196 results on '"Tünde Kovács"'
Search Results
152. Golyó/sík érintkezés elvén alapuló lokális kopásvizsgá- lati eljárás alkalmazási feltételeinek elemzése
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Tünde Kovács and László Dévényi
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- 2004
153. Kopásvizsgálatok tervezési szempontjai
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Tünde Kovács
- Published
- 2003
154. A Phenomenological Method for the Prediction of Damage Accumulation Processes under Varying External Conditions
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Tamás Réti and Tünde Kovács
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Materials science ,Creep ,Mechanics of Materials ,business.industry ,Mechanical Engineering ,Mechanical engineering ,General Materials Science ,Structural engineering ,Condensed Matter Physics ,business ,Phenomenological method - Published
- 2003
155. Expression of Endomembrane Calcium Pumps in Colon and Gastric Cancer Cells
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Jocelyne Enouf, Nathalie Rivard, Roosje van Gorp, Tünde Kovács, Pascal Gélébart, Jean-Philippe Brouland, Johannes Grossmann, Raymonde Bredoux, Yves Panis, Virginie Martin, and Béla Papp
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Calcium metabolism ,SERCA ,Calcium pump ,Endoplasmic reticulum ,Cellular differentiation ,chemistry.chemical_element ,Cell Biology ,Calcium ,Biology ,Biochemistry ,Calcium in biology ,Cell biology ,chemistry ,Cancer cell ,Molecular Biology - Abstract
Calcium mobilization from the endoplasmic reticulum (ER) into the cytosol is a key component of several signaling networks controlling tumor cell growth, differentiation, or apoptosis. Sarco/endoplasmic reticulum calcium transport ATPases (SERCA-type calcium pumps), enzymes that accumulate calcium in the ER, play an important role in these phenomena. We report that SERCA3 expression is significantly reduced or lost in colon carcinomas when compared with normal colonic epithelial cells, which express this enzyme at a high level. To study the involvement of SERCA enzymes in differentiation, in this work differentiation of colon and gastric cancer cell lines was initiated, and the change in the expression of SERCA isoenzymes as well as intracellular calcium levels were investigated. Treatment of the tumor cells with butyrate or other established differentiation inducing agents resulted in a marked and specific induction of the expression of SERCA3, whereas the expression of the ubiquitous SERCA2 enzymes did not change significantly or was reduced. A similar marked increase in SERCA3 expression was found during spontaneous differentiation of post-confluent Caco-2 cells, and this closely correlated with the induction of other known markers of differentiation. Analysis of the expression of the SERCA3 alternative splice isoforms revealed induction of all three known iso-SERCA3 variants (3a, 3b, and 3c). Butyrate treatment of the KATO-III gastric cancer cells led to higher resting cytosolic calcium concentrations and, in accordance with the lower calcium affinity of SERCA3, to diminished ER calcium content. These data taken together indicate a defect in SERCA3 expression in colon cancers as compared with normal colonic epithelium, show that the calcium homeostasis of the endoplasmic reticulum may be remodeled during cellular differentiation, and indicate that SERCA3 constitutes an interesting new differentiation marker that may prove useful for the analysis of the phenotype of gastrointestinal adenocarcinomas.
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- 2002
156. Three Novel Sarco/endoplasmic Reticulum Ca2+-ATPase (SERCA) 3 Isoforms
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Jocelyne Enouf, Roosje van Gorp, Elisabeth Corvazier, Raymonde Bredoux, Pascal Gélébart, Virginie Martin, and Tünde Kovács
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Gene isoform ,Retinoic acid receptor ,SERCA ,Endoplasmic reticulum ,HEK 293 cells ,STIM1 ,Cell Biology ,Signal transduction ,Biology ,Molecular Biology ,Biochemistry ,Molecular biology ,Protein kinase C - Abstract
Sarco/endoplasmic reticulum Ca2+-ATPases (SERCAs) pump Ca2+ into the endoplasmic reticulum. Recently, three human SERCA3 (h3a–c) proteins and a previously unknown rat SERCA3 (r3b/c) mRNA have been described. Here, we (i) document two novel human SERCA3 splice variants h3d and h3e, (ii) provide data for the expression and mechanisms regulating the expression of all known SERCA3 variants (r3a, r3b/c, and h3a–e), and (iii) show functional characteristics of the SERCA3 isoforms. h3d and h3e are issued from the insertion of an additional penultimate exon 22 resulting in different carboxyl termini for these variants. Distinct distribution patterns of the SERCA3 gene products were observed in a series of cell lines of hematopoietic, epithelial, embryonic origin, and several cancerous types, as well as in panels of rat and human tissues. Hypertension and protein kinase C, calcineurin, or retinoic acid receptor signaling pathways were found to differently control rat and human splice variant expression, respectively. Stable overexpression of each variant was performed in human embryonic kidney 293 cells, and the SERCA3 isoforms were fully characterized. All SERCA3 isoforms were found to pump Ca2+ with similar affinities. However, they modulated the cytosolic Ca2+ concentration ([Ca2+]c) and the endoplasmic reticulum Ca2+ content ([Ca2+]er) in different manners. A newly generated polyclonal antibody and a pan-SERCA3 antibody proved the endogenous expression of the three novel SERCA3 proteins, h3d, h3e, and r3b/c. All these data suggest that the SERCA3 gene products have a more widespread role in cellular Ca2+ signaling than previously appreciated.
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- 2002
157. Kopási károsodási folyamatok modellezése
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László Horváth, Tamás Réti, and Tünde Kovács
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- 2002
158. Plastic deformation effect of the corrosion resistance in case of austenitic stainless steel
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Tünde Kovács and F Haraszti
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Austenite ,High rate ,Materials science ,Metallurgy ,engineering ,Intergranular corrosion ,Deformation (engineering) ,Austenitic stainless steel ,engineering.material ,Corrosion ,Heat treating - Abstract
The corrosion forms are different in case of the austenitic steel than in case of carbon steels. Corrosion is very dangerous process, because that corrosion form is the intergranular corrosion. The austenitic stainless steel shows high corrosion resistance level. It knows that plastic deformation and the heat treating decrease it's resistance. The corrosion form in case of this steel is very special and the corrosion tests are difficult. We tested the selected steel about its corrosion behaviour after high rate deformation. We wanted to find a relationship between the corrosion resistance decreasing and the rate of the plastic deformation. We wanted to show this behaviour from mechanical and electrical changing.
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- 2017
159. High Energy Rate Forming Induced Phase Transition in Austenitic Steel
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Tünde Kovács and L. Kuzsella
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Austenite ,History ,Phase transition ,High energy ,Materials science ,Metallurgy ,Computer Science Applications ,Education - Published
- 2017
160. Equal values of standard counting polynomials
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Gyöngyvér Péter, Ákos Pintér, Tünde Kovács, and Kalman Gyory
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Pure mathematics ,Chebyshev polynomials ,Gegenbauer polynomials ,General Mathematics ,Discrete orthogonal polynomials ,Classical orthogonal polynomials ,symbols.namesake ,Macdonald polynomials ,Difference polynomials ,Természettudományok ,Orthogonal polynomials ,symbols ,Jacobi polynomials ,Matematika- és számítástudományok ,Mathematics - Published
- 2014
161. Reverzibilis ausztenit-ferrit átalakulási folyamat szimulációja
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Mihály Réger, Tamás Réti, and Tünde Kovács
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- 2001
162. ChemInform Abstract: Microwave-Assisted Solid-Liquid Phase Alkylation of Naphthols
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Orsolya Tünde Kovács, Erika Bálint, György Keglevich, and László Drahos
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inorganic chemicals ,Chemistry ,organic chemicals ,Phase (matter) ,Microwave irradiation ,heterocyclic compounds ,lipids (amino acids, peptides, and proteins) ,General Medicine ,Alkylation ,Photochemistry ,Microwave assisted ,Solid liquid - Abstract
Catalyst-free, selective O-alkylation of naphthols is achieved under microwave irradiation in the presence of K2CO3.
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- 2013
163. Lineage-Specific Modulation of Calcium Pump Expression During Myeloid Differentiation
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Fabien Zassadowski, Pascal Gélébart, Tünde Kovács, Sophie Launay, Raymonde Bredoux, Arlette Bruel, Christine Chomienne, Béla Papp, Jocelyne Enouf, and Maurizio Gianni
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SERCA ,Calcium pump ,Cellular differentiation ,Immunology ,Drug Resistance ,Retinoic acid ,Gene Expression ,Tretinoin ,Calcium-Transporting ATPases ,Biology ,Endoplasmic Reticulum ,Biochemistry ,chemistry.chemical_compound ,Leukemia, Promyelocytic, Acute ,Cyclic AMP ,Tumor Cells, Cultured ,Humans ,RNA, Messenger ,Calcium signaling ,Macrophages ,Endoplasmic reticulum ,Cell Differentiation ,Cell Biology ,Hematology ,Cell biology ,Kinetics ,Sarcoplasmic Reticulum ,Retinoic acid receptor ,chemistry ,Monocyte differentiation ,cardiovascular system ,Tetradecanoylphorbol Acetate ,Calcium ,Granulocytes - Abstract
Calcium is accumulated from the cytosol into the endoplasmic reticulum by sarco-endoplasmic reticulum calcium transport ATPase (SERCA) enzymes. Because calcium stored in the endoplasmic reticulum is essential for cell growth, differentiation, calcium signaling, and apoptosis and because different SERCA enzymes possess distinct functional characteristics, in the present report we explored SERCA expression during in vitro differentiation of the human myeloid/promyelocytic cell lines HL-60 and NB4 and of freshly isolated acute promyelocytic leukemia cells. Two SERCA species have been found to be coexpressed in these cells: SERCA 2b and another isoform, SERCAPLIM, which is recognized by the PLIM430 monoclonal antibody. Induction of differentiation along the neutrophil granulocytic lineage by all-trans retinoic acid or cyclic AMP analogs led to an increased expression of SERCAPLIM, whereas the expression of the SERCA 2b isoform was decreased. The modulation of SERCA expression was manifest also on the mRNA level. Experiments with retinoic acid receptor isoform-specific retinoids indicated that SERCA expression is modulated by retinoic acid receptor -dependent signaling. SERCA expression of retinoic acid-resistant cell variants was refractory to treatment. Differentiation along the monocyte/macrophage lineage by phorbol ester resulted in an increased expression of both SERCA isoforms. In addition, when cells were treated by phorbol ester in the presence of the glucocorticoid dexamethasone, a known inhibitor of monocyte differentiation, a selective blockage of the induction of SERCAPLIM was observed. Altered SERCA expression modified the functional characteristics of calcium transport into the endoplasmic reticulum. These observations show for the first time that the modulation of calcium pump expression is an integral component of the differentiation program of myeloid precursors and indicate that a lineage-specific remodelling of the endoplasmic reticulum occurs during cell maturation. In addition, these data show that SERCA isoforms may serve as useful markers for the study of myeloid differentiation.
