Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:2.7.10.2 (focal adhesion kinase)
 44,029 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

This contribution describes a new microscopic technique aimed at visualizing colloidal gold particles of 20-40 nm diameter as dynamic markers at the light microscopic level. The technique, called nanovid microscopy, is based on the use of contrast enhancement by video techniques and digital image processing. Two applications on living cells are discussed. The first is the receptor-mediated endocytosis of the transferrin receptor, which for the first time can be followed in real time. A second application documents the motion of cell-surface glycoproteins on PTK2-cells. In addition, nanovid microscopy allows the number of gold particles in immunogold staining to be quantified easily. With this tool, the translocation of specific molecules in living cells becomes one of the most enjoyable activities to observe.
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PMID:Detection of gold probes with video-enhanced contrast microscopy: nanovid microscopy. 247 23

Primary sites of subcellular destruction with photofrin II (PfII) and mono-L-aspartyl chlorin e6 (MACE) were determined by transmission electron microscopy (TEM). Potorous tridactylus (PTK2) cells were grown in Rose chambers and incubated for 24 hr with a sensitizer concentration sufficient to provide 100% mortality. Cells were irradiated with laser light and fixed and processed for electron microscopy at various times post-irradiation. The results indicate that PfII initially destroys mitochondria, whereas MACE destroys lysosomes. Both conclusions are consistent with fluorescence subcellular localization studies.
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PMID:In vitro photosensitization II. An electron microscopy study of cellular destruction with mono-L-aspartyl chlorin e6 and photofrin II. 252 92

The effects of the protein kinase C inhibitor H-7 on the actin cytoskeleton of cultured cells (Swiss 3T3 and PTK2) are described. As documented by fluorescence microscopy and the higher-resolution technique of photoelectron microscopy, the effects are rapid and dramatic; exposure to 30 microM H-7 in culture medium for less than 6 min is sufficient to induce a significant reduction in the numbers and thickness of actin microfilament bundles and alterations in the morphology of cell-cell boundaries in PTK2 cells. One-hour exposure to 30 microM H-7 results in nearly complete depletion of normal actin microfilament bundles from all of the cell types examined, without dramatic changes in overall cell shape. The intermediate filament and microtubule cytoskeletal networks did not appear to be affected to any extent over the times and doses examined. Forty-five minutes of exposure of Swiss 3T3 cells to 200 microM of either HA1004 (which is comparable to H-7 with respect to inhibition of cyclic nucleotide dependent kinases) or to the protein kinase C inhibitor sangivamycin did not induce the actin alterations characteristic of H-7. In addition, depletion of protein kinase C from Swiss 3T3 cells by means of phorbol ester-induced down-regulation did not prevent the effects of H-7 on the actin cytoskeleton. These results demonstrate that the protein kinase C inhibitor H-7 has a specific and rapid effect on the actin cytoskeleton, and furthermore H-7 may have biochemical effects beyond those mediated by inhibition of protein kinase C or the cyclic nucleotide dependent kinases.
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PMID:Protein kinase C inhibitor H-7 alters the actin cytoskeleton of cultured cells. 277 3

The in vivo biological activity of uroporphyrin I has been studied by determining the amount of necrosis produced in murine tumors exposed to various total doses of light at 615 nm. Similarly, the in vitro photosensitizing activity of uroporphyrin I was examined by measuring the percentage of cells killed in a cell culture system. Light doses used were 25-400 J/cm2. Mice that received uroporphyrin I at 40 mg/kg had only minimal superficial necrosis upon histological examination at doses of 400 J/cm2 (615 nm). Those tumors that received 300 J/cm2 or less showed no histological evidence of necrosis. Mice that received hematoporphyrin derivative (Photofrin II) at 10 mg/kg were completely necrotic at total doses of 100 J/cm2 (630 nm). PTK2 epithelial cells incubated with uroporphyrin I at either 40 micrograms/ml or 80 mu/ml and 10 J/cm2 (615 nm) showed no apparent damage and had 100% cell survival. By contrast, those cells treated with hematoporphyrin derivative (Photofrin II) at 25 micrograms/ml and 10 J/cm2 (630 nm) exhibited 100% cell kill. It is concluded that uroporphyrin I is a poor photosensitizer in both in vivo and in vitro systems compared to hematoporphyrin derivative (Photofrin II).
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PMID:Study of the in vivo and in vitro photosensitizing capabilities of uroporphyrin I compared to photofrin II. 294 30

