A novel mode of control of nickel uptake by a multifunctional metallochaperone.

Cellular metal homeostasis is a critical process for all organisms, requiring tight regulation. In the major pathogen Helicobacter pylori, the acquisition of nickel is an essential virulence determinant as this metal is a cofactor for the acid-resistance enzyme, urease. Nickel uptake relies on the NixA permease and the NiuBDE ABC transporter. Till now, bacterial metal transporters were reported to be controlled at their transcriptional level. Here we uncovered post-translational regulation of the essential Niu transporter in H. pylori. Indeed, we demonstrate that SlyD, a protein combining peptidyl-prolyl isomerase (PPIase), chaperone, and metal-binding properties, is required for the activity of the Niu transporter. Using two-hybrid assays, we found that SlyD directly interacts with the NiuD permease subunit and identified a motif critical for this contact. Mutants of the different SlyD functional domains were constructed and used to perform in vitro PPIase activity assays and four different in vivo tests measuring nickel intracellular accumulation or transport in H. pylori. In vitro, SlyD PPIase activity is down-regulated by nickel, independently of its C-terminal region reported to bind metals. In vivo, a role of SlyD PPIase function was only revealed upon exposure to high nickel concentrations. Most importantly, the IF chaperone domain of SlyD was shown to be mandatory for Niu activation under all in vivo conditions. These data suggest that SlyD is required for the active functional conformation of the Niu permease and regulates its activity through a novel mechanism implying direct protein interaction, thereby acting as a gatekeeper of nickel uptake. Finally, in agreement with a central role of SlyD, this protein is essential for the colonization of the mouse model by H. pylori.

Experimental and computational interaction studies of terbium (III) and lanthanide (III) complexes containing 2,2'-bipyridine with bovine serum albumin and their in vitro anticancer and antimicrobial activities.

To investigate the chemotherapeutic and pharmacokinetic aspects of two lanthanide complexes (Tb(III) and La(III) containing 2,2'-bipyridine ligand), in vitro binding studies were carried out with BSA by employing multiple biophysical methods and molecular modeling study. There are different techniques containing fluorescence, absorption spectroscopy and competitive experiments to determine the interaction mode between BSA and these complexes. These complexes efficiently quenched the BSA emission through a static procedure. The results showed that the terbium and lanthanum complexes exhibited a high propensity for BSA interaction via van der Waals force. Further, competitive examination and docking study showed that the interaction site of these complexes on BSA is site III. The results of docking calculations were in good agreement with experimental examinations. Also, the energy transfer from BSA to these complexes has happened with high possibility. Moreover, antimicrobial studies of different bacterial and fungi indicated its promising antibacterial activity. In vitro cytotoxicity of the Tb complex and La complex was carried out in MCF-7 and A-549 cell lines, which revealed significantly good activity.Communicated by Ramaswamy H. Sarma.

Evaluation of parent and nano-encapsulated terbium(III) complex toward its photoluminescence properties, FS-DNA, BSA binding affinity, and biological applications.

BACKGROUND: There is a crucial need for finding and developing new compounds as the anticancer and antimicrobial agents with better activity, specific target, and less toxic side effects. OBJECTIVES: Base on the potential anticancer properties of lanthanide complexes, in the paper, the biological applications of terbium (Tb) complex, containing 2,9-dimethyl- 1,10-phenanthroline (Me2Phen) such as anticancer, antimicrobial, DNA cleavage ability, the interaction with FS-DNA (Fish-Salmon DNA) and BSA (Bovine Serum Albumin) was examined. METHODS: The interaction of Tb-complex with BSA and DNA was studied by emission spectroscopy, absorption titration, viscosity measurement, CD spectroscopy, competitive experiments, and docking calculation. Also, the ability of this complex to cleave DNA was reported by gel electrophoresis. Tb-complex was concurrently screened for its antibacterial activities by different methods. Besides, the nanocarriers of Tb-complex (lipid nanoencapsulation (LNEP) and the starch nanoencapsulation (SNEP)), as active anticancer candidates, were prepared. MTT technique was applied to measure the antitumor properties of these compounds on human cancer cell lines. RESULTS: The experimental and docking results suggest significant binding between DNA as well as BSA with terbium-complex. Besides, groove binding plays the main role in the binding of this compound with DNA and BSA. The competitive experiment with hemin demonstrated that the terbium complex was bound at site III of BSA, which was confirmed by the docking study. Also, Tb-complex was concurrently screened for its DNA cleavage, antimicrobial, and anticancer activities. The anticancer properties of LNEP and SNEP are more than the terbium compound. CONCLUSIONS: Tb-complex can bond to DNA/BSA with high binding affinity. Base on biological applications of Tb-complex, it can be concluded that this complex and its nanocarriers can suggest as novel anticancer, antimicrobial candidates.

