Simultaneous detection of NADH and H₂O₂ using flow injection analysis based on a bifunctional poly(thionine)-modified electrode

We report here a novel detection scheme for simultaneous detection of NADH and H(2)O(2) based on a bifunctional poly(thionine)-modified electrode. Electropolymerization of thionine on a "preanodized" screen-printed carbon electrode effectively lowers the oxidation potential of NADH to 0.15 V (vs. Ag/AgCl). Since poly(thionine) is also a well known electrochemical mediator for H(2)O(2) reduction, we further developed a poly(thionine)-modified ring disk electrode for simultaneous measurement of nicotinamide adenine dinucleotide (NADH) and hydrogen peroxide (H(2)O(2)) by flow injection analysis. By applying the optimized detection potentials of 0.2V and -0.2V at disk and ring electrodes, respectively, this system allows the simultaneous measurement of both analytes with good sensitivity (0.13 μA/mM for H(2)O(2) and 0.34 μA/mM for NADH) and limit of detection (1.74 μM and 26.0 μM for NADH and H(2)O(2)). This opens the possibility of a whole series of biosensor applications.     

A tight coupling between β₂Y97 and β₂F200 of the GABA(A) receptor mediates GABA binding

The GABA(A) receptor is an oligopentameric chloride channel that is activated via conformation changes induced upon the binding of the endogenous ligand, GABA, to the extracellular inter-subunit interfaces. Although dozens of amino acid residues at the α/β interface have been implicated in ligand binding, the structural elements that mediate ligand binding and receptor activation are not yet fully described. In this study, double-mutant cycle analysis was employed to test for possible interactions between several arginines (α?R67, α?R120, α?R132, and β?R207) and two aromatic residues (β?Y97 and β?F200) that are present in the ligand-binding pocket and are known to influence GABA affinity. Our results show that neither α?R67 nor α?R120 is functionally coupled to either of the aromatics, whereas a moderate coupling exists between α?R132 and both aromatic residues. Significant functional coupling between β?R207 and both β?Y97 and β?F200 was found. Furthermore, we identified an even stronger coupling between the two aromatics, β?Y97 and β?F200, and for the first time provided direct evidence for the involvement of β?Y97 and β?F200 in GABA binding. As these residues are tightly linked, and mutation of either has similar, severe effects on GABA binding and receptor kinetics, we believe they form a single functional unit that may directly coordinate GABA.     

Bimetallic PtM (M=Pd, Ir) nanoparticle decorated multi-walled carbon nanotube enzyme-free, mediator-less amperometric sensor for H₂O₂

A new highly catalytic and intensely sensitive amperometric sensor based on PtM (where M=Pd, Ir) bimetallic nanoparticles (NPs) for the rapid and accurate estimation of hydrogen peroxide (H(2)O(2)) by electrooxidation in physiological conditions is reported. PtPd and PtIr NPs-decorated multiwalled carbon nanotube nanocatalysts (PtM/MWCNTs) were prepared by a modified Watanabe method, and were characterized by XRD, TEM, ICP, and XAS. The sensors were constructed by immobilizing PtM/MWCNTs nanocatalysts in a Nafion film on a glassy carbon electrode. Both PtPd/MWCNTs and PtIr/MWCNTs assemblies catalyzed the electrochemical oxidation of H(2)O(2). Cyclic voltammetry characterization measurements revealed that both the PtM (M=Pd, Ir)/MWCNTs/GCE possessed similar electrochemical surface areas (～0.55 cm(2)), and electron transfer rate constants (～1.23 × 10(-3)cms(-1)); however, the PtPd sensor showed a better performance in H(2)O(2) sensing than did the PtIr counterpart. Explanations were sought from XAS measurements to explain the reasons for differences in sensor activity. When applied to the electrochemical detection of H(2)O(2), the PtPd/MWCNTs/GC electrode exhibited a low detection limit of 1.2 μM with a wide linear range of 2.5-125 μM (R(2)=0.9996). A low working potential (0V (SCE)), fast amperometric response (<5s), and high sensitivity (414.8 μA mM(-1)cm(-2)) were achieved at the PtPd/MWCNTs/GC electrode. In addition, the PtPd/MWCNTs nanocatalyst sensor electrode also exhibited excellent reproducibility and stability. Along with these attractive features, the sensor electrode also displayed very high specificity to H(2)O(2) with complete elimination of interference from UA, AA, AAP and glucose.     