- Published
- 1999
164. Platelet sarco/endoplasmic reticulum Ca2+ATPase isoform 3b and Rap 1b: interrelation and regulation in physiopathology
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Christine Lacabaratz-Porret, Regis Bobe, Jocelyne Enouf, Béla Papp, Elisabeth Corvazier, Tünde Kovács, Raymonde Bredoux, and Sophie Launay
- Subjects
Blood Platelets ,inorganic chemicals ,SERCA ,Inositol Phosphates ,Protein subunit ,ATPase ,Calcium-Transporting ATPases ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Cell Line ,chemistry.chemical_compound ,GTP-Binding Proteins ,Rats, Inbred SHR ,Cyclic AMP ,Animals ,Humans ,Inositol ,RNA, Messenger ,Phosphorylation ,Rats, Wistar ,Protein kinase A ,Molecular Biology ,Endoplasmic reticulum ,fungi ,Cell Biology ,Rats ,Isoenzymes ,chemistry ,Hypertension ,cardiovascular system ,biology.protein ,Intracellular ,Research Article - Abstract
Platelet Ca2+ signalling involves intracellular Ca2+ pools, whose content is controlled by sarco/endoplasmic reticulum Ca2+ATPases (SERCAs). Among these, a key role is played by the inositol trisphosphate-sensitive Ca2+ pool, associated with the SERCA 3b isoform. We have investigated the control of this Ca2+ pool through the cAMP-dependent phosphorylation of the GTP-binding protein, Rap (Ras-proximate) 1b. We first looked for this Ca2+ pool target of regulation by studying the expression of the different SERCA and Rap 1 proteins in human platelets and various cell lines, by Western blotting and reverse transcription-PCR. Since co-expression of Rap 1b and SERCA 3b was obtained, we looked for their protein–protein interaction as a function of the cAMP-dependent phosphorylation of Rap 1b. Co-immunoprecipitations of SERCA 3b and Rap 1b proteins were found in the absence of phosphorylation, induced by the catalytic subunit of the cAMP-dependent protein kinase (csPKA). In contrast, upon pre-treatment of platelet membranes with csPKA, the SERCA 3b dissociated from the Rap 1b protein, in agreement with a role of its phosphorylated state in their interaction. Finally, we looked for adaptation of this complex in a platelet pathological model of hypertension. We investigated the expression of both proteins, as well as the cAMP-dependent phosphorylation of Rap 1b and SERCA 3b activity in platelets from control normotensive Wistar-Kyoto rats and from spontaneously hypertensive rats (SHRs). A decrease in SERCA 3b activity was associated with a decrease in Rap 1b endogenous phosphorylation in SHR platelets, consistent with a functional role in the regulation of the SERCA 3b-associated Ca2+ pool.
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- 1998
165. Expression of two isoforms of the third sarco/endoplasmic reticulum Ca2+ATPase (SERCA3) in platelets. Possible recognition of the SERCA3b isoform by the PL/IM430 monoclonal antibody1
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Béla Papp, Sophie Launay, Christine Lacabaratz-Porret, Elisabeth Corvazier, Raymonde Bredoux, Tünde Kovács, Virginie Martin, Anne Ozog, Regis Bobe, and Jocelyne Enouf
- Subjects
Gene isoform ,Messenger RNA ,SERCA ,Endoplasmic reticulum ,Biophysics ,RNA ,Cell Biology ,Biology ,Biochemistry ,Molecular biology ,Isozyme ,Exon ,Structural Biology ,Genetics ,Molecular Biology ,Gene - Abstract
Human platelets express several sarco/endoplasmic reticulum Ca2+ATPase (SERCA) isoenzymes: SERCA2b of 100 kDa apparent molecular mass and two distinct enzymes of 97 kDa, one of them identified as being the SERCA3a isoform. The molecular identity of the third enzyme specifically recognized by the PL/IM430 monoclonal antibody has remained elusive. First, the study of the 3′-end part of platelet SERCA3 mRNA, by means of RT-PCR amplification using sets of primers covering the N−3 to N (ultimate) exons of the human SERCA3 sequence, revealed the presence of two distinct mRNA sequences, SERCA3a and a longer variant. Second, this additional sequence was identified as SERCA3b and found to refer to the insertion of a new exon of 73 bp, located at bp 349 from the beginning of the intronic sequence, linking the penultimate (N−1) exon to the last exon (N) of the human SERCA3 gene. Third, a relationship between the expression of this SERCA3b mRNA and the PL/IM430 recognizable SERCA protein was observed. SERCA3b mRNA was found to be absent in epithelial HeLa cells not recognized by the PL/IM430 antibody and the expression of this SERCA3b RNA species correlated with that of the SERCA protein recognized by PL/IM430 which was down-modulated in the platelet precursor megakaryocytic CHRF 288-11 cell line as well as upon in vitro lymphocyte activation. Taken together, these results strongly support the notion of the presence of the SERCA3b protein in human cells by showing SERCA3b mRNA in platelets and the fact that the protein corresponding to this mRNA species is very likely the 97 kDa protein recognized by the PL/IM430 antibody.
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- 1998
166. Modulation of Endoplasmic Reticulum Calcium Pump Expression during T Lymphocyte Activation
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Tünde Kovács, Raymonde Bredoux, Christine Lacabaratz-Porret, Béla Papp, Jocelyne Enouf, Regis Bobe, and Sophie Launay
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SERCA ,T-Lymphocytes ,Calcium pump ,chemistry.chemical_element ,Calcium-Transporting ATPases ,Biology ,Calcium ,Endoplasmic Reticulum ,Lymphocyte Activation ,Biochemistry ,Gene Expression Regulation, Enzymologic ,Calcium in biology ,Cell Line ,Jurkat Cells ,chemistry.chemical_compound ,Humans ,Molecular Biology ,Calcium metabolism ,Ionomycin ,T-type calcium channel ,Cell Biology ,Cell biology ,Isoenzymes ,Calcium ATPase ,Sarcoplasmic Reticulum ,chemistry ,Cyclosporine ,Tetradecanoylphorbol Acetate - Abstract
Calcium mobilization from intracellular storage organelles is a key component of the second messenger system inducing cell activation. Calcium transport ATPases associated with intracellular calcium storage organelles play a major role in controlling this process by accumulating calcium from the cytosol into intracellular calcium pools. In this study the modulation of the expression of the sarco-endoplasmic reticulum calcium transport ATPase (SERCA) isoenzymes has been studied in lymphocytes undergoing phorbol myristate acetate and ionomycin-induced activation. In several T lymphocyte cell lines a combined treatment by the two drugs resulted in an approximately 90% decrease of the expression of the calcium pump isoform recognized by the PLIM430 isoform-specific antibody, whereas the expression of the SERCA 2b isoform was increased approximately 2-fold. Phorbol ester or ionomycin applied separately was ineffective. In Jurkat T cells the down-modulation of expression of the SERCA isoform recognized by the PLIM430 antibody appeared concomitantly with the induction of interleukin-2 expression and could be inhibited by the immunosuppressant drug cyclosporine-A. These data indicate that T cell activation induces a selective and cyclosporine-A-sensitive modulation of the expression of the SERCA calcium pump isoforms. This reflects a profound reorganization of the calcium homeostasis of T cells undergoing activation and may open new avenues in the understanding of the plasticity of the calcium homeostasis of differentiating cells and in the pharmacological modulation of lymphocyte function.
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- 1997
167. Immunolocalization of the multi‐sarco/endoplasmic reticulum Ca 2+ ATPase system in human platelets
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Regis Bobe, Tünde Kovács, Elisabeth M. Cramer, Béla Papp, Frank Wuytack, Elisabeth Corvazier, Katalin Pászty, Angie S. Brown, Gaëtan Berger, and Jocelyne Enouf
- Subjects
Blood Platelets ,inorganic chemicals ,Gene isoform ,animal structures ,SERCA ,ATPase ,Immunoelectron microscopy ,Blotting, Western ,Neuraminidase ,Calcium-Transporting ATPases ,Endoplasmic Reticulum ,Antibody Specificity ,Humans ,biology ,musculoskeletal, neural, and ocular physiology ,Endoplasmic reticulum ,Antibodies, Monoclonal ,Hematology ,Subcellular localization ,Molecular biology ,Microscopy, Electron ,Membrane ,cardiovascular system ,biology.protein ,tissues ,Intracellular - Abstract
We recently identified a multi-SERCA (sarco/endoplasmic reticulum Ca2+ ATPase) system in haemopoietic cells comprising the SERCA 2b, SERCA 3 and a new monoclonal anti-Ca2+ ATPase antibody (PL/IM 430) recognizable SERCA isoforms. We have now investigated the subcellular localization of these enzymes in human platelets by Western blotting of subcellular membrane fractions and by immunoelectron microscopy. We precisely defined the recognition specificity of the polyclonal anti-SERCA 2b, anti-SERCA 3, anti-SERCA 1 antibodies as well as of the monoclonal antibody PL/IM 430 by testing their recognition of the tryptic fragments of the SERCA isoforms. The analysis of fragmented membranes enriched in plasma membrane and intracellular membrane components by Western blotting showed that the SERCA 2b and the SERCA 3 isoforms were found in both the plasma membrane and the intracellular membrane fractions, whereas the PL/IM 430 recognizable SERCA isoform was restricted to membranes associated with the plasma membrane fraction. The immunoelectron microscopical study of the SERCA isoforms in resting platelets showed that: (i) the SERCA 2b isoform was expressed in membranes associated with the plasma membrane and open canalicular system, some alpha-granules and in unidentified membranes; (ii) the SERCA 3 isoform was found associated with plasma and intracellular membranes; and (iii) the PL/IM 430 recognizable SERCA isoform was observed only in structures associated with the cytoplasmic face of the plasma membranes, as confirmed by flow cytometry. Finally, since the PL/IM 430 antibody was raised against intracellular membranes, we looked for a potential membrane redistribution during the isolation procedure used for the preparation of the immunizing membranes. Neuraminidase treatment indeed induced a translocation of the PL/IM 430 recognizable SERCA isoform from plasma to intracellular membranes. Thus, the multi-SERCA system in platelets: (i) is distributed over different platelet membranes, (ii) presents a sub-compartmental organization with some overlapping, and (iii) is partly associated with motile membranes, reflecting an unrecognized level of complexity of Ca2+ stores in these cells.
