Determination of trace cobalt concentrations in human serum by adsorptive stripping voltammetry

The goal of our study was to develop an accurate and reliable method for determining trace cobalt concentrations in human serum. The method was used to determine cobalt in the sera of healthy persons and patients with orthopaedic implants containing cobalt - a possible source of systemic release of cobalt into the human body. This goal is of vital interest since cobalt and its compounds are classified by IARC as potentially carcinogenic to humans. We used an electrochemical method, adsorptive stripping voltammetry (AdSV), which made possible the low detection limit and high sensitivity needed for measurements in human serum. The serum was acid digested by a combination of H2SO4, HNO3 and H2O2 in a 10 mL Kjeldhal flask. The digested sample was then dissolved in 0.1 mol/L ammonia buffer, pH 9.0 +/- 0.2. The determination is based on the adsorptive collection of the complex of cobalt (II) with dimethylglyoxime on a hanging mercury drop electrode (HMDE). The optimum values of adsorption potential and time were determined to be -0.8 V and 60 s. The optimisation of the sample digestion protocol and measurement procedures ensured the reliable assessment of low cobalt concentrations, down to 0.03 microg/L. The mean concentration of serum cobalt in four healthy persons was 0.11 +/- 0.06 microg/L, and in four patients with total hip replacements 0.34 +/- 0.07 microg/L. This method will be used routinely for measuring serum cobalt levels in patients with total hip replacements.     

Adsorptive transfer stripping voltammetry: Determination of nanogram quantities of DNA immobilized at the electrode surface

In adsorptive transfer stripping voltammetry (AdTSV), DNA is first adsorbed at the electrode, the electrode is washed and transferred (with the adsorbed layer) in the medium not containing DNA, and voltammetric analysis is performed in this medium. Adsorption can be performed from a drop of DNA solution, which makes it possible to reduce the volume of the analyzed sample by two orders of magnitude as compared to that of conventional voltammetry. With the hanging mercury drop electrode the limit of detection of single-stranded DNA is below 0.1 micrograms/ml; thus if the adsorption is performed from a 10-microliter drop of DNA solution subnanogram quantities of single-stranded DNA are sufficient for the analysis. In AdTSV the behavior of single- and double-stranded DNAs markedly differ from each other in a manner similar to that in the conventional voltammetric or polarographic analysis; AdTSV can thus be used in DNA structure analysis. In AdTSV the DNA transport and its adsorption at the electrode are separated from the electrode process; due to this fact it is possible (a) to perform the voltammetric analysis of DNA from media not suitable for voltammetric analysis of the conventional type, (b) to study the interaction of immobilized DNA with other substances in solution without the results of the voltammetric analysis being influenced by DNA interactions in the bulk of solution, and (c) to exploit the differences of adsorbability of DNA and other substances in order to separate them on the electrode.     

Simultaneous determination of Zn,Cd, Pb,and Cu in urine of patients with blackfoot disease using anodic stripping voltammetry

Blackfoot disease (BFD) is an endemic peripheral vascular disorder resulting in gangrene of the lower extremities, especially the feet, among residents in a limited area on the southwest coast of Taiwan. In the present study, the concentrations of zinc, cadmium, lead, and copper in urine of BFD patients with matched normal controls are investigated by differential pulse anodic stripping voltammetry (DPASV) on a hanging mercury drop electrode (HMDE). The analytical results indicate that urinary copper, cadmium, and lead of the BFD patients are significantly higher than those of the controls. In addition, the patients showed a significantly lower concentration of zinc in the urine than the normal controls. The possible connection of these elements with the etiology of the disease is discussed.     

