RESUMEN
Mammalian two-pore channel proteins (TPC1, TPC2; TPCN1, TPCN2) encode ion channels in intracellular endosomes and lysosomes and were proposed to mediate endolysosomal calcium release triggered by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). By directly recording TPCs in endolysosomes from wild-type and TPC double-knockout mice, here we show that, in contrast to previous conclusions, TPCs are in fact sodium-selective channels activated by PI(3,5)P(2) and are not activated by NAADP. Moreover, the primary endolysosomal ion is Na(+), not K(+), as had been previously assumed. These findings suggest that the organellar membrane potential may undergo large regulatory changes and may explain the specificity of PI(3,5)P(2) in regulating the fusogenic potential of intracellular organelles.
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Canales de Calcio/metabolismo , Lisosomas/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Animales , Calcio/metabolismo , Canales de Calcio/genética , Línea Celular , Glucosa/metabolismo , Células Secretoras de Insulina/metabolismo , Islotes Pancreáticos/metabolismo , Ratones , Ratones Noqueados , NADP/análogos & derivados , NADP/metabolismo , Canales de Sodio/metabolismoRESUMEN
A plethora of growth factors regulate keratinocyte proliferation and differentiation that control hair morphogenesis and skin barrier formation. Wavy hair phenotypes in mice result from naturally occurring loss-of-function mutations in the genes for TGF-alpha and EGFR. Conversely, excessive activities of TGF-alpha/EGFR result in hairless phenotypes and skin cancers. Unexpectedly, we found that mice lacking the Trpv3 gene also exhibit wavy hair coat and curly whiskers. Here we show that keratinocyte TRPV3, a member of the transient receptor potential (TRP) family of Ca(2+)-permeant channels, forms a signaling complex with TGF-alpha/EGFR. Activation of EGFR leads to increased TRPV3 channel activity, which in turn stimulates TGF-alpha release. TRPV3 is also required for the formation of the skin barrier by regulating the activities of transglutaminases, a family of Ca(2+)-dependent crosslinking enzymes essential for keratinocyte cornification. Our results show that a TRP channel plays a role in regulating growth factor signaling by direct complex formation.
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Receptores ErbB/metabolismo , Cabello/crecimiento & desarrollo , Transducción de Señal , Piel/crecimiento & desarrollo , Canales Catiónicos TRPV/metabolismo , Animales , Calcio/metabolismo , Células Cultivadas , Cabello/metabolismo , Humanos , Queratinocitos/metabolismo , Ratones , Ratones Noqueados , Piel/metabolismo , Canales Catiónicos TRPV/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
OBJECTIVE: Our research aimed to investigate the therapeutic effects of Tubastatin-A, a glucocorticoid receptor (GR) mitochondrial translocation inhibitor, and mitoquinone (MitoQ), an antioxidant, on attenuating dexamethasone (DEX)-induced macrophage apoptosis. METHODS: We treated RAW264.7 macrophages with different combinations of DEX and either Tubastatin-A or MitoQ. Parameters such as mitochondrial GR translocation, mitochondrial reactive oxygen species levels, mitochondrial membrane potential, mitochondrial permeability transition pore opening, cytochrome C efflux to the cytosol, and apoptosis were subsequently evaluated in the different treatment groups via qRT-PCR, western blotting, and immunofluorescence assays. RESULTS: DEX intervention increased the translocation of GRs into the mitochondria, while reducing the expression of the mitochondrial gene MT-CO1 and the activity of mitochondrial respiratory chain complex IV in macrophages. In addition, DEX administration increased mtROS levels, mitochondrial permeability transition pore opening, and mitochondrial cytochrome C release in macrophages, which promoted their apoptosis. We found that Tubastatin-A inhibited mitochondrial GR translocation and reversed the DEX-induced increase in GR levels within the mitochondria. Furthermore, Tubastatin-A mitigated various mitochondrial changes induced by DEX, including reducing the efflux of mitochondrial cytochrome C and inhibiting macrophage apoptosis. Similarly, MitoQ exerted its effects on macrophage apoptosis by reducing mtROS levels through the mitochondrial pathway. CONCLUSIONS: The DEX-mediated translocation of GR into mitochondria disrupts the mitochondrial function of macrophages, which induces their apoptosis. By inhibiting mitochondrial translocation of GR and reducing mtROS levels, Tubastatin-A and MitoQ can effectively attenuate macrophage apoptosis, which has clinical implications for reducing the notable side effects associated with glucocorticoid use.