- Published
- 1997
168. AB0074 The Effect of Extracellular Vesicles on Human in Vitro Osteoclastogenesis in Health and in Arthritis
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Orsolya Tünde Kovács, Florian M. P. Meier, G.S. Nagy, Eszter Baricza, Iain B. McInnes, Carl S. Goodyear, David Gyori, Nikolett Marton, Edit I. Buzás, and Attila Mócsai
- Subjects
biology ,business.industry ,CD14 ,Immunology ,Cell ,Arthritis ,medicine.disease ,Peripheral blood mononuclear cell ,Molecular biology ,General Biochemistry, Genetics and Molecular Biology ,Microvesicles ,In vitro ,medicine.anatomical_structure ,Rheumatology ,Osteoclast ,RANKL ,medicine ,biology.protein ,Immunology and Allergy ,business - Abstract
Background Extracellular vesicles (EVs) like microvesicles (MVs) and exosomes (EXOs) are released in an evolutionary conserved manner by cells. EVs mediate intercellular communication with the transmission of molecules from their parent cell to their targets. Objectives Our objective was to investigate the effect of EVs on human in vitro osteoclastogenesis in healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected. EVs were isolated by filtration and differential centrifugation. CD14+ cells were isolated from PBMCs by using positive selection, and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs. Then the samples were treated with 50 ng/ml recombinant human RANKL and blood-derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartarate resistant acid phosphatase (TRAP). TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results Healthy and RA-derived exosomes significantly (p Conclusions Our data suggest that EXOs profoundly regulate human osteoclastogenesis. PsA-derived EXOs stimulate, while healthy and RA-derived EXOs inhibit the osteoclast differentiation. Disclosure of Interest None declared
- Published
- 2016
169. A7.21 The effect of extracellular vesicles on humanin vitroosteoclastogenesis
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Florian M. P. Meier, Gy Nagy, Iain B. McInnes, Orsolya Tünde Kovács, Carl S. Goodyear, Nikolett Marton, Attila Mócsai, Eszter Baricza, Edit I. Buzás, and Dávid Győri
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Pathology ,medicine.medical_specialty ,biology ,medicine.diagnostic_test ,Immunology ,Arthritis ,medicine.disease ,Molecular biology ,Peripheral blood mononuclear cell ,General Biochemistry, Genetics and Molecular Biology ,Microvesicles ,Flow cytometry ,Acid-citrate-dextrose ,Rheumatology ,RANKL ,biology.protein ,medicine ,Immunology and Allergy ,Antibody ,Tartrate-resistant acid phosphatase - Abstract
Introduction Extracellular vesicles (EV) are subcellular sized intercellular messengers, which are present in various biological fluids. EVs carry a wide variety of biomolecules and they may alter the recipient cells’ functions. The microvesicles are plasma membrane derived EVs; the exosomes, the smallest vesicles, originate from the endosome. Our objectives were to investigate the effect of EVs on the human in vitro osteoclastogenesis, and to characterise the serum EV profile of healthy donors, rheumatoid arthritis (RA) and psoriatic arthritis (PsA) patients. Methods Blood samples of healthy volunteers, RA patients and PsA patients with peripheral arthritis were collected into acid citrate dextrose vacutainer tubes. EVs were isolated by filtration and centrifugation. EV samples were stained with fluorescent anti-CD3, CD14, CD15, CD19, CD42b, CD235a antibodies (BioLegend) and detected by flow cytometry. CD14 + cells were extracted from PBMCs by using positive selection method (StemCell), and the cells were stimulated with 50 ng/ml recombinant human M-CSF for 24 hrs (PeproTech). Then the samples were treated with 50–50 ng/ml recombinant human M-CSF and RANKL (PeproTech) and with or without blood derived microvesicles or exosomes. After 7 days, the cells were fixed and stained for tartrate resistant acid phosphatase (TRAP) using a commercially available kit (Sigma) . TRAP-positive cells with at least 3 nuclei were considered as osteoclasts and counted by using the ImageJ software. Results RA and PsA EV samples showed increased expression of lymphocyte markers (CD3, CD19) compared to healthy donor-derived EVs (n = 6). Microvesicles did not alter the number of mature osteoclasts. By contrast, exosomes significantly (p Conclusion Our present data suggest that the plasma profile of EVs might be altered in active arthritis compared to physiological condition, and exosomes or their cargo can regulate the human osteoclastogenesis.
- Published
- 2016
170. The rat platelet 97-kDa Ca2+ATPase isoform is the sarcoendoplasmic reticulum Ca2+ATPase 3 protein
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Raymonde Bredoux, Clarice Magnier, Tünde Kovács, Elisabeth Corvazier, Regis Bobe, Béla Papp, Frank Wuytack, Jocelyne Enouf, and Rozenn Quarck
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Blood Platelets ,Gene isoform ,SERCA ,ATPase ,Blotting, Western ,Gene Expression ,Calcium-Transporting ATPases ,Endoplasmic Reticulum ,Rats, Inbred WKY ,Biochemistry ,Gene expression ,Animals ,RNA, Messenger ,Molecular Biology ,Messenger RNA ,biology ,Endoplasmic reticulum ,Cell Biology ,Molecular biology ,Rats ,Molecular Weight ,Blot ,Sarcoplasmic Reticulum ,Hypertension ,cardiovascular system ,biology.protein ,Immunostaining - Abstract
We recently showed that human and rat platelets express two types of SERCAs (Sarco Endoplasmic Reticulum Ca2+ATPases): a 100-kDa SERCA2b isoform and a 97-kDa SERCA isoform. Here, we explored the possibility that the rat 97-kDa isoform is identical to the SERCA3 protein. For this purpose, we first attempted to detect SERCA3 mRNA in rat platelet total RNA by reverse transcription-polymerase chain reaction using SERCA3-specific primers, and demonstrated the presence of this mRNA species by sequencing the amplification product. We then searched for a relationship between the expression of the SERCA3 mRNA and of the 97-kDa protein using either rat aortic smooth muscle cells, previously found not to express the 97-kDa SERCA isoform (negative model), or platelets of spontaneously hypertensive rats (SHR), which overexpress this isoform (overexpression model) but express the 100-kDa SERCA2b isoform normally. No expression of SERCA3 mRNA was detectable by analysis of smooth muscle cell RNA, but comparison by reverse transcription-polymerase chain reaction of the SERCA2b and SERCA3 mRNAs from the platelets of normotensive (Wistar-Kyoto, WKY) rats and SHR clearly demonstrated a 238 +/- 43% increase in the expression of the SERCA3 mRNA in SHR platelets only. Last, by comparative Western blotting of WKY rat and SHR platelet membranes using a recently developed polyclonal anti-SERCA3 antibody, we established that the 97-kDa SERCA and the SERCA3 protein are identical, as immunostaining of the 97-kDa protein revealed a 230 +/- 25% increase in the expression of this protein in SHR versus WKY rat platelets. It is concluded that the 97-kDa platelet SERCA isoform, which is up-regulated in SHR, is the SERCA3 protein. As far as we know, this constitutes the first demonstration of the actual presence of this Ca2+ATPase isoform in normal cells, in addition to the artificial transfection systems.
- Published
- 1994
171. Almost fifth powers in arithmetic progression
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Tünde Kovács and Lajos Hajdu
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Discrete mathematics ,Algebra and Number Theory ,Perfect power ,Genus 2 curves ,Perfect powers ,Chabauty method ,Mathematical proof ,Természettudományok ,Fifth power (algebra) ,Genus (mathematics) ,Product (mathematics) ,Arithmetic progression ,Algebraic number ,Matematika- és számítástudományok ,Perfect fifth ,Mathematics - Abstract
We prove that the product of k consecutive terms of a primitive arithmetic progression is never a perfect fifth power when 3 ⩽ k ⩽ 54 . We also provide a more precise statement, concerning the case where the product is an “almost” fifth power. Our theorems yield considerable improvements and extensions, in the fifth power case, of recent results due to Győry, Hajdu and Pinter. While the earlier results have been proved by classical (mainly algebraic number theoretical) methods, our proofs are based upon a new tool: we apply genus 2 curves and the Chabauty method (both the classical and the elliptic verison).
