Blue Tetrazolium Chloride Research Articles

Ultrasensitive surface-enhanced Raman scattering detection of alkaline phosphatase

In this study, an ultrasensitive surface-enhanced Raman scattering (SERS) detection of alkaline phosphatase (ALP) has been developed, in which nile blue A (NBA) was chosen to replace nitro blue tetrazolium chloride (NBT) in a reactive system of 5-bromo-4-chloro-3-indolyl phosphate (BCIP), NBT, and ALP.

Abstract B01: Impacts of inulin on bile acids induced colon cancer

Abstract Bile acids are normal components of the luminal contents of gastrointestinal tract but have been reported to cause oxidative damage by stimulating the generation of free oxygen radicals from the mitochondria. Several studies have demonstrated the contribution of reactive species to mutagenesis, carcinogenesis and tumor promotion. There is accumulating evidence to show that some carbohydrates and carbohydrate-containing molecules possess antioxidant ability by inhibiting the production of reactive species. Prebiotics are complex carbohydrates which have been shown to improve gut health when used in combination with probiotics. However, there is paucity of data on how prebiotics alone affect gut health and by which mechanism of action. The reactive oxygen scavenging ability of inulin, a prebiotic, has been shown in vitro in non-cell based system and in mice. The anti-inflammatory effect, through production of short chain fatty acids by fermentable inulin has also been shown. However, the mechanism of inulin in the amelioration of bile acid induced cancer remains unknown. Methods: Three different cell-lines (colon stem cells, human colon carcinoma cells-HCT-116 and control normal colon epithelial cells) will be examined. Cells will be treated separately with secondary bile acids, primary bile acids and prebiotics. Cells will also be treated with microorganisms' filtrate such as probiotics (bifidobacteria) in conjunction with secondary bile acids, primary bile acids or prebiotics. Production of free radicals will be measured using nitro blue tetrazolium chloride assay (NBT) and inflammatory proteins will be measured using RT-PCR. DNA damage will be assessed using Endonuclease 111 enzyme modified Comet assay. In addition, C1 Single-Cell auto pep system will be used for mRNA sequencing of individual cells within the well plate. This study hypothesized that normal cells exposed to secondary bile acids in the presence of inulin may be protected from transformation to cancer cells and this may correlate with decrease in production of reactive radicals and pro-inflammatory proteins. The anti inflammatory ability of short chain fatty acid and antioxidant ability of inulin may possibly be associated with AMP-activated protein kinase (AMPK), phosphatidylinositol 3-kinase (P13-K), NF-κB signaling pathways. Thus inulin and their fermentation/breakdown products may not only stimulate beneficial bacteria in the colon but may directly interfere with absorption and cellular metabolism processes in the colon. Citation Format: Bene Akromaa Afolabi, Sandra Appiah, Azra Pachenari, Lucy Ghali. Impacts of inulin on bile acids induced colon cancer. [abstract]. In: Proceedings of the Third AACR International Conference on Frontiers in Basic Cancer Research; Sep 18-22, 2013; National Harbor, MD. Philadelphia (PA): AACR; Cancer Res 2013;73(19 Suppl):Abstract nr B01.

Scanning of flat textile-based radiation dosimeters: Influence of parameters on the quality of results

Flat woven polyamide textiles were chosen for modification with nitro blue tetrazolium chloride (NBT) or 2,3,5-triphenyltetrazolium chloride (TTC). Such samples change colour from white to blue (NBT) or red (TTC) if exposed to ionizing radiation or UV light. When inhomogenously irradiated, a clear pattern of the absorbed dose distribution is visible to the naked eye. Performance of quantitative 2D analysis with the aid of a flat-bed document scanner was proposed. Most importantly, the application of a scanner is an easy method for the assessment of irradiated samples. Therefore, scanning parameters such as resolution, sharpness, scanning reproducibility and sample preparation were assessed in this work; and optimal parameters were chosen. The cause of uncertainty in the measurements is discussed.

