ABSTRACT
BACKGROUND: The incapacity to store lipids in adipose tissue in Congenital Generalized Lipodystrophy (CGL) causes hypoleptinemia, increased appetite, ectopic fat deposition and lipotoxicity. CGL patients experience shortened life expectancy. The plasma lipidomic profile has not been characterized fully in CGL, nor has the extent of dietary intake in its modulation. The present work investigated the plasma lipidomic profile of CGL patients in comparison to eutrophic individuals at the fasted state and after a breakfast meal. METHOD: Blood samples from 11 CGL patients and 10 eutrophic controls were collected after 12 h fasting (T0) and 90 min after an ad libitum fat-containing breakfast (T90). The lipidomic profile of extracted plasma lipids was characterized by non-target liquid chromatography mass spectrometry. RESULTS: Important differences between groups were observed at T0 and at T90. Several molecular species of fatty acyls, glycerolipids, sphingolipids and glycerophospholipids were altered in CGL. All the detected fatty acyl molecular species, several diacylglycerols and one triacylglycerol species were upregulated in CGL. Among sphingolipids, one sphingomyelin and one glycosphingolipid species showed downregulation in CGL. Alterations in the glycerophospholipids glycerophosphoethanolamines, glycerophosphoserines and cardiolipins were more complex. Interestingly, when comparing T90 versus T0, the lipidomic profile in CGL did not change as intensely as it did for control participants. CONCLUSIONS: The present study found profound alterations in the plasma lipidomic profile of complex lipids in CGL patients as compared to control subjects. A fat-containing breakfast meal did not appear to significantly influence the CGL profile observed in the fasted state. Our study may have implications for clinical practice, also aiding to a deeper comprehension of the role of complex lipids in CGL in view of novel therapeutic strategies.
Subject(s)
Lipodystrophy, Congenital Generalized , Humans , Breakfast , Lipidomics , Adipose Tissue , LipidsABSTRACT
The widely expressed two-pore homodimeric inward rectifier CLC-2 chloride channel regulates transepithelial chloride transport, extracellular chloride homeostasis, and neuronal excitability. Each pore is independently gated at hyperpolarized voltages by a conserved pore glutamate. Presumably, exiting chloride ions push glutamate outwardly while external protonation stabilizes it. To understand the mechanism of mouse CLC-2 opening we used homology modelling-guided structure-function analysis. Structural modelling suggests that glutamate E213 interacts with tyrosine Y561 to close a pore. Accordingly, Y561A and E213D mutants are activated at less hyperpolarized voltages, re-opened at depolarized voltages, and fast and common gating components are reduced. The double mutant cycle analysis showed that E213 and Y561 are energetically coupled to alter CLC-2 gating. In agreement, the anomalous mole fraction behaviour of the voltage dependence, measured by the voltage to induce half-open probability, was strongly altered in these mutants. Finally, cytosolic acidification or high extracellular chloride concentration, conditions that have little or no effect on WT CLC-2, induced reopening of Y561 mutants at positive voltages presumably by the inward opening of E213. We concluded that the CLC-2 gate is formed by Y561-E213 and that outward permeant anions open the gate by electrostatic and steric interactions.
Subject(s)
Chloride Channels/chemistry , Ion Channel Gating , Amino Acid Sequence , Animals , CLC-2 Chloride Channels , Cattle , Chloride Channels/genetics , Chloride Channels/metabolism , Chlorides/metabolism , Humans , Mice , Mutation , Protein Structure, Tertiary , Sequence Alignment , Structure-Activity RelationshipABSTRACT
It has been reported that muscarinic type-2 receptors (M2R) are voltage sensitive in an agonist-specific manner. In this work, we studied the effects of membrane potential on the interaction of M2R with the superagonist iperoxo (IXO), both functionally (using the activation of the ACh-gated K+ current (IKACh) in cardiomyocytes) and by molecular dynamics (MD) simulations. We found that IXO activated IKACh with remarkable high potency and clear voltage dependence, displaying a larger effect at the hyperpolarized potential. This result is consistent with a greater affinity, as validated by a slower (τ = 14.8 ± 2.3 s) deactivation kinetics of the IXO-evoked IKACh than that at the positive voltage (τ = 6.7 ± 1.2 s). The voltage-dependent M2R-IXO interaction induced IKACh to exhibit voltage-dependent features of this current, such as the 'relaxation gating' and the modulation of rectification. MD simulations revealed that membrane potential evoked specific conformational changes both at the external access and orthosteric site of M2R that underlie the agonist affinity change provoked by voltage on M2R. Moreover, our experimental data suggest that the 'tyrosine lid' (Y104, Y403, and Y426) is not the previously proposed voltage sensor of M2R. These findings provide an insight into the structural and functional framework of the biased signaling induced by voltage on GPCRs.
