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Voltammetric and amperometric methods are attractive for the quantification of adenylic nucleotides and nucleosides once their electrochemical behavior has been widely explored. However, in a solution containing both adenosine (ADO) and adenylic nucleotides, distinguishing between the oxidation peaks of nucleoside and all nucleotides is impracticable since the voltammetric peak potentials of adenine (ADE), adenosine (ADO), adenosine monophosphate (AMP), and adenosine triphosphate (ATP) are similar at physiological pH. This work describes the modification of carbon paste electrodes in a solution containing purine bases or their derivatives and the utilization of semi-derivative analysis of cyclic voltammograms to gain insights into the formation of these modified surfaces. The carbon paste electrode modified in the adenine solution (CPE/ADE), as well as in guanine and adenosine solutions, allowed the selective detection of ADO while suppressing nearly all signals from AMP, ADP, and ATP. The suppression of the electrochemical signal of the nucleotides was associated with a poor interaction of the modified surface with negatively charged species. Differential pulse voltammetry was used to determine ADO at CPE/ADE in the range of 1.0 × 10−5 mol L−1 to 7.0 × 10−5 mol L−1 in PBS, pH 7.4 with LOD and LOQ of 0.30 μM and 1.0 μM, respectively, or LOD and LOQ of 0.20 μM and 0.67 μM, respectively, at CPE/ADO modified electrode. CPE/ADE surface is the most recommended since the LOD and LOQ are very similar to those obtained with CPE/ADO, but adenine is less expensive than adenosine. [ABSTRACT FROM AUTHOR]

"Chemical Communications" is a journal published by The Royal Society of Chemistry, focusing on various topics in the field of chemistry. The latest issue includes articles on hydrogen production from seawater electrolysis, transition metal-catalyzed reactions, antimicrobial resistance surveillance, lanthanide chemistry, and high-pressure chemistry of functional materials. The journal also covers topics such as chiral synthetic hosts, substance migration in catalyst synthesis, and the development of rare earth ions for anti-counterfeiting applications. [Extracted from the article]

Herein we present a series of luminescent Tb(III)-probes ([Tb-Ltrp], [Tb-Ltyr], and [Tb-Lphe]) for sensing and discriminating purine nucleoside polyphosphates (NPP) based on a modified DTTA chelator appended to aromatic amino acids (Laa). The optically most effective luminescent [Tb-Ltrp] probe preferentially discriminates the guanine-NPPs over the adenine-NPPs via PeT-based modulation of Tb(III) luminescence within the biological concentration range. [ABSTRACT FROM AUTHOR]

Two-pore channels (TPCs) are adenine nucleotide and phosphoinositide regulated cation channels. NAADP activates and ATP blocks TPCs, while the endolysosomal phosphoinositide PI(3,5)P2 activates TPCs. TPCs are ubiquitously expressed including expression in the innate as well as the adaptive immune system. In the immune system TPCs are found, e.g. in macrophages, mast cells and T cells. In cytotoxic T cells, NAADP activates TPCs on cytolytic granules to stimulate exocytosis and killing. TPC inhibition or knockdown increases the number of regulator T cells in a transmembrane TNF/TNFR2 dependent manner, contributing to anti-inflammatory effects in a murine colitis model. TPC1 regulates exocytosis in mast cells in vivo and ex vivo , and TPC1 deficiency in mast cells augments systemic anaphylaxis in mice. In bone marrow derived macrophages NAADP regulates TPCs to control phagocytosis in a calcineurin/dynamin dependent manner, which was recently challenged by data, claiming no effect of TPCs on phagocytosis in macrophages but instead a role in phagosome resolution, a process thought to be mediated by vesiculation and tubulation. In this review we will discuss evidence and recent findings on the different roles of TPCs in immune cell function as well as evidence for adenine nucleotides being involved in these processes. Since the adenine nucleotide effects (NAADP, ATP) are mediated by auxiliary proteins, respectively, another major focus will be on the complex network of TPC regulatory proteins that have been discovered recently. [ABSTRACT FROM AUTHOR]

Background: Sepsis-induced cardiomyopathy (SICM) often occurs in the acute phase of sepsis and is associated with increased mortality due to cardiac dysfunction. The pathogenesis remains poorly understood, and no specific treatments are available. Although SICM is considered reversible, emerging evidence suggests potential long-term sequelae. We hypothesized that metabolic and inflammatory cardiac changes, previously observed in acute sepsis as potential drivers of SICM, partially persist in prolonged sepsis. Methods: In 24-week-old C57BL/6J mice, sepsis was induced by cecal ligation and puncture, followed by intravenous fluid resuscitation, subcutaneous analgesics and antibiotics, and, in the prolonged phase, by parenteral nutrition. Mice were killed after 5 days of sepsis (prolonged sepsis, n = 15). For comparison, we included acutely septic mice killed at 30 h (acute sepsis, n = 15) and healthy controls animals (HC, n = 15). Cardiac tissue was collected for assessment of inflammatory and metabolic markers through gene expression, metabolomic analysis and histological assessment. Results: In prolonged sepsis, cardiac expression of IL-1β and IL-6 and macrophage infiltration remained upregulated (p ≤ 0.05). In contrast, tissue levels of Krebs cycle intermediates and adenosine phosphates were normal, whereas NADPH levels were low in prolonged sepsis (p ≤ 0.05). Gene expression of fatty acid transporters and of the glucose transporter Slc2a1 was upregulated in prolonged sepsis (p ≤ 0.01). Lipid staining and glycogen content were elevated in prolonged sepsis together with increased gene expression of enzymes responsible for lipogenesis and glycogen synthesis (p ≤ 0.05). Intermediate glycolytic metabolites (hexose-phosphates, GADP, DHAP) were elevated (p ≤ 0.05), but gene expression of several enzymes for glycolysis and mitochondrial oxidation of pyruvate, fatty-acyl-CoA and ketone bodies to acetyl-CoA were suppressed in prolonged sepsis (p ≤ 0.05). Key metabolic transcription factors PPARα and PGC-1α were downregulated in acute, but upregulated in prolonged, sepsis (p ≤ 0.05 for both). Ketone body concentrations were normal but ketolytic enzymes remained suppressed (p ≤ 0.05). Amino acid metabolism showed mild, mixed changes. Conclusions: Our results suggest myocardial lipid and glycogen accumulation and suppressed mitochondrial oxidation, with a functionally intact Krebs cycle, in the prolonged phase of sepsis, together with ongoing myocardial inflammation. Whether these alterations have functional consequences and predispose to long-term sequelae of SICM needs further research. [ABSTRACT FROM AUTHOR]

Copyright of Retos: Nuevas Perspectivas de Educación Física, Deporte y Recreación is the property of Federacion Espanola de Asociaciones de Docentes de Educacion Fisica and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

ENPP1/CD203a is a membrane-bound ectonucleotidase capable of hydrolyzing ATP, cGAMP and other substrates. Its enzymatic activity plays an important role in the balance of extracellular adenine nucleotides and the modulation of purinergic signaling, in soft tissue calcification, and in the regulation of the cGAS/STING pathway. However, a detailed analysis of ENPP1 surface expression on human immune cells has not been performed. Here, we selected VHH domains from human ENPP1-immunized alpacas to generate heavy-chain antibodies targeting ENPP1, and analyzed cell surface expression on all circulating immune cell subsets using flow cytometry. We find high expression of ENPP1 in CD141high conventional dendritic cells (cDC1), while ENPP1 was not detectable on other dendritic cells and monocytes. In the lymphocytic compartment, only CD56bright natural killer cells and mucosal-associated invariant T cells (MAIT) express ENPP1. In contrast, all other T cell subpopulations, CD56dim natural killer cells and B lymphocytes do not or only minimally express ENPP1. In summary, we describe highly cell type-specific expression of ENPP1 in the immune system using a newly generated heavy-chain antibody. This reagent will help to decipher the function of ENPP1 in the regulation of the immune response, allow a quick identification of ENPP1-deficiency and of ENPP1-positive tumors, and constitutes the basis for targeted anti-tumor intervention. [ABSTRACT FROM AUTHOR]