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Apoptosis , Dexametasona , Glucocorticoides , Macrófagos , Mitocondrias , Receptores de Glucocorticoides , Receptores de Glucocorticoides/metabolismo , Animales , Ratones , Apoptosis/efectos de los fármacos , Células RAW 264.7 , Mitocondrias/efectos de los fármacos , Mitocondrias/metabolismo , Macrófagos/efectos de los fármacos , Macrófagos/metabolismo , Dexametasona/farmacología , Glucocorticoides/farmacología , Transporte de Proteínas/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Ubiquinona/farmacología , Ubiquinona/análogos & derivados , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Compuestos OrganofosforadosRESUMEN
Toxic and major elements, such as As and Fe, in watersheds can significantly impact the surrounding water environment and ecosystem. Thus, in this study, we conducted an investigation into the origins and spatial distribution of typical toxic trace elements (As and Mn) and crustal major elements (Al, Fe, and Ti) in suspended particulate matter (SPM) across various glacial watersheds located at different elevations in the northeastern Tibetan Plateau (NETP) from June to July in 2017. The results revealed that the mean value of each element followed the order of abundance in the samples, with Al having the highest mean value at 21307 µg/L, followed by Fe at 13366 µg/L, Ti at 1520 µg/L, Mn at 245 µg/L, and As at 66.6 µg/L. Moreover, our study identified high content of these elements from the Dabanshan Snowpack, Laohugou Glacier No.12, and Yuzhufeng Glacier in the upper reaches of the basin, which were found to be 9.9, 10.2, and 19.4 times higher, respectively, than that of the upper reaches of the Heihe River. We found that As and Mn exhibited clear indications of anthropogenic influence on a local and regional scale. The calculated enrichment factor (EF) demonstrated a significant As enrichment (EF>100) in the Qiyi and Lenglongling Glaciers, possibly resulting in the release of upstream glacier melt and anthropogenic-derived As deposition. Our findings suggested that the upstream region was primarily linked to glacier meltwater discharge. In contrast, the middle and lower reaches of the basin exhibited a more pronounced influence from local human activities. Based on the findings, the water environment of the glacier watershed appears to be in good condition overall. However, the presence of elevated levels of As element in the water system can be traced back to both anthropogenic and natural factors. As a result, ensuring the safety of the water supply for nearby residents is a matter of utmost concern. This study provides a comprehensive examination of hydrochemical variations and the overall water environment of high-altitude glacier basins in the NETP, offering valuable insights into the topic.
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Oligoelementos , Humanos , Tibet , Oligoelementos/análisis , Ecosistema , Material Particulado , Agua/química , Monitoreo del Ambiente/métodosRESUMEN
Amino acids, the building blocks of proteins in the cells and tissues, are of fundamental importance for cell survival, maintenance, and proliferation. The liver plays a critical role in amino acid metabolism and detoxication of byproducts such as ammonia. Urea cycle disorders with hyperammonemia remain difficult to treat and eventually necessitate liver transplantation. In this study, ornithine transcarbamylase deficient (Otcspf-ash ) mouse model was used to test whether knockdown of a key glutamine metabolism enzyme glutaminase 2 (GLS2, gene name: Gls2) or glutamate dehydrogenase 1 (GLUD1, gene name: Glud1) could rescue the hyperammonemia and associated lethality induced by a high protein diet. We found that reduced hepatic expression of Gls2 but not Glud1 by AAV8-mediated delivery of a short hairpin RNA in Otcspf-ash mice diminished hyperammonemia and reduced lethality. Knockdown of Gls2 but not Glud1 in Otcspf-ash mice exhibited reduced body weight loss and increased plasma glutamine concentration. These data suggest that Gls2 hepatic knockdown could potentially help alleviate risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle.