Synthesis, crystal structure and superoxide dismutase activity of two new bis(μ-acetato/μ-nitrato) bridged copper(II) complexes with N′-[phenyl(pyridin-2-yl)methylidene]benzohydrazone

Two homodinuclear copper(II) end-on bis(μ-acetato/μ-nitrato) bridged complexes with two NNO donor ligands, viz., [(L)Cu(μ-CH3COO)2Cu(L)] (1) and [(L)Cu(μ-NO3)2Cu(L)] (2) (where L=N′-[phenyl(pyridin-2-yl)methylidene]benzohydrazone) are reported. Both complexes are structurally characterized using single crystal X-ray diffraction studies and belong to monoclinic crystal system having space group P21/n. The distance between two copper centers is 3.344(8) and 3.406(7)Å respectively for 1 and 2. On the basis of density function theory (DFT) calculations, the electronic excitations involve transitions mainly from metal–ligand bonding MOs to the β-LUMO within the dominant Cu dxy character and to β-LUMO+1. Epr spectra for polycrystalline samples exhibited the copper(II) hyperfine structures as well as zero-field splitting which are appropriate for triplet state of such a dimmers. Complexes effectively catalyze the dismutation of superoxide (O2−) in alkaline nitro blue tetrazolium chloride assay and IC50 values were determined.

Determination of manganese(VII) as 3,3′-Dianisole-4,4′-bis(3,5-diphenyltetrazolium chloride) complex and its application

A simple, rapid, selective and sensitive spectrophotometric method is described for the determination of trace amounts of manganese using Blue tetrazolium chloride as a chromogenic reagent. The method is based on the formation of ion-associate complex between manganese(VII) and the cation of ditetrazolium salt, having an absorption maximum at 255 nm. Beer’s law is obeyed in the range of 0.1–1.6 μg mL−1. The molar absorptivity and Sandell’s sensitivity were found to be 4.97 × 104 L mol−1 cm−1 and 1.11 × 10−3 μg cm−2, respectively. Limit of deterction is 6.86 ng mL−1 Mn(VII) and limit of quantitation is 22.8 ng mL−1 Mn(VII). Optimum reaction conditions were evaluated. The effect of interfering ions on the determination is described. The extraction, distribution and association constants, and the recovery factor have been calculated.

Phenylmethoxybis(tetrazolium) ion-association complexes with an anionic indium(III) — 4-(2-pyridylazo)resorcinol chelate

Abstract Complex formation and liquid-liquid extraction were studied in systems containing indium(III), 4-(2-pyridylazo)resorcinol (PAR), phenylmethoxybis(tetrazolium) salt (MBT), water and chloroform. The following MBTs, which differ only by the number of -NO2 groups in their cationic parts, were used: 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis(2,5-diphenyl-2H-tetrazolium chloride) (Blue Tetrazolium chloride, BT), 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2-(4-nitrophenyl)-5-phenyl-2H-tetrazolium chloride] (Nitro Blue Tetrazolium chloride, NBT) and 3,3′-(3,3′-dimetoxy-4,4′-biphenylene)bis[2,5-di(4-nitrophenyl)-2H-tetrazolium chloride] (Tetranitro Blue Tetrazolium chloride, TNBT). The composition of the formed ternary complexes was determined, In:PAR:MBT=1:2:2, and the optimum conditions for their extraction found: pH, shaking time, concentration of the reagents and the sequence of their addition. Some key constants were estimated: constants of extraction (Kex), constants of association (β) and constants of distribution (KD). BT appears to be the best MBT for extraction of the In(III)-PAR species, [In3+(OH)3(PAR)2]4−, (Log Kex=10.9, Log β=9.8, Log KD=1.12, R%=92.7%). Several additional characteristics concerning its application as extraction-spectrophotometric reagent were calculated: limit of detection (LOD = 0.12 µg cm−3), limit of quantification (LOD = 0.40 µg cm−3) and Sandell’s sensitivity (SS =1.58 ng cm−2); Beer’s law is obeyed for In(III) concentrations up to 3.2 µg mL−1 with a molar absorptivity coefficient of 7.3×104 L mol−1 cm−1 at λmax=515 nm.

