Pál Gergely

Publications of Pál Gergely

• Poly(ADP-ribose) polymerase-2: emerging transcriptional roles of a DNA-repair protein.

  Authors: Magdolna Szántó, Attila Brunyánszki, Borbála Kiss, Lilla Nagy, Pál Gergely, László Virág, Péter Bai

  Cellular and molecular life sciences : CMLS. 05/2012;

  Poly(ADP-ribose) polymerase (PARP)-2 is a nuclear enzyme that belongs to the PARP family and PARP-2 is responsible for 5-15 % of total cellular PARP activity. PARP-2 was originally described inPoly(ADP-ribose) polymerase (PARP)-2 is a nuclear enzyme that belongs to the PARP family and PARP-2 is responsible for 5-15 % of total cellular PARP activity. PARP-2 was originally described in connection to DNA repair and in physiological and pathophysiological processes associated with genome maintenance (e.g., centromere and telomere protection, spermiogenesis, thymopoiesis, azoospermia, and tumorigenesis). Recent reports have identified important rearrangements in gene expression upon the knockout of PARP-2. Such rearrangements heavily impact inflammation and metabolism. Metabolic effects are mediated through modifying PPARγ and SIRT1 function. Altered gene expression gives rise to a complex phenotype characterized primarily by enhanced mitochondrial activity that results both in beneficial (loss of fat, enhanced insulin sensitivity) and in disadvantageous (pancreatic beta cell hypofunction upon high fat feeding) consequences. Enhanced mitochondrial biogenesis provides protection in oxidative stress-related diseases. Hereby, we review the recent developments in PARP-2 research with special attention to the involvement of PARP-2 in transcriptional and metabolic regulation.

• Synthesis of heterocyclic N-(β-d-glucopyranosyl)carboxamides for inhibition of glycogen phosphorylase.

  Authors: Bálint Kónya, Tibor Docsa, Pál Gergely, László Somsák

  Carbohydrate research. 04/2012; 351:56-63.

  In a DCC-mediated coupling 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine and propiolic acid gave N-propynoyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine which was transformed by 1,3-dipolarIn a DCC-mediated coupling 2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine and propiolic acid gave N-propynoyl-2,3,4,6-tetra-O-acetyl-β-d-glucopyranosylamine which was transformed by 1,3-dipolar cycloadditions with aromatic azides and nitrile-oxides to the corresponding O-peracetylated N-(β-d-glucopyranosyl)-1-substituted-1,2,3-triazole-4-carboxamides and N-(β-d-glucopyranosyl)-3-substituted-isoxazole-5-carboxamides, respectively. These compounds were O-deacetylated by Zemplén's protocol to be tested as inhibitors of rabbit muscle glycogen phosphorylase b. The best inhibitors of the two series were N-(β-d-glucopyranosyl)-1-(3,5-dimethyl-phenyl)-1,2,3-triazole-4-carboxamide (K(i)=34μM) and N-(β-d-glucopyranosyl)-3-(indol-2-yl)-isoxazole-5-carboxamide (K(i)=164μM).

• N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl)ureas as inhibitors of glycogen phosphorylase: Synthesis and evaluation by kinetic, crystallographic, and molecular modelling methods.

  Authors: Veronika Nagy, Nóra Felföldi, Bálint Kónya, Jean-Pierre Praly, Tibor Docsa, Pál Gergely, Evangelia D Chrysina, Costas Tiraidis, Magda N Kosmopoulou, Kyra-Melinda Alexacou, Maria Konstantakaki, Demetres D Leonidas, Spyros E Zographos, Nikos G Oikonomakos, Stanislav Kozmon, Igor Tvaroška, László Somsák

  Bioorganic & medicinal chemistry. 03/2012; 20(5):1801-16.

