scholarly journals A proposed enzyme-linked hydrogen peroxide radical assay system (HORAS)

2021 ◽  
Author(s):  
EunJee Park
Keyword(s):  
Oxidation Product ◽  
Colorimetric Method ◽  
Detection Methods ◽  
H2o2 Production ◽  
Peroxide Radical ◽  
Phagocytic Cells ◽  
Cellular Assay ◽  
Esi Ms ◽  
Amplex Red

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.

scholarly journals A proposed enzyme-linked hydrogen peroxide radical assay system (HORAS)

2021 ◽  
Author(s):  
EunJee Park
Keyword(s):  
Oxidation Product ◽  
Colorimetric Method ◽  
Detection Methods ◽  
H2o2 Production ◽  
Peroxide Radical ◽  
Phagocytic Cells ◽  
Cellular Assay ◽  
Esi Ms ◽  
Amplex Red

Reactive Oxygen Species (ROS) are chemically reactive molecules that contain oxygen. ROS are naturally generated as a byproduct during mitochondrial oxidative metabolism as well as by cellular responses to a variety of inflammatory stimuli. Intracellularly formed ROS plays an important role in maintaining homeostasis and in cell signaling but, ROS are challenging to quantify. Phagocytic cells such as macrophages may produce H2O2 during the action of bacterial engulfment. Here UV-Vis versus LC-ESI-MS detection methods for an enzyme-linked, cellular assay of H2O2 production in cultured macrophages are compared. In the presence of Horseradish Peroxidase (HRP), Amplex Red (AR) reacts with H2O2 in a 1:1 stoichiometry to produce the red-fluorescent oxidation product resorufin that can be measured by UV/Vis at an absorbance of 570 nm or by LC-ESI-MS at 214 m/z [M+H]+. RAW 264.7 macrophages were stimulated by microscopic foreign particles, with the addition of 0.1mM of Amplex Red substrate and 10 ng/mL of HRP to the cellular media to enzymatically detect H2O2 production. The oxidation product resorufin can be detected by the colorimetric method as low as 50 pmol while liquid chromatography with electrospray ionization and mass spectrometry (LC-ESI-MS) was able to detect as little as 0.2 pmol in vitro. Thus, it was possible to measure low levels of H2O2 released by cells using an enzyme coupled cellular assay with LC-ESI-MS.

scholarly journals Pharmacoinformatics and UPLC-QTOF/ESI-MS-Based Phytochemical Screening of Combretum indicum against Oxidative Stress and Alloxan-Induced Diabetes in Long–Evans Rats

Molecules ◽  
2021 ◽  
Vol 26 (15) ◽  
pp. 4634
Author(s):  
Md. Shaekh Forid ◽  
Md. Atiar Rahman ◽  
Mohd Fadhlizil Fasihi Mohd Aluwi ◽  
Md. Nazim Uddin ◽  
Tapashi Ghosh Roy ◽  
...  
Keyword(s):  
Blood Glucose ◽  
Immune Modulation ◽  
Computational Study ◽  
Density Lipoprotein ◽  
Control Group ◽  
Esi Ms ◽  
Long Evans ◽  
Antidiabetic Effects

This research investigated a UPLC-QTOF/ESI-MS-based phytochemical profiling of Combretum indicum leaf extract (CILEx), and explored its in vitro antioxidant and in vivo antidiabetic effects in a Long–Evans rat model. After a one-week intervention, the animals’ blood glucose, lipid profile, and pancreatic architectures were evaluated. UPLC-QTOF/ESI-MS fragmentation of CILEx and its eight docking-guided compounds were further dissected to evaluate their roles using bioinformatics-based network pharmacological tools. Results showed a very promising antioxidative effect of CILEx. Both doses of CILEx were found to significantly (p < 0.05) reduce blood glucose, low-density lipoprotein (LDL), and total cholesterol (TC), and increase high-density lipoprotein (HDL). Pancreatic tissue architectures were much improved compared to the diabetic control group. A computational approach revealed that schizonepetoside E, melianol, leucodelphinidin, and arbutin were highly suitable for further therapeutic assessment. Arbutin, in a Gene Ontology and PPI network study, evolved as the most prospective constituent for 203 target proteins of 48 KEGG pathways regulating immune modulation and insulin secretion to control diabetes. The fragmentation mechanisms of the compounds are consistent with the obtained effects for CILEx. Results show that the natural compounds from CILEx could exert potential antidiabetic effects through in vivo and computational study.