Subject(s)
Ion Channel Gating/drug effects , Isoxazoles/pharmacology , Molecular Dynamics Simulation , Quaternary Ammonium Compounds/pharmacology , Receptor, Muscarinic M2/physiology , Acetylcholine/pharmacology , Animals , Cats , Cells, Cultured , Electric Stimulation , Female , Ion Channel Gating/physiology , Male , Membrane Potentials/drug effects , Models, Molecular , Muscarinic Agonists/pharmacology , Myocytes, Cardiac/drug effects , Myocytes, Cardiac/physiology , Oocytes/drug effects , Oocytes/physiology , Patch-Clamp Techniques , Protein Conformation , Receptor, Muscarinic M2/chemistry , Receptor, Muscarinic M2/metabolism , Xenopus laevisABSTRACT
Resumen Antecedentes: Los procedimientos coronarios invasivos conllevan la administración de contraste y la exposición a radiaciones ionizantes, comportando un incremento de la morbimortalidad. La angiografía coronaria rotacional (ACR) permite adquirir múltiples proyecciones con una inyección de contraste. Hasta la fecha, no hay metaanálisis específicos comparando la ACR y la angiografía coronaria convencional (ACC) en pacientes en los que se realizan procedimientos coronarios invasivos, tanto diagnósticos como diagnósticos y terapéuticos. El objetivo de este metaanálisis es evaluar el impacto de la ACR en la cantidad de contraste, y la radiación ionizante en procedimientos coronarios invasivos. Métodos: Se realizó una búsqueda en las bases de datos PubMed y Ovid para identificar estudios tanto diagnósticos como diagnósticos y terapéuticos que comparasen ACR y ACC. Los estudios fueron evaluados sobre la calidad y los sesgos, y fueron incluidos si contemplaban alguna de las siguientes variables de valoración: volumen de contraste, radiación ionizante medida como producto dosis-área, Kerma-aire o tiempo de fluoroscopia. Resultados: Dieciséis estudios, totalizando 2,327 pacientes, fueron incluidos en el análisis final (1,146 pacientes recibieron ACR y 1,181, ACC), objetivándose diferencias significativas en volumen de contraste (diferencia estándar de medias (intervalo de confianza al 95%) −1.887 (−2.472 a −1.302); p < 0.001), producto dosis-área (−0.726 (−1.034 a −0.418); p < 0.001), Kerma-aire (−0.842 (−1.104 a −0.581); p < 0.001) y tiempo de fluoroscopia (0.263 (−0.496 a −0.030); p = 0.027). Conclusiones: La ACR permite reducir el volumen de contraste y la radiación, evaluada como producto dosis-área, Kerma-aire y tiempo de fluoroscopia en pacientes a los que se les realizan procedimientos coronarios invasivos.
Abstract Background: Invasive coronary procedures involve the administration of iodinated contrast and the exposure to ionising radiations, increasing morbidity and mortality. The rotational coronary angiography (RCA) allows acquiring multiple projections with a unique injection of iodinated contrast. To date, there are no meta-analyses specifically comparing RCA and conventional coronary angiography (CCA) in patients undergoing invasive coronary procedures, whether diagnostic or diagnostic and therapeutic. The aim of this meta-analysis is to assess the impact of RCA on the amount of iodinated contrast and the exposure to ionising radiations during invasive coronary procedures. Methods: A search in PubMed and Ovid databases was conducted to identify studies, including diagnostic and diagnostic and therapeutic studies, comparing RCA and CCA. The manuscripts were evaluated on quality and biases, and were included if they analysed any of the following endpoints: volume of contrast and exposure to ionising radiations measured as dose-area product, and Kerma-air or fluoroscopy time. Results: Sixteen studies, with a total of 2,327 patients, were included in the final analysis (1,146 patients underwent RCA and 1,181 patients underwent CCA), with significant differences being detected in volume of contrast (standard difference in means (95% confidence interval) −1.887 (−2.472 to −1.302); P < .001), dose-area product (−0.726 (−1.034 to −0.418); P < .001), Kerma-air (−0.842 (−1.104 to −0.581); P < .001), and fluoroscopy time (0.263 (−0.496 to −0.030); P = .027). Conclusions: RCA reduces the volume of contrast and the exposure to radiation, evaluated as dose-area product, Kerma-air, and fluoroscopy time, in patients undergoing invasive coronary procedures.