Polyamide woven fabrics with 2,3,5-triphenyltetrazolium chloride or nitro blue tetrazolium chloride as 2D ionizing radiation dosimeters

Abstract The development of flat woven fabric-based ionizing radiation 2D dosimeters is reported in this work. Polyamide fabric was surface modified with radiation-sensitive 2,3,5-triphenyltetrazolium chloride (TTC) or nitro blue tetrazolium chloride (NBT). These samples responded to gamma radiation of 60 Co through a colour change: red and blue for TTC and NBT, respectively. The tinge intensity was observed to depend on the absorbed dose, which allowed for the reflectance of light ( R ) measurements and calculation of the calibration parameters: dose range, quasi-linear range, threshold dose and sensitivity. Oxygen was shown to be an important factor determining the dose response of the samples. For this reason, a range of additional modifications to the TTC- and NBT-polyamide fabric was proposed which lead to a decrease in the threshold dose and increase in the sensitivity to irradiation of the samples. For instance, a dosimeter made of polyamide fabric modified with 10 g/dm 3 TTC, 0.5 mol/dm 3 tert-butyl alcohol, 7.5% gelatine hydrogel at pH 10 (vacuum packed) showed the lowest dose threshold (50 Gy), dose range up to 2.8 kGy and the highest sensitivity to irradiation (−0.0396%/Gy) among the compositions studied. In consequence, this dosimeter was examined in terms of response to inhomogeneous irradiation from a 192 Ir brachytherapy radiation source. The relative dose distribution profiles across the source's longitudinal axis were calculated. This showed potential of the textile dosimeters for 2D dose distribution measurements; however, further modifications towards improvement of the dosimeter's low dose response can be considered.

Spectrophotometric Determination of Carbon Disulphide in the Workplace Air

This papre describes a simple method of carbon disulphide determination in the air of working environment in the chemical companies and plants after its absorption into aprotic N,N‐dimethylformamide solvent. Carbon disulphide absorbed into aprotic solvent was transformed by using ammonium hydroxide on sulphides which were determined by spectrophotometry. 5,5′‐Dithiobis(2‐nitrobenzoic acid) and blue tetrazolium chloride were used as chromogenic sensing reagents. Colour‐reducing products were measured at the wavelength of 500, respectively 520 nm. Detection limits for determination of carbon disulphide in the air are 0.2, respectively 0.4 mg·m−3.

Embedding Embryos for High-Resolution Episcopic Microscopy (HREM)

Episcopic fluorescence image capturing (EFIC) and high-resolution episcopic microscopy (HREM) are related techniques that are used to generate digital volume data and create three-dimensional (3D) images. Both techniques require specimens that are embedded in an appropriate medium, and images are captured from successive sections before removal from the embedded tissue block. EFIC detects autofluorescence emitted from the embedded tissue, whereas HREM requires the tissue to be stained with a fluorescent dye such as eosin. Different procedures are therefore necessary for embedding tissue for EFIC or HREM imaging. For HREM, the choice of fixative appears to be of little consequence. If gene expression patterns are to be visualized in addition to tissue architecture, whole-mount staining for gene expression patterns using the 4-nitro blue tetrazolium chloride (NBT)/5-bromo-4-chloro-3-indolyl phosphate (BCIP) or LacZ detection system must be performed before embedding of samples. This protocol describes the procedure for embedding E11.5 mouse embryos for HREM imaging. Processing times should be adapted for the size and nature of other specimens.

L-Galactono-1,4-lactone dehydrogenase (GLDH) Forms Part of Three Subcomplexes of Mitochondrial Complex I in Arabidopsis thaliana

L-galactono-1,4-lactone dehydrogenase (GLDH) catalyzes the terminal step of the Smirnoff-Wheeler pathway for vitamin C (l-ascorbate) biosynthesis in plants. A GLDH in gel activity assay was developed to biochemically investigate GLDH localization in plant mitochondria. It previously has been shown that GLDH forms part of an 850-kDa complex that represents a minor form of the respiratory NADH dehydrogenase complex (complex I). Because accumulation of complex I is disturbed in the absence of GLDH, a role of this enzyme in complex I assembly has been proposed. Here we report that GLDH is associated with two further protein complexes. Using native gel electrophoresis procedures in combination with the in gel GLDH activity assay and immunoblotting, two mitochondrial complexes of 470 and 420 kDa were identified. Both complexes are of very low abundance. Protein identifications by mass spectrometry revealed that they include subunits of complex I. Finally, the 850-kDa complex was further investigated and shown to include the complete "peripheral arm" of complex I. GLDH is attached to a membrane domain, which represents a major fragment of the "membrane arm" of complex I. Taken together, our data further support a role of GLDH during complex I formation, which is based on its binding to specific assembly intermediates.

