Volume 44 (1998) No. 1
Cyclosporin A-Induced Autoimmunity: the Result of Defective de novo T-Cell Development
J. G. M. C. DAMOISEAUX, P. J. C. VAN BREDA VRIESMAN ........................1
Department of Immunology, Maastricht University, Maastricht, The Netherlands
Corresponding author: J. G. M. C. Damoiseaux, Department of Immunology, Maastricht University, P.O. Box 616, 6200 MD Maastricht, The Netherlands. Tel. +31-43-3881433; Fax +31-43-3671016.
A Structure-Activity Study of a C-Terminal Endothelin Analogue
E. CASSANO1, C. GALOPPINI1, L. GIUSTI2, M. HAMDAN3, M. MACCHIA4, M.R. MAZZONI2, E. MENCHINI4, S. PEGORARO1, P. ROVERO1 .................................................11
1Istituto di Mutagenesi e Differenziamento, CNR, Laboratorio Sintesi Peptidica, Pisa, Italy
2Istituto Policattedra di Discipline Biologiche, Universit¸ di Pisa, Pisa, Italy
3GlaxoWellcome Medicine Research Center, Verona, Italy
4Dipartimento di Scienze Farmaceutiche, Universit¸ di Pisa, Pisa, Italy
Corresponding author: Paolo Rovero, Istituto di Mutagenesi e Differenziamento, CNR, Laboratorio Sintesi Peptidica, via Svezia 2A, 56124 Pisa, Italy. Tel. +/39/50/574161; Fax +/39/50/576661; e-mail firstname.lastname@example.org
Saccharide-Binding Properties of Boar AQN Spermadhesins and DQH Sperm Surface Protein
M. TICHÁ1, M. KRAUS2, D. ÈECHOVÁ2 , V. JONÁKOVÁ2 ............................15
1Department of Biochemistry, Charles University, Prague, Czech Republic
2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Corresponding author: Vìra Jonáková, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Flemingovo nám. 2, 166 37 Prague 6, Czech Republic. Fax: 420 2 24310955, Tel: 420 2 20183347.
Seroprevalence of Human T Cell Leukaemia/Lymphoma Virus Type I (HTLV-I) in Pregnant Women, Patients Attending Venereological Outpatient Services and Intravenous Drug Users from Slovenia
M. POLJAK1, J. BEDNARIK1, K. REDNAK1, K. SEME1, L. KRISTANÈIÈ1, B. ÈELAN-LUCU2
1Slovenian AIDS Reference Center, Institute of Microbiology and Immunology, Medical Faculty, Ljubljana, Slovenia
2Primary Health Care Center, Ljubljana, Slovenia
Corresponding author: Mario Poljak, Slovenian AIDS Reference Center, Institute of Microbiology and Immunology, Medical Faculty of Ljubljana, Zaloška 4, 1105 Ljubljana, Slovenia. Tel. +(386 61) 1403042; Fax +(386 61) 302895; e-mail email@example.com.
Fine Specifity of Anti-Lex Monoclonal Antibody TEC-01
J. BOHATA1, F. ŠMÍD1, P. DRÁBER2 .....................................................27
11st Department of Medicine, 1st Medical Faculty, Charles University, Prague, Czech Republic 2Department of Mammalian Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Corresponding author: Petr Dráber, Department of Mammalian Gene Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague 4, Czech Republic.
Monoclonal Antibody Register
Novel Monoclonal Antibodies TU-08 and TU-16 Specific for Tubulin Subunits
E. DRÁBEROVÁ, P. DRÁBER
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic Corresponding author: Pavel Dráber, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague 4, Czech Republic. Fax (420-2) 475 2632; e-mail firstname.lastname@example.org.
Microtubules are dynamic cytoskeletal structures essential for a wide variety of cellular functions, including organization of cytoplasm, cell motility and cell division. The major component of microtubules is tubulin, an ðÐ-heterodimer protein with the molecular weight of each subunit approximately 50 kDa (Dustin, 1984). Each of the tubulin subunits is encoded by a family of tubulin genes (Joshi and Cleveland, 1990). Together with post-translational modifications this causes a high charge heterogeneity of the tubulin molecule (Linhartová et al., 1992). Monoclonal antibodies against ðÐ-tubulin heterodimers were used to study the dynamics of microtubules, post-translational modifications of tubulin subunits, orientation of tubulin dimers in microtubule wall and tissue distribution of the products of individual tubulin genes (Andreu and de Pereda, 1993). Monoclonal antibodies revealed a differential subcellular localization of tubulin epitopes (Nováková et al., 1997).
