Modification of the sugar specificity of a plant lectin: structural studies on a point mutant of Erythrina corallodendron lectin

A mutant of Erythrina corallodendron lectin was generated with the aim of enhancing its affinity for N-acetylgalactosamine. A tyrosine residue close to the binding site of the lectin was mutated to a glycine in order to facilitate stronger interactions between the acetamido group of the sugar and the lectin which were prevented by the side chain of the tyrosine in the wild-type lectin. The crystal structures of this Y106G mutant lectin in complex with galactose and N-acetylgalactosamine have been determined. A structural rationale has been provided for the differences in the relative binding affinities of the wild-type and mutant lectins towards the two sugars based on the structures. A hydrogen bond between the O6 atom of the sugars and the variable loop of the carbohydrate-binding site of the lectin is lost in the mutant complexes owing to a conformational change in the loop. This loss is compensated by an additional hydrogen bond that is formed between the acetamido group of the sugar and the mutant lectin in the complex with N-acetylgalactosamine, resulting in a higher affinity of the mutant lectin for N-acetylgalactosamine compared with that for galactose, in contrast to the almost equal affinity of the wild-type lectin for the two sugars. The structure of a complex of the mutant with a citrate ion bound at the carbohydrate-binding site that was obtained while attempting to crystallize the complexes with sugars is also presented.

Crystal structure of Salmonella typhimurium 2-methylcitrate synthase: Insights on domain movement and substrate specificity

2-Methylcitric acid (2-MCA) cycle is one of the well studied pathways for the utilization of propionate as a source of carbon and energy in bacteria such as Salmonella typhimurium and Escherichia coli. 2-Methylcitrate synthase (2-MCS) catalyzes the conversion of oxaloacetate and propionyl-CoA to 2-methylcitrate and CoA in the second step of 2-MCA cycle. Here, we report the X-ray crystal structure of S. typhimurium 2-MCS (StPrpC) at 2.4 A resolution and its functional characterization. StPrpC was found to utilize propionyl-CoA more efficiently than acetyl-CoA or butyryl-CoA. The polypeptide fold and the catalytic residues of StPrpC are conserved in citrate synthases (CSs) suggesting similarities in their functional mechanisms. In the triclinic P1 cell, StPrpC molecules were organized as decamers composed of five identical dimer units. In solution, StPrpC was in a dimeric form at low concentrations and was converted to larger oligomers at higher concentrations. CSs are usually dimeric proteins. In Gram-negative bacteria, a hexameric form, believed to be important for regulation of activity by NADH, is also observed. Structural comparisons with hexameric E. coil CS suggested that the key residues involved in NADH binding are not conserved in StPrpC. Structural comparison with the ligand free and bound states of CSs showed that StPrpC is in a nearly closed conformation despite the absence of bound ligands. It was found that the Tyr197 and Leu324 of StPrpC are structurally equivalent to the ligand binding residues His and Val, respectively, of CSs. These substitutions might determine the specificities for acyl-CoAs of these enzymes. (C) 2010 Elsevier Inc. All rights reserved.

Synthesis and characterization of barium bis(citrato) oxozirconate(iv) tetrahydrate--A new molecular precursor for fine particle BaZrO3

Barium metazirconate (BaZrO3) fine powder has been produced by thermally decomposing a molecular precursor, barium bis(citrato)oxozirconate(IV) tetrahydrate at about 700-degrees-C. The precursor, Ba[ZrO(C6H6O7)2] . 4H2O (BZO) has been synthesized and characterized by employing a combination of spectroscopic and thermoanalytical techniques. The precursor undergoes thermal decomposition in three major stages: (i) dehydration to give an anhydrous barium zirconyl citrate, (ii) decomposition of the anhydrous citrate in a multistep process to form an ionic oxycarbonate intermediate, Ba2Zr2O5CO3, and (iii) decomposition of the oxycarbonate to produce BaZrO3 fine powder. The particle size of the resultant BaZrO3 is about 0.2 mum, and the surface area is found to be 4.0 m2 g-1.

