RESUMEN
The Structural Maintenance of Chromosomes (SMC) protein complexes are DNA-binding molecular machines required to shape chromosomes into functional units and to safeguard the genome through cell division. These ring-shaped multi-subunit protein complexes, which are present in all kingdoms of life, achieve this by organizing chromosomes in three-dimensional space. Mechanistically, the SMC complexes hydrolyze ATP to either stably entrap DNA molecules within their lumen, or rapidly reel DNA into large loops, which allow them to link two stretches of DNA in cis or trans. In this chapter, the canonical structure of the SMC complexes is first introduced, followed by a description of the composition and general functions of the main types of eukaryotic and prokaryotic SMC complexes. Thereafter, the current model for how SMC complexes perform in vitro DNA loop extrusion is presented. Lastly, chromosome loop formation by SMC complexes is introduced, and how the DNA loop extrusion mechanism contributes to chromosome looping by SMC complexes in cells is discussed.
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Cromosomas , Cromosomas/química , Complejos Multiproteicos/metabolismo , Complejos Multiproteicos/química , Proteínas de Ciclo Celular/metabolismo , Proteínas de Ciclo Celular/genética , ADN/química , ADN/metabolismo , ADN/genética , Proteínas Cromosómicas no Histona/metabolismo , Proteínas Cromosómicas no Histona/química , Adenosina Trifosfato/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas de Unión al ADN/químicaRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Gelsemium dynamized dilutions (GDD) are known as a remedy for a wide range of behavioral and psychological symptoms of depression and anxiety at ultra-low doses, yet the underlying mechanisms of the mode of action of G. sempervirens itself are not well understood. AIM OF THE STUDY: The present study was designed to examine the neuroprotective effects of Gelsemium preparations in counteracting stress-related mitochondrial dysfunctions in neuronal cells. MATERIALS AND METHODS: We started by studying how serum deprivation affects the mitochondrial functions of human neuroblastoma (SH-SY5Y) cells. Next, we looked into the potential of various Gelsemium dilutions to improve cell survival and ATP levels. After identifying the most effective dilutions, 3C and 5C, we tested their ability to protect SH-SY5Y cells from stress-induced mitochondrial deficits. We measured total and mitochondrial superoxide anion radicals using fluorescent dyes dihydroethidium (DHE) and the red mitochondrial superoxide indicator (MitoSOX). Additionally, we assessed total nitric oxide levels with 4,5-diaminofluorescein diacetate (DAF-2DA), examined the redox state using pRA305 cells stably transfected with a plasmid encoding a redox-sensitive green fluorescent protein, and analyzed mitochondrial network morphology using an automated high-content analysis device, Cytation3. Furthermore, we investigated bioenergetics by measuring ATP production with a bioluminescence assay (ViaLighTM HT) and evaluated mitochondrial respiration (OCR) and glycolysis (ECAR) using the Seahorse Bioscience XF24 Analyzer. Finally, we determined cell survival using an MTT reduction assay. RESULTS: Our research indicates that Gelsemium dilutions (3C and 5C) exhibited neuroprotective effects by: - Normalizing total and mitochondrial superoxide anion radicals and total nitric oxide levels. - Regulating the mitochondrial redox environment and mitochondrial networks morphology. - Increasing ATP generation as well as OCR and ECAR levels, thereby reducing the viability loss induced by serum withdrawal stress. CONCLUSIONS: These findings highlight that dynamized Gelsemium preparations may have neuroprotective effects against stress-induced cellular changes in the brain by regulating mitochondrial functions, essential for the survival, plasticity, and function of neurons in depression.
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Supervivencia Celular , Mitocondrias , Neuronas , Fármacos Neuroprotectores , Humanos , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Adenosina Trifosfato/metabolismo , Estrés Oxidativo/efectos de los fármacos , Óxido Nítrico/metabolismo , Extractos Vegetales/farmacología , Relación Dosis-Respuesta a Droga , Superóxidos/metabolismoRESUMEN
The symptoms of fragile X syndrome (FXS), caused by a single gene mutation to Fmr1, have been increasingly linked to disordered astrocyte signalling within the cerebral cortex. We have recently demonstrated that the purinergic signalling pathway, which utilizes nucleoside triphosphates and their metabolites to facilitate bidirectional glial and glial-neuronal interactions, is upregulated in cortical astrocytes derived from the Fmr1 knockout (KO) mouse model of FXS. Heightened Fmr1 KO P2Y purinergic receptor levels were correlated with prolonged intracellular calcium release, elevated synaptogenic protein secretion, and hyperactivity of developing circuits. However, due to the relative lack of sensitive and reproducible quantification methods available for measuring purines and pyrimidines, determining the abundance of these factors in Fmr1 KO astrocytes was limited. We therefore developed a hydrophilic interaction liquid chromatography protocol coupled with mass spectrometry to compare the abundance of intracellular and extracellular purinergic molecules between wildtype and Fmr1 KO mouse astrocytes. Significant differences in the concentrations of UDP, ATP, AMP, and adenosine intracellular stores were found within Fmr1 KO astrocytes relative to WT. The extracellular level of adenosine was also significantly elevated in Fmr1 KO astrocyte-conditioned media in comparison to media collected from WT astrocytes. Glycosylation of the astrocyte membrane-bound CD39 ectonucleotidase, which facilitates ligand breakdown following synaptic release, was also elevated in Fmr1 KO astrocyte cultures. Together, these differences demonstrated further dysregulation of the purinergic signalling system within Fmr1 KO cortical astrocytes, potentially leading to significant alterations in FXS purinergic receptor activation and cellular pathology.