  N-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylatedN-(4-Substituted-benzoyl)-N'-(β-d-glucopyranosyl) ureas (substituents: Me, Ph, Cl, OH, OMe, NO(2), NH(2), COOH, and COOMe) were synthesised by ZnCl(2) catalysed acylation of O-peracetylated β-d-glucopyranosyl urea as well as in reactions of O-peracetylated or O-unprotected glucopyranosylamines and acyl-isocyanates. O-deprotections were carried out by base or acid catalysed transesterifications where necessary. Kinetic studies revealed that most of these compounds were low micromolar inhibitors of rabbit muscle glycogen phosphorylase b (RMGPb). The best inhibitor was the 4-methylbenzoyl compound (K(i)=2.3μM). Crystallographic analyses of complexes of several of the compounds with RMGPb showed that the analogues exploited, together with water molecules, the available space at the β-pocket subsite and induced a more extended shift of the 280s loop compared to RMGPb in complex with the unsubstituted benzoyl urea. The results suggest the key role of the water molecules in ligand binding and structure-based ligand design. Molecular docking study of selected inhibitors was done to show the ability of the binding affinity prediction. The binding affinity of the highest scored docked poses was calculated and correlated with experimentally measured K(i) values. Results show that correlation is high with the R-squared (R(2)) coefficient over 0.9.

• Cell Cycle Dependent Association of EBP50 with Protein Phosphatase 2A in Endothelial Cells.

  Authors: Anita Boratkó, Pál Gergely, Csilla Csortos

  PloS one. 01/2012; 7(4):e35595.

  Ezrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in theEzrin-radixin-moesin (ERM)-binding phosphoprotein 50 (EBP50) is a phosphorylatable PDZ domain-containing adaptor protein that is abundantly expressed in epithelium but was not yet studied in the endothelium. We report unusual nuclear localization of EBP50 in bovine pulmonary artery endothelial cells (BPAEC). Immunofluorescent staining and cellular fractionation demonstrated that EBP50 is present in the nuclear and perinuclear region in interphase cells. In the prophase of mitosis EBP50 redistributes to the cytoplasmic region in a phosphorylation dependent manner and during mitosis EBP50 co-localizes with protein phosphatase 2A (PP2A). Furthermore, in vitro wound healing of BPAEC expressing phospho-mimic mutant of EBP50 was accelerated indicating that EBP50 is involved in the regulation of the cell division. Cell cycle dependent specific interactions were detected between EBP50 and the subunits of PP2A (A, C, and Bα) with immunoprecipitation and pull-down experiments. The interaction of EBP50 with the Bα containing form of PP2A suggests that this holoenzyme of PP2A can be responsible for the dephosphorylation of EBP50 in cytokinesis. Moreover, the results underline the significance of EBP50 in cell division via reversible phosphorylation of the protein with cyclin dependent kinase and PP2A in normal cells.

• Poly(ADP-ribose) polymerase-2 depletion reduces doxorubicin-induced damage through SIRT1 induction.

  Authors: Magdolna Szántó, Ibolya Rutkai, Csaba Hegedus, Ágnes Czikora, Máté Rózsahegyi, Borbála Kiss, László Virág, Pál Gergely, Attila Tóth, Péter Bai

  Cardiovascular research. 09/2011; 92(3):430-8.

  Doxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turnDoxorubicin (DOX) is widely used in cytostatic treatments, although it may cause cardiovascular dysfunction as a side effect. DOX treatment leads to enhanced free radical production that in turn causes DNA strand breakage culminating in poly(ADP-ribose) polymerase (PARP) activation and mitochondrial and cellular dysfunction. DNA nicks can activate numerous enzymes, such as PARP-2. Depletion of PARP-2 has been shown to result in a protective phenotype against free radical-mediated diseases, suggesting similar properties in the case of DOX-induced vascular damage. PARP-2(+/+) and PARP-2(-/-) mice and aortic smooth muscle (MOVAS) cells were treated with DOX (25 mg/kg or 3 μM, respectively). Aortas were harvested 2-day post-treatment while MOVAS cells were treated with DOX for 7 hours. Aortas from PARP-2(-/-) mice displayed partial protection against DOX toxicity, and the protection depended on the conservation of smooth muscle but not on the conservation of endothelial function. DOX treatment evoked free radical production, DNA breakage and PARP activation. Importantly, depletion of PARP-2 did not quench any of these phenomena, suggesting an alternative mechanism. Depletion of PARP-2 prevented DOX-induced mitochondrial dysfunction through SIRT1 activation. Genetic deletion of PARP-2 resulted in the induction of the SIRT1 promoter and consequently increased SIRT1 expression both in aortas and in MOVAS cells. SIRT1 activation enhanced mitochondrial biogenesis, which provided protection against DOX-induced mitochondrial damage. Our data identify PARP-2 as a mediator of DOX toxicity by regulating vascular SIRT1 activity and mitochondrial biogenesis. Moreover, to the best of our knowledge, this is the first report of SIRT1 as a protective factor in the vasculature upon oxidative stress.

