György Vereb

Publications of György Vereb

• Expression of coagulation factor XIII subunit A in acute promyelocytic leukemia.

  Authors: Agnes Simon, Zsuzsa Bagoly, Zsuzsanna Hevessy, László Csáthy, Eva Katona, György Vereb, Anikó Ujfalusi, László Szerafin, László Muszbek, János Kappelmayer

  Cytometry. Part B, Clinical cytometry. 03/2012;

  Leukemic cells often express markers which are not characteristic of their particular cell lineage. In this study we identified the "A" subunit of coagulation factor XIII (FXIII-A) in leukemicLeukemic cells often express markers which are not characteristic of their particular cell lineage. In this study we identified the "A" subunit of coagulation factor XIII (FXIII-A) in leukemic promyelocytes in de novo AML M3 cases. The cytoplasmic presence of factor XIII-A has previously been shown only in platelets/megakaryocytes and monocytes/macrophages. Furthermore, more recently we described the presence of FXIII-A in leukemic lymphoblasts. We studied 14 patients with this rare type of acute leukemia in a period of 4 years and investigated their bone marrow samples by 3-color flow cytometry upon diagnosis, mainly focusing on FXIII-A expression of leukemic cells. We detected FXIII-A also by ELISA, Western-blot and confocal laser scanning microscopy. This was a homogenous group of AML M3 patients with translocation t(15;17)(q22;q21) detected by fluorescence in situ hybridization (FISH). In 10 out of 14 samples, FXIII-A was detectable by flow cytometry and was coexpressed with markers characteristic for leukemic promyleocytes (CD45dim/CD13+/CD33+/CD117+/cyMPO+ and HLA-DR-/CD34-/CD14-/CD15-). Staining for the markers GPIIb and GPIX were negative, and FXIII-A was identified in the cytoplasm of the cells by confocal microscopy in a relatively high quantity, as measured by ELISA. By Western blot analysis we could identify FXIII-A in the native 82 kD form and in cleaved forms corresponding to cleavage products observed when purified FXIII-A was treated by human neutrophil elastase. Since normal promyelocytes were FXIII-A negative, this novel expression site of FXIII-A in AML M3 can be considered as a leukemia associated immunophenotype and may have pathophysiological significance. © 2012 International Society for Advancement of Cytometry.

• ErbB protein modifications are secondary to severe cell membrane alterations induced by elisidepsin treatment.

  Authors: Tímea Váradi, Janos Roszik, Duarte Lisboa, György Vereb, José Manuel Molina-Guijarro, Carlos M Galmarini, János Szöllosi, Peter Nagy

  European journal of pharmacology. 06/2011; 667(1-3):91-9.

  Elisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points toElisidepsin is a marine-derived anti-tumor agent with unique mechanism of action. It has been suggested to induce necrosis associated with severe membrane damage. Since indirect evidence points to the involvement of ErbB receptor tyrosine kinases and lipid rafts in the mechanism of action of elisidepsin, we investigated the effect of the drug on the distribution of ErbB proteins and systematically compared the elisidepsin sensitivity of cell lines overexpressing ErbB receptors. Stable expression of a single member of the ErbB family (ErbB1-3) or co-transfection of ErbB2 and ErbB3 did not modify the elisidepsin sensitivity of CHO and A431 cells. However, elisidepsin induced the redistribution of ErbB3 and two GPI-anchored proteins (transfected GPI-anchored eGFP and placental alkaline phosphatase) from the plasma membrane to intracellular vesicles without comparable effects on ErbB1 and ErbB2. Elisidepsin increased the binding of a conformational sensitive anti-ErbB3 antibody without modifying the binding of other ErbB2 or ErbB3 antibodies, and it decreased the homoassociation of both ErbB2 and ErbB3. We also found that elisidepsin decreased the fluorescence anisotropy of a membrane specific fluorescent probe and induced a blue shift in the emission spectrum of Laurdan pointing to significant changes in the order of the plasma membrane possibly associated with the formation of liquid ordered domains. Although the distribution of ErbB proteins is preferentially altered by elisidepsin, our data question their role in determining sensitivity to the drug. We assume that induction of liquid ordered domains is the primary action of elisidepsin leading to all the other observed changes.

