RESUMEN
Mitochondrial DNA (mtDNA) encodes components essential for cellular respiration. Low levels of point mutations and deletions accumulate in mtDNA during normal aging. However, improper maintenance of mtDNA results in mitochondrial diseases, stemming from progressive loss of mitochondrial function through the accelerated formation of deletions and mutations in mtDNA. To better understand the molecular mechanisms underlying the creation and propagation of mtDNA deletions, we developed the LostArc next-generation DNA sequencing pipeline to detect and quantify rare mtDNA species in small tissue samples. LostArc procedures are designed to minimize PCR amplification of mtDNA and instead achieve enrichment of mtDNA by selective destruction of nuclear DNA. This approach leads to cost-effective, high-depth sequencing of mtDNA with a sensitivity sufficient to identify one mtDNA deletion per million mtDNA circles. Here, we describe detailed protocols for isolation of genomic DNA from mouse tissues, enrichment of mtDNA through enzymatic destruction of linear nuclear DNA, and preparation of libraries for unbiased next-generation sequencing of mtDNA.
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ADN Mitocondrial , Enfermedades Mitocondriales , Ratones , Animales , ADN Mitocondrial/genética , Mitocondrias/genética , Enfermedades Mitocondriales/genética , Mutación Puntual , Secuenciación de Nucleótidos de Alto Rendimiento/métodosRESUMEN
BACKGROUND: Previous studies showed that probiotics could improve glycemic control and attenuate some of the adverse effects of type 2 diabetes. However, whether the effects are generalizable to gestational diabetes mellitus (GDM) remains uncertain. OBJECTIVE: We conducted a systematic review and meta-analysis to evaluate the effects of probiotic supplement in GDM. METHOD: PubMed, EMBASE, the Cochrane Library, and EBSCO were systematically searched for relevant literature published through January 2019. Randomized controlled trials (RCTs) assessing the effects of probiotic supplement on one or more of the following in GDM were included: pregnancy outcome (the primary outcome), glycemic control, blood lipid profile, and inflammation and oxidative stress. Two reviewers independently extracted data and assessed the risk of bias in studies. Meta-analysis was conducted by using the fixed effects model unless substantial heterogeneity was found among studies. RESULTS: Eleven randomized trials involving 719 participants were included for analysis. Eight of the trials were from Iran. Probiotics were given alone in eight trials and synbiotics in three trials. Though the components of probiotics varied, Lactobacillus was included in all trials and Bifidobacterium in all except one. The duration of intervention ranged from 4 to 8 weeks. Almost all trials (10/11) had a low risk of bias. Probiotic supplementation reduced the risk of a newborn's hyperbilirubinemia by 74% and improved four biomarkers for glycemic control (fasting blood glucose, fasting serum insulin, homeostasis model assessment insulin resistance, and quantitative insulin sensitivity check index), two biomarkers for lipid profile (triglycerides and HDL-cholesterol), and four biomarkers for inflammation and oxidative stress (total glutathione, malondialdehyde, nitric oxide, and total antioxidant capacity). But significant heterogeneity was observed in the meta-analyses on the four biomarkers related to glycemic control and on triglycerides, which could not be explained by prespecified subgroup analyses according to the mean age of participants and intervention type (i.e., probiotics or synbiotics). The effects on the risk of preterm delivery, macrosomia and a newborns' hypoglycemia, gestational age, total cholesterol, and LDL-cholesterol were not statistically significant. CONCLUSION: Probiotic supplementation seemed to be able to reduce the risk of a newborn's hyperbilirubinemia and improve glycemic control, blood lipid profiles and inflammation and oxidative stress in pregnant women with GDM. However, due to the heterogeneity among existing studies, the surrogate nature of outcomes, and/or the fact that most studies were from Iran, the clinical significance and generalizability of the above findings remain uncertain. Further studies are warranted to address the limitations of existing evidence and better inform the management of GDM.
