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1.
Int J Mol Sci ; 22(6)2021 Mar 15.
Artículo en Inglés | MEDLINE | ID: mdl-33804129

RESUMEN

SARS-CoV-2 currently lacks effective first-line drug treatment. We present promising data from in silico docking studies of new Methisazone compounds (modified with calcium, Ca; iron, Fe; magnesium, Mg; manganese, Mn; or zinc, Zn) designed to bind more strongly to key proteins involved in replication of SARS-CoV-2. In this in silico molecular docking study, we investigated the inhibiting role of Methisazone and the modified drugs against SARS-CoV-2 proteins: ribonucleic acid (RNA)-dependent RNA polymerase (RdRp), spike protein, papain-like protease (PlPr), and main protease (MPro). We found that the highest binding interactions were found with the spike protein (6VYB), with the highest overall binding being observed with Mn-bound Methisazone at -8.3 kcal/mol, followed by Zn and Ca at -8.0 kcal/mol, and Fe and Mg at -7.9 kcal/mol. We also found that the metal-modified Methisazone had higher affinity for PlPr and MPro. In addition, we identified multiple binding pockets that could be singly or multiply occupied on all proteins tested. The best binding energy was with Mn-Methisazone versus spike protein, and the largest cumulative increases in binding energies were found with PlPr. We suggest that further studies are warranted to identify whether these compounds may be effective for treatment and/or prophylaxis.


Asunto(s)
Antivirales/química , Metales/química , Metisazona/química , Simulación del Acoplamiento Molecular , /química , Antivirales/metabolismo , Calcio/química , Calcio/metabolismo , /metabolismo , /metabolismo , /metabolismo , Diseño de Fármacos , Humanos , Hierro/química , Hierro/metabolismo , Magnesio/química , Magnesio/metabolismo , Manganeso/química , Manganeso/metabolismo , Metales/metabolismo , Metisazona/metabolismo , Modelos Moleculares , Simulación de Dinámica Molecular , Unión Proteica , Glicoproteína de la Espiga del Coronavirus/química , Glicoproteína de la Espiga del Coronavirus/metabolismo , Zinc/química , Zinc/metabolismo
2.
Sheng Li Xue Bao ; 73(1): 137-142, 2021 Feb 25.
Artículo en Chino | MEDLINE | ID: mdl-33665668

RESUMEN

Rapamycin (Rap) is an immunosuppressant, which is mainly used in the anti-rejection of organ transplantation. Meanwhile, it also shows great potential in the fields of anticancer, neuroprotection and anti-aging. Rap can inhibit the activity of mammalian target of Rap (mTOR). It activates the transcription factor EB (TFEB) to up-regulate lysosomal function and eliminates the inhibitory effect of mTOR on ULK1 (unc-51 like autophagy activating kinase 1) to promote autophagy. Recent research showed that Rap can directly activate the lysosomal cation channel TRPML1 in an mTOR-independent manner. TRPML1 activation releases lysosomal calcium. Calcineurin functions as the sensor of the lysosomal calcium signal and activates TFEB, thus promoting lysosome function and autophagy. This finding has greatly broadened and deepened our understanding of the pharmacological roles of Rap. In this review, we briefly introduce the canonical Rap-mTOR-ULK1/TFEB signaling pathway, and then discuss the discovery of TRPML1 as a new target of Rap and the pharmacological potential of this novel Rap-TRPML1-Calcineurin-TFEB pathway.


Asunto(s)
Canales de Calcio , Sirolimus , Autofagia , Calcio/metabolismo , Lisosomas/metabolismo , Transducción de Señal
3.
J Vis Exp ; (168)2021 02 13.
Artículo en Inglés | MEDLINE | ID: mdl-33645555

RESUMEN

The retina transforms light signals from the environment into electrical signals that are propagated to the brain. Diseases of the retina are prevalent and cause visual impairment and blindness. Understanding how such diseases progress is critical to formulating new treatments. In vivo microscopy in animal models of disease is a powerful tool for understanding neurodegeneration and has led to important progress towards treatments of conditions ranging from Alzheimer's disease to stroke. Given that the retina is the only central nervous system structure inherently accessible by optical approaches, it naturally lends itself towards in vivo imaging. However, the native optics of the lens and cornea present some challenges for effective imaging access. This protocol outlines methods for in vivo two-photon imaging of cellular cohorts and structures in the mouse retina at cellular resolution, applicable for both acute- and chronic-duration imaging experiments. It presents examples of retinal ganglion cell (RGC), amacrine cell, microglial, and vascular imaging using a suite of labeling techniques including adeno-associated virus (AAV) vectors, transgenic mice, and inorganic dyes. Importantly, these techniques extend to all cell types of the retina, and suggested methods for accessing other cellular populations of interest are described. Also detailed are example strategies for manual image postprocessing for display and quantification. These techniques are directly applicable to studies of retinal function in health and disease.