Description of the antibodies TU-08 and TU-16
Production. A hybridoma cell line producing the TU-08 was obtained after immunization of BALB/c mouse with porcine brain microtubule protein MTP-1 (Shelanski et al., 1973) and fusion with Sp2/0 myeloma cells. A hybridoma cell line producing the TU-16 was obtained after immunization of B10.A mouse with bovine brain microtubule protein MTP-2 (Shelanski et al., 1973) and fusion with Ag8 myeloma cells. The details of fusion, screening by ELISA, cloning and production of ascitic fluids have been described previously (Viklický et al., 1982; Dráber et al., 1988). Specificity. Immunoblotting analysis showed that TU-08 reacted with both ð- and Ð-tubulin subunits from porcine brain (stronger reaction with the ð-subunit), while TU-16 reacted only with the ð-tubulin subunit (Fig. 1). On PtK2 cells the antibodies recognized interphase microtubules and midbodies (Fig. 2), as well as mitotic spindles (not shown). The antibodies also reacted with taxol-stabilized unfixed microtubules (Dráber et al., 1989) of 3T3 cells. In vinblastine-treated cells the antibodies reacted with tubulin paracrystals (not shown). The antibodies reacted with tubulins of various species ranging from human to plants. Properties. Antibodies TU-08 (IgM) and TU-16 (IgM) can bind tubulin under denaturing and non-denaturing conditions and can therefore be used for detection of tubulins of various species by ELISA, immunofluorescence, immunoblotting and immunoprecipitatiton. Acknowledgements This work was supported by grant EU 1450 from the Ministry of Education of the Czech Republic.
Andreu, J. M., de Pereda, J. M. (1993) Site-directed antibodies to tubulin. Cell Motil. Cytoskel. 26, 1-6. Dráber, P., Lagunowich, L. A., Dráberová, E., Viklický, V., Damjanov, I. (1988) Heterogeneity of tubulin epitopes in mouse fetal tissues. Histochemistry 89, 485-492. Dráber, P., Dráberová, E., Linhartová, I., Viklický, V. (1989) Differences in the exposure of C- and N-terminal tubulin domains in cytoplasmic microtubules detected with domain-specific monoclonal antibodies. J. Cell Sci. 92, 519-528. Dráberová, E., Dráber, P. (1993) A microtubule-interacting protein involved in coalignment of vimentin intermediate filaments with microtubules. J. Cell Sci. 106, 1263-1273. Dustin, P. (1984) Microtubules. 2nd ed.. Springer-Verlag, Berlin. Joshi, H. C., Cleveland, D. W. (1990) Diversity among tubulin subunits: toward what functional end? Cell Motil. Cytoskel. 16, 159-163. Linhartová, I., Dráber, P., Dráberová, E., Viklický, V. (1992) Immunological discrimination of Ð-tubulin isoforms in developing mouse brain. Posttranslational modification of non-class III Ð-tubulins. Biochem. J. 288, 919-924. Nováková, M., Riederer, B. M., Viklický, V., Dráber, P. (1997) Distinct subcellular localization of Ð-tubulin epitopes in the adult mouse. Histochem. Cell Biol. 107, 337-344. Shelanski, M. L., Gaskin, F., Cantor, C. R. (1973) Microtubule assembly in the absence of added nucleotides. Proc. Natl. Acad. Sci. USA. 70, 765-768. Viklický, V., Dráber, P., Hašek, J., Bártek, J. (1982) Production and characterization of a monoclonal antitubulin antibody. Cell Biol. Int. Rep. 6, 725-731.
Fig. 1. Specificity of antibodies TU-08 and TU-16 as determined by immunoblotting. An immunoblot of the total extract of porcine brain with anti-tubulin monoclonal antibodies is shown. The immunoblotting detection of tubulin including development of blots with alkaline-labelled antibodies has been done as described (Dráber et al., 1988). Lane 1, Amido Black staining of proteins transferred to nitrocellulose. Lanes 2--5, immunoreactivity with antibodies TU-01, TU-06, TU-08 and TU-16. Antibodies TU-01 and TU-06 (Dráber et al., 1989) were used as markers of ð-tubulin and Ð-tubulin, respectively. Bars on the left margin indicate position, from top to bottom, of specific molecular weight markers (205 kDa, 116 kDa, 97.4 kDa, 66 kDa, 45 kDa, 29 kDa).