- Published
- 2011
172. Spontaneously hypertensive rats and platelet Ca2+-ATPases: specific up-regulation of the 97 kDa isoform
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P Poitevin, Sylviane Levy-Toledano, N Bourdeau, Tünde Kovács, Elisabeth Corvazier, Raymonde Bredoux, Frank Wuytack, Bernard I. Levy, Béla Papp, C Magnier, A M Lompré, and Jocelyne Enouf
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Blood Platelets ,Gene isoform ,medicine.medical_specialty ,Vascular smooth muscle ,Blotting, Western ,chemistry.chemical_element ,Calcium-Transporting ATPases ,Calcium ,Biology ,Endoplasmic Reticulum ,Rats, Inbred WKY ,Biochemistry ,Rats, Inbred SHR ,Internal medicine ,medicine ,Animals ,Myocyte ,Platelet ,Phosphorylation ,Molecular Biology ,Cell Membrane ,Autophosphorylation ,Muscle, Smooth ,Cell Biology ,Rats ,Molecular Weight ,Blot ,Kinetics ,Endocrinology ,chemistry ,Hypertension ,cardiovascular system ,Homeostasis ,Research Article - Abstract
The use of platelets instead of smooth muscle cells (SMC) to study the abnormal Ca2+ handling found in hypertension was investigated using spontaneously hypertensive rats (SHR). We studied the regulation of platelet Ca(2+)-ATPases, as we have recently demonstrated that human platelets, like SMC, contain the Ca(2+)-ATPase isoform termed SERCA2-b (sarco-endoplasmic reticulum Ca(2+)-ATPase). In mixed membranes isolated from platelets of normotensive Wistar-Kyoto (WKY) rats and SHR, total Ca(2+)-ATPase activity was found to be 43% higher in SHR than in WKY rats. By the use of autophosphorylation of rat platelet Ca(2+)-ATPases with [gamma-32P]ATP, followed by SDS/PAGE and Western blotting, we found that rat platelets express two distinct Ca(2+)-ATPases: a 100 kDa isoform, recognized by a SERCA2-b-specific anti-peptide antibody, and a 97 kDa isoform, specifically recognized by a polyclonal anti-SERCA antibody. Comparative analysis of platelet membrane Ca(2+)-ATPases from WKY rats and SHR demonstrated that the expression of the SERCA2-b isoform did not change significantly (128 +/- 22%), whereas that of the 97 kDa isoform reached 300 +/- 35% in SHR when compared with WKY rats. We concluded that the upregulation of total platelet Ca(2+)-ATPases in SHR is not due to the 100 kDa SERCA2-b isoform found in SMC, but is specific to the 97 kDa Ca(2+)-ATPase isoform which is not present in SMC. Therefore platelets should be used with extreme caution as a surrogate model of vascular smooth muscle Ca2+ homeostasis.
- Published
- 1993
173. Characterization of the inositol trisphosphate-sensitive and insensitive calcium stores by selective inhibition of the endoplasmic reticulum-type calcium pump isoforms in isolated platelet membrane vesicles
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Ágnes Enyedi, Katalin Pászty, Tünde Kovács, G. Gárdos, Balázs Sarkadi, Jocelyne Enouf, and Béla Papp
- Subjects
Blood Platelets ,Indoles ,Physiology ,Calcium pump ,chemistry.chemical_element ,Diaphragm pump ,Calcium-Transporting ATPases ,Inositol 1,4,5-Trisphosphate ,Calcium ,Endoplasmic Reticulum ,Calcium in biology ,chemistry.chemical_compound ,Benzoquinones ,Humans ,Inositol ,Inositol phosphate ,Molecular Biology ,chemistry.chemical_classification ,Terpenes ,Antibodies, Monoclonal ,Biological Transport ,Inositol trisphosphate ,Intracellular Membranes ,Cell Biology ,Cell Compartmentation ,Isoenzymes ,Calcium ATPase ,chemistry ,Biochemistry ,Thapsigargin ,Signal Transduction - Abstract
In mixed platelet membrane vesicles the presence of two distinct endoplasmic reticulum-type calcium pump enzymes of 100 and 97 kD molecular mass has been demonstrated. We have previously shown that both calcium pumps were recognized by polyclonal anti-sarcoplasmic reticulum calcium pump antisera [11]. In the present work we studied the effects of several calcium pump inhibitors on active calcium transport and inositol trisphosphate-induced calcium release in these vesicles in an attempt to assign the two calcium pump isoenzymes to specific calcium pools. The effect of the PL IM 430 inhibitory anti-calcium pump antibody was compared to that of other calcium pump inhibitors acting predominantly on the 100 and the 97 kD calcium pump isoforms, respectively. The PL IM 430 antibody, which recognized the 97 kD pump on Western blots and 2,5-di-(tert-butyl)-1,4-benzohydroquinone, which inhibited phosphoenzyme formation of the same pump isoform, inhibited calcium accumulation predominantly into an inositol trisphosphate-releasable calcium pool. On the other hand, low concentration of thapsigargin, which inhibited phosphoenzyme formation mainly of the 100 kD pump isozyme, had a more pronounced effect on calcium uptake into an inositol trisphosphate-resistant pool. These data suggest that in platelets the 97 kD calcium pump isoform is likely to be associated with the inositol trisphosphate-sensitive calcium storage organelle.
- Published
- 1993
174. Endoplasmic reticulum calcium pumps and cancer
- Author
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Jocelyne Enouf, Béla Papp, Jean-Philippe Brouland, Atousa Arbabian, Tünde Kovács, Regis Bobe, and Pascal Gélébart
- Subjects
Calcium metabolism ,SERCA ,Calcium pump ,Endoplasmic reticulum ,Clinical Biochemistry ,chemistry.chemical_element ,STIM1 ,Cell Differentiation ,General Medicine ,Biology ,Calcium ,Endoplasmic Reticulum ,Biochemistry ,Calcium in biology ,Cell biology ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,chemistry ,Neoplasms ,Molecular Medicine ,Animals ,Humans ,Calcium signaling - Abstract
Endoplasmic reticulum calcium homeostasis is involved in a multitude of signaling, as well as "house-keeping" functions that control cell growth, differentiation or apoptosis in every human/eukaryotic cell. Calcium is actively accumulated in the endoplasmic reticulum by Sarco/Endoplasmic Reticulum Calcium transport ATPases (SERCA enzymes). SERCA-dependent calcium transport is the only calcium uptake mechanism in this organelle, and therefore the regulation of SERCA function by the cell constitutes a key mechanism to adjust calcium homeostasis in the endoplasmic reticulum depending on the cell type and its state of differentiation. The direct pharmacological modulation of SERCA activity affects cell differentiation and survival. SERCA expression levels can undergo significant changes during cell differentiation or tumorigenesis, leading to modified endoplasmic reticulum calcium storage. In several cell types such as cells of hematopoietic origin or various epithelial cells, two SERCA genes (SERCA2 and SERCA3) are simultaneously expressed. Expression levels of SERCA3, a lower calcium affinity calcium pump are highly variable. In several cell systems SERCA3 expression is selectively induced during differentiation, whereas during tumorigenesis and blastic transformation SERCA3 expression is decreased. These observations point at the existence of a cross-talk, via the regulation of SERCA3 levels, between endoplasmic reticulum calcium homeostasis and the control of cell differentiation, and show that endoplasmic reticulum calcium homeostasis itself can undergo remodeling during differentiation. The investigation of the anomalies of endoplasmic reticulum differentiation in tumor and leukemia cells may be useful for a better understanding of the contribution of calcium signaling to the establishment of malignant phenotypes.
- Published
- 2010
175. ChemInform Abstract: Synthesis of a Difluoromethylenephosphonate Analogue of AZT 5′- Triphosphate and Its Inhibition of HIV-1 Reverse Transcriptase
- Author
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Tünde Kovács, Kenneth L. Kirk, D. Hebel, Paul F. Torrence, Jan Balzarini, Krystyna Lesiak, E. De Clercq, and Junei Kinjo
- Subjects
Coupling (electronics) ,Stereochemistry ,Chemistry ,Human immunodeficiency virus (HIV) ,medicine ,Nucleic acid ,virus diseases ,Ester hydrolysis ,General Medicine ,medicine.disease_cause ,Competitive inhibitor ,Reverse transcriptase - Abstract
Difluoromethylenebisphosphonic acid was prepared by acetyl hypofluorite-mediated fluorination of tetraisopropyl methylenebisphosphonate and ester hydrolysis. Coupling to 3'-azido-3'-deoxythymidine 5'-monophosphate gave the title compound. The difluoromethylenephosphonate was 30-fold less effective than AZT-triphosphate as a competitive inhibitor of HIV-1 reverse transcriptase but 10-fold more effective than the methylenephosphonate analogue.
- Published
- 2010
176. Modulation of B-cell endoplasmic reticulum calcium homeostasis by Epstein-Barr virus latent membrane protein-1
- Author
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Martin Rowe, Christine Chomienne, Irene Joab, Tünde Kovács, Béla Papp, Olivier Dellis, Atousa Arbabian, Jean-Philippe Brouland, Hématologie -Immunologie -Cibles thérapeutiques, Université Paris Diderot - Paris 7 (UPD7)-Institut National de la Santé et de la Recherche Médicale (INSERM), Service d'anatomie et cytologie pathologiques, Université Paris Diderot - Paris 7 (UPD7)-Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP), Membrane Research Group, Hungarian Academy of Sciences (MTA), Division of Cancer Studies, University of Birmingham Medical School, Greffes d'Epitheliums et Regulation de l'Activation Lymphocytaire, Université Paris-Sud - Paris 11 (UP11)-Institut National de la Santé et de la Recherche Médicale (INSERM), This work was supported by Inserm, the Association pour la Recherche sur le Cancer, the Ligue contre le Cancer, Fondation de France, the Association Laurette Fugain and by the Hungarian Academy of Sciences Grant OTKA T046814 (to T.K.), Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Hôpital Lariboisière-Fernand-Widal [APHP], Assistance publique - Hôpitaux de Paris (AP-HP) (AP-HP)-Université Paris Diderot - Paris 7 (UPD7), and BMC, Ed.
