scholarly journals A novel cycling assay for nicotinic acid–adenine dinucleotide phosphate with nanomolar sensitivity

2002 ◽  
Vol 367 (1) ◽  
pp. 163-168 ◽  
Author(s):  
Richard GRAEFF ◽  
Hon Cheung LEE
Keyword(s):  
Nicotinic Acid ◽  
Hydrolytic Enzymes ◽  
High Sensitivity ◽  
Adp Ribosyl Cyclase ◽  
High Throughput Method ◽  
Plate Reader ◽  
Coupled Reactions ◽  
High Concentrations ◽  
Micromolar Range ◽  
Fluorescence Plate Reader

Nicotinic acid—adenine dinucleotide phosphate (NAADP) is a novel nucleotide derived from NADP that has now been shown to be active in releasing Ca2+ from intracellular stores in a wide variety of cells ranging from plant to human. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, no assay has been reported for NAADP to date. In the present study, a widely applicable assay for NAADP with high sensitivity is described. NAADP was first dephosphorylated to nicotinic acid—adenine dinucleotide by treatment with alkaline phosphatase. The conversion was shown to be stoichiometric. NMN-adenylyltransferase was then used to convert nicotinic acid—adenine dinucleotide into NAD in the presence of high concentrations of NMN. The resultant NAD was amplified by a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD cycled through these coupled reactions, a molecule of highly fluorescent resorufin was generated. The reaction could be performed for hours, resulting in more than a 1000-fold amplification. Concentrations of NAADP over the 10—20nM range could be routinely measured. This novel cycling assay was combined with an enzymic treatment to provide the necessary specificity for the assay. NAADP was found to be resistant to NADase and apyrase. Pretreatment of samples with a combination of the hydrolytic enzymes completely eliminated the interference from common nucleotides. The versatility of the cycling assay can also be extended to measure nicotinic acid, which is a substrate in the synthesis of NAADP catalysed by ADP-ribosyl cyclase, over the micromolar range. All the necessary reagents for the cycling assay are widely available and it can be performed using a multi-well fluorescence plate reader, providing a high-throughput method. This is the first assay reported for NAADP and nicotinic acid, which should be valuable in elucidating the messenger functions of NAADP.

scholarly journals A novel cycling assay for cellular cADP-ribose with nanomolar sensitivity

2002 ◽  
Vol 361 (2) ◽  
pp. 379-384 ◽  
Author(s):  
Richard GRAEFF ◽  
Hon Cheung LEE
Keyword(s):  
High Sensitivity ◽  
Physiological Conditions ◽  
Adp Ribosyl Cyclase ◽  
High Throughput Method ◽  
Plate Reader ◽  
Coupled Reactions ◽  
High Concentrations ◽  
Nanomolar Range ◽  
Fluorescence Plate Reader

cADP-ribose (cADPR) is a novel cyclic nucleotide derived from NAD+ that has now been established as a general Ca2+ messenger in a wide variety of cells. Despite the obvious importance of monitoring its cellular levels under various physiological conditions, its measurement has been technically difficult and requires specialized reagents. In this study a widely applicable high-sensitivity assay for cADPR is described. ADP-ribosyl cyclase normally catalyses the synthesis of cADPR from NAD+, but the reaction can be reversed in the presence of high concentrations of nicotinamide, producing NAD+ from cADPR stoichiometrically. The resultant NAD+ can then be coupled to a cycling assay involving alcohol dehydrogenase and diaphorase. Each time NAD+ cycles through these coupled reactions, a molecule of highly fluorescent resorufin is generated. The reaction can be conducted for hours, resulting in more than a thousand-fold amplification of cADPR. Concentrations of cADPR in the nanomolar range can be measured routinely. The unique ability of ADP-ribosyl cyclase to catalyse the reverse reaction provides the required specificity. Using this assay, it is demonstrated that cADPR is present in all tissues tested and that the levels measured are directly comparable with those obtained using a radioimmunoassay. All the necessary reagents are widely available and the assay can be performed using a multiwell fluorescence plate reader, providing a high-throughput method for monitoring cADPR levels. This assay should be valuable in elucidating the messenger role of cADPR in cells.

scholarly journals Electroanalytical Techniques for the Detection of Selenium as a Biologically and Environmentally Significant Analyte—A Short Review

Molecules ◽  
2021 ◽  
Vol 26 (6) ◽  
pp. 1768
Author(s):  
Miroslav Rievaj ◽  
Eva Culková ◽  
Damiána Šandorová ◽  
Zuzana Lukáčová-Chomisteková ◽  
Renata Bellová ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Essential Element ◽  
Analytical Techniques ◽  
High Sensitivity ◽  
Stripping Voltammetry ◽  
Short Review ◽  
Electroanalytical Methods ◽  
High Concentrations ◽  
Speed Of Analysis

