A mass spectrometric investigation of non-covalent interactions between ruthenium complexes and DNA

Electrospray ionisation mass spectrometry was used to investigate reactions between six ruthenium compounds and three different non self-complementary duplex oligonucleotides containing 16 base pairs. Each of the compounds studied formed non-covalent complexes containing between one and five ruthenium molecules bound to DNA. Competition experiments involving duplex 16mers and pairs of ruthenium compounds were used to determine the order of relative binding affinities of the metal compounds. Other competition experiments involving ruthenium compounds, and the organic DNA binding agents daunomycin and distamycin, provided information about the sites and modes of DNA binding of the ruthenium compounds.     

A comparison of the binding of metal complexes to duplex and quadruplex DNA

Electrospray ionisation mass spectrometry (ESI-MS) and circular dichroism (CD) spectroscopy were used to compare the binding of mononuclear nickel, ruthenium and platinum complexes to double stranded DNA (dsDNA) and quadruplex DNA (qDNA). CD studies provided evidence for the binding of intact complexes of all three metal ions to qDNA. ESI mass spectra of solutions containing platinum or ruthenium complexes and qDNA showed evidence for the formation of non-covalent complexes consisting of intact metal molecules bound to DNA. However, the corresponding spectra of solutions containing nickel complexes principally contained ions consisting of fragments of the initial nickel molecule bound to qDNA. In contrast ESI mass spectra of solutions containing nickel, ruthenium or platinum complexes and dsDNA only showed the presence of ions attributable to intact metal molecules bound to DNA. The fragmentation observed in mass spectral studies of solutions containing nickel complexes and qDNA is attributable to the lower thermodynamic stability of the former metal complexes relative to those containing platinum or ruthenium, as well as the slightly harsher instrumental conditions required to obtain spectra of qDNA. This conclusion is supported by the results of tandem mass spectral studies, which showed that ions consisting of intact nickel complexes bound to qDNA readily undergo fragmentation by loss of one of the ligands initially bound to the metal. The ESI-MS results also demonstrate that the binding affinity of each of the platinum and ruthenium complexes towards qDNA is significantly less than that towards dsDNA.     

Arsenic determination in soils and hair from schools in past mining activity areas in Ron Phibun district,Nakhon Si Thammarat province,Thailand and relationship between soil and hair arsenic

Arsenic (As) in soils and hair collected from schools in Ron Phibun district, Nakhon Si Thammarat province, Thailand, where former tin mining operation were located, was determined by hydride generation atomic absorption spectrophotometry. The relationship between As content in soils and hair with distance from secured landfill was also investigated. Soil and hair samples were collected from 6 schools in summer (February) and rainy season (July). For soils, silt+clay (<45 µm) fraction and sand (45 µm–2 mm) fraction were analyzed. The average concentrations of arsenic in soils during summer (21.70 ± 16.79 mg/kg) and rainy season (22.45 ± 14.17 mg/kg) were at the same concentration level. The average arsenic content in hair samples was 2.24 ± 0.05 mg/kg in rainy season which was higher than 1.05 ± 0.04 mg/kg in summer. It was found that arsenic contents in hair and soils are correlated with the distance from the secured landfill. Most importantly, a positive relationship between arsenic content in hair and soil was obtained for rainy season, which indicated that arsenic in soil corresponded to arsenic in hair. The cancer risk from soils ranged from 4.48 × 10^?7 to 2.06 × 10^?6 indicating low carcinogenic risk to school children.     

ESI-MS and thermal melting studies of nanoscale platinum(II) metallomacrocycles with DNA

The hydrophilic, long-chain diamine PEGda (O,O'-bis(2-aminoethyl)octadeca(ethylene glycol)), when complexed with cis-protected Pt(II) ions afforded water-soluble complexes of the type [Pt(N,N)(PEGda)](NO(3))(2) (N,N = N,N,N',N'-tetramethyl-1,2-diaminoethane (tmeda), 1,2-diaminoethane (en), and 2,2'-bipyridine (2,2'-bipy)) featuring unusual 62-membered chelate rings. Equimolar mixtures containing either the 16-mer duplex DNA D2 or the single-stranded D2a and [Pt(N,N)(PEGda)](2+) were analyzed by negative-ion ESI-MS. Analysis of D2-Pt(II) mixtures showed the formation of 1 : 1 adducts of [Pt(en)(PEGda)](2+), [Pt(tmeda)(PEGda)](2+) and the previously-described metallomacrocycle [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(4)4,4'-bipy}(2)](8+) with D2; the dinuclear species bound to D2 most strongly, consistent with its greater charge and aromatic surface area. D2 formed 1 : 2 complexes with the acyclic species [Pt(2,2'-bipy)(Mebipy)(2)](4+) and [Pt(2,2'-bipy)(NH(3))(2)](2+). Analyses of D2a-Pt(II) mixtures gave results similar to those obtained with D2, although fragmentation was more pronounced, indicating that the nucleobases in D2a play more significant roles in mediating the decomposition of complexes than those in D2, in which they are paired in a complementary manner. Investigations were also conducted into the effects of selected platinum(II) complexes on the thermal denaturation of calf thymus DNA (CT-DNA) in buffered solution. Both [Pt(2)(2,2'-bipy)(2){4,4'-bipy(CH(2))(6)4,4'-bipy}(2)](8+) and [Pt(2,2'-bipy)(Mebipy)(2)](4+) stabilized CT-DNA. In contrast, [Pt(tmeda)(PEGda)](2+) and [Pt(en)(PEGda)](2+) (as well as free PEGda) caused negligible changes in melting temperature (ΔT(m)), suggesting that these species interact weakly with CT-DNA.     

