A two-state homology model of the hERG K+ channel: application to ligand binding

Homology models based on available K+ channel structures have been used to construct a multiple state representation of the hERG cardiac K+ channel. These states are used to capture the flexibility of the channel. We show that this flexibility is essential in order to correctly model the binding affinity of a set of diverse ligands. Using this multiple state approach, a binding affinity model was constructed for set of known hERG channel binders. The predicted pIC50s are in good agreement with experiment (RMSD: 0.56 kcal/mol). In addition, these calculations provide structures for the bound ligands that are consistent with published mutation studies. These computed ligand bound complex structures can be used to guide synthesis of analogs with reduced hERG liability.     

Dynamics of nucleotide incorporation: snapshots revealed by 2-aminopurine fluorescence studies

Formation of a noncanonical base pair between dFTP, a dTTP analogue that cannot form H bonds, and the fluorescent base analogue 2-aminopurine (2AP) was studied in order to discover how the bacteriophage T4 DNA polymerase selects nucleotides with high accuracy. Changes in 2AP fluorescence intensity provided a spectroscopic reporter of the nucleotide binding reactions, which were combined with rapid-quench, pre-steady-state reactions to measure product formation. These studies supported and extended previous findings that the T4 DNA polymerase binds nucleotides in multiple steps with increasing selectivity. With 2AP in the template position, initial dTTP binding was rapid but selective: K(d(dTTP)) (first step) = 31 microM; K(d(dCTP)) (first step) approximately 3 mM. In studies with dFTP, this step was revealed to have two components: formation of an initial preinsertion complex in which H bonds between bases in the newly forming base pair were not essential, which was followed by formation of a final preinsertion complex in which H bonds assisted. The second nucleotide binding step was characterized by increased discrimination against dTTP binding opposite template 2AP, K(d) (second step) = 367 microM, and additional conformational changes were detected in ternary enzyme-DNA-dTTP complexes, as expected for forming closed complexes. We demonstrate here that the second binding step occurs before formation of the phosphodiester bond. Thus, the high fidelity of nucleotide insertion by T4 DNA polymerase is accomplished by the sequential application of selectivity in first forming accurate preinsertion complexes, and then additional conformational changes are applied that further increase discrimination against incorrect nucleotides.     

Nitric oxide synthase regulation and diversity: Implications in Parkinson’s disease

Nitric oxide (NO) is a janus faced chemical messenger, which, in the recent years, has been the focus of neurobiologists for its involvement in neurodegenerative disorders in particular, Parkinson's disease (PD). Nitric oxide synthase, the key enzyme involved in NO production exists in three known isoforms. The neuronal and inducible isoforms have been implicated in the pathogenesis of PD. These enzymes are subject to complex expressional and functional regulation involving mRNA diversity, phosphorylation and protein interaction. In the recent years, mRNA diversity and polymorphisms have been identified in the NOS isoforms. Some of these genetic variations have been associated with PD, indicating an etiological role for the NOS genes. This review mainly focuses on the NOS genes - their differential regulation and genetic heterogeneity, highlighting their significance in the pathobiology of PD.     

Genetic regulation of amniotic fluid TNF-alpha and soluble TNF receptor concentrations affected by race and preterm birth

Racial disparity in spontaneous preterm birth (PTB) between African Americans and Caucasians in the US is unexplained, but is probably related to differences in amniotic fluid (AF) inflammatory cytokine profiles. Therefore, this study analyzed the association of 34 single nucleotide polymorphisms (SNPs) in TNF-α and its receptor genes (TNFR1 and TNFR2) with AF TNF-α and soluble TNF receptor (R1 and R2) concentrations in PTB. Samples consisted of African American and Caucasian cases (PTB), and controls (term birth) for which both cytokine, and maternal and fetal genotype data were available. Analyses were performed with genotype, case, and maker-status interaction in the model for log transformed cytokine concentrations. In Caucasians, two interactions between genotype and pregnancy outcome associated with cytokine concentrations, whereas 14 gene variants in African Americans showed interactions with pregnancy outcome, and 13 showed association with genetic markers. In conclusion, cytokine concentrations in African American preterm births can be partially explained by interactions between pregnancy outcome, SNPs and infection. This does not appear to be the case in Caucasians. These findings may be important in understanding disparity in rates of PTB between the two populations. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Application of numerical modeling for the development of optimized complex medium for D-hydantoinase production from Agrobacterium radiobacter NRRL B 11291