A new D₂ dopamine receptor agonist allosterically modulates A(2A) adenosine receptor signalling by interacting with the A(2A)/D₂ receptor heteromer

The structural and functional interaction between D₂ dopamine receptor (DR) and A_2A adenosine receptor (AR) has suggested these two receptors as a pharmacological target in pathologies associated with dopamine dysfunction, such as Parkinson's disease. In transfected cell lines it has been demonstrated the activation of D₂DR induces a significant negative regulation of A_2AAR-mediated responses, whereas few data are at now available about the regulation of A_2AAR by D₂DR agonists at receptor recognition site. In this work we confirmed that in A_2AAR/D₂DR co-transfected cells, these receptors exist as homo- and hetero-dimers. The classical D₂DR agonists were able to negatively modulate both A_2AAR affinity and functionality. These effects occurred even if any significant changes in A_2AAR/D₂DR energy transfer interaction could be detected in BRET experiments.Since the development of new molecules able to target A_2A/D₂ dimers may represent an attractive tool for innovative pharmacological therapy, we also identified a new small molecule, 3-(3,4-dimethylphenyl)-1-(2-piperidin-1-yl)ethyl)piperidine (compound 1), full agonist of D₂DR and modulator of A_2A-D₂ receptor dimer. This compound was able to negatively modulate A_2AAR binding properties and functional responsiveness in a manner comparable to classical D₂R agonists. In contrast to classical agonists, compound 1 led to conformational changes in the quaternary structure in D₂DR homomers and heteromers and induced A_2AAR/D₂DR co-internalization. These results suggest that compound 1 exerts a high control of the function of heteromers and could represent a starting point for the development of new drugs targeting A_2AAR/D₂ DR heteromers.     

Adenosine A₂a receptors and O₂ sensing in development

Reduced mitochondrial oxidative phosphorylation, via activation of adenylate kinase and the resulting exponential rise in the cellular AMP/ATP ratio, appears to be a critical factor underlying O? sensing in many chemoreceptive tissues in mammals. The elevated AMP/ATP ratio, in turn, activates key enzymes that are involved in physiologic adjustments that tend to balance ATP supply and demand. An example is the conversion of AMP to adenosine via 5'-nucleotidase and the resulting activation of adenosine A(?A) receptors, which are involved in acute oxygen sensing by both carotid bodies and the brain. In fetal sheep, A(?A) receptors associated with carotid bodies trigger hypoxic cardiovascular chemoreflexes, while central A(?A) receptors mediate hypoxic inhibition of breathing and rapid eye movements. A(?A) receptors are also involved in hypoxic regulation of fetal endocrine systems, metabolism, and vascular tone. In developing lambs, A(?A) receptors play virtually no role in O? sensing by the carotid bodies, but brain A(?A) receptors remain critically involved in the roll-off ventilatory response to hypoxia. In adult mammals, A(?A) receptors have been implicated in O? sensing by carotid glomus cells, while central A(?A) receptors likely blunt hypoxic hyperventilation. In conclusion, A(?A) receptors are crucially involved in the transduction mechanisms of O? sensing in fetal carotid bodies and brains. Postnatally, central A(?A) receptors remain key mediators of hypoxic respiratory depression, but they are less critical for O? sensing in carotid chemoreceptors, particularly in developing lambs.     