Adenine nucleotides (ANs)—adenosine 5′-triphosphate (ATP), adenosine 5′-diphosphate (ADP), and adenosine 5′-monophosphate (AMP)—are essential for energy transfer and the supply of countless processes within cellular metabolism. Their concentrations can be expressed as adenylate energy charge (AEC), a measure of cellular metabolic energy that directly correlates with the homeostasis of the organism. AEC index has broad diagnostic potential, as reduced ATP levels are associated to various conditions, such as inflammatory diseases, metabolic disorders, and cancer. We introduce a novel methodology for rapid isolation, purification, and quantification of ANs from a single drop of capillary blood. Of all the stationary phases tested, activated carbon proved to be the most efficient for the purification of adenine nucleotides, using an automated micro-solid phase extraction (µ-SPE) platform. An optimized µ-SPE method, coupled with RP-HPLC and a run time of 30 min, provides a reliable analytical framework for adenine nucleotide analysis of diverse biological samples. AN concentrations measured in capillary blood samples were 1393.1 µM, 254.8 µM, and 76.9 µM for ATP, ADP, and AMP molecules aligning with values reported in the literature. Overall, this study presents a streamlined and precise approach for analyzing ANs from microliters of blood, offering promising applications in clinical diagnostics. [ABSTRACT FROM AUTHOR]

The Pallas' spadefoot Pelobates vespertinus is a frog species native to eastern Europe and west Siberia. This species resists harsh winter conditions by moving up to 2 m underground. This amphibian is the first species known to withstand extreme air hypoxia. In this study, we investigated the metabolome of liver, heart, and brain of the Pallas' spadefoot after a month-long exposure of hypoxia, with oxygen levels reduced to approximately one-tenth of the air normal content. Surprisingly, our findings revealed a limited impact of hypoxia on the metabolomic profiles. Concentrations of glycolysis end products (lactate and alanine) increased only slightly compared to other amphibians under hypoxia, and no accumulation of succinate was observed. Furthermore, there were no notable changes in the content of adenosine phosphates. These results are consistent with a previous study, which indicated that the Pallas' spadefoot possesses relatively small glycogen and fat reserves before the winter compared to other frogs. It appears that this species conserves energy during winter by minimizing its metabolic activity. These findings corroborated the hypothesis that the survival of P. vespertinus under hypoxic conditions primarily relies on metabolic suppression rather than substantial energy reserves. [ABSTRACT FROM AUTHOR]

Diabetes mellitus is one of the major causes of ischemic and nonischemic heart failure. While hypertension and coronary artery disease are frequent comorbidities in patients with diabetes, cardiac contractile dysfunction and remodeling occur in diabetic patients even without comorbidities, which is referred to as diabetic cardiomyopathy. Investigations in recent decades have demonstrated that the production of reactive oxygen species (ROS), impaired handling of intracellular Ca2+, and alterations in energy metabolism are involved in the development of diabetic cardiomyopathy. AMP deaminase (AMPD) directly regulates adenine nucleotide metabolism and energy transfer by adenylate kinase and indirectly modulates xanthine oxidoreductase-mediated pathways and AMP-activated protein kinase-mediated signaling. Upregulation of AMPD in diabetic hearts was first reported more than 30 years ago, and subsequent studies showed similar upregulation in the liver and skeletal muscle. Evidence for the roles of AMPD in diabetes-induced fatty liver, sarcopenia, and heart failure has been accumulating. A series of our recent studies showed that AMPD localizes in the mitochondria-associated endoplasmic reticulum membrane as well as the sarcoplasmic reticulum and cytosol and participates in the regulation of mitochondrial Ca2+ and suggested that upregulated AMPD contributes to contractile dysfunction in diabetic cardiomyopathy via increased generation of ROS, adenine nucleotide depletion, and impaired mitochondrial respiration. The detrimental effects of AMPD were manifested at times of increased cardiac workload by pressure loading. In this review, we briefly summarize the expression and functions of AMPD in the heart and discuss the roles of AMPD in diabetic cardiomyopathy, mainly focusing on contractile dysfunction caused by this disorder. [ABSTRACT FROM AUTHOR]

Enzymatic mechanisms are typically inferred from structural data. However, understanding enzymes require unravelling the intricate dynamic interplay between dynamics, conformational substates, and multiple protein structures. Here, we use single-molecule nanopore analysis to investigate the catalytic conformational changes of adenylate kinase (AK), an enzyme that catalyzes the interconversion of various adenosine phosphates (ATP, ADP, and AMP). Kinetic analysis validated by hidden Markov models unravels the details of domain motions during catalysis. Our findings reveal that allosteric interactions between ligands and cofactor enable converting binding energies into directional conformational changes of the two catalytic domains of AK. These coordinated motions emerged to control the exact sequence of ligand binding and the affinity for the three different substrates, thereby guiding the reactants along the reaction coordinates. Interestingly, we find that about 10% of enzymes show altered allosteric regulation and ligand affinities, indicating that a subset of enzymes folds in alternative catalytically active forms. Since molecules or proteins might be able to selectively stabilize one of the folds, this observation suggests an evolutionary path for allostery in enzymes. In AK, this complex catalytic framework has likely emerged to prevent futile ATP/ADP hydrolysis and to regulate the enzyme for different energy needs of the cell. Single molecule nanopore analysis is used to reveal adenylate kinase global dynamics in real time. It was found that allosteric interactions guide domain motions and substrate affinity, with 10% of enzymes displaying alternative active forms, suggesting evolutionary paths for enzyme regulation. [ABSTRACT FROM AUTHOR]

Background: The decision to imbibe a blood meal is predominantly dependent on the sensitivity and specificity of haematophagous arthropods to blood-derived adenine nucleotides, in particular adenosine triphosphate (ATP). Despite previous efforts to identify and characterise the specificity and sensitivity to ATP and other adenine nucleotides, as well as the role of other blood-derived phagostimulants across the Culicidae, comparisons across species remain difficult. Methods: The feeding response of the yellow fever mosquito Aedes aegypti and the African malaria vector Anopheles gambiae to adenine nucleotides in the presence of a carbonate buffer was assessed using a membrane feeding assay. The proportion of mosquitoes engorged and the volume imbibed by all mosquitoes was scored visually and spectrophotometrically. In addition, the proportion of prediuresing An. gambiae, as well as the volume engorged and prediuresed, was examined. Results: Aedes aegypti was more sensitive to adenine nucleotides than An. gambiae, but both species maintained specificity to these phagostimulants, demonstrating a dose-dependent bimodal feeding pattern, thereby expanding our understanding of the all-or-none blood-feeding hypothesis. Feeding on the bicarbonate buffer by An. gambiae—but not that of Ae. aegypti—demonstrated a species-specific variation in how blood phagostimulants are encoded. Adenine nucleotides, with and without bovine serum albumin, were observed to dose-dependently regulate the proportion of An. gambiae prediuresing and the volumes prediuresed but not volumes engorged. Conclusions: Taken together, the results of this study expand our understanding of how mosquitoes differentially assess and respond to blood meal constituents, and provide a basis for further physiological and molecular studies. [ABSTRACT FROM AUTHOR]