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Glucemia/análisis , Diabetes Gestacional/sangre , Suplementos Dietéticos , Probióticos , Femenino , Humanos , Insulina/sangre , Resistencia a la Insulina/fisiología , Embarazo , Resultado del Embarazo , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
ORAI1 constitutes the store-operated Ca2+ release-activated Ca2+ (CRAC) channel crucial for life. Whereas ORAI1 activation by Ca2+-sensing STIM proteins is known, still obscure is how ORAI1 is turned off through Ca2+-dependent inactivation (CDI), protecting against Ca2+ toxicity. Here we identify a spatially-restricted Ca2+/cAMP signaling crosstalk critical for mediating CDI. Binding of Ca2+-activated adenylyl cyclase 8 (AC8) to the N-terminus of ORAI1 positions AC8 near the mouth of ORAI1 for sensing Ca2+. Ca2+ permeating ORAI1 activates AC8 to generate cAMP and activate PKA. PKA, positioned by AKAP79 near ORAI1, phosphorylates serine-34 in ORAI1 pore extension to induce CDI whereas recruitment of the phosphatase calcineurin antagonizes the effect of PKA. Notably, CDI shapes ORAI1 cytosolic Ca2+ signature to determine the isoform and degree of NFAT activation. Thus, we uncover a mechanism of ORAI1 inactivation, and reveal a hitherto unappreciated role for inactivation in shaping cellular Ca2+ signals and NFAT activation.
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Calcio/metabolismo , AMP Cíclico/metabolismo , Proteína ORAI1/metabolismo , Proteínas de Anclaje a la Quinasa A/metabolismo , Western Blotting , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Células HEK293 , Humanos , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Proteína ORAI1/genética , Fosforilación , Molécula de Interacción Estromal 1/genética , Molécula de Interacción Estromal 1/metabolismo , Molécula de Interacción Estromal 2/genética , Molécula de Interacción Estromal 2/metabolismoRESUMEN
BACKGROUND: Adiponectin (ADIPOQ) is an important factor involved in the regulation of both carbohydrate and lipid metabolism. Polymorphisms in the ADIPOQ gene are known to influence an individual's predisposition to metabolic syndrome and type 2 diabetes. Moreover, women with gestational diabetes mellitus (GDM) are at an increased risk of developing type 2 diabetes. Several studies have been conducted previously to assess the association between ADIPOQ polymorphisms and GDM; however, the results of the association are inconclusive. AIM: To quantitatively evaluate the association between ADIPOQ +45T/G, +276G/T, and -11377C/G polymorphisms and the risk of GDM. METHODS: A systematic search of EMBASE, PubMed, CNKI, Web of Science, and WANFANG DATA was conducted up to October 20, 2018. We calculated merged odds ratios (ORs) with 95% confidence intervals (CIs) using a fixed-effects or random-effects model depending on the between-study heterogeneity to evaluate the association between AIDPOQ +45T/G, +276G/T, and -11377C/G polymorphisms and the risk of GDM. Subgroup analysis was performed by ethnicity. Publication and sensitivity bias analyses were performed to test the robustness of the association. All statistical analyses were conducted using Stata12.0. RESULTS: Nine studies of +45T/G included 1024 GDM cases and 1059 controls, five studies of +276G/T included 590 GDM cases and 595 controls, and five studies of -11377C/G included 722 GDM cases and 791 controls. Pooled ORs indicated that +45T/G increased GDM risk in Asians (allelic model: OR = 1.47, 95%CI: 1.27-1.70, P = 0.000; dominant model: OR = 1.54, 95%CI: 1.27-1.85, P = 0.000; recessive model: OR=2.00, 95%CI: 1.43-2.85, P = 0.000), not in South Americans (allelic model: OR = 1.21, 95%CI: 0.68-2.41, P = 0.510; dominant model: OR = 1.13, 95%CI: 0.59-2.15, P = 0.710; recessive model: OR = 2.18, 95%CI: 0.43-11.07, P = 0.350). There were no significant associations between +276G/T (allelic model: OR = 0.88, 95%CI: 0.74-1.05, P = 0.158; dominant model: OR = 0.91, 95%CI: 0.65-1.26, P = 0.561; recessive model: OR = 0.82, 95%CI: 0.64-1.05, P = 0.118) or -11377C/G (allelic model: OR = 0.96, 95%CI: 0.72-1.26, P = 0.750; dominant model: OR = 1.00, 95%CI: 0.73-1.37, P = 0.980; recessive model: OR = 0.90, 95%CI: 0.61-1.32, P = 0.570) and the risk of GDM. CONCLUSION: Our meta-analysis shows the critical role of the ADIPOQ +45T/G polymorphism in GDM, especially in Asians. Studies focused on delineating ethnicity-specific factors with larger sample sizes are needed.
