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Emmanouilidou E.,Biomedical Research Foundation of the Academy of Athens BRFAA | Stefanis L.,Biomedical Research Foundation of the Academy of Athens BRFAA | Stefanis L.,National and Kapodistrian University of Athens | Vekrellis K.,Biomedical Research Foundation of the Academy of Athens BRFAA
Neurobiology of Aging | Year: 2010

Proteasomal dysfunction may play a role in neurodegenerative conditions and protein aggregation. Overexpression in neuronal cells of α-synuclein, a molecule linked to Parkinson's Disease, may lead to proteasomal dysfunction. Using PC12 cells stably expressing wild-type or mutant α-synuclein and gel filtration, we demonstrate that soluble, intermediate size oligomers of α-synuclein co-elute with the 26S proteasome. These soluble oligomers associate with the 26S proteasome and are significantly increased following treatment with proteasomal, but not lysosomal, inhibitors, indicating specific degradation of these particular species by the 26S proteasome. Importantly, expression of α-synuclein resulted in a significant inhibition of all proteasomal activities without affecting the levels or assembly of the 26S proteasome. Pharmacological dissociation of α-synuclein oligomers restored proteasomal function and reduced polyubiquitinated protein load in intact cells. Our findings suggest a model where only a subset of specific soluble cell-derived α-synuclein oligomers is targeted to the 26S proteasome for degradation, and simultaneously inhibit its function, likely by impeding access of other proteasomal substrates. © 2008 Elsevier Inc.


Botitsi H.V.,General Chemical State Laboratory | Garbis S.D.,Biomedical Research Foundation Of The Academy Of Athens Brfaa | Economou A.,National and Kapodistrian University of Athens | Tsipi D.F.,General Chemical State Laboratory
Mass Spectrometry Reviews | Year: 2011

Analysis of pesticides and their metabolites in food and water matrices continues to be an active research area closely related to food safety and environmental issues. This review discusses the most widely applied mass spectrometric (MS) approaches to pesticide residues analysis over the last few years. The main techniques for sample preparation remain solvent extraction and solid-phase extraction. The QuEChERS (Quick, Easy, Cheap, Effective, Rugged, Safe) approach is being increasingly used for the development of multi-class pesticide residues methods in various sample matrices. MS detectors-triple quadrupole (QqQ), ion-trap (IT), quadrupole linear ion trap (QqLIT), time-of-flight (TOF), and quadrupole time-of-flight (QqTOF)-have been established as powerful analytical tools sharing a primary role in the detection/quantification and/or identification/confirmation of pesticides and their metabolites. Recent developments in analytical instrumentation have enabled coupling of ultra-performance liquid chromatography (UPLC) and fast gas chromatography (GC) with MS detectors, and faster analysis for a greater number of pesticides. The newly developed "ambient-ionization" MS techniques (e.g., desorption electrospray ionization, DESI, and direct analysis in real time, DART) hyphenated with high-resolution MS platforms without liquid chromatography separation, and sometimes with minimum pre-treatment, have shown potential for pesticide residue screening. The recently introduced Orbitrap mass spectrometers can provide high resolving power and mass accuracy, to tackle complex analytical problems involved in pesticide residue analysis. © 2010 Wiley Periodicals, Inc.


Bitsika V.,Biomedical Research Foundation of the Academy of Athens BRFAA | Vlahou A.,Biomedical Research Foundation of the Academy of Athens BRFAA | Roubelakis M.G.,Biomedical Research Foundation of the Academy of Athens BRFAA | Roubelakis M.G.,National and Kapodistrian University of Athens
Current Stem Cell Research and Therapy | Year: 2013

There is compelling evidence that mesenchymal stem cells (MSCs) can be utilized as delivery vehicles for cancer therapeutics. During the last decade, bone marrow MSCs have been used as delivery vehicles for the local production of therapeutic proteins in multiple tumor types, taking advantage of their innate tropism to the tumor site and their low immunogenicity. More recently, MSCs have been isolated from fetal tissues during gestation or after birth. Fetal MSCs derived from amniotic fluid, amniotic membrane, umbilical cord matrix (Wharton's jelly) and umbilical cord blood are more advantageous than adult MSCs, as they can be isolated noninvasively in large numbers without the ethical reservations associated with embryo research. Several studies have documented that fetal MSCs harbor a therapeutic potential in cancer treatment, as they can home to the tumor site and reduce tumor burden. This natural tumor tropism together with their low immunogenicity renders fetal MSCs as powerful therapeutic tools in gene therapy-based cancer therapeutic schemes. This review summarizes various approaches where the tumor-homing capacity of fetal MSCs has been employed for the localized delivery of anti-tumor therapeutic agents. © 2013 Bentham Science Publishers.