- Subjects
Cancer Research ,Herpesvirus 4, Human ,Endoplasmic Reticulum ,Lymphocyte Activation ,Calcium in biology ,0302 clinical medicine ,hemic and lymphatic diseases ,Homeostasis ,Calcium signaling ,0303 health sciences ,B-Lymphocytes ,STIM1 ,lcsh:Neoplasms. Tumors. Oncology. Including cancer and carcinogens ,Burkitt Lymphoma ,Immunohistochemistry ,Cell biology ,Oncology ,MESH: Cell Transformation, Viral ,MESH: Homeostasis ,030220 oncology & carcinogenesis ,MESH: Calcium ,[SDV.BBM.GTP] Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Molecular Medicine ,MESH: Epstein-Barr Virus Nuclear Antigens ,SERCA ,MESH: Cell Line, Tumor ,Calcium pump ,Blotting, Western ,chemistry.chemical_element ,[SDV.CAN]Life Sciences [q-bio]/Cancer ,Biology ,Calcium ,lcsh:RC254-282 ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Viral Matrix Proteins ,03 medical and health sciences ,MESH: Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Viral Proteins ,[SDV.CAN] Life Sciences [q-bio]/Cancer ,MESH: Endoplasmic Reticulum ,MESH: B-Lymphocytes ,[SDV.BBM.GTP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Genomics [q-bio.GN] ,Cell Line, Tumor ,MESH: Blotting, Western ,Humans ,MESH: Lymphocyte Activation ,030304 developmental biology ,MESH: Viral Matrix Proteins ,MESH: Humans ,Endoplasmic reticulum ,Research ,MESH: Immunohistochemistry ,MESH: Herpesvirus 4, Human ,Cell Transformation, Viral ,MESH: Viral Proteins ,Calcium ATPase ,MESH: Burkitt Lymphoma ,chemistry ,Epstein-Barr Virus Nuclear Antigens - Abstract
BackgroundCalcium signaling plays an important role in B lymphocyte survival and activation, and is critically dependent on the inositol-1,4,5-tris-phosphate-induced release of calcium stored in the endoplasmic reticulum (ER). Calcium is accumulated in the ER by Sarco/Endoplasmic Reticulum Calcium ATPases (SERCA enzymes), and therefore these enzymes play an important role in ER calcium homeostasis and in the control of B of cell activation. Because Epstein-Barr virus (EBV) can immortalize B cells and contributes to lymphomagenesis, in this work the effects of the virus on SERCA-type calcium pump expression and calcium accumulation in the endoplasmic reticulum of B cells was investigated.ResultsTwo Sarco-Endoplasmic Reticulum Calcium transport ATPase isoforms, the low Ca2+-affinity SERCA3, and the high Ca2+-affinity SERCA2 enzymes are simultaneously expressed in B cells. Latency type III infection of Burkitt's lymphoma cell lines with immortalization-competent virus expressing the full set of latency genes selectively decreased the expression of SERCA3 protein, whereas infection with immortalization-deficient virus that does not express the EBNA2 or LMP-1 viral genes was without effect. Down-modulation of SERCA3 expression could be observed upon LMP-1, but not EBNA2 expression in cells carrying inducible transgenes, and LMP-1 expression was associated with enhanced resting cytosolic calcium levels and increased calcium storage in the endoplasmic reticulum. Similarly to virus-induced B cell immortalisation, SERCA3 expression was also decreased in normal B cells undergoing activation and blastic transformation in germinal centers of lymph node follicles.ConclusionThe data presented in this work indicate that EBV-induced immortalization leads to the remodelling of ER calcium homeostasis of B cells by LMP-1 that copies a previously unknown normal phenomenon taking place during antigen driven B cell activation. The functional remodelling of ER calcium homeostasis by down-regulation of SERCA3 expression constitutes a previously unknown mechanism involved in EBV-induced B cell immortalisation.
- Published
- 2009
177. Synthesis of a difluoromethylenephosphonate analogue of AZT 5'-triphosphate and its inhibition of HIV-1 reverse transcriptase
- Author
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Jan Balzarini, D. Hebel, Tünde Kovács, Krystyna Lesiak, Paul F. Torrence, Kenneth L. Kirk, Junei Kinjo, and E. De Clercq
- Subjects
Stereochemistry ,Chemistry ,Organic Chemistry ,Clinical Biochemistry ,Human immunodeficiency virus (HIV) ,virus diseases ,Pharmaceutical Science ,Ester hydrolysis ,medicine.disease_cause ,Biochemistry ,Reverse transcriptase ,Coupling (electronics) ,Drug Discovery ,medicine ,Molecular Medicine ,Molecular Biology ,Competitive inhibitor - Abstract
Difluoromethylenebisphosphonic acid was prepared by acetyl hypofluorite-mediated fluorination of tetraisopropyl methylenebisphosphonate and ester hydrolysis. Coupling to 3'-azido-3'-deoxythymidine 5'-monophosphate gave the title compound. The difluoromethylenephosphonate was 30-fold less effective than AZT-triphosphate as a competitive inhibitor of HIV-1 reverse transcriptase but 10-fold more effective than the methylenephosphonate analogue.
- Published
- 1991
178. External cell control polymerase chain reaction: replacing internal standards with an unbiased strategy for quantitative polymerase chain reaction normalization
- Author
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Polett Ribiczey, Attila Tordai, Tünde Kovács, Mária Magócsi, András Váradi, Gabriella Köblös, Tamás Arányi, András Bors, Anna Brózik, and Zsuzsanna Újfaludi
- Subjects
Normalization (statistics) ,Inverse polymerase chain reaction ,Biophysics ,Cell Biology ,Computational biology ,Biology ,Biochemistry ,Polymerase Chain Reaction ,Sensitivity and Specificity ,law.invention ,Cell Line ,Reverse transcription polymerase chain reaction ,Polymerase chain reaction optimization ,Real-time polymerase chain reaction ,law ,Multiplex polymerase chain reaction ,Animals ,Drosophila ,RNA, Messenger ,Molecular Biology ,Nested polymerase chain reaction ,Polymerase chain reaction - Published
- 2007
179. The loss of sarco/endoplasmic reticulum calcium transport ATPase 3 expression is an early event during the multistep process of colon carcinogenesis
- Author
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Jocelyne Enouf, Pascal Gélébart, Tünde Kovács, Béla Papp, Jean-Philippe Brouland, and Johannes Grossmann
- Subjects
Pathology ,medicine.medical_specialty ,Beta-catenin ,SERCA ,Adenomatous polyposis coli ,Cellular differentiation ,Adenomatous Polyposis Coli Protein ,Blotting, Western ,Colonic Polyps ,Calcium-Transporting ATPases ,Biology ,medicine.disease_cause ,Endoplasmic Reticulum ,Transfection ,Pathology and Forensic Medicine ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Cell Line, Tumor ,medicine ,Biomarkers, Tumor ,Humans ,Transgenes ,Intestinal Mucosa ,beta Catenin ,Endoplasmic reticulum ,TCF4 ,Immunohistochemistry ,digestive system diseases ,DNA-Binding Proteins ,Original Research Paper ,Cytoskeletal Proteins ,Cell Transformation, Neoplastic ,Hyperplastic Polyp ,Colonic Neoplasms ,biology.protein ,Cancer research ,Trans-Activators ,Calcium ,Carcinogenesis ,TCF Transcription Factors ,Transcription Factor 7-Like 2 Protein ,Transcription Factors - Abstract
Calcium accumulation in the endoplasmic reticulum is accomplished by sarco/endoplasmic reticulum calcium transport ATPases (SERCA enzymes). To better characterize the role of SERCA3 in colon carcinogenesis, its expression has been investigated in colonic epithelium, benign lesions, adenomas, and adenocarcinomas. In addition, the regulation of SERCA3 expression was analyzed in the context of the adenomatous polyposis coli/beta-catenin/T-cell factor 4 (TCF4) pathway and of specificity protein 1 (Sp1)-like factor-dependent transcription. We report that SERCA3 expression increased along the crypts as cells differentiated in normal colonic mucosa and in hyperplastic polyps, was moderately and heterogeneously expressed in colonic adenomas with expression levels inversely correlated with the degree of dysplasia, was barely detectable in well and moderately differentiated adenocarcinomas, and was absent in poorly differentiated tumors. Inhibition of Sp1-like factor-dependent transcription blocked SERCA3 expression during cell differentiation, and SERCA3 expression was induced by the expression of dominant-negative TCF4 in colon cancer cells. These data link SERCA3 expression to the state of differentiation of colonic epithelial cells, and relate SERCA3 expression, already decreased in adenomas, to enhanced adenomatous polyposis coli/beta-catenin/TCF4-dependent signaling and deficient Sp1-like factor-dependent transcription. In conclusion, intracellular calcium homeostasis becomes progressively anomalous during colon carcinogenesis as reflected by deficient SERCA3 expression.
- Published
- 2005
180. All three splice variants of the human sarco/endoplasmic reticulum Ca2+-ATPase 3 gene are translated to proteins: a study of their co-expression in platelets and lymphoid cells
- Author
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Tünde KOVÁCS, Ferenc FELFÖLDI, Béla PAPP, Katalin PÁSZTY, Raymonde BREDOUX, Ágnes ENYEDI, and Jocelyne ENOUF
- Subjects
Blood Platelets ,Transcription, Genetic ,T-Lymphocytes ,Molecular Sequence Data ,Genetic Variation ,Cell Biology ,Calcium-Transporting ATPases ,Endoplasmic Reticulum ,Transfection ,Biochemistry ,Peptide Fragments ,Recombinant Proteins ,Cell Line ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,Isoenzymes ,Alternative Splicing ,Epitopes ,Jurkat Cells ,Sarcoplasmic Reticulum ,Humans ,Amino Acid Sequence ,Molecular Biology ,Research Article - Abstract
The molecular cloning of two previously unknown human sarco/endoplasmic reticulum Ca2+-ATPase 3 (SERCA3) 3′-end transcripts, 3b and 3c, has been recently published. Data were lacking, however, for the presence of these SERCA3 variants in different tissue or cell types at the protein level. Here we report the co-expression of three human SERCA3 protein isoforms in platelets and T lymphoid Jurkat cells. Isoform-specific polyclonal anti-peptide antibodies have been generated that recognize specifically the SERCA3a, 3b or 3c splice variants at their C-termini, and this has been confirmed by peptide-competition experiments as well. None of these antibodies cross-reacted with the housekeeping SERCA2b isoform co-expressed endogenously with SERCA3 proteins in non-muscle cells. Although all three SERCA3 isoforms could be detected in platelets, the 3a form was the most abundantly expressed species. Its size matched the apparent size of SERCA3a over-expressed in HEK-293 cells. Immunoprecipitation of the SERCA3 variants from platelet membranes using a PL/IM 430-affinity matrix provided evidence that the putative pan-anti-SERCA3 antibody, PL/IM 430, recognizes all SERCA3 protein isoforms. The epitope for the PL/IM 430 antibody could be localized in a 40kDa N-terminal tryptic fragment common to all three SERCA3 variants. Comparative Western-blot analysis showed that the expression level of the SERCA3a, 3b and 3c isoforms was more than 10 times lower in Jurkat cells than in platelets, whereas expression of the ubiquitous SERCA2b was nearly identical. This work highlights new Ca2+-transporting proteins of haematopoietic cells and provides specific antibodies for their detection.