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AIM: To compare the accuracy of the scoring systems Child-Turcotte-Pugh (CTP), Model for End-stage Liver Disease score (MELD), MELD-Na, and MELD to Serum Sodium ratio (MESO) to predict the mortality in decompensated liver cirrhosis. METHODS: The PubMed, Web of Science, Cochrane Library, EMBASE, and Ovid databases were systematically searched from inception to September 2018 for relevant articles, and we evaluated the quality of the included studies. The accuracy of scoring systems was analyzed with Stata 12 and MetaDiSc 1.4. RESULTS: Sixteen studies involving 2337 patients were included. The pooled areas under the summary receiver operating characteristic curves (AUROCs) of CTP, MELD, MELD-Na, and MESO to predict mortality were 0.81, 0.78, 0.85, and 0.86, respectively. Within 3 mo, the AUROCs of CTP, MELD, and MELD-Na in predicting mortality were 0.78, 0.76, and 0.89, respectively. The AUROCs of CTP, MELD, and MELD-Na at 3 mo were 0.86, 0.78, and 0.86, respectively. The AUROCs of CTP, MELD, and MELD-Na at 6 mo were 0.91, 0.83, and 0.90, respectively. The AUROCs of CTP, MELD, and MELD-Na at 12 mo were 0.72, 0.75 and 0.84, respectively. In cirrhotic patients with bleeding, the AUROCs of CTP and MELD were 0.76 and 0.88, respectively. CONCLUSION: MESO has the highest AUROC in all assessed scoring systems. Considering the different time points, MELD-Na has good accuracy in predicting the mortality of decompensated liver cirrhosis. Compared to CTP, MELD is better in predicting variceal bleeding.
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Store-operated calcium channels provide calcium signals to the cytoplasm of a wide variety of cell types. The basic components of this signaling mechanism include a mechanism for discharging Ca2+ stores (commonly but not exclusively phospholipase C and inositol 1,4,5-trisphosphate), a sensor in the endoplasmic reticulum that also serves as an activator of the plasma membrane channel (STIM1 and STIM2), and the store-operated channel (Orai1, 2 or 3). The advent of mice genetically altered to reduce store-operated calcium entry globally or in specific cell types has provided important tools to understand the functions of these widely encountered channels in specific and clinically important physiological systems. This review briefly discusses the history and cellular properties of store-operated calcium channels, and summarizes selected studies of their physiological functions in specific physiological or pathological contexts. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.
