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Analytical Biochemistry Jul 2023This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (HO) and superoxide anion radicals (O), promoting...
This paper reports a sensitive method for assaying xanthine oxidase (XO) enzyme activity. XO produces hydrogen peroxide (HO) and superoxide anion radicals (O), promoting the development of oxidative stress-related diseases, and is inhibited by various plant extracts. XO activity is quantified by incubating enzyme samples with an appropriate xanthine concentration as the substrate. The proposed method requires XO activity to be quantified based on HO generation using a 3,3',5,5'-tetramethylbenzidine (TMB)-HO system catalyzed by cupric ions. After a 30-min incubation at 37 °C, sufficient cupric ion and TMB amounts are added. The assay produces optical signals that can be visually recognized or detected with a UV-visible spectrometer. A direct correlation was found between XO activity and the absorbance at 450 nm of the resulting di-imine (dication) yellow product. The proposed method uses sodium azide to prevent catalase enzyme interference. The new assay's function was confirmed using the TMB-XO assay and a Bland-Altman plot. The resulting correlation coefficient was 0.9976. The innovative assay was relatively precise and comparable to the comparison protocols. In conclusion, the presented method is very efficient at measuring XO activity.
Topics: Xanthine Oxidase; Hydrogen Peroxide; Superoxides; Oxidation-Reduction; Xanthine
PubMed: 37225068
DOI: 10.1016/j.ab.2023.115192 -
Pharmacological Research Dec 2023The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases....
The NLRP3 inflammasome is a supramolecular complex that is linked to sterile and pathogen-dependent inflammation, and its excessive activation underlies many diseases. Ion flux disturbance and cell volume regulation are both reported to mediate NLRP3 inflammasome activation, but the underlying orchestrating signaling remains not fully elucidated. The volume-regulated anion channel (VRAC), formed by LRRC8 proteins, is an important constituent that controls cell volume by permeating chloride and organic osmolytes in response to cell swelling. We now demonstrate that Lrrc8a, the essential component of VRAC, plays a central and specific role in canonical NLRP3 inflammasome activation. Moreover, VRAC acts downstream of K efflux for NLRP3 stimuli that require K efflux. Mechanically, our data demonstrate that VRAC modulates itaconate efflux and damaged mitochondria production for NLRP3 inflammasome activation. Further in vivo experiments show mice with Lrrc8a deficiency in myeloid cells were protected from lipopolysaccharides (LPS)-induced endotoxic shock. Taken together, this work identifies VRAC as a key regulator of NLRP3 inflammasome and innate immunity by regulating mitochondrial adaption for macrophage activation and highlights VRAC as a prospective drug target for the treatment of NLRP3 inflammasome and itaconate related diseases.
Topics: Mice; Animals; Membrane Proteins; Inflammasomes; NLR Family, Pyrin Domain-Containing 3 Protein; Anions; Mitochondria
PubMed: 38006980
DOI: 10.1016/j.phrs.2023.107016 -
Nature Communications Aug 2023Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels...
Chloride channels (CLCs) transport anion across membrane to regulate ion homeostasis and acidification of intracellular organelles, and are divided into anion channels and anion/proton antiporters. Arabidopsis thaliana CLCa (AtCLCa) transporter localizes to the tonoplast which imports NO and to a less extent Cl from cytoplasm. The activity of AtCLCa and many other CLCs is regulated by nucleotides and phospholipids, however, the molecular mechanism remains unclear. Here we determine the cryo-EM structures of AtCLCa bound with NO and Cl, respectively. Both structures are captured in ATP and PI(4,5)P bound conformation. Structural and electrophysiological analyses reveal a previously unidentified N-terminal β-hairpin that is stabilized by ATP binding to block the anion transport pathway, thereby inhibiting the AtCLCa activity. While AMP loses the inhibition capacity due to lack of the β/γ- phosphates required for β-hairpin stabilization. This well explains how AtCLCa senses the ATP/AMP status to regulate the physiological nitrogen-carbon balance. Our data further show that PI(4,5)P or PI(3,5)P binds to the AtCLCa dimer interface and occupies the proton-exit pathway, which may help to understand the inhibition of AtCLCa by phospholipids to facilitate guard cell vacuole acidification and stomatal closure. In a word, our work suggests the regulatory mechanism of AtCLCa by nucleotides and phospholipids under certain physiological scenarios and provides new insights for future study of CLCs.