Asunto(s)
Fotones , Pupila/fisiología , Retina/diagnóstico por imagen , Animales , Calcio/metabolismo , Dependovirus/metabolismo , Procesamiento de Imagen Asistido por Computador , Inyecciones Intravítreas , Ratones , Ratones Transgénicos , Microglía/citología , Células Ganglionares de la Retina/citología , Programas Informáticos
4.
J Vis Exp ; (168)2021 02 14.
Artículo en Inglés | MEDLINE | ID: mdl-33645583

RESUMEN

Recording of the electrical activity from one of the smallest cells of a mammalian organism- a sperm cell- has been a challenging task for electrophysiologists for many decades. The method known as "spermatozoan patch clamp" was introduced in 2006. It has enabled the direct recording of ion channel activity in whole-cell and cell-attached configurations and has been instrumental in describing sperm cell physiology and the molecular identity of various calcium, potassium, sodium, chloride, and proton ion channels. However, recording from single spermatozoa requires advanced skills and training in electrophysiology. This detailed protocol summarizes the step-by-step procedure and highlights several 'tricks-of-the-trade' in order to make it available to anyone who wishes to explore the fascinating physiology of the sperm cell. Specifically, the protocol describes recording from human and murine sperm cells but can be adapted to essentially any mammalian sperm cell of any species. The protocol covers important details of the application of this technique, such as isolation of sperm cells, selection of reagents and equipment, immobilization of the highly motile cells, formation of the tight (Gigaohm) seal between a recording electrode and the plasma membrane of the sperm cells, transition into the whole-spermatozoan mode (also known as break-in), and exemplary recordings of the sperm cell calcium ion channel, CatSper, from six mammalian species. The advantages and limitations of the sperm patch clamp method, as well as the most critical steps, are discussed.


Asunto(s)
Membrana Celular/fisiología , Fenómenos Electrofisiológicos , Espermatozoides/fisiología , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Tamaño de la Célula , Disección , Fenómenos Electrofisiológicos/efectos de los fármacos , Flagelos/efectos de los fármacos , Flagelos/fisiología , Humanos , Concentración de Iones de Hidrógeno , Transporte Iónico/efectos de los fármacos , Macaca mulatta , Masculino , Ratones Endogámicos C57BL , Técnicas de Placa-Clamp , Perfusión , Progesterona/farmacología , Soluciones , Espermatozoides/citología , Espermatozoides/efectos de los fármacos
5.
Biomed Environ Sci ; 34(3): 203-212, 2021 Mar 20.
Artículo en Inglés | MEDLINE | ID: mdl-33766216

RESUMEN

Objective: Exposure to microgravity results in postflight cardiovascular deconditioning in astronauts. Vascular oxidative stress injury and mitochondrial dysfunction have been reported during this process. To elucidate the mechanism for this condition, we investigated whether mitochondrial oxidative stress regulates calcium homeostasis and vasoconstriction in hindlimb unweighted (HU) rat cerebral arteries. Methods: Three-week HU was used to simulate microgravity in rats. The contractile responses to vasoconstrictors, mitochondrial fission/fusion, Ca 2+ distribution, inositol 1,4,5-trisphosphate receptor (IP 3R) abundance, and the activities of voltage-gated K + channels (K V) and Ca 2+-activated K + channels (BK Ca) were examined in rat cerebral vascular smooth muscle cells (VSMCs). Results: An increase of cytoplasmic Ca 2+ and a decrease of mitochondrial/sarcoplasmic reticulum (SR) Ca 2+ were observed in HU rat cerebral VSMCs. The abundance of fusion proteins (mitofusin 1/2 [MFN1/2]) and fission proteins (dynamin-related protein 1 [DRP1] and fission-mitochondrial 1 [FIS1]) was significantly downregulated and upregulated, respectively in HU rat cerebral VSMCs. The cerebrovascular contractile responses to vasoconstrictors were enhanced in HU rats compared to control rats, and IP 3R protein/mRNA levels were significantly upregulated. The current densities and open probabilities of K V and BK Ca decreased and increased, respectively. Treatment with the mitochondrial-targeted antioxidant mitoTEMPO attenuated mitochondrial fission by upregulating MFN1/2 and downregulating DRP1/FIS1. It also decreased IP 3R expression levels and restored the activities of the K V and BK Ca channels. MitoTEMPO restored the Ca 2+ distribution in VSMCs and attenuated the enhanced vasoconstriction in HU rat cerebral arteries. Conclusion: The present results suggest that mitochondrial oxidative stress enhances cerebral vasoconstriction by regulating calcium homeostasis during simulated microgravity.