Cytotoxic effect of HPD plus light irradiation on PTK2 and HeLa cells in vitro were studied by laser microbeam irradiation. The dynamic changes, in three different steps as observed in the cells ranged from visible damage to cell lysis, were recorded at the monocellular level. It was found that the speed of the cytotoxic process varied chiefly with the dose of light used. The cytotoxic progress of PTK2 and HeLa cells was compared and analyzed. In addition, the cytotoxic effects of three different laser wavelengths (6328A, 5145A and 4880A) plus HPD were compared. The most significant killing effect occurred at 6328A wavelength of red light.
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PMID:[Cytotoxic effect of hematoporphyrin derivative (HPD) plus light irradiation in vitro]. 294 80

Colloidal gold particles of 20 to 40 nm diameter stabilized with polyethylene glycol (PEG) were microinjected in PTK2 cells. Aggregates and individual particles, which are smaller than the theoretical limit of resolution of the optical microscope and invisible to the eye are discernible from organelles by reflection of polarized light. They are optimally visualized using transmitted light and electronic subtraction of diffuse background light. The gold particles show saltatory motion. The direction, speed, median distance travelled and frequency of saltations are indiscernible from measurements made on cell organelles in the same preparations. Because microtubule treadmilling has been implicated as a potential motor for organelle motility, gold particles coupled to monoclonal antibodies, recognizing the alpha-subunit of tubulin (Kilmartin et al., 1982), were injected. These particles, often forming linear arrays, assumed entirely fixed positions in the cell. The results suggest that there is a transport system associated with microtubules which can carry synthetic particles through the cell without the need for them being covered with specific proteins. Microtubule treadmilling does not seem to be involved. The possibility of following 20-40 nm particles and probably even smaller ones, that can be coupled to most proteins, within living cells provides a tool of wide applicability to study the fate and behaviour of such proteins. It is suggested that this new method be called nanoparticle video ultramicroscopy or nanovid ultramicroscopy.
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PMID:Probing microtubule-dependent intracellular motility with nanometre particle video ultramicroscopy (nanovid ultramicroscopy). 390 99

The sera of 103 patients with connective tissue diseases were studied for the presence of anticytoskeletal antibodies by using an indirect immunofluorescence method. PTK2 cells fixed with paraformaldehyde and digitonin were used as substrate. Antibodies to intermediate filaments were detected in sera of 85.7% of polymyositis/dermatomyositis (PM/DM), 62.8% of systemic sclerosis, 54.5% of rheumatoid arthritis, and 37.5% of systemic lupus erythematosus patients, and in 42.5% of normal sera. High titers of these antibodies, which were IgM, were present in 30% of patients' and 5% of normal sera. Antibodies to microfilaments were present in 11.6% of patients' sera and absent in all control sera. These antibodies were IgM or IgG. The switch from an IgM to an IgG antibody was observed in 1 patient. An IgG antibody to the spindle poles and midbody of mitotic cells was present in the serum of 1 patient with the CREST syndrome (calcinosis, Raynaud's phenomenon, esophageal dysmotility, sclerodactyly, telangiectasias). Antibodies to intermediate filaments and to microfilaments occur commonly in the connective tissue diseases, particularly in PM/DM, and are not detected with substrates or fixation methods used in routine antinuclear antibody testing.
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PMID:Anticytoskeletal autoantibodies in the connective tissue diseases. 404 Jul 59

