Structure Determination and Biochemical Characterization of a Putative HNH Endonuclease from Geobacter metallireducens GS-15

The crystal structure of a putative HNH endonuclease, Gmet_0936 protein from Geobacter metallireducens GS-15, has been determined at 2.6 Å resolution using single-wavelength anomalous dispersion method. The structure contains a two-stranded anti-parallel β-sheet that are surrounded by two helices on each face, and reveals a Zn ion bound in each monomer, coordinated by residues Cys38, Cys41, Cys73, and Cys76, which likely plays an important structural role in stabilizing the overall conformation. Structural homologs of Gmet_0936 include Hpy99I endonuclease, phage T4 endonuclease VII, and other HNH endonucleases, with these enzymes sharing 15–20% amino acid sequence identity. An overlay of Gmet_0936 and Hpy99I structures shows that most of the secondary structure elements, catalytic residues as well as the zinc binding site (zinc ribbon) are conserved. However, Gmet_0936 lacks the N-terminal domain of Hpy99I, which mediates DNA binding as well as dimerization. Purified Gmet_0936 forms dimers in solution and a dimer of the protein is observed in the crystal, but with a different mode of dimerization as compared to Hpy99I. Gmet_0936 and its N77H variant show a weak DNA binding activity in a DNA mobility shift assay and a weak Mn2+-dependent nicking activity on supercoiled plasmids in low pH buffers. The preferred substrate appears to be acid and heat-treated DNA with AP sites, suggesting Gmet_0936 may be a DNA repair enzyme

Overview of marine fisheries in India during 2007

Fisheries sector in India plays an important role in the country’s economy and it supports the livelihood of millions of people. India is having 8,129 km of coastal length with 2.02 million sq. km of Exclusive Economic Zone (upto 200 m depth) and 0.452 million sq. km of continental shelf area

The Roles of Transmembrane Domain Helix-III during Rhodopsin Photoactivation

Background: Rhodopsin, the prototypic member of G protein-coupled receptors (GPCRs), undergoes isomerization of 11- cis-retinal to all-trans-retinal upon photoactivation. Although the basic mechanism by which rhodopsin is activated is well understood, the roles of whole transmembrane (TM) helix-III during rhodopsin photoactivation in detail are not completely clear. Principal Findings: We herein use single-cysteine mutagenesis technique to investigate conformational changes in TM helices of rhodopsin upon photoactivation. Specifically, we study changes in accessibility and reactivity of cysteine residues introduced into the TM helix-III of rhodopsin. Twenty-eight single-cysteine mutants of rhodopsin (P107C-R135C) were prepared after substitution of all natural cysteine residues (C140/C167/C185/C222/C264/C316) by alanine. The cysteine mutants were expressed in COS-1 cells and rhodopsin was purified after regeneration with 11-cis-retinal. Cysteine accessibility in these mutants was monitored by reaction with 4, 49-dithiodipyridine (4-PDS) in the dark and after illumination. Most of the mutants except for T108C, G109C, E113C, I133C, and R135C showed no reaction in the dark. Wide variation in reactivity was observed among cysteines at different positions in the sequence 108–135 after photoactivation. In particular, cysteines at position 115, 119, 121, 129, 131, 132, and 135, facing 11-cis-retinal, reacted with 4-PDS faster than neighboring amino acids. The different reaction rates of mutants with 4-PDS after photoactivation suggest that the amino acids in different positions in helix-III are exposed to aqueous environment to varying degrees. Significance: Accessibility data indicate that an aqueous/hydrophobic boundary in helix-III is near G109 and I133. The lack of reactivity in the dark and the accessibility of cysteine after photoactivation indicate an increase of water/4-PDS accessibility for certain cysteine-mutants at Helix-III during formation of Meta II. We conclude that photoactivation resulted in water-accessible at the chromophore-facing residues of Helix-III.National Institutes of Health (U.S.) (grant GM28289)National Eye Institute (Grant Grant EY11716)National Science Foundation (U.S.) (grant EIA-0225609

Nonlinear deterministic equations in biological evolution

We review models of biological evolution in which the population frequency changes deterministically with time. If the population is self-replicating, although the equations for simple prototypes can be linearised, nonlinear equations arise in many complex situations. For sexual populations, even in the simplest setting, the equations are necessarily nonlinear due to the mixing of the parental genetic material. The solutions of such nonlinear equations display interesting features such as multiple equilibria and phase transitions. We mainly discuss those models for which an analytical understanding of such nonlinear equations is available.Comment: Invited review for J. Nonlin. Math. Phy

Particle Size Effects on the Quality of Flour Tortillas Enriched with Whole Grain Waxy Barley