Asunto(s)
Calcio/metabolismo , Homeostasis , Mitocondrias/fisiología , Miocitos del Músculo Liso/fisiología , Estrés Oxidativo , Vasoconstricción/fisiología , Simulación de Ingravidez , Animales , Arterias Cerebrales , Masculino , Ratas , Ratas Sprague-Dawley
6.
Food Chem ; 352: 129332, 2021 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-33690075

RESUMEN

In order to construct a novel and efficient calcium delivery system, a dextran- casein phosphopeptide (CPP) conjugates as calcium carrier was prepared by Maillard reaction of CPP and dextran. The preparation of the conjugates, construction of calcium delivery system and digestion in vitro were studied. The grafting rate of conjugates, which was confirmed by migration and intensity changes in the characteristic peaks using ultraviolet-visible and Fourier transform infrared spectroscopy, reached 48.88%. The microscopy showed CPP was coated with dextran, the conjugates with a kind of "shell-core" structure had excellent stability. Compared with CPP, the chelating rate of conjugates increased from 6.0% to 13.87%, and the calcium retention rate improved from 1.09% to 7.90% in vitro digestion. The calcium binding capacity and effect of controlled release of the conjugates were superior to those of CPP. Therefore, the conjugates could be used as an effective carrier for new calcium supplements.


Asunto(s)
Calcio/química , Caseínas/química , Dextranos/química , Digestión , Portadores de Fármacos/química , Fosfopéptidos/química , Calcio/metabolismo , Reacción de Maillard
7.
Life Sci ; 273: 119296, 2021 May 15.
Artículo en Inglés | MEDLINE | ID: mdl-33675897

RESUMEN

AIMS: Skeletal muscle mass and strength are reduced in asthma and contribute to compromised functional capacity in asthmatic patients. However, an effective pharmacological intervention remains elusive, partly because molecular mechanisms dictating muscle decline in asthma are not known. MATERIALS: We investigated the potential contribution(s) of skeletal muscle sarcoplasmic reticulum Ca2+ ATPase (SERCA) to muscle atrophy and weakness in asthmatic patients. Quadriceps muscle biopsies were taken from 58 to 72 years old male patients with mild and advanced asthma and the SERCA activity was analyzed in association with cellular redox environment and myonuclear domain (MND) size. KEY FINDINGS: Maximal SERCA activity was reduced in skeletal muscles of mild and advanced asthmatics and was associated with reduced expression of SERCA2 protein and upregulation of sarcolipin, a SERCA inhibitory lipoprotein. We also found downregulation of Ca2+ release protein calstabin and upregulation of Ca2+ buffer, calsequestrin in skeletal muscles of asthmatic patients. The atrophic single muscle fibers had smaller cytoplasmic domains per myonucleus possibly indicating the reduced transcriptional reserves of individual myonuclei. Plasma periostin and CAF22 levels were significantly elevated in asthmatic patients and showed a strong correlation with hand-grip strength. These changes were accompanied by substantially elevated markers of global oxidative stress including lipid peroxidation and mitochondrial ROS production. CONCLUSION: Taken together, our data suggest that muscle weakness and atrophy in asthma is in part driven by SERCA dysfunction and oxidative stress. The data propose SERCA dysfunction as a therapeutic intervention to address muscle decline in asthma.


Asunto(s)
Asma/complicaciones , Biomarcadores/sangre , Calcio/metabolismo , Músculo Esquelético/patología , Atrofia Muscular/patología , ATPasas Transportadoras de Calcio del Retículo Sarcoplásmico/metabolismo , Anciano , Estudios Transversales , Humanos , Masculino , Persona de Mediana Edad , Músculo Esquelético/enzimología , Atrofia Muscular/enzimología , Atrofia Muscular/etiología , Retículo Sarcoplasmático
8.
Nat Commun ; 12(1): 1423, 2021 03 03.
Artículo en Inglés | MEDLINE | ID: mdl-33658509

RESUMEN

In the mammalian hippocampus, adult-born granule cells (abGCs) contribute to the function of the dentate gyrus (DG). Disruption of the DG circuitry causes spontaneous recurrent seizures (SRS), which can lead to epilepsy. Although abGCs contribute to local inhibitory feedback circuitry, whether they are involved in epileptogenesis remains elusive. Here, we identify a critical window of activity associated with the aberrant maturation of abGCs characterized by abnormal dendrite morphology, ectopic migration, and SRS. Importantly, in a mouse model of temporal lobe epilepsy, silencing aberrant abGCs during this critical period reduces abnormal dendrite morphology, cell migration, and SRS. Using mono-synaptic tracers, we show silencing aberrant abGCs decreases recurrent CA3 back-projections and restores proper cortical connections to the hippocampus. Furthermore, we show that GABA-mediated amplification of intracellular calcium regulates the early critical period of activity. Our results demonstrate that aberrant neurogenesis rewires hippocampal circuitry aggravating epilepsy in mice.