A monoclonal antibody from clone T7 raised against total nuclear proteins from the Kc cell line of Drosophila melanogaster (Saumweber, H. Symmons. P. Kabisch, R. Will, H & Bonhoeffer, F, Chromosoma 80 (1980) 253) [1] showed positive immunofluorescent staining on interphase nuclei of HeLa and PTK2 cells. When this antibody was allowed to react with several nuclear protein fractions isolated from HeLa S3 cells, three polypeptides of molecular weights (MW) 44 000, 63 000 and 70 000 were identified as the corresponding antigens, all components of hnRNA containing ribonucleoprotein particles. Sucrose gradient fractionation of such particles after mild RNase treatment and subsequent analysis of the proteins by the immunoblotting method revealed that the 44 000 MW antigen was an integral part of the ribonuclease-resistant complex. The results support the view that hnRNA molecules are associated with certain proteins conserved during evolution which may play structural roles in the ribonucleoprotein organization.
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PMID:Cross-reaction of hnRNP-proteins of HeLa cells with nuclear proteins of Drosophila melanogaster demonstrated by a monoclonal antibody. 681 36

Serum from a patient with the CREST Syndrome and systemic lupus erythematosus contained an IgM antibody that reacted at dilutions up to 1:800 with a fibrous cytoplasmic network in several epithelioid and fibroblastic cell lines. The antibody was shown by immunofluorescence microscopy to label a specific subset of cytoskeletal polymers, the intermediate filaments. The reactive antigen from this biochemically heterogeneous group of filaments was established as the 58,000-mol wt protein, vimentin: (a) the patient's serum reacts with a range of cell lines that contain intermediate filaments composed of vimentin, but not with cells whose intermediate filaments are composed of different protein subunits; (b) in PTK2 epithelioid cells the serum reacts with the class of filaments that coils around the nucleus after colchicine treatment (vimentin) and not with the filaments that remain dispersed after colchicine (prekeratin); and (c) the component of reactive cells that combines with the serum is shown by immunoelectrophoresis to be a 58,000-mol wt protein antigen. A similar antibody that binds intermediate filaments of PTK2 cells was encountered at lower titer in some sera from other patients with connective tissue diseases and in control sera. Previous routine antinuclear antibody assays using mouse liver or commercially prepared HEp-2 cells have failed to reveal anticytoskeletal antibodies in patient sera, perhaps due to inadequate presentation or preservation of cytoplasmic antigens.
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PMID:Immunoglobulin M autoantibody to vimentin intermediate filaments. 703 55

The reassembly of microtubules is described in mitotic cells after release from nocodazole-induced block. The formation of microtubules was followed by light microscopic immunocytochemical staining using the PAP method, combined with toluidine blue staining of the chromatin. The light microscopic observations on whole cells were compared with ultrastructural observations on thin sections. This step is essential to ascertain complete destruction of microtubules during the nocodazole treatment and to correlate immunocytochemical staining with the presence of microtubules. Removal of nocodazole (10 or 1 micrograms/ml) after a sufficiently long incubation to induce a complete disappearance of microtubules resulted in the appearance of tubulin staining specifically associated with the centromeres and with one or two isolated points in the cytoplasm. Electron microscopy confirmed that the staining was due to the massive accumulation of small microtubules at the kinetochores and centrosomes. Kinetochore nucleation was seen only in association with condensed metaphase-stage chromosomes and not with the less-condensed prophase chromosomes. In a second type of experiment cells were allowed to enter mitosis in the presence of an incompletely active concentration of nocodazole (0.1 microgram/ml). The construction of the mitotic spindle was arrested; however, short microtubules were assembled at the kinetochores and centrosomes. These experiments demonstrate that in living mitotic PTK2 cells the kinetochores, as well as the centrosomes, exert a nucleating action on tubulin assembly. The further elongation of microtubules after removal of nocodazole was seen to occur preferentially along axes between the centrosomes and the kinetochores. This resulted in the construction of normal metaphases that evolved through anaphase and telophase. We have attempted to formulate a hypothesis that may explain the oriented assembly that seems to be essential in the construction of the spindle.
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PMID:Nucleated assembly of mitotic microtubules in living PTK2 cells after release from nocodazole treatment. 734 6


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