• Ecto-5'-nucleotidase (CD73) decreases mortality and organ injury in sepsis.

  Authors: György Haskó, Balázs Csóka, Balázs Koscsó, Rachna Chandra, Pál Pacher, Linda F Thompson, Edwin A Deitch, Zoltán Spolarics, László Virág, Pál Gergely, Rolando H Rolandelli, Zoltán H Németh

  Journal of immunology (Baltimore, Md. : 1950). 09/2011; 187(8):4256-67.

  The extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of theThe extracellular concentrations of adenosine are increased during sepsis, and adenosine receptors regulate the host's response to sepsis. In this study, we investigated the role of the adenosine-generating ectoenzyme, ecto-5'-nucleotidase (CD73), in regulating immune and organ function during sepsis. Polymicrobial sepsis was induced by subjecting CD73 knockout (KO) and wild type (WT) mice to cecal ligation and puncture. CD73 KO mice showed increased mortality in comparison with WT mice, which was associated with increased bacterial counts and elevated inflammatory cytokine and chemokine concentrations in the blood and peritoneum. CD73 deficiency promoted lung injury, as indicated by increased myeloperoxidase activity and neutrophil infiltration, and elevated pulmonary cytokine levels. CD73 KO mice had increased apoptosis in the thymus, as evidenced by increased cleavage of caspase-3 and poly(ADP-ribose) polymerase and increased activation of NF-κB. Septic CD73 KO mice had higher blood urea nitrogen levels and increased cytokine levels in the kidney, indicating increased renal dysfunction. The increased kidney injury of CD73 KO mice was associated with augmented activation of p38 MAPK and decreased phosphorylation of Akt. Pharmacological inactivation of CD73 in WT mice using α, β-methylene ADP augmented cytokine levels in the blood and peritoneal lavage fluid. These findings suggest that CD73-derived adenosine may be beneficial in sepsis.

• Effect of glucopyranosylidene-spiro-thiohydantoin on glycogen metabolism in liver tissues of streptozotocin-induced and obese diabetic rats.

  Authors: Tibor Docsa, Katalin Czifrák, Csaba Hüse, László Somsák, Pál Gergely

  Molecular medicine reports. 05/2011; 4(3):477-81.

  The major role of liver glycogen is to supply glucose to the circulation in order to maintain normal blood glucose levels. In the muscle and liver, the accumulation and breakdown of glycogen areThe major role of liver glycogen is to supply glucose to the circulation in order to maintain normal blood glucose levels. In the muscle and liver, the accumulation and breakdown of glycogen are regulated by the reciprocal activities of glycogen phosphorylase and glycogen synthase. Glycogen phosphorylase catalyses the key step of glycogen degradation and its activity is inhibited by glucose and its analogues. Thus, any readily accessible inhibitor of glycogen phosphorylase may serve as a potential therapy for non-insulin-dependent or type 2 diabetes. Hepatic glycogen phosphorylase has been identified as a novel target for drugs that control blood glucose concentration. Glucopyranosylidene-spiro-thiohydantoin (TH) was found to be one of the most potent glucose derivates, inhibiting the catalytic activity of both muscle and liver glycogen phosphorylase. Here, we demonstrated the co-ordinated regulation of glycogen phosphorylase and synthase by 50 µM TH in liver extracts of Wistar rats, resulting in the activation of synthase by a shortening of the latency compared to control animals. TH was also effective in lowering blood glucose levels and restoring hepatic glycogen content in streptozotocin-induced diabetic rats. Furthermore, intravenous administration of TH to Zucker diabetic fatty rats significantly decreased hepatic glycogen phosphorylase a levels, and the activation of synthase was initiated without any delay.

• PARP-2 regulates SIRT1 expression and whole-body energy expenditure.