The in vitro effect of kynurenic acid on the rainbow trout (Oncorhynchus mykiss) leukocyte and splenocyte activity

2014 ◽  
Vol 17 (3) ◽  
pp. 453-458 ◽  
Author(s):  
J. Małaczewska ◽  
A. K. Siwicki ◽  
R. Wójcik ◽  
W. a. Turski ◽  
E. Kaczorek
Keyword(s):  
Rainbow Trout ◽  
Immune Cells ◽  
Kynurenic Acid ◽  
Kynurenine Pathway ◽  
Phagocytic Cells ◽  
Mitogenic Response ◽  
Tryptophan Degradation ◽  
Selective Ligand ◽  
Vitro Effect

Abstract Kynurenic acid (KYNA), an endogenous neuroprotectant formed along the kynurenine pathway of tryptophan degradation, is a selective ligand of the GPR35 receptor, which can be found on the surface of various populations of human immune cells. In infections and inflammations, KYNA produces an anti-inflammatory effect through this receptor, by depressing the synthesis of reactive oxygen species and pro-inflammatory cytokines. However, it is still unrecognized whether receptors for kynurenic acid are also localized on immune cells of poikilothermic animals, or whether KYNA is able to affect these cells. The objective of this study has been to determine the effect of different concentrations of kynurenic acid (12.5 μM to 10 mM) on the viability and mitogenic response of lymphocytes and on the activity of phagocytic cells isolated from blood and the spleen of rainbow trout. The results imply low toxicity of kynurenic acid towards fish immune cells, and the proliferative effect observed at the two lowest concentrations of KYNA (12.5 μM and 25 μM) seems indicative of endogenous kynurenic acid being capable of activating fish lymphocytes. Non-toxic, micromole concentrations of KYNA, however, had no influence on the mitogenic response of lymphocytes nor on the activity of phagocytes in rainbow trout under in vitro conditions. There is some likelihood that such an effect could be observed at lower, nanomole concentrations of KYNA.

scholarly journals Macrophage oxygen-dependent antimicrobial activity. III. Enhanced oxidative metabolism as an expression of macrophage activation.

1980 ◽  
Vol 152 (6) ◽  
pp. 1596-1609 ◽  
Author(s):  
H W Murray ◽  
Z A Cohn
Keyword(s):  
Antimicrobial Activity ◽  
Oxidative Metabolism ◽  
Macrophage Activation ◽  
Systemic Infection ◽  
Peritoneal Macrophages ◽  
H2o2 Production ◽  
Oxygen Intermediates ◽  
Cultivation In Vitro

The capacity of 15 separate populations of mouse peritoneal macrophages to generate and release H2O2 (an index of oxidative metabolism) was compared with their ability to inhibit the intracellular replication of virulent Toxoplasma gondii. Resident macrophages and those elicited by inflammatory agents readily supported toxoplasma multiplication and released 4-20X less H2O2 than macrophages activated in vivo by systemic infection with Bacille Calmette-Guérin or T. gondii, or by immunization with Corynebacterium parvum. Immunologically activated cells consistently displayed both enhanced H2O2 production and antitoxoplasma activity. Exposure to lymphokines generated from cultures of spleen cells from T. gondii immune mice and toxoplasma antigen preserved both the antitoxoplasma activity and the heightened H2O2 release of toxoplasma immune and immune-boosted macrophages, which otherwise were lost after 48-72 h of cultivation. In vitro activation of resident and chemically-elicited cells by 72 h of exposure to mitogen- and antigen-prepared lymphokines, conditions that induce trypanocidal (5) and leishmanicidal activity (14), stimulated O2- and H2O2 release, and enhanced nitroblue tetrazolium reduction in response to toxoplasma ingestion. Such treatment, however, failed to confer any antitoxoplasma activity, indicating that intracellular pathogens may vary in their susceptibility to macrophage microbicidal mechanisms, including specific oxygen intermediates. In contrast, cocultivating normal macrophages with lymphokine plus heart infusion broth for 18H rendered these cells toxoplasmastatic. This in vitro-acquired activity was inhibited by scavengers of O2-, H2O2, OH., and 1O2, demonstrating a role for oxidative metabolites in lymphokine-induced enhancement of macrophage antimicrobial activity. These findings indicate that augmented oxidative metabolism is an consistent marker of macrophage activation, and that oxygen intermediates participate in the resistance of both in vivo- and vitro-activated macrophages toward the intracellular parasite, T. gondii.

Telithromycin: The First of the Ketolides

2002 ◽  
Vol 36 (3) ◽  
pp. 452-464 ◽  
Author(s):  
Christopher S Shain ◽  
Guy W Amsden
Keyword(s):  
Respiratory Tract ◽  
Clinical Efficacy ◽  
Respiratory Tract Infections ◽  
Safety Issue ◽  
Qtc Interval ◽  
Phagocytic Cells ◽  
Medline Search ◽  
Atypical Pathogens ◽  
Tract Infections

