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

Monoclonal antibody TEC-02, raised against mouse embryonal carcinoma cells, has been shown to react with murine preimplantation embryos and with a very limited number of adult mouse tissues. The target epitope, TEC-2, is a carbohydrate carried in mouse embryonal carcinoma cells by large glycoprotein-bound glycan. We report here the expression of TEC-2 epitope on human carcinoma-derived cell lines, HeLa and HS, and the properties of its carbohydrate carriers. Immunolabeling of Nonidet P-40 lysates of HeLa cells separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that TEC-2 antigens are polydispersed glycoconjugates of high molecular weight (mostly above 100,000). TEC-2 antigens detected by the two-site sandwich assay, in which the antigen is immobilized and detected with the same antibody, had a slightly higher molecular weight than those detected by the solid-phase assay. This suggests heterogeneity in the number of TEC-2 epitopes per carrier molecule. When the cells were lysed by Triton X-114 and the detergent and aqueous phases were separated by warming and centrifugation, most of the TEC-2 antigenic activity was found in the aqueous phase. TEC-2 antigens isolated by indirect precipitation from [3H]galactose-labeled HeLa cells were degraded by extensive pronase digestion or mild alkaline treatment to glycopeptides or oligosaccharides of low molecular weight. Thus, TEC-2 epitope in human HeLa cells is carried by carbohydrates of only several monosaccharide units. TEC-02 antibody was also found to bind to Tamm-Horsfall glycoprotein isolated from human urine and its binding was enhanced by desialylation. Combined data indicate that TEC-02 antibody recognizes the GalNAc beta 1----4Gal beta 1----4 structure which may be carried on different types of molecule, according to the site of their synthesis.
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PMID:Expression of mouse embryonic epitope TEC-2 on human carcinoma-derived cell lines and characterization of its glycoprotein carriers. 244 11

We have developed a rapid and direct method for determining urine nickel. The urine specimen is diluted (1 + 1) with 2.0% v/v nitric acid and 0.001% v/v Triton X-100 and absorbance measurements are made with Zeeman-effect graphite furnace atomic absorption. The method is sensitive enough to be used to evaluate "normal" subjects for baseline studies or to evaluate environmental or other nonoccupational exposure to nickel. The characteristic mass (pg/0.0044A.s) is 26 pg, which is comparable to that obtained for aqueous solutions. The observed absorbance is linear up to about 100 micrograms l-1, after which the calibration curve departs from linearity. Procedures are described to rigorously exclude nickel contamination. We evaluated precision and accuracy with a U.S. National Bureau of Standards urine reference material. SRM 2670, with an informational nickel value of 70 micrograms l-1, and with a multielement water reference material, SRM 1643b, with a certified nickel value of 49 ng g-1. Within- and among-run standard deviations for SRM 2670 were calculated to be 9.0 and 2.45 micrograms l-1, respectively, and 2.1 and 1.1 micrograms l-1 for SRM 1643b. The detection limit, calculated as 3 SD of a "low" concentration urine, is about 1.1 micrograms l-1. The proposed method was applied to the determination of nickel in urine of 258 workers in a magnet manufacturing plant, and the data obtained support the usefulness of urine nickel for biological monitoring.
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PMID:Determination of nickel in urine with graphite furnace AAS using Zeeman correction. 261 92