- Published
- 2001
181. Expression of hPMCA4b, the major form of the plasma membrane calcium pump in megakaryoblastoid cells is greatly reduced in mature human platelets
- Author
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Katalin Pászty, Béla Papp, John T. Penniston, Ágnes Enyedil, Adelaida G. Filoteo, Jocelyne Enouf, Tünde Kovács, and Christine Lacabaratz-Porret
- Subjects
Gene isoform ,Blood Platelets ,Erythrocytes ,Physiology ,medicine.drug_class ,Cell ,Calcium-Transporting ATPases ,Monoclonal antibody ,Sensitivity and Specificity ,Cell Line ,Plasma Membrane Calcium-Transporting ATPases ,medicine ,Animals ,Humans ,Platelet ,Molecular Biology ,Cation Transport Proteins ,biology ,Endoplasmic reticulum ,Antibodies, Monoclonal ,Cell Biology ,Cell biology ,Isoenzymes ,medicine.anatomical_structure ,Biochemistry ,Cell culture ,Polyclonal antibodies ,COS Cells ,biology.protein ,Plasma membrane Ca2+ ATPase ,Megakaryocytes - Abstract
Antibodies 5F10 and JA3 (raised against the erythrocyte Ca 2+ pump) were used to identify hPMCA4b as the major form of the plasma membrane Ca 2+ pump in human platelets and in three human megakaryoblastoid cell lines, MEG 01, DAMI and CHRF 288-11. 5F10 was used because it has been shown to recognize all known isoforms of the hPMCA and JA3 because it reacts exclusively with hPMCA4b [Caride A.J., Filoteo A.G., Enyedi A., Verma A.K., Penniston J.T Detection of isoform 4 of the plasma membrane calcium pump in human tissues by using isoform-specific monoclonal antibodies. Biochem J 1996; 316 : 353–359]. In addition to hPMCA4b, hPMCA1b was also detected in the megakaryoblastoid cells by using isoform-specific polyclonal antibodies. The apparent size of this isoform, however, was smaller than that seen in HeLa and COS-7 cell membranes indicating the presence of a modified form of hPMCA1 b. In platelets, no evidence of the expression of hPMCA1 b could be found. The amount of PMCA in these cells was compared with that of the constitutive form of the sarco/endoplasmic reticulum Ca 2+ pump in non-muscle cells (SERCA2b) and also with the amount of PMCA in human erythrocytes. A very low level of the plasma membrane Ca 2+ pump was found in platelets while in their precursor cells the expression of this Ca 2+ pump was much more abundant. Whereas the expression level of PMCA decreased dramatically in mature human platelets, the expression of SERCA2b did not change substantially upon megakaryocytic differentiation.
- Published
- 1998
182. The platelet Ca2+ transport ATPase system
- Author
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Regis Bobe, Raymonde Bredoux, Elisabeth Corvazier, Tünde Kovács, C. Lacabaratz-Porret, Béla Papp, and Jocelyne Enouf
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Gene isoform ,SERCA ,Endoplasmic reticulum ,ATPase ,P-type ATPase ,biology.protein ,Plasma membrane Ca2+ ATPase ,Hematology ,General Medicine ,Biology ,Intracellular ,Cellular localization ,Cell biology - Abstract
The Ca2+ signal accompanying cell function involves the activities of plasma membrane Ca2+ transport ATPases (PMCA) which transport Ca2+ ions out of the cell and those of sarco/endoplasmic reticulum Ca2+ transport ATPases (SERCA), which pump Ca2+ ions into intracellular Ca2+ pools. Although a platelet Ca2+ transport ATPase was described three decades ago, for a long time it remained poorly understood in terms of its cellular localization and identity. By integrating data obtained during recent years, including newly available information in the literature for the PMCAs and aspects of our work concerning the SERCAs, the present review will show how the overall view of the platelet Ca2+ATPase system has to be modified due to the presence of a number of Ca2+ATPases in these cells. These Ca2+ATPases include a typical 144 kDa PMCA protein, although its molecular identity still remains to be established, expressed together with a multi-SERCA system constituted by the ubiquitous 100 kDa SERCA 2b isoform, the 97 kDa SERCA 3 isoform and a new 97 kDa SERCA isoform recognized by the monoclonal antibody termed PL/IM 430 which also remains to be identified. The new paradigm of the platelet multi-Ca2+ATPase system will be discussed including: (i) the problems solved, as it has now become possible to reconciliate previous contradictory observations and (ii) those which still remain due to the fact that the platelet Ca2+ATPase system is more complex than previously assumed. Finally, to put this complexity of the platelet Ca2+ transport ATPase system into perspective, the biological significance of the multi-SERCA system in the context of Ca2+ signalling will be tentatively discussed in an attempt to produce a model of the organization of the intracellular Ca2+ pools in platelets.
- Published
- 1997
183. The PL/IM 430 and the N 89 antibodies recognize two distinct 97 kDa sarco/endoplasmic-reticulum Ca(2+)-ATPase proteins
- Author
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Elisabeth Corvazier, Christine Lacabaratz, Béla Papp, Regis Bobe, Frank Wuytack, Jocelyne Enouf, and Tünde Kovács
- Subjects
Blood Platelets ,Letter ,ATPase ,Blotting, Western ,Calcium-Transporting ATPases ,Endoplasmic Reticulum ,Biochemistry ,Antibodies monoclonal ,Tumor Cells, Cultured ,Humans ,Platelet ,Molecular Biology ,biology ,Chemistry ,Endoplasmic reticulum ,Antibodies, Monoclonal ,STIM1 ,Cell Biology ,Blotting western ,Molecular biology ,Peptide Fragments ,Molecular Weight ,Sarcoplasmic Reticulum ,Microsome ,biology.protein ,Antibody - Published
- 1996
184. The Effect of Microstructure on the Local Wear Behavior of Heat Treated Structural Steel
- Author
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Péter Pinke and Tünde Kovács-Coskun
- Subjects
Work (thermodynamics) ,Materials science ,Differential equation ,Bainite ,Mechanical Engineering ,Phase (matter) ,Martensite ,Metallurgy ,Wear coefficient ,Microstructure ,Tribometer - Abstract
It is known that the friction and wear properties of metals and alloys show a strong correlation with their chemical composition, hardness and microstructure.The aim of this work was to analyse and evaluate the correlations between the microstructure and the wear properties of low alloyed, heat treated structural steel during dry friction. Three kinds of hypoeutectoid structural steel with different microstructure were studied. For experimental purposes, a new type of micro-scale, ball-cratering tribometer and a proper wear-kinetic model based on an ordinary differential equation have been constructed. It was verified that if the process parameters (load, angular speed) are constant, the solution of the wear-kinetic differential equation could be expressed in a simple, closed form. Additionally, it was shown that (i) the heat treated steels have the highest wear resistance if the microstructure consists of only one hard martensitic phase, (ii) in case of microstructures consisting of two different phases (fer rite-pearlite, bainite, and spheroidite) the wear resistance decreases in the following order: bainite, ferrite-pearlite, and spheroidite.
- Published
- 2011
185. Membrane depolarization inhibits thrombin-induced calcium influx and aggregation in human platelets
- Author
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Balázs Sarkadi, Attila Tordai, Tünde Kovács, G. Gárdos, and Ilma Szász
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Blood Platelets ,Cytoplasm ,Platelet Aggregation ,Biophysics ,chemistry.chemical_element ,Calcium ,In Vitro Techniques ,Biochemistry ,Calcium in biology ,Membrane Potentials ,chemistry.chemical_compound ,Aggregation ,Receptor-operated calcium channel ,Structural Biology ,Calcium flux ,Genetics ,Humans ,Molecular Biology ,Egtazic Acid ,Membrane potential ,Voltage-dependent calcium channel ,Gramicidin ,Thrombin ,Depolarization ,Cell Biology ,Calcium ATPase ,chemistry ,Human platelet ,Signal Transduction - Abstract
The relationship between thrombin-evoked changes in intracellular calcium concentration ([Ca2+]i) and aggregation was examined in Indo-1-loaded human platelets. The stimulus-induced intracellular calcium release and external calcium influx, as well as platelet aggregation, were studied in the same cell preparation. A close correlation between the sustained high [Ca2+]i level, depending on calcium entry, and the aggregation response was found. Gramicidin, at a concentration high enough to induce membrane depolarization, strongly inhibited the calcium influx and aggregation, but did not influence the thrombin-induced intracellular calcium release. We conclude that calcium influx through depolarization-inhibited calcium channels is a prerequisite of thrombin-induced platelet aggregation.
- Published
- 1990
186. Conditions of formation of the heparin-fibronectin-collagen complex and the effect of plasmin
- Author
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István Léránt, Raymund Machovich, Tünde Kovács, Zsolt Nagy, and Béla Papp
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Plasmin ,Proteolysis ,Biophysics ,chemistry.chemical_element ,Sodium Chloride ,Calcium ,Binding, Competitive ,Biochemistry ,Glycosaminoglycan ,Extracellular matrix ,medicine ,Chemical Precipitation ,Humans ,Fibrinolysin ,Molecular Biology ,Ternary complex ,Hexadimethrine Bromide ,medicine.diagnostic_test ,biology ,Heparin ,Hydrolysis ,Fibronectins ,Fibronectin ,Kinetics ,Solubility ,chemistry ,biology.protein ,Collagen ,Protein Binding ,medicine.drug - Abstract
The formation and composition of the insoluble heparin-fibronectin-collagen complex and its degradation by proteolysis was investigated. At fixed concentrations of the other molecular components of the complex, the maximal rate of complex formation, measured turbidimetrically, was reached at a concentration of 4 microM heparin and 0.9 microM collagen, while the rate of complex formation was linearly related to concentrations of fibronectin as high as 3 microM. Heparin was incorporated into the complex in a saturable manner, and was released in active anticoagulant form by plasmin but not by urokinase. The complex formation was inhibited by 5 mM calcium or 250 mM NaCl as well as by polybrene or spermin. It is suggested that fibronectin binds both heparin and collagen cooperatively to form an insoluble ternary complex of the extracellular matrix.