Subject(s)
Humans , Coronary Artery Disease/diagnostic imaging , Coronary Angiography/methods , Contrast Media/administration & dosage , Radiation, Ionizing , Fluoroscopy , Iodine Compounds/administration & dosageABSTRACT
Phosphatidylinositol 4,5-bisphosphate (PIP2) is a membrane phospholipid that regulates the function of multiple ion channels, including some members of the voltage-gated potassium (Kv) channel superfamily. The PIP2 sensitivity of Kv channels is well established for all five members of the Kv7 family and for Kv1.2 channels; however, regulation of other Kv channels by PIP2 remains unclear. Here, we investigate the effects of PIP2 on Kv2.1 channels by applying exogenous PIP2 to the cytoplasmic face of excised membrane patches, activating muscarinic receptors (M1R), or depleting endogenous PIP2 using a rapamycin-translocated 5-phosphatase (FKBP-Inp54p). Exogenous PIP2 rescued Kv2.1 channels from rundown and partially prevented the shift in the voltage-dependence of inactivation observed in inside-out patch recordings. Native PIP2 depletion by the recruitment of FKBP-Insp54P or M1R activation in whole-cell experiments, induced a shift in the voltage-dependence of inactivation, an acceleration of the closed-state inactivation, and a delayed recovery of channels from inactivation. No significant effects were observed on the activation mechanism by any of these treatments. Our data can be modeled by a 13-state allosteric model that takes into account that PIP2 depletion facilitates inactivation of Kv2.1. We propose that PIP2 regulates Kv2.1 channels by interfering with the inactivation mechanism.
Subject(s)
Phosphatidylinositol 4,5-Diphosphate/metabolism , Shab Potassium Channels/metabolism , HEK293 Cells , Humans , Ion Channel Gating/physiology , Patch-Clamp Techniques/methods , Potassium Channels, Voltage-Gated/metabolism , Receptors, Muscarinic/metabolismABSTRACT
BACKGROUND: Invasive coronary procedures involve the administration of iodinated contrast and the exposure to ionising radiations, increasing morbidity and mortality. The rotational coronary angiography (RCA) allows acquiring multiple projections with a unique injection of iodinated contrast. To date, there are no meta-analyses specifically comparing RCA and conventional coronary angiography (CCA) in patients undergoing invasive coronary procedures, whether diagnostic or diagnostic and therapeutic. The aim of this meta-analysis is to assess the impact of RCA on the amount of iodinated contrast and the exposure to ionising radiations during invasive coronary procedures. METHODS: A search in PubMed and Ovid databases was conducted to identify studies, including diagnostic and diagnostic and therapeutic studies, comparing RCA and CCA. The manuscripts were evaluated on quality and biases, and were included if they analysed any of the following endpoints: volume of contrast and exposure to ionising radiations measured as dose-area product, and Kerma-air or fluoroscopy time. RESULTS: Sixteen studies, with a total of 2,327 patients, were included in the final analysis (1,146 patients underwent RCA and 1,181 patients underwent CCA), with significant differences being detected in volume of contrast (standard difference in means [95% confidence interval] -1.887 [-2.472 to -1.302]; P<.001), dose-area product (-0.726 [-1.034 to -0.418]; P<.001), Kerma-air (-0.842 [-1.104 to -0.581]; P<.001), and fluoroscopy time (0.263 [-0.496 to -0.030]; P=.027). CONCLUSIONS: RCA reduces the volume of contrast and the exposure to radiation, evaluated as dose-area product, Kerma-air, and fluoroscopy time, in patients undergoing invasive coronary procedures.
