Replication Independent Formation of Extrachromosomal Circular DNA in Mammalian Cell-Free System

Extrachromosomal circular DNA (eccDNA) is a pool of circular double stranded DNA molecules found in all eukaryotic cells and composed of repeated chromosomal sequences. It was proposed to be involved in genomic instability, aging and alternative telomere lengthening. Our study presents novel mammalian cell-free system for eccDNA generation. Using purified protein extract we show that eccDNA formation does not involve de-novo DNA synthesis suggesting that eccDNA is generated through excision of chromosomal sequences. This process is carried out by sequence- independent enzymes as human protein extract can produce mouse- specific eccDNA from high molecular weight mouse DNA, and vice versa. EccDNA production does not depend on ATP, requires residual amounts of Mg²⁺ and is enhanced by double strand DNA breaks.     

Incorporation of N‐amidino‐pyroglutamic acid into peptides using intramolecular cyclization of α‐guanidinoglutaric acid

N‐terminal modification of peptides by unnatural amino acids significantly affects their enzymatic stability, conformational properties and biological activity. Application of N‐amidino‐amino acids, positively charged under physiological conditions, can change peptide conformation and its affinity to the corresponding receptor. In this article, we describe synthesis of short peptides, containing a new building block—N‐amidino‐pyroglutamic acid. Although direct guanidinylation of pyroglutamic acid and oxidation of N‐amidino‐proline using RuO₄ did not produce positive results, N‐amidino‐Glp‐Phe‐OH was synthesized on Wang polymer by cyclization of α‐guanidinoglutaric acid residue. In the course of synthesis, it was found that literature procedure of selective Boc deprotection using TMSOTf/TEA reagent is accompanied by concomitant side reaction of triethylamine alkylation by polymer linker fragment. It should be mentioned that independently from cyclization time and coupling agent (DIC or HCTU), the lactam formation was incomplete. Separation of the cyclic product from the linear precursor was achieved by HPLC in ammonium formate buffer at pH 6. HPLC analysis showed N‐amidino‐Glp‐Phe‐OH stability at acidic and physiological pH and fast ring opening in water solution at pH 9. The suggested method of N‐amidino‐Glp residue formation can be applied in the case of short peptide chains, whereas synthesis of longer ones will require fragment condensation approach. Copyright © 2009 European Peptide Society and John Wiley & Sons, Ltd.     

A novel soluble protein factor with non-opioid dynorphin A-binding activity

A novel soluble non-opioid dynorphin A-binding factor (DABF) was identified and characterized in neuronal cell lines, rat spinal cord, and brain. DABF binds dynorphin A(1-17), dynorphin A(2-17), and the 32 amino acid prodynorphin fragment big dynorphin consisting of dynorphin A and B, but not other opioid and non-opioid peptides, opiates, and benzomorphans. The IC50 for dynorphin A(1-17), dynorphin A(2-17), and big dynorphin is in the 5-10 nM range. Using dynorphin A and big dynorphin fragments a binding epitope was mapped to dynorphin A(6-13). DABF has a molecular mass of about 70 kDa. SH-groups are apparently involved in the binding of dynorphin A since p-hydroxy-mercuribenzoic acid inhibited this process. Upon interaction with DABF dynorphin A was converted into Leu-enkephalin, which remained bound to the protein. These data suggest that DABF functions as an oligopeptidase that forms stable and specific complexes with dynorphin A. The presence of DABF in brain structures and other tissues with low level of prodynorphin expression suggests that DABF as an oligopeptidase may degrade other peptides. Dynorphin A at the sites of its release in the CNS may attenuate this degradation as a competitor when it specifically binds to the enzyme.     

High stability of the hinge region in the membrane-active peptide helix of zervamicin: paramagnetic relaxation enhancement studies

Zervamicin IIB is a 16 amino acid peptaibol that forms voltage dependent ion channels with multilevel conductance states in planar lipid bilayers and vesicular systems. Stability of the hinge region and intermolecular interactions were investigated in the N- and C-terminally spin-labelled peptide analogues. Intermolecular and intramolecular paramagnetic enhancement indicates that zervamicin behaves as a rigid helical rod in methanol solution. There are no high amplitude hinge-bending motions, and the peptaibol is monomeric up to concentration 1.5 mM. Stability of the hinge region illustrates the helix stabilising propensity of the Pro residue in membrane mimic environments and implies absence of significant conformational rearrangement due to voltage peptaibol activation.     

Interaction of ZPR1 with Translation Elongation Factor-1α in Proliferating Cells

The zinc finger protein ZPR1 is present in the cytoplasm of quiescent mammalian cells and translocates to the nucleus upon treatment with mitogens, including epidermal growth factor (EGF). Homologues of ZPR1 were identified in yeast and mammals. These ZPR1 proteins bind to eukaryotic translation elongation factor-1α (eEF-1α). Studies of mammalian cells demonstrated that EGF treatment induces the interaction of ZPR1 with eEF-1α and the redistribution of both proteins to the nucleus. In the yeast Saccharomyces cerevisiae, genetic analysis demonstrated that ZPR1 is an essential gene. Deletion analysis demonstrated that the NH₂-terminal region of ZPR1 is required for normal growth and that the COOH-terminal region was essential for viability in S. cerevisiae. The yeast ZPR1 protein redistributes from the cytoplasm to the nucleus in response to nutrient stimulation. Disruption of the binding of ZPR1 to eEF-1α by mutational analysis resulted in an accumulation of cells in the G2/M phase of cell cycle and defective growth. Reconstitution of the ZPR1 interaction with eEF-1α restored normal growth. We conclude that ZPR1 is essential for cell viability and that its interaction with eEF-1α contributes to normal cellular proliferation.     

Excitation delocalization in the bacteriochlorophyll c antenna of the green bacterium Chloroflexus aurantiacus as revealed by ultrafast pump-probe spectroscopy

Room temperature absorption difference spectra were measured on the femtosecond through picosecond time scales for chlorosomes isolated from the green bacterium Chloroflexus aurantiacus. Anomalously high values of photoinduced absorption changes were revealed in the BChl c Q_y transition band. Photoinduced absorption changes at the bleaching peak in the BChl c band were found to be 7–8 times greater than those at the bleaching peak in the BChl a band of the chlorosome. This appears to be the first direct experimental proof of excitation delocalization over many BChl c antenna molecules in the chlorosome.     

19F NMR study on the biodegradation of fluorophenols by various Rhodococcus species

Of all NMR observable isotopes 19F is the one perhaps most convenient for studies on biodegradation of environmental pollutants. The reasons underlying this potential of 19F NMR are discussed and illustrated on the basis of a study on the biodegradation of fluorophenols by four Rhodococcus strains. The results indicate marked differences between the biodegradation pathways of fluorophenols among the various Rhodococcus species. This holds not only for the level and nature of the fluorinated biodegradation pathway intermediates that accumulate, but also for the regioselectivity of the initial hydroxylation step. Several of the Rhodococcus species contain a phenol hydroxylase that catalyses the oxidative defluorination of ortho-fluorinated di- and trifluorophenols. Furthermore, it is illustrated how the 19F NMR technique can be used as a tool in the process of identification of an accumulated unknown metabolite, in this case most likely 5-fluoromaleylacetate. Altogether, the 19F NMR technique proved valid to obtain detailed information on the microbial biodegradation pathways of fluorinated organics, but also to provide information on the specificity of enzymes generally considered unstable and, for this reason, not much studied so far.