Volume 48 (2002) No. 2
DNA Vaccine against Friend Erythroleukaemia Virus
P. ŠÍMA, M. ŠMAHEL, F. JELÍNEK, V. VONKA...........................................43
Institute of Haematology and Blood Transfusion, Department of Experimental Virology, Prague, Czech Republic
Corresponding author: Vladimír Vonka, Institute of Haematology and Blood Transfusion, Department of Experimental Virology, U Nemocnice 1, 128 20 Prague 2, Czech Republic. Tel.: +420 (02) 21977383; fax: +420 (02) 21977392; e-mail: vonka@uhkt.cz.
Abstract.
Full text. 43-44 45-46 47-48 49-50
Antiapoptotic Cytokine IL-3 + SCF + FLT3L Influence on Proliferation of Gamma-Irradiated AC133+/CD34+ Progenitor Cells
J. VÁVROVÁ1, D. VOKURKOVÁ2, M. MAREKOVÁ3, M. BLÁHA4, L. JEBAVÝ5, S. FILIP6..................51
1Institute of Radiobiology and Immunology, Purkyně Military Medical Academy, Hradec Králové, Czech Republic
2Institute of Clinical Immunology and Allergology, University Hospital, Hradec Králové, Czech Republic
3Department of Medical Biochemistry, Faculty of Medicine Hradec Králové, Charles University Prague, Czech Republic
4Department of Clinical Haematology, University Hospital, Hradec Králové, Czech Republic
5Department of Field Internal Medicine, Purkyně Military Medical Academy, Hradec Králové, Czech Republic
6Department of Oncology and Radiotherapy, University Hospital, Hradec Králové, Czech Republic
Corresponding author: Jiřina Vávrová, Institute of Radiobiology and Immunology, Purkyně Military Medical Academy, Třebešská 1575, 500 01 Hradec Králové, Czech Republic. Tel: + 420 (49) 5253214; fax: + 420 (49) 5513018; e-mail: vavrova@pmfhk.cz.
Abstract.
Full text. 51-52 53-54 55-57
Apoptosis Induction in Lymphoma Cells: Thiol Deprivation versus Thiol Excess
J. KOVÁŘ, H. ŠTÝBROVÁ, J. TRUKSA, K. SPĚVÁKOVÁ, T. VALENTA............................58
Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic.
Corresponding author: Jan Kovář, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Prague 4, Czech Republic. Tel.: +420 (2) 4752637; fax: +420 (2) 44471707; e-mail: kovar@biomed.cas.cz.
Abstract.
Full text. 58-59 60-61 62 63-64 65-66 67-68
Short Communication
An ATP-Dependent Step Is Required for the Translocation of Microinjected Precursor mRNA into Nuclear Speckles
V. KOPSKÝ1,2,3,+, J. VEČEŘOVÁ1,2,+, I. MELČÁK1,2,4, A. PLISS1,2, J. ŠTULÍK2, K. KOBERNA1,2, L. TOMŠÍKOVÁ1,2, I. RAŠKA1,2........................................................69
1Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of Czech Republic, and 2Laboratory of Gene Expression, 1st Medical Faculty, Charles University, Prague, Czech Republic
3Present address: Department of Animal Physiology and Developmental Biology, Faculty of Science, Charles University, Viničná 7, Prague 2, 128 44, Czech Republic
4Present address: Laboratory of Cell Biology, Howard Hughes Medical Institute, The Rockefeller University, 1230 York Avenue, New York, NY 10021-6399, USA
+ V. K and J. V. were equally contributing authors
Corresponding author: Ivan Raška, Department of Cell Biology, Institute of Experimental Medicine, Academy of Sciences of Czech Republic, Albertov 4, 128 00 Prague, Czech Republic, Tel: +420 (2) 24910315, Fax: +420 (2) 24917418, e-mail: iraska(zavináč)lf1.cuni.cz.
Abstract.
Full text. 69-70 71-72
Expression of Protein Tyrosine Kinase pp60v-src Variants in Dictyostelium discoideum
J. BRÁBEK, D. MOJŽITA, F. PŮTA......................................................73
Department of Physiology, Charles University, Prague, Czech Republic
Corresponding author: František Půta, Department of Physiology, Charles University, Viničná 7, 128 00 Praha 2, Czech Republic, Tel.: +420 (2) 2195 3147; Fax: +420 (2) 2195 3242; e:mail: dicty@mbox.cesnet.cz.
Abstract.
Full text. 73-74 75-76
Monoclonal Antibody Register
Monoclonal Antibody KN-01 against the Heavy Chain of Kinesin
E. DRÁBEROVÁ1, L. MACUREK1, V. RICHTEROVÁ1, K. J. BOHM2, P. DRÁBER1..............77
1Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
2Institute of Molecular Biotechnology, Deparment of Molecular Cytology and Electron Microscopy, Jena, Germany
Corresponding author: Pavel Dráber, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic,
Vídeňská 1083, 142 20 Prague 4, Czech Republic. Fax: +420 (2) 41062758; e-mail: paveldra@biomed.cas.cz
Full text: 77-79
DNA Vaccine against Friend Erythroleukaemia Virus
P. ŠÍMA, M. ŠMAHEL, F. JELÍNEK, V. VONKA
Plasmids carrying DNA copies of the gag and env genes of FV, which causes erythroleukaemia in susceptible mouse strains, were prepared. Expression of the gene clones was confirmed by indirect immunofluoroscence in cells transfected in vitro. Immunization experiments were performed in DBA/2 mice. Animals were injected with three doses of plasmid either intramuscularly (100 µg DNA per dose) or intradermally (1 µg DNA per dose); in the latter case, a gene gun was used. The FV type A or P was used as a challenge. The immunization with gag- and env-derived vaccines resulted in protective immunity in a high proportion of mice.