  Authors: Péter Bai, Carles Canto, Attila Brunyánszki, Aline Huber, Magdolna Szántó, Yana Cen, Hiroyasu Yamamoto, Sander M Houten, Borbala Kiss, Hugues Oudart, Pál Gergely, Josiane Menissier-de Murcia, Valérie Schreiber, Anthony A Sauve, Johan Auwerx

  Cell metabolism. 04/2011; 13(4):450-60.

  SIRT1 is a NAD(+)-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here, we tested whether deletion of PARP-2, an alternativeSIRT1 is a NAD(+)-dependent enzyme that affects metabolism by deacetylating key transcriptional regulators of energy expenditure. Here, we tested whether deletion of PARP-2, an alternative NAD(+)-consuming enzyme, impacts on NAD(+) bioavailability and SIRT1 activity. Our results indicate that PARP-2 deficiency increases SIRT1 activity in cultured myotubes. However, this increase was not due to changes in NAD(+) levels, but to an increase in SIRT1 expression, as PARP-2 acts as a direct negative regulator of the SIRT1 promoter. PARP-2 deletion in mice increases SIRT1 levels, promotes energy expenditure, and increases mitochondrial content. Furthermore, PARP-2(-/-) mice were protected against diet-induced obesity. Despite being insulin sensitized, PARP-2(-/-) mice were glucose intolerant due to a defective pancreatic function. Hence, while inhibition of PARP activity promotes oxidative metabolism through SIRT1 activation, the use of PARP inhibitors for metabolic purposes will require further understanding of the specific functions of different PARP family members.

• Characterization of the effect of TIMAP phosphorylation on its interaction with protein phosphatase 1.

  Authors: István Czikora, Kyung-mi Kim, Anita Kása, Bálint Bécsi, Alexander D Verin, Pál Gergely, Ferenc Erdodi, Csilla Csortos

  Biochimie. 04/2011; 93(7):1139-45.

  TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin)TIMAP, TGF-β inhibited, membrane-associated protein, is highly abundant in endothelial cells (EC). We have shown earlier the involvement of TIMAP in PKA-mediated ERM (ezrin-radixin-moesin) dephosphorylation as part of EC barrier protection by TIMAP (Csortos et al., 2008). Emerging data demonstrate the regulatory role of TIMAP on protein phosphatase 1 (PP1) activity. We provide here evidence for specific interaction (K(a) = 1.80 × 10(6) M(-1)) between non-phosphorylated TIMAP and the catalytic subunit of PP1 (PP1c) by surface plasmon resonance based binding studies. Thiophosphorylation of TIMAP by PKA, or sequential thiophosphorylation by PKA and GSK3β slightly modifies the association constant for the interaction of TIMAP with PP1c and decreases the rate of dissociation. However, dephosphorylation of phospho-moesin substrate by PP1cβ is inhibited to different extent in the presence of non- (~60% inhibition), mono- (~50% inhibition) or double-thiophosphorylated (<10% inhibition) form of TIMAP. Our data suggest that double-thiophosphorylation of TIMAP has minor effect on its binding ability to PP1c, but considerably attenuates its inhibitory effect on the activity of PP1c. PKA activation by forskolin treatment of EC prevented thrombin evoked barrier dysfunction and ERM phosphorylation at the cell membrane (Csortos et al., 2008). With the employment of specific GSK3β inhibitor it is shown here that PKA activation is followed by GSK3β activation in bovine pulmonary EC and both of these activations are required for the rescuing effect of forskolin in thrombin treated EC. Our results suggest that the forskolin induced PKA/GSK3β activation protects the EC barrier via TIMAP-mediated decreasing of the ERM phosphorylation level.

• Synthesis of variously coupled conjugates of D-glucose, 1,3,4-oxadiazole, and 1,2,3-triazole for inhibition of glycogen phosphorylase.

  Authors: Sándor Kun, Gergo Z Nagy, Marietta Tóth, Laura Czecze, Albert Nguyen Van Nhien, Tibor Docsa, Pál Gergely, Maria-Despoina Charavgi, Paraskevi V Skourti, Evangelia D Chrysina, Tamás Patonay, László Somsák

  Carbohydrate research. 03/2011; 346(12):1427-38.

