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  • 1
    Electronic Resource
    Electronic Resource
    The use of Fluoresceincadaverine for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells (1997)
    Springer
    Journal of molecular histology 29 (1997), S. 593-606 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The use of Fluoresceincadaverine as a primary amine donor for detecting the endogenous substrates for active transglutaminase in living cells was studied. Fluoresceincadaverine was found to be suitable for labelling cells in culture as it did not induce cytotoxicity when used at 0.5 mSmD in culture media and diffused throughout the cell. After appropriate fixation using methanol, Fluoresceincadaverine-labelled cells were observed by direct fluorescence microscopy, allowing visualization of the substrates for active transglutaminase. Simultaneous detection of trans glutaminase and of Fluoresceincadaverine incorporated into proteins strongly suggested that cytosolic transglutaminase was inactive in these living cells. However, transglutaminase co- distributed with Fluoresceincadaverine-labelled structures, which resembled a lattice. Fluoresceincadaverine-labelled proteins detected by Western blotting using an anti-Fluorescein antibody showed that, in living cells, the major transglutaminase substrate migrated at an apparent molecular weight of 220 kDa, as does fibronectin. Fibronectin was found to co-distribute with Fluoresceincadaverine-labelled lattice. This confirmed that these lattice structures were extracellular and, therefore, that transglutaminase is in an active form in this compartment. This opportunity to perform morphological and biochemical analyses in the search for transglutaminase substrates in living cells should help in determining the specific function of transglutaminases in a particular cell type as well as in universal cellular events, such as apoptosis or cell growth.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026484227851
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  • 2
    Electronic Resource
    Electronic Resource
    Subculture of rabbit articular chondrocytes within a collagen gel: growth and analysis of differentiation (1988)
    Springer
    Cytotechnology 1 (1988), S. 123-132 
    Details
    ISSN: 1573-0778
    Keywords: collagen gel culture ; rabbit articular chondrocytes ; flow cytometry ; ultroser G
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Primary cultures of rabbit articular chondrocytes have been subcultured within three-dimensional (3D) collagen gels. Under these conditions, the cells remained viable and divided, but with a lower proliferation rate than that observed in control monolayer cultures. Flow cytometric analysis of progression of the cells into the cell cycle has confirmed and extended these findings. Also the cellular volume was decreased in 3D-culture, being in the same range as thein vivo size of cartilage cells. Specific staining for proteoglycans and type II collagen immunolocalization on sections of gels showed the expression of differentiated phenotypes and revealed the accumulation of these matrix components in the immediate surroundings of the cells. The use of Ultroser G (a serum substitute) improved the conditions for 3D- culture of rabbit articular chondrocytes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00146812
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  • 3
    Electronic Resource
    Electronic Resource
    Cytotoxicity of D-penicillamine in association with several heavy metals against cultured rabbit articular chondrocytes (1988)
    Springer
    Cytotechnology 1 (1988), S. 139-144 
    Details
    ISSN: 1573-0778
    Keywords: catalase ; cultured chondrocyte ; cytotoxicity ; heavy metals ; D-penicillamine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The potentiation or antagonistic effects of Cu, Hg, Pb and Cd salts in the presence of a long-acting anti-rheumatic drug, D-penicillamine (D.P.) were studied on cultured chondrocytes. CuSO4 (10−4M), HgCl2 (10−5M), Pb(CH3COO)2 (10−3M) and D.P. (10−3M) when used alone caused a small decrease in cell proliferation. The addition of D.P. with Cu, Hg or Pb salts resulted in a marked increase in the extent of growth inhibition. In contrast, CdCl2 (10−5M) produced an important growth inhibitory effect, and D.P. antagonized CdCl2 action. The CuSO4 D.P. toxicity was probably due to production of H2O2 in situ. To verify this hypothesis, catalase, responsible for H2O2 metabolism was used, and was found to partially reverse the inhibitory effect of CuSO4-D.P.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00146814
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  • 4
    Electronic Resource
    Electronic Resource
    Transfection of rabbit articular chondrocytes by the calcium phosphate procedure: Improvement of efficiency and reproducibility (1995)
    Springer
    Cytotechnology 19 (1995), S. 89-94 
    Details
    ISSN: 1573-0778
    Keywords: Chondrocytes ; transfection ; calcium-phosphate-DNA-coprecipitate ; hyaluronidase ; β-galactosidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The understanding of cartilage disorders relies on the possibility of studying mechanisms which monitor the regulation of matrix protein genes through introducing efficiently and in a reproducible manner these genes, or their regulatory regions, into cells. To this end, we attempted to improve the transfection efficiency of rabbit articular chondrocytes by the calcium phosphate procedure. Transfection efficiencies were assessed by measuring the expression of the Lac Z reporter gene encoding β-galactosidase using anin situ staining (X-gal staining) and an enzymatic assay (β-galactosidase assay). Results revealed that addition of 4 U ml−1 of hyaluronidase before and during transfection increases by 2 to 4-fold the transfection efficiency of rabbit articular chondrocytes. Furthermore, we demonstrated that the use of a “giant” calcium phosphate DNA coprecipitate gives a higher transfection efficiency and much more reproducible results than those obtained with classical small volumes of precipitates.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00749759
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  • 5
    Electronic Resource
    Electronic Resource
    In vitro pharmacotoxicology (1989)
    Springer
    Cytotechnology 2 (1989), S. 72-77 