Papanikolaou E.,Biomedical Research Foundation of the Academy of Athens BRFAA | Papanikolaou E.,National and Kapodistrian University of Athens | Anagnou N.P.,Biomedical Research Foundation of the Academy of Athens BRFAA | Anagnou N.P.,National and Kapodistrian University of Athens
Current Gene Therapy | Year: 2010

Gene therapy utilizing retroviral vectors is being postulated as a real therapeutic alternative for many hemopoietic inherited diseases, such as β-thalassemia or sickle cell disease. A major limitation of current vectors is their inability to achieve efficient gene transfer into quiescent cells, such as human CD34 +cells that reside in the Go phase of the cell cycle and are highly enriched in hemopoietic stem cells. For that reason, lentiviral vectors (LVs) were proven to be more efficient than oncoretroviral vectors. Additional problems of these vectors are a) the low titers observed due to regulatory elements of the β-globin locus, used for the improvement of the transgene's expression, b) the eventual silencing of the transgene and c) the toxicity posed on CD34 + cells due to the usage of VSV-G as an envelope protein. These facts hamper their application for gene therapy of hematopoietic cells. Thus, the major current drawbacks of the field affecting therapeutic efficacy, include 1) insufficient transduction efficiency of the target hemopoietic stem cells, 2) inconsistent expression of the transgene, 3) putative aberrant expression near integration sites raising safety issues and 4) lack of long term expression of the transgene exhibiting eventual silencing. This review presents the current status of globin gene therapy for the hemoglobin disorders, reviews the recent results and discusses how the knowledge gained from these trials can be used to develop a safe and effective gene therapy approach for the treatment of β-thalassemia and SCD. © 2010 Bentham Science Publishers Ltd.


Xilouri M.,Biomedical Research Foundation of the Academy of Athens BRFAA | Stefanis L.,Biomedical Research Foundation of the Academy of Athens BRFAA | Stefanis L.,National and Kapodistrian University of Athens
Expert Reviews in Molecular Medicine | Year: 2011

Macroautophagy and chaperone-mediated autophagy (CMA) are the two main mammalian lysosomal proteolytic systems. In macroautophagy, double-membrane structures engulf organelles and other intracellular constituents through a highly regulated process that involves the formation of autophagic vacuoles and their fusion with lysosomes. In CMA, selected proteins are targeted through a nonvesicular pathway to a transport complex at the lysosomal membrane, through which they are threaded into the lysosomes and degraded. Autophagy is important in development, differentiation, cellular remodelling and survival during nutrient starvation. Increasing evidence suggests that autophagic dysregulation causes accumulation of abnormal proteins or damaged organelles, which is a characteristic of chronic neurodegenerative conditions, such as Parkinson disease (PD). Evidence from post-mortem material, transgenic mice, and animal and cellular models of PD suggests that both major autophagic pathways are malfunctioning. Numerous connections exist between proteins genetically linked to autosomal dominant PD, in particular α-synuclein and LRRK2, and autophagic pathways. However, proteins involved in recessive PD, such as PINK1 and Parkin (PINK2), function in the process of mitophagy, whereby damaged mitochondria are selectively engulfed by macroautophagy. This wealth of new data suggests that both autophagic pathways are potential targets for therapeutic intervention in PD and other related neurodegenerative conditions. Copyright © 2011 Cambridge University Press.


Gazouli M.,National and Kapodistrian University of Athens | Roubelakis M.G.,National and Kapodistrian University of Athens | Roubelakis M.G.,Biomedical Research Foundation of the Academy of Athens BRFAA | Theodoropoulos G.E.,National and Kapodistrian University of Athens
Inflammatory Bowel Diseases | Year: 2014

The incidence and prevalence of inflammatory bowel disease is increasing in Western countries. Current therapies, ranging from antiinflammatory drugs, immunosuppressive regimens to new biological therapies, remain inadequate. Advances in our understanding of the pathophysiological mechanisms underlying the pathogenetic disease process and the recent findings on the regenerative and immunoregulatory potential of stem cells open new opportunities in the therapy of inflammatory bowel disease. Therapeutic modalities, including hematopoietic stem cells, adult mesenchymal stem/stromal cells, and the recently identified amniotic origin stem cells, attracted much attention in the recent years. The current review highlights the recent pivotal findings for stem cell-based approaches to inflammatory bowel disease therapy. Copyright © 2014 Crohn's & Colitis Foundation of America, Inc.


Chawla R.,ETH Zurich | Chawla R.,University of Zürich | Redon S.,Ecole Polytechnique Federale de Lausanne | Raftopoulou C.,Biomedical Research Foundation of the Academy of Athens BRFAA | And 3 more authors.
EMBO Journal | Year: 2011

Eukaryotic up-frameshift 1 (UPF1) is a nucleic acid-dependent ATPase and 5′-to-3′ helicase, best characterized for its roles in cytoplasmic RNA quality control. We previously demonstrated that human UPF1 binds to telomeres in vivo and its depletion leads to telomere instability. Here, we show that UPF1 is present at telomeres at least during S and G2/M phases and that UPF1 association with telomeres is stimulated by the phosphoinositide 3-kinase (PI3K)-related protein kinase ataxia telangiectasia mutated and Rad3-related (ATR) and by telomere elongation. UPF1 physically interacts with the telomeric factor TPP1 and with telomerase. Akin to UPF1 binding to telomeres, this latter interaction is mediated by ATR. Moreover, the ATPase activity of UPF1 is required to prevent the telomeric defects observed upon UPF1 depletion, and these defects stem predominantly from inefficient telomere leading-strand replication. Our results portray a scenario where UPF1 orchestrates crucial aspects of telomere biology, including telomere replication and telomere length homeostasis. © 2011 European Molecular Biology Organization | All Rights Reserved.


