Iron(III) Schiff base complexes of arginine and lysine as netropsin mimics showing AT-selective DNA binding and photonuclease activity

Iron(III) complexes [Fe(L)(2)]Cl (1-3), where L is monoanionic N-salicylidene-arginine (sal-argH for 1), hydroxynaphthylidene-arginine (nap-argH for 2) and N-salicylidene-lysine (sal-lysH for 3), were prepared and their DNA binding and photo-induced DNA cleavage activity studied. Complex 3 as its hexafluorophosphate salt [Fe(sal-lysH)(2)](PF6)center dot 6H(2)O (3a) was structurally characterized by single crystal Xray crystallography. The crystals belonged to the triclinic space group P-1. The complex has two tridentate ligands in FeN2O4 coordination geometry with two pendant cationic amine moieties. Complexes 1 and 2 with two pendant cationic guanidinium moieties are the structural models for the antitumor antibiotics netropsin. The complexes are stable and soluble in water. They showed quasi-reversible Fe(III)/Fe(II) redox couple near 0.6 V in H2O-0.1 M KCl. The high-spin 3d(5)-iron(III) complexes with mu(eff) value of similar to 5.9 mu(B) displayed ligand-to-metal charge transfer electronic band near 500 mm in Tris-HCl buffer. The complexes show binding to Calf Thymus (CT) DNA. Complex 2 showed better binding propensity to the synthetic oligomer poly(dA)center dot poly(dT) than to CT-DNA or poly(dG)center dot poly(dC). All the complexes displayed chemical nuclease activity in the presence of 3-mercaptopropionic acid as a reducing agent and cleaved supercoiled pUC19 DNA to its nicked circular form. They exhibited photo-induced DNA cleavage activity in UV-A light and visible light via a mechanistic pathway that involves the formation of reactive hydroxyl radical species. (C) 2010 Elsevier Inc. All rights reserved.

Immobilization of Aspergillus oryzae alpha-galactosidase in gelatin and its application in removal of flatulence-inducing sugars in soymilk

Raffinose oligosaccharides (RO) are the major factors responsible for flatulence following ingestion of soybean-derived products. Removal of RO from seeds or soymilk would then have a positive impact on the acceptance of soy-based foods. In this study, alpha-galactosidase from Aspergillus oryzae was entrapped in gelatin using formaldehyde as the hardener. The immobilization yield was 64.3% under the optimum conditions of immobilization. The immobilized alpha-galactosidase showed a shift in optimum pH from 4.8 to 5.4 in acetate buffer. The optimum temperature also shifted from 50 degrees C to 57 degrees C compared with soluble enzyme. Immobilized alpha-galactosidase was used in batch, repeated batch and continuous mode to degrade RO present in soymilk. In the repeated batch, 45% reduction of RO was obtained in the fourth cycle. The performance of immobilized alpha-galactosidase was tested in a fluidized bed reactor at different flow rates and 86% reduction of RO in soymilk was obtained at 25 ml h(-1) flow rate. The study revealed that immobilized alpha-galactosidase in continuous mode is efficient in reduction of RO present in soymilk.

Synthesis of BaSO4 nanoparticles by precipitation method using sodium hexa metaphosphate as a stabilizer

We describe the synthesis and structure of Barium sulfate nanoparticles by precipitation method in the presence of water soluble inorganic stabilizing agent, sodium hexametaphosphate, (NaPO3)(6). The structural parameters were refined by the Rietveld refinement method using powder X-ray diffraction data. Barium sulfate nanoparticles were crystallized in the orthorhombic structure with space group Pbnm (No. 62) having the lattice parameters a = 7.215(1) (angstrom), b = 8.949(1) (angstrom) and c = 5.501 (1) (angstrom) respectively. Transmission electron microscopy study reveals that the nanoparticles are size range, 30-50 nm. Fourier transform infrared spectra showed distinct absorption due to the SO42- moiety at 1115 and 1084 cm(-1) indicating formation of barium sulfate nanoparticles free from the phosphate group from the stabilizer used in the synthesis. (C) 2009 Elsevier Ltd. All rights reserved.