Asunto(s)
Astrocitos , Corteza Cerebral , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil , Síndrome del Cromosoma X Frágil , Ratones Noqueados , Transducción de Señal , Animales , Astrocitos/metabolismo , Ratones , Síndrome del Cromosoma X Frágil/genética , Síndrome del Cromosoma X Frágil/metabolismo , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/genética , Proteína de la Discapacidad Intelectual del Síndrome del Cromosoma X Frágil/metabolismo , Corteza Cerebral/metabolismo , Corteza Cerebral/citología , Apirasa/genética , Apirasa/metabolismo , Células Cultivadas , Adenosina Trifosfato/metabolismo , Medios de Cultivo Condicionados , Adenosina/metabolismo , Adenosina/análogos & derivados , Receptores Purinérgicos P2Y/metabolismo , Receptores Purinérgicos P2Y/genética , Ratones Endogámicos C57BL , Antígenos CDRESUMEN
Dynein is the primary molecular motor responsible for retrograde intracellular transport of a variety of cargoes, performing successive nanometer-sized steps within milliseconds. Due to the limited spatiotemporal precision of established methods for molecular tracking, current knowledge of dynein stepping is essentially limited to slowed-down measurements in vitro. Here, we use MINFLUX fluorophore localization to directly track CRISPR/Cas9-tagged endogenous dynein with nanometer/millisecond precision in living primary neurons. We show that endogenous dynein primarily takes 8 nm steps, including frequent sideways steps but few backward steps. Strikingly, the majority of direction reversals between retrograde and anterograde movement occurred on the time scale of single steps (16 ms), suggesting a rapid regulatory reversal mechanism. Tug-of-war-like behavior during pauses or reversals was unexpectedly rare. By analyzing the dwell time between steps, we concluded that a single rate-limiting process underlies the dynein stepping mechanism, likely arising from just one adenosine 5'-triphosphate hydrolysis event being required during each step. Our study underscores the power of MINFLUX localization to elucidate the spatiotemporal changes underlying protein function in living cells.
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Dineínas , Neuronas , Dineínas/metabolismo , Neuronas/metabolismo , Animales , Sistemas CRISPR-Cas , Adenosina Trifosfato/metabolismo , RatonesRESUMEN
In the realm of this study, obtaining a comprehensive understanding of ischemic brain injury and its molecular foundations is of paramount importance. Our study delved into single-cell data analysis, with a specific focus on sub-celltypes and differentially expressed genes in the aftermath of ischemic injury. Notably, we observed a significant enrichment of the "ATP METABOLIC PROCESS" and "ATP HYDROLYSIS ACTIVITY" pathways, featuring pivotal genes such as Pbx3, Dguok, and Kif21b. A remarkable finding was the consistent upregulation of genes like Fabp7 and Bcl11a within the MCAO group, highlighting their crucial roles in regulating the pathway of mitochondrial ATP synthesis coupled proton transport. Furthermore, our network analysis unveiled pathways like "Neuron differentiation" and "T cell differentiation" as central in the regulatory processes of sub-celltypes. These findings provide valuable insights into the intricate molecular responses and regulatory mechanisms that govern brain injury. The shared differentially expressed genes among sub-celltypes emphasize their significance in orchestrating responses post-ischemic injury. Our research, viewed from the perspective of a medical researcher, contributes to the evolving understanding of the molecular landscape underlying ischemic brain injury, potentially paving the way for targeted therapeutic strategies and improved patient outcomes.