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Glutaminasa/metabolismo , Hiperamonemia , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa , Trastornos Innatos del Ciclo de la Urea , Amoníaco , Animales , Modelos Animales de Enfermedad , Glutaminasa/genética , Glutamina/metabolismo , Humanos , Hiperamonemia/metabolismo , Hígado/metabolismo , Ratones , Ornitina Carbamoiltransferasa/genética , Enfermedad por Deficiencia de Ornitina Carbamoiltransferasa/metabolismo , Urea/metabolismo , Trastornos Innatos del Ciclo de la Urea/genética , Trastornos Innatos del Ciclo de la Urea/metabolismoRESUMEN
NASH is a chronic liver disease that affects 3%-6% of individuals and requires urgent therapeutic developments. Isolating the key cell types in the liver is a necessary step towards understanding their function and roles in disease pathogenesis. However, traditional isolation methods through gradient centrifugation can only collect one or a few cell types simultaneously and pose technical difficulties when applied to NASH livers. Taking advantage of identified cell surface markers from liver single-cell RNAseq, here we established the combination of gradient centrifugation and antibody-based cell sorting techniques to isolate five key liver cell types (hepatocytes, endothelial cells, stellate cells, macrophages and other immune cells) from a single mouse liver. This method yielded high purity of each cell type from healthy and NASH livers. Our five-in-one protocol simultaneously isolates key liver cell types with high purity under normal and NASH conditions, enabling for systematic and accurate exploratory experiments such as RNA sequencing.
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Separación Celular , Hígado/citología , Enfermedad del Hígado Graso no Alcohólico/patología , Animales , Biomarcadores , Separación Celular/métodos , Modelos Animales de Enfermedad , Células Endoteliales/metabolismo , Técnica del Anticuerpo Fluorescente , Hepatocitos/metabolismo , Inmunofenotipificación , Macrófagos/metabolismo , Masculino , Ratones , Enfermedad del Hígado Graso no Alcohólico/etiologíaRESUMEN
BACKGROUND: Elucidation of the genetic factors underlying chronic liver disease may reveal new therapeutic targets. METHODS: We used exome sequence data and electronic health records from 46,544 participants in the DiscovEHR human genetics study to identify genetic variants associated with serum levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST). Variants that were replicated in three additional cohorts (12,527 persons) were evaluated for association with clinical diagnoses of chronic liver disease in DiscovEHR study participants and two independent cohorts (total of 37,173 persons) and with histopathological severity of liver disease in 2391 human liver samples. RESULTS: A splice variant (rs72613567:TA) in HSD17B13, encoding the hepatic lipid droplet protein hydroxysteroid 17-beta dehydrogenase 13, was associated with reduced levels of ALT (P=4.2×10-12) and AST (P=6.2×10-10). Among DiscovEHR study participants, this variant was associated with a reduced risk of alcoholic liver disease (by 42% [95% confidence interval {CI}, 20 to 58] among heterozygotes and by 53% [95% CI, 3 to 77] among homozygotes), nonalcoholic liver disease (by 17% [95% CI, 8 to 25] among heterozygotes and by 30% [95% CI, 13 to 43] among homozygotes), alcoholic cirrhosis (by 42% [95% CI, 14 to 61] among heterozygotes and by 73% [95% CI, 15 to 91] among homozygotes), and nonalcoholic cirrhosis (by 26% [95% CI, 7 to 40] among heterozygotes and by 49% [95% CI, 15 to 69] among homozygotes). Associations were confirmed in two independent cohorts. The rs72613567:TA variant was associated with a reduced risk of nonalcoholic steatohepatitis, but not steatosis, in human liver samples. The rs72613567:TA variant mitigated liver injury associated with the risk-increasing PNPLA3 p.I148M allele and resulted in an unstable and truncated protein with reduced enzymatic activity. CONCLUSIONS: A loss-of-function variant in HSD17B13 was associated with a reduced risk of chronic liver disease and of progression from steatosis to steatohepatitis. (Funded by Regeneron Pharmaceuticals and others.).