OBJECTIVE: To review the chemistry, spectrum of activity, pharmacology, clinical efficacy, and safety of telithromycin. DATA SOURCES: A MEDLINE search from 1966 to December 2000 was performed via OVID and PubMed using the following search terms: HMR 3647, HMR3647, Ketek, RU 66647, and telithromycin. An extensive review of retrieved literature, abstracts from international scientific conferences, and minutes from regulatory authority meetings was also performed. DATA EXTRACTION: Medicinal chemistry, in vitro, animal, and human trials were reviewed for information on the antimicrobial activity, clinical efficacy, pharmacology, and safety of telithromycin. DATA SYNTHESIS: Several chemical modifications to the macrolide structure have led to the development of telithromycin, the first ketolide antimicrobial that demonstrates improved activity against penicillin- and macrolide/azalide-resistant Streptococcus pneumoniae due to its unique binding to the ribosomal target site. Although telithromycin may be useful in the treatment of community-acquired respiratory tract infections due to its activity against common typical and atypical pathogens, questions concerning its reliable activity against Haemophilus influenzae need to be addressed. Telithromycin's pharmacokinetics permit once-daily dosing for abbreviated periods and good distribution into lung tissue and phagocytic cells. Clinical and bacteriologic cure rates have been similar to those of comparator agents in human efficacy trials; however, the incidence of adverse gastrointestinal events were generally higher with telithromycin patients. Like other macrolides and many newer fluoroquinolones, telithromycin's ability to prolong the QTc interval is a potential safety issue, especially in elderly patients with predisposing conditions or those who are concurrently receiving drugs that are substrates for CYP2D6 and 3A4. Liver function test elevations demonstrated during clinical trials, although not overtly severe, may warrant monitoring in some patients taking multiple hepatically metabolized/cleared agents. CONCLUSIONS: Telithromycin offers potential advantages over traditional macrolides/azalides for community-acquired respiratory tract infections caused by macrolide-resistant pathogens. Further studies are needed to elucidate its clinical efficacy against H. influenzae, potential drug interactions, and safety in various subpopulations.

GC-MS and LC-ESI-MS analysis of biologically active fractions from Verbascum letourneuxii; efficient protocol for in vitro propagation

2020 ◽  
Vol 29 ◽  
pp. 101817
Author(s):  
Mai M. Farid ◽  
Alia Y. Ragheb ◽  
Mona El-Shabrawy ◽  
Mona M. Marzouk ◽  
Sameh R. Hussein ◽  
...  
Keyword(s):  
In Vitro Propagation ◽  
Biologically Active ◽  
Esi Ms ◽  
Ms Analysis

Cigarette smoking, emphysema, and damage to alpha 1-proteinase inhibitor

1994 ◽  
Vol 266 (6) ◽  
pp. L593-L611 ◽  
Author(s):  
M. D. Evans ◽  
W. A. Pryor
Keyword(s):  
Cigarette Smoke ◽  
Proteinase Inhibitor ◽  
Inflammatory Cells ◽  
Lavage Fluid ◽  
Lung Lavage ◽  
Tissue Destruction ◽  
Phagocytic Cells ◽  
Lung Fluid

The proteinase-antiproteinase theory for the pathogenesis of emphysema proposes that the connective tissue destruction associated with emphysema arises from excessive proteinase activity in the lower respiratory tract. For this reason, the relative activities of neutrophil elastase and alpha 1-proteinase inhibitor (alpha 1-PI) are considered important. Most emphysema is observed in smokers; therefore, alpha 1-PI has been studied as a target for smoke-induced damage. Damage to alpha 1-PI in lung fluid could occur by several mechanisms involving species delivered to the lung by cigarette smoke and/or stimulated inflammatory cells. Oxidative damage to alpha 1-PI has received particular attention, since both cigarette smoke and inflammatory cells are rich sources of oxidants. In this article we review almost two decades of research on mechanistic studies of damage to alpha 1-PI by cigarette smoke and phagocytic cells in vitro, studies emphasizing the importance of elastinolytic activity in the pathogenesis of emphysema in vivo and studies of human lung lavage fluid to detect defects in alpha 1-PI at the molecular and functional levels.

scholarly journals Triple-Helical DNA in Drosophila Heterochromatin

Cells ◽  
2018 ◽  
Vol 7 (12) ◽  
pp. 227 ◽  
Author(s):  
Eduardo Gorab
Keyword(s):  
Nucleic Acids ◽  
Dna Sequences ◽  
Detection Methods ◽  
Triplex Dna ◽  
Thiazole Orange ◽  
Chromosomal Dna ◽  
Mitotic Chromosomes ◽  
Satellite Repeats