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Canales de Calcio/fisiología , Animales , Calcio/metabolismo , Señalización del Calcio , Retículo Endoplásmico/metabolismo , Humanos , Ratones , Ratones TransgénicosRESUMEN
In mammals exclusively, the pore-forming Ca(2+) release-activated Ca(2+) (CRAC) channel subunit Orai1 occurs in two forms because of alternative translation initiation. The longer, mammal-specific Orai1α contains an additional 63 amino acids upstream of the conserved start site for Orai1ß, which occurs at methionine 64 in Orai1α. Orai1 participates in the generation of three distinct Ca(2+) currents, including two store-operated currents: Icrac, which involves activation of Orai1 channels by the Ca(2+)-sensing protein STIM1 (stromal interaction molecule 1), and Isoc, which involves an interaction among Orai1, the transient receptor potential (TRP) family member TRPC1 (TRP canonical 1), and STIM1. Orai1 is also a pore-forming subunit of an arachidonic acid (or leukotriene C4)-regulated current Iarc that involves interactions among Orai1, Orai3, and STIM1. We evaluated the roles of the two Orai1 forms in the Ca(2+) currents Icrac, Isoc, and Iarc. We found that Orai1α and Orai1ß were largely interchangeable for Icrac and Isoc, although Orai1α exhibited stronger inhibition by Ca(2+). Only the mammalian-specific Orai1α functioned in the arachidonic acid-regulated current Iarc. Thus, alternative translation initiation of the Orai1 message produces at least three types of Ca(2+) channels with distinct signaling and regulatory properties.
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Canales de Calcio/biosíntesis , Señalización del Calcio/fisiología , Iniciación de la Cadena Peptídica Traduccional/fisiología , ARN Mensajero/metabolismo , Animales , Ácido Araquidónico/farmacología , Canales de Calcio/genética , Canales de Calcio/metabolismo , Señalización del Calcio/efectos de los fármacos , Células HEK293 , Humanos , Ratones , Proteína ORAI1 , Iniciación de la Cadena Peptídica Traduccional/efectos de los fármacos , Isoformas de Proteínas/biosíntesis , Isoformas de Proteínas/genética , ARN Mensajero/genética , Molécula de Interacción Estromal 1 , Canales Catiónicos TRPC/genética , Canales Catiónicos TRPC/metabolismoRESUMEN
Using immortalized [3H]inositol-labelled S3 cells, we demonstrated in the present study that various elements of the inositol phosphate signalling cascade are recruited by a Drosophila homologue from a cytokine family of so-called GBPs (growth-blocking peptides). HPLC analysis revealed that dGBP (Drosophila GBP) elevated Ins(1,4,5)P3 levels 9-fold. By using fluorescent Ca2+ probes, we determined that dGBP initially mobilized Ca2+ from intracellular pools; the ensuing depletion of intracellular Ca2+ stores by dGBP subsequently activated a Ca2+ entry pathway. The addition of dsRNA (double-stranded RNA) to knock down expression of the Drosophila Ins(1,4,5)P3 receptor almost completely eliminated mobilization of intracellular Ca2+ stores by dGBP. Taken together, the results of the present study describe a classical activation of PLC (phospholipase C) by dGBP. The peptide also promoted increases in the levels of other inositol phosphates with signalling credentials: Ins(1,3,4,5)P4, Ins(1,4,5,6)P4 and Ins(1,3,4,5,6)P5. These results greatly expand the regulatory repertoire of the dGBP family, and also characterize S3 cells as a model for studying the regulation of inositol phosphate metabolism and signalling by endogenous cell-surface receptors. We therefore created a cell-line (S3ITPK1) in which heterologous expression of human ITPK (inositol tetrakisphosphate kinase) was controlled by an inducible metallothionein promoter. We found that dGBP-stimulated S3ITPK1 cells did not synthesize Ins(3,4,5,6)P4, contradicting a hypothesis that the PLC-coupled phosphotransferase activity of ITPK1 [Ins(1,3,4,5,6)P5+Ins(1,3,4)P3âIns(3,4,5,6)P4+Ins(1,3,4,6)P4] is driven solely by the laws of mass action [Chamberlain, Qian, Stiles, Cho, Jones, Lesley, Grabau, Shears and Spraggon (2007) J. Biol. Chem. 282, 28117-28125]. This conclusion represents a fundamental breach in our understanding of ITPK1 signalling.