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17-Hidroxiesteroide Deshidrogenasas/genética , Hígado Graso/genética , Predisposición Genética a la Enfermedad , Hepatopatías/genética , Mutación con Pérdida de Función , 17-Hidroxiesteroide Deshidrogenasas/metabolismo , Alanina Transaminasa/sangre , Aspartato Aminotransferasas/sangre , Biomarcadores/sangre , Enfermedad Crónica , Progresión de la Enfermedad , Femenino , Variación Genética , Genotipo , Humanos , Modelos Lineales , Hígado/patología , Hepatopatías/patología , Masculino , Análisis de Secuencia de ARN , Secuenciación del ExomaRESUMEN
OBJECTIVES: We sought to analyse the expression characteristics of cytochrome C oxidase subunit I in mitochondrial of MRL/lpr lupus mice. METHODS: The whole blood of MRL/lpr lupus mice was detected for whole mitochondrial genome sequencing performed by Illumina HiSeq PE150 instrument, compared with house mouse (NC_005089.1) and screened for the maximum difference gene, MT-CO1. Quantitative real-time polymerase chain reaction (qRT-PCR) and western blot were used to detect the mRNA and protein expression of MT-CO1 in lupus mice and control mice. The total antioxidant capacities of lupus mice and control mice were measured using the rapid 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) (ABTS) method. RESULTS: The mitochondrial genome sequencing showed that five mitochondrial genes had base differences and MT-CO1 was the maximum difference gene, 31 in total. Among the 31 base difference sites, 2 were missense mutations and 29 were synonymous_variant. qRT-PCR test results showed that the MT-CO1 expression in lupus mouse blood was statistically lower than that in control mice blood (t=4.333; p=0.0003). Western blot test results revealed that the expression of MT-CO1 was lower in the lupus mice compared with the control mice at the protein level. Serum total antioxidant capacity testing showed that: the serum total antioxidant capacity of lupus mice was statistically lower than that of the control mice (t=9.957; p<0.0001). CONCLUSIONS: High mutation rate and decreased expression of MT-CO1 in MRL/lpr lupus mice accompanied the decrease of antioxidant capacity, which indicated that abnormal MT-CO1 might be involved in the pathogenesis of SLE and the production of anti-dsDNA antibodies.
Asunto(s)
Complejo IV de Transporte de Electrones , Lupus Eritematoso Sistémico , Animales , Western Blotting , Complejo IV de Transporte de Electrones/genética , Lupus Eritematoso Sistémico/genética , Ratones , Ratones Endogámicos MRL lpr , ARN Mensajero/genéticaRESUMEN
Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal function. Wnt/ß-catenin signaling controls metabolic zonation by activating genes in the perivenous hepatocytes, while suppressing genes in the periportal counterparts. We now demonstrate that glucagon opposes the actions of Wnt/ß-catenin signaling on gene expression and metabolic zonation pattern. The effects were more pronounced in the periportal hepatocytes where 28% of all genes were activated by glucagon and inhibited by Wnt/ß-catenin. The glucagon and Wnt/ß-catenin receptors and their signaling pathways are uniformly distributed in periportal and perivenous hepatocytes and the expression is not regulated by the opposing signal. Collectively, our results show that glucagon controls gene expression and metabolic zonation in the liver through a counterplay with the Wnt/ß-catenin signaling pathway.
Asunto(s)
Regulación de la Expresión Génica/fisiología , Glucagón/metabolismo , Hepatocitos/metabolismo , Hígado/metabolismo , Vía de Señalización Wnt/fisiología , Animales , Glucagón/genética , Ratones , Ratones NoqueadosRESUMEN
Inactivating mutations in the insulin receptor results in extreme insulin resistance. The resulting hyperglycemia is very difficult to treat, and patients are at risk for early morbidity and mortality from complications of diabetes. We used the insulin receptor antagonist S961 to induce severe insulin resistance, hyperglycemia, and ketonemia in mice. Using this model, we show that glucagon receptor (GCGR) inhibition with a monoclonal antibody normalized blood glucose and ß-hydroxybutyrate levels. Insulin receptor antagonism increased pancreatic ß-cell mass threefold. Normalization of blood glucose levels with GCGR-blocking antibody unexpectedly doubled ß-cell mass relative to that observed with S961 alone and 5.8-fold over control. GCGR antibody blockage expanded α-cell mass 5.7-fold, and S961 had no additional effects. Collectively, these data show that GCGR antibody inhibition represents a potential therapeutic option for treatment of patients with extreme insulin-resistance syndromes.