Asunto(s)
Epilepsia/fisiopatología , Hipocampo/fisiopatología , Neurogénesis/fisiología , Animales , Calcio/metabolismo , Clozapina/análogos & derivados , Clozapina/farmacología , Modelos Animales de Enfermedad , Electroencefalografía , Epilepsia del Lóbulo Temporal/fisiopatología , Femenino , Ratones Endogámicos C57BL , Ratones Transgénicos , Neurogénesis/efectos de los fármacos , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Pilocarpina/farmacología , Retroviridae/genética , Convulsiones/fisiopatología , Ácido gamma-Aminobutírico/metabolismo
9.
Biomed Khim ; 67(1): 17-33, 2021 Jan.
Artículo en Ruso | MEDLINE | ID: mdl-33645519

RESUMEN

Ca2+-activated chloride channels (CaCC) are a class of intracellular calcium activated chloride channels that mediate numerous physiological functions. In 2008, the molecular structure of CaCC was determined. CaCC are formed by the protein known as anoctamine 1 (ANO1 or TMEM16A). CaCC mediates the secretion of Cl- in secretory epithelia, such as the airways, salivary glands, intestines, renal tubules, and sweat glands. The presence of CaCC has also been recognized in the vascular muscles, smooth muscles of the respiratory tract, which control vascular tone and hypersensitivity of the respiratory tract. TMEM16A is activated in many cancers; it is believed that TMEM16A is involved in carcinogenesis. TMEM16A is also involved in cancer cells proliferation. The role of TMEM16A in the mechanisms of hypertension, asthma, cystic fibrosis, nociception, and dysfunction of the gastrointestinal tract has been determined. In addition to TMEM16A, its isoforms are involved in other physiological and pathophysiological processes. TMEM16B (or ANO2) is involved in the sense of smell, while ANO6 works like scramblase, and its mutation causes a rare bleeding disorder, known as Scott syndrome. ANO5 is associated with muscle and bone diseases. TMEM16A interacts with various cellular signaling pathways including: epidermal growth factor receptor (EGFR), mitogen-activated protein kinases (MAPK), calmodulin (CaM) kinases, transforming growth factor TGF-ß. The review summarizes existing information on known natural and synthetic compounds that can block/modulate CaCC currents and their effect on some pathologies in which CaCC is involved.


Asunto(s)
Carcinogénesis , Canales de Cloruro , Anoctamina-1 , Anoctaminas , Calcio/metabolismo , Canales de Cloruro/genética , Humanos
10.
Molecules ; 26(5)2021 Feb 26.
Artículo en Inglés | MEDLINE | ID: mdl-33652901

RESUMEN

Slow-channel congenital myasthenic syndromes (SCCMSs) are rare genetic diseases caused by mutations in muscle nicotinic acetylcholine receptor (nAChR) subunits. Most of the known SCCMS-associated mutations localize at the transmembrane region near the ion pore. Only two SCCMS point mutations are at the extracellular domains near the acetylcholine binding site, α1(G153S) being one of them. In this work, a combination of molecular dynamics, targeted mutagenesis, fluorescent Ca2+ imaging and patch-clamp electrophysiology has been applied to G153S mutant muscle nAChR to investigate the role of hydrogen bonds formed by Ser 153 with C-loop residues near the acetylcholine-binding site. Introduction of L199T mutation to the C-loop in the vicinity of Ser 153 changed hydrogen bonds distribution, decreased acetylcholine potency (EC50 2607 vs. 146 nM) of the double mutant and decay kinetics of acetylcholine-evoked cytoplasmic Ca2+ rise (τ 14.2 ± 0.3 vs. 34.0 ± 0.4 s). These results shed light on molecular mechanisms of nAChR activation-desensitization and on the involvement of such mechanisms in channelopathy genesis.