Synthesis, characterization, and biological activity of nickel(II) complexes with a tridentate Schiff base derived from heterocyclic aldehyde

A series of nickel(II) complexes, namely [Ni(L)2] (1), [Ni(L)(HL)](ClO4)(H2O) (2), [Ni(HL)(bipy)(H2O)](NO3)(ClO4)(H2O) (3), and [Ni(HL)(dien)](ClO4)2(H2O) (4) have been synthesized with Schiff base (L) derived from 2-pyridinecarboxaldehyde and benzoylhydrazine. The elemental analyses of the complexes indicate stoichiometry ML2 and M(L)(B), where L = N-[(1)-pyridin-2-ylmethylidene]benzohydrazide, B = diethylenetriamine/2,2′-bipyridine. L is a deprotonated as well as neutral tridentate ligand. Single-crystal X-ray structures of 1–4 reveal distorted octahedral geometry in the complexes. The molecules are connected by various hydrogen-bonding interactions. Magnetic susceptibility measurements at room temperature were 2.79–2.91 MB. The electrochemical behavior, superoxide dismutase, and antibacterial activities of these complexes were made by cyclic voltammetry, the alkaline DMSO-nitro blue tetrazolium chloride assay, and the paper disc diffusion method, respectively.

Enhancing Effect of Alcoholic Solvent on Hydrosulfite–Hydrogen Peroxide Chemiluminescence System

In this work, a chemiluminescence (CL) reaction between hydrogen peroxide (H(2)O(2)) and sodium hydrosulfite (NaHSO(3)) was developed. Hydroxyl radical ((•)OH) and sulfite radical ((•)SO(3)(-)) were the main intermediates generated in the NaHSO(3)-H(2)O(2) CL system. Inhibition effects of radical scavengers such as thiourea, chloride ion, nitro blue tetrazolium chloride (NBT), and 5,5-dimethyl-1-pyrroline N-oxide (DMPO) indicated the existence of these two radicals. Singlet oxygen ((1)O(2)) and excited sulfur dioxide (SO(2)*) were emitting species involved in NaHSO(3)-H(2)O(2) CL system. (1)O(2) were confirmed by 1,4-diazobicyclo[2,2,2]octane (DABCO) and sodium azide (NaN(3)), which were specific (1)O(2) scavengers. In addition, electron spin resonance (ESR) spectra clearly show the existence of (1)O(2) and (•)OH. Alcoholic solvent, especially n-butanol, enhanced the ultraweak CL emission more than 40 times. The enhancing effect of alcoholic solvent on NaHSO(3)-H(2)O(2) CL system was ascribed to the formation of solvent cage, which can accelerate the reaction rate and protect the emitting species from quenching by water. The CL emission of the NaHSO(3)-n-butanol-H(2)O(2) system was measured by cutoff filters. The maximum wavelength was located around 490 nm, which belongs to (1)O(2). The wide peak from 400 to 600 nm is the characteristic peak of SO(2)*.

Combined postconditioning with ischemia and α7nAChR agonist produces an enhanced protection against rat myocardial ischemia reperfusion injury.

