Beyond crisis: constitutional change in Greece after the Memoranda

Can financial crises bring constitutional change? Has Greece become a prominent such example? Already 7 years into recession, crisis-hit Greece is experiencing a tremendous institutional change. This change, which takes place informally rather than formally, is affecting state institutions- primarily its parliament, the executive and courts. Everyday practice, political or otherwise, changes the norms, vital checks and balances and brings about a new reading and ethos to our Constitution. With my upcoming paper, I aim to explore the idea of constitutional change and to connect it to parliamentary and judicial activity after the Memoranda. How have state institutions reacted to the financial crisis? How do economic emergency and external players interplay with local institutions and interests? Finally, will this change have a longer-lasting impact on the country, beyond its economic recovery

Brexit and the ECJ: If the UK plays in EU territory, it has to accept EU rules and referees

Theresa May was adamant that the UK would not accept the jurisdiction of the European Court of Justice after Brexit. But as reality has sunk in, that red line has begun to blur. LSE Fellow Anna Tsiftsoglou explains why the ECJ is such a vital issue in the exit negotiations. To reverse David Davis’ footballing metaphor, if the UK plays in EU territory, it has to accept EU rules and referees

Why May can't have it all: the ECJ and the Brexit rules of (dis-)engagement

Theresa May was adamant that the UK would not accept the jurisdiction of the European Court of Justice after Brexit. But as reality has sunk in, that red line has begun to blur. LSE Fellow Anna Tsiftsoglou explains why the ECJ is such a vital issue in the exit negotiations. To reverse David Davis' footballing metaphor, if the UK plays in EU territory, it has to accept EU rules and referees

Greece after the memoranda: a constitutional retrospective

Can financial crises bring constitutional change? Has Greece become a prominent example? Over eight years into recession and having undergone several programmes of fiscal adjustment, Greece is gradually experiencing an institutional change. This change, which takes place informally rather than formally, is affecting state institutions, primarily its parliament, as well as the executive and the courts. Everyday practice, political or otherwise, has changed the norms, vital checks and balances and has brought about a new ethos to our Constitution. With this paper, I aim to explore the idea of constitutional change and to connect it primarily to parliamentary and judicial activity after the Memoranda. How have state institutions reacted to the crisis? How do external players interplay with local institutions and interests? Will this crisis have a longer-term impact on the country, beyond its economic recovery

Analysis of binding sites on complement factor I using artificial N-linked glycosylation.

Factor I (FI) is a serine protease that inhibits all complement pathways by degrading activated complement components C3b and C4b. FI functions only in the presence of several cofactors such as factor H, C4b-binding protein, complement receptor 1 and membrane cofactor protein. FI is composed of two chains linked by a disulphide bridge; the light chain comprises only the serine protease (SP) domain, while the heavy chain contains FI membrane attack complex domain (FIMAC), CD5 domain, low-density lipoprotein receptor 1 (LDLr1) and LDLr2 domains. To better understand how FI inhibits complement, we used homology-based 3D models of FI domains in an attempt to identify potential protein-protein interaction sites. Specific amino acids were then mutated to yield 20 recombinant mutants of FI carrying additional surface exposed N-glycosylation sites that were expected to sterically hinder interactions. The Michaelis constant (Km) of all FI mutants toward a small substrate was not increased. We found that many mutations in the FIMAC and SP domains nearly abolished ability of FI to degrade C4b and C3b in the fluid phase and on the surface, irrespectively of the cofactor used. In the other hand only few alterations in the CD5 and LDLr1/2 domains impaired this activity. In conclusion, all analyzed cofactors form similar trimolecular complexes with FI and C3b/C4b, and the accessibility of FIMAC and SP domains is crucial for the function of FI

Intracellular delivery of full length recombinant human mitochondrial L-Sco2 protein into the mitochondria of permanent cell lines and SCO2 deficient patient's primary cells

AbstractMutations in human SCO2 gene, encoding the mitochondrial inner membrane Sco2 protein, have been found to be responsible for fatal infantile cardioencephalomyopathy and cytochrome c oxidase (COX) deficiency. One potentially fruitful therapeutic approach for this mitochondrial disorder should be considered the production of human recombinant full length L-Sco2 protein and its deliberate transduction into the mitochondria. Recombinant L-Sco2 protein, fused with TAT, a Protein Transduction Domain (PTD), was produced in bacteria and purified from inclusion bodies (IBs). Following solubilisation with l-arginine, this fusion L-Sco2 protein was transduced in cultured mammalian cells of different origin (U-87 MG, T24, K-562, and patient's primary fibroblasts) and assessed for stability, transduction into mitochondria, processing and impact on recovery of COX activity. Our results indicate that: a) l-Arg solution was effective in solubilising recombinant fusion L-Sco2 protein, derived from IBs; b) fusion L-Sco2 protein was delivered successfully via a time- and concentration-dependent process into the mitochondria of human U-87 MG and T24 cells; c) fusion L-Sco2 protein was also transduced in human K-562 cells, transiently depleted of SCO2 transcripts and thus COX deficient; transduction of this fusion protein led to partial recovery of COX activity in such cells; d) [35S]Methionine-labelled fusion L-Sco2 protein, produced in a cell free transcription/translation system and incubated with intact isolated mitochondria derived from K-562 cells, was efficiently processed to yield the corresponding mature Sco2 protein, thus justifying the potential of the transduced fusion L-Sco2 protein to successfully activate COX holoenzyme; and finally, e) recombinant fusion L-Sco2 protein was successfully transduced into the mitochondria of primary fibroblasts derived from SCO2/COX deficient patient and facilitated recovery of COX activity. These findings provide the rationale of delivering recombinant proteins via PTD technology as a model for therapeutic approach of mitochondrial disorders