Strontium and calcium zirconyl citrates as precursors for the low-temperature synthesis of SrZrO3 and CaZrO3 fine powders

Synthesis and the thermal decomposition behavior of new molecular precursors, strontium, and calcium zirconyl citrates are presented. The pathway to the metazirconate formation has been found to proceed through a multistep process. The precursors yield SrZrO3 and CaZrO3 fine powders at temperatures as low as 650 degrees C. Physico-chemical, spectroscopic, thermoanalytical, and microscopic techniques have enabled the identification of the sequence of events leading to the perovskite formation and proposition of a thermolysis scheme. Retention of the molecular level mixing of the metal ions during the course of the precursor decomposition is supported by these techniques. Prior to the formation of MZrO3 (M = Sr and Ca) an ionic oxycarbonate, M2Zr2O5CO3 (M = SI. and Ca), intermediate is produced by the thermal decomposition of the citrate precursors.

Role of spacer lengths of gemini surfactants in the synthesis of silver nanorods in micellar media

In this work, we have prepared Ag-nanorods using biscationic gemini surfactant micelles as the media by a seed-mediated wet synthesis method. Towards this end, we first synthesized Ag-nanoseeds of diameter similar to 7 nm stabilized by trisodium citrate (as the capping agent). Then these Ag-nanoseeds were used to synthesize Ag-nanorods of different aspect ratios. With decreasing Ag-nanoseed concentration, the aspect ratios of the Ag-nanorods stabilized by these gemini surfactants increased gradually. Various Ag-nanoseeds and Ag-nanospecies were characterized using UV-Vis spectroscopy (to know the surface plasmon bands), transmission electron microscopy (to find out their particle sizes and distribution), energy-dispersive X-ray spectroscopy and X-ray diffraction. When we used micelles derived from gemini surfactants of shorter spacer-(CH(2))(n)-(n = 2 or 4) to stabilize the Ag-nanorods, the lambda(max) of the longitudinal band shifted more towards the blue region compared to that of the gemini surfactant micelles with a longer spacer-(CH(2))(n)-(n = 5, 12) at a given amount of the Ag-nanoseed solution. So, the growth of Ag-nanorods in the gemini micellar solutions depends on the spacer-chain length of gemini surfactants employed.

Monodisperse sub-10 nm gold nanoparticles by reversing the order of addition in Turkevich method - The role of chloroauric acid

The Turkevich method for synthesizing gold nanoparticles, using sodium citrate as the reducing agent, is renowned for its ability to produce biocompatible colloids with mean size >10 nm. Here we show that monodisperse gold nanoparticles in the 5-10 nm size range can be synthesized by simply reversing the order of addition of reactants, i.e. adding chloroauric acid to citrate solution. Kinetic studies and electron microscopic characterization revealed that the reactivity of chloroauric acid, initial molar ratio of citrate to chloroauric acid (MR), and reaction mixture pH play an important role in producing monodisperse gold nanoparticles. Reversing the order of addition also enhanced the stabilization of nanoparticles at high MR values. Remarkably, the system exhibits a `memory' of the order of addition, even when the timescale of mixing is much shorter than the timescale of synthesis. (C) 2011 Elsevier Inc. All rights reserved.

Graphene as a Nanocarrier for Tamoxifen Induces Apoptosis in Transformed Cancer Cell Lines of Different Origins

A cationic amphiphile, cholest-5en-3 beta-oxyethyl pyridinium bromide (PY(+)-Chol), is able to efficiently disperse exfoliated graphene (GR) in water by the physical adsorption of PY(+)-Chol on the surface of GR to form stable, dark aqueous suspensions at room temperature. The GRPY(+)-Chol suspension can then be used to solubilize Tamoxifen Citrate (TmC), a breast cancer drug, in water. The resulting TmCGRPY(+)-Chol is stable for a long time without any precipitation. Fluorescence emission and UV absorption spectra indicate the existence of noncovalent interactions between TmC, GR, and PY(+)-Chol in these suspensions. Electron microscopy shows the existence of segregated GR sheets and TmC ribbons in the composite suspensions. Atomic force microscopy indicates the presence of extended structures of GRPY(+)-Chol, which grows wider in the presence of TmC. The slow time-dependent release of TmC is noticed in a reconstituted cell culture medium, a property useful as a drug carrier. TmCGRPY(+)-Chol selectively enhanced the cell death (apoptosis) of the transformed cancer cells compared to normal cells. This potency is found to be true for a wide range of transformed cancer cells viz. HeLa, A549, ras oncogene-transformed NIH3T3, HepG2, MDA-MB231, MCF-7, and HEK293T compared to the normal cell HEK293 in vitro. Confocal microscopy confirmed the high efficiency of TmCGRPY(+)-Chol in delivering the drug to the cells, compared to the suspensions devoid of GR.