Parent and nano-encapsulated ytterbium(iii) complex toward binding with biological macromolecules, in vitro cytotoxicity, cleavage and antimicrobial activity studies.

To determine the chemotherapeutic and pharmacokinetic aspects of an ytterbium complex containing 2,9-dimethyl-1,10-phenanthroline (Me2Phen), in vitro binding studies were carried out with FS-DNA/BSA by employing multiple biophysical methods and a molecular modeling study. There are different techniques including absorption spectroscopy, fluorescence spectroscopy, circular dichroism studies, viscosity experiments (only in the case of DNA), and competitive experiments used to determine the interaction mode between DNA/BSA and the ytterbium-complex. The results showed that the Yb-complex exhibited a high propensity for the interaction of BSA and DNA via hydrophobic interactions and van der Waals forces. Further, a competitive examination and docking study showed that the interaction site of the ytterbium complex on BSA is site III. The results of docking calculations for DNA/BSA were in good agreement with experimental findings. The complex displays efficient DNA cleavage in the presence of hydrogen peroxide. Moreover, antimicrobial studies of different bacteria and fungi indicated its promising antibacterial activity. In vitro cytotoxicity studies of the Yb-complex, starch nano-encapsulated, and lipid nano-encapsulated were carried out in MCF-7 and A-549 cell lines, which revealed significantly good activity. The results of anticancer activity studies showed that the cytotoxic activity of the Yb-complex was increased when encapsulated with nanocarriers. Based on biological applications of the Yb-complex, it can be concluded that this complex and its nanocarriers can act as novel anticancer and antimicrobial candidates.

Bimodal Nickel-Binding Site on Escherichia coli [NiFe]-Hydrogenase Metallochaperone HypA.

[NiFe]-hydrogenase enzymes catalyze the reversible oxidation of hydrogen at a bimetallic cluster and are used by bacteria and archaea for anaerobic growth and pathogenesis. Maturation of the [NiFe]-hydrogenase requires several accessory proteins to assemble and insert the components of the active site. The penultimate maturation step is the delivery of nickel to a primed hydrogenase enzyme precursor protein, a process that is accomplished by two nickel metallochaperones, the accessory protein HypA and the GTPase HypB. Recent work demonstrated that nickel is rapidly transferred to HypA from GDP-loaded HypB within the context of a protein complex in a nickel selective and unidirectional process. To investigate the mechanism of metal transfer, we examined the allosteric effects of nucleotide cofactors and partner proteins on the nickel environments of HypA and HypB by using a combination of biochemical, microbiological, computational, and spectroscopic techniques. We observed that loading HypB with either GDP or a nonhydrolyzable GTP analogue resulted in a similar nickel environment. In addition, interaction with a mutant version of HypA with disrupted nickel binding, H2Q-HypA, does not induce substantial changes to the HypB G-domain nickel site. Instead, the results demonstrate that HypB modifies the acceptor site of HypA. Analysis of a peptide maquette derived from the N-terminus of HypA revealed that nickel is predominately coordinated by atoms from the N-terminal Met-His motif. Furthermore, HypA is capable of two nickel-binding modes at the N-terminus, a HypB-induced mode and a binding mode that mirrors the peptide maquette. Collectively, these results reveal that HypB brings about changes in the nickel coordination of HypA, providing a mechanism for the HypB-dependent control of the acquisition and release of nickel by HypA.