• Anti-angiogenic effects of purine inhibitors of cyclin dependent kinases.

  Authors: Johanna Liebl, Vladimir Krystof, György Vereb, Lili Takács, Miroslav Strnad, Paul Pechan, Libor Havlicek, Marek Zatloukal, Robert Fürst, Angelika M Vollmar, Stefan Zahler

  Angiogenesis. 04/2011; 14(3):281-91.

  Small molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitorSmall molecular inhibitors of Cyclin dependent kinases (Cdks) are currently being developed as anticancer therapeutics due to their antiproliferative properties. The purine Cdk specific inhibitor (R)-roscovitine (seliciclib, CYC202) represents one of the most promising of these compounds. It is currently evaluated in clinical trials concerning cancer therapy. Recently, we have shown that roscovitine exerts potent antiangiogenic effects and elucidated Cdk5 as a new player in angiogenesis. These findings introduce Cdk5 as novel target for antiangiogenic therapy, and Cdk5 inhibitors as an attractive therapeutic approach. Here, we present the antiangiogenic profile of 15 derivatives of roscovitine in vitro and in vivo and provide structure activity relationships of the roscovitine analogs. The (S)-isomer LGR561 and the respective (R)- and (S)-isomers LGR848 and LGR849 strongly inhibited proliferation and cell cycle progression, induced cell death, and reduced migration of endothelial cells in vitro. In comparison to roscovitine, these compounds showed an increased potency to inhibit Cdk2, Cdk5, Cdk7, and Cdk9. By analyzing the effects of LGR561, LGR848, and LGR849 on endothelial cell tube formation, mouse aortic ring sprouting, angiogenesis in the chick chorioallantoic membrane, and neovessel formation in the mouse cornea, we elucidate the two (S)-isomers LGR561 and LGR849 as highly potent inhibitors of angiogenesis. This study provides first information on how to modify roscovitine to develop Cdk inhibitors with increased antiangiogenic activity and suggests the application of existing and the development of new Cdk inhibitors to inhibit both, cancer cell proliferation and angiogenesis.

• T-cell synapse formation depends on antigen recognition but not CD3 interaction: studies with TCR:ζ, a candidate transgene for TCR gene therapy.

  Authors: János Roszik, Zsolt Sebestyén, Coen Govers, Yakir Guri, Arpád Szöor, Zsuzsanna Pályi-Krekk, György Vereb, Peter Nagy, János Szöllosi, Reno Debets

  European journal of immunology. 03/2011; 41(5):1288-97.

  T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ,T-cell receptors (TCRs) can be genetically modified to improve gene-engineered T-cell responses, a strategy considered critical for the success of clinical TCR gene therapy to treat cancers. TCR:ζ, which is a heterodimer of TCRα and β chains each coupled to complete human CD3ζ, overcomes issues of mis-pairing with endogenous TCR chains, shows high surface expression and mediates antigen-specific T-cell functions in vitro. In the current study, we further characterized TCR:ζ in gene-engineered T cells and assessed whether this receptor is able to interact with surface molecules and drive correct synapse formation in Jurkat T cells. The results showed that TCR:ζ mediates the formation of synaptic areas with antigen-positive target cells, interacts closely with CD8α and MHC class I (MHCI), and co-localizes with CD28, CD45 and lipid rafts, similar to WT TCR. TCR:ζ did not closely associate with endogenous CD3ε, despite its co-presence in immune synapses, and TCR:ζ showed enhanced synaptic accumulation in T cells negative for surface-expressed TCR molecules. Notably, synaptic TCR:ζ demonstrated lowered densities when compared with TCR in dual TCR T cells, a phenomenon that was related to both extracellular and intracellular CD3ζ domains present in the TCR:ζ molecule and responsible for enlarged synapse areas.

• Flow cytometric FRET analysis of protein interaction.

  Authors: György Vereb, Péter Nagy, János Szöllosi

  Methods in molecular biology (Clifton, N.J.). 01/2011; 699:371-92.

