Volume 47 (2001) No. 6
Genetic Variation in the MHC II Promoter: Lessons for Regulation and for Comparative Genomics
N. A. MITCHISON............................................183
Windeyer Institute of University College London, London, U.K.
Corresponding author: N. Avrion Mitchison, Windeyer Institute, University College London, 46 Cleveland Street, W1T 4JF, U.K.
Abstract.
Full text. 183-186
Articles
Expression of Human Erythropoietin Gene in the Mammary Gland of a Transgenic Mouse
T. MIKUŠ1,2, P. MALÝ2, M. POPLŠTEIN1, V. LANDA2, P. TREFIL1, J. LIDICKÝ1................................187
1Biopharm Research Institute of Biopharmacy and Veterinary Drugs, Inc., Pohoří-Chotouň, Jílové u Prahy, Czech Republic
2Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Czech Republic
Corresponding author: Tomáš Mikuš, Biopharm Research Institute of Biopharmacy and Veterinary Drugs, Inc., Pohoří-Chotouň, 254 46 Jílové u Prahy, Czech Republic
Abstract.
Full text. 187-188 189 190 191 192-193 194-195
Allosteric Modulation of GABAA Receptor by Somatostatin Is Altered undeer Stress in Rat Brainstem
F. CHIGR1,2, S. B. M'HAMED2, M. NAJIMI1.................................196
1Unité Génie Biologique, Faculty of Sciences & Techniques, Beni-Mellal, Morocco
2Unité Neurosciences du Comportement, Départment de Biologie, Faculté des Sciences Semlalia, Marrakesh, Morocco
Corresponding author: Fatiha Chigr, Unité Génie Biologique, Faculty of Sciences & Techniques, P.O.Box: 523, 23000 Beni-Mellal, Morocco. Tel: 212-23485112/212-23485122; fax: 212-23485201; e-mail: mnajimi1@fstbm.ac.ma.
Abstract.
Full text. 196-197 198-199
Cells of Porcine Epidermis and Corneal Epithelium Are Not Recognized by Human Natural Anti-alfa-galactoside IgG
E. HRDLIČKOVÁ-CELA1, 2, K. SMETANA, Jr.1, 3, J. PLZÁK1, 4, Z. HOLÍKOVÁ1, 3, 5, S. ANDRÉ6, M. HŘEBÍČEK7, K. HODAŇOVÁ7, B. DVOŘÁNKOVÁ2, 8, J. MOTLÍK3, 9, H.-J. GABIUS6...............200
1Charles University, 1st Faculty of Medicine, Institute of Anatomy, Prague, Czech Republic
2Charles University, 1st Faculty of Medicine, Department of Ophthalmology, Prague, Czech Republic
3Research Center for Cell Therapy and Tissue Repair, Prague, Czech Republic
4Charles University, 1st Faculty of Medicine, Department of Otorhinolaryngology, Head and Neck Surgery, Prague, Czech Republic
5Charles University, 2nd Faculty of Medicine, Department of Dermatology, Prague, Czech Republic
6Ludwig-Maximilians-University, Faculty of Veterinary Medicine, Institute of Physiological Chemistry, Munich, Germany
7Charles University, 1st Faculty of Medicine, Institute of Inherited Metabolic Disorders, Prague, Czech Republic
8Charles University, 3rd Faculty of Medicine, Department of Burn Surgery, Prague, Czech Republic
9Institute of Animal Physiology and Genetics, Academy of Sciences of the Czech Republic, Liběchov, Czech Republic
Corresponding author: Karel Smetana, Jr., Charles University, 1st Faculty of Medicine, Institute of Anatomy, U Nemocnice 3, 128 00 Prague 2, Czech Republic. E-mail: ksmet(zavináč)lf1.cuni.cz.
Abstract.
Full text. 200-201 202 203 204-205
Detection of Minimal Bone Marrow Infiltration in Patients with Localized and Metastatic Ewing Sarcoma Using RT-PCR
D. SUMERAUER, A. VÍTEK, H. KUČEROVÁ, R. KODET1, J. HOUSKOVÁ2, J. BEDRNÍČEK, T. ECKSCHLAGER ..........................................................206
Department of Paediatric Oncology, 1Pathology and 2Haematology, 2nd Medical School, Charles University, Prague, Czech Republic
Corresponding author: Tomáš Eckschlager, Department of Paediatric Oncology, V Úvalu 84, University Hospital Motol, 150 06 Prague 5, Czech Republic.
Abstract.
