Thessaloníki, Greece
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Bergh A.-C.,Linköping University | Evaldsson C.,Linköping University | Pedersen L.B.,Rigshospitalet | Geisler C.,Rigshospitalet | And 5 more authors.
Haematologica | Year: 2014

Chronic lymphocytic leukemia B cells express auto/xeno antigen-reactive antibodies that bind to self-epitopes and resemble natural IgM antibodies in their repertoire. One of the antigenic structures recognized is oxidation induced malonedialdehyde that is present on low-density lipoprotein, apoptotic blebs, and on certain microbes. The poor-prognostic stereotyped subset #1 (Clan I IGHV genes-IGKV1(D)-39) express IgM B-cell receptors that bind oxidized low-density lipoprotein. In this study, we have used for the first time this authentic cognate antigen for analysis of downstream B-cell receptor-signal transduction events, since it is more faithful to B-cell physiology than anti-IgM. Multivalent oxidized low-density lipoprotein showed specific binding to subset #1 IgM/IgD B-cell receptors, whereas native low-density lipoprotein did not. The antigen binding induced prompt receptor clustering followed by internalization. However, the receptor-signal transduction was silenced, revealing no Ca2+ mobilization or cell-cycle entry, while phosphorylated extracellular-regulated kinase 1/2 basal levels were high and could not be elevated further by oxidized low-density lipoprotein. Interestingly, B-cell receptor responsiveness was recovered after 48-h culture in the absence of antigen in half of the cases. Toll-like receptor 9-ligand was found to breach the B-cell receptor-signaling incompetence in 5 of 12 cases pointing to intra-subset heterogeneity. Altogether, this study supports B-cell receptor unresponsiveness to cognate self-antigen on its own in poor-prognostic subset #1 chronic lymphocytic leukemia, indicating that these cells proliferate by other mechanisms that may override B-cell receptor silencing brought about in a context of self-tolerance/anergy. These novel findings have implications for the understanding of chronic lymphocytic leukemia pathobiology and therapy. © 2014 Ferrata Storti Foundation.


Agathangelidis A.,San Raffaele Scientific Institute | Vardi A.,G Papanicolaou Hospital | Baliakas P.,Uppsala University | Stamatopoulos K.,G Papanicolaou Hospital | And 2 more authors.
Leukemia and Lymphoma | Year: 2014

Over the last decade, immunogenetic analysis of B-cell receptor immunoglobulins (BcR IGs) has proved to be a particularly fruitful field in chronic lymphocytic leukemia (CLL), not only for understanding disease pathogenesis but also for discriminating clinical subgroups with markedly distinct course and outcome. Of utmost importance was the identification of quasi-identical BcR IGs among unrelated patients with CLL, fittingly coined as "stereotypy," that set the wheels in motion for unraveling the role of antigen(s) in the selection and expansion of the leukemic clones. The categorization of CLL clones into "subsets" according to shared BcR IG structural characteristics provided a compartmentalized view of this otherwise heterogeneous disease, which eventually led to defining strikingly homogeneous groups of patients in terms of: (i) functional properties of the clonal BcR IGs, e.g. BcR reactivity and signaling; (ii) clonal genetic landscape, e.g. genomic aberrations, gene expression/methylation profiles, microRNA signatures; and (iii) clinical course and outcome. The remarkable restriction of the CLL IG gene repertoire, resulting to a great degree from the high impact of BcR IG stereotypy, may also prompt speculations regarding CLL ontogenesis. Overall, the BcR IG molecule justifiably lies at the heart of CLL clinical research, holding the promise of subset-tailored therapies. © 2014 Informa UK, Ltd.