Inflammation is one of important mechanisms for myocardial ischemia reperfusion injury (IRI). Ischemia postconditioning (IPOC) can protect the heart against IRI by inhibiting inflammation, but its cardioprotection is weaker than that of ischemia preconditioning. Recently, the α7 subunit-containing nicotinic acetylcholine receptor (α7nAChR) agonist has shown anti-inflammatory effects in many diseases related to inflammation. This randomized controlled experiment was designed to evaluate whether combined postconditioning with IPOC and the α7nAChR agonist could produce an enhanced cardioprotection in a rat in vivo model of acute myocardial IRI. Fifty Sprague-Dawley rats were randomly divided into five equal groups: sham group, control group, IPOC group, α7nAChR agonist postconditioning group (APOC group) and combined postconditioning with IPOC and α7nAChR agonist group (combined group). Hemodynamic parameters were recorded during the periods of ischemia and reperfusion. Serum concentrations of troponin I (TnI), tumor necrosis factor α (TNF-α) and high-mobility group box 1 (HMGB-1) at 180 minutes after reperfusion were assayed in all groups. At the end of the experiment, the infarct size was assessed from excised hearts by Evans blue and triphenyl tetrazolium chloride staining. As compared to the sham group, the infarct size in the other four groups was significantly increased, serum levels of TnI, TNF-α and HMGB1 in the control group and TNF-α, HMGB1 in the IPOC group were significantly increased. The infarct size and serum concentrations of TNF-α, HMGB1 and TnI in the IPOC, APOC and combined groups were significantly lower than those in the control group. As compared to the IPOC group, the infarct size in the combined group was significantly decreased, serum concentrations of TnI, TNF-α and HMGB1 in the APOC and combined groups were significantly reduced. Although the infarct size was significantly smaller in the combined group than in the APOC group, serum levels of TNF-α and HMGB1 were significantly higher in the combined group than in the APOC group. In a rat in vivo model of acute myocardial IRI, combined postconditioning with IPOC and the α7nAChR agonist can produce enhanced protection against myocardial IRI by increasing the anti-inflammatory effect.

Abstract P302: CaMKII Is Not Critical for Ischemia/Reperfusion Injury

Objectives: The role of the Calcium/calmodulin-dependent protein kinase II (CaMKII) in Ischemia/Reperfusion of the heart (I/R) has not completely been clarified yet - partly due to lacking evidence from animal studies with specific gene knockout (KO) strategies. Moreover, the contribution of the nuclear and cytosolic CaMKIIδ splice variants δB and δC to I/R-induced myocardial damage have been controversially discussed. To investigate the role of CaMKII in myocardial I/R we used several KO mice with genetic deletion of the two major isoforms expressed in the heart, CaMKIIδ and CaMKIIγ, respectively. Both single and double KO models were examined. In addition, we developed a reliable approach to express CaMKII δB and δC splice variants in CaMKIIδ-null mice using adeno-associated-virus serotype 9 (AAV9) to investigate their specific roles in I/R. Methods and Results: A surgical in vivo I/R model was used following two protocols: 30 minutes ischemia with 48 hours of subsequent reperfusion (30/48) and 60 minutes ischemia followed by a 24 hour reperfusion period (60/24). Planimetric measurements of infarct size after staining with Evans Blue and Triphenyl tetrazolium chloride showed no significant difference between CaMKII KO and control groups (infarct-zone/area-at-risk ratio for 30/48-protocol: CaMKIIγ −/− vs. C57/Bl6: 0.49 ± 0.04 vs. 0.43 ± 0.05, p=0.43, n≥6; CaMKIIδ −/− vs. C57/Bl6: 0.42 ± 0.04 vs. 0.44 ± 0.04, p=0.69, n≥19; infarct-zone/area-atrisk ratio for 60/24-protocol: CaMKIIδ −/− vs. C57/Bl6: 0.52 ± 0.02 vs. 0.56 ± 0.03, p=0.33, n≥8; CaMKIIγ loxP/loxP δ loxP/loxP ; alphaMHC-Cre vs. CaMKIIγ loxP/loxP δ loxP/loxP : 0.52 ± 0.03 vs. 0.52 ± 0.04, p=0.86, n≥9). Consistently, no differences in high-sensitive serum troponin T levels 24 hours after onset of ischemia could be detected. Neither histologic TUNEL-staining nor Caspase3/7-acivity assays revealed any significant differences in apoptotic cell death processes. Importantly, specific expression of CaMKII δB or δC splice variants via AAV9 gene transfer - at approximate endogenous protein levels - in CaMKIIδ −/− mice had no effect on infarct size and necrotic or apoptotic markers. Conclusion: Our findings indicate that CaMKII is not critical for myocardial infarct size and apoptosis in response to I/R.

