Remarkable visible light-triggered cytotoxicity of mitochondria targeting mixed-ligand cobalt(III) complexes of curcumin and phenanthroline bases binding to human serum albumin

Six new mixed-ligand cobalt(III) complexes of formulation Co(N-N)(2)(O-O)](ClO4)(2) (1-6), where N-N is a N,N-donor phenanthroline base, namely, 1,10-phenanthroline (phen in 1, 2), dipyrido3,2-d:2',3'-f] quinoxaline (dpq in 3, 4), and dipyrido3,2-a:2',3'-c]phenazine (dppz in 5, 6), O-O is acetylacetonate (acac in 1, 3, 5) or curcumin (bis(4-hydroxy-3-methoxyphenyl)-1,6-diene-3,5-dione, cur in 2, 4, 6), have been synthesized and characterized. The X-ray crystal structures of complex 1 (as PF6- salt, 1a) and 3 show distorted octahedral geometries formed by the CoN4O2 core. The complexes 1, 3 and 5 having the simple acac ligand are prepared as control species to understand the role of curcumin. The optimized geometries and the frontier orbitals of the curcumin complexes 2, 4, and 6 are obtained from the DFT calculations. The complexes 2, 4, and 6 having the photoactive curcumin moiety display an absorption band in the visible region near 420 nm and show remarkable photocytotoxicity in HeLa cancer cells with respective IC50 values of 7.4 mu M, 5.1 mu M and 1.6 mu M while being much less toxic in dark. MTT assay using complex 6 shows that it is not significantly photocytotoxic to MCF-10A normal cells. The control complexes having the acac ligand are non-toxic both in the presence and absence of light. The cell death is apoptotic in nature and triggered by the photogeneration of reactive oxygen species. Fluorescence imaging experiments on HeLa cells reveals that complex 6 accumulated primarily inside the mitochondria. Human serum albumin (HSA) binding experiments show that the complexes bind HSA with good affinity, but 6 binds with the highest affinity, with a K-b value of 9.8 x 10(5) M-1. Thus, complex 6 with its negligible toxicity in the dark and in normal cells but remarkable toxicity in visible light holds significant photochemotherapeutic potential.

Water Dispersible, Positively and Negatively Charged MoS2 Nanosheets: Surface Chemistry and the Role of Surfactant Binding

Stable aqueous dispersions of atomically thin layered MoS2 nanosheets have been obtained by sonication in the presence of ionic surfactants. The dispersions are stabilized by electrostatic repulsion between the sheets, and we show that the sign of the charge on the MoS2 nanosheets, either positive or negative, can be can be controlled by the choice of the surfactant. Using techniques from solution NMR, we show that the surfactant chains are weakly bound to the MoS2 sheets and undergo rapid exchange with free surfactant chains present in the dispersion. In situ nuclear Overhauser effect spectroscopic measurements provide direct evidence that the surfactant chains lie flat, arranged randomly on the basal plane of the MoS2 nanosheets with their charged headgroup exposed. These results provide a chemical perspective for understanding the stability of these inorganic nanosheets in aqueous dispersions and the origin of the charge on the sheets.

Remarkable enhancement in photocytotoxicity and hydrolytic stability of curcumin on binding to an oxovanadium(IV) moiety

Oxovanadium(IV) complexes of polypyridyl and curcumin-based ligands, viz. VO(cur)(L)Cl] (1, 2) and VO(scur)(L)Cl] (3, 4), where L is 1,10-phenanthroline (phen in 1 and 3), dipyrido3,2-a:2',3'-c]phenazine (dppz in 2 and 4), Hcur is curcumin and Hscur is diglucosylcurcumin, were synthesized and characterized and their cellular uptake, photocytotoxicity, intracellular localization, DNA binding, and DNA photo-cleavage activity studied. Complex VO(cur)(phen)Cl] (1) has (VN2O3Cl)-N-IV distorted octahedral geometry as evidenced from its crystal structure. The sugar appended complexes show significantly higher uptake into the cancer cells compared to their normal analogues. The complexes are remarkably photocytotoxic in visible light (400-700 nm) giving an IC50 value of <5 mu M in HeLa, HaCaT and MCF-7 cells with no significant dark toxicity. The green emission of the complexes was used for cellular imaging. Predominant cytosolic localization of the complexes 1-4 to a lesser extent into the nucleus was evidenced from confocal imaging. The complexes as strong binders of calf thymus DNA displayed photocleavage of supercoiled pUC19 DNA in red light by generating (OH)-O-center dot radicals as the ROS. The cell death is via an apoptotic pathway involving the ROS. Binding to the VO2+ moiety has resulted in stability against any hydrolytic degradation of curcumin along with an enhancement of its photocytotoxicity.