Full text. 206-208 209-210
Monoclonal Antibody Register
New Monoclonal Antibodies Recognizing the p53 Tumour Suppressor Protein Homologue p73
P. ČEŠKOVÁ1, R. NENUTIL2, S. BRAY3, M. SVITÁKOVÁ1, S. BABČANOVÁ1, S. ULDRIJAN1,
B. VOJTĚŠEK1...................................................................211
1Department of Experimental Oncology, Masaryk Memorial Cancer Institute, Brno, Czech Republic
2Department of Pathology, Faculty Woman´s Hospital Brno, Brno, Czech Republic
3Department of Molecular and Cellular Pathology, Ninewells Medical School, The University of Dundee, Scotland, UK
Corresponding author: Bořivoj Vojtěšek, Department of Experimental Oncology, Masaryk Memorial Cancer Institute,
Žlutý kopec 7, 656 53 Brno, Czech Republic. Tel.: +420 (5) 43133303; Fax: +420 (5) 43211696; e-mail: vojtesek@mou.cz.
Abstract.
Full text. 211-212 213-214
New Monoclonal Antibodies Recognizing the Adaptor Protein LAT
P. TOLAR, M. TŮMOVÁ, P. DRÁBER.............................................215
Department of Mammalian Genes Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Prague, Czech Republic
Corresponding author: Petr Dráber, Department of Mammalian Genes Expression, Institute of Molecular Genetics, Academy of Sciences of the Czech Republic, Vídeňská 1083, 142 20 Praha, Czech Republic. Fax: (420-2) 4147 0339; e-mail: draberpe@biomed.cas.cz.
Abstract.
Full text. 215 216-217
Genetic Variation in the MHC II Promoter: Lessons for Regulation and for Comparative Genomics
N. A. MITCHISON
Sequence data have been accumulating that reveal variation in gene promoters of the immune system, notably in MHC
class II, cytokines and chemokines. The variation is non-random: it occurs most often in proximity to and within certain regulatory elements such as CRE and NFY (in MHC class II these are respectively the X2 and Y boxes). These are elements that are widely used elsewhere in the genome, and appear to act as rheostats (modulators of expression) in contrast to the type of on-off switch operated by the RFX element that is unique to a single family of promoters such as MHC class II. It is proposed that a complex mouse phenotype described in Prague and elsewhere may reflect this pattern of variation in/around CRE. Such rheostats are expected to operate in other promoters. Their identification will be facilitated by short-range comparisons (e.g. human – chimp), and indeed this is a motive for extending comparative genomics.
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Expression of Human Erythropoietin Gene in the Mammary Gland of a Transgenic Mouse
T. MIKUŠ, P. MALÝ, M. POPLŠTEIN, V. LANDA, P. TREFIL, J. LIDICKÝ
WAP is being recognized as the principal milk protein expressed in pregnant or lactating females of several mammalian species. Recently, it has been shown that the 6.3-kb 5’ untranslated region of the rWAP gene is able to control, and almost completely restrict, the expression of the transgene into the mammary gland of the transgenic animal. We cloned the genomic fragment carrying the rWAP gene locus from the rabbit phage genomic library and used the 8.5-kb long 5’ untranslated part of the rWAP gene to target the expression of hEPO, cloned from the human phage genomic library, into the mammary gland of the mouse. The vectors, carrying either the hEPO gene or the rWAP-hEPO hybrid gene, were injected into the mouse ova, and 12 transgenic animals were identified by PCR and Southern blot from the progeny of 168 tested littermates. Transgenic mice were viable, fertile and displayed a normal development. Recombinant human erythropoietin was produced in the milk of a transgenic mouse female at a secretion level of 5.3 mIU/ml, as detected by ELISA. Despite the low production of the transgenic glycoprotein in the milk we demonstrate that the hybrid gene can be expressed in the mammary gland of the host animal. Thus, WAP-based recombinant vectors, with additional optimizing modifications, can be useful for production of therapeutic proteins in the transgenic mammals.
