Induction-resonance energy transfer between the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP

Two novel FRET-pairs: Tb³⁺-binding peptide-DsRed2 and Tb³⁺-binding peptide-TagRFP have been produced based on the terbium-binding peptide and the red fluorescent proteins DsRed2 and TagRFP. Two induction-resonance energy transfer processes in both FRET-pairs have been registered, from tryptophan of the terbium-binding peptide to Tb³⁺ and from sensitized Tb³⁺ to the acceptor, the chromophore, DsRed2 or TagRFP. The lifetimes of terbium in the presence and absence of the acceptor have been determined. It has been shown that the lifetime of Tb³⁺ in the presence of 150 mM NaCl decreases in both FRET-pairs. The efficiency of fluorescent resonance transfer from Tb³⁺ to the acceptor proteins is 28 and 35% for Tb³⁺-binding peptide-DsRed2 and Tb³⁺-binding peptide-TagRFP, respectively.     

Tissue distribution of a plasmid DNA encoding Hsp65 gene is dependent on the dose administered through intramuscular delivery

In order to assess a new strategy of DNA vaccine for a more complete understanding of its action in immune response, it is important to determine the in vivo biodistribution fate and antigen expression. In previous studies, our group focused on the prophylactic and therapeutic use of a plasmid DNA encoding the Mycobacterium leprae 65-kDa heat shock protein (Hsp65) and achieved an efficient immune response induction as well as protection against virulent M. tuberculosis challenge. In the present study, we examined in vivo tissue distribution of naked DNA-Hsp65 vaccine, the Hsp65 message, genome integration and methylation status of plasmid DNA. The DNA-Hsp65 was detectable in several tissue types, indicating that DNA-Hsp65 disseminates widely throughout the body. The biodistribution was dose-dependent. In contrast, RT-PCR detected the Hsp65 message for at least 15 days in muscle or liver tissue from immunized mice. We also analyzed the methylation status and integration of the injected plasmid DNA into the host cellular genome. The bacterial methylation pattern persisted for at least 6 months, indicating that the plasmid DNA-Hsp65 does not replicate in mammalian tissue, and Southern blot analysis showed that plasmid DNA was not integrated. These results have important implications for the use of DNA-Hsp65 vaccine in a clinical setting and open new perspectives for DNA vaccines and new considerations about the inoculation site and delivery system.     

Different mechanisms of telomere length regulation

Eukaryotic chromosomes are linear and have their, ends formed by DNA-protein structures, telomeres. At present more and more facts demonstrate the diversity of telomere functions. Telomeres protect the chromosome ends from degradation, fusion, recombination, and from the repair system that recognizes nicks in DNA strands. As shown recently, shortening of the telomeres is a cause of cell aging. In most organisms, telomeres are elongated by means of a special ribonucleoprotein complex; however, in some insects this takes place by either gene conversion or transposition of mobile elements. Evolutionary relations between different types of telomeres are discussed.     

Analysis of binding properties and specificity through identification of the interface forming residues (IFR) for serine proteases <Emphasis Type="Italic">in silico</Emphasis> docked to different inhibitors

Background  

Enzymes belonging to the same super family of proteins in general operate on variety of substrates and are inhibited by wide selection of inhibitors. In this work our main objective was to expand the scope of studies that consider only the catalytic and binding pocket amino acids while analyzing enzyme specificity and instead, include a wider category which we have named the Interface Forming Residues (IFR). We were motivated to identify those amino acids with decreased accessibility to solvent after docking of different types of inhibitors to sub classes of serine proteases and then create a table (matrix) of all amino acid positions at the interface as well as their respective occupancies. Our goal is to establish a platform for analysis of the relationship between IFR characteristics and binding properties/specificity for bi-molecular complexes.