Series-connected substrate-integrated lead-carbon hybrid ultracapacitors with voltage-management circuit

Cell voltage for a fully charged-substrate-integrated lead-carbon hybrid ultracapacitor is about 2.3 V. Therefore, for applications requiring higher DC voltage, several of these ultracapacitors need to be connected in series. However, voltage distribution across each series-connected ultracapacitor tends to be uneven due to tolerance in capacitance and parasitic parallel-resistance values. Accordingly, voltage-management circuit is required to protect constituent ultracapacitors from exceeding their rated voltage. In this study, the design and characterization of the substrate-integrated lead-carbon hybrid ultracapacitor with co-located terminals is discussed. Voltage-management circuit for the ultracapacitor is presented, and its effectiveness is validated experimentally.

Influence of substrate temperature on structure, microstructure and magnetic properties of sputtered Fe-Ga thin films

This paper reports the structure, microstructure and magnetic properties of Fe-Ga thin films deposited using DC magnetron sputtering technique on Si(100) substrate kept at different temperatures. Structural studies employing X-ray diffraction and TEM revealed the presence of only disordered A2 phase in the film. Columnar growth of nanocrystalline grains from the substrate was observed in the film deposited at room temperature. With increase in substrate temperature the grain size as well as surface roughness was found to increase. The magnetization of the films deposited at higher substrate temperatures were Found to saturate at lower magnetic held as compared to the room temperature deposited Film. Coercivity was found to decrease with increasing substrate temperature upto a minimum value of similar to 2 Oe for the film deposited at 450 degrees C and with further increase in substrate temperature it was found to increase. A maximum magnetostriction of 200 mu-strains was also observed for the film deposited at 450 degrees C. (C) 2015 Elsevier B.V. All rights reserved

Magnetic field assisted stem cell differentiation - role of substrate magnetization in osteogenesis

Among the multiple modulatory physical cues explored to regulate cellular processes, the potential of magneto-responsive substrates in magnetic field stimulated stem cell differentiation is still unperceived. In this regard, the present work demonstrates how an external magnetic field can be applied to direct stem cell differentiation towards osteogenic commitment. A new culture methodology involving periodic delivery of 100 mT static magnetic field (SMF) in combination with HA-Fe3O4 magnetic substrates possessing a varying degree of substrate magnetization was designed for the study. The results demonstrate that an appropriate combination of weakly ferromagnetic substrates and SMF exposure enhanced cell viability, DNA synthesis and caused an early switchover to osteogenic lineage as supported by Runx2 immunocytochemistry and ALP expression. However, the mRNA expression profile of early osteogenic markers (Runx2, ALP, Col IA) was comparable despite varying substrate magnetic properties (diamagnetic to ferromagnetic). On the contrary, a remarkable upregulation of late bone development markers (OCN and OPN) was explicitly detected on weak and strongly ferromagnetic substrates. Furthermore, SMF induced matrix mineralization with elevated calcium deposition on similar substrates, even in the absence of osteogenic supplements. More specifically, the role of SMF in increasing intracellular calcium levels and in inducing cell cycle arrest at G0/G1 phase was elucidated as the major molecular event triggering osteogenic differentiation. Taken together, the above results demonstrate the competence of magnetic stimuli in combination with magneto-responsive biomaterials as a potential strategy for stem cell based bone tissue engineering.

Cyclic nucleotide binding and structural changes in the isolated GAF domain of Anabaena adenylyl cyclase, CyaB2

GAF domains are a large family of regulatory domains, and a subset are found associated with enzymes involved in cyclic nucleotide (cNMP) metabolism such as adenylyl cyclases and phosphodiesterases. CyaB2, an adenylyl cyclase from Anabaena, contains two GAF domains in tandem at the N-terminus and an adenylyl cyclase domain at the C-terminus. Cyclic AMP, but not cGMP, binding to the GAF domains of CyaB2 increases the activity of the cyclase domain leading to enhanced synthesis of cAMP. Here we show that the isolated GAFb domain of CyaB2 can bind both cAMP and cGMP, and enhanced specificity for cAMP is observed only when both the GAFa and the GAFb domains are present in tandem(GAFab domain). In silico docking and mutational analysis identified distinct residues important for interaction with either cAMP or cGMP in the GAFb domain. Structural changes associated with ligand binding to the GAF domains could not be detected by bioluminescence resonance energy transfer (BRET) experiments. However, amide hydrogen-deuterium exchange mass spectrometry (HDXMS) experiments provided insights into the structural basis for cAMP-induced allosteric regulation of the GAF domains, and differences in the changes induced by cAMP and cGMP binding to the GAF domain. Thus, our findings could allow the development of molecules that modulate the allosteric regulation by GAF domains present in pharmacologically relevant proteins.