Schreck I.,Karlsruhe Institute of Technology | Marko D.,Institute of Applied Biosciences | Marko D.,University of Vienna | Deigendesch U.,Karlsruhe Institute of Technology | And 3 more authors.
Archives of Toxicology | Year: 2012

The Alternaria mycotoxins alternariol (AOH) and alternariol methyl ether (AME) are potential carcinogens. As planar compounds, AOH and AME are preferentially metabolized by cytochrome P450 (CYP) 1A1 and 1A2. The most prominent regulator of CYP1A1 is the dimeric transcription factor complex AhR/ARNT, which is activated by planar ligands. Therefore, we studied the activation of AhR/ARNT by AOH and AME and monitored CYP1A1 induction in murine hepatoma cells (Hepa-1c1c7). Indeed, AOH and AME enhanced the levels of CYP1A1 in Hepa1c1c7 cells but not in cells with inactivated AhR (Hepa1c1c12) or ARNT (Hepa-1c1c4). AOH and AME did not increase the production of reactive oxygen species but reduced cell counts in Hepa-1c1c7 cells after 24 and 48 h. This effect, however, was independent of AhR/ARNT. At 48 h, AOHand AME increased apoptosis dependent on AhR and ARNT. In conclusion, AOH and AME are novel inducers of the AhR/ARNT pathway, which mediates induction of CYP1A1 and apoptosis and might thereby contribute to the toxicity of these mycotoxins. © Springer-Verlag 2011.


Chronopoulou E.,Agricultural University of Athens | Madesis P.,Institute of Applied Biosciences | Tsaftaris A.,Institute of Applied Biosciences | Tsaftaris A.,Aristotle University of Thessaloniki | Labrou N.E.,Agricultural University of Athens
Applied Biochemistry and Biotechnology | Year: 2014

Glutathione transferases (GSTs, EC 2.5.1.18) are ubiquitous proteins in plants that play important roles in stress tolerance and in the detoxification of toxic chemicals and metabolites. In this study, we systematically examined the catalytic diversification of a GST isoenzyme from Phaseolus vulgaris (PvGST) which is induced under biotic stress treatment (Uromyces appendiculatus infection). The full-length cDNA of this GST isoenzyme (termed PvGSTU3-3) with complete open reading frame, was isolated using RACE-RT and showed that the deduced amino acid sequence shares high homology with the tau class plant GSTs. PvGSTU3-3 catalyzes several different reactions and exhibits wide substrate specificity. Of particular importance is the finding that the enzyme shows high antioxidant catalytic function and acts as hydroperoxidase, thioltransferase, and dehydroascorbate reductase. In addition, its K m for GSH is about five to ten times lower compared to other plant GSTs, suggesting that PvGSTU3-3 is able to perform efficient catalysis under conditions where the concentration of reduced glutathione is low (e.g.; oxidative stress). Its ability to conjugate GSH with isothiocyanates may provide an additional role for this enzyme to act as a regulator of the released isothiocyanates from glucosinolates as a response of biotic stress. Molecular modeling showed that PvGSTU3-3 shares the same overall fold and structural organization with other plant cytosolic GSTs, with major differences at their hydrophobic binding sites (H-sites) and some differences at the level of C-terminal domain and the linker between the C- and N-terminal domains. PvGSTU3-3, in general, exhibits restricted ability to bind xenobiotics in a nonsubstrate manner, suggesting that the biological role of PvGSTU3-3, is restricted mainly to the catalytic function. Our findings highlight the functional and catalytic diversity of plant GSTs and demonstrate their pivotal role for addressing biotic stresses in Phaseolus vulgaris. © 2013 Springer Science+Business Media New York.