Asunto(s)
Acetilcolina/genética , Secuencia de Aminoácidos/genética , Síndromes Miasténicos Congénitos/genética , Receptores Nicotínicos/genética , Acetilcolina/metabolismo , Sitios de Unión/genética , Calcio/metabolismo , Humanos , Cinética , Síndromes Miasténicos Congénitos/patología , Técnicas de Placa-Clamp , Mutación Puntual/genética , Unión Proteica/genética
11.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671444

RESUMEN

Lipid rafts are a primary target in studies of amyloid ß (Aß) cytotoxicity in neurons. Exogenous Aß peptides bind to lipid rafts, which in turn play a key role in Aß uptake, leading to the formation of neurotoxic intracellular Aß aggregates. On the other hand, dysregulation of intracellular calcium homeostasis in neurons has been observed in Alzheimer's disease (AD). In a previous work, we showed that Aß(1-42), the prevalent Aß peptide found in the amyloid plaques of AD patients, binds with high affinity to purified calmodulin (CaM), with a dissociation constant ≈1 nM. In this work, to experimentally assess the Aß(1-42) binding capacity to intracellular CaM, we used primary cultures of mature cerebellar granule neurons (CGN) as a neuronal model. Our results showed a large complexation of submicromolar concentrations of Aß(1-42) dimers by CaM in CGN, up to 120 ± 13 picomoles of Aß(1-42) /2.5 × 106 cells. Using fluorescence microscopy imaging, we showed an extensive co-localization of CaM and Aß(1-42) in lipid rafts in CGN stained with up to 100 picomoles of Aß(1-42)-HiLyteTM-Fluor555 monomers. Intracellular Aß(1-42) concentration in this range was achieved by 2 h incubation of CGN with 2 µM Aß(1-42), and this treatment lowered the resting cytosolic calcium of mature CGN in partially depolarizing 25 mM potassium medium. We conclude that the primary cause of the resting cytosolic calcium decrease is the inhibition of L-type calcium channels of CGN by Aß(1-42) dimers, whose activity is inhibited by CaM:Aß(1-42) complexes bound to lipid rafts.


Asunto(s)
Péptidos beta-Amiloides/metabolismo , Calcio/metabolismo , Calmodulina/metabolismo , Cerebelo/metabolismo , Citosol/metabolismo , Homeostasis , Microdominios de Membrana/metabolismo , Neuronas/metabolismo , Fragmentos de Péptidos/metabolismo , Animales , Canales de Calcio Tipo L/metabolismo , Supervivencia Celular , Transferencia Resonante de Energía de Fluorescencia , Humanos , Unión Proteica , Ratas Wistar
12.
Int J Mol Sci ; 22(4)2021 Feb 17.
Artículo en Inglés | MEDLINE | ID: mdl-33671466

RESUMEN

During capacitation, sperm undergo a myriad of changes, including remodeling of plasma membrane, modification of sperm motility and kinematic parameters, membrane hyperpolarization, increase in intracellular calcium levels, and tyrosine phosphorylation of certain sperm proteins. While potassium channels have been reported to be crucial for capacitation of mouse and human sperm, their role in pigs has not been investigated. With this purpose, sperm samples from 15 boars were incubated in capacitation medium for 300 min with quinine, a general blocker of potassium channels (including voltage-gated potassium channels, calcium-activated potassium channels, and tandem pore domain potassium channels), and paxilline (PAX), a specific inhibitor of calcium-activated potassium channels. In all samples, acrosome exocytosis was induced after 240 min of incubation with progesterone. Plasma membrane and acrosome integrity, membrane lipid disorder, intracellular calcium levels, mitochondrial membrane potential, and total and progressive sperm motility were evaluated after 0, 120, and 240 min of incubation, and after 5, 30, and 60 min of progesterone addition. Although blocking potassium channels with quinine and PAX prevented sperm to elicit in vitro capacitation by impairing motility and mitochondrial function, as well as reducing intracellular calcium levels, the extent of that inhibition was larger with quinine than with PAX. Therefore, while our data support that calcium-activated potassium channels are essential for sperm capacitation in pigs, they also suggest that other potassium channels, such as the voltage-gated, tandem pore domain, and mitochondrial ATP-regulated ones, are involved in that process. Thus, further research is needed to elucidate the specific functions of these channels and the mechanisms underlying its regulation during sperm capacitation.


Asunto(s)
Acrosoma/metabolismo , Exocitosis/efectos de los fármacos , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio/metabolismo , Progesterona/farmacología , Capacitación Espermática/efectos de los fármacos , Acrosoma/efectos de los fármacos , Animales , Calcio/metabolismo , Membrana Celular/efectos de los fármacos , Membrana Celular/metabolismo , Supervivencia Celular/efectos de los fármacos , Espacio Intracelular/metabolismo , Masculino , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Paxillin/farmacología , Quinina/farmacología , Motilidad Espermática/efectos de los fármacos , Porcinos
13.
Cell Prolif ; 54(4): e13022, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33686740