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Allosteric Modulation of GABAA Receptor by Somatostatin Is Altered undeer Stress in Rat Brainstem
F. CHIGR, S. B. M'HAMED, M. NAJIMI
This study was conducted to investigate somatostatin modulation of GABAA receptor binding in several rat brainstem structures, located principally in the mesencephalon, after exposure to acute immobilization stress (single 1- hour session). Animals were randomly assigned to either control or stress conditions and changes in specific binding of the GABAA receptor as labelled with TBPS were assessed by in vitro quantitative autoradiography with the aid of a computer-assisted image analysis system. Exposure to immobilization stress led to a significant increase in [35S]TBPS binding sites density in the SN of stressed rats compared to controls. In the other brainstem structures analysed, specific binding of [35S]TBPS remained unchanged in stressed rats. Furthermore, the results of the present in vitro study demonstrate an alteration of the modulatory effect of somatostatin on the GABAA receptor complex in the SN of stressed rats as compared to controls. This apparent alteration of allosteric effects of GABA receptor-somatostatin in the SN of stressed rats was eliminated in the presence of 1 micromolar concentration of GABA. Taken together, these data provide the first evidence of stress-induced alteration of allosteric effects of GABA-somatostatin in the rat mesencephalon. Furthermore, they also demonstrate that the tetradecapeptide somatostatin is particularly effective in modifying the [35S]TBPS binding to the GABAA receptor in this cerebral region.
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E. HRDLIČKOVÁ-CELA, K. SMETANA, Jr., J. PLZÁK, Z. HOLÍKOVÁ, S. ANDRÉ, M. HŘEBÍČEK, K. HODAŇOVÁ, B. DVOŘÁNKOVÁ, J. MOTLÍK, H.-J. GABIUS
Human natural antibodies against Galalfa1,3Gal-R are mainly responsible for hyperacute rejection of xenografts transplanted to the human host. In addition to the anti-alfa-Gal activity, human serum also contains anti-b-Gal IgG fractions. Employing biotinylated IgG subfractions with anti-alfa- and anti-beta-Gal activity purified from human natural IgG, we have studied expression of reactive epitopes in porcine and human skin, porcine cultured keratinocytes and porcine and human cornea, porcine liver and human lacrimal gland, tear fluid and capillaries. No reactivity of porcine and human epidermis as well as anterior corneal epithelium was observed for human anti-alfa-Gal IgG. Serving as positive control, porcine capillaries gave the expected signal with the anti-alfa-Gal antibody. The anti-beta-Gal subfraction recognized cell nuclei in the epidermis of both these species. The pig liver cells interacted with antibodies against alfa- and beta-galactosides like cells of the human lacrimal gland. alfa-galactoside-reactive glycoproteins were also detected in the human tear fluid. The carbohydrate specificity of the reaction was ascertained by using melibiose as competitive sugar for alfa-galactoside-mediated binding. These results reveal the presentation of Galalfa1,3Gal in epithelial cells of human lacrimal gland, its biosynthetic origin being unclear. With respect to a potential clinical perspective, the given results facilitate consideration of the use of porcine epidermal cells in engineering of non-permanent wound covers to improve treatment.
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D. SUMERAUER, A. VÍTEK, H. KUČEROVÁ, R. KODET, J. HOUSKOVÁ, J. BEDRNÍČEK, T. ECKSCHLAGER
Ewing sarcoma and related neoplasias are characterized by the presence of specific chromosomal translocations resulting in EWS/ETS gene rearrangements. Created EWS/ETS-oncogene fusion transcripts can be detected in up to 98% of ESFT and provide tumour-specific markers useful in diagnostics. Using RT-PCR for detection of this aberration we can reveal minimal amounts of tumour cells contaminating BM, blood or apheresis products. We have examined BM samples from 22 patients (21 newly diagnosed and one recurrent disease) with histologically confirmed ESFT for the presence of contaminating tumour cells in BM at the time of diagnosis. Sixteen patients presented with localized disease, six had distant metastases at the first presentation. Ewing sarcoma cells were detected in the BM of 5/16 (31%) patients with localized disease and 3/6 (50%) with clinically detectable metastases at diagnosis. BM smears prepared from the same aspirates evaluated by light microscopy were all negative, even in two patients with multiple bone disease. We have confirmed the high sensitivity of the RT-PCR assay for detection of minimal BM infiltration in localized and metastatic ESFT. We have found that more than a quarter of patients with localized ESFT have minimal BM infiltration. Although the clinical significance of the minimal disease detected at the molecular level remains unknown, RT-PCR evaluation may enable better stratification of patients into risk groups in the future.
