On the magnitude of the electrostatic contribution to ligand-DNA interactions.

A model based on the nonlinear Poisson-Boltzmann equation is used to study the electrostatic contribution to the binding free energy of a simple intercalating ligand, 3,8-diamino-6-phenylphenanthridine, to DNA. We find that the nonlinear Poisson-Boltzmann model accurately describes both the absolute magnitude of the pKa shift of 3,8-diamino-6-phenylphenanthridine observed upon intercalation and its variation with bulk salt concentration. Since the pKa shift is directly related to the total electrostatic binding free energy of the charged and neutral forms of the ligand, the accuracy of the calculations implies that the electrostatic contributions to binding are accurately predicted as well. Based on our results, we have developed a general physical description of the electrostatic contribution to ligand-DNA binding in which the electrostatic binding free energy is described as a balance between the coulombic attraction of a ligand to DNA and the disruption of solvent upon binding. Long-range coulombic forces associated with highly charged nucleic acids provide a strong driving force for the interaction of cationic ligands with DNA. These favorable electrostatic interactions are, however, largely compensated for by unfavorable changes in the solvation of both the ligand and the DNA upon binding. The formation of a ligand-DNA complex removes both charged and polar groups at the binding interface from pure solvent while it displaces salt from around the nucleic acid. As a result, the total electrostatic binding free energy is quite small. Consequently, nonpolar interactions, such as tight packing and hydrophobic forces, must play a significant role in ligand-DNA stability.

Distal enhancer regulation by promoter derepression in topologically constrained DNA in vitro

Long-range promoter–enhancer interactions are a crucial regulatory feature of many eukaryotic genes yet little is known about the mechanisms involved. Using cloned chicken βA-globin genes, either individually or within the natural chromosomal locus, enhancer-dependent transcription is achieved in vitro at a distance of 2 kb with developmentally staged erythroid extracts. This occurs by promoter derepression and is critically dependent upon DNA topology. In the presence of the enhancer, genes must exist in a supercoiled conformation to be actively transcribed, whereas relaxed or linear templates are inactive. Distal protein–protein interactions in vitro may be favored on supercoiled DNA because of topological constraints. In this system, enhancers act primarily to increase the probability of rapid and efficient transcription complex formation and initiation. Repressor and activator proteins binding within the promoter, including erythroid-specific GATA-1, mediate this process.

Direct Involvement of N-Cadherin–mediated Signaling in Muscle Differentiation

Cell–cell interactions, mediated by members of the cadherin family of Ca2+-dependent adhesion molecules, play key roles in morphogenetic processes as well as in the transduction of long-range growth and differentiation signals. In muscle differentiation cell adhesion is involved in both early stages of myogenic induction and in later stages of myoblast interaction and fusion. In this study we have explored the involvement of a specific cadherin, namely N-cadherin, in myogenic differentiation. For that purpose we have treated different established lines of cultured myoblasts with beads coated with N-cadherin–specific ligands, including a recombinant N-cadherin extracellular domain, and anti-N-cadherin antibodies. Immunofluorescent labeling for cadherins and catenins indicated that treatment with the cadherin-reactive beads for several hours enhances the assembly of cell–cell adherens-type junctions. Moreover, immunofluorescence and immunoblotting analyses indicated that treatment with the beads for 12–24 h induces myogenin expression and growth arrest, which are largely independent of cell plating density. Upon longer incubation with the beads (2–3 d) a major facilitation in the expression of several muscle-specific sarcomeric proteins and in cell fusion into myotubes was observed. These results suggest that surface clustering or immobilization of N-cadherin can directly trigger signaling events, which promote the activation of a myogenic differentiation program.

Direct interaction of lignin and lignin peroxidase from Phanerochaete chrysosporium

Binding properties of lignin peroxidase (LiP) from the basidiomycete Phanerochaete chrysosporium against a synthetic lignin (dehydrogenated polymerizate, DHP) were studied with a resonant mirror biosensor. Among several ligninolytic enzymes, only LiP specifically binds to DHP. Kinetic analysis revealed that the binding was reversible, and that the dissociation equilibrium constant was 330 μM. The LiP–DHP interaction was controlled by the ionization group with a pKa of 5.3, strongly suggesting that a specific amino acid residue plays a role in lignin binding. A one-electron transfer from DHP to oxidized intermediates LiP compounds I and II (LiPI and LiPII) was characterized by using a stopped-flow technique, showing that binding interactions of DHP with LiPI and LiPII led to saturation kinetics. The dissociation equilibrium constants for LiPI–DHP and LiPII–DHP interactions were calculated to be 350 and 250 μM, and the first-order rate constants for electron transfer from DHP to LiPI and to LiPII were calculated to be 46 and 16 s−1, respectively. These kinetic and spectral studies strongly suggest that LiP is capable of oxidizing lignin directly at the protein surface by a long-range electron transfer process. A close look at the crystal structure suggested that LiP possesses His-239 as a possible lignin-binding site on the surface, which is linked to Asp-238. This Asp residue is hydrogen-bonded to the proximal His-176. This His–Asp⋅⋅⋅proximal-His motif would be a possible electron transfer route to oxidize polymeric lignin.

