Theodora Choli-Papadopoulou

Fast Biosynthesis of GFP Molecules: A Single‐Molecule Fluorescence Study

Its not easy being green: Real-time visualization of labeled ribosomes and de novo synthesized green fluorescent protein molecules using single-molecule-sensitive fluorescence microscopy demonstrates that the mutant GFPem is produced with a characteristic time of five minutes. Fluorescence of the fastest GFP molecules appears within one minute (see picture).

Protein Structure Analysis

The SCOP database aims to provide a detailed and comprehensive description of the structural and evolutionary relationships between all proteins whose structure is known. Proteins are classified to reflect both structural and evolutionary relatedness. Many levels exist in the hierarchy; the principal levels are family, superfamily and fold Family: Clear evolutionarily relationship Superfamily: Probable common evolutionary origin Fold: Major structural similarity SCOP: Structural Classification of Proteins

Redox activity and in vitro bioactivity of the water-soluble fraction of urban particulate matter in relation to particle size and chemical composition.

Chemical and toxicological characterization of the water-soluble fraction of size-segregated urban particulate matter (PM) (<0.49, 0.49-0.97, 0.97-1.5, 1.5-3.0, 3.0-7.2 and >7.2 μm) was carried out at two urban sites, traffic and urban background, during the cold and the warm period. Chemical analysis of the water-soluble PM fraction included ionic species (NO3(-), SO4(2-), Cl(-), Na(+), NH4(+), K(+), Mg(2+), Ca(2+)), water-soluble organic carbon (WSOC), and trace elements (Al, As, Ba, Cd, Cr, Cu, Fe, Pb, Mn, Ni, Zn, Pt, Pd, Rh, Ru, Ir, Ca, and Mg). The dithiothreitol (DTT) assay was employed for the abiotic assessment of the oxidative PM activity. Cytotoxic responses were investigated in vitro by applying the mitochondrial dehydrogenase (MTT) and the lactate dehydrogenase (LDH) bioassays on human lung cells (MRC-5), while DNA damage was estimated by the single cell gel electrophoresis assay, known as Comet assay. The correlations between the observed bioactivity responses and the concentrations of water-soluble chemical PM constituents in the various size ranges were investigated. The results of the current study corroborate that short-term bioassays using lung human cells and abiotic assays, such as the DTT assay, could be relevant to complete the routine chemical analysis and to obtain a preliminary screening of the potential effects of PM-associated airborne pollutants on human health.

Helicobacter pylori neutrophil-activating protein activates neutrophils by its C-terminal region even without dodecamer formation, which is a prerequisite for DNA protection – novel approaches against Helicobacter pylori inflammation

Helicobacter pylori neutrophil‐activating protein (HP‐NAP) protects DNA from free radicals as a dodecamer through its ferroxidase activity without, however, directly binding to it. The retardation that was observed at pH 7.5 could be easily attributed to an iron effect, as it was revealed by experiments in the absence of HP‐NAP. A total loss of ferroxidase activity, dodecamer formation and DNA protection in environments rich in free radicals was observed after replacement of His25, His37, Asp52 and Lys134, which are located within the ferroxidase site, with Ala. Molecular dynamics simulations revealed that dimer formation is highly unlikely following mutation of the above amino acids, as the Fe2+ is no longer attracted with equal strength by both subunits. These findings probably indicate that iron plays an important role in the conformation of HP‐NAP by initiating the formation of stable dimers that are indispensable for the ensuing dodecamer structure. Very surprisingly, neutrophil activation appeared to be stimulated by structural elements that are localized within the C‐terminal region of both mutant HP‐NAP and wild‐type dodecamer HP‐NAP. In particular, the dodecamer conformation does not seem to be necessary for activation, and helices H3 (Leu69–Leu75) and H4 (Lys89–Leu114) or the linking coils (His63–Thr68 and Thr76–Ser88) are probably critical in stimulating neutrophil activation.

Helicobacter pylori neutrophil activating protein as target for new drugs against H. pylori inflammation

Helicobacter pylori (H. pylori) infection is among the most common human infections and the major risk factor for peptic ulcer disease and gastric cancer. Within this work we present the implication of C-terminal region of H. pylori neutrophil activating protein in the stimulation of neutrophil activation as well as the evidence that the C-terminal region of H. pylori activating protein is indispensable for neutrophil adhesion to endothelial cells, a step necessary to H. pylori inflammation. In addition we show that arabino galactan proteins derived from chios mastic gum, the natural resin of the plant Pistacia lentiscus var. Chia inhibit neutrophil activation in vitro.

Towards Atomic Resolution of Prokaryotic Ribosomes: Crystallographic, Genetic and Biochemical Studies

The studies reported here were initiated and inspired by the late Prof. H.G. Wittmann. From the early stages of this project, when it was widely believed that even the initial steps in determining the molecular structure of ribosomes are impossible, until his last days, Prof. Wittmann was actively involved in the experimental design and in the actual studies. We have no doubt that without his motivation, optimism, guidance and support, this project would not have reached its current stage.