  Investigation of protein-protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modelingInvestigation of protein-protein interactions in situ in living or intact cells gains expanding importance as structure/function relationships proposed from bulk biochemistry and molecular modeling experiments require demonstration at the cellular level. Fluorescence resonance energy transfer (FRET)-based methods are excellent tools for determining proximity and supramolecular organization of biomolecules at the cell surface or inside the cell. This could well be the basis for the increasing popularity of FRET; in fact, the number of publications exploiting FRET has doubled in the past 5 years. In this chapter, we intend to provide a generally useable protocol for measuring FRET in flow cytometry. After a concise theoretical introduction, recipes are provided for successful labeling techniques and measurement approaches. The simple, quenching-based population-level measurement; the classic ratiometric, intensity-based technique providing cell-by-cell actual FRET efficiencies, and a more advanced version of the latter, allowing for cell-by-cell autofluorescence correction, are described. Finally, points of caution are given to help design proper experiments and critically interpret the results.

• Cyclin-dependent kinase 5 regulates endothelial cell migration and angiogenesis.

  Authors: Johanna Liebl, Sabine B Weitensteiner, György Vereb, Lili Takács, Robert Fürst, Angelika M Vollmar, Stefan Zahler

  The Journal of biological chemistry. 11/2010; 285(46):35932-43.

  Angiogenesis contributes to various pathological conditions. Due to the resistance against existing antiangiogenic therapy, an urgent need exists to understand the molecular basis of vessel growthAngiogenesis contributes to various pathological conditions. Due to the resistance against existing antiangiogenic therapy, an urgent need exists to understand the molecular basis of vessel growth and to identify new targets for antiangiogenic therapy. Here we show that cyclin-dependent kinase 5 (Cdk5), an important modulator of neuronal processes, regulates endothelial cell migration and angiogenesis, suggesting Cdk5 as a novel target for antiangiogenic therapy. Inhibition or knockdown of Cdk5 reduces endothelial cell motility and blocks angiogenesis in vitro and in vivo. We elucidate a specific signaling of Cdk5 in the endothelium; in contrast to neuronal cells, the motile defects upon inhibition of Cdk5 are not caused by an impaired function of focal adhesions or microtubules but by the reduced formation of lamellipodia. Inhibition or down-regulation of Cdk5 decreases the activity of the small GTPase Rac1 and results in a disorganized actin cytoskeleton. Constitutive active Rac1 compensates for the inhibiting effects of Cdk5 knockdown on migration, suggesting that Cdk5 exerts its effects in endothelial cell migration via Rac1. Our work elucidates Cdk5 as a pivotal new regulator of endothelial cell migration and angiogenesis. It suggests Cdk5 as a novel, pharmacologically accessible target for antiangiogenic therapy and provides the basis for a new therapeutic application of Cdk5 inhibitors as antiangiogenic agents.

• Differentially expressed genes associated with human limbal epithelial phenotypes: new molecules that potentially facilitate selection of stem cell-enriched populations.

  Authors: Lili Takács, Eniko Tóth, Gergely Losonczy, Attila Szanto, Tomi Bähr-Ivacevic, Vladimir Benes, András Berta, György Vereb

  Investigative ophthalmology & visual science. 11/2010; 52(3):1252-60.

  The aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. Total RNA isolates of human limbal and central corneal epithelia were usedThe aim of this study was to identify differentially expressed genes in the human limbal epithelium by microarray analysis. Total RNA isolates of human limbal and central corneal epithelia were used after transcription for hybridization on whole human genome expression microarrays. A set of differentially expressed genes detected by both microarrays was established. In the case of eight selected molecules, microarray results were confirmed by qRT-PCR, and protein expression in the cornea was examined by confocal immunofluorescence microscopy. Colocalization with the putative stem cell marker C/EBPδ was also examined. The authors established a database of 126 limbal overexpressed genes. qRT-PCR confirmed microarray results in all examined cases (SPON1, IFITM1, ITM2A, PHLDA1, CXCR4, FZD7, DCT, DKK4). Limbal localization of the protein product of SPON1, IFITM1, ITM2A, CXCR4, and DKK4 was shown with confocal immunofluorescence microscopy. SPON1, IFITM1, and ITM2A signals mostly colocalized with C/EBPδ-positive putative resting limbal stem cells. By detecting several new differentially expressed genes in the human corneal limbus, this study further expands current knowledge on the molecular signature of limbal epithelial stem cells. Plasma membrane localization of IFITM1 and ITM2A suggests their potential usefulness as targets to select stem cell-enriched populations from the limbal epithelium.