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J. VÁVROVÁ, D. VOKURKOVÁ, M. MAREKOVÁ, M. BLÁHA, L. JEBAVÝ, S. FILIP
Recovery from radiation-induced bone marrow aplasia depends on appropriate cytokine support. The aim of our work was to find a cytokine combination allowing in vitro gamma-irradiated (2.5 Gy) CD34+/AC133+ haematopoietic stem cells to evade radiation-induced apoptosis and to enhance damage reparation, which should enable proliferation and ex vivo expansion of cells. Cells were isolated using separation in a Cobe separator followed by immunomagnetic selection by antibody against the AC133 antigen. Thus isolated cells were 80% AC133+/CD34+ and 10% of them expressed the CD33+ antigen. Ten thousand of AC133+ cells formed 1146 CFU-GM and 304 BFU-E. We proved a high expansion efficiency of cytokine combination SCF + IL3 + FLT3L in comparison with the combination SCF + IL-3 + IL-11 in both, non-irradiated cells and cells irradiated with a dose of 2.5 Gy.
The D0 value for AC133+ cells was determined by the clonogeneity test. The D0 value for CFU-GM was estimated to be 1.08 Gy and for BFU-E 0.95 Gy.
The results of DNA analysis showed that the majority of isolated AC133+ cells were in G0/G1 phase of the cell cycle. We proved that the dose of 2.5 Gy induced massive apoptosis (80%) of these cells without progression through the cell cycle, which indicates interphase cell death. Under the influence of cytokine combination (SCF + IL3 + FLT3L), the surviving 20% of cells entered the cell cycle and, similarly to non-irradiated control cells, on 7th day 35% of cells were in S phase.
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J. KOVÁŘ, H. ŠTÝBROVÁ, J. TRUKSA, K. SPĚVÁKOVÁ, T. VALENTA
We studied the effects of thiol availability on apoptosis induction in B-cell lymphoma 38C13, T-cell lymphoma EL4, and also other cells. Compounds with a free SH group are required for survival and growth of 38C13 cells but not of EL4 cells. Thiol deprivation (2-mercaptoethanol concentrations about 0.3 microM and lower) induced apoptosis in 38C13 cells. On the other hand, thiol excess (2-mercaptoethanol concentrations higher than 300 microM) induced apoptosis in 38C13 cells and EL4 cells as well as in other cells (e.g. Raji, HeLa). L-cystine and non-thiol antioxidant ascorbic acid were unable to support survival of 38C13 cells. Ascorbic acid induced cell death at concentrations higher than 600 microM. Thiol cross-linking compound diamide (100 microM and higher) abrogated the survival-supporting effect of 2-mercaptoethanol (50 microM). Apoptosis induction by thiol deprivation and by thiol excess was not directly related to a specific significant change in the p53 level or p53 activation. Apoptosis induction by thiol excess was associated with a certain decrease in the Bcl-2 level while the Bax level did not change. We conclude that both thiol deprivation and thiol excess can induce apoptosis in lymphoma cells. Apoptosis induction by thiol deprivation is specifically related to the presence of a free SH group. However, apoptosis induction by thiol excess does not seem to be specifically related to the presence of a free SH group. It probably results from the excess of a reductant. Apoptotic control protein p53 does not seem to play a significant role in apoptosis induction either by thiol deprivation or by thiol excess.
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An ATP-Dependent Step Is Required for the Translocation of Microinjected Precursor mRNA into Nuclear Speckles
V. KOPSKÝ, J. VEČEŘOVÁ, I. MELČÁK, A. PLISS, J. ŠTULÍK, K. KOBERNA, L. TOMŠÍKOVÁ,
I. RAŠKA
Nuclear speckles (speckles) represent a distinct nuclear compartment within the interchromatin space and are enriched in splicing factors. In a previous study (Melčák et al., 2001), it has been shown that the pre-spliceosomal assembly on microinjected splicing-competent precursor mRNA takes place in the speckles, and it has been suggested that the targeting of RNA into speckles consists of two interdependent steps, namely the movement of RNA towards the speckles, probably as a diffusion process, followed by the energy-dependent translocation of RNA into the speckles. In the present study, we confirm the existence of these two steps and show that the translocation is ATP dependent.
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J. BRÁBEK, D. MOJŽITA, F. PŮTA
We achieved production of v?Src of the low-oncogenic PRC and its variant proviral structure H19 in Dictyostelium discoideum, an emerging host system suitable for synthesis of heterologous proteins. To accomplish their expression, the first six codons of the N?terminus of v-src had to be changed according to the D. discoideum codon preference. Alternatively, N-terminal fusions of 6xHis-tag or GFP were sufficient to overcome the incompatibility in codon usage. D. discoideum-expressed v-Src kinases of the expected molecular weight were recognized by Src-specific antibodies; GFP-PRC was distributed uniformly in the cytosol. In contrast to other lower eukaryotes, where the accumulation of v-Src leads to growth inhibition, D. discoideum cells silenced the kinase activity of PRC-derived v-Src and showed no developmental or growth defects.
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