Biochemical analysis of the kinetics of assembly of two cytoplasmic plaque proteins of the desmosome, desmoplakins I (250,000 Mr) and II (215,000 Mr), in Madin-Darby canine kidney (MDCK) epithelial cells, demonstrated that these proteins exist in a soluble and insoluble pool, as defined by their extract ability in a Triton X-100 high salt buffer (CSK buffer). Upon cell-cell contact, there is a rapid increase in the capacity of the insoluble pool at the expense of the soluble pool; subsequently, the insoluble pool is stabilized, while proteins remaining in the soluble pool continue to be degraded rapidly (Pasdar, M., and W. J. Nelson. 1988. J. Cell Biol. 106:677-685). In this paper, we have sought to determine the spatial distribution of the soluble and insoluble pools of desmoplakins I and II, and their organization in the absence and presence of cell-cell contact by using differential extraction procedures and indirect immunofluorescence microscopy. In the absence of cell-cell contact, two morphologically and spatially distinct patterns of staining of desmoplakins I and II were observed: a pattern of discrete spots in the cytoplasm and perinuclear region, which is insoluble in CSK buffer; and a pattern of diffuse perinuclear staining, which is soluble in CSK buffer, but which is preserved when cells are fixed in 100% methanol at -20 degrees C. Upon cell-cell contact, in the absence or presence of protein synthesis, the punctate staining pattern of desmoplakins I and II is cleared rapidly and efficiently from the cytoplasm to the plasma membrane in areas of cell-cell contact (less than 180 min). The distribution of the diffuse perinuclear staining pattern remains relatively unchanged and becomes the principal form of desmoplakins I and II in the cytoplasm 180 min after induction of cell-cell contact. Thereafter, the relative intensity of staining of the diffuse pattern gradually diminishes and is completely absent 2-3 d after induction of cell-cell contact. Significantly, double immunofluorescence shows that during desmosome assembly on the plasma membrane both staining patterns coincide with a subpopulation of cytokeratin intermediate filaments. Taken together with the preceding biochemical analysis, we suggest that the assembly of desmoplakins I and II in MDCK epithelial cells is regulated at three discrete stages during the formation of desmosomes.
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PMID:Kinetics of desmosome assembly in Madin-Darby canine kidney epithelial cells: temporal and spatial regulation of desmoplakin organization and stabilization upon cell-cell contact. II. Morphological analysis. 327 50

We recently cloned a partial cDNA (35H) for a protein kinase C (PKC) binding protein from a rat kidney cDNA library and demonstrated that it is a PKC substrate in vitro (Chapline, C., Ramsay, K., Klauck, T., and Jaken, S. (1993) J. Biol. Chem. 268, 6858-6861). Additional library screening and 5' rapid amplification of cDNA ends were used to obtain the complete open reading frame. Amino acid sequence analysis, DNA sequence analysis, and Northern analysis indicate that 35H is a unique cDNA related to alpha-and beta-adducins. Antisera prepared to the 35H bacterial fusion protein recognized two polypeptides of 80 and 90 kDa on immunoblots of kidney homogenates and cultured renal proximal tubule epithelial cell extracts. The 35H-related proteins were similar to alpha- and beta-adducins in that they were preferentially recovered in the Triton X-100-insoluble (cytoskeletal, CSK) fraction of cell extracts and were predominantly localized to cell borders. Phorbol esters stimulated phosphorylation of CSK 35H proteins, thus emphasizing that sequences isolated according to PKC binding activity in vitro are also PKC substrates in vivo. The phosphorylated forms of the 35H proteins were preferentially recovered in the soluble fraction, thus demonstrating that phosphorylation regulates their CSK association and, thereby, their function in regulating cytoskeletal assemblies. We have isolated another PKC binding protein partial cDNA (clone 45) from a rat fibroblast library with substantial homology to alpha-adducin. Antisera raised against this expressed sequence recognized a protein of 120 kDa, the reported size of alpha-adducin, on immunoblots of renal proximal tubule epithelial cell extracts. A 120-kDa protein that cross-reacts with the clone 45 (alpha-adducin) antisera coprecipitated with 35H immunecomplexes, indicating that alpha-adducin associates with 35H proteins in vivo. Taken together, these results indicate that 35H is a new, widely expressed form of adducin capable of forming heterodimers with alpha-adducin. We propose naming this adducin homologue gamma-adducin.
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PMID:35H, a sequence isolated as a protein kinase C binding protein, is a novel member of the adducin family. 759 23