    Details
    ISSN: 1573-0778
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02279730
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  • 6
    Electronic Resource
    Electronic Resource
    Detection and Characterization, using Fluoresceincadaverine, of Amine Acceptor Protein Substrates Accessible to Active Transglutaminase Expressed by Rabbit Articular Chondrocytes (1998)
    Springer
    Journal of molecular histology 30 (1998), S. 499-508 
    Details
    ISSN: 1573-6865
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The purpose of this study was to investigate the implication of transglutaminases in the biology of articular chondrocytes. Transglutaminase activity measurements performed on cell lysates showed that a transglutaminase was present in chondrocytes in primary culture and that it was strongly activated by limited proteolysis. In chondrocytes dedifferentiated by subculture or retinoic acid treatment, this transglutaminase appeared to be downregulated, while type II transglutaminase expression was induced. However, protein levels, mRNA steady-state levels or transglutaminase activity in whole-cell lysates do not necessarily reflect the activity present in living cells, as it is strongly regulated. Therefore, Fluoresceincadaverine, a fluorescent polyamine, was used for detecting amine acceptor protein substrates accessible to active transglutaminase in living cells. After incubation of chondrocytes with Fluoresceincadaverine, dedifferentiated cells exhibited an extracellula r labelling, while chondrocytes in primary culture did not, unless thrombin was added to the culture medium. In contrast, Fluoresceincadaverine labelling was not detected in the cytosol, although the transglutaminases were also partly cytosolic. By confocal microscopy and Western blot analysis of labelled cells in culture, fibronectin was shown to be the main substrate for both transglutaminases. The transglutaminases present in articular chondrocytes may, therefore, contribute to the organization and the stabilization of their extracellular matrix.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1003251705197
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  • 7
    Electronic Resource
    Electronic Resource
    Flow cytometric assessment of cell viability: a multifaceted analysis (1989)
    Springer
    Cytotechnology 2 (1989), S. 27-37 
    Details
    ISSN: 1573-0778
    Keywords: cell viability ; flow cytometry ; light absorption ; light scattering ; exclusion dyes ; supravital dyes ; fluorescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Flow cytometry offers the possibility to simultaneously analyze, on a cell by cell basis, different parameters related to cell viability i.e. cell size, morphology and incorporation of dyes. Different types of analysis: light absorption of unstained/stained cells, forward angle light scattering (FALS), right angle light scattering (RALS) or both, cell fluorescence based on dye retention or dye exclusion (due to erythrosin B, ethidium bromide, fluorescein diacetate, rhodamine 123) were tested and compared, with the classical Trypan blue exclusion test, for their effectiveness in the determination of cell viability. Two types of cells in monolayer cultures (L929, SIRC) and a freshly isolated suspension of mouse splenocytes were used. For each dye, the optimal dose, incubation time and conditions for analysis were determined. Viability indications by different techniques for the three type of cell line and their reliability as compared with Trypan blue were analyzed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00365412
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  • 8
    Electronic Resource
    Electronic Resource
    Cartilage and bone culture (1989)
    Springer
    Cytotechnology 2 (1989), S. 54-56 
    Details
    ISSN: 1573-0778
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF02279725
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  • 9
    Electronic Resource
    Electronic Resource
    Effects of retinoic acid on the growth of cultured rabbit articular chondrocytes: Relation with alkaline phosphatase activity and beta receptor (1989)
    Springer
    Cytotechnology 2 (1989), S. 233-237 
    Details
    ISSN: 1573-0778
    Keywords: rabbit articular chondrocytes ; retinoic acid ; β-blocker ; alkaline phosphatase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract The effects of retinoic acid (RA) on rabbit articular cartilage cells were studied for concentrations ranging from 5.10-5 M to 10-7 M; the treatment with RA over three days resulted in dose dependent inhibition of chondrocyte proliferation between 5.10-5 and 10-5 M with persistence of the inhibitory effect until 10-6 M. RA until 10-7 M induced a slight, but significant, enhancement of cell proliferation. This growth stimulating effect seems to be related to the Beta receptor system because Beta blockers, such as sotalol and DL propranolol, were able to suppress the stimulating action of agonist type isoprenaline. The activity of alkaline phosphatase (AP) was also determined. The highest dose of RA (5.10-5 M) induced an increase (x 3) of AP activity, and 10-7 M RA decreased it (x 0.4).
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00133248
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  • 10
    Electronic Resource
    Electronic Resource
    Cross contamination associated with the use of multiwell culture plates for cytotoxicity assessment of volatile chemicals (1991)
    Springer
    Cytotechnology 6 (1991), S. 79-82 
    Details
    ISSN: 1573-0778
    Keywords: Cytotoxicity multi-well plate ; evaporation ; volatile chemical
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract In vitro toxicity testing can involve technical problems due to the evaporation of volatile test chemicals. The cytotoxicity of two volatile chemicals (butanol and ethanol has been assessed with neutral red assay in conventional microtiter plates. The non volatile DMSO chemical is used as a negative control. Under these conditions, an important cross contamination between test concentration groups has been observed. This affects cytotoxicity estimation which is overestimated. This cross contamination is prevented when special plates containing removalbe bars are used.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00353705
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