Development of an effective extraction process for coenzyme Q10 from Artemia

A method for extracting coenzyme Q₁₀ (CoQ₁₀) from Artemia was developed. 1 g of fresh Artemia was incubated with 75 % acetic acid at (30 ± 2)°C for 24 h, followed by three consecutive extractions with a mixture of 5 mL of hexane and 5 mL of ethanol, then analysis by a validated high-performance liquid chromatography with a diode-array detector. The calibration curve for CoQ₁₀ was linear in a range of 1–50 μg mL^?1. The limits of detection and quantification were 0.3 μg mL^?1 and 1.1 μg mL^?1, respectively. Mean recoveries were 94–100 % with a high precision of below 10 %. The method developed was found to be simple, efficient and the time required for releasing CoQ₁₀ from Artemia was short. The method provides not only low energy consumption but is also practical for industrial applications.     

Binding studies of nNOS-active amphibian peptides and Ca2+ calmodulin, using negative ion electrospray ionisation mass spectrometry

Amphibian peptides which inhibit the formation of nitric oxide by neuronal nitric oxide synthase (nNOS) do so by binding to the protein cofactor, Ca2+calmodulin (Ca2+CaM). Complex formation between active peptides and Ca2+CaM has been demonstrated by negative ion electrospray ionisation mass spectrometry using an aqueous ammonium acetate buffer system. In all cases studied, the assemblies are formed with a 1:1:4 calmodulin/peptide/Ca2+ stoichiometry. In contrast, the complex involving the 20-residue binding domain of the plasma Ca2+ pump C20W (LRRGQILWFRGLNRIQTQIK-OH) with CaM has been shown by previous two-dimensional nuclear magnetic resonance (2D NMR) studies to involve complexation of the C-terminal end of CaM. Under identical conditions to those used for the amphibian peptide study, the ESI complex between C20W and CaM shows specific 1:1:2 stoichiometry. Since complex formation with the studied amphibian peptides requires Ca2+CaM to contain its full complement of four Ca2+ ions, this indicates that the amphibian peptides require both ends of the CaM to effect complex formation. Charge-state analysis and an H/D exchange experiment (with caerin 1.8) suggest that complexation involves Ca2+CaM undergoing a conformational change to a more compact structure.     

Multiple oligomeric forms of Escherichia coli DnaB helicase revealed by electrospray ionisation mass spectrometry

The Escherichia coli DnaB protein (DnaB(6)) is the hexameric helicase that unwinds genomic DNA so it can be copied by the DNA replication machinery. Loading of the helicase onto DNA requires interactions of DnaB(6) with six molecules of its loading partner protein, DnaC. Nano-electrospray ionisation mass spectrometry (nanoESI-MS) of mutant proteins was used to examine the roles of the residues Phe102 (F102) and Asp82 (D82) in the N-terminal domain of DnaB in the assembly of the hexamer. When the proteins were prepared in 1 M ammonium acetate containing magnesium and adenosine triphosphate (ATP) at pH 7.6, both hexameric and heptameric forms of wild-type and F102W, F102E and D82N mutant DnaBs were observed in mass spectra. The spectra of the D82N mutant also showed substantial amounts of a decameric species and small amounts of a dodecamer. In contrast, the F102H DnaB mutant was incapable of forming oligomers of order higher than the hexamer. Thus, although Phe102 is not the only determinant of hexamer assembly, this residue has a role in oligomerisation. NanoESI mass spectra were obtained of mixtures of DnaB(6) with DnaC. The DnaB(6)(DnaC)(6) complex (calculated M(r) 481 164) was observed only when the two proteins were present in equimolar amounts. The data are consistent with cooperative assembly of the complex. ESI mass spectra of mixtures containing DnaC and ATP showed that DnaC slowly hydrolysed ATP to ADP as indicated by ions corresponding to DnaC/ATP and DnaC/ADP complexes. These experiments show that E. coli DnaB can form a heptameric complex and that nanoESI-MS can be used to probe assembly of large (>0.5 MDa) macromolecular complexes.     