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Monoclonal Antibody Register
New Monoclonal Antibodies Recognizing the p53 Tumour Suppressor Protein Homologue p73
P. ČEŠKOVÁ, R. NENUTIL, S. BRAY, M. SVITÁKOVÁ, S. BABČANOVÁ, S. ULDRIJAN,
B. VOJTĚŠEK
The p53 tumour suppressor gene is the most frequent target for genetic alterations in human tumours (Nigro et al., 1989; Hollstein et al., 1991) and encodes a nuclear phosphoprotein which plays a key role in cell-cycle regulation. This protein protects cells from undergoing tumorigenic alterations by inducing either cell-cycle arrest or programmed cell death in response to a variety of cellular stress signals (Ko and Prives, 1996; Levine, 1997) and also plays an important role in maintaining the integrity of the genome (Lane, 1992). Attempts to find p53 homologues analogous to the pRb family of tumour suppressors were successful and a new gene, p73, has been identified on the short arm of chromosome 1 in a region frequently deleted in neuroblastoma (Kaghad et al., 1997; Yang et all 1998). This gene encodes several proteins with structural and functional homology to p53 (De Laurenzi et al., 1998) that can activate transcription of the p21WAF1 gene responsible for cell-cycle arrest, and can also induce apoptosis when overexpressed (Jost et al., 1997; Kaghad et al., 1997). There are several differentially spliced variants of mRNAs, which are translated into different p73 proteins. This splicing occurs at the 3´ end of the sequence and translated proteins have differing C-termini in which p73alfa is a full-length protein and the beta and delta isoforms are truncated forms of this protein. The delta isoform lacking the major part of the C-terminal region is most similar to p53. The gamma isoform contains a different 75-residue C-terminus compared to the alfa isoform due to a long alternative reading frame in this region (Kaghad et al., 1997; De Laurenzi et al., 1998; De Laurenzi et al., 1999; Ueda et al., 1999). p73 as well as p53 contains a transactivation domain, a DNA-binding domain and an oligomerization domain. The highest level of homology is reached in the DNA-binding domain (63% identity between the p53 and p73) and this finding suggests that both these proteins can bind to the same DNA sequences and transactivate the same target genes. The conservation of high homology in the oligomerization domain suggests that the members of this family could form mixed oligomers. Although the existence of these mixed oligomers in vivo is still open to question, the availability of specific antibodies could help to address this problem.
The identification of a family of p53-related transcription factors that can be potentially redundant in their ability to activate similar cellular responses (i.e. cell-cycle arrest and apoptosis) has encouraged a study into the basis for their similarities and differences in terms of their physical and functional interactions with one another, their mechanism of activation and their regulation. Monoclonal antibodies specific to different forms of the p73 protein therefore provide a powerful tool to study these proteins both in vivo and in vitro.
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P. TOLAR, M. TŮMOVÁ, P. DRÁBER
LAT, the linker for activation of T cells, is a 36–38 kDa transmembrane signalling adaptor protein expressed exclusively on T lymphocytes, NK cells, mast cells, megakaryocytes and platelets. The molecule consists of a short, 5 amino acid extracellular stretch, and a transmembrane helix followed by a cytoplasmic tail containing 9 conserved tyrosines. Though no modular protein-binding domains have been identified in LAT, several of the tyrosines lie within motifs predicted to bind important downstream signalling molecules (Weber et al, 1998; Zhang et al., 1998a). Due to posttranslational palmitoylation of two membrane-proximal cysteins, LAT preferentially sublocalizes to lipid rafts, small regions of the plasma membrane distinct in lipid composition and enriched in signalling molecules (Zhang et al., 1998b).
LAT has been demonstrated to function centrally in the propagation of signals from surface antigen receptors of immune cells (Finco et al., 1998; Zhang et al., 1999a; Saitoh et al., 2000). These receptors include the T-cell receptor and certain Fc receptors and belong to a structurally defined family that has been termed the multichain immune recognition receptors (MIRRs). Following the engagement of MIRRs on LAT-expressing cells, LAT tyrosine residues are phosphorylated by receptor-associated and activated Syk/ZAP-70 kinases, and directly recruit Src homology 2 domain-containing signalling molecules, including PLCgamma, Grb2, Gads, Grap, 3BP2, Shb. Translocation and/or susceptibility to further phosphorylation of these molecules and their binding partners is a key step that regulates the pathways ultimately leading to most of the cellular responses. Thus, LAT forms a membrane raft-confined signaling complex, which links the proximal, cell-type specific receptor apparatus with the subsequent, more ubiquitous signaling pathways (reviewed in Wange, 2000). Antigen-driven responses of T cells and mast cells lacking functional LAT are manifestly abrogated (Finco et al., 1998; Zhang et al., 1999b; Saitoh et al., 2000).
Given the essential role of LAT in the development and function of the immune system, it has been proposed that the reduced signalling from antigen receptors under many pathological situations may be related to the impairment of LAT localization and function (Gringhuis et al., 2000; Wange, 2000). Therefore, monoclonal antibodies specifically recognizing LAT are obligatory tools for the investigation of signalling phenotypes of immunocompetent cells under various circumstances.
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