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.     

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.     

Spectral analysis of photoplethysmographic signals: The importance of preprocessing

Heart rate variability (HRV) is an important and useful index to assess the responses of the autonomic nervous system (ANS). HRV analysis is performed using electrocardiography (ECG) or photoplethysmography (PPG) signals which are typically subject to noise and trends. Therefore, the elimination of these undesired conditions is very important to achieve reliable ANS activation results. The purpose of this study was to analyze and compare the effects of preprocessing on the spectral analysis of HRV signals obtained from PPG waveform. Preprocessing consists of two stages: filtering and detrending. The performance of linear Butterworth filter is compared with nonlinear weighted Myriad filter. After filtering, two different approaches, one based on least squares fitting and another on smoothness priors, were used to remove trends from the HRV signal. The results of two filtering and detrending methods were compared for spectral analysis accomplished using periodogram, Welch's periodogram and Burg's method. The performance of these methods is presented graphically and the importance of preprocessing clarified by comparing the results. Although both filters have almost the same performance in the results, the smoothness prior detrending approach was found more successful in removing trends that usually appear in the low frequency bands of PPG signals. In conclusion, the results showed that trends in PPG signals are altered during spectral analysis and must be removed prior to HRV analysis.     

Calmodulin binding to G protein-coupling domain of opioid receptors.

The ubiquitous intracellular Ca(2+) sensor calmodulin (CaM) regulates numerous proteins involved in cellular signaling of G protein-coupled receptors, but most known interactions between GPCRs and CaM occur downstream of the receptor. Using a sequence-based motif search, we have identified the third intracellular loop of the opioid receptor family as a possible direct contact point for interaction with CaM, in addition to its established role in G protein activation. Peptides derived from the third intracellular loop of the mu-opioid (OP(3)) receptor strongly bound CaM and were able to reduce binding interactions observed between CaM and immunopurified OP(3) receptor. Functionally, CaM reduced basal and agonist-stimulated (35)S-labeled guanosine 5'-3-O-(thio)triphosphate incorporation, a measure of G protein activation, in membranes containing recombinant OP(3) receptor. Changes in CaM membrane levels as a result of overexpression or antisense CaM suppression inversely affected basal and agonist-induced G protein activation. The ability of CaM to abolish high affinity binding sites of an agonist at OP(3) further supports the hypothesis of a direct interaction between CaM and opioid receptors. An OP(3) receptor mutant with a Lys(273) --> Ala substitution (K273A-OP(3)), an amino acid predicted to play a critical role in CaM binding based on motif structure, was found to be unaffected by changes in CaM levels but coupled more efficiently to G proteins than the wild-type receptor. Stimulation of both the OP(1) (delta-opioid) and OP(3) wild-type receptors, but not the K273A-OP(3) mutant, induced release of CaM from the plasma membrane. These results suggest that CaM directly competes with G proteins for binding to opioid receptors and that CaM may itself serve as an independent second messenger molecule that is released upon receptor stimulation.     

Ecological specialization promotes diversity and diversification in the Eastern Mediterranean genus Ricotia (Brassicaceae)

Despite its amazing biodiversity, the Eastern Mediterranean remains a highly understudied region when compared withthe Western Mediterranean, restricting our understanding of diversity across the entire Mediterranean. Here we use a combination of molecular markers and presence/absence data from all species of the Eastern Mediterranean genus Ricotia collected across its full geographic range to determine historical, ecological, and evolutionary factors responsible for lineage-specific diversification in the Eastern Mediterranean. Network analysis based on molecular data revealed a high genetic structure within all lineages, and phylogenetic reconstructions based on the multispecies coalescent showed that within-lineage diversification corresponded to the onset of the Mediterranean climate. Reconstruction of ancestral histories indicates that the genus originated within Anatolia and spread across the Eastern Mediterranean and Levant using the Taurus mountains. Ecological niche models suggest that local populations did not go through any major distributional shifts and have persisted in present-day habitats since the Last Glacial Maximum. Furthermore, niche differentiation tests revealed significant differences between closely related species and showed the main variables predicting species limits to be different for each species. Our results give crucial information on the patterns and processes shaping diversity in the Eastern Mediterranean and show the main factors promoting diversification to be local environmental dynamics and ecological specialization and not large-scale latitudinal movements, as often reported for southern Europe. By determining local and regional patterns of diversification in an Eastern Mediterranean genus, we further our understanding of the major trends influencing plant diversity in the Mediterranean basin as a whole.