  5-(O-Perbenzoylated-β-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-β-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl-5-(O-Perbenzoylated-β-D-glucopyranosyl)tetrazole was obtained from O-perbenzoylated-β-D-glucopyranosyl cyanide by Bu(3)SnN(3) or Me(3)SiN(3)-Bu(2)SnO. This tetrazole was transformed into 5-ethynyl- as well as 5-chloromethyl-2-(O-perbenzoylated-β-D-glucopyranosyl)-1,3,4-oxadiazoles by acylation with propiolic acid-DCC or chloroacetyl chloride, respectively. The chloromethyl oxadiazole gave the corresponding azidomethyl derivative on treatment with NaN(3). These compounds were reacted with several alkynes and azides under Cu(I) catalysed cycloaddition conditions to give, after removal of the protecting groups by the Zemplén protocol, β-D-glucopyranosyl-1,3,4-oxadiazolyl-1,2,3-triazole, β-D-glucopyranosyl-1,2,3-triazolyl-1,3,4-oxadiazole, and β-D-glucopyranosyl-1,3,4-oxadiazolylmethyl-1,2,3-triazole type compounds. 5-Phenyltetrazole was also transformed under the above conditions into a series of aryl-1,3,4-oxadiazolyl-1,2,3-triazoles, aryl-1,2,3-triazolyl-1,3,4-oxadiazoles, and aryl-1,3,4-oxadiazolylmethyl-1,2,3-triazoles. The new compounds were assayed against rabbit muscle glycogen phosphorylase b and the best inhibitors had inhibition constants in the upper micromolar range (2-phenyl-5-[1-(β-D-glucopyranosyl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 36: K(i)=854μM, 2-(β-D-glucopyranosyl)-5-[1-(naphthalen-2-yl)-1,2,3-triazol-4-yl]-1,3,4-oxadiazole 47: K(i)=745μM).

• PKCdelta is a positive regulator of chondrogenesis in chicken high density micromass cell cultures.

  Authors: Csaba Matta, Tamás Juhász, Zsolt Szíjgyártó, Bernadett Kolozsvári, Csilla Somogyi, Georgina Nagy, Pál Gergely, Róza Zákány

  Biochimie. 02/2011; 93(2):149-59.

  We aimed to elucidate the role of the Ca-independent PKC isoenzyme PKCdelta in the regulation of spontaneous in vitro chondrogenesis occurring in a 6-day-long culturing period in chicken limbWe aimed to elucidate the role of the Ca-independent PKC isoenzyme PKCdelta in the regulation of spontaneous in vitro chondrogenesis occurring in a 6-day-long culturing period in chicken limb bud-derived high density cell cultures (HDC). PKCdelta expression and activity were detectable throughout the entire culturing period with a peak on days 2 and 3, when most of the chondroblasts differentiate. To inhibit the activity of PKCdelta, either the natural compound rottlerin was transiently applied to the culture medium of HDC in 2.5, 5 or 10 μM concentrations, or gene silencing was performed by using PKCdelta shRNA. Rottlerin significantly reduced the overall PKC activity in enzyme activity assays of cell-free samples of untreated control HDC, probably via the inhibition of PKCdelta. On the contrary, we were unable to detect any consistent change of PKC enzyme activity assayed in samples of HDC treated with rottlerin during culturing. PKCdelta gene silencing resulted in a significantly lower PKC activity. Both rottlerin and PKCdelta shRNA caused a severe reduction in cartilage formation, furthermore protein and phospho-protein levels of Sox9, the key transcription factor of chondrogenesis, were also significantly decreased. Rottlerin lowered, while PKCdelta gene silencing elevated the phosphorylation status of ERK1/2. Our data suggest that PKCdelta stimulates chondrogenesis via influencing Sox9 and ERK1/2 phosphorylation, but the inhibition of cartilage formation in the rottlerin-treated HDC is probably PKCdelta independent and rottlerin might have different effects when applied to cells or to an in vitro enzyme activity assay.

• Synthesis of 1-(D-glucopyranosyl)-1,2,3-triazoles and their evaluation as glycogen phosphorylase inhibitors.

  Authors: Eva Bokor, Tibor Docsa, Pál Gergely, László Somsák

  Bioorganic & medicinal chemistry. 02/2010; 18(3):1171-80.