Multilayer assemblies of polyelectrolyte-gold nanoparticles for the electrocatalytic oxidation and detection of arsenic(III)

Multilayers of poly(diallyldimethylammonium chloride) (PDDA) and citrate capped Au nanoparticles (AuNPs) anchored on sodium 3-mercapto-1-propanesulfonate modified gold electrode by electrostatic layer-by-layer assembly (LbL) technique are shown to be an excellent architecture for the direct electrochemical oxidation of As(III) species. The growth of successive layers in the proposed LbL architecture is followed by atomic force microscopy, UV-vis spectroscopy, quartz crystal microbalance with energy dissipation, and electrochemistry. The first bilayer is found to show rather different physico-chemical characteristics as compared to the subsequent bilayers, and this is attributed to the difference in the adsorption environments. The analytical utility of the architecture with five bilayers is exploited for arsenic sensing via the direct electrocatalytic oxidation of As(III), and the detection limit is found to be well below the WHO guidelines of 10 ppb. When the non-redox active PDDA is replaced by the redoxactive Os(2,2'-bipyridine)(2)Cl-poly(4-vinylpyridine) polyelectrolyte (PVPOs) in the LbL assembly, the performance is found to be inferior, demonstrating that the redox activity of the polyelectrolyte is futile as far as the direct electro-oxidation of As(III) is concerned. (C) 2012 Elsevier Inc. All rights reserved.

Structural and mechanistic investigations on Salmonella typhimurium acetate kinase (AckA): identification of a putative ligand binding pocket at the dimeric interface

Background: Bacteria such as Escherichia coli and Salmonella typhimurium can utilize acetate as the sole source of carbon and energy. Acetate kinase (AckA) and phosphotransacetylase (Pta), key enzymes of acetate utilization pathway, regulate flux of metabolites in glycolysis, gluconeogenesis, TCA cycle, glyoxylate bypass and fatty acid metabolism. Results: Here we report kinetic characterization of S. typhimurium AckA (StAckA) and structures of its unliganded (Form-I, 2.70 angstrom resolution) and citrate-bound (Form-II, 1.90 angstrom resolution) forms. The enzyme showed broad substrate specificity with k(cat)/K-m in the order of acetate > propionate > formate. Further, the K-m for acetyl-phosphate was significantly lower than for acetate and the enzyme could catalyze the reverse reaction (i.e. ATP synthesis) more efficiently. ATP and Mg2+ could be substituted by other nucleoside 5'-triphosphates (GTP, UTP and CTP) and divalent cations (Mn2+ and Co2+), respectively. Form-I StAckA represents the first structural report of an unliganded AckA. StAckA protomer consists of two domains with characteristic beta beta beta alpha beta alpha beta alpha topology of ASKHA superfamily of proteins. These domains adopt an intermediate conformation compared to that of open and closed forms of ligand-bound Methanosarcina thermophila AckA (MtAckA). Spectroscopic and structural analyses of StAckA further suggested occurrence of inter-domain motion upon ligand-binding. Unexpectedly, Form-II StAckA structure showed a drastic change in the conformation of residues 230-300 compared to that of Form-I. Further investigation revealed electron density corresponding to a citrate molecule in a pocket located at the dimeric interface of Form-II StAckA. Interestingly, a similar dimeric interface pocket lined with largely conserved residues could be identified in Form-I StAckA as well as in other enzymes homologous to AckA suggesting that ligand binding at this pocket may influence the function of these enzymes. Conclusions: The biochemical and structural characterization of StAckA reported here provides insights into the biochemical specificity, overall fold, thermal stability, molecular basis of ligand binding and inter-domain motion in AckA family of enzymes. Dramatic conformational differences observed between unliganded and citrate-bound forms of StAckA led to identification of a putative ligand-binding pocket at the dimeric interface of StAckA with implications for enzymatic function.