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Adenosina Trifosfato , Infarto de la Arteria Cerebral Media , Cinesinas , Mitocondrias , Células Precursoras de Oligodendrocitos , Transducción de Señal , Animales , Transducción de Señal/genética , Infarto de la Arteria Cerebral Media/patología , Infarto de la Arteria Cerebral Media/metabolismo , Mitocondrias/metabolismo , Adenosina Trifosfato/metabolismo , Adenosina Trifosfato/biosíntesis , Cinesinas/genética , Cinesinas/metabolismo , Células Precursoras de Oligodendrocitos/metabolismo , Humanos , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Masculino , Isquemia Encefálica/genética , Isquemia Encefálica/metabolismo , Isquemia Encefálica/patología , Ratas , Proteínas Proto-OncogénicasRESUMEN
Bioenergy decline occurs with reperfusion following acute ischemic stroke. However, the molecular mechanisms that limit energy metabolism and their impact on post-stroke cognitive and emotional complications are still unclear. In the present study, we demonstrate that the p53 transcriptional response is responsible for neuronal adenosine triphosphate (ATP) deficiency and progressively neuropsychiatric disturbances, involving the downregulation of mitochondrial voltage-dependent anion channels (VDACs). Neuronal p53 transactivated the promoter of microRNA-183 (miR-183) cluster, thereby upregulating biogenesis of miR-183-5p (miR-183), miR-96-5p (miR-96), and miR-182-5p. Both miR-183 and miR-96 directly targeted and post-transcriptionally suppressed VDACs. Neuronal ablation of p53 protected against ATP deficiency and neurological deficits, whereas post-stroke rescue of miR-183/VDAC signaling reversed these benefits. Interestingly, cyclin-dependent kinase 9 (CDK9) was found to be enriched in cortical neurons and upregulated the p53-induced transcription of the miR-183 cluster in neurons after ischemia. Post-treatment with the CDK9 inhibitor oroxylin A promoted neuronal ATP production mainly through suppressing the miR-183 cluster/VDAC axis, further improved long-term sensorimotor abilities and spatial memory, and alleviated depressive-like behaviors in mice following stroke. Our findings reveal an intrinsic CDK9/p53/VDAC pathway that drives neuronal bioenergy decline and underlies post-stroke cognitive impairment and depression, thus highlighting the therapeutic potential of oroxylin A for better outcomes.
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Metabolismo Energético , Ratones Endogámicos C57BL , MicroARNs , Neuronas , Transducción de Señal , Accidente Cerebrovascular , Proteína p53 Supresora de Tumor , Animales , Proteína p53 Supresora de Tumor/metabolismo , Proteína p53 Supresora de Tumor/genética , Ratones , Neuronas/metabolismo , Neuronas/patología , MicroARNs/genética , MicroARNs/metabolismo , Masculino , Accidente Cerebrovascular/metabolismo , Accidente Cerebrovascular/complicaciones , Adenosina Trifosfato/metabolismoRESUMEN
A key player in energy metabolism is phosphofructokinase-1 (PFK1) whose activity and behavior strongly influence glycolysis and thus have implications in many areas. In this research, PFK1 assays were performed to convert F6P and ATP into F-1,6-P and ADP for varied pH and ATP concentrations. PFK1 activity was assessed by evaluating F-1,6-P generation velocity in two ways: (1) directly calculating the time slope from the first two or more datapoints of measured product concentration (the initial-velocity method), and (2) by fitting all the datapoints with a differential equation explicitly representing the effects of ATP and pH (the modeling method). Similar general trends of inhibition were shown by both methods, but the former gives only a qualitative picture while the modeling method yields the degree of inhibition because the model can separate the two simultaneous roles of ATP as both a substrate of reaction and an inhibitor of PFK1. Analysis based on the model suggests that the ATP affinity is much greater to the PFK1 catalytic site than to the inhibitory site, but the inhibited ATP-PFK1-ATP complex is much slower than the uninhibited PFK1-ATP complex in product generation, leading to reduced overall reaction velocity when ATP concentration increases. The initial-velocity method is simple and useful for general observation of enzyme activity while the modeling method has advantages in quantifying the inhibition effects and providing insights into the process.