- Published
- 1987
187. Incorporation of the carbocyclic analogue of (E)-5-(2-bromovinyl)-2′-deoxyuridine 5′-triphosphate into a synthetic DNA
- Author
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Tünde Kovács, Janos Sagi, Á. H. Csárnyi, A. Szemző, L. Ötvös, and E. De Clercq
- Subjects
Stereochemistry ,DNA polymerase ,Biophysics ,Nucleic Acid Denaturation ,Biochemistry ,Substrate Specificity ,chemistry.chemical_compound ,Thymine Nucleotides ,Molecular Biology ,Chromatography, High Pressure Liquid ,Polymerase ,Klenow fragment ,DNA clamp ,biology ,Chemistry ,Spectrum Analysis ,DNA ,Templates, Genetic ,Cell Biology ,DNA Polymerase I ,Peptide Fragments ,Deoxyuridine ,Bromodeoxyuridine ,biology.protein ,Nucleic Acid Conformation ,Primer (molecular biology) ,DNA polymerase I ,Deoxyuracil Nucleotides - Abstract
The carbocyclic analogue of (E)-5-(2-bromovinyl)-2'-deoxyuridine, C-BVDU, is a very potent and selective anti-herpes-virus compound. In order to synthesize and study the properties of a DNA that contains C-BVDU, the 5'-triphosphate, C-BVDUTP was prepared and evaluated as a potential substrate of the E. coli Klenow DNA polymerase enzyme. Although C-BVDUTP proved to be a very poor substrate also of this enzyme, it could be incorporated up to 3.6% into the synthetic DNA, poly(dA-dT, C-BVDU). This level of substitution decreased significantly the template activity for DNA and RNA polymerases, as compared to that of poly(dA-dT).
- Published
- 1987
188. Expression of sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) 3 proteins in two major conformational states in native human cell membranes
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Raymonde Bredoux, Tünde Kovács, Jocelyne Enouf, and Elisabeth Corvazier
- Subjects
Blood Platelets ,Gene isoform ,SERCA ,Isoform ,Protein Conformation ,Molecular Sequence Data ,Biophysics ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Cell Line ,Sarcoplasmic Reticulum Calcium-Transporting ATPases ,law.invention ,Fluorides ,chemistry.chemical_compound ,HEK-293 cell ,law ,Humans ,Trypsin ,Amino Acid Sequence ,Aluminum Compounds ,Site-directed mutagenesis ,Egtazic Acid ,Gene ,Cell Membrane ,Mutagenesis ,Platelet ,tBHQ ,Cell Biology ,Sarco/endoplasmic reticulum Ca2+ ATPase ,Peptide Fragments ,Hydroquinones ,Isoenzymes ,Blot ,EGTA ,Ca2+ ,SERCA3 ,chemistry ,Recombinant DNA ,Thapsigargin ,Calcium ,TG ,Proteolysis trypsin - Abstract
The SERCA family includes 3 genes (SERCA1-3), each of which giving rise to various isoforms. To date, detailed structural data is only available for the SERCA1a isoform. Here, limited trypsinolysis of either human platelet membranes or recombinant SERCA3a in HEK-293 cells followed by Western blotting using antibodies covering different regions of the SERCA3(a) protein revealed two, kinetically distinct, Early (ETF) and Late (LTF) Tryptic Fragmentations. The ETF uses many tryptic sites while the LTF uses a unique tryptic site. Using site-directed mutagenesis: i) Arg(334), Arg(396) and Arg(638) were directly assigned to the ETF and ii) Arg(198) was assigned as the only tryptic site to the LTF. Arg(671), Lys(712)/Lys(713) and Lys(728) were also found to modulate the ETF. SERCA inhibitors Tg and tBHQ induced modest inhibition of the ETF. In contrast, the addition of CaCl(2), EGTA or AlF(4)(-) strikingly modified the ETF without any effect on the LTF. Trypsinolysis of the other recombinant SERCA3b-3f isoforms revealed: i) same ETF and LTF as SERCA3a, with variations of the length of the C-terminal fragments; ii) Arg(1002) as an additional tryptic site in SERCA3b-3e isoforms. Taken together, the two distinct SERCA3 fragmentation profiles sign the co-expression of SERCA3 proteins in two conformational states in cell membranes.
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189. Internal Structures of the Developing Brain Assessed by HDlive Imaging
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Florin Stamatian, Dan Boitor-Borza, and Tunde Kovacs
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HDlive, Silhouette mode, developing brain, ganglionic eminences. ,Gynecology and obstetrics ,RG1-991 - Abstract
OBJECTIVES: The aim of this observational descriptive study of morphological research is to assess the nervous structures within the embryonic and early fetal brains not previously documented in literature by HDlive and Silhouette® modes. STUDY DESIGN: A total of 26 subjects were examined in vivo, i.e. 15 embryos and 11 fetuses in the first trimester of pregnancy (7 to 13 gestational weeks (GW)), using a transvaginal ultrasound using a Voluson E10, BT 15 scanner (GE Healthcare, Zipf, Austria). RESULTS: The clear visualization of the brain structures by HDlive rendering mode was possible in all 26 selected optimal volumes. The most representative images for each week of gestation are shown. At 7 GW the ultrasound semiology of the brain is simple. The choroid plexuses can be seen in the 4th ventricle at 8 GW and in the lateral ventricles at 9 GW by HDlive mode. At 9 GW the brain is developed enough so that the walls of the cerebral hemispheres, the ventricular system and the rhombic lips could be visualized by HDlive mode. At 10 GW we depicted the ganglionic eminences within the brain by HDlive mode. At 11 GW the thalamus was noticed. At 12 GW and 13 GW, HDlive images of choroid plexus asymmetry and choroid plexuses cysts are shown. CONCLUSION: The HDlive combined with Silhouette® mode can provide almost natural images of the internal structures of the embryonic and early fetal brain. Small-sized nervous structures such as the
- Published
- 2016
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190. Thrombin-induced activation of calcium transport pathways and their role in platelet functions
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Balázs Sarkadi, Tünde Kovács, Mária Magócsi, and G. Gárdos
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Blood Platelets ,Platelet Aggregation ,Biophysics ,Calcium ion transport ,chemistry.chemical_element ,Calcium ,Indo-1 ,Phosphatidylinositols ,Biochemistry ,Calcium in biology ,chemistry.chemical_compound ,Adenosine Triphosphate ,Humans ,Voltage-dependent calcium channel ,Calcium channel ,T-type calcium channel ,Thrombin ,Biological Transport ,Neomycin ,Cell Biology ,Calcium Channel Blockers ,Calcium ATPase ,chemistry - Abstract
In human platelets thrombin-induced calcium release from intracellular stores, the consequent influx of extracellular calcium, as well as their role in the aggregation and ATP-secretion reactions were examined. In indo-1-loaded platelets intracellular calcium release was studied in the presence of excess EGTA in the incubation medium, while calcium influx was followed after a rapid repletion of external calcium. After thrombin-stimulation both calcium release and calcium influx produced about the same peak levels of cytoplasmic free calcium but in the first case it was only a transient response, while in the latter one a sustained calcium signal was observed. Increased calcium influx could be evoked for several minutes after the addition of thrombin, it was selectively inhibited by Mg2+ (20 mM) and Ni2+ (1 mM) ions, by neomycin and by PCMB, a non-penetrating SH-group reagent. This calcium influx was practically insensitive to organic calcium channel blockers. Thrombin-induced platelet aggregation was only partial in the absence of external calcium, even if excess magnesium was present in the media, while the aggregation response became complete if external calcium was repleted. A significantly reduced aggregation could be seen in calcium-containing media if calcium influx was selectively inhibited. Platelet ATP-secretion under the same conditions did not depend on external calcium or on calcium influx. These data indicate that in thrombin-stimulated platelets the opening of specific plasma membrane calcium channels can be selectively modulated and these channels play a major role in the development of a full-scale aggregation.
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- 1989
191. Interaction of thrombin, antithrombin III and their complex with hepatocytes: comparison of the molecular components of human and mouse origin
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Miklós Péter Kalapos, Tamás Garzó, Ferenc A. Antoni, Tünde Kovács, Zoltan Spolarics, József Mandl, and Raymund Machovich
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Antithrombin III ,Mice, Inbred Strains ,Binding, Competitive ,Iodine Radioisotopes ,Mice ,Thrombin ,Species Specificity ,medicine ,Animals ,Humans ,Hemostasis ,Binding Sites ,biology ,Thrombin/Antithrombin III ,Hematology ,Molecular biology ,Molecular Weight ,medicine.anatomical_structure ,Biochemistry ,Liver ,Enzyme inhibitor ,Hepatocyte ,biology.protein ,medicine.drug ,Protein Binding - Published
- 1987
192. Interaction of antithrombin III and thrombin-antithrombin III complex with cultured aortic endothelial cells
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Bella Papp, Katalin Bartha, Tünde Kovács, Raymund Machovich, István Léránt, Éva Csonka, and Krasimir Kolev
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Binding Sites ,Chemistry ,Antithrombin ,Antithrombin III ,Thrombin ,Hematology ,Heparin ,biological factors ,Receptor–ligand kinetics ,carbohydrates (lipids) ,Endothelial stem cell ,Kinetics ,Biochemistry ,Cell culture ,hemic and lymphatic diseases ,medicine ,Animals ,cardiovascular diseases ,Endothelium, Vascular ,Binding site ,Cells, Cultured ,circulatory and respiratory physiology ,medicine.drug ,Binding domain - Abstract
The binding of antithrombin III, thrombin, thrombin-antithrombin III complex to endothelial cells was investigated. While the rate of the binding of thrombin to these cells was very rapid, that of antithrombin III was relatively slow and the thrombin-antithrombin III complex was intermediate. Binding kinetics indicated that antithrombin III, like thrombin, showed high affinity to endothelial cells; with a Kd of 3×10 −8 M and with 5×10 4 binding sites per cell. The dissociation of the inhibitor molecule was also rapid, i.e., approximately 70% bound antithrombin III was released in 2 minutes. Heparin, in a 100-fold molar excess to antithrombin III, or the modification of lysine residues of the inhibitor involved in the interaction with heparin, did not influence the association of antithrombin III with endothelial cells. In addition, antithrombin III did not compete with thrombin blocked in its active center for binding to endothelial cells. It is suggested that the binding sites of endothelial cells are different for thrombin and antithrombin III, and antithrombin III does not bind to these cells through its heparin binding domain.
