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 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.

Role of endothelium and hyperpolarization in CGRP-induced vasodilation of rabbit ophthalmic artery

1992 ◽  
Vol 263 (2) ◽  
pp. H359-H365 ◽  
Author(s):  
A. Zschauer ◽  
H. Uusitalo ◽  
J. E. Brayden
Keyword(s):  
Ophthalmic Artery ◽  
Muscle Membrane ◽  
Physiological Conditions ◽  
Calcitonin Gene ◽  
Electromechanical Effects ◽  
Smooth Muscle Membrane ◽  
Endothelium Derived Relaxing Factor ◽  
High Concentrations ◽  
Ophthalmic Arteries

The electromechanical effects of calcitonin gene-related peptide (CGRP) on intact and endothelium-denuded rabbit ophthalmic arteries were studied. CGRP inhibited norepinephrine (NE)-induced contractions. In intact arteries after washout of CGRP the contractile sensitivity to NE was increased. Conversely, in endothelium-denuded arteries, the relaxation induced by CGRP was prolonged, and after washout of CGRP the contractile sensitivity to NE was diminished. In intact arteries NE contractions were enhanced by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of endothelium-derived relaxing factor (EDRF) synthesis, and in the presence of L-NMMA, CGRP-induced relaxations resembled those seen in endothelium-denuded arteries. This result suggests that there is an increased EDRF synthesis in intact arteries during NE stimulation and that CGRP may inhibit either the synthesis or the activity of EDRF. High concentrations of CGRP hyperpolarized the smooth muscle membrane both in intact and endothelium-denuded arteries. Hyperpolarizations were blocked by glibenclamide, indicating that they are mediated by activation of ATP-sensitive K+ channels. However, glibenclamide had little effect on the CGRP-induced relaxation. These results suggest that in normal physiological conditions CGRP-induced relaxation of the rabbit ophthalmic artery is mediated mainly by mechanisms other than hyperpolarization.

scholarly journals Hydrogen Peroxide: Its Role in Plant Biology and Crosstalk with Signalling Networks

2018 ◽  
Vol 19 (9) ◽  
pp. 2812 ◽  
Author(s):  
Martin Černý ◽  
Hana Habánová ◽  
Miroslav Berka ◽  
Markéta Luklová ◽  
Břetislav Brzobohatý
Keyword(s):  
Hydrogen Peroxide ◽  
Meta Analysis ◽  
Sources And Sinks ◽  
Signalling Network ◽  
Signalling Molecule ◽  
Transcriptomics Data ◽  
High Concentrations ◽  
Nanomolar Range ◽  
Biology Research

Hydrogen peroxide (H2O2) is steadily gaining more attention in the field of molecular biology research. It is a major REDOX (reduction–oxidation reaction) metabolite and at high concentrations induces oxidative damage to biomolecules, which can culminate in cell death. However, at concentrations in the low nanomolar range, H2O2 acts as a signalling molecule and in many aspects, resembles phytohormones. Though its signalling network in plants is much less well characterized than are those of its counterparts in yeast or mammals, accumulating evidence indicates that the role of H2O2-mediated signalling in plant cells is possibly even more indispensable. In this review, we summarize hydrogen peroxide metabolism in plants, the sources and sinks of this compound and its transport via peroxiporins. We outline H2O2 perception, its direct and indirect effects and known targets in the transcriptional machinery. We focus on the role of H2O2 in plant growth and development and discuss the crosstalk between it and phytohormones. In addition to a literature review, we performed a meta-analysis of available transcriptomics data which provided further evidence for crosstalk between H2O2 and light, nutrient signalling, temperature stress, drought stress and hormonal pathways.

Role of high sensitivity C - Reactive protein and some of heavy metals in patients with rheumatoid arthritis

2020 ◽  
Vol 23 (16) ◽  
Author(s):  
Nashwan S. Albabawaty ◽  
Ali Y. Majid ◽  
Mohammed H. Alosami ◽  
Halla G. Mahmood
Keyword(s):  
Rheumatoid Arthritis ◽  
Heavy Metals ◽  
High Sensitivity ◽  
C Reactive Protein ◽  
Reactive Protein

Involvement of Heme Oxygenase-1 in Neuropsychiatric and Neurodegenerative Diseases

2018 ◽  
Vol 24 (20) ◽  
pp. 2283-2302 ◽  
Author(s):  
Vivian B. Neis ◽  
Priscila B. Rosa ◽  
Morgana Moretti ◽  
Ana Lucia S. Rodrigues
Keyword(s):  
Neurodegenerative Diseases ◽  
Heme Oxygenase ◽  
Therapeutic Potential ◽  
Redox Homeostasis ◽  
Antioxidant Defenses ◽  
Heme Oxygenase 1 ◽  
Physiological Conditions ◽  
And Oxidative Stress ◽  
Defensive Mechanism

Heme oxygenase (HO) family catalyzes the conversion of heme into free iron, carbon monoxide and biliverdin. It possesses two well-characterized isoforms: HO-1 and HO-2. Under brain physiological conditions, the expression of HO-2 is constitutive, abundant and ubiquitous, whereas HO-1 mRNA and protein are restricted to small populations of neurons and neuroglia. HO-1 is an inducible enzyme that has been shown to participate as an essential defensive mechanism for neurons exposed to oxidant challenges, being related to antioxidant defenses in certain neuropathological conditions. Considering that neurodegenerative diseases (Alzheimer’s Disease (AD), Parkinson’s Disease (PD) and Multiple Sclerosis (MS)) and neuropsychiatric disorders (depression, anxiety, Bipolar Disorder (BD) and schizophrenia) are associated with increased inflammatory markers, impaired redox homeostasis and oxidative stress, conditions that may be associated with alterations in HO-levels/activity, the purpose of this review is to present evidence on the possible role of HO-1 in these Central Nervous System (CNS) diseases. In addition, the possible therapeutic potential of targeting brain HO-1 is explored in this review.