• PPARγ modulated inflammatory response of human dendritic cell subsets to engulfed apoptotic neutrophils.

  Authors: Gyöngyike Majai, Péter Gogolák, Csilla Ambrus, György Vereb, Judit Hodrea, László Fésüs, Eva Rajnavölgyi

  Journal of leukocyte biology. 11/2010; 88(5):981-91.

  The means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by humanThe means of how phagocytes handle apoptotic cells has a great impact on the outcome of immune responses. Here, we show that phagocytosis of allogeneic, apoptotic neutrophils by human monocyte-derived DCs is slow and less efficient than that of macrophages, and CD1a(-) DCs are more active in the engulfment of apoptotic neutrophils than CD1a(+) DCs. Blocking DC-SIGN function partially interferes with the uptake of apoptotic cells, and long-term interaction of apoptotic neutrophils with DCs makes them prone to proinflammatory cytokine responses. Engulfment of apoptotic cells sensitizes CD1a(-) DCs for high IL-8, TNF-α, IL-6, and CD1a(+) cells for IL-12 and IL-10 cytokine secretion elicited by additional inflammatory stimuli, which also result in the polarization of autologous T lymphocytes to Th1 effector cells. Ligand-induced activation of PPARγ by RSG results in enhanced phagocytosis, but the proinflammatory response and the capacity to trigger Th1 cell activation of CD1a(-) DCs are not enhanced. These results demonstrate that DCs are able to respond to allogeneic, apoptotic neutrophils with inflammatory cytokines and T cell responses in a subtype-specific manner that is modulated by the anti-inflammatory effects of PPARγ.

• Cisplatin and a potent platinum(IV) complex-mediated enhancement of TRAIL-induced cancer cells killing is associated with modulation of upstream events in the extrinsic apoptotic pathway.

  Authors: Olga Vondálová Blanárová, Iva Jelínková, Arpád Szöor, Belma Skender, Karel Soucek, Viktor Horváth, Alena Vaculová, Ladislav Andera, Petr Sova, János Szöllosi, Jirina Hofmanová, György Vereb, Alois Kozubík

  Carcinogenesis. 10/2010; 32(1):42-51.

  TRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediatedTRAIL (tumor necrosis factor-related apoptosis-inducing ligand) can selectively trigger apoptosis in various cancer cell types. However, many cancer cells are resistant to death receptor-mediated apoptosis. Combination therapy with platinum complexes may affect TRAIL-induced signaling via modulation of various steps in apoptotic pathways. Here, we show that cisplatin or a more potent platinum(IV) complex LA-12 used in 20-fold lower concentration enhanced killing effects of TRAIL in human colon and prostate cancer cell lines via stimulation of caspase activity and overall apoptosis. Both platinum complexes increased DR5 surface expression in colon cancer cells. Small interfering RNA-mediated DR5 silencing rescued cells from sensitizing effects of platinum drugs on TRAIL-induced caspase-8 activation and apoptosis, showing the functional importance of DR5 in the effects observed. In addition, both cisplatin and LA-12 triggered the relocalization of DR4 and DR5 receptors to lipid rafts and accelerated internalization of TRAIL, which may also affect TRAIL signaling. Collectively, modulations of the initial steps of the extrinsic apoptotic pathway at the level of DR5 and plasma membrane are important for sensitization of colon and prostate cancer cells to TRAIL-induced apoptosis mediated by LA-12 and cisplatin.

• Urokinase down-regulation by aprotinin in rabbit corneal cells after photorefractive keratectomy.

  Authors: Adrienne Csutak, David M Silver, Tamás Sperka, János Kádas, György Vereb, András Berta, József Tozsér

  Current eye research. 09/2010; 35(9):806-11.