Structural basis for the catalytic mechanism of homoserine dehydrogenase

Homoserine dehydrogenase (HSD) is an oxidoreductase in the aspartic acid pathway. This enzyme coordinates a critical branch point of the metabolic pathway that leads to the synthesis of bacterial cell-wall components such as L-lysine and m-DAP in addition to other amino acids such as L-threonine, L-methionine and L-isoleucine. Here, a structural rationale for the hydride-transfer step in the reaction mechanism of HSD is reported. The structure of Staphylococcus aureus HSD was determined at different pH conditions to understand the basis for the enhanced enzymatic activity at basic pH. An analysis of the crystal structure revealed that Lys105, which is located at the interface of the catalytic and cofactor-binding sites, could mediate the hydride-transfer step of the reaction mechanism. The role of Lys105 was subsequently confirmed by mutational analysis. Put together, these studies reveal the role of conserved water molecules and a lysine residue in hydride transfer between the substrate and the cofactor.

Calcium binding properties of calcium dependent protein kinase 1 (CaCDPK1) from Cicer arietinum

Calcium plays a crucial role as a secondary messenger in all aspects of plant growth, development and survival. Calcium dependent protein kinases (CDPKs) are the major calcium decoders, which couple the changes in calcium level to an appropriate physiological response. The mechanism by which calcium regulates CDPK protein is not well understood. In this study, we investigated the interactions of Ca2+ ions with the CDPK1 isoform of Cicer arietinum (CaCDPK1) using a combination of biophysical tools. CaCDPK1 has four different EF hands as predicted by protein sequence analysis. The fluorescence emission spectrum of CaCDPK1 showed quenching with a 5 nm red shift upon addition of calcium, indicating conformational changes in the tertiary structure. The plot of changes in intensity against calcium concentrations showed a biphasic curve with binding constants of 1.29 mu M and 120 mu M indicating two kinds of binding sites. Isothermal calorimetric (ITC) titration with CaCl2 also showed a biphasic curve with two binding constants of 0.027 mu M and 1.7 mu M. Circular dichroism (CD) spectra showed two prominent peaks at 208 and 222 nm indicating that CaCDPK1 is a alpha-helical rich protein. Calcium binding further increased the alpha-helical content of CaCDPK1 from 75 to 81%. Addition of calcium to CaCDPK1 also increased fluorescence of 8-anilinonaphthalene-1-sulfonic acid (ANS) indicating exposure of hydrophobic surfaces. Thus, on the whole this study provides evidence for calcium induced conformational changes, exposure of hydrophobic surfaces and heterogeneity of EF hands in CaCDPK1. (C) 2015 Elsevier GmbH. All rights reserved.

Molecular and Functional Characterization of RecD, a Novel Member of the SF1 Family of Helicases, from Mycobacterium tuberculosis

Background: The heterotrimeric M. tuberculosis RecBCD complex, or each of its individual subunits, remains uncharacterized. Results: MtRecD exists as a homodimer in solution, catalyzes ssDNA-dependent ATP hydrolysis, unwinding of DNA replication/recombination intermediates, and interacts with RecA. Conclusion: MtRecD possesses strong 5 3- and weak 3 5-helicase activities. Significance: These findings provide insights into the mechanism underlying DSB repair and homologous recombination in mycobacteria. The annotated whole-genome sequence of Mycobacterium tuberculosis revealed the presence of a putative recD gene; however, the biochemical characteristics of its encoded protein product (MtRecD) remain largely unknown. Here, we show that MtRecD exists in solution as a stable homodimer. Protein-DNA binding assays revealed that MtRecD binds efficiently to single-stranded DNA and linear duplexes containing 5 overhangs relative to the 3 overhangs but not to blunt-ended duplex. Furthermore, MtRecD bound more robustly to a variety of Y-shaped DNA structures having 18-nucleotide overhangs but not to a similar substrate containing 5-nucleotide overhangs. MtRecD formed more salt-tolerant complexes with Y-shaped structures compared with linear duplex having 3 overhangs. The intrinsic ATPase activity of MtRecD was stimulated by single-stranded DNA. Site-specific mutagenesis of Lys-179 in motif I abolished the ATPase activity of MtRecD. Interestingly, although MtRecD-catalyzed unwinding showed a markedly higher preference for duplex substrates with 5 overhangs, it could also catalyze significant unwinding of substrates containing 3 overhangs. These results support the notion that MtRecD is a bipolar helicase with strong 5 3 and weak 3 5 unwinding activities. The extent of unwinding of Y-shaped DNA structures was approximate to 3-fold lower compared with duplexes with 5 overhangs. Notably, direct interaction between MtRecD and its cognate RecA led to inhibition of DNA strand exchange promoted by RecA. Altogether, these studies provide the first detailed characterization of MtRecD and present important insights into the type of DNA structure the enzyme is likely to act upon during the processes of DNA repair or homologous recombination.