Asunto(s)
Adenosina Trifosfato , Fosfofructoquinasa-1 , Adenosina Trifosfato/metabolismo , Fosfofructoquinasa-1/metabolismo , Concentración de Iones de Hidrógeno , Cinética , Fructosafosfatos/metabolismo , Adenosina Difosfato/metabolismo , GlucólisisRESUMEN
Rationale: Tissue regeneration of skin and bone is an energy-intensive, ATP-consuming process that, if impaired, can lead to the development of chronic clinical pictures. ATP levels in the extracellular space including the exudate of wounds, especially chronic wounds, are low. This deficiency can be compensated by inorganic polyphosphate (polyP) supplied via the blood platelets to the regenerating site. Methods: The contribution of the different forms of energy derived from polyP (metabolic energy, mechanical energy and heat) to regeneration processes was dissected and studied both in vitro and in patients. ATP is generated metabolically during the enzymatic cleavage of the energy-rich anhydride bonds between the phosphate units of polyP, involving the two enzymes alkaline phosphatase (ALP) and adenylate kinase (ADK). Exogenous polyP was administered after incorporation into compressed collagen or hydrogel wound coverages to evaluate its regenerative activity for chronic wound healing. Results: In a proof-of-concept study, fast healing of chronic wounds was achieved with the embedded polyP, supporting the crucial regeneration-promoting activity of ATP. In the presence of Ca2+ in the wound exudate, polyP undergoes a coacervation process leading to a conversion of fibroblasts into myofibroblasts, a crucial step supporting cell migration during regenerative tissue repair. During coacervation, a switch from an endothermic to an exothermic, heat-generating process occurs, reflecting a shift from an entropically- to an enthalpically-driven thermodynamic reaction. In addition, mechanical forces cause the appearance of turbulent flows and vortices during liquid-liquid phase separation. These mechanical forces orient the cellular and mineralic (hydroxyapatite crystallite) components, as shown using mineralizing SaOS-2 cells as a model. Conclusion: Here we introduce the energetic triad: metabolic energy (ATP), thermal energy and mechanical energy as a novel theranostic biomarker, which contributes essentially to a successful application of polyP for regeneration processes.
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Adenosina Trifosfato , Polifosfatos , Cicatrización de Heridas , Polifosfatos/metabolismo , Polifosfatos/farmacología , Humanos , Cicatrización de Heridas/efectos de los fármacos , Adenosina Trifosfato/metabolismo , Metabolismo Energético/efectos de los fármacos , Calor , Fosfatasa Alcalina/metabolismo , Adenilato Quinasa/metabolismo , MasculinoRESUMEN
Potency assessment of monoclonal antibodies or corresponding biosimilars in cell-based assays is an essential prerequisite in biopharmaceutical research and development. However, cellular bioassays are still subject to limitations in sample throughput, speed, and often need costly reagents or labels as they are based on an indirect readout by luminescence or fluorescence. In contrast, whole-cell Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry (MS) has emerged as a direct, fast and label-free technology for functional drug screening being able to unravel the molecular complexity of cellular response to pharmaceutical reagents. However, this approach has not yet been used for cellular testing of biologicals. In this study, we have conceived, developed and benchmarked a label-free MALDI-MS based cell bioassay workflow for the functional assessment of complement-dependent cytotoxicity (CDC) of Rituximab antibody. By computational evaluation of response profiles followed by subsequent m/z feature annotation via fragmentation analysis and trapped ion mobility MS, we identified adenosine triphosphate and glutathione as readily MS-assessable metabolite markers for CDC and demonstrate that robust concentration-response characteristics can be obtained by MALDI-TOF MS. Statistical assay performance indicators suggest that whole-cell MALDI-TOF MS could complement the toolbox for functional cellular testing of biopharmaceuticals.
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Rituximab , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción/métodos , Humanos , Rituximab/farmacología , Proteínas del Sistema Complemento/metabolismo , Bioensayo/métodos , Anticuerpos Monoclonales , Glutatión/metabolismo , Adenosina Trifosfato/metabolismoRESUMEN
The ATP-independent chaperone SurA protects unfolded outer membrane proteins (OMPs) from aggregation in the periplasm of Gram-negative bacteria, and delivers them to the ß-barrel assembly machinery (BAM) for folding into the outer membrane (OM). Precisely how SurA recognises and binds its different OMP clients remains unclear. Escherichia coli SurA comprises three domains: a core and two PPIase domains (P1 and P2). Here, by combining methyl-TROSY NMR, single-molecule Förster resonance energy transfer (smFRET), and bioinformatics analyses we show that SurA client binding is mediated by two binding hotspots in the core and P1 domains. These interactions are driven by aromatic-rich motifs in the client proteins, leading to SurA core/P1 domain rearrangements and expansion of clients from collapsed, non-native states. We demonstrate that the core domain is key to OMP expansion by SurA, and uncover a role for SurA PPIase domains in limiting the extent of expansion. The results reveal insights into SurA-OMP recognition and the mechanism of activation for an ATP-independent chaperone, and suggest a route to targeting the functions of a chaperone key to bacterial virulence and OM integrity.