Replacement of histidine in position 105 in the α₅ subunit by cysteine stimulates zolpidem sensitivity of α₅β₂γ₂ GABA(A) receptors

Zolpidem is a positive allosteric modulator of GABA_A receptors with sensitivity to subunit composition. While it acts with high affinity and efficacy at GABA_A receptors containing the α₁ subunit, it has a lower affinity to GABA_A receptors containing α₂, α₃, or α₅ subunits and has a very weak efficacy at receptors containing the α₅ subunit. Here, we show that replacing histidine in position 105 in the α₅ subunit by cysteine strongly stimulates the effect of zolpidem in receptors containing the α₅ subunit. The side chain volume of the amino acid residue in this position does not correlate with the modulation by zolpidem. Interestingly, serine is not able to promote the potentiation by zolpidem. The homologous residues to α₅H105 in α₁, α₂, and α₃ are well-known determinants of the action of classical benzodiazepines. Other studies have shown that replacement of these histidines α₁H101, α₂H101, and α₃H126 by arginine, as naturally present in α₄ and α₆, leads to benzodiazepine insensitivity of these receptors. Thus, the nature of the amino acid residue in this position is not only crucial for the action of classical benzodiazepines but in α₅ containing receptors also for the action of zolpidem.     

Effects of H₂O₂ on electrical membrane properties of skeletal myotubes

Reactive oxygen species (ROS), normally generated in skeletal muscles, could control excitability of muscle fibers through redox modulation of membrane ion channels. However, the mechanisms of ROS action remain largely unknown. To investigate the action of ROS on electrical properties of muscle cells, patch-clamp recordings were performed after application of hydrogen peroxide (H₂O₂) to skeletal myotubes. H₂O₂ facilitated sodium spikes after a hyperpolarizing current pulse, by decreasing the latency for spike initiation. Importantly, the antioxidant N-acetylcysteine induced the opposite effect, suggesting the redox control of muscle excitability. The effect of H₂O₂ was abolished in the presence of catalase. The kinetics of sodium channels were not affected by H₂O₂. However, the fast inward rectifier K⁺ (K_IR) currents, activated by hyperpolarization, were reduced by H₂O₂, similar to the action of the potassium channel blockers Ba²⁺ and Cs⁺. The block of the outward tail current contributing to K_IR deactivation can explain the shorter latency for spike initiation. We propose that the K_IR current is an important target for ROS action in myotubes. Our data would thus suggest that ROS are involved in the control of the excitability of myotubes and, possibly, in the oscillatory behavior critical for the plasticity of developing muscle cells.     

Synthesis of functional SiO₂-coated graphene oxide nanosheets decorated with Ag nanoparticles for H₂O₂ and glucose detection

In this paper, we report on the first preparation of well-defined SiO(2)-coated graphene oxide (GO) nanosheets (SiO(2)/GO) without prior GO functionalization by combining sonication with sol-gel technique. The functional SiO(2)/GO nanocomposites (F-SiO(2)/GO) obtained by surface functionalization with NH(2) group were subsequently employed as a support for loading Ag nanoparticles (AgNPs) to synthesize AgNP-decorated F-SiO(2)/GO nanosheets (AgNP/F-SiO(2)/GO) by two different routes: (1) direct adsorption of preformed, negatively charged AgNPs; (2) in situ chemical reduction of silver salts. The morphologies of these nanocomposites were characterized by transmission electron microscopy (TEM) and scanning electron microscopy (SEM). It is found that the resultant AgNP/F-SiO(2)/GO exhibits remarkable catalytic performance for H(2)O(2) reduction. This H(2)O(2) sensor has a fast amperometric response time of less than 2s. The linear range is estimated to be from 1×10(-4) M to 0.26 M (r=0.998) and the detection limit is estimated to be 4 × 10(-6) M at a signal-to-noise ratio of 3, respectively. We also fabricated a glucose biosensor by immobilizing glucose oxidase (GOD) into AgNP/F-SiO(2)/GO nanocomposite-modified glassy carbon electrode (GCE) for glucose detection. Our study demonstrates that the resultant glucose biosensor can be used for the glucose detection in human blood serum.     