In this simple, quick procedure for determining copper in human serum and urine, the serum and urine specimens were analyzed directly after dilution with a solution of HNO3 and Triton X-100, 1 mL/L each. We calibrated with aqueous standards for quantitation in Zeeman background atomic absorption spectrometry. By modifying the drying and pyrolysis stages of the graphite furnace atomic absorption spectrometer, we reduced the analytical time to 30 s per determination. The within-run imprecision (CV) is 2.6% and 3.4% and the between-run imprecision is 0.9% and 2.5% for serum and urine copper at concentrations of 30.4 and 2.70 mumol/L, respectively. The accuracy of this fast method was verified by analyzing the National Institute of Standards and Technology Standard Reference Materials SRM 1598 bovine serum and SRM 2670 urine (agreement with certified values within 0.1 mumol/L for serum and within 0.02 mumol/L for urine), by analytical recovery studies (98% recovered in serum, 100% recovered in urine), and by comparison with our normal routine method. We also used the Quebec Interlaboratory Comparison Program to validate the analytical performance. From the precision and accuracy studies, we conclude that this fast-furnace program is a rapid, simple, and reliable method for determining copper in serum and urine.
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PMID:Rapid Zeeman atomic absorption determination of copper in serum and urine. 837 70

N-dodecylimidazole is a compound which acquires detergent properties under acidic conditions and might be useful in killing selectively cells in those regions of solid tumours which have a reduced extracellular pH (pHe). We have therefore studied the effects of N-dodecylimidazole against malignant cells in tissue culture. N-dodecylimidazole displayed pHe-dependent cytotoxicity against EMT-6 and MGH U1 cells; cell killing was dose dependent and was 100-fold greater at pHe 6.0 than pHe 7.0. Reduced toxicity of N-dodecylimidazole was observed at higher cell concentrations (> 10(6) cells ml-1), and only minor effects were observed against multicellular tumour spheroids. Potential mechanisms of action of N-dodecylimidazole include detergent-mediated lysis of the cell membrane at low pHe, and selective uptake into lysosomes where detergent activity leads to rupture of the lysosomal membrane and release of cytolytic enzymes. Inhibition of activity of cysteine proteases by the inhibitor E-64 did not protect cells against the toxicity of N-dodecylimidazole, suggesting that these lysosomal enzymes do not play a major role in the mechanism of action of this compound. Lysis of erythrocytes (which contain no lysosomes) was observed with low concentrations of N-dodecylimidazole. Dependence of cell lysis on cell concentration was similar to that observed for two other detergents that act on the plasma membrane, Triton X-100 and sodium dodecyl sulfate. We conclude that N-dodecylimidazole causes pHe dependent cell killing in two cultured tumour cell lines, and that its mechanism of action is probably due to acid mediated production of detergent activity which acts primarily on the cell plasma membrane.
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PMID:pH dependent cytotoxicity of N-dodecylimidazole: a compound that acquires detergent properties under acidic conditions. 842 83

B16 melanoma is characterized by high content of GM3 ganglioside, which has been recognized as a melanoma-associated antigen defined by specific monoclonal antibodies. We report now that GM3 is present predominantly (>90%) in the 1% Triton X-100-insoluble, low-density microvesicular fraction ("detergent-insoluble glycosphingolipid-enriched microdomain"; DIGEM) separated on sucrose density-gradient centrifugation. Associated with DIGEM, many signal transducer molecules such as c-Src, FAK, and the low-molecular-weight G-proteins Rho A and H-Ras were also found. Rho A and FAK were found in part, and PLC-beta2 and G alphas were found exclusively, in the high-density fraction. Immunoprecipitation of GM3 present in DIGEM by anti-GM3 antibody DH2, followed by Western blotting, revealed co-precipitation of Rho A and c-Src with GM3. These findings suggest (i) a specific organization of GM3 in close association with Rho A and c-Src within DIGEM at the melanoma cell surface; and (ii) such organizational units may be directly involved in signal transduction, in which glycosphingolipids receive signals which are subsequently transduced by associated transducer molecules.
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PMID:A close association of GM3 with c-Src and Rho in GM3-enriched microdomains at the B16 melanoma cell surface membrane: a preliminary note. 922 55