Polynucleotide chains obeying Watson-Crick pairing are apt to form non-canonical complexes such as triple-helical nucleic acids. From early characterization in vitro, their occurrence in vivo has been strengthened by increasing evidence, although most remain circumstantial particularly for triplex DNA. Here, different approaches were employed to specify triple-stranded DNA sequences in the Drosophila melanogaster chromosomes. Antibodies to triplex nucleic acids, previously characterized, bind to centromeric regions of mitotic chromosomes and also to the polytene section 59E of mutant strains carrying the brown dominant allele, indicating that AAGAG tandem satellite repeats are triplex-forming sequences. The satellite probe hybridized to AAGAG-containing regions omitting chromosomal DNA denaturation, as expected, for the intra-molecular triplex DNA formation model in which single-stranded DNA coexists with triplexes. In addition, Thiazole Orange, previously described as capable of reproducing results obtained by antibodies to triple-helical DNA, binds to AAGAG repeats in situ thus validating both detection methods. Unusual phenotype and nuclear structure exhibited by Drosophila correlate with the non-canonical conformation of tandem satellite arrays. From the approaches that lead to the identification of triple-helical DNA in chromosomes, facilities particularly provided by Thiazole Orange use may broaden the investigation on the occurrence of triplex DNA in eukaryotic genomes.

scholarly journals Mycobacterium tuberculosis Alters the Metalloprotease Activity of the COP9 Signalosome

mBio ◽  
2014 ◽  
Vol 5 (4) ◽  
Author(s):  
Lia Danelishvili ◽  
Lmar Babrak ◽  
Sasha J. Rose ◽  
Jamie Everman ◽  
Luiz E. Bermudez
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Cop9 Signalosome ◽  
Virulence Determinants ◽  
Bacterial Survival ◽  
Phagocytic Cells ◽  
Content Type ◽  
Metalloprotease Activity ◽  
Macrophage Protein

ABSTRACT Inhibition of apoptotic death of macrophages by Mycobacterium tuberculosis represents an important mechanism of virulence that results in pathogen survival both in vitro and in vivo. To identify M. tuberculosis virulence determinants involved in the modulation of apoptosis, we previously screened a transposon bank of mutants in human macrophages, and an M. tuberculosis clone with a nonfunctional Rv3354 gene was identified as incompetent to suppress apoptosis. Here, we show that the Rv3354 gene encodes a protein kinase that is secreted within mononuclear phagocytic cells and is required for M. tuberculosis virulence. The Rv3354 effector targets the metalloprotease (JAMM) domain within subunit 5 of the COP9 signalosome (CSN5), resulting in suppression of apoptosis and in the destabilization of CSN function and regulatory cullin-RING ubiquitin E3 enzymatic activity. Our observation suggests that alteration of the metalloprotease activity of CSN by Rv3354 possibly prevents the ubiquitin-dependent proteolysis of M. tuberculosis-secreted proteins. IMPORTANCE Macrophage protein degradation is regulated by a protein complex called a signalosome. One of the signalosomes associated with activation of ubiquitin and protein labeling for degradation was found to interact with a secreted protein from M. tuberculosis, which binds to the complex and inactivates it. The interference with the ability to inactivate bacterial proteins secreted in the phagocyte cytosol may have crucial importance for bacterial survival within the phagocyte.

scholarly journals Models and methods to characterise levonorgestrel release from intradermally administered contraceptives

2021 ◽  
Author(s):  
Adnan Al Dalaty ◽  
Benedetta Gualeni ◽  
Sion A. Coulman ◽  
James C. Birchall
Keyword(s):  
Ex Vivo ◽  
Extraction Procedure ◽  
In Vitro Release ◽  
Reversed Phase ◽  
Experimental Models ◽  
Detection Methods ◽  
Hormonal Contraceptives ◽  
Limit Of Quantification ◽  
Long Acting

AbstractMicroneedle (MN)-based technologies have been proposed as a means to facilitate minimally invasive sustained delivery of long-acting hormonal contraceptives into the skin. Intradermal administration is a new route of delivery for these contraceptives and therefore no established laboratory methods or experimental models are available to predict dermal drug release and pharmacokinetics from candidate MN formulations. This study evaluates an in vitro release (IVR) medium and a medium supplemented with ex vivo human skin homogenate (SH) as potential laboratory models to investigate the dermal release characteristics of one such hormonal contraceptive that is being tested for MN delivery, levonorgestrel (LNG), and provides details of an accompanying novel two-step liquid–liquid drug extraction procedure and sensitive reversed-phase HPLC–UV assay. The extraction efficiency of LNG was 91.7 ± 3.06% from IVR medium and 84.6 ± 1.6% from the medium supplemented with SH. The HPLC–UV methodology had a limit of quantification of 0.005 µg/mL and linearity between 0.005 and 25 µg/mL. Extraction and detection methods for LNG were exemplified in both models using the well-characterised, commercially available sustained-release implant (Jadelle®). Sustained LNG release from the implant was detected in both media over 28 days. This study reports for the first time the use of biologically relevant release models and a rapid, reliable and sensitive methodology to determine release characteristics of LNG from intradermally administered long-acting drug delivery systems. Graphical abstract

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