the 2 h incubation and the binding of annexin V on the cell membrane, as estimated by FITC fluorescence. The dimly and brightly fluorescent populations were sorted on the basis of annexin V binding, and assayed for 1) DNA breaks by in situ nick translation assay and DNA content by DNA-propidium iodine fluorescence in a bivariate analysis, 2) membrane integrity by dye exclusion, and 3) morphological characteristics of apoptosis. The dimly fluorescent cell population appeared to represent apoptotic cells in the early phase of the death process, as demonstrated by intact cell membranes, normal DNA content, few DNA breaks, and chromatin condensation. The brightly fluorescent cells predominantly had sub-G1 DNA content, nuclear fragmentation, leaky cell membranes, and probably represent late apoptotic cells. These results demonstrate that cytotoxic drug-induced apoptosis can be quantitated by annexin V binding and that by using this assay early and late apoptotic cells can be identified.
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PMID:Quantification of apoptotic cells with fluorescein isothiocyanate-labeled annexin V in chinese hamster ovary cell cultures treated with cisplatin. 872 61

Early during the process of apoptosis, cells lose their phospholipid membrane asymmetry and expose phosphatidylserine (PS) at the cell surface while maintaining their plasma membrane integrity intact. This process can be monitored for suspended cell types by using annexin V-FITC, which is a Ca(2+)-dependent, phospholipid-binding protein with high affinity for PS, and flow cytometry. If adherent cell types are to be studied for this apoptosis-associated phenomenon, then a problem is encountered, in that specific membrane damage occurs during harvesting. In this paper, a flow cytometric-based method is described that allows the measurement of loss of phospholipid asymmetry during apoptosis of adherent cells in culture. The method relies on the phospholipid binding property of biotinylated annexin V. Furthermore, the use of this conjugate allows tricolor flow cytometric analysis of apoptosis. Employing the method to MR65 cells, which were initiated by olomoucine to enter apoptosis, it is shown that PS exposure occurs early after the onset of apoptosis and, at the prevalent time-resolution, that PS exposure is accompanied by loss of both cytokeratin and DNA. The annexin V+ cells appear as a characteristic sub-G1 peak in the DNA histogram.
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PMID:A novel assay to measure loss of plasma membrane asymmetry during apoptosis of adherent cells in culture. 872 62

Anti-Thy-1 nephritis is a model of mesangial proliferative glomerulonephritis. It has been suggested that apoptosis, which is a counteracting regulatory mechanism against undesired cell proliferation, is involved in sequential histological changes in this model. In the present study, we investigated whether IgG2a anti-Thy-1 monoclonal antibody (ER4) or its F(ab')2 fragments are able to induce apoptosis of rat glomerular mesangial cells (GMC) in vitro. After co-culture with ER4 or its F(ab')2 fragments, apoptosis was assessed by morphological studies with Hoechst 33258 stain and FITC-annexin V. The latter detects the dislocation of negatively charged phospholipid, phosphatidylserine, from the inner to the outer leaflet of the membrane during apoptosis. This is a sensitive method for the detection of apoptosis. Under fluorescent microscopy, distinct nuclear condensation and positive reactivity with FITC-annexin V were observed in cells co-cultured with ER4 or its F(ab')2 fragments. The results obtained by FACS analysis with annexin V showed a direct correlation with the detection of apoptosis with the terminal deoxynucleotidyl transferase reaction (TDT). Up to 19% and 23% of rat GMC, which were co-cultured for 24 hours with 1 microgram/ml (0.5 microgram/l x 10(5) cells) of ER4 or its F(ab')2 fragments, were labeled by TDT, respectively. With annexin V, up to 34% and 31% cells displaying apoptosis were seen. The degree of apoptosis as measured by the annexin V method was dependent on the concentration of ER4 and time of incubation in the presence of ER4. Finally, apoptosis was confirmed by gel electrophoresis of DNA isolated from the cells co-cultured with each monoclonal antibody (MAb). DNA extracts from cells co-cultured with ER4 or its F(ab')2 fragments demonstrated typical internucleosomal DNA fragmentation. Medium alone, controls of anti-human C3bi receptor MAb (IB4) and anti-rat MHC class I MAb (OX18) showed neither nuclear changes nor significant labeling of the cells with the TDT reaction or with the annexin V. Taken together, these results demonstrate for the first time that anti-Thy-1 MAb is able to induce apoptosis of rat GMC in vitro. The Thy-1 antigen on rat GMC, therefore, seems to function as one of the molecules regulating cell death and thereby may determine the degree of mesangial alteration in Thy-1 nephritis.
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PMID:Apoptosis of cultured rat glomerular mesangial cells induced by IgG2a monoclonal anti-Thy-1 antibodies. 882 24


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