Effect of high gold salt concentrations on the size and polydispersity of gold nanoparticles prepared by an extended Turkevich-Frens method

The Turkevich-Frens synthesis starting conditions are expanded, ranging the gold salt concentrations up to 2 mM and citrate/gold(III) molar ratios up to 18:1. For each concentration of the initial gold salt solution, the citrate/gold(III) molar ratios are systematically varied from 2:1 to 18:1 and both the size and size distribution of the resulting gold nanoparticles are compared. This study reveals a different nanoparticle size evolution for gold salt solutions ranging below 0.8 mM compared to the case of gold salt solutions above 0.8 mM. In the case of Au3+]<0.8 mM, both the size and size distribution vary substantially with the citrate/gold(III) ratio, both displaying plateaux that evolve inversely to Au3+] at larger ratios. Conversely, for Au3+]>= 0.8 mM, the size and size distribution of the synthesized gold nanoparticles continuously rise as the citrate/gold(III) ratio is increased. A starting gold salt concentration of 0.6 mM leads to the formation of the most monodisperse gold nanoparticles (polydispersity index<0.1) for a wide range of citrate/gold(III) molar ratios (from 4:1 to 18:1). Via a model for the formation of gold nanoparticles by the citrate method, the experimental trends in size could be qualitatively predicted:the simulations showed that the destabilizing effect of increased electrolyte concentration at high initial Au3+] is compensated by a slight increase in zeta potential of gold nanoparticles to produce concentrated dispersion of gold nanoparticles of small sizes.

Preparation and characterization chemistry of nano-crystalline Ni-Cu-Zn ferrite

In submitted research; nanocrystalline powders having elements Ni0.5Cu0.25Zn0.25Fe2 xInxO4 with varied amounts of indium ( x = 0.0, 0.1, 0.2, 0.3 and 0.4) were grown-up by modified citrate to nitrate alchemy. The realism of single phase cubic spinel creation of the synthesized ferrite samples was studied by the DTA-TGA, XRD, SEM, EDX, FT-IR, VSM and dielectric measurements. SEM was applied to inspect the morphological variations and EDX was used to determine the compositional mass ratios. The studies on the dielectric constant (epsilon'), dielectric loss (epsilon `'), loss tangent (tan delta), ac conductivity (sigma(ac)), resistive and reactive parts of the impedance analysis (Z' and Z `') at room temperature were also carried out. The saturation magnetizations (Ms) were determined using the vibrating sample magnetometer (VSM). Ms. decreased with the increase In3+ doping content, as Fe3+ of 5(mu B) ions are replaced by In3+ of 5 mu(B) ions. (C) 2012 Elsevier B. V. All rights reserved.

Rapid synthesis and characterization of an oxygen-deficient defect perovskite La4BaCu5O13+d phase

Oxygen-deficient defect perovskite La4BaCu5O13+d phase has been synthesized by the nitrate-citrate gel combustion method at 950 C for 2 h. Structural parameters were refined by the Rietveld refinement method using room-temperature powder XRD data. The La4BaCu5O13+d crystallizes in the tetragonal structure with space group P4/m (no. 83) and having the lattice parameters a=8.6508 c=3.8606 (1) Å and (2) Å, respectively. Oxygen content was determined by the iodometric titration. Low-temperature resistivity result reveals that La4BaCu5O13+d compound exhibit metallic behavior up to 15 K.

Overexpression of the Rv0805 phosphodiesterase elicits a cAMP-independent transcriptional response

The Rv0805 gene in Mycobacterium tuberculosis encodes a metallophosphoesterase which shows cAMP-hydrolytic activity. Overexpression of Rv0805 has been used as a tool to lower intracellular cAMP levels and thereby elucidate the roles of cAMP in mycobacteria. Here we show that levels of cAMP in M. tuberculosis were lowered by only similar to 30% following overexpression of Rv0805, and transcript levels of a number of genes, which include those associated with virulence and the methyl citrate cycle, were altered. The genes that showed altered expression were distinct from those differentially regulated in a strain deleted for the cAMP-receptor protein (CRPMt), consistent with the relatively low dependence on cAMP of CRPMt binding to DNA. Using mutants of Rv0805 we show that the transcriptional signature of Rv0805 overexpression is a combination of catalysis-dependent and independent effects, and that the structurally flexible C-terminus of Rv0805 is crucial for the catalysis-independent effects of the protein. Our study demonstrates the dissociation of Rv0805 and cAMP-regulated gene expression, and reveals alternate functions for this phosphodiesterase from M. tuberculosis.

