Recognition of the Structural Distortions at the Junctions Between B and Z Segments in Negatively Supercoiled DNA by Osmium Tetroxide

Abstract

It has been shown for the first time that conformational junction between contiguous right- handed B and left-handed Z segments can be recognized by a chemical probe. Plasmid pRW751 containing (dC-dG)₁₃ and (dC-dG)₁₆ blocks was treated with osmium tetroxide, pyridine (a reagent known to be single-strand selective) at physiological ionic conditions (0.1 and 0.2 M NaCl) and neutral pH. Mapping of the osmium binding sites by restriction enzyme digestion followed by nuclease SI cleavage has revealed selective binding of osmium at, or near to, the end of the (dC-dG)_n segments proximal to the 95 bp lac sequence. The junction of the shorter (dC-dG)₁₃ segment was modified to a substantially greater extent than that of the longer segment. Partial inhibition of DNA cleavage by BamHI was observed at the restriction sites neighbouring to the both (dC-dG)_n segments as a result of DNA modification by osmium tetroxide. The site-selective modification occurred only in supercoiled and not in relaxed molecules. Differences in the sensitivity of the B/Z junctions in pRW751 to the osmium tetroxide were explained by different structural features of these junctions.     

Population increase of forest birds in the Czech Republic between 1982 and 2003

Capsule Populations of most forest bird species increased between 1982 and 2003, probably due to increased forest cover and changes in forest age-class composition.

Aims To determine population changes of forest birds in the Czech Republic and to determine their possible causes.

Methods Population data were collected via the Breeding Bird Monitoring Programme, which is based on skilled volunteers counting birds at point transects using a standardized technique. Population trends and indices for the period 1982–2003 were calculated for 47 species using log-linear models. Published data on development of forest cover and forest age composition in the Czech Republic were used to indicate environmental change over the same period.

Results Populations of most forest species increased between 1982 and 2003. There was also an increase in forest cover and an increase in the proportion of older forest age-classes. The increase in forest specialist birds was positively correlated with the average increase in forest coverage.

Conclusions The populations of Czech forest birds have increased in the last two decades. This contrasts with widely reported declines of farmland bird populations throughout Europe. The correlation between populations of specialized forest species and extent of forest habitat suggests that changes in land-use are an important factor. However, increasing cover of mature forests could have a similar effect on the populations of specialist species.     

An immunoaffinity purification method for the proteomic analysis of ubiquitinated protein complexes

Protein ubiquitination plays an important role in the regulation of many cellular processes, including protein degradation, cell cycle regulation, apoptosis, and DNA repair. To study the ubiquitin proteome we have established an immunoaffinity purification method for the proteomic analysis of endogenously ubiquitinated protein complexes. A strong, specific enrichment of ubiquitinated factors was achieved using the FK2 antibody bound to protein G-beaded agarose, which recognizes monoubiquitinated and polyubiquitinated conjugates. Mass spectrometric analysis of two FK2 immunoprecipitations (IPs) resulted in the identification of 296 FK2-specific proteins in both experiments. The isolation of ubiquitinated and ubiquitination-related proteins was confirmed by pathway analyses (using Ingenuity Pathway Analysis and Gene Ontology-annotation enrichment). Additionally, comparing the proteins that specifically came down in the FK2 IP with databases of ubiquitinated proteins showed that a high percentage of proteins in our enriched fraction was indeed ubiquitinated. Finally, assessment of protein–protein interactions revealed that significantly more FK2-specific proteins were residing in protein complexes than in random protein sets. This method, which is capable of isolating both endogenously ubiquitinated proteins and their interacting proteins, can be widely used for unraveling ubiquitin-mediated protein regulation in various cell systems and tissues when comparing different cellular states.     

Synthesis of 2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes via LiAlH4-induced reductive cyclization of 2-(4-chloro-2-cyano-2-phenylbutyl)aziridines and evaluation of their antimalarial activity

2-(4-Chloro-2-cyano-2-phenylbutyl)aziridines were employed for the one-step stereoselective construction of both endo- and exo-2-aminomethyl-4-phenyl-1-azabicyclo[2.2.1]heptanes as new azaheterobicyclic scaffolds via a double LiAlH₄-induced reductive cyclization protocol. Antiplasmodial assessment of these 1-azabicyclo[2.2.1]heptanes revealed moderate to good activities in the micromolar range, with the exo-isomers being the most promising structures. Furthermore, the proposed mode of action was supported by ligand docking studies, pointing to a strong binding interaction with the enzyme plasmepsin II.     

A combinatorial approach to the structure elucidation of a pyoverdine siderophore produced by a Pseudomonas putida isolate and the use of pyoverdine as a taxonomic marker for typing P. putida subspecies

The structure of a pyoverdine produced by Pseudomonas putida, W15Oct28, was elucidated by combining mass spectrometric methods and bioinformatics by the analysis of non-ribosomal peptide synthetase genes present in the newly sequenced genome. The only form of pyoverdine produced by P. putida W15Oct28 is characterized to contain α-ketoglutaric acid as acyl side chain, a dihydropyoverdine chromophore, and a 12 amino acid peptide chain. The peptide chain is unique among all pyoverdines produced by Pseudomonas subspecies strains. It was characterized as –l-Asp-l-Ala-d-AOHOrn-l-Thr-Gly-c[l-Thr(O-)-l-Hse-d-Hya-l-Ser-l-Orn-l-Hse-l-Ser-O-]. The chemical formula and the detected and calculated molecular weight of this pyoverdine are: C₆₅H₉₃N₁₇O₃₂, detected mass 1624.6404 Da, calculated mass 1624.6245. Additionally, pyoverdine structures from both literature reports and bioinformatics prediction of the genome sequenced P. putida strains are summarized allowing us to propose a scheme based on pyoverdines structures as tool for the phylogeny of P. putida. This study shows the strength of the combination of in silico analysis together with analytical data and literature mining in determining the structure of secondary metabolites such as peptidic siderophores.     

Quinacrine reactivity with prion proteins and prion-derived peptides

Quinacrine is a drug that is known to heal neuronal cell culture infected with prions, which are the causative agents of neurodegenerative diseases called transmissible spongiform encephalopathies. However, the drug fails when it is applied in vivo. In this work, we analyzed the reason for this failure. The drug was suggested to “covalently” modify the prion protein via an acridinyl exchange reaction. To investigate this hypothesis more closely, the acridine moiety of quinacrine was covalently attached to the thiol groups of cysteines belonging to prion-derived peptides and to the full-length prion protein. The labeled compounds were conveniently monitored by fluorescence and absorption spectroscopy in the ultraviolet and visible spectral regions. The acridine moiety demonstrated characteristic UV–vis spectrum, depending on the substituent at the C-9 position of the acridine ring. These results confirm that quinacrine almost exclusively reacts with the thiol groups present in proteins and peptides. The chemical reaction alters the prion properties and increases the concentration of the acridine moiety in the prion protein.