Ectophosphatases catalyse the hydrolysis of phosphorylated molecules, such as phospho‐amino acids, in the extracellular environment. Nevertheless, the hydrolysis of nucleotides in the extracellular environment is typically catalysed by ectonucleotidases. Studies have shown that acid ectophosphatase, or transmembrane‐prostatic acid phosphatase (TM‐PAP), a membrane‐bound splice variant of prostatic acid phosphatase, has ecto‐5′‐nucleotidase activity. Furthermore, it was demonstrated that ectophosphatase cannot hydrolyse ATP, ADP, or AMP in triple‐negative breast cancer cells. In contrast to previous findings in MDA‐MB‐231 cells, the ectophosphatase studied in the present work displayed a remarkable capacity to hydrolyse AMP in luminal A breast cancer cells (MCF‐7). We showed that AMP dose‐dependently inhibited p‐nitrophenylphosphate (p‐NPP) hydrolysis. The p‐NPP and AMP hydrolysis showed similar biochemical behaviours, such as increased hydrolysis under acidic conditions and comparable inhibition by NiCl2, ammonium molybdate, and sodium orthovanadate. In addition, this ectophosphatase with ectonucleotidase activity was essential for the release of adenosine and inorganic phosphate from phosphorylated molecules available in the extracellular microenvironment. This is the first study to show that prostatic acid phosphatase on the membrane surface of breast cancer cells (MCF‐7) is correlated with cell adhesion and migration. Highlights: A transmembrane prostatic acid phosphatase (TM‐PAP) is expressed in luminal A MCF‐7 breast cancer cells.Ectonucleotidase activity by TM‐PAP was observed in MCF‐7 cells.The presence of prostatic acid phosphatase on the membrane surface is correlated with cell adhesion and migration. [ABSTRACT FROM AUTHOR]

Copyright of Journal of Medical Clinics / Tıp Fakültesi Klinikleri Dergisi is the property of Journal of Medical Clinics / Tip Fakültesi Klinikleri Dergisi and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

Dercas nina Mell 1913 (Pieridae) is a little-studied butterfly species endemic to China that flies primarily in the forest canopy. Genome skimming by Illumina sequencing allowed assembly of 146,702 reads for complete 1471.3-fold mean coverage of the circular 15,264 bp mitogenome from D. nina consisting of 82.1% AT nucleotides. A gene order typical of butterflies was recovered consisting of 13 protein-coding genes, 22 tRNAs, two rRNAs, and a predicted control region. The Dercas nina COX1 open reading frame begins with atypical start codon CGA. Six protein-coding genes (COX1, COX2, ND2, ND3, ND4, ND5) with single-nucleotide (T) stop codons, and two protein-coding genes (ATP6, ATP8) with two-nucleotide (TA) stop codons encoded in the DNA were inferred to be completed by adenine nucleotides from the Poly-A tail of the mRNA. Bayesian's phylogenetic reconstruction places the D. nina and D. lycorias mitogenomes as sister clades. Dercas mitogenomes were sister to those from genus Colias in the monophyletic subfamily Coliadinae. The mitogenome phylogeny is consistent with previous molecular phylogenetic hypotheses based on other markers, but differs somewhat from a morphology-based hypothesis that suggested that Dercas was more closely related to genus Gonepteryx. This may falsify the hypothesis or may instead reflect mitochondrial-nuclear phylogenetic discordance. [ABSTRACT FROM AUTHOR]

Adenosine phosphates (adenosine 5′-monophosphate (AMP), adenosine 5′-diphosphate (ADP), and adenosine 5′-triphosphate (ATP)) play important roles in energy storage and signal transduction in the human body. Thus, a measurement method that simultaneously recognizes and detects adenosine phosphates is necessary to gain insight into complex energy-relevant biological processes. Surface-enhanced Raman scattering (SERS) is a powerful technique for this purpose. However, the similarities in size, charge, and structure of adenosine phosphates (APs) make their simultaneous recognition and detection difficult. Although approaches that combine SERS and machine learning have been studied, they require massive quantities of training data. In this study, limited AP spectral data were obtained using fabricated gold nanostructures for SERS measurements. The training data were created by feature selection and data augmentation after preprocessing the small amount of acquired spectral data. The performances of several machine learning models trained on these generated training data were compared. Multilayer perceptron model successfully detected the presence of AMP, ADP, and ATP with an accuracy of 0.914. Consequently, this study establishes a new measurement system that enables the highly accurate recognition and detection of adenosine phosphates from limited SERS spectral data. [ABSTRACT FROM AUTHOR]

Background/Objective: Maintaining intracellular adenosine triphosphate (ATP) levels is essential for numerous cellular functions, including energy metabolism, muscle contraction, and nerve impulse transmission. ATP is primarily synthesized in mitochondria through oxidative phosphorylation. It is also generated in the cytosol under anaerobic conditions using phosphocreatine (PCr) as a phosphate donor to adenosine diphosphate. However, the intracellular delivery of exogenous PCr is challenging as it does not readily cross the plasma membrane. This complicates the use of PCr as a therapeutic agent to maintain energy homeostasis or to treat conditions like cerebral creatine deficiency syndrome (CDS), which results from defective creatine transporters. Methods: This study employs the use of fusogenic liposomes to deliver PCr directly into the cytosol, bypassing membrane impermeability issues. We engineered various 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP)-based fusogenic liposomes, incorporating phospholipids such as 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) in combination with phospholipid-aromatic dye components to facilitate membrane fusion and to enhance the delivery of the PCr cargo. Liposomal formulations were co-loaded with membrane-impermeable chromophores and PCr and studied on live cells using confocal microscopy. Conclusions: We demonstrated the successful intracellular delivery of these agents and observed a 23% increase in intracellular ATP levels in cells treated with PCr-loaded liposomes. This increase was not observed with free PCr, confirming the effectiveness of the liposome-based delivery system. Additionally, cell viability assays showed minimal toxicity from the liposomes. Our results indicate that fusogenic liposomes are a promising method for the delivery of PCr (and potentially other cell-impermeable therapeutic agents) to the cellular cytosol. The approach demonstrated here could be advantageous for treating energy-related disorders and improving cellular energy homeostasis. [ABSTRACT FROM AUTHOR]

Parkinson's disease (PD) affects movement; however, most patients with PD also develop nonmotor symptoms, such as hyposmia, sleep disorder, and depression. Dopamine levels in the brain have a critical influence on movement control, but other neurotransmitters are also involved in the progression of PD. This study analyzed the fluctuation of neurotransmitters in PC12 cells during neurogenesis and neurodegeneration by performing mass spectrometry. We found that the dopaminergic metabolism pathway of PC12 cells developed vigorously during the neuron differentiation process and that the neurotransmitters were metabolized into 3-methoxytyramine, which was released from the cells. The regulation of the intracellular and extracellular concentrations of adenosine indicated that adenine nucleotides were actively utilized in neural differentiation. Moreover, we exposed the differentiated PC12 cells to rotenone, which is a suitable material for modeling PD. The cells exposed to rotenone in the early stage of differentiation exhibited stimulated serotoninergic metabolism, and the contents of the serotoninergic neurotransmitters returned to their normal levels in the late stage of differentiation. Interestingly, the nondifferentiated cells can resist the toxicant rotenone and produce normal dopaminergic metabolites. However, when differentiated neuron cells were exposed to rotenone, they were seriously damaged, leading to a failure to produce dopaminergic neurotransmitters. In the low-dosage damage process, the amino acids that functioned as dopaminergic pathway precursors could not be absorbed by the cells, and dopamine and L-dopa were secreted and unable to be reuptaken to trigger the cell damage. [ABSTRACT FROM AUTHOR]

This correction notice is for an article titled "Establishment, characterization, and genetic profiling of patient-derived osteosarcoma cells from a patient with retinoblastoma" published in Scientific Reports. The correction addresses errors in the original article, including a mistake in the unit 'μg/mL' and inaccuracies in the reporting of a frameshift insertion in the RB1 gene. The correction also includes revisions to the discussion section and the author contributions section. The corrected version of the article is now available. [Extracted from the article]