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- 1987
193. Demonstration of two forms of calcium pumps by thapsigargin inhibition and radioimmunoblotting in platelet membrane vesicles
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Tünde Kovács, Balázs Sarkadi, Sylviane Levy-Toledano, Ole Thastrup, Raymonde Bredoux, Ágnes Enyedi, Béla Papp, Jocelyne Enouf, Frank Wuytack, and G. Gárdos
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Blood Platelets ,Thapsigargin ,Calcium pump ,ATPase ,Blotting, Western ,chemistry.chemical_element ,Diaphragm pump ,Calcium-Transporting ATPases ,Calcium ,Biochemistry ,Antibodies ,chemistry.chemical_compound ,Humans ,Trypsin ,Molecular Biology ,biology ,Terpenes ,Chemistry ,Hydrolysis ,Endoplasmic reticulum ,Cell Membrane ,Biological membrane ,Cell Biology ,Isoenzymes ,Calcium ATPase ,Sarcoplasmic Reticulum ,biology.protein ,Autoradiography - Abstract
In mixed membrane vesicles prepared from human platelets, the presence of two distinct calcium pump enzymes (molecular mass 100 and 97 kDa) was demonstrated by 32P autoradiography, immunoblotting, and thapsigargin inhibition. Both the 100- and 97-kDa membrane proteins showed calcium-dependent phosphoenzyme formation and reacted with a polyclonal anti-sarcoplasmic reticulum calcium pump antiserum, while only the 100-kDa protein reacted with the antiserum specific for the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform. Thapsigargin, inhibiting active calcium transport in platelet membrane vesicles, predominantly blocked the phosphoenzyme formation of the 100-kDa isoform and of the tryptic calcium pump fragments of 55 and 35 kDa, while lanthanum specifically increased the phosphoenzyme formation of the 97-kDa enzyme and of the tryptic fragment of 80 kDa. These results indicate the presence of the sarco-endoplasmic reticulum-type calcium transport ATPase 2b isoform and of a yet unidentified, 97-kDa calcium pump protein in human platelet membranes.
194. Simultaneous presence of two distinct endoplasmic-reticulum-type calcium-pump isoforms in human cells. Characterization by radio-immunoblotting and inhibition by 2,5-di-(t-butyl)-1,4-benzohydroquinone
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Jocelyne Enouf, G. Gárdos, Ágnes Enyedi, Tünde Kovács, Béla Papp, Frank Wuytack, Katalin Pászty, C Magnier, and Balázs Sarkadi
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Blood Platelets ,Calcium pump ,ATPase ,Immunoblotting ,Diaphragm pump ,Calcium-Transporting ATPases ,Endoplasmic Reticulum ,Biochemistry ,Tumor Cells, Cultured ,Humans ,Trypsin ,Lymphocytes ,Phosphorylation ,Molecular Biology ,Molecular mass ,biology ,Myocardium ,Endoplasmic reticulum ,Autophosphorylation ,Antibodies, Monoclonal ,Muscle, Smooth ,Cell Biology ,Molecular biology ,Peptide Fragments ,Hydroquinones ,Molecular Weight ,Calcium ATPase ,biology.protein ,Megakaryocytes ,Intracellular ,Research Article - Abstract
Phosphorylation, immunoblotting, limited proteolysis and drug-sensitivity analysis were used to characterize the sarcoendoplasmic-reticulum Ca2+ ATPases in a variety of human cell types. In platelets, several megakaryoblastoid and lymphoblastoid cell lines two distinct autophosphorylated forms of these ATPases with molecular mass of 100 and 97 kDa could be observed, whereas in several other cell types the 97 kDa form was absent. On immunoblots the 97 kDa species was specifically recognized by an inhibitory monoclonal antibody raised against the Ca2+ pump of platelet internal membranes, yielded on trypsinolysis a major fragment of 80 kDa, exhibited a distinct electrophoretic migration pattern as compared with the skeletal-, cardiac- and smooth-muscle Ca2+ pumps, and its autophosphorylation was strongly inhibited by the Ca(2+)-mobilizing agent 2,5-di-(t-butyl)-1,4-benzohydroquinone (tBHQ). The 100 kDa species reacted with an antibody specific for the cardiac- and smooth-muscle Ca2+ pumps, yielded on trypsinolysis fragments of 55 and 35 kDa, and its autophosphorylation was much less sensitive to tBHQ inhibition. These findings indicate the simultaneous presence of two different endoplasmic-reticulum Ca2+ pumps in a variety of human cell types, and may explain the previously observed differences in the Ca(2+)-handling characteristics of different intracellular Ca2+ pools and cell types.
195. Controlled proteolysis of Ca(2+)-ATPases in human platelet and non-muscle cell membrane vesicles. Evidence for a multi-sarco/endoplasmic reticulum Ca(2+)-ATPase system
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Jocelyne Enouf, Ágnes Enyedi, Elisabeth Corvazier, Béla Papp, Balázs Sarkadi, Raymonde Bredoux, Clarice Magnier, and Tünde Kovács
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Blood Platelets ,SERCA ,Proteolysis ,ATPase ,Calcium-Transporting ATPases ,Biology ,Endoplasmic Reticulum ,Biochemistry ,Peptide Mapping ,Cell membrane ,medicine ,Tumor Cells, Cultured ,Humans ,Trypsin ,Molecular Biology ,Cells, Cultured ,medicine.diagnostic_test ,Endoplasmic reticulum ,Hydrolysis ,Cell Membrane ,Cell Biology ,Molecular biology ,Trypsinization ,Isoenzymes ,Kinetics ,Sarcoplasmic Reticulum ,medicine.anatomical_structure ,Membrane protein ,biology.protein ,medicine.drug - Abstract
Two sarco/endoplasmic reticulum Ca(2+)-ATPases (SERCAs) have been previously identified in platelets: the 100-kDa SERCA2b and the 97-kDa SERCA3 isoforms. Analysis of the acylphosphate intermediate (E-P) formation and the immunoreactivity of the platelet Ca(2+)-ATPases and their proteolytic fragments upon controlled trypsinolysis revealed the presence of an additional 97-kDa Ca(2+)-ATPase that comigrates with SERCA3 on SDS-polyacrylamide gels. At a trypsin/membrane protein ratio of 0.025 at 4 degrees C, tryptic fragments of 73-, 68- and 40-kDa, previously unknown in the SERCA family, could be detected by using the PL/IM 430 anti-Ca(2+)-ATPase antibody that had been shown to recognize a 97-kDa Ca(2+)-ATPase. The 73- and 68-kDa fragments were precursors of the 40-kDa one. Ca(2+)-dependent phospholabeling of the 73-kDa fragment and immunostaining of all these proteolytic products by another antibody raised against SERCA1 established the SERCA nature of the 97-kDa parent enzyme. The SERCA3-related E-P-forming 80-kDa tryptic fragment appeared during trypsinolysis with a different time course from that of the 73-, 68-, and 40-kDa ones. At a trypsin/membrane protein ratio of 0.125 at 37 degrees C, it reached its maximum level at 5 min of digestion, while the 73-, 68-, and 40-kDa fragments were fully degraded at 2 min of trypsinization. This 80-kDa species was immunostained neither with the PL/IM 430, nor with the anti-SERCA1 antibodies. Similar results were found in some megakaryoblastoid and lymphoblastoid cell lines. All these data indicate the presence of two distinct tryptic fragmentation patterns attributed to two 97-kDa SERCA isoforms and point to the existence of a multi-SERCA system in different human non-muscle cells.
196. Impact of Medium-Sized Extracellular Vesicles on the Transduction Efficiency of Adeno-Associated Viruses in Neuronal and Primary Astrocyte Cell Cultures
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Orsolya Tünde Kovács, Eszter Soltész-Katona, Nikolett Marton, Eszter Baricza, László Hunyady, Gábor Turu, and György Nagy
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adeno-associated virus ,AAV ,extracellular vesicles ,microvesicles ,N2A ,primary astrocyte cells ,Biology (General) ,QH301-705.5 ,Chemistry ,QD1-999 - Abstract
(1) Adeno-associated viruses (AAV) are safe and efficient gene therapy vectors with promising results in the treatment of several diseases. Extracellular vesicles (EV) are phospholipid bilayer-surrounded structures carrying several types of lipids, proteins, and nucleic acids with the ability to cross biological barriers. EV-associated AAVs might serve as new and efficient gene therapy vectors considering that they carry the benefits of both AAVs and EVs. (2) We tested vesicle-associated AAVs and vesicles mixed with AAVs on two major cell types of the central nervous system: a neural cell line (N2A) and primary astrocyte cells. (3) In contrast to previously published in vivo observations, the extracellular vesicle packaging did not improve but, in the case of primary astrocyte cells, even inhibited the infection capacity of the AAV particles. The observed effect was not due to the inhibitory effects of the vesicles themselves, since mixing the AAVs with extracellular vesicles did not change the effectiveness. (4) Our results suggest that improvement of the in vivo efficacy of the EV-associated AAV particles is not due to the enhanced interaction between the AAV and the target cells, but most likely to the improved delivery of the AAVs through tissue barriers and to the shielding of AAVs from neutralizing antibodies.
- Published
- 2021
- Full Text
- View/download PDF
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