Asunto(s)
Diabetes Mellitus Experimental/genética , Glucagón/metabolismo , Resistencia a la Insulina/genética , Receptor de Insulina/genética , Receptores de Glucagón/genética , Ácido 3-Hidroxibutírico/metabolismo , Animales , Glucemia/genética , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/patología , Glucagón/genética , Células Secretoras de Glucagón/metabolismo , Células Secretoras de Glucagón/patología , Humanos , Hiperglucemia/genética , Hiperglucemia/metabolismo , Hiperglucemia/patología , Insulina/genética , Insulina/metabolismo , Células Secretoras de Insulina/metabolismo , Células Secretoras de Insulina/patología , Cetosis/genética , Cetosis/metabolismo , Cetosis/patología , Ratones , Mutación , Péptidos/farmacología , Receptores de Glucagón/antagonistas & inhibidoresRESUMEN
OBJECTIVE: This study presented common lesions with systemic toxicities and uncommon adverse cutaneous reactions such as anaphylactic dermatitis in patients undergoing treatment with erlotinib for the benefit of practicing dermatologists and oncologists. METHODS: Adverse cutaneous reactions associated with erlotinib were reported in 20 Chinese patients with cancer. RESULTS: Adverse cutaneous reactions reported included six cases of anaphylactic dermatitis, 12 cases of acneiform rash, nine cases of xerosis, five cases of nail changes and four cases of hair changes. One case of anaphylactic dermatitis manifested as erythema with swelling on the face and neck, and others as erosive and scaly erythema on the fold of skin, or red macules, papules, plaques and pigmentation on the whole body. Clinical details indicated anaphylactic reactions, including a high percentage of eosinophils in the peripheral blood, eosinophilic infiltration in the dermis layer and good response to antihistamines and topical steroids. Systemic toxicities accompanied by cutaneous reactions occurred in five patients including one case of anaphylactic dermatitis and four cases of acneiform rash. Elevated hepatic enzymes were observed among all the patients with grade-3 or grade-4 acneiform rashes. One patient with anaphylactic dermatitis and one with acneiform rash discontinued erlotinib administration due to severe lesions, high fever or severe elevation of hepatic enzymes. CONCLUSIONS: Anaphylactic cutaneous reactions caused by erlotinib are rarely described hitherto. Systemic toxicities should be emphasized especially in cases with severe skin disorders. Timely detection and appropriate early intervention in patients who develop severe cutaneous reaction while on erlotinib therapy should be considered clinically.
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Anafilaxia/inducido químicamente , Antineoplásicos/efectos adversos , Clorhidrato de Erlotinib/efectos adversos , Inhibidores de Proteínas Quinasas/efectos adversos , Enfermedades de la Piel/inducido químicamente , Adulto , Anciano , Antineoplásicos/uso terapéutico , Pueblo Asiatico , Clorhidrato de Erlotinib/uso terapéutico , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neoplasias/tratamiento farmacológico , Inhibidores de Proteínas Quinasas/uso terapéuticoRESUMEN
Resealing allows cells to mend damaged membranes rapidly when plasma membrane (PM) disruptions occur. Models of PM repair mechanisms include the "lipid-patch", "endocytic removal", and "macro-vesicle shedding" models, all of which postulate a dependence on local increases in intracellular Ca(2+) at injury sites. Multiple calcium sensors, including synaptotagmin (Syt) VII, dysferlin, and apoptosis-linked gene-2 (ALG-2), are involved in PM resealing, suggesting that Ca(2+) may regulate multiple steps of the repair process. Although earlier studies focused exclusively on external Ca(2+), recent studies suggest that Ca(2+) release from intracellular stores may also be important for PM resealing. Hence, depending on injury size and the type of injury, multiple sources of Ca(2+) may be recruited to trigger and orchestrate repair processes. In this review, we discuss the mechanisms by which the resealing process is promoted by vesicular Ca(2+) channels and Ca(2+) sensors that accumulate at damage sites.
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Señalización del Calcio , Membrana Celular/fisiología , Animales , Exocitosis , Humanos , Lisosomas/fisiología , Canales de Potencial de Receptor Transitorio/metabolismo , Cicatrización de HeridasRESUMEN
Neuronal polarization is pivotal for neural network formation during brain development. Axon differentiation is a hallmark of initial neuronal polarization. Here, we report that the leucine-rich repeat-containing protein netrin-G ligand-2 (NGL-2) as a polarity regulator that localizes asymmetrically in rat hippocampal neurons and is required for differentiation of the future axon. NGL-2 was associated with PAR complex, and this interaction resulted in local stabilization of axonal microtubules. Further study showed that the C terminal of NGL-2 binds to the PDZ domain of PAR6, and NGL-2 interacts with PAR3 and atypical PKCζ (aPKCζ), with PAR6 acting as a bridge or modifier. Then, NGL-2 regulates the local stabilization of microtubules and promotes axon differentiation by the aPKCζ/microtubule affinity-regulating kinase 2 pathway. These findings reveal the critical role of NGL-2 in regulating axon differentiation in rat hippocampal neurons and reveal a novel partner of the PAR complex.