Electrochemical studies of DNA immobilization onto the azide-terminated monolayers and its interaction with taxol

A surface modification procedure for the creation of self-assembled monolayers (SAMs) that can be used as a scaffold for double-stranded DNA (dsDNA) incorporation onto the gold surfaces is described. The SAMs of an azidohexane thiol derivative were prepared on the Au electrode and then used for the immobilization of dsDNA. The electrochemical characteristics of dsDNA onto the SAM-modified gold electrode were investigated by cyclic voltammetry and electrochemical impedance spectroscopy, and the surface concentration of dsDNA onto the SAMs surface was estimated. The interaction of dsDNA with the anticancer drug, taxol (paclitaxel), was also studied on the surface of DNA/SAM/Au electrode. The observed decrease in the guanine oxidation peak current was used to monitor the interaction of taxol with DNA. The resulting Langmuir isotherm for taxol binding to DNA at the modified electrode was used to evaluate the binding constant of taxol-DNA. The results obtained supported the groove binding interaction of taxol with DNA. The modified electrode was used as a sensitive sensor for quantification of taxol in human serum sample.     

EPR study on the interaction of hexavalent chromium with glutathione or cysteine: Production of pentavalent chromium and its stability

The reduction of hexavalent chromium (Cr(VI)) by glutathione was studied by EPR spectrometry and comparing it with that by cysteine. The characteristics of production of the pentavalent chromium (Cr(V)) species were studied. Two Cr(V) species, which were characterized by g values of 1.995–1.996 and 1.985–1.986, were detected at pH values above 5.0, whereas at pH 3.0 and 4.0 a single species of Cr(V) with a g value of 1.989–1.990 was found. The Cr(V) species were relatively long-lived and most stable at pH 7.0, where signal of two Cr(V) species were observed for more than 30 min. The intensities of the Cr(V) signals were pH-dependent, increasing with an increase in pH from 3.0 to 8.0. At neutral pH, the signal corresponding to the species of the larger g value increased markedly with an increase in glutathione concentration. Stable production of Cr(V) by glutathione was also confirmed by EPR measurements at 77 K. On the other hand, the characteristics of Cr(V) generation by cysteine were quite different. Production of the Cr(V) species was confirmed by a sharp single signal with a g value of 1.984–1.987. The signal intensity corresponding to Cr(V) generation did not change much with a change in pH from 3.0 to 6.0; at pH 7.0 only a small signal was observed, and at pH 8.0 no signal was observed. Moreover, the life-time of the Cr(V) signal was shorter than that observed for the reduction with glutathione. These results suggest that Cr(V) may be stabilized in glutathione solution by its suitable redox potential and ligand structure.     

Diversity of spore germination in response to inosine and L-alanine and its interaction with NaCl and pH in the Bacillus cereus group

Aims: Our aim was to assess the diversity of the nutrient germination response of Bacillus cereus spores. Methods and Results: B. cereus spore germination was monitored by decrease in optical density using a Bioscreen C analyser in response to the major germinant substances inosine and l -alanine. Spores of a set of 12 strains taken to illustrate the diversity of the B. cereus group showed ranging germination capacities. Two strains never germinated in the presence of l -alanine, at any of the germinant concentrations tested. Both the extent and rate of spore germination were affected by low pH and high NaCl concentration, but differently according to the strain. Conclusions: A broad diversity was observed in nutrient-triggered spore germination among the members of the B. cereus group. Spore germination of some strains occurred at low concentrations of inosine or l -alanine, suggesting high receptor sensitivity to germinants. The activity of these receptors was also affected by pH or high NaCl concentration. Significance and Impact of the Study: The greater ability of some strains to germinate in response to l -alanine and inosine is one criterion among others for B. cereus strain selection in food processing or storage studies, before confirmation in complex food or laboratory media. The diversity in response to germinants found among the B. cereus strains suggests a differential expression and (or) absence of some germination genes involved in the response, mainly to l -alanine.     