Ferrous iron (Fe2+) is required for the growth and virulence of many pathogenic bacteria, including Vibrio cholerae (Vc), the causative agent of the disease cholera. For this bacterium, Feo is the primary system that transports Fe2+ into the cytosol. FeoB, the main component of this system, is regulated by a soluble cytosolic domain termed NFeoB. Recent reanalysis has shown that NFeoBs can be classified as either GTP-specific or NTP-promiscuous, but the structural and mechanistic bases for these differences were not known. To explore this intriguing property of FeoB, we solved the X-ray crystal structures of VcNFeoB in both the apo and the GDP-bound forms. Surprisingly, this promiscuous NTPase displayed a canonical NFeoB G-protein fold like GTP-specific NFeoBs. Using structural bioinformatics, we hypothesized that residues surrounding the nucleobase could be important for both nucleotide affinity and specificity. We then solved the X-ray crystal structures of N150T VcNFeoB in the apo and GDP-bound forms to reveal H-bonding differences surrounding the guanine nucleobase. Interestingly, isothermal titration calorimetry revealed similar binding thermodynamics of the WT and N150T proteins to guanine nucleotides, while the behavior in the presence of adenine nucleotides was dramatically different. AlphaFold models of VcNFeoB in the presence of ADP and ATP showed important conformational changes that contribute to nucleotide specificity among FeoBs. Combined, these results provide a structural framework for understanding FeoB nucleotide promiscuity, which could be an adaptive measure utilized by pathogens to ensure adequate levels of intracellular iron across multiple metabolic landscapes. [ABSTRACT FROM AUTHOR]

Ecto‐5′‐nucleotidase (CD73) hydrolyses 5′AMP to adenosine and inorganic phosphate. Breast cancer cells (MDA‐MB‐231) express high CD73 levels, and this enzyme has been found to play a tumour‐promoting role in breast cancer. However, no studies have sought to investigate whether CD73 has differential affinity or substrate preferences between noncancerous and cancerous breast cells. In the present study, we aimed to biochemically characterise ecto‐5′‐nucleotidase in breast cancer cell lines and assess whether its catalytic function and tumour progression are correlated in breast cancer cells. The results showed that compared to nontumoral breast MCF‐10A cells, triple‐negative breast cancer MDA‐MB‐231 cells had a higher ecto‐5′‐nucleotidase expression level and enzymatic activity. Although ecto‐5′‐nucleotidase activity in the MDA‐MB‐231 cell line showed no selectivity among monophosphorylated substrates, 5′AMP was preferred by the MCF‐10A cell line. Compared to the MCF‐10A cell line, the MDA‐MB‐231 cell line has better hydrolytic ability, lower substrate affinity, and high inhibitory potential after treatment with a specific CD73 inhibitor α,β‑methylene ADP (APCP). Therefore, we demonstrated that a specific inhibitor of the ecto‐5‐nucleotidase significantly reduced the migratory and invasive capacity of MDA‐MB‐231 cells, suggesting that ecto‐5‐nucleotidase activity might play an important role in metastatic progression. [ABSTRACT FROM AUTHOR]

The purinergic signaling system is an evolutionarily conserved and critical regulatory circuit that maintains homeostatic balance across various organ systems and cell types by providing compensatory responses to diverse pathologies. Despite cardiovascular diseases taking a leading position in human morbidity and mortality worldwide, pulmonary diseases represent significant health concerns as well. The endothelium of both pulmonary and systemic circulation (bronchial vessels) plays a pivotal role in maintaining lung tissue homeostasis by providing an active barrier and modulating adhesion and infiltration of inflammatory cells. However, investigations into purinergic regulation of lung endothelium have remained limited, despite widespread recognition of the role of extracellular nucleotides and adenosine in hypoxic, inflammatory, and immune responses within the pulmonary microenvironment. In this review, we provide an overview of the basic aspects of purinergic signaling in vascular endothelium and highlight recent studies focusing on pulmonary microvascular endothelial cells and endothelial cells from the pulmonary artery vasa vasorum. Through this compilation of research findings, we aim to shed light on the emerging insights into the purinergic modulation of pulmonary endothelial function and its implications for lung health and disease. [ABSTRACT FROM AUTHOR]

The bacterial stringent response, which responds to several types of stress, including nutritional deprivation, is controlled via hyperphosphorylated guanosine nucleotides collectively referred to as (p)ppGpp. Synthesis of (p)ppGpp from ATP and either GTP or GDP is catalysed by one of several alarmone synthetases, some of which are also capable of removing the alarmones by hydrolysis. A special class of small alarmone hydrolases (SAHs) consists of single-domain hydrolases. SAHs have been functionally characterised from a range of bacterial species and this shows that they can potentially hydrolyse both (p)ppGpp and (p)ppApp. Structural analysis of SAHs has further shown that they often form dimers but that the protein–protein interface varies between species, which may reflect differences in regulation. Little is currently known about the regulation of SAH activity and specificity towards various alarmones and other modified nucleotides. Comparison with the class of small alarmone synthetases (SASs), which form tetramers and are allosterically regulated, suggested that similar principles may govern the regulation of SAHs. Guanosine tetra- and pentaphosphate nucleotides, (p)ppGpp, function as central secondary messengers and alarmones in bacterial cell biology, signalling a range of stress conditions, including nutrient starvation and exposure to cell-wall-targeting antibiotics, and are critical for survival. While activation of the stringent response and alarmone synthesis on starved ribosomes by members of the RSH (Rel) class of proteins is well understood, much less is known about how single-domain small alarmone synthetases (SASs) and their corresponding alarmone hydrolases, the small alarmone hydrolases (SAHs), are regulated and contribute to (p)ppGpp homeostasis. The substrate spectrum of these enzymes has recently been expanded to include hyperphosphorylated adenosine nucleotides, suggesting that they take part in a highly complex and interconnected signalling network. In this review, we provide an overview of our understanding of the SAHs and discuss their structure, function, regulation, and phylogeny. [ABSTRACT FROM AUTHOR]

Ozone therapy's efficacy might stem from the regulated and mild oxidative stress resulting from ozone's interactions with various biological elements. The present work aimed to characterize the hepatic mitochondrial response to ozone treatment and its relationship with the antioxidant system response. Two groups of mice were used: one control group and another injected intraperitoneally with an O 3 /O 2 mixture (80 ml/kg) for 5 days. Mitochondrial respiration supported by different substrates was significantly inhibited, as well as complexes I and II/III, but not complex IV. The analysis of the electron transport chain complex activity showed significant inhibitions in complexes I and II/III but not in complex IV. These inhibitions can prevent mitochondrial reactive oxygen species (ROS) production. Additionally, there was a decline in glutathione content, unaccompanied by a rise in its oxidized form. The ozone-treated groups showed a significant increase in the activity of superoxide dismutase and glutathione peroxidase, while catalase and glutathione reductase experienced no significant alterations. Adenine nucleotides increased in the ozone group, but only the increase in adenosine diphosphate is significant, so the cell's energy charge is unaffected. This study shows that mitochondria may play a crucial role in ozone treatment. However, it also highlights the need for further studies to understand the molecular mechanism. • Ozone boosts liver mitochondria function and modulates antioxidant response. • Ozone alters mitochondrial respiration by inhibiting the main sites of ROS formation (complexes I and II/III). • Ozone treatment increases superoxide dismutase and glutathione peroxidase activity. • The adenine nucleotides are increased by the ozone treatment, but the energy charge is not affected. [ABSTRACT FROM AUTHOR]