Investigation of inclusion complexes of citronella oil, citronellal and citronellol with β-cyclodextrin for mosquito repellent

The aim of this study was to prepare the inclusion complexes of citronella oil, citronellal or citronellol with β-cyclodextrin and evaluate their physicochemical properties using scanning electron microscopy (SEM), Fourier transform infrared spectroscopy (FTIR) and differential scanning calorimetry (DSC). A kneading method was employed to prepare the inclusion complexes and weight ratios of each of the active substance to β-cyclodextrin were 1:1 (1:1 CPX) and 1:2 (1:2 CPX). For comparison purposes, physical mixtures of these active compounds and β-cyclodextrin were also prepared and investigated. Unlike the physical mixtures, the SEM technique revealed drastic changes in the shapes and morphologies of the particles for the inclusion complexes. Furthermore, the FTIR and DSC results seemed to reveal some interactions between the active substance and β-cyclodextrin. The o/w lotions, which contained 10% w/w citronella oil (normal citronella oil; 1:1 CPX or 1:2 CPX), were formulated using Cremophors as emulsifiers. With modified Franz diffusion cell and synthetic membrane, the release rates of citronella oil from the lotions containing the inclusion complexes were significantly lower than that from the prepared lotion containing normal citronella oil. The mosquito (Aedes aegypti) repellent efficacy of the lotions containing citronella oil, citronellal or citronellol (both normal and inclusion complexes) was further evaluated by human-bait technique. The highest mosquito repellent activity was observed in the formulation which contained citronella oil–β-cyclodextrin inclusion complex at weight ratio of 1:1.     

Enhancement of DNA hybridization under acoustic streaming with three-piezoelectric-transducer system

Recently, we have demonstrated that DNA hybridization using acoustic streaming induced by two piezoelectric transducers provides higher DNA hybridization efficiency than the conventional method. In this work, we refine acoustic streaming system for DNA hybridization by inserting an additional piezoelectric transducer and redesigning the locations of the transducers. The Comsol? Multiphysics was used to design and simulate the velocity field generated by the piezoelectric agitation. The simulated velocity vector followed a spiral vortex flow field with an average direction outward from the center of the transducers. These vortices caused the lower signal intensity in the middle of the microarray for the two-piezoelectric disk design. On the contrary, the problem almost disappeared in the three-piezoelectric-disk system. The optimum condition for controlling the piezoelectric was obtained from the dye experiments with different activation settings for the transducers. The best setting was to activate the side disks and middle disk alternatively with 1 second activating time and 3 second non-activating time for both sets of transducers. DNA hybridization using microarrays for the malaria parasite Plasmodium falciparum from the optimized process yielded a three-fold enhancement of the signal compared to the conventional method. Moreover, a greater number of spots passed quality control in the optimized device, which could greatly improve biological interpretation of DNA hybridization data.     

Flavylium-Based Hypoxia-Responsive Probe for Cancer Cell Imaging

A hypoxia-responsive probe based on a flavylium dye containing an azo group (AZO-Flav) was synthesized to detect hypoxic conditions via a reductase-catalyzed reaction in cancer cells. In in vitro enzymatic investigation, the azo group of AZO-Flav was reduced by a reductase in the presence of reduced nicotinamide adenine dinucleotide phosphate (NADPH) followed by fragmentation to generate a fluorescent molecule, Flav-NH₂. The response of AZO-Flav to the reductase was as fast as 2 min with a limit of detection (LOD) of 0.4 μM. Moreover, AZO-Flav displayed high enzyme specificity even in the presence of high concentrations of biological interferences, such as reducing agents and biothiols. Therefore, AZO-Flav was tested to detect hypoxic and normoxic environments in cancer cells (HepG2). Compared to the normal condition, the fluorescence intensity in hypoxic conditions increased about 10-fold after 15 min. Prolonged incubation showed a 26-fold higher fluorescent intensity after 60 min. In addition, the fluorescence signal under hypoxia can be suppressed by an electron transport process inhibitor, diphenyliodonium chloride (DPIC), suggesting that reductases take part in the azo group reduction of AZO-Flav in a hypoxic environment. Therefore, this probe showed great potential application toward in vivo hypoxia detection.