Proposed amino acid sequence and the 1.63 A X-ray crystal structure of a plant cysteine protease,ervatamin B: some insights into the structural basis of its stability and substrate specificity

The crystal structure of a cysteine protease ervatamin B, isolated from the medicinal plant Ervatamia coronaria, has been determined at 1.63 A. The unknown primary structure of the enzyme could also be traced from the high-quality electron density map. The final refined model, consisting of 215 amino acid residues, 208 water molecules, and a thiosulfate ligand molecule, has a crystallographic R-factor of 15.9% and a free R-factor of 18.2% for F > 2sigma(F). The protein belongs to the papain superfamily of cysteine proteases and has some unique properties compared to other members of the family. Though the overall fold of the structure, comprising two domains, is similar to the others, a few natural substitutions of conserved amino acid residues at the interdomain cleft of ervatamin B are expected to increase the stability of the protein. The substitution of a lysine residue by an arginine (residue 177) in this region of the protein may be important, because Lys --> Arg substitution is reported to increase the stability of proteins. Another substitution in this cleft region that helps to hold the domains together through hydrogen bonds is Ser36, replacing a conserved glycine residue in the others. There are also some substitutions in and around the active site cleft. Residues Tyr67, Pro68, Val157, and Ser205 in papain are replaced by Trp67, Met68, Gln156, and Leu208, respectively, in ervatamin B, which reduces the volume of the S2 subsite to almost one-fourth that of papain, and this in turn alters the substrate specificity of the enzyme.     

Positive Charges on Lysine Residues of the Extrinsic 18 kDa Protein Are Important to Its Electrostatic Interaction with Spinach Photosystem Ⅱ Membranes

To determine the contribution of charged amino acids to binding with the photosystem II complex （PSII）, the amino or carboxyl groups of the extrinsic 18 kDa protein were modified with N- succinimidyl propionate （NSP） or glycine methyl ester （GME） in the presence of a water-soluble carbodiimide, respectively. Based on isoelectric point shift, 4-10 and 10-14 amino groups were modified in the presence of 2 and 4 mM NSP, respectively. Similarly, 3-4 carboxyl groups were modified by reaction with 100 mM GME. Neutralization of negatively charged carboxyl groups with GME did not alter the binding activity of the extrinsic 18 kDa protein. However, the NSP-modified 18 kDa protein, in which the positively charged amino groups had been modified to uncharged methyl esters, failed to bind with the PSII membrane in the presence of the extrinsic 23 kDa protein. This defect can not be attributed to structural or conformational alterations imposed by chemical modification, as the fluorescence and circular dichroism spectra among native, GME- and NSP-modified extrinsic 18 kDa proteins were similar. Thus, we have concluded that the positive charges of lysyl residues in the extrinsic 18 kDa protein are important for its interaction with PSII membranes in the presence of the extrinsic 23 kDa protein. Furthermore, it was found that the negative charges of carboxyl groups of this protein did not participate in binding with the extrinsic 23 kDa protein associated with PSII membranes.     

Treatment of YAC128 mice and their wild-type littermates with cystamine does not lead to its accumulation in plasma or brain: implications for the treatment of Huntington disease

Cystamine is beneficial to Huntington disease (HD) transgenic mice. To elucidate the mechanism, cystamine metabolites were determined in brain and plasma of cystamine-treated mice. A major route for cystamine metabolism is thought to be: cystamine --> cysteamine --> hypotaurine --> taurine. Here we describe an HPLC system with coulometric detection that can rapidly measure underivatized cystamine, cysteamine and hypotaurine, as well as cysteine and glutathione in the same deproteinized tissue sample. A method is also described for the coulometric estimation of taurine as its isoindole-sulfonate derivative. Using this new methodology we showed that cystamine and cysteamine are undetectable (< or = 0.2 nmol/100 mg protein) in the brains of 3-month-old HD transgenic (YAC128) mice (or their wild-type littermates) treated daily for 2 weeks with cystamine (225 mg/kg) in their drinking water. No significant changes were observed in brain glutathione and taurine but significant increases were observed in brain cysteine. Cystamine and cysteamine were not detected in the plasma of YAC128 mice treated daily with cystamine between the ages of 4 and 12 or 7 and 12 months. These findings suggest that cystamine is not directly involved in mitigating HD but that increased brain cysteine or uncharacterized sulfur metabolites may be responsible.     

