Real-time oligonucleotide hybridization kinetics monitored by resonant mirror technique

The kinetics of hybridization of 11-meric and 14-meric oligonucleotides, dTGGGAAGAGGG (ODN-11) and dTGGGAAGAGG GTCA (ODN-14), with 14-meric oligonucleotide dpTGACCCTCT TCCCA (p14) attached to the surface of a cuvette was studied by the resonant mirror method. The treatment of the experimental curves with exponential equations leads to the following values for association (kas) and dissociation (kdis) rate constants at 25 degrees C: kas = 219 +/- 39 and 183 +/- 162 M-1 s-1, kdis = (2.0 +/- 0.4) x 10(-3) and (4 +/- 1) x 10(-4) s-1 for the duplexes (p14) x (ODN-11) and p14 x (ODN-14), respectively. The oligonucleotide dTGCCTTGAATGGGAA GAGGGTCA (ODN-23), which forms a hairpin structure, does not associate with p14. The data were compared with the results of melting curve detection and temperature-jump experiments. The association rate constants for ODN-11 and ODN-14 are much slower than those values in homogeneous aqueous solution. The dissociation rate constants have the same magnitude values as estimated by using association constants measured from melting curves but differ from the values estimated in temperature-jump experiments.     

Thermodynamics of interaction of phthalocyanine-oligonucleotide conjugates with single- and double-stranded DNA

Thermodynamics of interaction of phthalocyanine-oligonucleotide conjugates with single- and double-stranded DNA resulting in formation of duplexes and triplexes was measured by UV melting method. It was shown that a phthalocyanine moiety of conjugates stabilized the formation of duplexes and triplexes.     

Cell-cell interactions in regulating lung function

Tight junction barrier formation and gap junctional communication are two functions directly attributable to cell-cell contact sites. Epithelial and endothelial tight junctions are critical elements of the permeability barrier required to maintain discrete compartments in the lung. On the other hand, gap junctions enable a tissue to act as a cohesive unit by permitting metabolic coupling and enabling the direct transmission of small cytosolic signaling molecules from one cell to another. These components do not act in isolation since other junctional elements, such as adherens junctions, help regulate barrier function and gap junctional communication. Some fundamental elements related to regulation of pulmonary barrier function and gap junctional communication were presented in a Featured Topic session at the 2004 Experimental Biology Conference in Washington, DC, and are reviewed in this summary.     

Cu(2+)-dependent DNA-cleaving activity of arenes

In the presence of Cu(II) ions, plasmid DNA is cleaved under physiological condition by different arenes at low concentrations. The cleavage was dependent on the presence of O2. The DNA cleavage efficiency of the designed system arene-Cu is comparable to that of the well-known DNA cleaving reagents such as phenanthroline-Cu and ascorbic acid-Cu. However in contrast to the mentioned reagents, the system arene-Cu does not require external reducing agents or H2O2.     

Connexin45 interacts with zonula occludens-1 in osteoblastic cells

Connexin43 (Cx43) and Cx45 are co-expressed in a number of different tissues. Studies demonstrated that Cx45 transfected ROS (ROS/Cx45) cells, were less permeable to low molecular weight dyes than untransfected ROS cells, that have gap junctions made of Cx43. This suggests that there may be a functionally important interaction between Cx43 and Cx45 in these cells. One way in which these proteins may interact is by associating with the same set of proteins. In order to isolate connexin interacting proteins, we isolated Cx45 from Cx45 transfected ROS cells (ROS/Cx45 cells) under mild detergent conditions. These studies showed that Cx45 co-purified with the tight junction protein, ZO-1. Immunofluorescence studies of ROS/Cx45 cells simultaneously stained with polyclonal Cx45 antibody and a monoclonal ZO-1 antibody showed that Cx45 and ZO-1 colocalized in ROS/Cx45 cells. Furthermore we found that ZO-1 could bind to peptides derived from the carboxyl terminal of Cx45 that had been covalently bound to an agarose resin. These data suggests that Cx45 and ZO-1 directly interact in ROS/Cx45 cells.     

Sharing signals: connecting lung epithelial cells with gap junction channels

Gap junction channels enable the direct flow of signaling molecules and metabolites between cells. Alveolar epithelial cells show great variability in the expression of gap junction proteins (connexins) as a function of cell phenotype and cell state. Differential connexin expression and control by alveolar epithelial cells have the potential to enable these cells to regulate the extent of intercellular coupling in response to cell stress and to regulate surfactant secretion. However, defining the precise signals transmitted through gap junction channels and the cross talk between gap junctions and other signaling pathways has proven difficult. Insights from what is known about roles for gap junctions in other systems in the context of the connexin expression pattern by lung cells can be used to predict potential roles for gap junctional communication between alveolar epithelial cells.     

Multiharmonic cubic-nonlinear theory of plasma-beam superheterodyne free-electron lasers of the dopplertron type

A multiharmonic cubic-nonlinear theory of a plasma-beam superheterodyne free-electron laser of the dopplertron type is constructed. A retarded electromagnetic wave propagating in the magnetized plasma-beam system toward the electron beam is used for pumping. The multiharmonic interaction of waves which plays an important role is taken into account. Saturation levels and mechanisms are analyzed. The promising application of such systems for generating high-power electromagnetic radiation in the millimeter wavelength range is demonstrated.     

Europium-labeled GTP as a general nonradioactive substitute for [S]GTPγS in high-throughput G protein studies

[³⁵S]GTPγS, the nonhydrolyzable radioactive GTP analog, has been a powerful tool in G protein studies and has set the standards in this field of research. However, its radioactive nature imposes clear limitations to its use in regular laboratory practice and in high-throughput experimentation. The europium-labeled GTP analog (Eu-GTP) has been used as an alternative in the analysis of G protein activation by G protein-coupled receptors in cellular membrane preparations. Here we expand the usage of Eu-GTP and show that it can be applied in other types of assays where [³⁵S]GTPγS has been previously utilized. We demonstrate the applicability of the modified Eu-GTP binding technology to analysis of heterotrimeric and monomeric G proteins of natural and recombinant sources, from different organisms, in assays with soluble proteins and membrane-containing assays of a high-throughput format. The deci-nanomolar K_D of Eu-GTP for the tested G proteins is similar to that of other fluorescent-modified GTP analogs, while the sensitivity achieved in time-resolved fluorescence analysis of Eu-GTP exceeds that of the radioactive measurements. Overall, the results of our modified Eu-GTP binding assay present Eu-GTP as a general nonradioactive alternative for G protein studies, especially attractive in high-throughput experiments.     

Improving the clinical assessment of consciousness with advances in electrophysiological and neuroimaging techniques

In clinical neurology, a comprehensive understanding of consciousness has been regarded as an abstract concept - best left to philosophers. However, times are changing and the need to clinically assess consciousness is increasingly becoming a real-world, practical challenge. Current methods for evaluating altered levels of consciousness are highly reliant on either behavioural measures or anatomical imaging. While these methods have some utility, estimates of misdiagnosis are worrisome (as high as 43%) - clearly this is a major clinical problem. The solution must involve objective, physiologically based measures that do not rely on behaviour. This paper reviews recent advances in physiologically based measures that enable better evaluation of consciousness states (coma, vegetative state, minimally conscious state, and locked in syndrome). Based on the evidence to-date, electroencephalographic and neuroimaging based assessments of consciousness provide valuable information for evaluation of residual function, formation of differential diagnoses, and estimation of prognosis.