Replacement of water on a steel substrate by a surrounding oil reservoir

With an objective to replace a water droplet from a steel surface by oil we study here the impact of injecting a hydrophilic/lipophilic surfactant into the droplet or into the surrounding oil reservoir. Contact angle goniometery, Grazing angle FTIR spectroscopy and Atomic force microscopy are used to record the oil/water interfacial tension, surface energetics of the substrate under the oil and water phases as well as the corresponding physical states of the substrates. Such energetics reflect the rate at which the excess surfactant molecules accumulate at the water/oil interface and desorb into the phases. The molecules diffuse into the substrate from the phases and build up specific molecular configurations which, with the interfacial tension, control the non-equilibrium progress of and the equilibrium status of the contact line. The study shows that the most efficient replacement of water by the surrounding oil happens when a surfactant is sparingly soluble in the supplier oil phase and highly soluble in the recipient water phase.

Trap-State Dynamics in Visible-Light-Emitting ZnO:MgO Nanocrystals

Oleate-capped ZnO:MgO nanocrystals have been synthesized that are soluble in nonpolar solvents and which emit strongly in the visible region (450−600 nm) on excitation by UV radiation. The visible emission involves recombination of trap states of the nanocrystalline ZnO core and has a higher quantum yield than the band gap UV exciton emission. The spectrally resolved dynamics of the trap states have been investigated by time-resolved emission spectroscopy. The time-evolution of the photoluminescence spectra show that there are, in fact, two features in the visible emission whose relative importance and efficiencies vary with time. These features originate from recombination involving trapped electrons and holes, respectively, and with efficiencies that depend on the occupancy of the trap density of states.

Structure and assembly of Sesbania mosaic virus

Sesbania mosaic virus (SeMV) is a ss-RNA (4149 nt) plant sobemovirus isolated from farmer's field around Tirupathi, Andhra Pradesh. The viral capsid (30 nm diameter) consists of 180 copies of protein subunits (MW 29 kDa) organized with icosahedral symmetry. In order to understand the mechanism of assembly of SeMV, a large number of deletion and substitution mutants of the coat protein (CP) were constructed. Recombinant SeMV CP (rCP) as well as the N-terminal rCP deletion mutant Delta N22 were found to assemble in E. coli into virus-like particles (VLPs). Delta N36 and Delta N65 mostly formed smaller particles consisting of 60 protein subunits. Although particlem assembly was not affected due to the substitution of aspartates (D14 and D149) that coordinate calcium ions by asparagines, the stability of the resulting capsids was drastically reduced. Deletion of residues forming a characteristic beta-annulus at the icosahedral 3-folds did not affect the assembly of VLPs. Mutation of a single tryptophan, which occurs near the icosahedral fivefold axis to glutamate or lysine, resulted in the disruption of the capsid leading to soluble dimers that resembled the quasi-dimer structure of the native virus. Replacement of positively charged residues in the amino terminal segment of CP resulted in the formation of empty shells. Based on these observations, a plausible mechanism of assembly is proposed.

Covalent tethering of the dimer interface annuls aggregation in thymidylate synthase

Thymidylate synthase (TS), a dimeric enzyme, forms large soluble aggregates at concentrations of urea (3.3-5 M), well below that required for complete denaturation, as established by fluorescence and size-exclusion chromatography. In contrast to the wild-type enzyme, an engineered mutant of TS (T155C/E188C/C244T), TSMox, in which two subunits are crosslinked by disulfide bridges between residues 155-188' and 188-155', does not show this behavior. Aggregation behavior is restored upon disulfide bond reduction in the mutant protein, indicating the involvement of interface segments in forming soluble associated species. Intermolecular disulfide crosslinking has been used as a probe to investigate the formation of larger non-native aggregates. The studies argue for the formation of large multimeric species via a sticky patch of polypeptide from the dimer interface region that becomes exposed on partial unfolding. Covalent reinforcement of relatively fragile protein-protein interfaces may be a useful strategy in minimizing aggregation of non-native structures in multimeric proteins.

