Thermodynamic contributions of the reactions of DNA intramolecular structures with their complementary strands

One focus of our research is to further our understanding of the physico-chemical properties of unusual DNA structures and their interaction with complementary oligonucleotides. We have investigated three types of reactions involving the interaction of intramolecular DNA complexes with their complementary single strands of varied length. Specifically, we have used a combination of isothermal titration (ITC) and differential scanning (DSC) calorimetry and spectroscopy techniques to determine standard thermodynamic profiles for the reaction of an i-motif, G-quadruplex, and triplex with their complementary strands. The enthalpies for each reaction are measured directly in ITC titrations and compared with those obtained indirectly from Hess cycles using DSC unfolding data. All reactions investigated yielded favorable free energy contributions, indicating that each single strand is able to invade and disrupt the corresponding intramolecular DNA complex. These favorable free energy terms are enthalpy driven, which result from a compensation of exothermic contributions, due to the formation of additional base-pair stacks (or base-triplet stacks) in the duplex product (or triplex product), immobilization of electrostricted water by the base-pair and base-triplet stacks, and the removal of structural water from the reactant single strands; and endothermic contributions from the disruption of base-base stacking interactions of the reactant single strands. This investigation of nucleic acid reactions has provided new methodology, based on physico-chemical principles, to determine the molecular forces involved in the interactions between DNA nucleic acid structures. This methodology may be used in targeting reactions for the control of gene expression.     

Fetal mesenchymal stem cells derived from human umbilical cord sustain primitive characteristics during extensive expansion

Stem cells of fetal origin lie between embryonic and adult stem cells in terms of potentiality. Because of the ethical controversy surrounding embryonic stem cells and the relatively inferior quality of adult stem cells, the use of fetal stem cells would be an attractive option in future therapeutic applications. Here, we have investigated primitive characteristics of human umbilical-cord-derived fetal mesenchymal stem cells (UC fMSCs) during extensive expansion. We have successfully isolated and cultured UC fMSCs from all UC samples, but with two early fungal contaminations. UC fMSCs proliferated without significant evidence of morphological changes, and the average cumulative population-doubling level was over 25 for about 3 months. UC fMSCs showed the positive expression of several CD markers, known to be related to MSCs, including CD73 (SH-3, 4), CD90 (Thy-1), CD105 (SH-2), CD117 (c-kit), and CD166 (ALCAM). They demonstrated primitive properties throughout the expansion period: multilineage differentiation potentials examined by functional assays, a variety of pluripotent stem cell markers including Nanog, Oct-4, Sox-2, Rex-1, SSEA-3, SSEA-4, Tra-1–60, and Tra-1–81, minimal evidence of senescence as shown by β-galactosidase staining, and the consistent expression of telomerase activity. These results suggest that UC fMSCs have more primitive properties than adult MSCs, which might make them a useful source of MSCs for clinical applications. This work was supported by the Seoul R&BD Program (10548).     

Plaques from different individuals yield different microbiota responses to oral-antiseptic treatment

Dental caries is a polymicrobial disease and complicated to treat. Understanding the microbiota responses to treatment from different individuals is a key factor in developing effective treatments. The aim of this study was to investigate the 24-h posttreatment effect of two oral antiseptics (chlorhexidine and Listerine) on species composition of microplate plaque biofilms that had been initiated from the saliva of five different donors and grown in both 0.15% and 0.5% sucrose. Plaque composition was analyzed using checkerboard DNA : DNA hybridization analysis, which comprised of a panel of 40 species associated with oral health and disease. The supernatant pH of the plaques grown in 0.15% sucrose ranged from 4.3 to 6 and in 0.5% sucrose, it ranged from 3.8 to 4. Plaque biomass was largely unaffected by either antiseptic. Each donor had a different salivary microbial profile, differentiating according to the prevalence of either caries or periodontal/anaerobic pathogens. Despite similar plaque microbiota compositions being elicited through the sucrose growth conditions, microbiota responses to chlorhexidine and Listerine differentiated according to the donor. These findings indicate that efficacious caries treatments would depend on the responses of an individual's microbiota, which may differ from person to person.     

Peroxidase activity of hemoglobin towards ascorbate and urate: a synergistic protective strategy against toxicity of Hemoglobin-Based Oxygen Carriers (HBOC)

Acellular hemoglobins developed as oxygen bridging agents with volume expanding properties ("blood substitutes") are prone to autoxidation and oxidant-mediated structural changes in circulation. In the presence of hydrogen peroxide and either ascorbate or urate we show that ferric hemoglobin functions as a true enzymatic peroxidase. The activity saturates with both substrates and is linearly dependent on protein concentration. The activity is enhanced at low pH with a pKa of 4.7, consistent with protonation of the ferryl species (Fe(IV)-OH) as the active intermediate. To test whether these redox reactions define its behaviour in vivo we exchanged transfused guinea pigs with 50% polymerized bovine Hb (PolyHbBv) and monitored plasma levels of endogenous ascorbate and urate. Immediately after transfusion, met PolyHbBv levels increased up to 30% of total Hb and remained at this level during the first 24 h post transfusion. Plasma ascorbate decreased by 50% whereas urate levels remained unchanged after transfusion. A simple kinetic model, assuming that ascorbate was a more active ferric heme reductase and peroxidase substrate than urate, was consistent with the in vivo data. The present finding confirms the primary and secondary roles of ascorbate and urate respectively in maintaining the oxidative stability of infused Hb.     

Redox control in actinobacteria

As most actinobacteria are obligate aerobes, they have to cope with endogenously generated reactive oxygen species, and actinobacterial pathogens have to resist oxidative attack by phagocytes. Actinobacteria also have to survive long periods under low oxygen tension; for example, Mycobacterium tuberculosis can persist in the host for years under apparently hypoxic conditions in a latent, non-replicative state. Here we focus on the regulatory switches that control actinobacterial responses to peroxide stress, disulfide stress and low oxygen tension. Other unique aspects of their redox biology will be highlighted, including the use of the pseudodisaccharide mycothiol as their major low-molecular-weight thiol buffer, and the [4Fe-4S]-containing WhiB-like proteins, which play diverse, important roles in actinobacterial biology, but whose biochemical role is still controversial.     

Periplasmic export machines for outer membrane assembly

The cell envelope of Gram-negative bacteria protects the organism from environmental stresses, components of the innate immune response, and the actions of other antagonistic molecules. However, the complexity of the cell envelope dictated by these protective roles creates a significant challenge for assembly of the outer membrane. Extensive research has focused on the export and assembly of outer membrane proteins and there is continuing progress in this area. By contrast, knowledge of the export and assembly of complex glycoconjugates in the outer membrane has been limited until recently. New structural and biochemical information identifies an envelope-spanning molecular scaffold for the export of group 1 capsular polysaccharides and provides insight into a complex molecular machine.