This short review deals with the properties and significance of the determination of selenium, which is in trace amounts an essential element for animals and humans, but toxic at high concentrations. It may cause oxidative stress in cells, which leads to the chronic disease called selenosis. Several analytical techniques have been developed for its detection, but electroanalytical methods are advantageous due to simple sample preparation, speed of analysis and high sensitivity of measurements, especially in the case of stripping voltammetry very low detection limits even in picomoles per liter can be reached. A variety of working electrodes based on mercury, carbon, silver, platinum and gold materials were applied to the analysis of selenium in various samples. Only selenium in oxidation state + IV is electroactive therefore the most of voltammetric determinations are devoted to it. However, it is possible to detect also other forms of selenium by indirect electrochemistry approach.

scholarly journals The Double Face of Ketamine—The Possibility of Its Identification in Blood and Beverages

Molecules ◽  
2021 ◽  
Vol 26 (4) ◽  
pp. 813
Author(s):  
Magdalena Świądro ◽  
Paweł Stelmaszczyk ◽  
Irena Lenart ◽  
Renata Wietecha-Posłuszny
Keyword(s):  
Date Rape ◽  
Signal To Noise Ratio ◽  
High Sensitivity ◽  
Heroin Addiction ◽  
Assisted Extraction ◽  
Low Concentrations ◽  
High Concentrations ◽  
Rosé Wine ◽  
Tof Ms

The purpose of this study was to develop and validate a high-sensitivity methodology for identifying one of the most used drugs—ketamine. Ketamine is used medicinally to treat depression, alcoholism, and heroin addiction. Moreover, ketamine is the main ingredient used in so-called “date-rape” pills (DRP). This study presents a novel methodology for the simultaneous determination of ketamine based on the Dried Blood Spot (DBS) method, in combination with capillary electrophoresis coupled with a mass spectrometer (CE-TOF-MS). Then, 6-mm circles were punched out from DBS collected on Whatman DMPK-C paper and extracted using microwave-assisted extraction (MAE). The assay was linear in the range of 25–300 ng/mL. Values of limits of detection (LOD = 6.0 ng/mL) and quantification (LOQ = 19.8 ng/mL) were determined based on the signal to noise ratio. Intra-day precision at each determined concentration level was in the range of 6.1–11.1%, and inter-day between 7.9–13.1%. The obtained precision was under 15.0% (for medium and high concentrations) and lower than 20.0% (for low concentrations), which are in accordance with acceptance criteria. Therefore, the DBS/MAE/CE-TOF-MS method was successfully checked for analysis of ketamine in matrices other than blood, i.e., rose wine and orange juice. Moreover, it is possible to identify ketamine in the presence of flunitrazepam, which is the other most popular ingredient used in DRP. Based on this information, the selectivity of the proposed methodology for identifying ketamine in the presence of other components of rape pills was checked.

scholarly journals Arginine Inhibits Serpins

2007 ◽  
Vol 13 (2) ◽  
pp. 213-218
Author(s):  
Thomas W. Stief
Keyword(s):  
Esterase Activity ◽  
Serine Proteases ◽  
Biological Fluid ◽  
Microtiter Plate ◽  
Human Albumin ◽  
Amidolytic Activity ◽  
Peptide Substrates ◽  
Plate Reader ◽  
High Concentrations ◽  
Very High

Serine protease inactivators (serpins) are important regulators in biochemistry. Often it is necessary to block the serpin action, that is, to stabilize the sample. The guanidine group of arginine is the ligand for the active center pocket of many serine proteases. Arginine or guanidine inhibits serine proteases, and arginine belongs to the reactive P1-P1' center of many serpins. The plasmatic antithrombin, antiplasmin, or anti-C1-esterase activity was determined: A total of 20 µL of pooled normal plasma or 7% human albumin was added to 100 µL of 0—2.67 M arginine, pH 8.6, 10 µL of 26 mIU/mL thrombin in 7% human albumin, and 30 µL of 1.7 mM CHG-Ala-Arg-pNA (37°C). ΔA at 405 nm was determined, by using a microtiter plate reader. Thrombin was substituted by plasmin or C1-esterase, and the chromogenic peptide substrates <Glu-Phe-Lys-pNA or MeOC-Lys(eCBO)-Gly-Arg-pNA, respectively, were used. The IC50 of arginine against plasmatic antithrombin activity is 580 mM; the IC 25 is 440 mM. The IC25 of arginine against plasmatic α 2-antiplasmin or C1-inactivator is 1650 mM. The amidolytic activity of thrombin, plasmin, and C1-esterase is inhibited similarly by arginine: the IC50 for arginine against the amidolytic activity of these proteases is about 400 mM. Arginine at very high concentrations inhibits serpins. This is important, if stabilization of a biological fluid is a prerequisite for valid activities of serine proteases. In addition, these high concentrations of arginine might be a new gentle principle to inhibit pathogens that need serpins for their pathophysiology.