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Citocinas/metabolismo , Proteínas de Drosophila/metabolismo , Fosfatos de Inositol/metabolismo , Proteínas de Insectos/metabolismo , Fosfotransferasas (Aceptor de Grupo Alcohol)/metabolismo , Fosfolipasas de Tipo C/metabolismo , Animales , Secuencia de Bases , Señalización del Calcio , Línea Celular , Cartilla de ADN/genética , Drosophila , Activación Enzimática , Humanos , Modelos Biológicos , Fosfotransferasas (Aceptor de Grupo Alcohol)/genética , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Transducción de SeñalRESUMEN
The endoplasmic reticulum (ER) undergoes significant reorganization between interphase and mitosis, but the underlying mechanisms are unknown. Stromal interaction molecule 1 (STIM1) is an ER Ca(2+) sensor that activates store-operated Ca(2+) entry (SOCE) and also functions in ER morphogenesis through its interaction with the microtubule +TIP protein end binding 1 (EB1). We previously demonstrated that phosphorylation of STIM1 during mitosis suppresses SOCE. We now show that STIM1 phosphorylation is a major regulatory mechanism that excludes ER from the mitotic spindle. In mitotic HeLa cells, the ER forms concentric sheets largely excluded from the mitotic spindle. We show that STIM1 dissociates from EB1 in mitosis and localizes to the concentric ER sheets. However, a nonphosphorylatable STIM1 mutant (STIM1(10A)) colocalized extensively with EB1 and drove ER mislocalization by pulling ER tubules into the spindle. This effect was rescued by mutating the EB1 interaction site of STIM1(10A), demonstrating that aberrant association of STIM1(10A) with EB1 is responsible for the ER mislocalization. A STIM1 phosphomimetic exhibited significantly impaired +TIP tracking in interphase but was ineffective at inhibiting SOCE, suggesting different mechanisms of regulation of these two STIM1 functions by phosphorylation. Thus, ER spindle exclusion and ER-dependent Ca(2+) signaling during mitosis require multimodal STIM1 regulation by phosphorylation.
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Retículo Endoplásmico/fisiología , Proteínas de la Membrana/metabolismo , Proteínas Asociadas a Microtúbulos/metabolismo , Proteínas de Neoplasias/metabolismo , Huso Acromático/fisiología , Calcio/metabolismo , Células HeLa , Humanos , Mitosis , Fosforilación , Molécula de Interacción Estromal 1RESUMEN
Phosphorylation plays vital roles in the regulation and function of the V2 vasopressin receptor (V2R), a G protein-coupled receptor (GPCR) that is responsible for maintaining water homeostasis in the kidney. Through a combination of immunoaffinity purification, immobilized metal affinity chromatography, and nanoflow liquid chromatography tandem mass spectrometry, we identified a novel phosphorylation site (Ser(255)) in the third intracellular loop of human V2R. We showed that the third intracellular loop could be phosphorylated in vitro by protein kinase A, but not by Akt kinase, although sequence motif analysis predicated otherwise. The analytical procedures and methodologies described in this study should be generally applicable for identifying the endogenous phosphorylation sites in other GPCRs, overcoming the limitations of conventional approaches such as sequence motif analysis and site-directed mutagenesis.