Thermodynamic study of the BRCT domain of BARD1 and its interaction with the -pSER-X-X-Phe- motif-containing BRIP1 peptide

The BRCA1-associated RING domain protein 1 (BARD1) is the heterodimeric partner of BRCA1. The BRCA1/BARD1 complex demonstrates ubiquitin ligase activity and has been implicated in genomic stability and tumor suppression. Both proteins possess a structurally conserved C-terminal domain (BRCT). While BRCA1–BRCT has been shown to mediate BRCA1 interactions with phosphoproteins such as BRIP1 by recognizing the pSer-X-X-Phe motif, attempts to demonstrate analogous interactions of its dimeric counterpart BARD1–BRCT, have so far been unsuccessful. In this study, chemical-denaturation experiments of BARD1–BRCT domain suggest that its low thermodynamic stability (ΔG = 2.5 kcal/mol) at room temperature, may affect some of its biochemical properties, such as its interaction with phosphopeptides. The stability of BARD1–BRCT domain at 10 °C, increases to 7.5 kcal/mol and isothermal titration calorimetry (ITC) experiments at this lower temperature showed binding to the BRIP1 phosphopeptide via an enthalpy-driven interaction, which appears to be specific to the pSer-X-X-Phe peptide-binding motif. Substitution of either pSer at position 0 with Ser (non-phosphorylated peptide) or Phe with Val at position + 3, leads to no-binding ITC results. While these findings are indicative that BRIP1 is a potential BARD1 binding partner, it becomes evident that in vitro binding assays involving the entire BARD1 protein and in vivo experiments are also needed to establish its binding partners and its potential role in tumor suppression pathways.     

The interaction of water with the phospholipid head group and its relationship to the lipid electrical conductivity

We have studied the interaction of water with the lipid head group by gravimetrically measuring the lipid water adsorption and the lateral dc electrical conductivity increase resulting from this hydration. We have done this for dimyristoyl phosphatidylcholine (DMPC) having protonated or deuterated hydrocarbon chains. These studies were also done for two cationic lipids having rather different polar head groups. All three lipids behave as strong water adsorbers and all three display a steep, logarithmic increase in the conductivity as the first 1-3 waters per lipid are adsorbed. This increase is usually 5-6 orders of magnitude. After the initial 1-3 waters are adsorbed, the conductivity increases much more gradually, upon additional water adsorption. This electrical behavior is also found for weak water adsorbers and appears to be independent of the head group composition. The conductivity behavior suggests two types of water interacting with the head group. Our studies also indicate that a choline-like component is responsible for the strong water binding nature of the lipids, although, both phosphate and choline make significant contributions to the total amount of adsorbed water. The conductivity behavior, however, does not depend on the presence of both these head group components.     

Preliminary assignments of the aromatic and some methyl group resonances of the 1H-NMR spectrum of the oxidized form of uteroglobin. Application to the interaction of oxidized uteroglobin with progesterone

Two-dimensional NMR methods have been used to assign aromatic and methyl group resonances in the 1H-NMR spectrum of oxidized uteroglobin. Assignments to specific amino acids are based on X-ray-determined structures of two crystal forms (C222(1) and P2(1] and on an energy-minimized X-ray structure of the C222(1) form of uteroglobin. These preliminary assignments are sufficient to probe the interaction of oxidized uteroglobin with progesterone in solution. The protein global structure is unmodified but some direct or indirect conformational changes are induced in the H1H4(H1'H4') pockets and close to Phe28 by progesterone.     