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Proteínas de la Membrana Bacteriana Externa , Proteínas de Escherichia coli , Escherichia coli , Chaperonas Moleculares , Proteínas de Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/química , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/química , Escherichia coli/metabolismo , Escherichia coli/genética , Sitios de Unión , Chaperonas Moleculares/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Unión Proteica , Adenosina Trifosfato/metabolismo , Dominios Proteicos , Pliegue de Proteína , Isomerasa de Peptidilprolil/metabolismo , Isomerasa de Peptidilprolil/genética , Transportadoras de Casetes de Unión a ATP/metabolismo , Transportadoras de Casetes de Unión a ATP/química , Transportadoras de Casetes de Unión a ATP/genética , Modelos Moleculares , Proteínas PortadorasRESUMEN
We previously reported that normothermic ex vivo kidney perfusion (NEVKP) is superior in terms of organ protection compared to static cold storage (SCS), which is still the standard method of organ preservation, but the mechanisms are incompletely understood. We used a large animal kidney autotransplant model to evaluate mitochondrial function during organ preservation and after kidney transplantation, utilizing live cells extracted from fresh kidney tissue. Male porcine kidneys stored under normothermic perfusion showed preserved mitochondrial function and higher ATP levels compared to kidneys stored at 4 °C (SCS). Mitochondrial respiration and ATP levels were further enhanced when AP39, a mitochondria-targeted hydrogen sulfide donor, was administered during warm perfusion. Correspondingly, the combination of NEVKP and AP39 was associated with decreased oxidative stress and inflammation, and with improved graft function after transplantation. In conclusion, our findings suggest that the organ-protective effects of normothermic perfusion are mediated by maintenance of mitochondrial function and enhanced by AP39 administration. Activation of mitochondrial function through the combination of AP39 and normothermic perfusion could represent a new therapeutic strategy for long-term renal preservation.
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Trasplante de Riñón , Riñón , Mitocondrias , Preservación de Órganos , Perfusión , Isquemia Tibia , Animales , Mitocondrias/metabolismo , Riñón/metabolismo , Preservación de Órganos/métodos , Masculino , Porcinos , Perfusión/métodos , Sulfuro de Hidrógeno/metabolismo , Sulfuro de Hidrógeno/farmacología , Adenosina Trifosfato/metabolismo , Estrés Oxidativo , Compuestos Organofosforados , TionasRESUMEN
P2X receptors, a subfamily of ligand-gated ion channels activated by extracellular ATP, are implicated in various physiopathological processes, including inflammation, pain perception, and immune and respiratory regulations. Structural determinations using crystallography and cryo-EM have revealed that the extracellular three-dimensional architectures of different P2X subtypes across various species are remarkably identical, greatly advancing our understanding of P2X activation mechanisms. However, structural studies yield paradoxical architectures of the intracellular domain (ICD) of different subtypes (e.g., P2X3 and P2X7) at the apo state, and the role of the ICD in P2X functional regulation remains unclear. Here, we propose that the P2X3 receptor's ICD has an apo state conformation similar to the open state but with a less tense architecture, containing allosteric sites that influence P2X3's physiological and pathological roles. Using covalent occupancy, engineered disulfide bonds and voltage-clamp fluorometry, we suggested that the ICD can undergo coordinated motions with the transmembrane domain of P2X3, thereby facilitating channel activation. Additionally, we identified a novel P2X3 enhancer, PSFL77, and uncovered its potential allosteric site located in the 1α3ß domain of the ICD. PSFL77 modulated pain perception in P2rx3+/+, but not in P2rx3-/-, mice, indicating that the 1α3ß, a "tunable" region implicated in the regulation of P2X3 functions. Thus, when P2X3 is in its apo state, its ICD architecture is fairly ordered rather than an unstructured outward folding, enabling allosteric modulation of the signaling of P2X3 receptors.
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Sitio Alostérico , Dominios Proteicos , Receptores Purinérgicos P2X3 , Animales , Receptores Purinérgicos P2X3/metabolismo , Receptores Purinérgicos P2X3/química , Receptores Purinérgicos P2X3/genética , Humanos , Ratones , Células HEK293 , Adenosina Trifosfato/metabolismo , Masculino , Ratones Endogámicos C57BL , Regulación AlostéricaRESUMEN
Glioblastoma multiforme (GBM) is the most aggressive type of cancer in the brain and has an inferior prognosis because of the lack of suitable medicine, largely due to its tremendous invasion. GBM has selfish metabolic pathways to promote migration, invasion, and proliferation compared to normal cells. Among various metabolic pathways, NAD (nicotinamide adenine dinucleotide) is essential in generating ATP and is used as a resource for cancer cells. LbNOX (Lactobacillus brevis NADH oxidase) is an enzyme that can directly manipulate the NAD+/NADH ratio. In this study, we found that an increased NAD+/NADH ratio by LbNOX or mitoLbNOX reduced intracellular glutamate and calcium responses and reduced invasion capacity in GBM. However, the invasion was not affected in GBM by rotenone, an ETC (Electron Transport Chain) complex I inhibitor, or nicotinamide riboside, a NAD+ precursor, suggesting that the crucial factor is the NAD+/NADH ratio rather than the absolute quantity of ATP or NAD+ for the invasion of GBM. To develop a more accurate and effective GBM treatment, our findings highlight the importance of developing a new medicine that targets the regulation of the NAD+/NADH ratio, given the current lack of effective treatment options for this brain cancer.