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Axones/metabolismo , Proteínas Portadoras/metabolismo , Diferenciación Celular/fisiología , Proteínas Ligadas a GPI/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Proteínas Adaptadoras Transductoras de Señales , Animales , Células Cultivadas , Femenino , Células HEK293 , Hipocampo/citología , Hipocampo/metabolismo , Humanos , Masculino , Netrinas , RatasRESUMEN
Global disruption of transient receptor potential-melastatin-like 7 (Trpm7) in mice results in embryonic lethality before embryonic day 7. Using tamoxifen-inducible disruption of Trpm7 and multiple Cre recombinase lines, we show that Trpm7 deletion before and during organogenesis results in severe tissue-specific developmental defects. We find that Trpm7 is essential for kidney development from metanephric mesenchyme but not ureteric bud. Disruption of neural crest Trpm7 at early stages results in loss of pigment cells and dorsal root ganglion neurons. In contrast, late disruption of brain-specific Trpm7 after embryonic day 10.5 does not alter normal brain development. We developed induced pluripotent stem cells and neural stem (NS) cells in which Trpm7 disruption could be induced. Trpm7(-/-) NS cells retained the capacities of self-renewal and differentiation into neurons and astrocytes. During in vitro differentiation of induced pluripotent stem cells to NS cells, Trpm7 disruption prevents the formation of the NS cell monolayer. The in vivo and in vitro results demonstrate a temporal requirement for the Trpm7 channel kinase during embryogenesis.
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Desarrollo Embrionario/fisiología , Canales Catiónicos TRPM/fisiología , Animales , Diferenciación Celular , Femenino , Proteínas de Filamentos Intermediarios/fisiología , Masculino , Ratones , Ratones Transgénicos , Proteínas del Tejido Nervioso/fisiología , Nestina , Células Madre Pluripotentes/citologíaRESUMEN
TRPML1 (mucolipin 1, also known as MCOLN1) is predicted to be an intracellular late endosomal and lysosomal ion channel protein that belongs to the mucolipin subfamily of transient receptor potential (TRP) proteins. Mutations in the human TRPML1 gene cause mucolipidosis type IV disease (ML4). ML4 patients have motor impairment, mental retardation, retinal degeneration and iron-deficiency anaemia. Because aberrant iron metabolism may cause neural and retinal degeneration, it may be a primary cause of ML4 phenotypes. In most mammalian cells, release of iron from endosomes and lysosomes after iron uptake by endocytosis of Fe(3+)-bound transferrin receptors, or after lysosomal degradation of ferritin-iron complexes and autophagic ingestion of iron-containing macromolecules, is the chief source of cellular iron. The divalent metal transporter protein DMT1 (also known as SLC11A2) is the only endosomal Fe(2+) transporter known at present and it is highly expressed in erythroid precursors. Genetic studies, however, suggest the existence of a DMT1-independent endosomal and lysosomal Fe(2+) transport protein. By measuring radiolabelled iron uptake, by monitoring the levels of cytosolic and intralysosomal iron and by directly patch-clamping the late endosomal and lysosomal membrane, here we show that TRPML1 functions as a Fe(2+) permeable channel in late endosomes and lysosomes. ML4 mutations are shown to impair the ability of TRPML1 to permeate Fe(2+) at varying degrees, which correlate well with the disease severity. A comparison of TRPML1(-/- )ML4 and control human skin fibroblasts showed a reduction in cytosolic Fe(2+) levels, an increase in intralysosomal Fe(2+) levels and an accumulation of lipofuscin-like molecules in TRPML1(-/-) cells. We propose that TRPML1 mediates a mechanism by which Fe(2+) is released from late endosomes and lysosomes. Our results indicate that impaired iron transport may contribute to both haematological and degenerative symptoms of ML4 patients.