Topics: Arabidopsis; Nucleotides; Protons; Nitrates; Phospholipids; Arabidopsis Proteins; Anions; Adenosine Triphosphate; Chloride Channels
PubMed: 37573431
DOI: 10.1038/s41467-023-40624-z -
Biochemistry. Biokhimiia Oct 2023Electrical signals (ESs) appearing in plants under the action of various external factors play an important role in adaptation to changing environmental conditions.... (Review)
Review
Electrical signals (ESs) appearing in plants under the action of various external factors play an important role in adaptation to changing environmental conditions. Generation of ES in higher plant cells is associated with activation of Ca2+, K+, and anion fluxes, as well as with changes in the activity of plasma membrane H+-ATPase. In the present review, molecular nature of the ion channels contributing to ESs transmission in higher plants is analyzed based on comparison of the data from molecular-genetic and electrophysiological studies. Based on such characteristics of ion channels as selectivity, activation mechanism, and intracellular and tissue localization, those ion channels that meet the requirements for potential participation in ES generation were selected from a wide variety of ion channels in higher plants. Analysis of the data of experimental studies performed on mutants with suppressed or enhanced expression of a certain channel gene revealed those channels whose activation contributes to ESs formation. The channels responsible for Ca2+ flux during generation of ESs include channels of the GLR family, for K+ flux - GORK, for anions - MSL. Consideration of the prospects of further studies suggests the need to combine electrophysiological and genetic approaches along with analysis of ion concentrations in intact plants within a single study.
Topics: Calcium; Ion Channels; Signal Transduction; Cell Membrane; Plants; Anions
PubMed: 38105018
DOI: 10.1134/S000629792310005X -
The Journal of Biological Chemistry Nov 2023Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is...
Chloride intracellular channels (CLICs) are a family of proteins that exist in soluble and transmembrane forms. The newest discovered member of the family CLIC6 is implicated in breast, ovarian, lung gastric, and pancreatic cancers and is also known to interact with dopamine-(D(2)-like) receptors. The soluble structure of the channel has been resolved, but the exact physiological role of CLIC6, biophysical characterization, and the membrane structure remain unknown. Here, we aimed to characterize the biophysical properties of this channel using a patch-clamp approach. To determine the biophysical properties of CLIC6, we expressed CLIC6 in HEK-293 cells. On ectopic expression, CLIC6 localizes to the plasma membrane of HEK-293 cells. We established the biophysical properties of CLIC6 by using electrophysiological approaches. Using various anions and potassium (K) solutions, we determined that CLIC6 is more permeable to chloride-(Cl) as compared to bromide-(Br), fluoride-(F), and K ions. In the whole-cell configuration, the CLIC6 currents were inhibited after the addition of 10 μM of IAA-94 (CLIC-specific blocker). CLIC6 was also found to be regulated by pH and redox potential. We demonstrate that the histidine residue at 648 (H648) in the C terminus and cysteine residue in the N terminus (C487) are directly involved in the pH-induced conformational change and redox regulation of CLIC6, respectively. Using qRT-PCR, we identified that CLIC6 is most abundant in the lung and brain, and we recorded the CLIC6 current in mouse lung epithelial cells. Overall, we have determined the biophysical properties of CLIC6 and established it as a Cl channel.
Topics: Animals; Humans; Mice; Anions; Chloride Channels; Chlorides; Epithelial Cells; HEK293 Cells
PubMed: 37838179
DOI: 10.1016/j.jbc.2023.105349 -
Frontiers in Immunology 2024The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been... (Review)
Review
The innate immune response represents the first-line of defense against invading pathogens. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) have been implicated in various aspects of innate immune function, which involves respiratory bursts and inflammasome activation. These reactive species widely distributed within the cellular environment are short-lived intermediates that play a vital role in cellular signaling and proliferation and are likely to depend on their subcellular site of formation. NADPH oxidase complex of phagocytes is known to generate superoxide anion radical (O ) that functions as a precursor for antimicrobial hydrogen peroxide (HO) production, and HO is utilized by myeloperoxidase (MPO) to generate hypochlorous acid (HOCl) that mediates pathogen killing. HO modulates the expression of redox-responsive transcriptional factors, namely NF-kB, NRF2, and HIF-1, thereby mediating redox-based epigenetic modification. Survival and function of immune cells are under redox control and depend on intracellular and extracellular levels of ROS/RNS. The current review focuses on redox factors involved in the activation of immune response and the role of ROS in oxidative modification of proteins in macrophage polarization and neutrophil function.
Topics: Hydrogen Peroxide; Oxidation-Reduction; Superoxides; Oxidative Stress; Hypochlorous Acid; Immunity, Innate
PubMed: 38515749
DOI: 10.3389/fimmu.2024.1359600 -
Biochemical Society Transactions Oct 2023Inorganic polyphosphate (polyP), the polymeric form of phosphate, is attracting ever-growing attention due to the many functions it appears to perform within mammalian... (Review)
Review
Inorganic polyphosphate (polyP), the polymeric form of phosphate, is attracting ever-growing attention due to the many functions it appears to perform within mammalian cells. This essay does not aim to systematically review the copious mammalian polyP literature. Instead, we examined polyP synthesis and functions in various microorganisms and used an evolutionary perspective to theorise key issues of this field and propose solutions. By highlighting the presence of VTC4 in distinct species of very divergent eucaryote clades (Opisthokonta, Viridiplantae, Discoba, and the SAR), we propose that whilst polyP synthesising machinery was present in the ancestral eukaryote, most lineages subsequently lost it during evolution. The analysis of the bacteria-acquired amoeba PPK1 and its unique polyP physiology suggests that eukaryote cells must have developed mechanisms to limit cytosolic polyP accumulation. We reviewed the literature on polyP in the mitochondria from the perspective of its endosymbiotic origin from bacteria, highlighting how mitochondria could possess a polyP physiology reminiscent of their 'bacterial' beginning that is not yet investigated. Finally, we emphasised the similarities that the anionic polyP shares with the better-understood negatively charged polymers DNA and RNA, postulating that the nucleus offers an ideal environment where polyP physiology might thrive.