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Glioblastoma , Metaboloma , NAD , Glioblastoma/metabolismo , Glioblastoma/patología , NAD/metabolismo , Humanos , Línea Celular Tumoral , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/patología , Complejos Multienzimáticos/metabolismo , Levilactobacillus brevis/metabolismo , Invasividad Neoplásica , Calcio/metabolismo , Ácido Glutámico/metabolismo , Movimiento Celular , Adenosina Trifosfato/metabolismo , NADH NADPH OxidorreductasasRESUMEN
In conventional metal-organic framework (MOF) luminophore-involved electrochemiluminescence (ECL) systems, the aggregation-caused quenching commonly exists for the organic luminescent ligands, limiting the ECL efficiency and detection sensitivity. Herein, by employing the aggregation-induced emission luminogen (AIEgen) 1,1,2,2-tetra(4-carboxylbiphenyl)ethylene (H4TCBPE) as a ligand, one high-efficiency ECL emitter (Zr-MOF) was synthesized through a simple hydrothermal reaction. Compared with H4TCBPE monomers and their aggregates, the resultant Zr-MOF possesses the strongest ECL emission, which is mainly attributed to the framework-induced ECL enhancement. Specifically, the heterostructure was prepared by the deposition of silver nanoparticles on TiO2 microflowers and utilized as an efficient coreaction accelerator. Remarkably, the formative heterojunction can increase the interfacial charge transfer efficiency and promote the carrier separation, facilitating the oxidation of coreactant tripropylamine. In this way, a novel aptamer-mediated ECL sensing platform is constructed, achieving the sensitive analysis of adenosine triphosphate with a low detection limit of 0.17 nM. As a proof-of-concept study, this work may enlighten the rational design of new-type MOF-based ECL materials and expand the application scope of the ECL technology.
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Técnicas Biosensibles , Técnicas Electroquímicas , Mediciones Luminiscentes , Nanopartículas del Metal , Estructuras Metalorgánicas , Plata , Titanio , Titanio/química , Plata/química , Estructuras Metalorgánicas/química , Técnicas Biosensibles/métodos , Nanopartículas del Metal/química , Límite de Detección , Adenosina Trifosfato/análisis , Aptámeros de Nucleótidos/química , Circonio/químicaRESUMEN
Accurate and rapid imaging of tumor cells is of vital importance for early cancer diagnosis and intervention. Aptamer-based fluorescence sensors have become a potent instrument for bioimaging, while false positives and on-target off-tumors linked to single-biomarker aptasensors compromise the specificity and sensitivity of cancer imaging. In this paper, we describe a sequential response aptasensor for precise cancer cell identification that is based on a DNA "AND" logic gate. Specifically, the sensor consists of three single-stranded DNA, including the P-strand that can sensitively respond to an acid environment, the L-strand containing the ATP aptamer sequence, and the R-strand for target cell anchoring. These DNA strands hybridize with one another to create a Y-shaped structure (named Y-ALGN). The aptamer in the R-strand is utilized to anchor the sensor to the target cell membrane primarily. Responding to the extracellular acidic environment of the tumor (input 1), the I-motif sequence forms a tetramer structure so that the P-strand is released from the Y-shaped structure and exposes the ATP binding sites in the L-strand. Extracellular ATP, as input 2, continuously operates the DNA aptasensor to complete the logic computation. Upon the sequential response of both protons and ATP molecules, the aptasensor is activated with restored fluorescence on a particular cancer cell membrane. Benefiting from the precise computation capacity of the "AND" logic gate, the Y-ALGN aptasensor can distinguish between MCF-7 cells and normal cells with high accuracy. As a simple and dual-stimuli-responsive strategy, this nanodevice would offer a fresh approach for accurately diagnosing tumor cells.