  To examine the effect of aprotinin eye drops on urokinase-type plasminogen activator (uPA) gene expression in rabbit corneal cells during wound healing after photorefractive keratectomy (PRK). BothTo examine the effect of aprotinin eye drops on urokinase-type plasminogen activator (uPA) gene expression in rabbit corneal cells during wound healing after photorefractive keratectomy (PRK). Both eyes of 22 rabbits were subjected to PRK. One eye of each rabbit was treated with antibiotic eye drops five times while the contralateral eye was treated at the same time with antibiotic eye drops and a serine protease inhibitor. The animals were sacrificed at different time frames, and 2-4 rabbits were used for each time point. Half of each cornea was used for the determination of the amount of uPA mRNA after reverse transcription and real-time quantitative polymerase chain reaction, while frozen sections were prepared from the other halves for in situ zymography to detect uPA activity. The level of uPA mRNA in corneal cells was markedly increased in corneal samples obtained hours after PRK. The time-dependent up-regulation of uPA mRNA was strongly dependent on the diameter of the area from which the epithelial cells were removed before the surgery. Independently of the time scale of uPA up-regulation, application of eye drops containing aprotinin significantly diminished the uPA expression. In situ zymography confirmed that aprotinin has also decreased overall uPA activity. Aprotinin down-regulates uPA gene expression in corneal cells during the wound-healing phase after PRK.

• Rafts and the battleships of defense: the multifaceted microdomains for positive and negative signals in immune cells.

  Authors: Arpád Szöőr, János Szöllősi, György Vereb

  Immunology letters. 12/2009;

  Recognition of the heterogeneity of the cell membrane was one of the most important scientific achievements in the last decades. Since coining the term "lipid rafts", continuous development ofRecognition of the heterogeneity of the cell membrane was one of the most important scientific achievements in the last decades. Since coining the term "lipid rafts", continuous development of advanced microscopic and spectroscopic techniques has vastly expanded our view on these cell membrane microdomains that appear to have almost as many faces as researchers that look at them; they are variable in stability, size and composition that can change in a highly dynamic manner both by recruiting and expelling components as well as by coalescing and breaking up into smaller units. They have, however, one common feature: all eukaryotic cells present some variation of lipid rafts. Cells of the immune system are not exception to this, regardless of their lymphoid or myeloid origin their membranes show a domain structure and these domains serve to condense or reject particular transmembrane, GPI-linked and intracellularly membrane-anchored proteins as function requires. Here we provide a concise overview about the various weapons and shields that immune cells concentrate into their rafts, which have come into sight during the past years. The positive and negative regulatory roles of these microdomains are essential both in the functions of innate immunity and processes concatenated in the adaptive immune response.

• Measurement of molecular mobility with fluorescence correlation spectroscopy.

  Authors: György Vámosi, Sándor Damjanovich, János Szöllosi, György Vereb

  Current protocols in cytometry / editorial board, J. Paul Robinson, managing editor ... [et al.]. 10/2009; Chapter 2:Unit2.15.

  Fluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations ofFluorescence correlation spectroscopy (FCS) is a fluctuation method established three decades ago, whose application to cellular systems became popular in the last decade. Fluctuations of fluorescence emission are observed from a small, femtoliter to sub-femtoliter, usually confocal volume at high time resolution. A time-dependent autocorrelation function is generated and evaluated to obtain time constants of photophysical and photochemical reactions, as well as of molecular diffusion and in the observation volume. Molecules in various subcellular compartments-including the nucleus, the cytoplasm, and the membrane-can be observed after labeling them with antibodies, ligands, or fluorescent proteins. The anomaly of diffusion, the local concentration, and the average fluorescence per diffusing particle can also be determined, all of which can be characteristic of molecular interactions. A two-color version of FCS, fluorescence cross-correlation spectroscopy, can also be applied to observe co-diffusion, i.e., stable association of two distinct molecular species in their cellular environment.

• [Expression pattern of invasion-related molecules in cerebral tumors of different origin.]

  Authors: Miklós Petrás, Gábor Hutóczki, Imre Varga, György Vereb, János Szöllosi, László Bognár, Péter Ruszthi, Annamária Kenyeres, Judit Tóth, Zoltán Hanzély, Beáta Scholtz, Almos Klekner

  Magyar onkologia. 09/2009; 53(3):253-8.

  Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection and successful treatment, with tumor recurrence as microdisseminated disease.Tumor cell invasion into the surrounding brain tissue is mainly responsible for the failure of radical surgical resection and successful treatment, with tumor recurrence as microdisseminated disease. Epidermal growth factor receptors (EGFRs), integrins and their ligands in the extracellular matrix (ECM) predominantly participate in the invasion process, including the cell adhesion to the surrounding microenvironment and cell migration. The extent of infiltration of the surrounding brain tissue by malignant tumors strongly depends on the tumor cell type. Malignant gliomas show much more intensive peritumoral invasion than do metastatic tumors. In this study, the mRNA expression of 29 invasion-related molecules (18 cell membrane receptors or receptor subunits (EGFRs and integrins) and 11 ECM components: collagens, laminins and fibronectin) was investigated by quantitative reverse transcriptase-polymerase chain reaction. Fresh frozen human tissue samples from glioblastoma (GBM) and intracerebral bronchial adenocarcinoma metastases (five pieces from each) were evaluated. Significant differences were established in six of the 29 molecules (ErbB1, 2, 3, integrins alpha3, 7 and beta1). To confirm our results at the protein level, immunohistochemical analysis of nine molecules was performed. The staining intensity differed definitely in the case of ErbB1, 2 and integrins alpha3 and beta1. Determining the differences in invasion-related molecules in tumors of different origin can help identify the exact molecular mechanisms that facilitate peritumoral infiltration by glioblastoma cells. These results should allow the selection of target molecules for potential chemotherapeutic agents directed against highly invasive malignant gliomas.

• Over-expression of integrin beta3 can partially overcome the defect of integrin beta3 signaling in transglutaminase 2 null macrophages.

  Authors: Beáta Tóth, Zsolt Sarang, György Vereb, Ailiang Zhang, Sakae Tanaka, Gerry Melino, László Fésüs, Zsuzsa Szondy

  Immunology letters. 08/2009;

  Transglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2(-/-) mice the in vivo clearance of apoptotic cells isTransglutaminase 2 (TG2) is a protein crosslinking enzyme with many additional biological functions. We have previously shown that in TG2(-/-) mice the in vivo clearance of apoptotic cells is defective leading to autoimmunity. TG2 contributes to the formation of phagocytic portals by binding to both integrin beta(3), a known phagocytic receptor, and its bridging molecule, MFG-E8. In TG2 null macrophages integrin beta(3) cannot accumulate around the apoptotic cells and its signaling is impaired. In the present study we describe a subline of TG2 null mice, in which a compensatory increase in integrin beta(3) expression, which resulted alone in a high receptor concentration around the apoptotic cells without the requirement for accumulation, partially corrected the defect in integrin beta(3) signaling. Our data provide a proof for the concept that the function of TG2 is to stabilize accumulated integrin beta(3) concentration in the phagocytic cup.

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• Evaluation of intensity-based ratiometric FRET in image cytometry-Approaches and a software solution.

  Authors: János Roszik, Duarte Lisboa, János Szöllősi, György Vereb

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 08/2009;

  The intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based onThe intensity-based ratiometric FRET (fluorescence resonance energy transfer) method is a powerful technique for following molecular interactions in living cells. Since it is not based on irreversibly destroying the donor or the acceptor fluorophores, the time course of changes in FRET efficiency values can be monitored by this method. ImageJ, a sophisticated software tool for many types of image processing allows users to extend it with programs for various purposes. Implementing intensity-based ratiometric FRET with ImageJ vastly enhances the applicability of the FRET method. We developed an efficient ImageJ plugin, RiFRET, which calculates FRET efficiency on a pixel-by-pixel basis from ratiometric FRET images. It allows the user to correct for channel cross-talk (bleed-through) and to calculate FRET from image stacks, i.e., from 3D data sets. Semiautomatic processing for larger datasets is also included in the program. Furthermore, several options for calibrating FRET efficiency calculations were tested and their applicability to various expression systems is discussed. Although the ratiometric FRET method is widely applied, our plugin is the first freely available software for evaluating such FRET data. The program is user friendly and provides reliable, standardized results. (c) 2009 International Society for Advancement of Cytometry.

• Cylindrospermopsin induces alterations of root histology and microtubule organization in common reed (Phragmites australis) plantlets cultured in vitro.