Optoelectronic Properties of Graphene on Silicon Substrate: Effect of Defects in Graphene

Engineering of electronic energy band structure in graphene based nanostructures has several potential applications. Substrate induced bandgap opening in graphene results several optoelectronic properties due to the inter-band transitions. Various defects like structures, including Stone-Walls and higher-order defects are observed when a graphene sheet is exfoliated from graphite and in many other growth conditions. Existence of defect in graphene based nanostructures may cause changes in optoelectronic properties. Defect engineered graphene on silicon system are considered in this paper to study the tunability of optoelectronic properties. Graphene on silicon atomic system is equilibrated using molecular dynamics simulation scheme. Based on this study, we confirm the existence of a stable super-lattice. Density functional calculations are employed to determine the energy band structure for the super-lattice. Increase in the optical energy bandgap is observed with increasing of order of the complexity in the defect structure. Optical conductivity is computed as a function of incident electromagnetic energy which is also increasing with increase in the defect order. Tunability in optoelectronic properties will be useful in understanding graphene based design of photodetectors, photodiodes and tunnelling transistors.

Functional characterization of heat-shock protein 90 from Oryza sativa and crystal structure of its N-terminal domain

Heat-shock protein 90 (Hsp90) is an ATP-dependent molecular chaperone that is essential for the normal functioning of eukaryotic cells. It plays crucial roles in cell signalling, cell-cycle control and in maintaining proteome integrity and protein homeostasis. In plants, Hsp90s are required for normal plant growth and development. Hsp90s are observed to be upregulated in response to various abiotic and biotic stresses and are also involved in immune responses in plants. Although there are several studies elucidating the physiological role of Hsp90s in plants, their molecular mechanism of action is still unclear. In this study, biochemical characterization of an Hsp90 protein from rice (Oryza sativa; OsHsp90) has been performed and the crystal structure of its N-terminal domain (OsHsp90-NTD) was determined. The binding of OsHsp90 to its substrate ATP and the inhibitor 17-AAG was studied by fluorescence spectroscopy. The protein also exhibited a weak ATPase activity. The crystal structure of OsHsp90-NTD was solved in complex with the nonhydrolyzable ATP analogue AMPPCP at 3.1 angstrom resolution. The domain was crystallized by cross-seeding with crystals of the N-terminal domain of Hsp90 from Dictyostelium discoideum, which shares 70% sequence identity with OsHsp90-NTD. This is the second reported structure of a domain of Hsp90 from a plant source.

The crystal structure of a lectin from Butea monosperma: Insight into its glycosylation and binding of ligands

Crystal structure of a lectin purified from Butea monosperma seeds was determined by Molecular Replacement method. Its primary structure was determined by Tandem Mass Spectroscopy and electron density maps from X-ray diffraction data. Its quaternary structure was tetrameric, formed of two monomers, alpha and beta, beta appearing as truncated alpha. The occurrence of two tetramers in the asymmetric unit of the crystal might be a consequence of asymmetric contacts due to difference in glycosylation and variable loops structures, to form an `octamer-structure'. The crystal structure showed binding pockets for gamma Abu, having a proposed role in plant defense, at the interface of canonical dimer-partners. Hemagglutination studies, enzyme kinetics, isothermal titration calorimetry and molecular dynamics showed that the lectin is specific to N-acetyl D-galactosamine, galactose and lactose in decreasing order, and alpha-amylase inhibitor. (C) 2014 Elsevier B.V. All rights reserved.

Effects of Patterned Substrate on Thin Films Simulated by Family Model

Patterned substrate growth has been a subject of much interest. In this work, characteristics of some statistical properties of a film grown on triangular and vicinal substrates using the Family model are studied. Substrate size and tilt angle are varied. It is found that the interface width and the correlation function increase as the roughness of the pattern is increased. The new scaling exponents are calculated and anomalous scaling is obtained. The transient persistence probability does not show a power law relation when the initial surface is sufficiently rough. The initial rough surface also causes multifractal behavior in the model.