  1-(D-Glucopyranosyl)-1,2,3-triazoles were prepared from per-O-acetylated alpha- and beta-D-glucopyranosyl azides as well as per-O-benzoylated (beta-D-gluco-hept-2-ulopyranosylazide)onamide and onic1-(D-Glucopyranosyl)-1,2,3-triazoles were prepared from per-O-acetylated alpha- and beta-D-glucopyranosyl azides as well as per-O-benzoylated (beta-D-gluco-hept-2-ulopyranosylazide)onamide and onic acid methylester by using azide-alkyne cycloaddition catalysed by in situ generated Cu(I) under aqueous conditions. The O-acyl protecting groups were removed by the Zemplén protocol. The test compounds were assayed against rabbit muscle glycogen phosphorylase b to show that the beta-D-glucopyranosyl derivatives were superior inhibitors as compared to the two other series of triazoles.

• Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

  Authors: Katalin Erdélyi, Péter Bai, István Kovács, Eva Szabó, Gábor Mocsár, Annamária Kakuk, Csaba Szabó, Pál Gergely, László Virág

  The FASEB journal : official publication of the Federation of American Societies for Experimental Biology. 08/2009;

  Activation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible forActivation of poly(ADP-ribose) polymerase-1 (PARP1) has been shown to mediate cell death induced by genotoxic stimuli. The role of poly(ADP-ribose) glycohydrolase (PARG), the enzyme responsible for polymer degradation, has been largely unexplored in the regulation of cell death. Using lentiviral gene silencing we generated A549 lung adenocarcinoma cell lines with stably suppressed PARG and PARP1 expression (shPARG and shPARP1 cell lines, respectively) and determined parameters of apoptotic and necrotic cell death following hydrogen peroxide exposure. shPARG cells accumulated large amounts of poly(ADP-ribosyl)ated proteins and exhibited reduced PARP activation. Hydrogen peroxide-induced cell death is regulated by PARG in a dual fashion. Whereas the shPARG cell line (similarly to shPARP1 cells) was resistant to the necrotic effect of high concentrations of hydrogen peroxide, these cells exhibited stronger apoptotic response. Both shPARP1 and especially shPARG cells displayed a delayed repair of DNA breaks and exhibited reduced clonogenic survival following hydrogen peroxide treatment. Translocation of apoptosis-inducing factor could not be observed, but cells could be saved by methyl pyruvate and alpha-ketoglutarate, indicating that energy failure may mediate cytotoxicity in our model. These data indicate that PARG is a survival factor at mild oxidative damage but contributes to the apoptosis-necrosis switch in severely damaged cells.-Erdélyi, K., Bai, P., Kovács, I., Szabó, E., Mocsár, G., Kakuk, A., Szabó, C., Gergely, P., Virág, L. Dual role of poly(ADP-ribose) glycohydrolase in the regulation of cell death in oxidatively stressed A549 cells.

• Synthesis and glycogen phosphorylase inhibitory activity of N-(beta-d-glucopyranosyl)amides possessing 1,4-benzodioxane moiety.

  Authors: Zoltán Czakó, László Juhász, Agnes Kenéz, Katalin Czifrák, László Somsák, Tibor Docsa, Pál Gergely, Sándor Antus

  Bioorganic & medicinal chemistry. 08/2009;

  A series of N-(beta-d-glucopyranosyl)amides 5d-i were synthesized by PMe(3) mediated Staudinger reaction of O-peracetylated beta-d-glucopyranosyl azide (1) followed by acylation with carboxylic acidsA series of N-(beta-d-glucopyranosyl)amides 5d-i were synthesized by PMe(3) mediated Staudinger reaction of O-peracetylated beta-d-glucopyranosyl azide (1) followed by acylation with carboxylic acids 3d-i and subsequent Zemplén deacetylation. The new compounds were tested for their inhibitory activity against rabbit muscle glycogen phosphorylase and the structure-activity relationships of these compounds are also discussed in this paper.

• Glucose-based spiro-heterocycles as potent inhibitors of glycogen phosphorylase.