Interaction between like-charged colloidal spheres in electrolyte solutions

How colloidal particles interact with each other is one of the key issues that determines our ability to interpret experimental results for phase transitions in colloidal dispersions and our ability to apply colloid science to various industrial processes. The long-accepted theories for answering this question have been challenged by results from recent experiments. Herein we show from Monte-Carlo simulations that there is a short-range attractive force between identical macroions in electrolyte solutions containing divalent counterions. Complementing some recent and related results by others, we present strong evidence of attraction between a pair of spherical macroions in the presence of added salt ions for the conditions where the interacting macroion pair is not affected by any other macroions that may be in the solution. This attractive force follows from the internal-energy contribution of counterion mediation. Contrary to conventional expectations, for charged macroions in an electrolyte solution, the entropic force is repulsive at most solution conditions because of localization of small ions in the vicinity of macroions. Both Derjaguin–Landau–Verwey–Overbeek theory and Sogami–Ise theory fail to describe the attractive interactions found in our simulations; the former predicts only repulsive interaction and the latter predicts a long-range attraction that is too weak and occurs at macroion separations that are too large. Our simulations provide fundamental “data” toward an improved theory for the potential of mean force as required for optimum design of new materials including those containing nanoparticles.

Subthreshold facilitation and suppression in primary visual cortex revealed by intrinsic signal imaging.

Neurons in primary visual cortex (area 17) respond vigorously to oriented stimuli within their receptive fields; however, stimuli presented outside the suprathreshold receptive field can also influence their responses. Here we describe a fundamental feature of the spatial interaction between suprathreshold center and subthreshold surround. By optical imaging of intrinsic signals in area 17 in response to a stimulus border, we show that a given stimulus generates activity primarily in iso-orientation domains, which extend for several millimeters across the cortical surface in a manner consistent with the architecture of long-range horizontal connections in area 17. By mapping the receptive fields of single neurons and imaging responses from the same cortex to stimuli that include or exclude the aggregate suprathreshold receptive field, we show that intrinsic signals strongly reveal the subthreshold surround contribution. Optical imaging and single-unit recording both demonstrate that the relative contrast of center and surround stimuli regulates whether surround interactions are facilitative or suppressive: the same surround stimulus facilitates responses when center contrast is low, but suppresses responses when center contrast is high. Such spatial interactions in area 17 are ideally suited to contribute to phenomena commonly regarded as part of "higher-level" visual processing, such as perceptual "popout" and "filling-in."

Large electrostatic differences in the binding thermodynamics of a cationic peptide to oligomeric and polymeric DNA.

Results presented here demonstrate that the thermodynamics of oligocation binding to polymeric and oligomeric DNA are not equivalent because of long-range electrostatic effects. At physiological cation concentrations (0.1-0.3 M) the binding of an oligolysine octacation KWK6-NH2 (+8 charge) to single-stranded poly(dT) is much stronger per site and significantly more salt concentration dependent than the binding of the same ligand to an oligonucleotide, dT(pdT)10 (-10 charge). These large differences are consistent with Poisson-Boltzmann calculations for a model that characterizes the charge distributions with key preaveraged structural parameters. Therefore, both the experimental and the theoretical results presented here show that the polyelectrolyte character of a polymeric nucleic acid makes a large contribution to both the magnitude and the salt concentration dependence of its binding interactions with simple oligocationic ligands.

Spatial integration and cortical dynamics.

Cells in adult primary visual cortex are capable of integrating information over much larger portions of the visual field than was originally thought. Moreover, their receptive field properties can be altered by the context within which local features are presented and by changes in visual experience. The substrate for both spatial integration and cortical plasticity is likely to be found in a plexus of long-range horizontal connections, formed by cortical pyramidal cells, which link cells within each cortical area over distances of 6-8 mm. The relationship between horizontal connections and cortical functional architecture suggests a role in visual segmentation and spatial integration. The distribution of lateral interactions within striate cortex was visualized with optical recording, and their functional consequences were explored by using comparable stimuli in human psychophysical experiments and in recordings from alert monkeys. They may represent the substrate for perceptual phenomena such as illusory contours, surface fill-in, and contour saliency. The dynamic nature of receptive field properties and cortical architecture has been seen over time scales ranging from seconds to months. One can induce a remapping of the topography of visual cortex by making focal binocular retinal lesions. Shorter-term plasticity of cortical receptive fields was observed following brief periods of visual stimulation. The mechanisms involved entailed, for the short-term changes, altering the effectiveness of existing cortical connections, and for the long-term changes, sprouting of axon collaterals and synaptogenesis. The mutability of cortical function implies a continual process of calibration and normalization of the perception of visual attributes that is dependent on sensory experience throughout adulthood and might further represent the mechanism of perceptual learning.