Surflex-Dock employs an empirically derived scoring function to rank putative protein-ligand interactions by flexible docking of small molecules to proteins of known structure. The scoring function employed by Surflex was developed purely on the basis of positive data, comprising noncovalent protein-ligand complexes with known binding affinities. Consequently, scoring function terms for improper interactions received little weight in parameter estimation, and an ad hoc scheme for avoiding protein-ligand interpenetration was adopted. We present a generalized method for incorporating synthetically generated negative training data, which allows for rigorous estimation of all scoring function parameters. Geometric docking accuracy remained excellent under the new parametrization. In addition, a test of screening utility covering a diverse set of 29 proteins and corresponding ligand sets showed improved performance. Maximal enrichment of true ligands over nonligands exceeded 20-fold in over 80% of cases, with enrichment of greater than 100-fold in over 50% of cases.

Neuronal activity-dependent processes are believed to mediate the formation of synaptic connections during neocortical development, but the underlying intracellular mechanisms are not known. In the visual system, altering the pattern of visually driven neuronal activity by monocular deprivation induces cortical synaptic rearrangement during a postnatal developmental window, the critical period. Here, using transgenic mice carrying a CRE-lacZ reporter, we demonstrate that a calcium- and cAMP-regulated signaling pathway is activated following monocular deprivation. We find that monocular deprivation leads to an induction of CRE-mediated lacZ expression in the visual cortex preceding the onset of physiologic plasticity, and this induction is dramatically downregulated following the end of the critical period. These results suggest that CRE-dependent coordinate regulation of a network of genes may control physiologic plasticity during postnatal neocortical development.

The adult cerebral cortex can adapt to environmental change. Using monocular deprivation as a paradigm, we find that rapid experience-dependent plasticity exists even in the mature primary visual cortex. However, adult cortical plasticity differs from developmental plasticity in two important ways. First, the effect of adult, but not juvenile monocular deprivation is strongly suppressed by administration of barbiturate just prior to recording visual evoked potentials, suggesting that the effect of adult experience can be inactivated acutely. Second, the effect of deprivation is less persistent over time in adults than in juveniles. This correlates with the known decline in CREB function during maturation of the visual cortex. To compensate for this decline in CREB function, we expressed persistently active VP16-CREB and find that it causes adult plasticity to become persistent. These results suggest that in development and adulthood, the regulation of a trans-synaptic signaling pathway controls the adaptive potential of cortical circuits.A primary function of the brain is to integrate the individual into a continually changing environment. Some aspects of this integration are accomplished through developmental processes, other aspects through learning. Although learning can occur throughout life, many behaviors, from language to sexual behavior, are shaped profoundly by early life experience. In this study, we have examined how the adaptive capacity of the cerebral cortex changes with maturation.A classical model of developmental plasticity is ocular dominance plasticity (Wiesel and Hubel 1963;Hubel and Wiesel 1998). Hubel and Wiesel showed that closing one eye of an infant cat produced a visual cortex dominated by the nondeprived eye. Closing an eye of an adult cat was ineffective. Single-unit studies in a number of mammalian systems, ranging from rodents to primates, have found that ocular dominance plasticity is restricted to a period prior to puberty (Hubel and Wiesel 1970;Blakemore et al. 1978;Olson and Freeman 1980;Issa et al. 1999;Fagiolini and Hensch 2000). The amount of deprivation required to alter the responses of visual cortical neurons depends on the animal's age. In the cat, during the peak of the critical period (4-5 wk of age), as little as 1 d of deprivation is sufficient to cause ocular dominance changes (Olson and Freeman 1975). Near the age of puberty, weeks or months of deprivation are necessary to induce changes observable by single-unit recordings, and the changes are thought to occur only in layers 2 and 3 of the visual cortex (Daw et al. 1992).In the clinical literature, however, there are reports suggesting that improvement of visual acuity can occur in adult patients with amblyopia, a central disorder of visual acuity, following patching of the normal eye (Selenow and Ciuffreda 1986; Saulles 1987; Rutstein and Fuhr 1992; Wick and Wingard 1992). Furthermore, a lengthy period of monocular occlusion caused by a dense cataract (Sloper and Collins 1995) can cause significant changes in visu...

The development of precise connections in the mammalian brain proceeds through refinement of initially diffuse patterns, a process that occurs largely within critical developmental windows. To elucidate the molecular pathways that orchestrate these early periods of circuit remodeling, we have examined the role of a calcium- and cAMP-regulated transcriptional pathway. We show that there is a window of CRE/CREB-mediated gene expression in the developing thalamus, which precedes neocortical expression. In the LGN, this wave of gene expression occurs prior to visual experience, but requires retinal function. Mutant mice with reduced CREB expression show loss of refinement of retinogeniculate projections. These results suggest an important role of the CRE/CREB transcriptional pathway in the coordination of experience-independent circuit remodeling during forebrain development.

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Superparamagnetic iron oxide nanoparticles (SPIONs) are currently unavailable as MRI contrast agents for detecting atherosclerosis in the clinical setting because of either low signal enhancement or safety concerns. Therefore, a new generation of SPIONs with increased circulation time, enhanced image contrast, and less cytotoxicity is essential. In this study, monodisperse SPIONs were synthesized and coated with polyethylene glycol (PEG) of varying molecular weights. The resulting PEGylated SPIONs were characterized, and their interactions with vascular smooth muscle cells (VSMCs) were examined. SPIONs were tested at different concentrations (100 and 500 ppm Fe) for stability, T2 contrast, cytotoxicity, and cellular uptake to determine an optimal formulation for in vivo use. We found that at 100 ppm Fe, the PEG 2K SPIONs showed adequate stability and magnetic contrast, and exhibited the least cytotoxicity and nonspecific cellular uptake. An increase in cell viability was observed when the SPION-treated cells were washed with PBS after one hour incubation compared to 5 and 24 hour incubation without washing. Our investigation provides insight into the potential safe application of SPIONs in the clinic.

The estrogen receptor (ER) is a transcription factor involved in steroid hormone signal transduction in higher eukaryotes. The receptor also functions as a ligand-dependent transcriptional activator when introduced into Saccharomyces cerevisiae (baker's yeast), which suggests that at least some of the components of the signal transduction pathway are conserved between yeast and mammalian cells, and, moreover, allows the possibility of using this simple eukaryotic organism to dissect receptor function. However, whether the ER actually activates transcription in a mechanistically similar fashion in yeast and mammalian cells is unclear, since it has been reported that the transactivation function within the hormone binding domain (TAF-2) does not function in yeast. In this report, we have characterized the activity of the transactivation functions of the ER in yeast. Our results indicate that both TAF-2 and the N-terminal transactivation region (TAF-1) are functional in yeast and contribute synergistically to the receptor's total activity. These results are consistent with those obtained in mammalian cells. Furthermore, we show that in yeast the antagonistic effects of the antiestrogen nafoxidine arise from a modulation of the synergistic interactions of TAF-1 and TAF-2, and not simply from an inactivation of TAF-2 by antihormone. Finally, we characterize the effect of ER deletion mutants on chromatin structure in yeast. Our data lend support to the view that the formation of competent transcriptional initiation complexes requires a precise disruption of chromatin structure.