  Authors: Veronika Nagy, Mahmoud Benltifa, Sébastien Vidal, Eszter Berzsényi, Cathie Teilhet, Katalin Czifrák, Gyula Batta, Tibor Docsa, Pál Gergely, László Somsák, Jean-Pierre Praly

  Bioorganic & medicinal chemistry. 07/2009;

  Glucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cyclization of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogousGlucopyranosylidene-spiro-1,4,2-oxathiazoles were prepared in high yields by NBS-mediated spiro-cyclization of the corresponding glucosyl-hydroximothioates. In an effort to synthesize analogous glucopyranosylidene-spiro-1,2,4-oxadiazolines, with a nitrogen atom instead of the sulphur, attempted cyclizations resulted in aromatization of the heterocycle with opening of the pyranosyl ring. Enzymatic measurements showed that some of the glucose-based inhibitors were active in the micromolar range. The 2-naphthyl-substituted 1,4,2-oxathiazole displayed the best inhibition against RMGPb (K(i)=160nM), among glucose-based inhibitors known to date.

• Inhibition of calcineurin by cyclosporine A exerts multiple effects on human melanoma cell lines HT168 and WM35.

  Authors: Tamás Juhász, Csaba Matta, Gábor Veress, Georgina Nagy, Zsolt Szíjgyártó, Zsanett Molnár, János Fodor, Róza Zákány, Pál Gergely

  International journal of oncology. 05/2009; 34(4):995-1003.

  The immunosuppressant cyclosporine A (CsA) is a specific pharmacological inhibitor of calcineurin, the Ca2+-calmodulin activated phospho-Ser/Thr-specific protein phosphatase. AlthoughThe immunosuppressant cyclosporine A (CsA) is a specific pharmacological inhibitor of calcineurin, the Ca2+-calmodulin activated phospho-Ser/Thr-specific protein phosphatase. Although calcineurin-inhibiting compounds are applied for local treatment of psoriasis or atopic dermatitis in dermatological practice, little is known about the functions of calcineurin in epidermis-derived malignancies. We investigated the effects of CsA on two human melanoma cell lines, the metastasis forming HT168 and WM35 established from an RGP primary lesion. CsA of 2 microM lowered the enzyme activity by 50% and caused elevation in both mRNA and protein expression of calcineurin. Cell proliferation was diminished, as well as the cellular morphology and the actin organization were altered in both cell lines. CsA increased cell death moderately in both cell lines and reduced the metabolic activity of HT168 cells, but not that of WM35 cells. CsA also elevated the expressions of both Bcl-2 and ERK1/2. Fibronectin guided migration of HT168 cells was stimulated under the effect of CsA, while that of WM35 cells was reduced, moreover, HT168 cells switched from the expression of beta3 to beta1 integrin, but WM35 cells continued to express beta3. Based on our results we propose a multiple, partly malignancy-dependent role of calcineurin in these melanoma cell lines.

• Synthesis and structure-activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase.

  Authors: Marietta Tóth, Sándor Kun, Eva Bokor, Mahmoud Benltifa, Gaylord Tallec, Sébastien Vidal, Tibor Docsa, Pál Gergely, László Somsák, Jean-Pierre Praly

  Bioorganic & medicinal chemistry. 05/2009;

  A series of per-O-benzoylated 5-beta-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(beta-d-glucopyranosyl)tetrazole. As an alternative, oxidationA series of per-O-benzoylated 5-beta-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(beta-d-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated beta-d-glucopyranosyl cyanide gave the corresponding 5-beta-d-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-beta-d-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K(i)=8.8 and 11.6muM, respectively). A detailed analysis of the structure-activity relationships is presented.

• Ionotropic purinergic receptor P2X(4) is involved in the regulation of chondrogenesis in chicken micromass cell cultures.

  Authors: János Fodor, Csaba Matta, Tamás Juhász, Tamás Oláh, Mónika Gönczi, Zsolt Szíjgyártó, Pál Gergely, László Csernoch, Róza Zákány

  Cell calcium. 04/2009;