Enhancement of Ultraweak Chemiluminescence from Reaction of Hydrogen Peroxide and Bisulfite by Water-Soluble Carbon Nanodots

In this work, carbon nanodots were synthesized through a novel solvothermal route, and the effects of carbon nanodots on the ultraweak chemiluminescence (CL) reaction of hydrogen peroxide (H2O2) and sodium bisulfite (NaHSO3) were explored for the first time. It was found that the CL emission intensity of H2O2–HSO3– was significantly enhanced by carbon nanodots: about 60-fold increase in the CL intensity was obtained. The enhanced CL was induced by the excited-state carbon nanodots (CD*), which could be produced from the electron-transfer annihilation of positively charged carbon nanodots (CD•+) and negatively charged carbon nanodots (CD•–). Radical scavengers such as nitro blue tetrazolium chloride (NBT), sodium azide, thiourea, 5,5-dimethyl-1-pyrroline N-oxide, and ascorbic acid were used to study the intermediate species. The intermediate radicals generated during the reaction of H2O2 and NaHSO3, such as hydroxide radical (•OH), sulfate anionic radical (SO4•–), superoxide anionic radical (•O2–), and sul...

UV dosimeter based on polyamide woven fabric and nitro blue tetrazolium chloride as an active compound

This paper reports on the preparation and features of a UV light dosimeter composed of nitro blue tetrazolium chloride (NBT) and polyamide woven fabric. This textile dosimeter is based on the conversion reaction of NBT into formazan, which was initially examined in aerated aqueous solutions through steady state UV irradiation. Irradiated solutions change their colour as a consequence of the formation of polydisperse NBT formazan particles. This was analysed in relation to the absorbed dose of UV light through UV–VIS spectrophotometry and dynamic laser light scattering measurements. When NBT substrate molecules are embedded in polyamide textile, UV irradiation leads to similar effects as in solution. However, the tinge intensity changes at much lower absorbed doses. The dependence of the tinge intensity on the absorbed dose was followed by measurements of the remission of light from the NBT-polyamide samples. Consequently, the calibration parameters were calculated such as the dose sensitivity, dose range, and quasi-linear dose range. An improvement of the NBT-polyamide samples by application of a colour levelling agent and improvement of their resistance to humidity is presented. Finally, the samples were used for estimation of absorbed UV energy distribution showing their capability as new dosimeters for in-plane high resolution radiation dose measurements. ► Preparation of a textile dosimeter with nitro blue tetrazolium chloride is shown. ► The dosimeter responds to UV light by a colour change. ► 2D radiation dose measurements are possible. ► PC scanners can be employed for measurements of the dosimeter.

N-Acetylaspartylglutamate Synthetase II Synthesizes N-Acetylaspartylglutamylglutamate

N-Acetylaspartylglutamate (NAAG) is found at high concentrations in the vertebrate nervous system. NAAG is an agonist at group II metabotropic glutamate receptors. In addition to its role as a neuropeptide, a number of functions have been proposed for NAAG, including a role as a non-excitotoxic transport form of glutamate and a molecular water pump. We recently identified a NAAG synthetase (now renamed NAAG synthetase I, NAAGS-I), encoded by the ribosomal modification protein rimK-like family member B (Rimklb) gene, as a member of the ATP-grasp protein family. We show here that a structurally related protein, encoded by the ribosomal modification protein rimK-like family member A (Rimkla) gene, is another NAAG synthetase (NAAGS-II), which in addition, synthesizes the N-acetylated tripeptide N-acetylaspartylglutamylglutamate (NAAG(2)). In contrast, NAAG(2) synthetase activity was undetectable in cells expressing NAAGS-I. Furthermore, we demonstrate by mass spectrometry the presence of NAAG(2) in murine brain tissue and sciatic nerves. The highest concentrations of both, NAAG(2) and NAAG, were found in sciatic nerves, spinal cord, and the brain stem, in accordance with the expression level of NAAGS-II. To our knowledge the presence of NAAG(2) in the vertebrate nervous system has not been described before. The physiological role of NAAG(2), e.g. whether it acts as a neurotransmitter, remains to be determined.