Fast, But Not Slow, Effects of Olivocochlear Activation Are Resistant to Apamin

2001 ◽  
Vol 85 (1) ◽  
pp. 84-88 ◽  
Author(s):  
Naohiro Yoshida ◽  
M. Charles Liberman ◽  
M. Christian Brown ◽  
William F. Sewell
Keyword(s):  
Hair Cells ◽  
Auditory Nerve ◽  
Calcium Release ◽  
High Sensitivity ◽  
High Concentrations ◽  
Efferent Suppression ◽  
In Vivo Effects ◽  
Very High ◽  
Small Conductance

Olivocochlear (OC) efferent suppression of auditory-nerve responses comprises a fast effect lasting tens of milliseconds and a slow effect building and decaying over tens of seconds. Both fast and slow effects are mediated by activation of the same alpha 9 nicotinic receptor. We have hypothesized that fast effects are generated at the OC synapse, but that slow effects reflect activation of calcium-activated potassium (KCa) channels by calcium release from the subsurface cisternae on the basolateral wall of the hair cells. We measured in vivo effects of apamin, a blocker of small-conductance (SK) KCa channels, and charybdotoxin, a blocker of large-conductance KCa channels, perfused through scala tympani, on fast and slow effects evoked by electrical stimulation of the OC bundle in anesthetized guinea pigs. Apamin selectively and reversibly reduced slow-effect amplitude without altering fast effects or baseline amplitude of the auditory-nerve response, but only when perfused at concentrations of 100 μM. In contrast, the effects of charybdotoxin were noted at 30 nM, but were not specific, reducing both afferent and efferent responses. The very high concentrations of apamin needed to block efferent effects contrasts with the high sensitivity of isolated hair cells to apamin's block of acetylcholine's effects. The results suggest that in vivo fast OC effects are dominated by a conductance that is not apamin sensitive.

Use of a Fluorescence Plate Reader for Measuring Kinetic Parameters with Inner Filter Effect Correction

1999 ◽  
Vol 267 (2) ◽  
pp. 331-335 ◽  
Author(s):  
Yaya Liu ◽  
Warren Kati ◽  
Chih-Ming Chen ◽  
Rakesh Tripathi ◽  
Akhter Molla ◽  
...  
Keyword(s):  
Kinetic Parameters ◽  
Inner Filter Effect ◽  
Filter Effect ◽  
Plate Reader ◽  
Fluorescence Plate Reader

scholarly journals Channelling of substrate promiscuity of the skeletal-muscle ADP-ribosyl cyclase isoform

2004 ◽  
Vol 381 (1) ◽  
pp. 147-154 ◽  
Author(s):  
Ingrid BACHER ◽  
Andreas ZIDAR ◽  
Martin KRATZEL ◽  
Martin HOHENEGGER
Keyword(s):  
Skeletal Muscle ◽  
Nicotinic Acid ◽  
Exchange Reaction ◽  
Second Messengers ◽  
Cyclization Reaction ◽  
Regulation Mechanism ◽  
Base Exchange ◽  
Adp Ribosyl Cyclase ◽  
Highly Sensitive ◽  
Cyclic Adp Ribose

The novel Ca2+-mobilizing second messengers cADPr (cyclic ADP-ribose) and NAADP (nicotinic acid–adenine dinucleotide phosphate) are both synthesized by ADP-ribosyl cyclases. Using HSR (heavy sarcoplasmic reticulum) fractions from rabbit skeletal muscle, NAADP-induced Ca2+ release was observed. In the present paper, we show in HSR membranes the formation of authentic cADPr, cGDPr (cyclic GDP-ribose) and NAADP. The cyclization reaction to form cADPr and cGDPr as well as the base-exchange reaction to form NAADP were strictly dependent on pH. Although the formation of cGDPr is optimized at pH 6, the synthesis of NAADP was most pronounced at a pH below 5. A novel regulation mechanism is provided for nicotinic acid, the co-substrate for NAADP synthesis. Nicotinic acid had virtually no influence on the cyclization reaction, but increased the affinity of NADP at an acidic pH and had the opposite effect at alkaline pH. Nicotinamide, the side product of cADPr synthesis, is an inhibitor of the cyclization reaction (IC50, 0.7±0.1 mM) and was 30-fold more potent at suppressing the base-exchange reaction. Although the synthesis of NAADP was highly sensitive to nicotinamide inhibition, this was not via a competition with the nicotinic-acid-binding site. In contrast with the ecto-ADP-ribosyl cyclase (CD38), the cyclization and base-exchange reaction of the skeletal muscle isoform was inhibited by Cu2+ and Zn2+, while other bivalent cations such as Ca2+, Mg2+ and Mn2+ had virtually no effect. These findings allow for the prediction of a novel ADP-ribosyl cyclase isoform in skeletal muscle HSR, other than CD38. Hence the enzymic prerequisite for cADPr- and NAADP-mediated Ca2+ signalling is present.

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