(Arene)Cl₂Ru(II) complexes with N-coordinated estrogen and androgen isonicotinates: interaction with sex hormone binding globulin and anticancer activity

(Arene)dichloridoruthenium(II) complexes with N-coordinated isonicotinates of androgens (6) and estrogens (9) were prepared and tested for affinity to the estrogen receptor (ERα) and sex hormone binding globulin (SHBG), as well as for cytotoxicity in cancer cells. None of the new complexes bound noticeably to the ER and most of them also bound less strongly to SHBG than the corresponding unmetallated steroids 7. In MTT assays the Ru(p-cymene) complexes 9 of 2-substituted estrones were equally or even more cytotoxic than the metal-free steroids against hormone-dependent (MCF-7 breast and KB-V1 cervix carcinomas) and hormone-independent (518A2 melanoma) cells. The addition of external SHBG to MTT assays lowered the cytotoxicities of the complexes 9 and distinctly more so those of some steroids 7, probably by the way of sequestration and reduction of the cellular uptake. In the absence of SHBG the estrogen complexes 9 were internalized by 518A2 melanoma cells and ruthenated their DNA as quantified by ICP-OES. They also ruthenated salmon sperm DNA but did not change the topology of plasmid DNA in EMSA experiments. In addition, the Ru(p-cymene) complex of 2-ethoxyestrone (9c) was shown to reduce the motility of 518A2 melanoma cells in a wound-healing assay.     

Site-specific radical formation in DNA induced by Cu(II)-H₂O₂ oxidizing system, using ESR, immuno-spin trapping, LC-MS, and MS/MS

Oxidative stress-related damage to the DNA macromolecule produces a multitude of lesions that are implicated in mutagenesis, carcinogenesis, reproductive cell death, and aging. Many of these lesions have been studied and characterized by various techniques. Of the techniques that are available, the comet assay, HPLC-EC, GC-MS, HPLC-MS, and especially HPLC-MS/MS remain the most widely used and have provided invaluable information on these lesions. However, accurate measurement of DNA damage has been a matter of debate. In particular, there have been reports of artifactual oxidation leading to erroneously high damage estimates. Further, most of these techniques measure the end product of a sequence of events and thus provide only limited information on the initial radical mechanism. We report here a qualitative measurement of DNA damage induced by a Cu(II)–H₂O₂ oxidizing system using immuno-spin trapping (IST) with electron paramagnetic resonance (EPR), MS, and MS/MS. The radical generated is trapped by DMPO immediately upon formation. The DMPO adduct formed is initially EPR active but subsequently is oxidized to the stable nitrone, which can then be detected by IST and further characterized by MS and MS/MS.     

The first stereoselective total synthesis of (Z)-cryptomoscatone D2, a natural G₂ checkpoint inhibitor

The first stereoselective synthesis of (Z)-cryptomoscatone D2, a naturally occurring G(2) checkpoint inhibitor, was accomplished using propane-1,3-diol as the starting material. The Maruoka asymmetric allylation, ring closing metathesis and the hydrogenation of the triple bond employing Lindlar's catalyst were involved as the key steps.     

Optical detection of choline and acetylcholine based on H₂O₂-sensitive quantum dots

In this paper, we have constructed a simple, rapid and sensitive biosensor for detection of choline and acetylcholine (ACh) based on the hydrogen peroxide (H(2)O(2))-sensitive quantum dots (QDs). The detection limit for choline was 0.1 μM and the linear range was 0.1-0.9 μM and 5-150 μM, respectively. The detection limit for ACh was found to be 10 μM and the linear range was 10-5000 μM. The wide linear ranges were shown to be suitable for routine analyses of choline and ACh. Possible mechanism of the fluorescence of QDs quenched by H(2)O(2) was an electron transfer (ET) process. The experimental conditions of biosensors were optimized, and anti-interference ability was also presented. We also detected the choline in milk samples and the linear range was 5-150 μM. The detection linear range of ACh in serum was 10-140 μM. Most importantly, the recovery of choline in milk and ACh in serum samples were both close to 99%. The excellent performance of this biosensor showed that the method can be used in practice detection of choline and ACh.     

CO₂ utilizing microbes--a comprehensive review

CO₂ fixing microbes are the species primarily engaged in complexing the inorganic carbon dioxide to organic carbon compounds. There are many microorganisms from archaeal and bacterial domain that can fix carbon dioxide through six known CO₂ fixing pathways. These organisms are ubiquitous and can survive in wide range of aerobic and anaerobic habitats. This review focuses on the prior research, that has been conducted in this field and presents a summarized overview of all the mechanisms (along with their genes and enzymes) used by these microbes for CO₂ incorporation. In addition, this review provides a better understanding of diversity and taxonomy of CO₂ fixing microorganisms. The information presented here will motivate researchers to further explore the diversity of CO₂ fixing microorganisms as well as to decipher the underlying mechanisms of CO₂ utilization.     