Physicochemical studies on the gelation of hen egg yolk; separation of gelling protein components from yolk plasma

A study of the component(s) in egg yolk responsible for gelation of yolk on freezing and thawing has shown that granule-free yolk plasma, obtained by high-speed centrifugation of yolk, has the capacity to gel. As with the whole yolk, gelation of yolk plasma on freezing and thawing could be inhibited by additives such as sugars, sodium chloride, proteolytic enzymes, and phospholipase-A. Phospholipase-C, which induces gelation of whole yolk at room temperature, has a similar effect on yolk plasma. Yolk plasma has been separated into aggregating (gelling) and soluble fractions by delipidation, using formic acid. Each of these fractions consists of three or four protein components, as observed by gel filtration, ultracentrifugation, and agar electrophoresis. The proteins are glycoproteins and contain bound hexoses, hexosamine, and sialic acid. The gelation of yolk has been attributed to the interactions between protein molecules following disruption of lipid-protein bonds.

Indoleacetaldoxime hydro-lyase : II. Purification and properties

The purification and some properties of the enzyme indoleacetaldoxime hydrolyase (EC 4.2.1.29) from the fungus Gibberella fujikuroi, which dehydrates indoleacetaldoxime (IAOX) to indoleacetonitrile (IAN), are described. The enzyme activity in the fungus is present only under certain culture conditions. It is a soluble enzyme, has an optimum pH at 7, shows an energy of activation of —15,670 cal/mole, and has a Michaelis constant of 1.7 × 10−4 Image at 30 °. It appears to be specific for IAOX, and 1 mole of IAN is produced per mole of IAOX utilized. The enzyme is inhibited by a number of aldoximes of which phenylacetaldoxime (PAOX) is the most potent inhibitor. Inhibition by PAOX is competitive (Ki = 2.2 × 10−8 Image ). The enzyme is inhibited by SH reagents such as p-hydroxymercuribenzoate and N-ethylmaleimide, and by a number of SH compounds such as cysteine, β-mercaptoethanol, and 2,3-dimercaptopropanol (BAL). However, glutathione activates the enzyme. Metal chelating agents such as 8-OH-quinoline and diethyl dithiocarbamate inhibit the enzyme; the inhibition is partly reversed by ferric citrate. Ascorbic acid, and particularly dehydroascorbic acid (DHA), are good activators of the enzyme. Several other biological oxidants had either no action or had a slight effect. Potassium cyanide activates the enzyme at low concentration but inhibits at higher concentrations. Reduction of the enzyme with NaBH4 reduces activity, and the effect is partly reversed by pyridoxal phosphate and also by DHA. The above properties indicate that both an SH function and an oxidized function are required for activity.

Anthranilic acid hydroxylase from Aspergillus niger

The in vivo conversion of radioactive tryptophan to anthranilic acid and 2,3-dihydroxybenzoic acid by submerged cultures of Claviceps paspali was shown by Groeger and his co-workers (1965). More recently, Subba Rao et al. (1967a) reported that washed mycelial felts of Aspergillus niger incorporate the radioactivity from DL-tryptophan-C14 (benzene ring-labeled) into anthranilic acid, 3-hydroxyanthranilic acid, 2,3-dihydroxybenzoic acid and catechol. However, the conversion of anthranilic acid to 2,3-dihydroxybenzoic acid by cell-free preparations has not been demonstrated. In the present paper we report the demonstration of a soluble anthranilic acid hydroxylase from Aspergillus niger which is different from the anthranilic acid hydroxylases reported so far from microbes and higher plants.

Physicochemical studies on the gelation of hen's egg yolk: Delipidation of yolk plasma by treatment with phospholipase-C and extraction with solvents

A method for the delipidation of egg yolk plasma using phospholipase-C, n-heptane, and 1-butanol has been described. An aggregating protein fraction and a soluble protein fraction were separated by the action of phospholipase-C. The aggregating protein fraction freed of most of the lipids by treatment with n-heptane and 1-butanol was shown to be the apolipoproteins of yolk plasma, whereas the soluble proteins were identified as the livetins. Carbohydrate and the N-terminal amino acid analysis of these protein fractions are reported. A comparison of these protein fractions with the corresponding fractions obtained by formic acid delipidation of yolk plasma has been made. The gelation of yolk plasma by the action of phospholipase-C has been interpreted as an aggregation of lipoproteins caused by ionic interactions. The role of lecithin in maintaining the structural integrity of lipoproteins has been discussed.