RESUMEN

OBJECTIVES: This study aimed to investigate the protective effect of SCARF1 on acute rejection (AR), phagocytic clearance of Kupffer cells (KCs), M2 polarization and the exact mechanism underlying these processes. METHODS: AAV was transfected into the portal vein of rats, and AR and immune tolerance (IT) models of liver transplantation were established. Liver tissue and blood samples were collected. The level of SCARF1 was detected via WB and immunohistochemical staining. Pathological changes in liver tissue were detected using HE staining. Apoptotic cells were detected using TUNEL staining. KC polarization was assessed via immunohistochemical staining. Primary KCs were isolated and co-cultured with apoptotic T lymphocytes. Phagocytosis of apoptotic cells and polarization of KCs were both detected using immunofluorescence. Calcium concentration was determined using immunofluorescence and a fluorescence microplate reader. The levels of PI3K, p-AKT and P-STAT3 were assessed via WB and immunofluorescence. RESULTS: Compared to the IT group, the level of SCARF1 was significantly decreased in the AR group. Overexpression of SCARF1 in KCs improved AR and liver function markers. Enhanced phagocytosis mediated by SCARF1 is beneficial for improving the apoptotic clearance of AR and promoting M2 polarization of KCs. SCARF1-mediated enhancement of phagocytosis promotes increased calcium concentration in KCs, thus further activating the PI3K-AKT-STAT3 signalling pathway. CONCLUSIONS: SCARF1 promotes the M2 polarization of KCs by promoting phagocytosis through the calcium-dependent PI3K-AKT-STAT3 signalling pathway.


Asunto(s)
Calcio/metabolismo , Trasplante de Hígado , Receptores Depuradores de Clase F/metabolismo , Transducción de Señal , Animales , Apoptosis , Polaridad Celular , Células Cultivadas , Técnicas de Cocultivo , Macrófagos del Hígado/citología , Macrófagos del Hígado/metabolismo , Hígado/metabolismo , Hígado/patología , Masculino , Fagocitosis , Fosfatidilinositol 3-Quinasas/metabolismo , Proteínas Proto-Oncogénicas c-akt/metabolismo , Ratas , Ratas Endogámicas Lew , Factor de Transcripción STAT3/metabolismo , Receptores Depuradores de Clase F/genética , Linfocitos T/citología , Linfocitos T/metabolismo
14.
J Vis Exp ; (168)2021 02 11.
Artículo en Inglés | MEDLINE | ID: mdl-33645563

RESUMEN

Within the last ten years, advances in genetically encoded calcium indicators (GECIs) have promoted a revolution in in vivo functional imaging. Using calcium as a proxy for neuronal activity, these techniques provide a way to monitor the responses of individual cells within large neuronal ensembles to a variety of stimuli in real time. We, and others, have applied these techniques to image the responses of individual geniculate ganglion neurons to taste stimuli applied to the tongues of live anesthetized mice. The geniculate ganglion is comprised of the cell bodies of gustatory neurons innervating the anterior tongue and palate as well as some somatosensory neurons innervating the pinna of the ear. Imaging the taste-evoked responses of individual geniculate ganglion neurons with GCaMP has provided important information about the tuning profiles of these neurons in wild-type mice as well as a way to detect peripheral taste miswiring phenotypes in genetically manipulated mice. Here we demonstrate the surgical procedure to expose the geniculate ganglion, GCaMP fluorescence image acquisition, initial steps for data analysis, and troubleshooting. This technique can be used with transgenically encoded GCaMP, or with AAV-mediated GCaMP expression, and can be modified to image particular genetic subsets of interest (i.e., Cre-mediated GCaMP expression). Overall, in vivo calcium imaging of geniculate ganglion neurons is a powerful technique for monitoring the activity of peripheral gustatory neurons and provides complementary information to more traditional whole-nerve chorda tympani recordings or taste behavior assays.


Asunto(s)
Calcio/metabolismo , Ganglio Geniculado/fisiología , Neuronas/fisiología , Gusto/fisiología , Anestesia , Animales , Ganglio Geniculado/cirugía , Inmovilización , Ratones , Estimulación Física , Papilas Gustativas/fisiología , Traqueotomía
15.
Adv Exp Med Biol ; 1303: 305-317, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33788199

RESUMEN

Ca2+/calmodulin-dependent protein kinase II (CaMKII) is a multifunctional protein kinase and has been recently recognized to play a vital role in pathological events in the pulmonary system. CaMKII has diverse downstream targets that promote vascular disease, asthma, and cancer, so improved understanding of CaMKII signaling has the potential to lead to new therapies for lung diseases. Multiple studies have demonstrated that CaMKII is involved in redox modulation of ryanodine receptors (RyRs). CaMKII can be directly activated by reactive oxygen species (ROS) which then regulates RyR activity, which is essential for Ca2+-dependent processes in lung diseases. Furthermore, both CaMKII and RyRs participate in the inflammation process. However, their role in the pulmonary physiology in response to ROS is still an ambiguous one. Because CaMKII and RyRs are important in pulmonary biology, cell survival, cell cycle control, and inflammation, it is possible that the relationship between ROS and CaMKII/RyRs signal complex will be necessary for understanding and treating lung diseases. Here, we review roles of CaMKII/RyRs in lung diseases to understand with how CaMKII/RyRs may act as a transduction signal to connect prooxidant conditions into specific downstream pathological effects that are relevant to rare and common forms of pulmonary disease.