Application of electrospray ionization mass spectrometry to study the hydrophobic interaction between the epsilon and theta subunits of DNA polymerase III

The interactions between the N-terminal domain of the epsilon (epsilon186) and theta subunits of DNA polymerase III of Escherichia coli were investigated using electrospray ionization mass spectrometry. The epsilon186-theta complex was stable in 9 M ammonium actetate (pH 8), suggesting that hydrophobic interactions have a predominant contribution to the stability of the complex. Addition of primary alkanols to epsilon186-theta in 0.1 M ammonium acetate (pH 8), led to dissociation of the complex, as observed in the mass spectrometer. The concentrations of methanol, ethanol, and 1-propanol required to dissociate 50% of the complex were 8.9 M, 4.8 M, and 1.7 M, respectively. Closer scrutiny of the effect of alkanols on epsilon186, theta, and epsilon186-theta showed that epsilon186 formed soluble aggregates prior to precipitation, and that the association of epsilon186 with theta stabilized epsilon186. In-source collision-induced dissociation experiments and other results suggested that the epsilon186-theta complex dissociated in the mass spectrometer, and that the stability (with respect to dissociation) of the complex in vacuo was dependent on the solution from which it was sampled.     

Limnological Observations on Rihand Reservoir (Uttar Pradesh) with Reference to the Physical and Chemical Parameters of its Water

Observations in respect of Rihand Reservoir based on monsoon inflow, reservoir level, evaporation loss, air and water temperature, rainfall and wind velocity have been discussed in relation to the physical and chemical parameters of the water body. A weak thermocline has been observed, and the water showed a biogenic chemical stratification during the warmer months. Rihand is a monomictic tropical lake with a moderate nutrient supply, but is not very productive from the limnological point of view. The nutrient content of this lake declined from 1972 to 1974, but improved in 1975 and 1976 with the increase in rain and inflow of water. The productivity of the reservoir depends greatly on the nutrient level of the inflowing water from the catchment area, so that the monsoon inflow largely influences the water quality of the reservoir.     

Simultaneous determination of didanosine and its amino acid prodrug,valdidanosine by hydrophilic interaction chromatography coupled with electrospray ionization tandem mass spectrometry: Application to a pharmacokinetic study in rats

A rapid, sensitive and selective ultra-performance liquid chromatography–tandem mass spectrometry (UPLC–MS/MS) method with hydrophilic interaction chromatography has been developed and validated for the simultaneous determination of didanosine and valdidanosine (L-valine amino acid ester prodrug of didanosine) in rat plasma. Solid-phase extraction (SPE) column was employed to extract the analytes from rat plasma, with high extraction recovery (>85%) for both didanosine and valdidanosine. The analytes were then separated by hydrophilic interaction chromatography (HILIC column) and detected by a triple-quadrupole mass spectrometry equipped with an electrospray ionization (ESI) source. The method was linear over the concentration ranges of 2–20,000 ng/mL for didanosine and 4–300 ng/mL for valdidanosine. The lower limit of quantitation (LLOQ) of didanosine and valdidanosine was 2 and 4 ng/mL, respectively. The intra-day and inter-day relative standard deviation (RSD) were less than 15% and the relative errors (RE) were all within 15%. Finally, the validated UPLC–MS/MS method was successfully applied to the pharmacokinetic study after either didanosine or valdidanosine orally administrated to the Sprague–Dawley rats.     

Hydrophilic interaction liquid chromatography-tandem mass spectrometry for the determination of adefovir in human plasma and its application to a pharmacokinetic study