Role of electrolyte chemistry on electronic and in vitro electrochemical properties of micro-arc oxidized titania films on Cp Ti

The primary objective of the present work was to study the electronic and in vitro electrochemical properties of micro-arc oxidized titania films on Cp Ti, fabricated independently in various electrolyte solutions consisting of anions such as phosphate (PO43-), borate (B4O72-), citrate (C6H5O73-) and silicate (SiO32-). Further the role of anions on the structural, morphological and compositional properties of the fabricated films was studied. All the titania films were developed by micro-arc oxidation (MAO) technique for a fixed treatment time of 8 min under constant current mode. The surface morphology, elemental distribution, composition and structural characteristics of the films were assessed by scanning electron microscope (SEM) equipped with energy dispersive spectroscopy (EDS) and X-ray diffraction (XRD) techniques. The thermodynamic and kinetic corrosion properties of the films were studied under simulated body fluid (SBF) conditions (pH 7.4 and 37 degrees C) by conducting chronopotentiometric and potentiodynamic polarization tests. Electrochemical impedance spectroscopy (EIS) coupled with equivalent circuit modelling was carried out to analyse the frequency response and Mott-Schottky analysis was performed to study the semiconducting (electronic) properties of the films. Salt spray fog accelerated corrosion test was conducted for 168h as per ASTM B117 standard to corroborate the corrosion and semiconducting properties of the samples based on the visual examination. The XRD results showed that the transformation from the metastable anatase phase to the thermodynamically stable rutile phase and the crystalline growth of the respective phases were strongly influenced by the addition of anions. The SEM-EDS results demonstrated that the phosphorous (P) content in the films varied from 2.4 at% to 5.0 at% indicating that the amount of P in the films could be modified by adding an appropriate electrolyte additive. The electrochemical corrosion test results showed that the film fabricated in citrate (C6H5O73-) containing electrolyte is thermodynamically and kinetically more stable compared to that of all the others. The results of the Mott-Schottky analysis indicated that all the fabricated films showed an n-type semiconducting behaviour and the film developed in citrate (C6H5O73-) containing electrolyte exhibited the lowest donor concentration and the most negative flat band potential that contributed to its highest corrosion resistance in SBF solution. The results of the salt spray accelerated corrosion tests were in agreement with those obtained from the electrochemical and Mott-Schottky analysis.

Interaction of Silver Nanoparticles with Serum Proteins Affects Their Antimicrobial Activity In Vivo

The emergence of multidrug-resistant bacteria is a global threat for human society. There exist recorded data that silver was used as an antimicrobial agent by the ancient Greeks and Romans during the 8th century. Silver nanoparticles (AgNPs) are of potential interest because of their effective antibacterial and antiviral activities, with minimal cytotoxic effects on the cells. However, very few reports have shown the usage of AgNPs for antibacterial therapy in vivo. In this study, we deciphered the importance of the chosen methods for synthesis and capping of AgNPs for their improved activity in vivo. The interaction of AgNPs with serum albumin has a significant effect on their antibacterial activity. It was observed that uncapped AgNPs exhibited no antibacterial activity in the presence of serum proteins, due to the interaction with bovine serum albumin (BSA), which was confirmed by UV-Vis spectroscopy. However, capped AgNPs with citrate or poly(vinylpyrrolidone)] exhibited antibacterial properties due to minimized interactions with serum proteins. The damage in the bacterial membrane was assessed by flow cytometry, which also showed that only capped AgNPs exhibited antibacterial properties, even in the presence of BSA. In order to understand the in vivo relevance of the antibacterial activities of different AgNPs, a murine salmonellosis model was used. It was conclusively proved that AgNPs capped with citrate or PVP exhibited significant antibacterial activities in vivo against Salmonella infection compared to uncapped AgNPs. These results clearly demonstrate the importance of capping agents and the synthesis method for AgNPs in their use as antimicrobial agents for therapeutic purposes.