Fig. 2. Specificity of TU-16 antibody as determined by indirect immunofluorescence on PtK2 cells extracted with Triton X-100 and fixed with formaldehyde (Dráberová and Dráber, 1993). The cells were stained as described. Bar, 20 Þm.@LH 6
Monoclonal Antibody Register
A New Monoclonal Antibody Against p34CDC28
J. PALEÈEK1, P. VAVØIÈKOVÁ2, H. JIØINCOVÁ1, J. HAŠEK2
1Department of Developmental Biology, Charles University, Prague, Czech Republic
2Institute of Microbiology, Academy of Sciences of the Czech Republic, Prague, Czech Republic Corresponding author: Jiøí Hašek, Institute of Microbiology, Academy of Sciences of the Czech Republic, Vídeòská 1083, 142 20 Prague 4, Czech Republic. Fax (420-2)-4722257; e-mail email@example.com.
Biochemical and genetic analysis of an activity called maturation-promoting factor capable of causing frog extracts to enter the M phase (Masui and Markert, 1971) revealed that it is composed of 34 kDa kinase. The homologues of this kinase, Cdc28 and cdc2, from two distant yeasts (Saccharomyces cerevisiae and Schizosaccharomyces pombe, respectively) had previously been shown through genetic analysis to have key roles in regulating cell division (Hartwell, 1974; Nurse et al., 1976). To obtain a useful tool for elucidation of behaviour of the Cdc28 kinase during the cell cycle in various mutants, we have generated a new Cdc28-specific monoclonal antibody CHJ-28.
Description of the new monoclonal antibody, CHJ-28
Production. BALBc mice were immunized subcutaneously with a recombinant protein Cdc28 produced in E. coli (the plasmid containing the CDC28 coding region fused to the promoter region of gene 10 of the bacteriophage T7 was kindly provided by K. Nasmyth, Institute of Molecular Pathology, Vienna, Austria) blotted onto a strip of nitrocellulose membrane. Myeloma cell line Sp2/0-Ag14 was used to establish hybridomas according to the procedure described previously (Harlow and Lane, 1988). Production of antibodies was tested by ELISA and immunoblotting. Specificity. A hybridoma cell line producing a specific antibody CHJ-28 against S. cerevisiae protein Cdc28 expressed in E. coli was established after screening the produced antibodies by ELISA against inclusion bodies that contained recombinant Cdc28p using the lysate of E. coli with an empty plasmid as a negative control. The specificity to recombinant Cdc28p was confirmed by immunoblotting. In S. cerevisiae protein extract the antibody reacted with a 34 kDa protein band corresponding to Cdc28p (Fig. 1). Besides this protein, the antibody weakly reacted with a protein at position of approx. 66 kDa. The antibody specifically reacts with the Cdc28p precipitated with Suc1-beads (data not shown). Properties. CHJ-28 (IgM) binds either to native or denatured epitope of Cdc28p. For immunoblotting the antibody (crude ascitic fluid) can be diluted up to 1:106. The antibody precipitates Cdc28p from the native lysates of S. cerevisiae (Fig. 2) but the immunoprecipitate does not form any H1-histone kinase activity. This indicates that the antibody can not be used for the immunoprecipitation of the kinase in cell cycle studies.
Harlow, E., Lane, D. (1988) Laboratory Manual of Antibodies. Cold Spring Harbor Laboratory Press, New York. Hartwell, L. H. (1974) Saccharomyces cerevisiae cell cycle. Bacteriol. Rev. 38, 164-198. Masui, Y., Markert, C. (1971) Cytoplasmic control of nuclear behaviour during meiotic maturation of frog oocytes. J. Exp. Zool. 177, 129-146. Nurse, P., Thuriaux, P., Nasmyth, K. (1976) Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe. Mol. Gen. Genet. 146, 167-178.
Cyclosporin A-Induced Autoimmunity: the Result of Defective de novo T-Cell Development
J. G. M. C. DAMOISEAUX, P. J. C. VAN BREDA VRIESMAN
Cyclosporin A-induced autoimmunity (CsA-AI) is an autoimmune disease, caused by the combinatory treatment with irradiation and cyclosporin A (CsA). CsA-AI is the result of defective T-cell maturation leading to disturbed T-cell balances in the periphery. Increases in Th1 cells and reduction of autoregulatory cells eventually enables the enumerated autoreactive CD4 and CD8 T cells to disturb the homeostasis in the target organs. In unravelling the effect of CsA on T-cell maturation and the role of T cells in CsA-AI many pieces have been put in their places; nevertheless, some remain the topic of debate. The identity of the autoantigen(s) remains elusive, the working mechanism of the autoregulatory cells still is to be determined, and the interplay between CD4 and CD8 T-cell subsets in relation to type-1 and type-2 responses is a matter of interest. In spite of all these unknowns, the CsA-AI autoimmune model is, in contrast to many autoimmune models induced by immunization with a foreign protein in adjuvant, an interesting physiological model based on defective T-cell development including aberrant selection in the thymus and disturbed T-cell balances in the periphery.
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