A selective, rapid and sensitive hydrophilic interaction liquid chromatography–tandem mass spectrometry (HILIC–MS/MS) method was developed for the first time to determine adefovir in human plasma and applied to a pharmacokinetic study. Plasma samples were prepared by protein precipitation with methanol followed by a further cleaning using dichloromethane. The chromatographic separation was carried out on an ACQUITY UPLC™ BEH HILIC column with the mobile phase of methanol–water–formic acid (85:15:0.2, v/v/v). The detection was performed on a triple-quadrupole tandem mass spectrometer with multiple reaction monitoring (MRM) mode via electrospray ionization (ESI) source. The method was rapid with a run time of 3 min per sample. The linear calibration curves were obtained in the concentration range of 1.02–102 ng/mL (r² ≥ 0.99) with the lower limit of quantification (LLOQ) of 1.02 ng/mL. The intra- and inter-day precision (relative standard deviation, R.S.D.) values were below 12% and the accuracy (relative error, R.E.) was from 0.6% to 3.2% at all quality control (QC) levels. The method was applicable to clinical pharmacokinetic study of adefovir in healthy volunteers after oral administration of adefovir dipivoxil tablet.     

Temporal analysis of brown eye spot of coffee and its response to the interaction of irrigation with phosphorous levels

Brown eye spot (BES—Cercospora coffeicola) is a major disease of coffee, and its occurrence is affected by water supply and nutritional balance. Little is known about the effect of phosphorous (P) on coffee fields under irrigation. Thus, this study evaluated the effect of the interaction between different water application levels and phosphorus levels (5 irrigation levels × 4 phosphorus levels) on the intensity of this disease. The area under the incidence progress curve (AUIPC) was calculated and subjected to analysis of variance. The progress curve of the average incidence of BES varied in both evaluation years. In Year 1 (November 2011 to December 2012), the incidence peaked on August 12, 2012 (22.45%), while in Year 2 (January 2013 to January 2014), the incidence reached its highest level on September 12, 2013 (16.29%). The exponential nonlinear model was adjusted for the two years. There was an interaction between irrigation and phosphorus levels on October 07, 2012. The incidence interacted significantly with the harvest dates. On the first evaluation date, an increase in phosphorus levels at shallower irrigation depths and an absence of phosphate fertilizer at higher irrigation levels caused higher incidences.     

Studies on the formation of a complex of Cu(II) with sodium 1,4-dihydroxy-9,10-anthraquinone-2-sulphonate - An analogue of the core unit of anthracycline anticancer drugs and its interaction with calf thymus DNA

Copper(II) forms a complex with sodium 1,4-dihydroxy-9,10-anthraquinone-2-sulphonate (sodium quinizarin-2-sulphonate, NaQSH₂), an analogue of the core unit of anthracycline antibiotics used in the treatment of cancer. The 1:2 metal-ligand complex is formed in aqueous solution at neutral and acidic pH while in alkaline pH both 1:1 and 1:2 species are formed. The effective stability constant of the 1:2 metal-ligand complex is 9.64 × 10¹⁶ while that of the 1:1 metal-ligand complex is 9.4 × 10⁹. The 1:2 complex Cu(NaQSH)₂(H₂O)₂ was synthesized and characterized by different techniques in solid state and in solution. The complex Cu(NaQSH)₂(H₂O)₂ interacts with calf thymus DNA which was studied by fluorescence spectroscopy. The binding constant and site size for the interaction with DNA were determined.     

The binding of skeletal muscle C-protein to F-actin, and its relation to the interaction of actin with myosin subfragment-1.

C-protein is a component of thick filaments of skeletal muscle myofibrils. It is bound to the assembly of myosin tails that forms the filament backbone. We report here that C-protein can also bind to F-actin, with a limiting stoichiometry of approximately one C-protein molecule per 3 to 5 actin subunits and a dissociation constant in the micromolar range at ionic strength 0·07. The binding is not significantly affected by ATP, calcium ions or temperature, or by the presence of tropomyosin on the actin, but it is weakened by increasing ionic strength. Myosin subfragment-1 (S-1) competes with C-protein for binding to actin. In the absence of ATP, S-1 displaces nearly all bound C-protein from actin, while in the presence of ATP, C-protein inhibits the actin activation of S-1 ATPase. Although there is no direct evidence that interaction of C-protein with actin is physiologically significant, the lenght of the C-protein molecule is sufficient so that it could make contact with the thin filaments in muscle while remaining attached to the thick filaments.