Ignea C.,Mediterranean Agronomic Institute of Chania | Trikka F.A.,Institute of Applied Biosciences | Kourtzelis I.,Aristotle University of Thessaloniki | Argiriou A.,Institute of Applied Biosciences | And 4 more authors.
Microbial Cell Factories | Year: 2012

Background: Terpenoids and isoprenoids are an important class of natural products, which includes currently used drugs, high value bioactive and industrial compounds, and fuel candidates. Due to their industrial application, there is increasing interest in the development of S. cerevisiae strains capable of producing high levels of terpenoids. Results: Aiming to identify new gene targets which can be manipulated to increase sesquiterpene production, a set of HMG2 positive genetic interactors were assessed as single and digenic heterozygous deletions in the presence or absence of stable HMG2(K6R) overexpression. Upon single allele deletion, most genes examined led to increased sesquiterpene production in yeast cells. Tandem heterozygous deletion of a set of three genes, the ubiquitin ligases ubc7 and ssm4/doa10, and the ER resident protein pho86, led to an 11-fold increase in caryophyllene yields (125 mg/L in shake flasks) compared to cells lacking these modifications. The effect of the heterozygous deletions appears to be due to Hmg1p and Hmg2p stabilization.Conclusion: Heterozygous deletions cause significant reductions in protein levels but do not lead to growth impediments frequently seen in haploid strains. By exploiting desirable haploinsufficiencies in yeast, we identified a new set of genes that can be disrupted in tandem and cause significant stabilization of Hmgp and a substantial increase in sesquiterpene production. The approach presented here allows new genetic perturbations to be compiled on yeast cell factory strains without negatively impacting cell growth and viability. © 2012 Ignea et al.; licensee BioMed Central Ltd.


Chatzidimopoulos M.,University of Thessaly | Ganopoulos I.,Institute of Applied Biosciences | Madesis P.,Institute of Applied Biosciences | Vellios E.,University of Thessaly | And 2 more authors.
Plant Pathology | Year: 2014

A novel, high-resolution melting (HRM) analysis was developed to detect single nucleotide polymorphisms (SNPs) associated with resistance to fenhexamid (hydroxyanilides) and boscalid (succinate dehydrogenase inhibitors) in Botrytis cinerea isolates. Thirty-six single-spore isolates arising from 13 phenotypes were selected and tested for fungicide sensitivity. Germ tube elongation assays showed two distinct sensitivity levels for each fungicide. Sequencing revealed that resistance to fenhexamid was due to a nucleotide change in the erg27 gene, resulting in an amino acid replacement of phenylalanine (F) with serine (S) or valine (V) at position 412 of the protein, whereas in isolates resistant to boscalid, a nucleotide change in the sdhB gene resulted in the replacement of histidine (H) with arginine (R) or tyrosine (Y) at position 272 of the respective protein. In each case, melting curve analysis generated three distinct profiles corresponding to the presence of each nucleotide in the targeted areas. HRM analysis successfully detected and differentiated the substitutions associated with resistance to both fungicides. In vitro bioassays, direct sequencing and high-resolution melting analysis showed a 100% correlation with detection of resistance. The results demonstrate the utility of HRM analysis as a potential molecular tool for routine detection of fungicide resistance using known polymorphic genes of B. cinerea populations. © 2014 British Society for Plant Pathology.


Sakaridis I.,Institute of Applied Biosciences | Ganopoulos I.,Institute of Applied Biosciences | Argiriou A.,Institute of Applied Biosciences | Tsaftaris A.,Institute of Applied Biosciences
Meat Science | Year: 2013

The substitution of high priced meat with low cost ones and the fraudulent labeling of meat products make the identification and traceability of meat species and their processed products in the food chain important. A polymerase chain reaction followed by a High Resolution Melting (HRM) analysis was developed for species specific detection of buffalo; it was applied in six commercial meat products. A pair of specific 12S and universal 18S rRNA primers were employed and yielded DNA fragments of 220. bp and 77. bp, respectively. All tested products were found to contain buffalo meat and presented melting curves with at least two visible inflection points derived from the amplicons of the 12S specific and 18S universal primers. The presence of buffalo meat in meat products and the adulteration of buffalo products with unknown species were established down to a level of 0.1%. HRM was proven to be a fast and accurate technique for authentication testing of meat products. © 2012 Elsevier Ltd.