Pathophysiology of Thrombosis in Peripheral Artery Disease

2020 ◽  
Vol 18 (3) ◽  
pp. 204-214 ◽  
Author(s):  
Aida Habib ◽  
Giovanna Petrucci ◽  
Bianca Rocca
Keyword(s):  
Vascular Tone ◽  
Platelet Reactivity ◽  
Coagulation Factors ◽  
Peripheral Artery ◽  
Antithrombotic Agents ◽  
Pharmacological Interventions ◽  
Physiological Conditions ◽  
Vasoactive Mediators ◽  
Artery Disease

<P>Under physiological conditions, peripheral arteries release endogenous vascular-protective and antithrombotic agents. Endothelial cells actively synthesize vasoactive mediators, which regulate vascular tone and platelet reactivity thus preventing thrombosis. Atherosclerosis disrupts homeostasis and favours thrombosis by triggering pro-thrombotic responses in the vessels, platelet activation, aggregation as well as vasoconstriction, phenomena that ultimately lead to symptomatic lumen restriction or complete occlusion. <P> In the present review, we will discuss the homeostatic role of arterial vessels in releasing vascular-protective agents, such as nitric oxide and prostacyclin, the role of pro- and anti-thrombotic vascular receptors as well as the contribution of circulating platelets and coagulation factors in triggering the pro-thrombotic response(s). We will discuss the pathological consequences of disrupting the protective pathways in the arteries and the pharmacological interventions along these pathways.</P>

Recent Progress in the Analysis of Captopril Using Electrochemical Methods: A Review

2019 ◽  
Vol 15 (3) ◽  
pp. 198-206 ◽  
Author(s):  
Sarfaraz Ahmed Mahesar ◽  
Saeed Ahmed Lakho ◽  
Syed Tufail Hussain Sherazi ◽  
Hamid Ali Kazi ◽  
Kamran Ahmed Abro ◽  
...  
Keyword(s):  
Enzyme Inhibitor ◽  
Modified Electrodes ◽  
High Sensitivity ◽  
Pharmaceutical Formulations ◽  
Inorganic Materials ◽  
Electrochemical Methods ◽  
Nano Particles ◽  
Moderate Heart Failure ◽  
Analytical Instrumentation

Background: Captopril is the synthetic dipeptide used as an angiotensin converting enzyme inhibitor. Captopril is used to treat hypertension as well as for the treatment of moderate heart failure. Analytical instrumentation and methodology plays an important role in pharmaceutical analysis. Methods: This review presents some important applications of electrochemical modes used for the analysis of captopril. So far captopril has been analyzed by using different bare and modified working electrodes with a variety of modifiers from organic and inorganic materials to various types of nano particles/materials. Results: This paper presents some of the methods which have been published in the last few years i.e. from 2003 to 2016. This review highlights the role of the analytical instrumentation, particularly electrochemical methods in assessing captopril using various working electrodes. Conclusion: A large number of studies on voltammetry noted by means of various bare and modified electrodes. Among all of the published voltammetric methods, DPV, SWV, CV and miscellaneous modes were trendy techniques used to analyze captopril in pharmaceutical formulations as well as biological samples. Electrodes modified with nanomaterials are promising sensing tools as this showed high sensitivity, good accuracy with precision as well as selectivity. In comparison to chromatographic methods, the main advantages of electrochemical methods are its cheaper instrumentation, lower detection limit and minimal or no sample preparation.

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 Role of MRI evaluation in acute secondary inability to walk in children

2021 ◽  
Vol 52 (1) ◽  
Author(s):  
K. H. Sedeek ◽  
K. Aboualfotouh ◽  
S. M. Hassanein ◽  
N. M. Osman ◽  
M. H. Shalaby
Keyword(s):  
Transverse Myelitis ◽  
High Sensitivity ◽  
Acute Disseminated Encephalomyelitis ◽  
Limb Weakness ◽  
Non Invasive ◽  
Highly Sensitive ◽  
Common Causes ◽  
Bilateral Lower Limb ◽  
Severity Of The Disease

Abstract Background Acute bilateral lower limb weakness is a common problem in children which necessitates a rapid method for diagnosis. MRI is a non-invasive imaging technique that produces high-quality images of the internal structure of the brain and spinal cord. Results MRI was very helpful in reaching rapid and prompt diagnosis in children with acute inability to walk. Acute disseminated encephalomyelitis (ADEM), Guillain–Barré syndrome (GBS), and acute transverse myelitis (ATM) were the most common causes in our study. MRI proved to be of high sensitivity in detecting the lesions and reaching the diagnosis in ADEM and GBS; however, there was no significant relation between the lesions’ size, enhancement pattern, and severity of the disease or prognosis, yet in ATM the site of the lesion and number of cord segment affection were significantly related to the severity of the disease and prognosis. Conclusion MRI is a quick tool to reach the diagnosis of children with acute secondary inability to walk, and to eliminate other differential diagnosis which is essential for proper treatment and rapid full recovery. It is highly sensitive in detecting the lesions, their site and size.

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