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Endosomas/metabolismo , Hierro/metabolismo , Lisosomas/metabolismo , Mucolipidosis/metabolismo , Canales Catiónicos TRPM/metabolismo , Animales , Línea Celular , Permeabilidad de la Membrana Celular , Fibroblastos , Fluorescencia , Humanos , Transporte Iónico , Hierro/análisis , Ratones , Protones , Canales Catiónicos TRPM/deficiencia , Canales Catiónicos TRPM/genética , Transfección , Canales de Potencial de Receptor TransitorioRESUMEN
Microglia are resident macrophages within the central nervous system, serving as the first responders to neuroinflammation. Glucocorticoids (GCs) may cause damage to brain tissue, but the specific mechanism remains unclear. This study was divided into two parts: a glucocorticoid receptor (GR) mitochondrial translocation intervention experiment and a mitochondrial oxidative stress inhibition experiment. BV-2 microglia were stimulated with dexamethasone (DEX) and treated with either tubastatin-A or mitoquinone (MitoQ) for 24 h. Our results showed that DEX increased the translocation of GRs to mitochondria, and this effect was accompanied by decreases in the expression of mitochondrially encoded cytochrome c oxidase 1 (MT-CO1) and mitochondrially encoded cytochrome c oxidase 3 (MT-CO3) and increases in the expression of NOD-like receptor thermal protein domain-associated protein 3 (NLRP3), caspase-1, and Gasdermin D (GSDMD). The level of mitochondrial respiratory chain complex IV (MRCC IV) and adenosine triphosphate (ATP) was decreased. An elevation in the level of mitochondrial oxidative stress and the opening of the mitochondrial permeability transition pore (mPTP) was also observed. Mechanistically, tubastatin-A significantly suppressed the mitochondrial translocation of GRs, improved the expression of mitochondrial genes, promoted the restoration of mitochondrial function, and inhibited pyroptosis. MitoQ significantly prevented mitochondrial oxidative stress, improved mitochondrial function, and reduced apoptosis and pyroptosis. Both tubastatin-A and MitoQ suppressed DEX-induced pyroptosis. This study substantiates that the increase in the mitochondrial translocation of GRs mediated by GCs exacerbates oxidative stress and pyroptosis in microglia, which indicates that the regulation of mitochondrial pathways by GCs is pathogenic to microglia.
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Glucocorticoides , Piroptosis , Glucocorticoides/farmacología , Glucocorticoides/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Microglía/metabolismo , Complejo IV de Transporte de Electrones/metabolismo , Estrés Oxidativo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismoRESUMEN
Liver zonation characterizes the separation of metabolic pathways along the lobules and is required for optimal hepatic function. Wnt signaling is a master regulator of spatial liver zonation. A perivenous-periportal Wnt activity gradient orchestrates metabolic zonation by activating gene expression in perivenous hepatocytes, while suppressing gene expression in their periportal counterparts. However, the understanding as to the liver gene zonation and zonation regulators in diseases is limited. Non-alcoholic steatohepatitis (NASH) is a chronic liver disease characterized by fat accumulation, inflammation, and fibrosis. Here, we investigated the perturbation of liver gene zonation in a mouse NASH model by combining spatial transcriptomics, bulk RNAseq and in situ hybridization. Wnt-target genes represented a major subset of genes showing altered spatial expression in the NASH liver. The altered Wnt-target gene expression levels and zonation spatial patterns were in line with the up regulation of Wnt regulators and the augmentation of Wnt signaling. Particularly, we found that the Wnt activator Rspo3 expression was restricted to the perivenous zone in control liver but expanded to the periportal zone in NASH liver. AAV8-mediated RSPO3 overexpression in controls resulted in zonation changes, and further amplified the disturbed zonation of Wnt-target genes in NASH, similarly Rspo3 knockdown in Rspo3+/- mice resulted in zonation changes of Wnt-target genes in both chow and HFD mouse. Interestingly, there were no impacts on steatosis, inflammation, or fibrosis NASH pathology from RSPO3 overexpression nor Rspo3 knockdown. In summary, our study demonstrated the alteration of Wnt signaling in a mouse NASH model, leading to perturbed liver zonation.