Kaltezioti V.,Biomedical Research Foundation of the Academy of Athens BRFAA | Antoniou D.,Biomedical Research Foundation of the Academy of Athens BRFAA | Stergiopoulos A.,Biomedical Research Foundation of the Academy of Athens BRFAA | Rozani I.,Biomedical Research Foundation of the Academy of Athens BRFAA | And 2 more authors.
Journal of Neuroscience | Year: 2014

Specification of spinal cord neurons depends on gene regulation networks that impose distinct fates in neural progenitor cells (NPCs). Olig2 is a key transcription factor in these networks by inducing motor neuron (MN) specification and inhibiting interneuron identity. Despite the critical role of Olig2 in nervous system development and cancer progression, the upstream molecular mechanisms that control Olig2 gene transcription are not well understood. Here we demonstrate that Prox1, a transcription repressor and downstream target of proneural genes, suppresses Olig2 expression and therefore controls ventral spinal cord patterning. In particular, Prox1 is strongly expressed in V2 interneuron progenitors and largely excluded from Olig2+ MN progenitors (pMN). Gain- and loss-of-function studies in mouse NPCs and chick neural tube show that Prox1 is sufficient and necessary for the suppression of Olig2 expression and proper control of MN versus V2 interneuron identity. Mechanistically, Prox1 interacts with the regulatory elements of Olig2 gene locus in vivo and it is critical for proper Olig2 transcription regulation. Specifically, chromatin immunoprecipitation analysis in the mouse neural tube showed that endogenous Prox1 directly binds to the proximal promoter of the Olig2 gene locus, as well as to the K23 enhancer, which drives Olig2 expression in the pMN domain. Moreover, plasmid-based transcriptional assays in mouse NPCs suggest that Prox1 suppresses the activity of Olig2 gene promoter and K23 enhancer. These observations indicate that Prox1 controls binary fate decisions between MNs and V2 interneurons in NPCs via direct repression of Olig2 gene regulatory elements. © 2014 the authors.


Gouti M.,Biomedical Research Foundation of the Academy of Athens BRFAA | Briscoe J.,UK National Institute for Medical Research | Gavalas A.,Biomedical Research Foundation of the Academy of Athens BRFAA
Stem Cells | Year: 2011

Hox genes play a central role in neural crest (NC) patterning particularly in the cranial region of the body. Despite evidence that simultaneous loss of Hoxa1 and Hoxb1 function resulted in NC specification defects, the role of Hox genes in NC specification has remained unclear due to extended genetic redundancy among Hox genes. to circumvent this problem, we expressed anterior Hox genes in the trunk neural tube of the developing chick embryo. this demonstrated that anterior Hox genes play a central role in NC cell specification by rapidly inducing the key transcription factors Snail2 and Msx1/2 and a neural progenitor to NC cell fate switch characterized by cell adhesion changes and an epithelial-to-mesenchymal transition (EMT). Cells delaminated from dorsal and medial neural tube levels and generated ectopic neurons, glia progenitors, and melanocytes. The mobilization of the NC genetic cascade was dependent upon bone morphogenetic protein signaling and optimal levels of Notch signaling. Therefore, anterior Hox patterning genes participate in NC specification and EMT by interacting with NC-inducing signaling pathways and regulating the expression of key genes involved in these processes. © AlphaMed Press.


Xilouri M.,Biomedical Research Foundation of the Academy of Athens BRFAA
Molecular neurobiology | Year: 2013

An increasing wealth of data indicates a close relationship between the presynaptic protein alpha-synuclein and Parkinson's disease (PD) pathogenesis. Alpha-synuclein protein levels are considered as a major determinant of its neurotoxic potential, whereas secreted extracellular alpha-synuclein has emerged as an additional important factor in this regard. However, the manner of alpha-synuclein degradation in neurons remains contentious. Both the ubiquitin-proteasome system (UPS) and the autophagy-lysosome pathway (ALP)-mainly macroautophagy and chaperone-mediated autophagy-have been suggested to contribute to alpha-synuclein turnover. Additionally, other proteases such as calpains, neurosin, and metalloproteinases have been also proposed to have a role in intracellular and extracellular alpha-synuclein processing. Both UPS and ALP activity decline with aging and such decline may play a pivotal role in many neurodegenerative conditions. Alterations in these major proteolytic pathways may result in alpha-synuclein accumulation due to impaired clearance. Conversely, increased alpha-synuclein protein burden promotes the generation of aberrant species that may impair further UPS or ALP function, generating thus a bidirectional positive feedback loop leading to neuronal death. In the current review, we summarize the recent findings related to alpha-synuclein degradation, as well as to alpha-synuclein-mediated aberrant effects on protein degradation systems. Identifying the factors that regulate alpha-synuclein association to cellular proteolytic pathways may represent potential targets for therapeutic interventions in PD and related synucleinopathies.

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