Asunto(s)
Enfermedades Pulmonares , Canal Liberador de Calcio Receptor de Rianodina , Calcio/metabolismo , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/genética , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina/metabolismo , Humanos , Inflamación , Rianodina , Canal Liberador de Calcio Receptor de Rianodina/genética
16.
Adv Exp Med Biol ; 1303: 319-331, 2021.
Artículo en Inglés | MEDLINE | ID: mdl-33788200

RESUMEN

Asthma is a chronic disease characterized by airway hyperresponsiveness, which can be caused by exposure to an allergen, spasmogen, or be induced by exercise. Despite its prevalence, the exact mechanisms by which the airway becomes hyperresponsive in asthma are not fully understood. There is evidence that myosin light-chain kinase is overexpressed, with a concomitant downregulation of myosin light-chain phosphatase in the airway smooth muscle, leading to sustained contraction. Additionally, the sarco/endoplasmic reticulum ATPase may be affected by inflammatory cytokines, such as IL-4, IL-5, IL-13, and TNF-α, which are all associated with asthmatic airway inflammation. IL-13 and TNF-α seem to promote sodium/calcium exchanger 1 overexpression as well. Anyhow, the exact mechanisms beyond these dysregulations need to be clarified. Of note, multiple studies show an association between asthma and the ORMLD3 gene, opening new perspectives to future potential gene therapies. Currently, several treatments are available for asthma, although many of them have systemic side effects, or are not effective in patients with severe asthma. Furthering our knowledge on the molecular and pathophysiological mechanisms of asthma plays a pivotal role for the development of new and more targeted treatments for patients who cannot totally benefit from the current therapies.


Asunto(s)
Asma , Hipersensibilidad Respiratoria , Asma/genética , Asma/metabolismo , Calcio/metabolismo , Señalización del Calcio , Humanos , Músculo Liso , Sistema Respiratorio/metabolismo
17.
Nat Commun ; 12(1): 1648, 2021 03 12.
Artículo en Inglés | MEDLINE | ID: mdl-33712605

RESUMEN

Cardiomyocytes undergo significant structural and functional changes after birth, and these fundamental processes are essential for the heart to pump blood to the growing body. However, due to the challenges of isolating single postnatal/adult myocytes, how individual newborn cardiomyocytes acquire multiple aspects of the mature phenotype remains poorly understood. Here we implement large-particle sorting and analyze single myocytes from neonatal to adult hearts. Early myocytes exhibit wide-ranging transcriptomic and size heterogeneity that is maintained until adulthood with a continuous transcriptomic shift. Gene regulatory network analysis followed by mosaic gene deletion reveals that peroxisome proliferator-activated receptor coactivator-1 signaling, which is active in vivo but inactive in pluripotent stem cell-derived cardiomyocytes, mediates the shift. This signaling simultaneously regulates key aspects of cardiomyocyte maturation through previously unrecognized proteins, including YAP1 and SF3B2. Our study provides a single-cell roadmap of heterogeneous transitions coupled to cellular features and identifies a multifaceted regulator controlling cardiomyocyte maturation.


Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Miocitos Cardíacos/metabolismo , Receptores Activados del Proliferador del Peroxisoma/metabolismo , Factores de Empalme de ARN/metabolismo , Factores de Transcripción/metabolismo , Animales , Calcio/metabolismo , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Diferenciación Celular , Redes Reguladoras de Genes , Humanos , Células Madre Pluripotentes Inducidas/metabolismo , Ratones , Receptores Activados del Proliferador del Peroxisoma/genética , Células Madre Pluripotentes/metabolismo , Transducción de Señal , Factores de Transcripción/genética , Transcriptoma
18.
Plant Sci ; 306: 110876, 2021 May.
Artículo en Inglés | MEDLINE | ID: mdl-33775371