Topics: Animals; Polyphosphates; Mammals; Eukaryotic Cells; Mitochondria; Biology
PubMed: 37844192
DOI: 10.1042/BST20230483 -
Chemistry (Weinheim An Der Bergstrasse,... May 2024Fluorescence imaging-guided photodynamic therapy (PDT) has attracted extensive attention due to its potential of real-time monitoring the lesion locations and... (Review)
Review
Fluorescence imaging-guided photodynamic therapy (PDT) has attracted extensive attention due to its potential of real-time monitoring the lesion locations and visualizing the treatment process with high sensitivity and resolution. Aggregation-induced emission luminogens (AIEgens) show enhanced fluorescence and reactive oxygen species (ROS) generation after cellular uptake, giving them significant advantages in bioimaging and PDT applications. However, most AIEgens are unfavorable for the application in organisms due to their severe hydrophobicity. Anion-π type AIEgens carry intrinsic charges that can effectively alleviate their hydrophobicity and improve their binding capability to cells, which is expected to enhance the bioimaging quality and PDT performance. This concept summarizes the applications of anion-π type AIEgens in fluorescence imaging, fluorescence imaging-guided photodynamic anticancer and antimicrobial therapy in recent years, hoping to provide some new ideas for the construction of robust photosensitizers. Finally, the current problems and future challenges of anion-π AIEgens are discussed.
Topics: Photochemotherapy; Photosensitizing Agents; Humans; Optical Imaging; Anions; Reactive Oxygen Species; Fluorescent Dyes; Hydrophobic and Hydrophilic Interactions; Neoplasms
PubMed: 38418406
DOI: 10.1002/chem.202400378 -
International Journal of Molecular... Oct 2023The increasing concerns over the environment and the growing demand for sustainable water treatment technologies have sparked substantial interest in the field of... (Review)
Review
The increasing concerns over the environment and the growing demand for sustainable water treatment technologies have sparked substantial interest in the field of photocatalytic dye removal. Polyoxometalates (POMs), known for their intricate metal-oxygen anion clusters, have received considerable attention due to their versatile structures, compositions, and efficient facilitation of photo-induced electron transfers. This paper provides an overview of the ongoing research progress in the realm of photocatalytic dye degradation utilizing POMs and their derivatives. The details encompass the compositions of catalysts, catalytic efficacy, and light absorption propensities, and the photocatalytic mechanisms inherent to POM-based materials for dye degradation are exhaustively expounded upon. This review not only contributes to a better understanding of the potential of POM-based materials in photocatalytic dye degradation, but also presents the advancements and future prospects in this domain of environmental remediation.
Topics: Electrons; Metals; Anions
PubMed: 37894924
DOI: 10.3390/ijms242015244 -
Journal of Chromatography. A Sep 2023Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic...
Novel grafted anion exchangers with covalently bonded hyperbranched functional layers were prepared and evaluated for the separation of monovalent standard inorganic anions and oxyhalides. Preparation of base coating included grafting highly polar N-vinylformamide to the ethylvinylbenzene-divinylbenzene (EVB-DVB) substrate surface in highly polar solvent (methanol) with subsequent hydrolysis of grafted amide polymer in basic media, which resulted in preparation of polymer chains with multiple primary amino groups. Those amino groups were used as attachment points for forming hyperbranched anion-exchange layers using 1,4-butanediol diglycidyl ether and primary mono- or diamine (methylamine or 1,3-diaminopropane, respectively). The effects of hyperbranching reaction cycles number on selectivity were evaluated which revealed that selectivity and capacity can be controlled independently for the covalently bonded stationary phases in contrast to electrostatically bonded phases. It was demonstrated that unlike for electrostatically bonded phases, the intentional increase of crosslink by using primary diamine instead of primary monoamine doesn't cause the shift of selectivity coefficients. It was also shown that crosslink distribution throughout the hyperbranched layer is an important factor determining selectivity of hyperbranched anion exchangers.
Topics: Chromatography, Ion Exchange; Anions; Amines
PubMed: 37523909
DOI: 10.1016/j.chroma.2023.464218