Iron overload is a significant complication commonly observed in individuals with ß-thalassemia, resulting from enhanced iron absorption due to ineffective erythropoiesis and frequent blood transfusions. Iron overload can lead to severe tissue damage and organ dysfunction, significantly impacting the quality of life for those affected. Additionally, recent research indicates that iron overload may also adversely impact mitochondrial function, further exacerbating the pathophysiology of this disease. Excessive iron accumulation in mitochondria can impair the electron transport chain, reduce adenosine tri phosphate synthesis, and increase the generation of reactive oxygen species, resulting in elevated tissue damage and clinical complications. Emerging evidence suggests that specific mitochondrial DNA (mtDNA) mutations may further contribute to the severity of iron overload in ß-thalassemia patients. Currently, the clinical management of iron overload in patients with ß-thalassemia primarily relies on conventional iron chelation therapies, aiming to reduce iron burden and prevent tissue damage. However, cases involving mtDNA mutations introduce additional complexities, necessitating personalized treatment approaches. Advances in gene therapy and mitochondrial replacement strategies offer promising avenues for potential targeted interventions. This review provides a comprehensive overview of the mechanisms underlying iron overload in ß-thalassemia and its association with mtDNA mutations. It discusses the clinical manifestations, diagnostic challenges, and current treatment options for managing iron overload, while also highlighting emerging research directions and potential therapeutic targets for improved patient care. Ultimately, a better understanding of the complex interplay between iron overload and mtDNA mutations in ß-thalassemia will pave the way for innovative strategies to alleviate the disease burden. [ABSTRACT FROM AUTHOR]

Regulatory cystathionine β-synthase (CBS) domains are widespread in proteins; however, difficulty in structure determination prevents a comprehensive understanding of the underlying regulation mechanism. Tetrameric microbial inorganic pyrophosphatase containing such domains (CBS-PPase) is allosterically inhibited by AMP and ADP and activated by ATP and cell alarmones diadenosine polyphosphates. Each CBS-PPase subunit contains a pair of CBS domains but binds cooperatively to only one molecule of the mono-adenosine derivatives. We used site-directed mutagenesis of Desulfitobacterium hafniense CBS-PPase to identify the key elements determining the direction of the effect (activation or inhibition) and the "half-of-the-sites" ligand binding stoichiometry. Seven amino acid residues were selected in the CBS1 domain, based on the available X-ray structure of the regulatory domains, and substituted by alanine and other residues. The interaction of 11 CBS-PPase variants with the regulating ligands was characterized by activity measurements and isothermal titration calorimetry. Lys100 replacement reversed the effect of ADP from inhibition to activation, whereas Lys95 and Gly118 replacements made ADP an activator at low concentrations but an inhibitor at high concentrations. Replacement of these residues for alanine increased the stoichiometry of mono-adenosine phosphate binding by twofold. These findings identified several key protein residues and suggested a "two non-interacting pairs of interacting regulatory sites" concept in CBS-PPase regulation. [ABSTRACT FROM AUTHOR]

Protein kinases are crucial cellular enzymes that facilitate the transfer of phosphates from adenosine triphosphate (ATP) to their substrates, thereby regulating numerous cellular activities. Dysfunctional kinase activity often leads to oncogenic conditions. Chosen by using structural similarity to 5UG9, we selected 79 crystal structures from the PDB and based on the position of the phenylalanine side chain in the DFG motif, we classified these 79 crystal structures into 5 group clusters. Our approach applies our kinematic flexibility analysis (KFA) to explore the flexibility of kinases in various activity states and examine the impact of the activation loop on kinase structure. KFA enables the rapid decomposition of macromolecules into different flexibility regions, allowing comprehensive analysis of conformational structures. The results reveal that the activation loop of kinases acts as a "lock" that stabilizes the active conformation of kinases by rigidifying the adjacent α‐helices. Furthermore, we investigate specific kinase mutations, such as the L858R mutation commonly associated with non‐small cell lung cancer, which induces increased flexibility in active‐state kinases. In addition, through analyzing the hydrogen bond pattern, we examine the substructure of kinases in different states. Notably, active‐state kinases exhibit a higher occurrence of α‐helices compared to inactive‐state kinases. This study contributes to the understanding of biomolecular conformation at a level relevant to drug development. [ABSTRACT FROM AUTHOR]

The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status. When activated by increases in ADP:ATP and/or AMP:ATP ratios (signalling energy deficit), AMPK acts to restore energy balance. Binding of AMP to one or more of three CBS repeats (CBS1, CBS3, CBS4) on the AMPK-γ subunit activates the kinase complex by three complementary mechanisms: (i) promoting a-subunit Thr172 phosphorylation by the upstream kinase LKB1; (ii) protecting against Thr172 dephosphorylation; (iii) allosteric activation. Surprisingly, binding of ADP has been reported to mimic the first two effects, but not the third. We now show that at physiologically relevant concentrations of Mg.ATP2-(above those used in the standard assay) ADP binding does cause allosteric activation. However, ADP causes only a modest activation because (unlike AMP), at concentrations just above those where activation becomes evident, ADP starts to cause competitive inhibition at the catalytic site. Our results cast doubt on the physiological relevance of the effects of ADP and suggest that AMP is the primary activator in vivo. We have also made mutations to hydrophobic residues involved in binding adenine nucleotides at each of the three γ subunit CBS repeats of the human α2β2γ1 complex and examined their effects on regulation by AMP and ADP. Mutation of the CBS3 site has the largest effects on all three mechanisms of AMP activation, especially at lower ATP concentrations, while mutation of CBS4 reduces the sensitivity to AMP. All three sites appear to be required for allosteric activation by ADP. [ABSTRACT FROM AUTHOR]

The non-stop provision of chemical energy is of critical importance to normal cardiac function, requiring the rapid turnover of ATP to power both relaxation and contraction. Central to this is the creatine kinase (CK) phosphagen system, which buffers local ATP levels to optimise the energy available from ATP hydrolysis, to stimulate energy production via the mitochondria and to smooth out mismatches between energy supply and demand. In this review, we discuss the changes that occur in high-energy phosphatemetabolism (i.e., in ATP and phosphocreatine) during ischaemia and reperfusion, which represents an acute crisis of energy provision. Evidence is presented from preclinical models that augmentation of the CK system can reduce ischaemia-reperfusion injury and improve functional recovery. Energetic impairment is also a hallmark of chronic heart failure, in particular, down-regulation of the CK system and loss of adenine nucleotides, which may contribute to pathophysiology by limiting ATP supply. Herein, we discuss the evidence for this hypothesis based on preclinical studies and in patients using magnetic resonance spectroscopy. We conclude that the correlative evidence linking impaired energetics to cardiac dysfunction is compelling; however, causal evidence from loss-of-function models remains equivocal. Nevertheless, proof-of-principle studies suggest that augmentation of CK activity is a therapeutic target to improve cardiac function and remodelling in the failing heart. Further work is necessary to translate these findings to the clinic, in particular, a better understanding of the mechanisms by which the CK system is regulated in disease. [ABSTRACT FROM AUTHOR]

Adenine nucleotides play a critical role in maintaining essential functions of red blood cells (RBCs), including energy metabolism, redox status, shape fluctuations and RBC-dependent endothelial and microvascular functions. Recently, it has been shown that infection with the severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) might lead to morphological and metabolic alterations in erythrocytes in both mild and severe cases of coronavirus disease (COVID-19). However, little is known about the effects of COVID-19 on the nucleotide energetics of RBCs nor about the potential contribution of nucleotide metabolism to the long COVID syndrome. This study aimed to analyze the levels of adenine nucleotides in RBCs isolated from patients 12 weeks after mild SARS-CoV-2 infection who suffered from long COVID symptoms and to relate them with the endothelial and microvascular function parameters as well as the rate of peripheral tissue oxygen supply. Although the absolute quantities of adenine nucleotides in RBCs were rather slightly changed in long COVID individuals, many parameters related to the endothelial and microcirculatory function showed significant correlations with RBC adenosine triphosphate (ATP) and total adenine nucleotide (TAN) concentration. A particularly strong relationship was observed between ATP in RBCs and the serum ratio of arginine to asymmetric dimethylarginine—an indicator of endothelial function. Consistently, a positive correlation was also observed between the ATP/ADP ratio and diminished reactive hyperemic response in long COVID patients, assessed by the flow-mediated skin fluorescence (FMSF) technique, which reflected decreased vascular nitric oxide bioavailability. In addition, we have shown that patients after COVID-19 have significantly impaired ischemic response parameters (IR max and IR index), examined by FMSF, which revealed diminished residual bioavailability of oxygen in epidermal keratinocytes after brachial artery occlusion. These ischemic response parameters revealed a strong positive correlation with the RBC ATP/ADP ratio, confirming a key role of RBC bioenergetics in peripheral tissue oxygen supply. Taken together, the outcomes of this study indicate that dysregulation of metabolic processes in erythrocytes with the co-occurring endothelial and microvascular dysfunction is associated with diminished intracellular oxygen delivery, which may partly explain long COVID-specific symptoms such as physical impairment and fatigue. [ABSTRACT FROM AUTHOR]