RESUMEN

Acid rain, as a typical abiotic stress, damages plant growth and production. Calcium (Ca) mediates plant growth and links the signal transduction in plants for adapting to abiotic stresses. To understand the effect of Ca2+ on plant adaptable response to acid rain, we investigated changes in activities and gene expression of antioxidative enzymes and fatty acid composition of membrane lipid in rice seedlings treated with exogenous Ca2+ (5 mM) or/and simulated acid rain (SAR, pH 3.5 / 2.5). Exogenous Ca2+ enhanced activities of superoxide dismutase, catalase and peroxidase isozymes in rice leaves under SAR stress by promoting activation of existing isoforms and up-regulation of Cu/Zn-SOD1, Cu/Zn-SOD2, Cu/Zn-SOD3, CAT1, CAT2 and POD1. Compared to SAR treatment alone, exogenous Ca2+ alleviated SAR-induced oxidative damage to cell membrane by enhancing antioxidative capacity, as shown by the decrease in concentrations of H2O2, O2- and malondialdehyde in rice leaves. Meanwhile, Ca2+ alleviated SAR-induced decrease in unsaturation of membrane lipid for maintaining membrane fluidity. Finally, exogenous Ca2+ alleviated SAR-induced inhibition on relative growth rate of rice. Therefore, Ca2+ could play a role in regulating activities of antioxidative enzymes as well as maintaining unsaturation of membrane lipid for enhancing tolerance in rice seedlings to acid rain stress.


Asunto(s)
Lluvia Ácida/efectos adversos , Adaptación Fisiológica , Antioxidantes/metabolismo , Calcio/metabolismo , Isoenzimas/metabolismo , Oryza/enzimología , Oryza/crecimiento & desarrollo , Estrés Fisiológico/fisiología , Productos Agrícolas/enzimología , Productos Agrícolas/crecimiento & desarrollo
19.
Nat Commun ; 12(1): 1814, 2021 03 22.
Artículo en Inglés | MEDLINE | ID: mdl-33753734

RESUMEN

The self-assembly of α-synuclein (αS) into intraneuronal inclusion bodies is a key characteristic of Parkinson's disease. To define the nature of the species giving rise to neuronal damage, we have investigated the mechanism of action of the main αS populations that have been observed to form progressively during fibril growth. The αS fibrils release soluble prefibrillar oligomeric species with cross-ß structure and solvent-exposed hydrophobic clusters. αS prefibrillar oligomers are efficient in crossing and permeabilize neuronal membranes, causing cellular insults. Short fibrils are more neurotoxic than long fibrils due to the higher proportion of fibrillar ends, resulting in a rapid release of oligomers. The kinetics of released αS oligomers match the observed kinetics of toxicity in cellular systems. In addition to previous evidence that αS fibrils can spread in different brain areas, our in vitro results reveal that αS fibrils can also release oligomeric species responsible for an immediate dysfunction of the neurons in the vicinity of these species.


Asunto(s)
Amiloide/metabolismo , Cuerpos de Inclusión/metabolismo , Neuronas/metabolismo , alfa-Sinucleína/metabolismo , Amiloide/química , Animales , Calcio/metabolismo , Línea Celular Tumoral , Células Cultivadas , Humanos , Cinética , Microscopía Confocal , Enfermedad de Parkinson/metabolismo , Agregación Patológica de Proteínas , Multimerización de Proteína , Ratas Sprague-Dawley , alfa-Sinucleína/química
20.
Nat Commun ; 12(1): 1932, 2021 03 26.
Artículo en Inglés | MEDLINE | ID: mdl-33771998

RESUMEN

The physical distance between presynaptic Ca2+ channels and the Ca2+ sensors triggering the release of neurotransmitter-containing vesicles regulates short-term plasticity (STP). While STP is highly diversified across synapse types, the computational and behavioral relevance of this diversity remains unclear. In the Drosophila brain, at nanoscale level, we can distinguish distinct coupling distances between Ca2+ channels and the (m)unc13 family priming factors, Unc13A and Unc13B. Importantly, coupling distance defines release components with distinct STP characteristics. Here, we show that while Unc13A and Unc13B both contribute to synaptic signalling, they play distinct roles in neural decoding of olfactory information at excitatory projection neuron (ePN) output synapses. Unc13A clusters closer to Ca2+ channels than Unc13B, specifically promoting fast phasic signal transfer. Reduction of Unc13A in ePNs attenuates responses to both aversive and appetitive stimuli, while reduction of Unc13B provokes a general shift towards appetitive values. Collectively, we provide direct genetic evidence that release components of distinct nanoscopic coupling distances differentially control STP to play distinct roles in neural decoding of sensory information.


Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de la Membrana/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Plasticidad Neuronal/fisiología , Sinapsis/fisiología , Transmisión Sináptica/fisiología , Animales , Animales Modificados Genéticamente , Conducta Apetitiva/fisiología , Calcio/metabolismo , Canales de Calcio/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Femenino , Interneuronas/metabolismo , Interneuronas/fisiología , Proteínas de la Membrana/genética , Microscopía Confocal , Proteínas del Tejido Nervioso/genética , Plasticidad Neuronal/genética , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Interferencia de ARN , Sinapsis/metabolismo , Transmisión Sináptica/genética , Vesículas Sinápticas/metabolismo
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