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Receptores de Vasopresinas/química , Sitios de Unión , Cromatografía , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Humanos , Fosfoproteínas/análisis , Fosfoproteínas/química , Fosforilación , Receptores Acoplados a Proteínas G/química , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Vasopresinas/metabolismo , Serina , Espectrometría de Masas en TándemRESUMEN
Protein kinase D (PKD) isoforms are protein kinase C (PKC) effectors in diacylglycerol (DAG)-regulated signaling pathways. Key physiological processes are placed under DAG control by the distinctive substrate specificity and intracellular distribution of PKDs. Comprehension of the roles of PKDs in homeostasis and signal transduction requires further knowledge of regulatory interplay among PKD and PKC isoforms, analysis of PKC-independent PKD activation, and characterization of functions controlled by PKDs in vivo. Caenorhabditis elegans and mammals share conserved signaling mechanisms, molecules, and pathways Thus, characterization of the C. elegans PKDs could yield insights into regulation and functions that apply to all eukaryotic PKDs. C. elegans DKF-1 (D kinase family-1) contains tandem DAG binding (C1) modules, a PH (pleckstrin homology) domain, and a Ser/Thr protein kinase segment, which are homologous with domains in classical PKDs. DKF-1 and PKDs have similar substrate specificities. Phorbol 12-myristate 13-acetate (PMA) switches on DKF-1 catalytic activity in situ by promoting phosphorylation of a single amino acid Thr(588) in the activation loop. DKF-1 phosphorylation and activation are unaffected when PKC activity is eliminated by inhibitors. Both phosphorylation and kinase activity of DKF-1 are extinguished by substituting Ala for Thr(588) or Gln for Lys(455) ("kinase dead") or incubating with protein phosphatase 2C. Thus, DKF-1 is a PMA-activated, PKC-independent D kinase. In vivo, dkf-1 gene promoter activity is evident in neurons. Both dkf-1 gene disruption (null phenotype) and RNA interference-mediated depletion of DKF-1 protein cause lower body paralysis. Targeted DKF-1 expression corrected this locomotory defect in dkf-1 null animals. Supraphysiological expression of DKF-1 limited C. elegans growth to approximately 60% of normal length.
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Proteínas de Caenorhabditis elegans/genética , Proteínas de Caenorhabditis elegans/metabolismo , Caenorhabditis elegans/enzimología , Caenorhabditis elegans/genética , Proteína Quinasa C/genética , Proteína Quinasa C/metabolismo , Secuencia de Aminoácidos , Animales , Tamaño Corporal/fisiología , Caenorhabditis elegans/crecimiento & desarrollo , Proteínas de Caenorhabditis elegans/química , Línea Celular Tumoral , Cricetinae , ADN Complementario , Activación Enzimática/fisiología , Regulación del Desarrollo de la Expresión Génica , Regulación Enzimológica de la Expresión Génica , Humanos , Riñón/citología , Datos de Secuencia Molecular , Movimiento/fisiología , Fenotipo , Fosforilación , Proteína Quinasa C/química , Estructura Terciaria de ProteínaRESUMEN
The mTOR protein kinase is known to control cell cycle progression and cell growth through regulation of translation, transcription, membrane traffic and protein degradation. Known interactions of mTOR do not account for the multiple functions of this protein. Using a non-catalytic segment of mTOR (1-670) as bait in a yeast two-hybrid screen for interacting proteins, ubiquilin 1 (NM013438) was identified. Ubiquilin 1 is a member of a phylogenetically conserved gene family of unknown function, characterized by an N-terminal ubiquitin-like (Ubq) domain, a C-terminal ubiquitin associated (Uba) domain and a central region containing numerous NPXvar phi motifs (X, any; phi, hydrophobic amino acid). GST-ubiquilin 1 binds specifically to FLAG-mTOR (residues 1-670) in mammalian cells; residues 570-670 of mTOR and 226-323 of ubiquilin 1 are required for this interaction. Both mTOR and ubiquilin immunoreactivity appear as fine speckles throughout the cytoplasm; significant colocalization with cytoskeletal elements, early endosomes or proteasomes is not observed. As assessed by cell fractionation, mTOR is predominantly associated with low density membranes, along with 10% of ubiquilin 1. Ubiquilin 1 is a rapamycin-insensitive phosphoprotein. Overexpression of ubiquilin 1 does not alter the kinase activity of cotransfected mTOR or the phosphorylation of the mTOR target, p70 S6 kinase, in the presence or absence of rapamycin. Our data suggest that we have identified a novel mTOR interactor, ubiquilin 1. The biological significance of this, presumably membrane based, interaction, requires further study.