Kanduri M.,Gothenburg University | Sander B.,Karolinska University Hospital | Ntoufa S.,G Papanicolaou Hospital | Ntoufa S.,Institute of Applied Biosciences | And 7 more authors.
Epigenetics | Year: 2013

The chromatin modifier EZH2 is overexpressed and associated with inferior outcome in mantle cell lymphoma (MCL). Recently, we demonstrated preferential DNA methylation of HOX genes in MCL compared with chronic lymphocytic leukemia (CLL), despite these genes not being expressed in either entity. Since EZH2 has been shown to regulate HOX gene expression, to gain further insight into its possible role in differential silencing of HOX genes in MCL vs. CLL, we performed detailed epigenetic characterization using representative cell lines and primary samples. We observed significant overexpression of EZH2 in MCL vs. CLL. Chromatin immune precipitation (ChIP) assays revealed that EZH2 catalyzed repressive H3 lysine 27 trimethylation (H3K27me3), which was sufficient to silence HOX genes in CLL, whereas in MCL H3K27me3 is accompanied by DNA methylation for a more stable repression. More importantly, hypermethylation of the HOX genes in MCL resulted from EZH2 overexpression and subsequent recruitment of the DNA methylation machinery onto HOX gene promoters. The importance of EZH2 upregulation in this process was further underscored by siRNA transfection and EZH2 inhibitor experiments. Altogether, these observations implicate EZH2 in the long-term silencing of HOX genes in MCL, and allude to its potential as a therapeutic target with clinical impact. © 2013 Landes Bioscience.


Mansouri L.,Uppsala University | Papakonstantinou N.,Uppsala University | Papakonstantinou N.,Institute of Applied Biosciences | Ntoufa S.,Uppsala University | And 4 more authors.
Seminars in Cancer Biology | Year: 2016

The nuclear factor-κB (NF-κB) pathway is constitutively activated in chronic lymphocytic leukemia (CLL) patients, and hence plays a major role in disease development and evolution. In contrast to many other mature B-cell lymphomas, only a few recurrently mutated genes involved in canonical or non-canonical NF-κB activation have been identified in CLL (i.e. BIRC3, MYD88 and NFKBIE mutations) and often at a low frequency. On the other hand, CLL B cells seem ‘addicted’ to the tumor microenvironment for their survival and proliferation, which is primarily mediated by interaction through a number of cell surface receptors, e.g. the B-cell receptor (BcR), Toll-like receptors and CD40, that in turn activate downstream NF-κB. The importance of cell-extrinsic triggering for CLL pathophysiology was recently also highlighted by the clinical efficacy of novel drugs targeting microenvironmental interactions through the inhibition of BcR signaling. In other words, CLL can be considered a prototype disease for studying the intricate interplay between external triggers and intrinsic aberrations and their combined impact on disease evolution. In this review, we will discuss the current understanding of mechanisms underlying NF-κB deregulation in CLL, including micro-environmental, genetic and epigenetic events, and summarize data generated in murine models resembling human CLL. Finally, we will also discuss different strategies undertaken to intervene with the NF-κB pathway and its upstream mediators. © 2016 Elsevier Ltd


Kampranis S.C.,University of Crete | Makris A.M.,Institute of Applied Biosciences
Computational and Structural Biotechnology Journal | Year: 2012

Technological developments over the past century have made microbes the work-horses of large scale industrial production processes. Current efforts focus on the metabolic engineering of microbial strains to produce high levels of desirable end-products. The arsenal of the contemporary metabolic engineer contains tools that allow either targeted rational interventions or global screens that combine classical approaches with -omics technologies. Production of terpenoids in S. cerevisiae presents a characteristic example of contemporary biotechnology that integrates all the variety of novel approaches used in metabolic engineering. Terpenoids have attracted significant interest as pharmaceuticals, flavour and fragrance additives, and, more recently, biofuels. The ongoing metabolic engineering efforts, combined with the continuously increasing number of terpene biosynthetic enzymes discovered will enable the economical and environmentally friendly production of a wide range of compounds. © 2012 Bernstein and Carlson.

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