Complex formation between the Escherichia coli [NiFe]-hydrogenase nickel maturation factors.

The biosynthesis of the dinuclear metal cluster at the active sites of the [NiFe]-hydrogenase enzymes is a multi-step process executed by a suite of accessory proteins. Nickel insertion during maturation of Escherichia coli [NiFe]-hydrogenase 3 is achieved by the metallochaperones HypA, SlyD and the GTPase HypB, but how these proteins cooperate to ensure nickel delivery is not known. In this study, the complexes formed between the individual purified proteins were examined by using several methods. Size exclusion chromatography (SEC) indicated that SlyD and HypB interact primarily in a 1:1 complex. The affinity of HypB-SlyD was measured by using surface plasmon resonance, which revealed a KD of 24 ± 10 nM in the absence of nucleotide and an interaction several fold tighter in the presence of GDP. A ternary complex between all three proteins was not detected, and instead SlyD blocked the interaction of HypA with HypB in competitive binding experiments. Furthermore, cross-linking experiments suggest a weak interaction between HypA and SlyD, which is not detectable by SEC. Electrochemical analysis confirmed each of the pairwise interactions and that the relative affinities of these complexes are on the order of HypB-SlyD > HypB-HypA > HypA-SlyD. These results indicate a hierarchy of interactions, as opposed to a single multiprotein complex, and provide insight into the nickel delivery process during hydrogenase enzyme maturation.

In vitro cytotoxicity studies of parent and nanoencapsulated Holmium-2,9-dimethyl-1,10-phenanthroline complex toward fish-salmon DNA-binding properties and antibacterial activity.

In this study, the interaction of Holmium (Ho) complex including 2, 9-dimethyl-1,10-phenanthroline, also called Neocuproine (Neo), [Ho(Neo)2Cl3.H2O], as fluorescence probe with fish-salmon DNA (FS-DNA) is studied during experimental investigations. Multi-spectroscopic methods are utilized to determine the affinity binding constants (Kb) of complex-FS-DNA. It is found that fluorescence of Ho complex is strongly quenched by the FS-DNA through a static quenching procedure. Under optimal conditions in Tris(trishydroxymethyl-aminomethane)-HCl buffer at 25 °C with pH ≈ 7.2, intrinsic binding constant Kb of Ho complex is 6.12 ± 0.04 × 105 M-1. Also, the binding site number and Stern-Volmer quenching constant are calculated. There are different approaches, including iodide quenching assay, salt effect and thermodynamical assessment to determine the features of the binding mode between Ho complex and FS-DNA. Also, the parent and starch and lipid nanoencapsulated Ho complex, as potent antitumor candidates, were synthesized. The main structure of Ho complex is maintained after encapsulation using starch and lipid nanoparticles. 3-[4,5-Dimethylthiazole-2-yl]-2,5-diphenyltetrazolium bromide (MTT) method was used to assess the anticancer properties of Ho complex and its encapsulated forms on human cancer cell lines of human lung carcinoma cell line and breast cancer cell line. In conclusion, these compounds could be considered as new antitumor candidates. Communicated by Ramaswamy H. Sarma.

Simultaneous and sensitive determination of a quaternary mixture of AA, DA, UA and Trp using a modified GCE by iron ion-doped natrolite zeolite-multiwall carbon nanotube.

The evaluation of a novel modified glassy carbon electrode modified with iron ion-doped natrolite zeolite-multiwalled carbon nanotube for the simultaneous and sensitive determination of ascorbic acid (AA), dopamine (DA), uric acid (UA) and tryptophan (Trp) has been described. The measurements were carried out using cyclic voltammetry in buffer solution with pH 1. This modified electrode exhibits potent and persistent electroxidation behavior followed by well-separated oxidation peaks towards AA, DA, UA and Trp with increasing of the oxidation current. For the quaternary mixture containing AA, DA, UA and Trp, the 4 compounds can well separate from each other at the scan rate of 100 mVs(-1) with a potential difference of 270 mV, 150 mV and 260 mV for the oxidation peak potentials of AA-DA, DA-UA and UA-Trp, respectively, which was large enough to simultaneous determine AA, DA, UA and Trp. The catalytic peak current obtained, was linearly dependent on the AA, DA, UA and Trp concentrations in the range of 7.77-833 µM, 7.35-833 µM, 0.23-83.3 µM and 0.074-34.5 µM and the detection limits for AA, DA, UA and Trp were 1.11, 1.05, 0.033 and 0.011 µM, respectively. The analytical performance of this sensor has been evaluated for simultaneous detection of AA, DA, UA and Trp in human serum and urine samples.