D-Hydantoinases (E.C.3.5.2.2) are commercially valuable enzymes involved in the production of D-amino acids. However, commercial exploitation of the biological process is rare, mainly because sufficient details are not available on the efficient production of these enzymes by microorganisms. In the present study, Agrobacterium radiobacter was used as the source of D-hydantoinase and its production was optimized with inexpensive carbon and nitrogen sources. The four media components selected to study their effect on biomass and/or enzyme activities were molasses, ammonium nitrate, sodium di-hydrogen orthophosphate, and manganese chloride. With the use of an empirical modeling technique (response surface method), we have optimized both biomass and enzyme production in this organism, with a minimal number of batches. Experiments were performed with optimized media components to validate the model. The maximum level of enzyme and biomass obtained was 35 U/mL and 1.69 mg/mL, respectively. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 55: 148-154, 1997.     

A gene-trap strategy identifies quiescence-induced genes in synchronized myoblasts

Cellular quiescence is characterized not only by reduced mitotic and metabolic activity but also by altered gene expression. Growing evidence suggests that quiescence is not merely a basal state but is regulated by active mechanisms. To understand the molecular programme that governs reversible cell cycle exit, we focused on quiescence-related gene expression in a culture model of myogenic cell arrest and activation. Here we report the identification of quiescence-induced genes using a gene-trap strategy. Using a retroviral vector, we generated a library of gene traps in C2C12 myoblasts that were screened for arrest-induced insertions by live cell sorting (FACS-gal). Several independent gene- trap lines revealed arrest-dependent induction of betagal activity, confirming the efficacy of the FACS screen.The locus of integration was identified in 15 lines. In three lines,insertion occurred in genes previously implicated in the control of quiescence, i.e. EMSY - a BRCA2--interacting protein, p8/com1 - a p300HAT -- binding protein and MLL5 - a SET domain protein. Our results demonstrate that expression of chromatin modulatory genes is induced in G0, providing support to the notion that this reversibly arrested state is actively regulated.     

Highly Stable Plasmonic Nanostructures on a Nickel-Sputtered Glass and Polymeric Optical Fiber Sensors

Plasmonics - This study shows development of highly sensitive and stable localized surface plasmon resonance (LSPR)-active U-bent glass and polymeric optical fiber (GOF and POF) sensor probes by a...     

Twisted Perylene Diimides with Tunable Redox Properties for Organic Sodium‐Ion Batteries

Organic rechargeable batteries gain huge scientific interest owing to the design flexibility and resource renewability of the active materials. However, the low reduction potentials still remain a challenge to compete with the inorganic cathodes. This study demonstrates a simple and efficient approach to tune the redox properties of perylene diimides (PDIs) as high voltage cathodes for organic‐based sodium‐ion batteries (SIBs). With appropriate electron‐withdrawing groups as substituents on perylene diimides, this study shows a remarkable tunability in the discharge potential from 2.1 to 2.6 V versus Na⁺/Na with a sodium intake of ≈1.6 ions per molecule. Further, this study explores tuning the shape of the voltage profiles by systematically tuning the dihedral angle in the perylene ring and demonstrates a single plateau discharge profile for tetrabromo‐substituted perylene diimide (dihedral angles θ₁ & θ₂ = 38°). Detailed structural analysis and electrochemical studies on substituted PDIs unveil the correlation between molecular structure and voltage profile. The results are promising and offer new avenues to tailor the redox properties of organic electrodes, a step closer toward the realization of greener and sustainable electrochemical storage devices.