5-HT₂c receptor selectivity and structure-activity relationship of N-methyl-N-(1-methylpiperidin-4-yl)benzenesulfonamide analogs

Agonists of the 5-HT(2C) receptor have attracted much attention as therapeutic agents for the treatment of obesity. Subtype selectivity against other 5-HT(2) receptors is one of the most important prerequisites for reducing side effects. We present the synthesis of N-methyl-N-(1-methylpiperidin-4-yl)benzenesulfonamide analogs and their structure-activity relationship studies on 5-HT(2A) and 5-HT(2C) receptors. Although the compounds showed nanomolar activity to the 5-HT(2C) receptor, their selectivity against the 5-HT(2A) receptor was modest to low. Molecular modeling studies using homology modeling and docking simulation revealed that selectivity originated from subtype specific residues. The observed binding modes and receptor-ligand interactions provided us a clue for optimizing the selectivity against the 5-HT(2A) receptor.     

VO₂ requirements of boxing exercises

The purpose of this study was to quantify the physiological requirements of various boxing exercises such as sparring, pad work, and punching bag. Because it was not possible to measure the oxygen uptake (VO?) of "true" sparring with a collecting gas valve in the face, we developed and validated a method to measure VO? of "true" sparring based on "postexercise" measurements. Nine experienced male amateur boxers (Mean ± SD: age = 22.0 ± 3.5 years, height = 176.0 ± 8.0 cm, weight = 71.4 ± 10.9 kg, number of fights = 13.0 ± 9.5) of regional and provincial level volunteered to participate in 3 testing sessions: (a) maximal treadmill test in the LAB, (b) standardized boxing training in the GYM, and (c) standardized boxing exercises in the LAB. Measures of VO?, heart rate (HR), blood lactate concentration [LA], rated perceived exertion level, and punching frequencies were collected. VO? values of 43.4 ± 5.9, 41.1 ± 5.1, 24.7 ± 6.1, 30.4 ± 5.8, and 38.3 ± 6.5 ml·kg?1·min?1 were obtained, which represent 69.7 ± 8.0, 66.1 ± 8.0, 39.8 ± 10.4, 48.8 ± 8.5, and 61.7 ± 10.3%VO?peak for sparring, pad work, and punching bag at 60, 120, and 180 b·min?1, respectively. Except for lower VO? values for punching the bag at 60 and 120 b·min?1 (p < 0.05), there was no VO? difference between exercises. Similar pattern was obtained for %HRmax with respective values of 85.5 ± 5.9, 83.6 ± 6.3, 67.5 ± 3.5, 74.8 ± 5.9, and 83.0 ± 6.0. Finally, sparring %HRmax and [LA] were slightly higher in the GYM (91.7 ± 4.3 and 9.4 ± 2.2 mmol·L?1) vs. LAB (85.5 ± 5.9 and 6.1 ± 2.3 mmol·L?1). Thus, in this study simulated LAB sparring and pad work required similar VO? (43-41 ml·kg?1·min?1, respectively), which corresponds to ~70%VO?peak. These results underline the importance of a minimum of aerobic fitness for boxers and draw some guidelines for the intensity of training.     

Yersinia entry into host cells requires Rab5-dependent dephosphorylation of PI(4,5)P₂ and membrane scission

Some bacteria invade host cells by triggering a process akin to phagocytosis. We analyzed the mechanisms underlying invasion vacuole formation by the bacterium Yersinia pseudotuberculosis. After engaging integrins on host cells, Yersinia resided in PI(4,5)P?-rich, membrane-bound compartments called "prevacuoles" that were inaccessible to extracellular antibodies but remained connected to the plasma membrane. The scission of prevacuoles to form separate, sealed compartments coincided with and required PI(4,5)P? hydrolysis. At the time of scission, the inositol 5-phosphatases OCRL and Inpp5b were recruited to prevacuoles. This recruitment and subsequent PI(4,5)P? hydrolysis required the association of the GTPase Rab5 with prevacuoles. Phosphatidylinositol 3-kinase activity was required for fusion of Rab5-positive vesicles with prevacuoles and scission of nascent vacuoles from the plasma membrane. Thus, Yersinia invasion involves a multistep process in which the bacteria form a protective prevacuole and then recruit host factors to induce membrane fission, allowing the bacteria to invade the cell.     