Cyanide uptake from wastewater by modified natrolite zeolite-iron oxyhydroxide system: application of isotherm and kinetic models.

A method for the removal of cyanides from wastewater is described. The method involves the adsorption of cyanides by a modified natural zeolite (natrolite) using batch technique. A new iron oxyhydroxide-natrolite system was used in this study. A combination of XRD, XRF and FTIR spectroscopies, as well as TG/DSC thermal analyses was used for characterization of zeolitic materials. Effects of parameters such as pH, amount of adsorbent and contact time on the cyanide removing yield are studied. It was observed that the yield increases by increasing dosage of adsorbent and contact time at a fixed pH 7.5. A yield of 82% was achieved at optimum conditions for removing cyanide from industrial wastewaters. The experimental data obtained for optimum conditions were selected for modeling the adsorption behavior of the materials using six isotherm equations (Freundlich, Langmuir, Langmuir-Freundlich, Dubinin-Radushkevich, Redlich-Peterson and Toth). The obtained modeling results indicated that, although the three-parameter models, taking into account the surface heterogeneity, provided the closest approach to the measurement data, the parameters estimates could be highly biased. The kinetic studies proved that the second-order kinetic was the applicable model.

Pneumatic flow injection analysis-tandem spectrometer system for iron speciation.

A pneumatic flow injection-tandem spectrometer system, without a delivery pump was used for the speciation of iron. In this system, the suction force of a pneumatic nebulizer of a flame absorption spectrometer was used for solution delivery through the manifold. The Fe(III) and total Fe concentrations were determined using thiocyanate ion in a UV-Vis spectrometer and a FAAS, respectively. The Fe(II) was determined by the difference. The calibration curves were linear up to 18 microg mL(-1) and 25 microg mL(-1) with detection limits of 0.09 microg mL(-1) and 0.07 microg mL(-1) for Fe(III) and Fe(II), respectively. The mid-range precision and accuracy were <2.5% and +/-3% for the two species, respectively, at a sampling rate of 120 h(-1). This system was applied for the determination of Fe(III) and Fe(II) in industrial water, natural water and spiked samples.

New class of verdoheme analogues with weakly coordinating anions: the structure of (mu-oxo)bis[(octaethyloxoporphinato)iron(III)] hexafluorophosphate.

Three new verdoheme analogues with weakly coordinating anions, [OEOPFe(II)X], where OEOP is the monoanion of octaethyloxoporphyrin and X = PF(6), ClO(4), and BF(4), have been synthesized and characterized by spectroscopic methods. (1)H NMR spectroscopy reveals that the [OEOPFe(II)X] species are paramagnetic, and the iron is five-coordinate (S = 2). The oxidation of [OEOPFe(II)PF(6)] with dioxygen yields [(OEOPFe)(2)O](PF(6))(2). The structure of (mu-oxo)bis[(octaethyloxoporphinato)iron(III)] has been determined by X-ray diffraction analysis. The eight Fe-N bond distances have an average value of 2.077(3) Angstroms. The oxygen atom sits on the inversion center, and the average axial Fe-O bond length is 1.756(3) Angstroms. The average displacement of the iron(III) atom from the mean porphinato core is 0.60 Angstroms. Crystal data: crystal system, monoclinic; a = 8.7114(10) Angstroms; b = 26.102(4) Angstroms; c = 15.8323(14) Angstroms; beta = 104.134(6) degrees ; space group P2(1)/c; V = 3491.1(7) Angstroms (3); Z = 2; R1 = 0.0546, wR2 =0.1145 for data with I > 2sigma(I).