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Aptámeros de Nucleótidos , Membrana Celular , Aptámeros de Nucleótidos/química , Humanos , Membrana Celular/química , Membrana Celular/metabolismo , Técnicas Biosensibles/métodos , Adenosina Trifosfato/análisis , Adenosina Trifosfato/metabolismo , Imagen Óptica , Colorantes Fluorescentes/química , ADN de Cadena Simple/química , Células MCF-7RESUMEN
Purinergic signaling has emerged as an important paracrine-autocrine intercellular system that regulates physiological and pathological processes in practically all organs of the body. Although this system has been thoroughly defined since the nineties, recent research has made substantial advances regarding its role in aspects of liver physiology. However, most studies have mainly targeted the entire organ, 70% of which is made up of parenchymal cells or hepatocytes. Because of its physiological role, the liver is exposed to toxic metabolites, such as xenobiotics, drugs, and fatty acids, as well as to pathogens such as viruses and bacteria. Under injury conditions, all cell types within the liver undergo adaptive changes. In this context, the concentration of extracellular ATP has the potential to increase dramatically. Indeed, this purinergic response has not been studied in sufficient detail in non-parenchymal liver cells. In the present review, we systematize the physiopathological adaptations related to the purinergic system in chronic liver diseases of non-parenchymal liver cells, such as hepatic stellate cells, Kupffer cells, sinusoidal endothelial cells, and cholangiocytes. The role played by non-parenchymal liver cells in these circumstances will undoubtedly be strategic in understanding the regenerative activities that support the viability of this organ under stressful conditions.
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Hígado , Receptores Purinérgicos , Transducción de Señal , Humanos , Animales , Hígado/metabolismo , Receptores Purinérgicos/metabolismo , Macrófagos del Hígado/metabolismo , Células Estrelladas Hepáticas/metabolismo , Adenosina Trifosfato/metabolismo , Hepatopatías/metabolismo , Hepatopatías/patología , Hepatocitos/metabolismoRESUMEN
Human dental tissue mesenchymal stem cells (DT-MSCs) constitute an attractive alternative to bone marrow-derived mesenchymal stem cells (BM-MSCs) for potential clinical applications because of their accessibility and anti-inflammatory capacity. We previously demonstrated that DT-MSCs from dental pulp (DP-MSCs), periodontal ligaments (PDL-MSCs), and gingival tissue (G-MSCs) show immunosuppressive effects similar to those of BM, but to date, the DT-MSC-mediated immunoregulation of T lymphocytes through the purinergic pathway remains unknown. In the present study, we compared DP-MSCs, PDL-MSCs, and G-MSCs in terms of CD26, CD39, and CD73 expression; their ability to generate adenosine (ADO) from ATP and AMP; and whether the concentrations of ADO that they generate induce an immunomodulatory effect on T lymphocytes. BM-MSCs were included as the gold standard. Our results show that DT-MSCs present similar characteristics among the different sources analyzed in terms of the properties evaluated; however, interestingly, they express more CD39 than BM-MSCs; therefore, they generate more ADO from ATP. In contrast to those produced by BM-MSCs, the concentrations of ADO produced by DT-MSCs from ATP inhibited the proliferation of CD3+ T cells and promoted the generation of CD4+CD25+FoxP3+CD39+CD73+ Tregs and Th17+CD39+ lymphocytes. Our data suggest that DT-MSCs utilize the adenosinergic pathway as an immunomodulatory mechanism and that this mechanism is more efficient than that of BM-MSCs.
Asunto(s)
5'-Nucleotidasa , Adenosina , Apirasa , Pulpa Dental , Células Madre Mesenquimatosas , Ligamento Periodontal , Linfocitos T , Células Madre Mesenquimatosas/metabolismo , Células Madre Mesenquimatosas/inmunología , Humanos , Adenosina/metabolismo , Pulpa Dental/citología , Pulpa Dental/inmunología , Pulpa Dental/metabolismo , Linfocitos T/inmunología , Linfocitos T/metabolismo , 5'-Nucleotidasa/metabolismo , Apirasa/metabolismo , Ligamento Periodontal/citología , Ligamento Periodontal/metabolismo , Adenosina Trifosfato/metabolismo , Células Cultivadas , Encía/citología , Encía/metabolismo , Encía/inmunología , Antígenos CD/metabolismo , Inmunomodulación , Diferenciación Celular , Proliferación Celular , Dipeptidil Peptidasa 4/metabolismo , Transducción de Señal , Linfocitos T Reguladores/inmunología , Linfocitos T Reguladores/metabolismo , Proteínas Ligadas a GPIRESUMEN
Epilepsy is characterized by hypersynchronous neuronal discharges, which are associated with an increased cerebral metabolic rate of oxygen and ATP demand. Uncontrolled seizure activity (status epilepticus) results in mitochondrial exhaustion and ATP depletion, which potentially generate energy mismatch and neuronal loss. Many cells can adapt to increased energy demand by increasing metabolic capacities. However, acute metabolic adaptation during epileptic activity and its relationship to chronic epilepsy remains poorly understood. We elicited seizure-like events (SLEs) in an in vitro model of status epilepticus for eight hours. Electrophysiological recording and tissue oxygen partial pressure recordings were performed. After eight hours of ongoing SLEs, we used proteomics-based kinetic modeling to evaluate changes in metabolic capacities. We compared our findings regarding acute metabolic adaptation to published proteomic and transcriptomic data from chronic epilepsy patients. Epileptic tissue acutely responded to uninterrupted SLEs by upregulating ATP production capacity. This was achieved by a coordinated increase in the abundance of proteins from the respiratory chain and oxidative phosphorylation system. In contrast, chronic epileptic tissue shows a 25-40% decrease in ATP production capacity. In summary, our study reveals that epilepsy leads to dynamic metabolic changes. Acute epileptic activity boosts ATP production, while chronic epilepsy reduces it significantly.