Studies on some metal monothiobenzoates

Monothiobenzoate (MTB) (Chemical Equation Presented) complexes with the molecular formulas Cr(MTB)3, [Ni(MTB)2]n, [Zn(MTB)2]n, [Cd(MTB)2]n, [Hg(MTB)2]n, [Cu(MTB)]n, and [Ag(MTB)]n have been prepared and studied. All the complexes are nonionic in acetonitrile. Only the chromium complex is soluble in nitrobenzene and found to be monomeric cryoscopically. The thiobenzoate ligand appears to be asymmetrically chelated in Cr(III) and Cd(II) complexes, with stronger oxygen and sulfur coordination, respectively, while practically symmetrically coordinated in Ni(II) and Zn(II) complexes. These four complexes are assigned distorted octahedral structures around the metal ion. The coordination in Hg(II), Cu(I), and Ag(I) complexes is mainly through sulfur indicating the monodentate nature of the thiobenzoate ligand in these complexes. The coordination of monothiobenzoate ion in the complexes has been rationalized in terms of "hard" and "soft" acid-base concept.

Cloning,overexpression and purification of functionally active Saccharomyces cerevisiae Hop1 protein from scherichia coli

One of the major limitations to the application of high-resolution biophysical techniques such as X-crystallography and spectroscopic analyses to structure-function studies of Saccharomyces cerevisiae Hop1 protein has been the non-availability of sufficient quantities of functionally active pure protein. This has, indeed, been the case of many proteins, including yeast synaptonemal complex proteins. In this study, we have performed expression screening in Escherichia coli host strains, capable of high-level expression of soluble S. cerevisiae Hop1 protein. A new protocol has been developed for expression and purification of S. cerevisiae Hop1 protein, based on the presence of hexa-histidine tag and double-stranded DNA-Cellulose chromatography. Recombinant S. cerevisiae Hop1 protein was >98% pure and exhibited DNA-binding activity with high-affinity to the Holliday junction. The availability of the recombinant HOP1 expression vector and active Hop1 protein would facilitate structure-function investigations as well as the generation of appropriate truncated and site-directed mutant proteins, respectively. (C) 2010 Elsevier Inc. All rights reserved.

Alkaline and acid phosphatases of the silkworm, Bombyx mori L.

The variations in the activities of the alkaline and acid phosphatases of the silkworm, Bombyx mori, were studied in all stages of the life cycle. From hatching until the spinning stage a steady increase was recorded in the activity of both the enzymes followed with a conspicuous decrease at each moult. During the pupal stage the alkaline phosphatase was almost absent, whereas the acid phosphatase maintained a high and constant value. Increase or decrease of the activity of the enzymes during larval development was reflected in a decrease or increase in the acid-soluble phosphorus content. Acid phosphatase activity slowly increased from laying of the eggs to hatching of the larvae with a concomitant decrease in the acid-soluble phosphorus. Tissue analysis showed a high concentration of the alkaline enzyme in the intestines, but the haemolymph was almost free of both enzymes. Feeding of inorganic phosphate increased the alkaline enzyme in the intestines, whereas glucose had no effect on either of the enzymes in the intestines.

An immunochemical study of ovine pituitary follicle-stimulating hormone (FSH)

An immunochemical study of ovine follicle-stimulating hormone and its antibody carried out by using precipitin, agglutinating and complement-fixation systems, has suggested that the follicle-stimulating hormone, possibly by virtue of it being a univalent antigen, forms a soluble complex with its specific antibody. This antiserum is species nonspecific in that it is able to neutralize the follicle-stimulating activity of rat, mouse, hamster, guinea pig pituitary extracts, and pregnant mare serum gonadotropin. Human chorionic gonadotropin, however, has been shown not to form a complex with the follicle-stimulating hormone specific antibody.