  Authors: Dániel Beyer, Gyula Surányi, Gábor Vasas, János Roszik, Ferenc Erdődi, Márta M-Hamvas, István Bácsi, Róbert Bátori, Zoltán Serfőző, Zsuzsa M Szigeti, György Vereb, Zita Demeter, Sándor Gonda, Csaba Máthé

  Toxicon : official journal of the International Society on Toxinology. 06/2009;

  We aimed to study the histological and cytological alterations induced by cylindrospermopsin (CYN), a protein synthesis inhibitory cyanotoxin in roots of common reed (Phragmites australis). Reed isWe aimed to study the histological and cytological alterations induced by cylindrospermopsin (CYN), a protein synthesis inhibitory cyanotoxin in roots of common reed (Phragmites australis). Reed is an ecologically important emergent aquatic macrophyte, a model for studying cyanotoxin effects. We analyzed the histology and cytology of reed roots originated from tissue cultures and treated with 0.5-40mugml(-1) (1.2-96.4muM) CYN. The cyanotoxin decreased root elongation at significantly lower concentrations than the elongation of shoots. As general stress responses of plants to phytotoxins, CYN increased root number and induced the formation of a callus-like tissue and necrosis in root cortex. Callus-like root cortex consisted of radially swollen cells that correlated with the reorientation of microtubules (MTs) and the decrease of MT density in the elongation zone. Concomitantly, the cyanotoxin did not decrease, rather it increased the amount of beta-tubulin in reed plantlets. CYN caused the formation of double preprophase bands; the disruption of mitotic spindles led to incomplete sister chromatid separation and disrupted phragmoplasts in root tip meristems. This work shows that CYN alters reed growth and anatomy through the alteration of MT organization.

• Transglutaminase 2 is needed for the formation of an efficient phagocyte portal in macrophages engulfing apoptotic cells.

  Authors: Beáta Tóth, Eva Garabuczi, Zsolt Sarang, György Vereb, György Vámosi, Daniel Aeschlimann, Bernadett Blaskó, Bálint Bécsi, Ferenc Erdõdi, Adam Lacy-Hulbert, Ailiang Zhang, Laura Falasca, Raymond B Birge, Zoltán Balajthy, Gerry Melino, László Fésüs, Zsuzsa Szondy

  Journal of immunology (Baltimore, Md. : 1950). 03/2009; 182(4):2084-92.

  Transglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop anTransglutaminase 2 (TG2), a protein cross-linking enzyme with many additional biological functions, acts as coreceptor for integrin beta(3). We have previously shown that TG2(-/-) mice develop an age-dependent autoimmunity due to defective in vivo clearance of apoptotic cells. Here we report that TG2 on the cell surface and in guanine nucleotide-bound form promotes phagocytosis. Besides being a binding partner for integrin beta(3), a receptor known to mediate the uptake of apoptotic cells via activating Rac1, we also show that TG2 binds MFG-E8 (milk fat globulin EGF factor 8), a protein known to bridge integrin beta(3) to apoptotic cells. Finally, we report that in wild-type macrophages one or two engulfing portals are formed during phagocytosis of apoptotic cells that are characterized by accumulation of integrin beta(3) and Rac1. In the absence of TG2, integrin beta(3) cannot properly recognize the apoptotic cells, is not accumulated in the phagocytic cup, and its signaling is impaired. As a result, the formation of the engulfing portals, as well as the portals formed, is much less efficient. We propose that TG2 has a novel function to stabilize efficient phagocytic portals.

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• Stem cells of the adult cornea: From cytometric markers to therapeutic applications.

  Authors: Lili Takács, Enikö Tóth, András Berta, György Vereb

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 01/2009;