Ventralization of an indirect developing hemichordate by NiCl₂ suggests a conserved mechanism of dorso-ventral (D/V) patterning in Ambulacraria (hemichordates and echinoderms)

One of the earliest steps in embryonic development is the establishment of the future body axes. Morphological and molecular data place the Ambulacraria (echinoderms and hemichordates) within the Deuterostomia and as the sister taxon to chordates. Extensive work over the last decades in echinoid (sea urchins) echinoderms has led to the characterization of gene regulatory networks underlying germ layer specification and axis formation during embryogenesis. However, with the exception of recent studies from a direct developing hemichordate (Saccoglossus kowalevskii), very little is known about the molecular mechanism underlying early hemichordate development. Unlike echinoids, indirect developing hemichordates retain the larval body axes and major larval tissues after metamorphosis into the adult worm. In order to gain insight into dorso-ventral (D/V) patterning, we used nickel chloride (NiCl?), a potent ventralizing agent on echinoderm embryos, on the indirect developing enteropneust hemichordate, Ptychodera flava. Our present study shows that NiCl? disrupts the D/V axis and induces formation of a circumferential mouth when treated before the onset of gastrulation. Molecular analysis, using newly isolated tissue-specific markers, shows that the ventral ectoderm is expanded at expense of dorsal ectoderm in treated embryos, but has little effect on germ layer or anterior-posterior markers. The resulting ventralized phenotype, the effective dose, and the NiCl? sensitive response period of Ptychodera flava, is very similar to the effects of nickel on embryonic development described in larval echinoderms. These strong similarities allow one to speculate that a NiCl? sensitive pathway involved in dorso-ventral patterning may be shared between echinoderms, hemichordates and a putative ambulacrarian ancestor. Furthermore, nickel treatments ventralize the direct developing hemichordate, S. kowalevskii indicating that a common pathway patterns both larval and adult body plans of the ambulacrarian ancestor and provides insight in to the origin of the chordate body plan.     

Effects of olive leaf polyphenols against H₂O₂ toxicity in insulin secreting β-cells

In pancreatic β-cells, although H?O? is a metabolic signal for glucose stimulated insulin secretion, it may induce injury in the presence of increased oxidative stress (OS) as in the case of diabetic chronic hyperglycemia. Olea europea L. (olive) leaves contain polyphenolic compounds that may protect insulin-secreting cells against OS. The major polyphenolic compound in ethanolic olive leaf extract (OLE) is oleuropein (about 20%), thus we compared the effects of OLE with the effects of standard oleuropein on INS-1 cells. The cells were incubated with increasing concentrations of OLE or oleuropein for 24 h followed by exposure to H?O? (0.035 mM) for 45 min. H?O? alone resulted in a significantly decreased viability (MTT assay), depressed glucose-stimulated insulin secretion, increased apoptotic and necrotic cell death (AO/EB staining), inhibited glutathione peroxidase activity (GPx) and stimulated catalase activity that were associated with increased intracellular generation of reactive oxygen species (ROS) (fluorescence DCF). OLE and oleuropein partly improved the viability, attenuated necrotic and apoptotic death, inhibited the ROS generation and improved insulin secretion in H?O?-exposed cells. The effects of oleuropein on insulin secretion were more pronounced than those of OLE, while OLE exerted a stronger anti-cytotoxic effect than oleuropein. Unlike OLE, oleuropein had no significant preserving effect on GPx; however, both compounds stimulated the activity of catalase in H?O?-exposed cells. These findings indicate different modulatory roles of polyphenolic constituents of olive leaves on redox homeostasis that may have a role in the maintenance of β-cell physiology against OS.