Filamentous fungi display allorecognition genes that trigger regulated cell death (RCD) when strains of unlike genotype fuse. Podospora anserina is one of several model species for the study of this allorecognition process termed heterokaryon or vegetative incompatibility. Incompatibility restricts transmission of mycoviruses between isolates. In P. anserina, genetic analyses have identified nine incompatibility loci, termed het loci. Here we set out to clone the genes controlling het-B incompatibility. het-B displays two incompatible alleles, het-B1 and het-B2. We find that the het-B locus encompasses two adjacent genes, Bh and Bp that exist as highly divergent allelic variants (Bh1/Bh2 and Bp1/Bp2) in the incompatible haplotypes. Bh encodes a protein with an N-terminal HET domain, a cell death inducing domain bearing homology to Toll/interleukin-1 receptor (TIR) domains and a C-terminal domain with a predicted lectin fold. The Bp product is homologous to PII-like proteins, a family of small trimeric proteins acting as sensors of adenine nucleotides in bacteria. We show that although the het-B system appears genetically allelic, incompatibility is in fact determined by the non-allelic Bh1/Bp2 interaction while the reciprocal Bh2/Bp1 interaction plays no role in incompatibility. The highly divergent C-terminal lectin fold domain of BH determines recognition specificity. Population studies and genome analyses indicate that het-B is under balancing selection with trans-species polymorphism, highlighting the evolutionary significance of the two incompatible haplotypes. In addition to emphasizing anew the central role of TIR-like HET domains in fungal RCD, this study identifies novel players in fungal allorecognition and completes the characterization of the entire het gene set in that species. Author summary: Many cellular life forms display genetic systems that protect individuality and discriminate conspecific self from non-self. In filamentous fungi, cell fusion events between strains are under check by specific allorecognition genes that trigger regulated cell death upon detection of non-self. The role of incompatibility is to restrict mycovirus transmission and conspecific parasitism. Podospora anserina, a good model for the study of this form of allorecognition, harbors nine incompatibility het loci. Previous studies have revealed that these genes can be homologous to genes with immune functions in other phyla including bacteria, plants and animals. We have cloned het-B, the last of the nine het genes that remained to be identified and found that it is a complex locus comprising two adjacent genes Bh and Bp. BH displays an N-terminal HET domain (related to TIR domains) and a C-terminal domain with a predicted lectin fold. BP is homologous to PII-like proteins, known bacterial metabolite sensors. Intriguingly, despite apparent genetic allelism, incompatibility is dictated by the non-allelic Bh/Bp interaction. This study stresses the reoccurring involvement of HET domains in fungal RCD and signs completion of the characterization of the entire set of het loci in that species, enabling a comparative analysis of the different genetic architectures underlying allorecognition. [ABSTRACT FROM AUTHOR]

TRPM2 is a Ca2+ permeable, non-selective cation channel in the plasma membrane that is involved in the innate immune response regulating, for example, chemotaxis in neutrophils and cytokine secretion in monocytes and macrophages. The intracellular adenine nucleotides ADP-ribose (ADPR) and 2'-deoxy-ADPR (2dADPR) activate the channel, in combination with their coagonist Ca2+. Interestingly, activation of human TRPM2 (hsTRPM2) by 2dADPR is much more effective than activation by ADPR. However, the underlying mechanism of the nucleotides' differential effect on the channel is not yet fully understood. In this study, we performed whole-cell patch clamp experiments with HEK293 cells heterologously expressing hsTRPM2. We show that 2dADPR has an approx. 4-fold higher Ca2+ sensitivity than ADPR (EC50 = 190 and 690 nM). This allows 2dADPR to activate the channel at lower and thus physiological intracellular Ca2+ concentrations. Kinetic analysis of our data reveals that activation by 2dADPR is faster than activation by ADPR. Mutation in a calmodulin binding N-terminal IQ-like motif in hsTRPM2 completely abrogated channel activation by both agonists. However, mutation of a single amino acid residue (W1355A) in the C-terminus of hsTRPM2, at a site of extensive interdomain interaction, resulted in slower activation by 2dADPR and neutralized the difference in rate of activation between the two agonists. Taken together, we propose a mechanism by which 2dADPR induces higher hsTRPM2 currents than ADPR by means of faster channel activation. The finding that 2dADPR has a higher Ca2+ sensitivity than ADPR may indicate that 2dADPR rather than ADPR activates hsTRPM2 in physiological contexts such as the innate immune response. [ABSTRACT FROM AUTHOR]

Adenosine triphosphate (ATP) is the main energy currency of all cells, while creatine phosphate (CrP) is considered as a buffer of high energy-bond phosphate that facilitates rapid regeneration of ATP from adenosine diphosphate (ADP). Astrocyte-rich primary cultures contain ATP, ADP and adenosine monophosphate (AMP) in average specific contents of 36.0 ± 6.4 nmol/mg, 2.9 ± 2.1 nmol/mg and 1.7 ± 2.1 nmol/mg, respectively, which establish an adenylate energy charge of 0.92 ± 0.04. The average specific cellular CrP level was found to be 25.9 ± 10.8 nmol/mg and the CrP/ATP ratio was 0.74 ± 0.28. The specific cellular CrP content, but not the ATP content, declined with the age of the culture. Absence of fetal calf serum for 24 h caused a partial loss in the cellular contents of both CrP and ATP, while application of creatine for 24 h doubled the cellular CrP content and the CrP/ATP ratio, but did not affect ATP levels. In glucose-deprived astrocytes, the high cellular ATP and CrP contents were rapidly depleted within minutes after application of the glycolysis inhibitor 2-deoxyglucose and the respiratory chain inhibitor antimycin A. For those conditions, the decline in CrP levels always preceded that of ATP contents. In contrast, incubation of glucose-fed astrocytes for up to 30 min with antimycin A had little effect on the high cellular ATP content, while the CrP level was significantly lowered. These data demonstrate the importance of cellular CrP for maintaining a high cellular ATP content in astrocytes during episodes of impaired ATP regeneration. [ABSTRACT FROM AUTHOR]

BACKGROUND: Water‐free transportation (WFT), as a novel strategy for express delivery of live shrimp (Litopenaeus vannamei), was developed recently. However, air exposure during this transportation arouses a series of abiotic stress to the shrimp. In the present study, the influences of WFT stress on glycolysis and lipolysis metabolism and meat quality (umami flavor and drip loss) were investigated in comparison with conventional water transportation (WT). RESULTS: The results showed that type II muscle fibers with the feature of anaerobic metabolism were dominated in shrimp flesh. In addition, the increments of intracellular Ca2+ was detected in WFT and WT, which then activated the AMP‐activated protein kinase pathway and promoted the consumption of glycogen, as well as the accumulation of lactate and lipolysis, under the enzymolysis of hexokinase, pyruvate kinase, lactate dehydrogenase and adipose triglyceride lipase. Glycogen glycolyzed to latate. Meanwhile, ATP degraded along with glycolysis resulting in the generation of ATP‐related adenosine phosphates such as inosine monophosphate with umami flavor and phosphoric acid. More remarkable (P < 0.05) physiological changes (except lactate dehydrogenase and lactate) were observed in WFT compared to WT. Additionally, the fatty acid profile also slightly changed. CONCLUSION: The transport stress induced significant energy metabolism changes of shrimp flesh and therefore effected the flesh quality. The intensifications of freshness (K‐value) of shrimp flesh were detected as a result of ATP degradation, which were more pronounced after WFT. However, the drip loss of shrimp flesh was more significantly increased (P < 0.05) after WFT compared to WT. © 2023 Society of Chemical Industry. [ABSTRACT FROM AUTHOR]