Asunto(s)
Enfermedad del Hígado Graso no Alcohólico , Ratones , Animales , Enfermedad del Hígado Graso no Alcohólico/metabolismo , Hígado/metabolismo , Hepatocitos/metabolismo , Inflamación/metabolismo , Modelos Animales de Enfermedad , Fibrosis , Ratones Endogámicos C57BLRESUMEN
Objectives: This study aims to investigate the expression patterns of mitochondrially encoded cytochrome c oxidase 1 (MT-CO1) in different organs and tissues of MRL/lpr mice aged six and 18 weeks. Materials and methods: Six-week-old female MRL/lpr mice (n=10) were considered young lupus model mice, and 18-week-old MRL/lpr mice (n=10) were considered old lupus model mice. Additionally, six-week-old (n=10) and 39-week-old (n=10) female Balb/c mice were used as the young and old controls, respectively. The messenger ribonucleic acid (mRNA) and protein expression levels of MT-CO1 in nine organs/tissues were detected via quantitative polymerase chain reaction (qPCR) and Western blot. Malondialdehyde (MDA) levels were determined with thiobarbituric acid colorimetry. The correlation coefficient of MT-CO1 mRNA levels and MDA levels in each organ/tissue at different ages was analyzed by Pearson correlation analysis. Results: The results showed that most non-immune organs/tissues (heart, lung, liver, kidneys, and intestines) showed increased MT-CO1 expression levels in younger MRL/lpr mice (p<0.05) and decreased MT-CO1 expression in older mice (p<0.05). Expression of MT-CO1 in the lymph nodes was low in younger mice but high in older mice. In other immune organs (spleen and thymus), MT-CO1 expression was low in older MRL/lpr mice. Lower mRNA expression and higher MDA levels were observed in the brains of MRL/lpr mice. However, all MRL/lpr mice showed higher MDA levels than Balb/c mice in every organ no matter younger or older MRL/lpr mice. Conclusion: Our study results suggest that lymphoid mitochondrial hyperfunction at organ level may be an important intrinsic pathogenesis in systemic lupus erythematosus activity, which may affect mitochondrial dysfunction in non-immune organs.
RESUMEN
Alpine grasslands harbor diverse groups of flora and fauna, provide important ecosystem functions, and yield essential ecosystem goods and services, especially for the development of nature-based tourism. However, they are experiencing increasing anthropogenic perturbations such as tourist trampling. Although negative effects of tourist trampling on alpine vegetation have been frequently reported, previous studies have focused mainly on changes in taxonomic diversity after trampling, and rarely provide a mechanistic elucidation of trampling effects from a trait-based perspective. The present study evaluates the impacts of simulated trampling on taxonomic and functional diversity of a typical alpine grassland community in Shangri-La, China using a standardized protocol. The results showed that although taxonomic diversity was not statistically significantly affected by trampling, some functional attributes responded rapidly to trampling disturbance. Specifically, functional divergence decreased with an increase in trampling intensity, and characteristics of community-weighted mean trait values changed towards shorter species with reduced leaf area and lower leaf dry matter content. Such strong shifts in functional attributes may further affect ecosystem goods and services provided by alpine grasslands. Our inclusion of functional diversity in the analysis thus adds an important caution to previous studies predominantly focusing on taxonomic diversity, and it is urgent to keep alpine grasslands well managed and ecologically coherent so that their valuable functions and services can be safeguarded.
RESUMEN
The liver plays a critical role in maintaining ammonia homeostasis. Urea cycle defects, liver injury, or failure and glutamine synthetase (GS) deficiency result in hyperammonemia, serious clinical conditions, and lethality. In this study we used a mouse model with a defect in the urea cycle enzyme ornithine transcarbamylase (Otcspf-ash) to test the hypothesis that glucagon receptor inhibition using a monoclonal blocking antibody will reduce the hyperammonemia and associated lethality induced by a high-protein diet, which exacerbates disease. We found reduced expression of glutaminase, which degrades glutamine and increased expression of GS in livers of Otcspf-ash mice treated with the glucagon receptor blocking antibody. The gene expression changes favor ammonia consumption and were accompanied by increased circulating glutamine levels and diminished hyperammonemia. Otcspf-ash mice treated with the glucagon receptor-blocking antibody gained lean and body mass and had increased survival. These data suggest that glucagon receptor inhibition using a monoclonal antibody could reduce the risk for hyperammonemia and other clinical manifestations of patients suffering from defects in the urea cycle, liver injury, or failure and GS deficiency.