  The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins:The cornea is a major protective shield of the interior of the eye and represents two thirds of its refractive power. It is made up of three tissue layers that have different developmental origins: the outer, epithelial layer develops from the ectoderm overlying the lens vesicle, whereas the stroma and the endothelium have mesenchymal origin. In the adult organism, the outermost corneal epithelium is the most exposed to environmental damage, and its constant renewal is assured by the epithelial stem cells that reside in the limbus, the circular border of the cornea. Cell turnover in the stromal layer is very slow and the endothelial cells probably do not reproduce in the adult organism. However, recent experimental evidence indicates that stem cells may be found in these layers. Damage to any of the corneal layers leads to loss of transparency and low vision. Corneal limbal stem cell deficiency results in severe ocular surface disease and its treatment by transplantating ex vivo expanded limbal epithelial cells is becoming widely accepted today. Stromal and endothelial stem cells are potential tools of tissue engineering and regenerative therapies of corneal ulcers and endothelial cell loss. In the past few years, intensive research has focused on corneal stem cells aiming to improve the outcomes of the current corneal stem cell transplantation techniques. This review summarizes the current state of knowledge on corneal epithelial, stromal and endothelial stem cells. Special emphasis is placed on the molecular markers that may help to identify these cells, and the recently revealed mechanisms that could maintain their "stemness" or drive their differentiation. The techniques for isolating and culturing/expanding these cells are also described. (c) 2008 International Society for Advancement of Cytometry.

• Die hard: Are cancer stem cells the Bruce Willises of tumor biology?

  Authors: Akos Fábián, Márk Barok, György Vereb, János Szöllősi

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 01/2009;

  In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer:In recent years, an exponentially growing number of studies have focused on identifying cancer stem cells (CSC) in human malignancies. The rare CSCs could be crucial in controlling and curing cancer: through asymmetric division CSCs supposedly drive tumor growth and evade therapy with the help of traits shared with normal stem cells such as quiescence, self-renewal ability, and multidrug resistance pump activity. Here, we give a brief overview of techniques used to confirm the stem cell-like behavior of putative CSCs and discuss markers and methods for identifying, isolating, and culturing them. We touch on the limitations of each marker and why the combined use of CSC markers, in vitro and in vivo assays may still fail to identify all relevant CSC populations. Finally, the various experimental findings supporting and contradicting the CSC hypothesis are summarized. The large number of tumor types thus far with a subpopulation of uniquely tumorigenic and therapy resistant cells suggests that despite the unanswered questions and inconsistencies, the CSC hypothesis has a legitimate role to play in tumor biology. At the same time, experimental evidence supporting the established alternative theory of clonal evolution can be found as well. Therefore, a model that describes cancer initiation and progression should combine elements of clonal evolution and CSC theory. (c) 2008 International Society for Advancement of Cytometry.

• Two-sided fluorescence resonance energy transfer for assessing molecular interactions of up to three distinct species in confocal microscopy.

  Authors: Zsolt Fazekas, Miklós Petrás, Akos Fábián, Zsuzsanna Pályi-Krekk, Péter Nagy, Sándor Damjanovich, György Vereb, János Szöllosi

  Cytometry. Part A : the journal of the International Society for Analytical Cytology. 04/2008; 73(3):209-19.

  The role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis ofThe role of the expression patterns of proteins involved in oncogenesis can be understood after characterizing their multimolecular interactions. Conventional FRET methods permit the analysis of interaction between two molecular species at the most, which necessitates the introduction of new approaches for studying multicomponent signaling complexes. Flow cytometric as well as microscopic donor (dbFRET) and acceptor (abFRET) photobleaching FRET measurements were performed to determine the association states of ErbB2, beta1-integrin, and CD44 receptors. Based on consecutively applied abFRET and dbFRET methods (two-sided FRET), the relationship of beta1-integrin-ErbB2 heteroassociation to ErbB2 homoassociation and of beta1-integrin-ErbB2 heteroassociation to ErbB2-CD44 heteroassociation was studied by correlating pixel-by-pixel FRET values of the corresponding abFRET and dbFRET images in contour plots. Anticorrelation was observed between beta1-integrin-ErbB2 heteroassociation and ErbB2 homoassociation on trastuzumab sensitive N87 and SK-BR-3 cells, while modest positive correlation was found between beta1-integrin-ErbB2 and ErbB2-CD44 heteroassociation on trastuzumab resistant MKN-7 cells. The FRET efficiency values of beta1-integrin-ErbB2 heteroassociation were markedly higher at the focal adhesion regions on attached cells than those measured by flow cytometry on detached cells. In conclusion, we implemented an experimental set-up termed two-sided FRET for correlating two pairwise interactions of three arbitrarily chosen molecular species. On the basis of our results, we assume that the homoassociation state of ErbB2 is dynamically modulated by its interaction with beta1-integrins.