Next to white and brown adipocytes present in white and brown adipose tissue (WAT, BAT), vascular endothelial cells, tissue-resident macrophages and other immune cells have important roles in maintaining adipose tissue homeostasis but also contribute to the etiology of obesity-associated chronic inflammatory metabolic diseases. In addition to hormonal signals such as insulin and norepinephrine, extracellular adenine nucleotides modulate lipid storage, fatty acid release and thermogenic responses in adipose tissues. The complex regulation of extracellular adenine nucleotides involves a network of ectoenzymes that convert ATP via ADP and AMP to adenosine. However, in WAT and BAT the processing of extracellular adenine nucleotides and its relevance for intercellular communications are still largely unknown. Based on our observations that in adipose tissues the adenosine-generating enzyme CD73 is mainly expressed by vascular endothelial cells, we studied glucose and lipid handling, energy expenditure and adaptive thermogenesis in mice lacking endothelial CD73 housed at different ambient temperatures. Under conditions of thermogenic activation, CD73 expressed by endothelial cells is dispensable for the expression of thermogenic genes as well as energy expenditure. Notably, thermoneutral housing leading to a state of low energy expenditure and lipid accumulation in adipose tissues resulted in enhanced glucose uptake into WAT of endothelial CD73-deficient mice. This effect was associated with elevated expression levels of de novo lipogenesis genes. Mechanistic studies provide evidence that extracellular adenosine is imported into adipocytes and converted to AMP by adenosine kinase. Subsequently, activation of the AMP kinase lowers the expression of de novo lipogenesis genes, most likely via inactivation of the transcription factor carbohydrate response element binding protein (ChREBP). In conclusion, this study demonstrates that endothelial-derived extracellular adenosine generated via the ectoenzyme CD73 is a paracrine factor shaping lipid metabolism in WAT. [ABSTRACT FROM AUTHOR]

Objective: Several studies have shown that mitochondrial metabolism may be disrupted if the rate of the specific 4,977 bp deletion of mitochondrial DNA (mtDNA) reaches a threshold. This study aimed to investigate the possible associations between the mtDNA4977 deletion load and obesity-related metabolic abnormalities in the adipose tissue. Methods: The study included thirty obese individuals, who underwent bariatric surgery, and twelve control subjects. mtDNA4977 deletion, adenine nucleotides, and lactate levels, which show the bioenergetic status were evaluated in visceral adipose tissues. Fourier transform infrared (FTIR) spectroscopy was used to investigate the structural variations and composition of adipose tissues in the context of deletion load. Results: There were no differences between the two groups in terms of mtDNA4977 deletion, adenine nucleotides, and lactate levels. The FTIR spectra indicated a few obesity-related alterations in adipose tissues that were not related to the mtDNA deletion load. Also, statistical analysis showed a correlation between the deletion load and a band shift of 1,744 cm−1, which assigns C = O stretching of the carbonyl group of the ester group in triglycerides and other esterified fatty acids, although it is not associated with obesity. Conclusions: Our data suggest that the mtDNA4977 deletion in visceral adipose tissues of obese individuals do not have a significant impact on the bioenergetic status. However, the increased accumulation of deletion may be associated with a specific change in the ester bond, indicating structural differences in the lipids. These findings shed light on our understanding of the tissue-specific distribution of mtDNA deletions and obesity-related adipose tissue pathogeneses. [ABSTRACT FROM AUTHOR]

Regulatory adenine nucleotide-binding cystathionine β-synthase (CBS) domains are widespread in proteins; however, information on the mechanism of their modulating effects on protein function is scarce. The difficulty in obtaining structural data for such proteins is ascribed to their unusual flexibility and propensity to form higher-order oligomeric structures. In this study, we deleted the most movable domain from the catalytic part of a CBS domain-containing bacterial inorganic pyrophosphatase (CBS-PPase) and characterized the deletion variant both structurally and functionally. The truncated CBS-PPase was inactive but retained the homotetrameric structure of the full-size enzyme and its ability to bind a fluorescent AMP analog (inhibitor) and diadenosine tetraphosphate (activator) with the same or greater affinity. The deletion stabilized the protein structure against thermal unfolding, suggesting that the deleted domain destabilizes the structure in the full-size protein. A "linear" 3D structure with an unusual type of domain swapping predicted for the truncated CBS-PPase by Alphafold2 was confirmed by single-particle electron microscopy. The results suggest a dual role for the CBS domains in CBS-PPase regulation: they allow for enzyme tetramerization, which impedes the motion of one catalytic domain, and bind adenine nucleotides to mitigate or aggravate this effect. [ABSTRACT FROM AUTHOR]

Purinergic signaling is important for normal bladder function, as it is thought to initiate the voiding reflex and modulate smooth muscle tone. The availability of adenine nucleotides and nucleosides (aka purines) at receptor sites of various cell types in the bladder wall is regulated by ectonucleotidases (ENTDs). ENTDs hydrolyze purines such as adenosine 5'-triphosphate (ATP) and adenosine 5'-diphosphate (ADP) with varying preference for the individual substrate. Therefore, the end effect of extracellular purines may depend significantly on the type of ENTD that is expressed in close proximity to the target cells. ENTDs likely have distinct cellular associations, but the specific locations of individual enzymes in the bladder wall are poorly understood. We used RNAscope™, an RNA in situ hybridization (ISH) technology, to visualize the distribution and measure the levels of gene expression of the main recognized ectonucleotidases in large high-resolution images of murine bladder sections. The relative gene expression of ENTDs was Entpd3 > Alpl » Enppl = Entpd2 » Enpp3 > Entpdl (very low to no signal) in the urothelium, Entpdl ≥Entpd2 » Enpp3 > Enppl = Alpl ≥ Nt5e (very low to no signal) in the lamina propria, and Entpdl » Nt5e = Entpd2 » Enppl > Alpl = Enpp3 in the detrusor. These layer-specific differences might be important in compartmentalized regulation of purine availability and subsequent functions in the bladder wall and may explain reported asymmetries in purine availability in the bladder lumen and suburothelium/lamina propria spaces. [ABSTRACT FROM AUTHOR]

Purine nucleosides (adenosine) and nucleotides such as adenosine mono/di/triphosphate (AMP/ADP/ATP) may produce complex cardiovascular responses. For example, adenosine-5′-(β-thio)-diphosphate (ADPβS; a stable synthetic analogue of ADP) can induce vasodilatation/vasodepressor responses by endothelium-dependent and independent mechanisms involving purinergic P2Y receptors; however, the specific subtypes participating in these responses remain unknown. Therefore, this study investigated the receptor subtypes mediating the blood pressure changes induced by intravenous bolus of ADPβS in male Wistar rats in the absence and presence of central mechanisms with the antagonists MRS2500 (P2Y1), PSB0739 (P2Y12), and MRS2211 (P2Y13). For this purpose, 120 rats were divided into 60 anaesthetised rats and 60 pithed rats, and further subdivided into four groups (n = 30 each), namely: (a) anaesthetised rats, (b) anaesthetised rats with bilateral vagotomy, (c) pithed rats, and (d) pithed rats continuously infused (intravenously) with methoxamine (an α1-adrenergic agonist that restores systemic vascular tone). We observed, in all four groups, that the immediate decreases in diastolic blood pressure produced by ADPβS were exclusively mediated by peripheral activation of P2Y1 receptors. Nevertheless, the subsequent increases in systolic blood pressure elicited by ADPβS in pithed rats infused with methoxamine probably involved peripheral activation of P2Y1, P2Y12, and P2Y13 receptors. [ABSTRACT FROM AUTHOR]