Proteome and Protein Analysis

Selected papers presented at the MPSA 98 are covering new, sensitive and rapid methods for the analysis of proteins, with special emphasis on the total cell proteins, the proteome. In addition to the experimental details, the advantages and limitations of the methodological approaches are discussed. Topics included are: Protein sequencing analysis, protein and peptide sample preparation, mass spectrometry, NMR, analysis of post-translational modifications, purification of recombinant proteins, protein-protein and protein-DNA interactions, structure prediction, modeling and protein folding, functional implications of protein domains and newly emerging methods for the investigation of the proteome, allowing to analyse the expression of genes.

Investigating the entire course of telithromycin binding to Escherichia coli ribosomes

Applying kinetics and footprinting analysis, we show that telithromycin, a ketolide antibiotic, binds to Escherichia coli ribosomes in a two-step process. During the first, rapidly equilibrated step, telithromycin binds to a low-affinity site (KT = 500 nM), in which the lactone ring is positioned at the upper portion of the peptide exit tunnel, while the alkyl–aryl side chain of the drug inserts a groove formed by nucleotides A789 and U790 of 23S rRNA. During the second step, telithromycin shifts slowly to a high-affinity site (KT* = 8.33 nM), in which the lactone ring remains essentially at the same position, while the side chain interacts with the base pair U2609:A752 and the extended loop of protein L22. Consistently, mutations perturbing either the base pair U2609:A752 or the L22-loop hinder shifting of telithromycin to the final position, without affecting the initial step of binding. In contrast, mutation Lys63Glu in protein L4 placed on the opposite side of the tunnel, exerts only a minor effect on telithromycin binding. Polyamines disfavor both sequential steps of binding. Our data correlate well with recent crystallographic data and rationalize the changes in the accessibility of ribosomes to telithromycin in response to ribosomal mutations and ionic changes.

Effects of mastic gum Pistacia lentiscus var. Chia on innate cellular immune effectors.

Background The essential oil and Chios mastic gum (CMG) are natural antimicrobial agents currently broadly used in medicine owing to their antimicrobial, antioxidant, and hepatoprotective properties. The aim of this study was to investigate the effect of CMG-extracted arabinogalactan proteins (AGPs/CMG) both in vitro and in vivo, under the presence of Helicobacter pylori neutrophil-activating protein (HP-NAP), on the innate cellular immune effectors (neutrophils activations) comparing H. pylori-infected patients and healthy controls. Patients and methods The in-vivo effect of AGPs/CMG under the presence of HP-NAP in neutrophil activation was investigated in five H. pylori-infected patients and three healthy volunteers who received 1 g daily consumption of CMG for 2 months. All participants did not receive any immunosuppressive medication before or during the trial; patients with infectious diseases that could modify their immunologic status were excluded. In-vitro studies with pull-down experiments to assess the effect of AGPs/CMG under the presence of HP-NAP on the neutrophil activation were also carried out. Neutrophil activation was estimated by nicotinamide adenine dinucleotide phosphate-oxidase assays and optical microscopy methods by measurement of cytochrome C reduction. Results Neutrophil activation was reduced when incubated in vitro with HP-NAP (P=0.0027) and AGP plus HP-NAP (P=0.0004) in H. pylori-positive patients who consumed AGP for 2 months. Similar results were also obtained when neutrophils were incubated with AGP plus HP-NAP (P=0.0038) in controls. Pull-down experiments showed a specific binding of AGPs to two membrane proteins of neutrophils, possibly suggesting inhibition of neutrophil activation. Conclusion AGPs/CMG inhibit neutrophil activation in the presence of HP-NAP, playing a crucial role in H. pylori-associated pathologies in gastric mucosa.

The Potential Role of Ribosomal Protein S5 on Cell Cycle Arrest and Initiation of Murine Erythroleukemia Cell Differentiation

Evidence now exists to indicate that some ribosomal proteins besides being structural components of the ribosomal subunits are involved in the regulation of cell differentiation and apoptosis. As we have shown earlier, initiation of erythroid differentiation of murine erythroleukemia (MEL) cells is associated with transcriptional inactivation of genes encoding ribosomal RNAs and ribosomal proteins S5 (RPS5) and L35a. In this study, we extended these observations and investigated whether transfection of MEL cells with RPS5 cDNA affects the onset of initiation of erythroid maturation and their entrance in cell cycle arrest. Stably transfected MEL cloned cells (MEL‐C14 and MEL‐C56) were established and assessed for their capacity to produce RPS5 RNA transcript and its translated product. The impact of RPS5 cDNA transfection on the RPS5 gene expression patterns and the accumulation of RPS5 protein in inducible transfected MEL cells were correlated with their ability to: (a) initiate differentiation, (b) enter cell cycle arrest at G1/G0 phase, and (c) modulate the level of cyclin‐dependent kinases CDK2, CDK4, and CDK6. The data presented indicate that deregulation of RPS5 gene expression (constitutive expression) affects RPS5 protein level and delays both the onset of initiation of erythroid maturation and entrance in cell cycle arrest in inducer‐treated MEL cells. J. Cell. Biochem. 104: 1477–1490, 2008.