  We have previously demonstrated that elevation of free cytosolic Ca(2+) concentration at the time of differentiation of chondroblasts was mainly due to a Ca(2+) influx and it was indispensable toWe have previously demonstrated that elevation of free cytosolic Ca(2+) concentration at the time of differentiation of chondroblasts was mainly due to a Ca(2+) influx and it was indispensable to cartilage formation in chicken high density mesenchymal cell cultures (HDC) [C. Matta, J. Fodor, Z. Szijgyarto, T. Juhasz, P. Gergely, L. Csernoch, R. Zakany, Cytosolic free Ca(2+) concentration exhibits a characteristic temporal pattern during in vitro cartilage differentiation: a possible regulatory role of calcineurin in Ca-signalling of chondrogenic cells, Cell Calcium 44 (2008) 310-323]. Here, we report that chondrogenic cells secreted ATP and administration of ATP to the culture medium evoked Ca(2+) transients exclusively in the presence of extracellular Ca(2+) and only on day 3 of culturing, when the final commitment of chondroblasts occurs. Moreover, ATP caused elevated protein expression of the chondrogenic transcription factor Sox9 and stimulated cartilage matrix production. Expression pattern of different types of both ionotropic and metabotropic purinergic receptors was detected. Agonists of metabotropic receptors, ADP and UDP did not evoke any Ca(2+) transients and had no influence on cartilage formation, while UTP caused transient elevation of cytosolic Ca(2+) concentration in 3-day-old HDC without stimulating matrix production. Suramin, which blocks all P2X receptors but not P2X(4) did not impede the effects of ATP, furthermore, P2X(4) appeared in the plasma membrane fraction and gave signals with immunocytochemistry only from day 3. In summary, we suggest a role of ionotropic purinergic signalling of P2X(4) in the generation of ATP-dependent Ca(2+) transients of differentiating chondroblasts.

• Role of calcineurin in thrombin-mediated endothelial cell contraction.

  Authors: Bernadett Kolozsvári, Zsolt Szíjgyártó, Péter Bai, Pál Gergely, Alexander Verin, Joe G N Garcia, Eva Bakó

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 03/2009;

  Barrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gapBarrier function and shape changes of endothelial cells (EC) are regulated by phosphorylation/dephosphorylation of key signaling and contractile elements. EC contraction results in intercellular gap formation and loss of the selective vascular barrier to circulating macromolecules. EC dysfunction elicited by thrombin was found to correlate with actin microfilament redistribution. It is known that calcineurin (Cn) is involved in thrombin-induced EC dysfunction because inhibition of Cn potentiates PKC activity and the phosphorylation state of EC myosin light chain is also affected by Cn activity. Immunofluorescent detection of Cn catalytic subunit (CnA) isoforms coexpressed with GFP was visualized on paraformaldehyde (PFA) fixed bovine pulmonary artery endothelial cells (BPAEC). Actin microfilaments were stained with Texas Red-phalloidin. Cytotoxic effects of transfections or treatments and the efficiency of transfections were assessed by flow cytometry. Treatment of BPAEC with Cn inhibitors (cyclosporin A and FK506) hindered recovery of the cells from thrombin-induced EC dysfunction. Inhibition of Cn in the absence of thrombin had no effect on cytoskeletal actin filaments. We detected attenuated thrombin-induced stress fiber formation and changes in cell shape only when cells were transfected with constitutively active CnA and not with various CnA isoforms. Flow cytometry (FCM) analysis has proved that cytotoxic effect of treatments is negligible. We observed that Cn is involved in the recovery from thrombin-induced EC dysfunction. Inhibition of Cn caused prolonged contractile effect, while overexpression of constitutively active CnA resulted in reduced thrombin-induced stress fiber formation. (c) 2009 International Society for Advancement of Cytometry.

• Synthesis of a C-glucosylated cyclopropylamide and evaluation as a glycogen phosphorylase inhibitor.

  Authors: Philippe Bertus, Jan Szymoniak, Erwann Jeanneau, Tibor Docsa, Pál Gergely, Jean-Pierre Praly, Sébastien Vidal

  Bioorganic & medicinal chemistry letters. 10/2008; 18(17):4774-8.

  The synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylatedThe synthesis of carbohydrate-based glycogen phosphorylase inhibitors is attractive for potential applications in the treatment of type 2 diabetes. A titanium-mediated synthesis led to a benzoylated C-glucosylated cyclopropylamine intermediate, which underwent a benzoyl migration to afford the corresponding 2-hydroxy-C-glycoside. X-ray crystallographic studies revealed a unit cell composed of four molecules as pairs of dimers connected through two hydrogen bonds. The deprotection of the benzoate esters under Zemplén conditions afforded a glycogen phosphorylase inhibitor candidate displaying weak inhibition toward glycogen phosphorylase (16% at 2.5mM).