Wheat tortillas were enriched with whole barley flour (WBF) of different particle sizes including 237 µm (regular [RD. 131 µm (intermediate [IM]). and 68 µm (microground [MG], Topographical and fluorescent microstructure images of flours, doughs, and tortillas were examined, Flours and tortillas were analyzed for color, protein, ash, starch, moisture. and β-glucan content. Farinograph testing was conducted on the flour blends, Water activity and texture analyses of tortillas were conducted, A 9-point hedonic scale was used by 95 untrained panelists to evaluate tortilla appearance, color, flavor, texture, and overall acceptability, Two commercial products (CP) were included in some analyses, As WBF particle size decreased, color was lighter; protein, moisture content and mixing stability decreased; ash, starch content, water absorption and farinograph peak time increased: and β-glucan content was constant. WBF tortillas were darker than the control (C), while 1M and MG tortillas had lower peak forces than C, No flavor differences were reported among C, R, and MG tortillas but higher scores were given to both CP in all attributes tested, Tortillas made with the largest WBF particle size (R) were the most similar in protein content, texture and flavor when compared with C tortillas made with refined bread flour

Measurement of thermophysical properties of liquid metallic alloys in a ground- and microgravity based research program. The Thermolab Project

An outline of the Thermolab Project is reported with the aim of informing on the wide range of properties which are becoming available for some industrial alloys. Selected examples of experiments and properties are provided

Nanomolecular singlet oxygen photosensitizers based on hemiquinonoid-resorcinarenes, the fuchsonarenes

Singlet oxygen sensitization involving a class of hemiquinonoid-substituted resorcinarenes prepared from the corresponding 3,5-di-t-butyl-4-hydroxyphenyl-substituted resorcinarenes is reported. Based on variation in the molecular structures, quantum yields comparable with that of the well-known photosensitizing compound meso-tetraphenylporphyrin were obtained for the octabenzyloxy-substituted double hemiquinonoid resorcinarene reported herein. The following classes of compounds were studied: benzyloxy-substituted resorcinarenes, acetyloxy-substituted resorcinarenes and acetyloxy-substituted pyrogallarenes. Single crystal X-ray crystallographic analyses revealed structural variations in the compounds with conformation (i.e., rctt, rccc, rcct) having some influence on the identity of hemiquinonoid product available. Multiplicity of hemiquinonoid group affects singlet oxygen quantum yield with those doubly substituted being more active than those containing a single hemiquinone. Compounds reported here lacking hemiquinonoid groups are inactive as photosensitizers. The term ‘fuchsonarene’ (fuchson + arene of resorcinarene) is proposed for use to classify the compounds

The Carbohydrate-Binding Site in Galectin-3 Is Preorganized To Recognize a Sugarlike Framework of Oxygens: Ultra-High-Resolution Structures and Water Dynamics

The recognition of carbohydrates by proteins is a fundamental aspect of communication within and between living cells. Understanding the molecular basis of carbohydrate-protein interactions is a prerequisite for the rational design of synthetic ligands. Here we report the high- to ultrahigh-resolution crystal structures of the carbohydrate recognition domain of galectin-3 (Gal3C) in the ligand-free state (1.08 angstrom at 100 K, 1.25 angstrom at 298 K) and in complex with lactose (0.86 angstrom) or glycerol (0.9 angstrom). These structures reveal striking similarities in the positions of water and carbohydrate oxygen atoms in all three states, indicating that the binding site of Gal3C is preorganized to coordinate oxygen atoms in an arrangement that is nearly optimal for the recognition of beta-galactosides. Deuterium nuclear magnetic resonance (NMR) relaxation dispersion experiments and molecular dynamics simulations demonstrate that all water molecules in the lactose-binding site exchange with bulk water on a time scale of nanoseconds or shorter. Nevertheless, molecular dynamics simulations identify transient water binding at sites that agree well with those observed by crystallography, indicating that the energy landscape of the binding site is maintained in solution. All heavy atoms of glycerol are positioned like the corresponding atoms of lactose in the Gal3C complexes. However, binding of glycerol to Gal3C is insignificant in solution at room temperature, as monitored by NMR spectroscopy or isothermal titration calorimetry under conditions where lactose binding is readily detected. These observations make a case for protein cryo-crystallography as a valuable screening method in fragment-based drug discovery and further suggest that identification of water sites might inform inhibitor design

A relevant in vitro rat model for the evaluation of blood-brain barrier translocation of nanoparticles

Poly(MePEG2000cyanoacrylate-co-hexadecylcyanoacrylate) (PEG-PHDCA) nanoparticles have demonstrated their capacity to reach the rat central nervous system after intravenous injection. For insight into the transport of colloidal systems across the blood-brain barrier (BBB), we developed a relevant in vitro rat BBB model consisting of a coculture of rat brain endothelial cells (RBECs) and rat astrocytes. The RBECs used in our model displayed and retained structural characteristics of brain endothelial cells, such as expression of P-glycoprotein, occludin and ZO-1, and immunofluorescence studies showed the specific localization of occludin and ZO1. The high values of transendothelial electrical resistance and low permeability coefficients of marker molecules demonstrated the functionality of this model. The comparative passage of polyhexadecylcyanoacrylate and PEG-PHDCA nanoparticles through this model was investigated, showing a higher passage of PEGylated nanoparticles, presumably by endocytosis. This result was confirmed by confocal microscopy. Thanks to a good in vitro/in vivo correlation, this rat BBB model will help in understanding the mechanisms of nanoparticle translocation and in designing new types of colloidal carriers as brain delivery systems