Asunto(s)
Adenosina Trifosfato , Epilepsia , Adenosina Trifosfato/metabolismo , Humanos , Epilepsia/metabolismo , Animales , Adaptación Fisiológica , Masculino , Metabolismo Energético , Proteómica/métodos , Mitocondrias/metabolismo , Enfermedad Crónica , Fosforilación Oxidativa , Estado Epiléptico/metabolismoRESUMEN
The AMP-activated protein kinase (AMPK) is a sensor of cellular energy status that is expressed in almost all eukaryotic cells. In the canonical activation mechanism, it is activated by increases in AMP:ATP and ADP:ATP ratios that signify declining cellular energy status. Once activated, AMPK phosphorylates numerous targets that promote catabolic pathways generating ATP, while inhibiting anabolic and other processes that consume ATP, thus acting to restore energy homeostasis. Pharmacological agents that activate AMPK have been useful in identifying downstream targets and have potential as drugs for treatment of metabolic disorders such as Type 2 diabetes and non-alcoholic fatty liver disease. One such agent is C13, a pro-drug with a phosphonate bis(isobutyryloxymethyl) ester moiety, with the isobutyryloxymethyl groups increasing membrane permeability. Following cellular uptake, C13 is cleaved to release C2, an AMP analogue and potent AMPK activator that is specific for complexes containing the α1 (but not the α2) catalytic subunit isoform. This has previously been assumed to be the sole mechanism by which C13 activates AMPK, with potential roles for the isobutyryloxymethyl groups being ignored. We now report that, following cleavage from C13, these protective groups are metabolized to formaldehyde, an agent that inhibits mitochondrial function and increases cellular AMP:ATP ratios, thus providing additional AMPK activation by the canonical mechanism.
Asunto(s)
Proteínas Quinasas Activadas por AMP , Proteínas Quinasas Activadas por AMP/metabolismo , Humanos , Activación Enzimática/efectos de los fármacos , Adenosina Monofosfato/metabolismo , Adenosina Monofosfato/farmacología , Animales , Fosforilación/efectos de los fármacos , Adenosina Trifosfato/metabolismoRESUMEN
L-Lactate is a commodity chemical used in various fields. Microorganisms have produced L-lactate via lactic fermentation using saccharides derived from crops as carbon sources. Recently, L-lactate production using microalgae, whose carbon source is carbon dioxide, has been spotlighted because the prices of the crops have increased. A red alga Cyanidioschyzon merolae produce L-lactate via lactic fermentation under dark anaerobic conditions. The L-lactate titer of C. merolae is higher than those of other microalgae but lower than those of heterotrophic bacteria. Therefore, an increase in the L-lactate titer is required in C. merolae. L-Lactate dehydrogenase (L-LDH) catalyzes the reduction of pyruvate to L-lactate during lactic fermentation. C. merolae possesses five isozymes of L-LDH. The results of previous transcriptome analysis suggested that L-LDHs are the key enzymes in the lactic fermentation of C. merolae. However, their biochemical characteristics, such as catalytic efficiency and tolerance for metabolites, have not been revealed. We compared the amino acid sequences of C. merolae L-LDHs (CmLDHs) and characterized one of the isozymes, CmLDH1. BLAST analysis revealed that the sequence similarities of CmLDH1 and the other isozymes were above 99%. The catalytic efficiency of CmLDH1 under its optimum conditions was higher than those of L-LDHs of other organisms. ATP decreased the affinity and turnover number of CmLDH1 for NADH. These findings contribute to understanding the characteristics of L-LDHs of microalgae and the regulatory mechanisms of lactic fermentation in C. merolae.