Knockdown of ZNF268, which Is Transcriptionally Downregulated by GATA-1, Promotes Proliferation of K562 Cells

The human ZNF268 gene encodes a typical KRAB-C2H2 zinc finger protein that may participate in hematopoiesis and leukemogenesis. A recent microarray study revealed that ZNF268 expression continuously decreases during erythropoiesis. However, the molecular mechanisms underlying regulation of ZNF268 during hematopoiesis are not well understood. Here we found that GATA-1, a master regulator of erythropoiesis, repressed the promoter activity and transcription of ZNF268. Electrophoretic mobility shift assays and chromatin immunoprecipitation assays showed that GATA-1 directly bound to a GATA binding site in the ZNF268 promoter in vitro and in vivo. Knockdown of ZNF268 in K562 erythroleukemia cells with specific siRNA accelerated cellular proliferation, suppressed apoptosis, and reduced expression of erythroid-specific developmental markers. It also promoted growth of subcutaneous K562-derived tumors in nude mice. These results suggest that ZNF268 is a crucial downstream target and effector of GATA-1. They also suggest the downregulation of ZNF268 by GATA-1 is important in promoting the growth and suppressing the differentiation of K562 erythroleukemia cells

Late local recurrence of dermatofibrosarcoma protuberans in the skin of female breast

Dermatofibrosarcoma protuberans (DFSP) of the breast is exceptionally obscure and late local recurrence of this entity on this site is even more uncommon. We describe such a case in a 48-year-old woman, who at the age of 35 had a DFSP excised from her right breast. Thirteen years later, she developed an ovoid mass in her right breast over the postsurgical scar area. Wide local excision of the tumor with generous tissue margin was performed and microscopic and immunohistochemical findings established the diagnosis of recurrent DFSP. No further treatment was administered and she remains well 18 months later, without tumor recurrence. We report an exceptionally rare case of local recurrence of DFSP in the female breast and discuss in detail the diagnostic and therapeutic implications of this pathology

Induction of endogenous γ-globin gene expression with decoy oligonucleotide targeting Oct-1 transcription factor consensus sequence

Human β-globin disorders are relatively common genetic diseases cause by mutations in the β-globin gene. Increasing the expression of the γ-globin gene has great benefits in reducing complications associated with these diseases. The Oct-1 transcription factor is involved in the transcriptional regulation of the γ-globin gene. The human γ-globin genes (both Aγ and Gγ-globin genes) carry three Oct-1 transcription factor consensus sequences within their promoter regions. We have studied the possibility of inducing γ-globin gene expression using decoy oligonucleotides that target the Oct-1 transcription factor consensus sequence. A double-stranded 22 bp decoy oligonucleotide containing the Oct-1 consensus sequence was synthesized. The results obtained from our in vitro binding assay revealed a strong competitive binding of the decoy oligonucleotide for the Oct-1 transcription factor. When K562 human erythroleukemia cells were treated with the Oct-1 decoy oligonucleotide, significant increases in the level of the γ-globin mRNA were observed. The results of our western blots further demonstrated significant increases of the fetal hemoglobin (HbF, α2γ2) in the Oct-1 decoy oligonucleotide-treated K562 cells. The results of our immunoprecipitation (IP) studies revealed that the treatment of K562 cells with the Oct-1 decoy oligonucleotide significantly reduced the level of the endogenous γ-globin gene promoter region DNA co-precipitated with the Oct-1 transcription factor. These results suggest that the decoy oligonucleotide designed for the Oct-1 transcription factor consensus sequence could induce expression of the endogenous γ-globin gene through competitive binding of the Oct-1 transcription factor, resulting in activation of the γ-globin genes. Therefore, disrupting the bindings of the Oct-1 transcriptional factors with the decoy oligonucleotide provides a novel approach for inducing expression of the γ-globin genes. It also provides an innovative strategy for the treatment of many disease conditions, including sickle cell anemia and β-thalassemia