Enhanced Colorimetric Detection on Porous Microarrays Using in Situ Substrate Production

A new technique is reported for the enhanced colorimetric detection of multiplexed hybridization onto porous membrane-based microarrays. This approach combines the use of horseradish peroxidase (HRP) as a label together with a chromogen substrate and a local production of the hydrogen peroxide required for substrate oxidation. This in situ production of coreagent is obtained using glucose oxidase (GOx) directly immobilized within the microarray porous membrane mesh. The oxidation of glucose by the immobilized GOx produces hydrogen peroxide which itself enables the oxidation of TMB (3,3',5,5'-tetramethylbenzidine) by HRP and yields a blue precipitate on positive spots. Thanks to a coreagent overconcentration within the membrane, this design drastically surpasses the performances of the standard TMB/H(2)O(2) kit used for peroxidase label detection. The obtained target limit of detection is then 50 times lower (20 pM) than the one obtained with the standard kit approach, and the dynamic range expands at least one decade. Furthermore, the developed method was shown to compete well with the widely used alkaline phosphatase-BCIP (5-bromo-4-chloro-3-indolyl phosphate)/NBT (nitro blue tetrazolium chloride) readout while minimizing background signal. The method was finally successfully applied to the multiplex detection of single nucleotide polymorphisms (SNPs) in complex PCR samples. The background lowering was impacted here positively on the SNPs' detection by increasing the complementary/noncomplementary signal ratio.

Functional and developmental expression of a zebrafish Kir1.1 (ROMK) potassium channel homologue Kcnj1

The zebrafish, Danio rerio, is emerging as an important model organism for the pathophysiological study of some human kidney diseases, but the sites of expression and physiological roles of a number of protein orthologues in the zebrafish nephron remain mostly undefined. Here we show that a zebrafish potassium channel is orthologous to the mammalian kidney potassium channel, ROMK. The cDNA (kcnj1) encodes a protein (Kcnj1) that when expressed in Xenopus laevis oocytes displayed pH- and Ba2+-sensitive K+-selective currents, but unlike the mammalian channel, was completely insensitive to the peptide inhibitor tertiapin-Q. In the pronephros, kcnj1 transcript expression was restricted to a distal region and overlapped with that of sodium–chloride cotransporter Nkcc, chloride channel ClC-Ka, and ClC-Ka/b accessory subunit Barttin, indicating the location of the diluting segment. In a subpopulation of surface cells, kcnj1 was coexpressed with the a1a.4 isoform of the Na+/K+-ATPase, identifying these cells as potential K+ secretory cells in this epithelium. At later stages of development, kcnj1 appeared in cells of the developing gill that also expressed the a1a.4 subunit.Morpholino antisense-mediated knockdown of kcnj1 was accompanied by transient tachycardia followed by bradycardia, effects consistent with alterations in extracellular K+ concentration in the embryo.Our findings indicate that Kcnj1 is expressed in cells associated with osmoregulation and acts as a K+ efflux pathway that is important in maintaining extracellular levels of K+ in the developing embryo.

Evaluation of antioxidant potential of Amalakayas Rasayana: A polyherbal Ayurvedic formulation

Amalakayas Rasayana (AR) is a polyherbal formulation mentioned in Ayurveda to treat aging and age-associated diseases. Being an antiaging drug, AR may have antioxidants and free radical scavenging activity to minimize free radical-induced damage which is a key cause of aging. The methanolic extract of AR was evaluated in vitro for total phenolic and tannin content, free radical scavenging activity, superoxide radical scavenging activity, and reducing power. The total phenolic content was measured using Folin-ciocalteu reagent against gallic acid [relative standard deviation (R2) = 0.998]. Total tannin was estimated using the Stephen method and was found to be 2.82% w/w. Free radical scavenging activity was measured by 2,2-diphenyl-1-picryl hydrazyl assay and R2 was 1. Superoxide radical scavenging activity was done by ethylene diamine tetra acetate and Nitro Blue Tetrazolium Chloride assays against ascorbic acid and R2 was 0.976 (EC50= 77.5 μg/ml). Ferrous reducing power was evaluated by Oyaizu method where R2 was 0.986. All studies showed that AR possesses antioxidant activity. The results of this study suggest that the antioxidant and free radical scavenging activity of AR may explain its rasayana effect and justify its use as a medicine for age associated diseases.