Mouse melanoma B16 cells are characterized by the predominant presence of ganglioside GM3 and adhere to lactosylceramide- or Gg3-coated plates through interaction of GM3 with lactosylceramide or Gg3, whereby not only adhesion but also spreading and enhancement of cell motility occur (Kojima, N., Hakomori, S. (1991) J. Biol. Chem. 266, 17552-17558). We now report that the adhesion process is based essentially on a glycosphingolipid-enriched microdomain (GEM) at the B16 cell surface, since >90% of GM3 present in the original cells is found in GEM, and GEM is also enriched in several signal transducer molecules, e.g. c-Src, Ras, Rho, and focal adhesion kinase (FAK). GEM was isolated as a low density membranous fraction by homogenization of B16 cells in lysis buffer under two different conditions (i.e. buffer containing 1% Triton X-100, or hypertonic sodium carbonate without detergent), followed by sucrose density gradient centrifugation. A close association of GM3 with c-Src, Rho, and FAK was indicated by co-immunoprecipitation of GM3 present in GEM by anti-GM3 monoclonal antibody DH2, followed by Western blotting with antibodies directed to these transducer molecules. The following data indicate that GEM is a structural and functional unit for initiation of GM3-dependent cell adhesion coupled with signal transduction. 1) Tyrosine phosphorylation in FAK was greatly enhanced in B16 cells adhered to Gg3-coated plates but was minimal in cells adhered to GM3-coated, GlcCer-coated, or noncoated plates. 2) GTP loading on Ras and Rho increased significantly when cells were adhered to Gg3-coated plates, compared with GM3-coated, GlcCer-coated, or noncoated plates. Since Ras and Rho are closely associated with GM3 in GEM, cell adhesion/stimulation through GM3 in GEM may induce activation of Ras and Rho through enhanced GTP binding.
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PMID:GM3-enriched microdomain involved in cell adhesion and signal transduction through carbohydrate-carbohydrate interaction in mouse melanoma B16 cells. 953 3

Reverse micelle formation is presented as a new strategy for improving the extraction of polar species with supercritical (SC) CO2. The addition of a reverse-micelleforming reagent prior to SFE accelerates the quantitative extraction of the analyte. The effect was used to develop a method for the determination of cholesterol in solid foods. The proposed method involves the addition of a microemulsion of a nonionic surfactant (Triton X-100) to the sample and dynamic extraction with SC-CO2 at 383 bar and 40 degrees C for 20-40 min. The method was validated using a certified reference material (NIST-SRM 1845) and was subsequently used to analyze low-cholesterol (whole bread, oat biscuits, orange biscuits) and high-cholesterol foods (semiskimmed and whole milk) with excellent results (RSD < 11% in all instances).
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PMID:Reverse micelle formation for acceleration of the supercritical fluid extraction of cholesterol from food samples. 960 48

Osteoclast activation is initiated by adhesion to the bone surface, followed by cytoskeletal rearrangement, the formation of the sealing zone, and a polarized ruffled membrane. This study shows that PYK2/CAKbeta/RAFTK, a cytoplasmic kinase related to the focal adhesion kinase, is highly expressed in rat osteoclasts in vivo. Using murine osteoclast-like cells (OCLs) or their mononuclear precursors (pOCs), generated in a coculture of bone marrow and osteoblastic MB1.8 cells, we show: (a) tyrosine phosphorylation of PYK2 upon ligation of beta3 integrins or adhesion of pOCs to serum, vitronectin, osteopontin, or fibronectin but not to laminin or collagen; (b) coimmunoprecipitation of PYK2 and c-Src from OCLs; (c) PYK2 binding to the SH2 domains of Src; (d) marked reduction in tyrosine phosphorylation and kinase activity of PYK2 in OCLs derived from Src (-/-) mice, which do not form actin rings and do not resorb bone; (e) PYK2 phosphorylation by exogeneous c-Src; (f) translocation of PYK2 to the Triton X-100 insoluble cytoskeletal fraction upon adhesion; (g) localization of PYK2 in podosomes and the ring-like structures in OCLs plated on glass and in the sealing zone in OCLs plated on bone; and (h) activation of PYK2, in the presence of MB1.8 cells, parallels the formation of sealing zones and pit resorption in vitro and is reduced by echistatin or calcitonin and cytochalasin D. Taken together, these findings suggest that Src-dependent tyrosine phosphorylation of PYK2 is involved in the adhesion-induced formation of the sealing zone, required for osteoclastic bone resorption.
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PMID:PYK2 in osteoclasts is an adhesion kinase, localized in the sealing zone, activated by ligation of alpha(v)beta3 integrin, and phosphorylated by src kinase. 972 56


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