Formaldehyde (FA), as the simplest endogenous carbonyl molecule, participates in many biosynthesis and metabolism in living organisms, such as nucleotides and adenosine triphosphate (ATP). FA concentrations are sub-millimolar in the normal healthy body, but can rise significantly in a number of disease pathologies. As a result, detecting endogenous FA is critical for illness diagnosis and rehabilitation therapy monitoring. Recent studies have focused on the FA-responsive turn-on fluorescence probe, which has huge promise in the detection and visualization of FA in living cells and organisms, as well as exceptional use in disease diagnosis and therapeutic monitoring. This review summarizes the fluorescence luminescence mechanism and design concepts of FA fluorescent probes, as well as their recent applications in bioimaging and illness diagnostics. Additionally, this article indicates the present dilemma of FA-responsive fluorescent probe, including selectivity, specificity, and detection mode, which may provide references for the development of FA-responsive fluorescent probes. [ABSTRACT FROM AUTHOR]

Adenine nucleotide translocase (ANT) is the prototypical member of the mitochondrial carrier protein family, primarily involved in ADP/ATP exchange across the inner mitochondrial membrane. Several carrier proteins evolutionarily related to ANT, including SLC25A24 and SLC25A25, are believed to promote the exchange of cytosolic ATP‐Mg2+ with phosphate in the mitochondrial matrix. They allow a net accumulation of adenine nucleotides inside mitochondria, which is essential for mitochondrial biogenesis and cell growth. In the last two decades, mutations in the heart/muscle isoform 1 of ANT (ANT1) and the ATP‐Mg2+ transporters have been found to cause a wide spectrum of human diseases by a recessive or dominant mechanism. Although loss‐of‐function recessive mutations cause a defect in oxidative phosphorylation and an increase in oxidative stress which drives the pathology, it is unclear how the dominant missense mutations in these proteins cause human diseases. In this review, we focus on how yeast was productively used as a model system for the understanding of these dominant diseases. We also describe the relationship between the structure and function of ANT and how this may relate to various pathologies. Particularly, mutations in Aac2, the yeast homolog of ANT, were recently found to clog the mitochondrial protein import pathway. This leads to mitochondrial precursor overaccumulation stress (mPOS), characterized by the toxic accumulation of unimported mitochondrial proteins in the cytosol. We anticipate that in coming years, yeast will continue to serve as a useful model system for the mechanistic understanding of mitochondrial protein import clogging and related pathologies in humans. [ABSTRACT FROM AUTHOR]

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions. [ABSTRACT FROM AUTHOR]

Among the multiple antiviral defense mechanisms found in prokaryotes, CRISPR-Cas systems stand out as the only known RNA-programmed pathways for detecting and destroying bacteriophages and plasmids. Class 1 CRISPR-Cas systems, the most widespread and diverse of these adaptive immune systems, use an RNA-guided multiprotein complex to find foreign nucleic acids and trigger their destruction. In this review, we describe how these multisubunit complexes target and cleave DNA and RNA and how regulatory molecules control their activities. We also highlight similarities to and differences from Class 2 CRISPR-Cas systems, which use a single-protein effector, as well as other types of bacterial and eukaryotic immune systems. We summarize current applications of the Class 1 CRISPR-Cas systems for DNA/RNA modification, control of gene expression, and nucleic acid detection.

The hyporheic zone (HZ) is a sensitive ecotone in river ecosystems because of its biodiversity and susceptibility to human activities. Hyporheic fauna are exposed to multiple stressors that affect the physiology and metabolism of organisms and ultimately ecosystem functioning and biodiversity. This study aimed to quantify adenine nucleotides in the stygophilous freshwater amphipod Synurella ambulans and to evaluate the potential of adenylate energy charge (AEC) as a physiological biomarker of general stress in the HZ of the Sava River at sites with different intensities of environmental and anthropogenic stress. Field studies were conducted seasonally (in December 2018 and April, July, and October 2019) at two sampling sites, one upstream (1-UP) and the other downstream (2-DOWN) of the discharge from the wastewater treatment plant using the standard piston pump. The amphipod population from site 1-UP had significantly higher AEC values than the population from site 2-DOWN in all seasons except summer. Coordinated changes in AEC values with the ATP/ADP ratio indicated differences in energy status between the two populations. However, no changes in the apparent equilibrium constant of adenylate kinase were observed, except in spring at site 1-UP. Multiple linear regression models showed the strongest associations of AEC with Fe and Zn accumulated in amphipods, followed by environmental factors (conductivity, dissolved O2, and concentrations of nitrites and phosphates in the interstitial water). AEC was shown to be a useful index of environmental stress in S. ambulans because it can directly measure the change in available energy and thus the metabolic stress to which the organism is exposed. Finally, seasonal and spatial variations in AEC values reflected ecological status in the HZ. [ABSTRACT FROM AUTHOR]

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A β−galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis. [ABSTRACT FROM AUTHOR]

Adenine nucleotides (AN) are ubiquitous metabolites that regulate cellular energy metabolism and modulate cell communication and inflammation. To understand how disturbances in AN balance arise and affect cellular function, robust quantification techniques for these metabolites are crucial. However, due to their hydrophilicity, simultaneous quantification of AN across various biological samples has been challenging. Here we present a hydrophilic interaction high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS/MS) based method for the quantification of 26 adenosine nucleotides and precursors as well as metabolic products of nicotinamide adenine dinucleotide (NAD) in plasma, liver, and adipose tissue samples as well as cell culture supernatants and cells. Method validation was performed with regard to linearity, accuracy, precision, matrix effects, and carryover. Finally, analysis of cell culture supernatants derived from intestinal organoids and RAW 264.7 cells illustrates that the here described method is a reliable and easy-to-use tool to quantify AN and opens up new avenues to understand the role of AN generation and breakdown for cellular functions.

Introduction: Essential genes are essential for the survival of various species. These genes are a family linked to critical cellular activities for species survival. These genes are coded for proteins that regulate central metabolism, gene translation, deoxyribonucleic acid replication, and fundamental cellular structure and facilitate intracellular and extracellular transport. Essential genes preserve crucial genomics information that may hold the key to a detailed knowledge of life and evolution. Essential gene studies have long been regarded as a vital topic in computational biology due to their relevance. An essential gene is composed of adenine, guanine, cytosine, and thymine and its various combinations. Methods: This paper presents a novel method of extracting information on the stationary patterns of nucleotides such as adenine, guanine, cytosine, and thymine in each gene. For this purpose, some co-occurrencematrices are derived that provide the statistical distribution of stationary patterns of nucleotides in the genes, which is helpful in establishing the relationship between the nucleotides. For extracting discriminant features from each co-occurrence matrix, energy, entropy, homogeneity, contrast, and dissimilarity features are computed, which are extracted from all co-occurrence matrices and then concatenated to form a feature vector representing each essential gene. Finally, supervised machine learning algorithms are applied for essential gene classification based on the extracted fixed-dimensional feature vectors. Results: For comparison, some existing state-of-the-art feature representation techniques such as Shannon entropy (SE), Hurst exponent (HE), fractal dimension (FD), and their combinations have been utilized. Discussion: An extensive experiment has been performed for classifying the essential genes of five species that show the robustness and effectiveness of the proposed methodology. [ABSTRACT FROM AUTHOR]