Subject(s)
Contrast Media/administration & dosage , Coronary Angiography/methods , Coronary Artery Disease/diagnostic imaging , Fluoroscopy , Humans , Iodine Compounds/administration & dosage , Radiation, IonizingABSTRACT
The TMEM16A-mediated Ca2+-activated Cl- current drives several important physiological functions. Membrane lipids regulate ion channels and transporters but their influence on members of the TMEM16 family is poorly understood. Here we have studied the regulation of TMEM16A by phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2), cholesterol, and fatty acids using patch clamp, biochemistry and fluorescence microscopy. We found that depletion of membrane PI(4,5)P2 causes a decline in TMEM16A current that is independent of cytoskeleton, but is partially prevented by removing intracellular Ca2+. On the other hand, supplying PI(4,5)P2 to inside-out patches attenuated channel rundown and/or partially rescued activity after channel rundown. Also, depletion (with methyl-ß-cyclodextrin M-ßCD) or restoration (with M-ßCD+cholesterol) of membrane cholesterol slows down the current decay observed after reduction of PI(4,5)P2. Neither depletion nor restoration of cholesterol change PI(4,5)P2 content. However, M-ßCD alone transiently increases TMEM16A activity and dampens rundown whereas M-ßCD+cholesterol increases channel rundown. Thus, PI(4,5)P2 is required for TMEM16A function while cholesterol directly and indirectly via a PI(4,5)P2-independent mechanism regulate channel function. Stearic, arachidonic, oleic, docosahexaenoic, and eicosapentaenoic fatty acids as well as methyl stearate inhibit TMEM16A in a dose- and voltage-dependent manner. Phosphatidylserine, a phospholipid whose hydrocarbon tails contain stearic and oleic acids also inhibits TMEM16A. Finally, we show that TMEM16A remains in the plasma membrane after treatment with M-ßCD, M-ßCD+cholesterol, oleic, or docosahexaenoic acids. Thus, we propose that lipids and fatty acids regulate TMEM16A channels through a membrane-delimited protein-lipid interaction.
Subject(s)
Anoctamin-1/metabolism , Calcium Signaling/physiology , Cell Membrane/metabolism , Cholesterol/metabolism , Fatty Acids/metabolism , Neoplasm Proteins/metabolism , Phosphatidylinositol 4,5-Diphosphate/metabolism , Anoctamin-1/genetics , Calcium/metabolism , Cell Membrane/genetics , Cholesterol/genetics , Fatty Acids/genetics , HEK293 Cells , Humans , Neoplasm Proteins/genetics , Phosphatidylinositol 4,5-Diphosphate/geneticsABSTRACT
The objective of this study was to evaluate the presence of organochlorine pesticides in samples of forage, soil, water, and milk in four units of an organic production system for cow´s milk (samples of forage, milk, soil, and water) in Tecpatan, Chiapas, Mexico. The organochlorine pesticides were extracted from forage, soil and water based on the USEPA (2005) guideline and from milk based on the IDF 1991 guideline. The pesticides were identified and quantified by gas chromatography with electron capture detector (CG-ECD). In general, the highest average concentration of total pesticides was found in the samples of milk and forage (311 ± 328 and 116.5 ±77 ng g(-1) respectively). Although, the production systems analyzed are organic, organochlorine pesticides were detected in all environmental samples (forage, soil, water, and organic milk). Although no values surpassed the defined limits of Mexican and International regulation it is advisable that a monitoring program of contaminants in these production systems is continued.
Subject(s)
Food Contamination/analysis , Hydrocarbons, Chlorinated/analysis , Milk/chemistry , Organic Agriculture , Pesticides/analysis , Animal Feed/analysis , Animals , Cattle , Chromatography, Gas , Environmental Monitoring/methods , Mexico , Pesticide Residues/analysis , Soil/chemistry , Water/chemistryABSTRACT
Ammonium nitrate fuel oil (ANFO) is an explosive used in many civil applications. In Brazil, ANFO has unfortunately also been used in criminal attacks, mainly in automated teller machine (ATM) explosions. In this paper, we describe a detailed characterization of the ANFO composition and its two main constituents (diesel and a nitrate explosive) using high resolution and accuracy mass spectrometry performed on an FT-ICR-mass spectrometer with electrospray ionization (ESI(±)-FTMS) in both the positive and negative ion modes. Via ESI(-)-MS, an ion marker for ANFO was characterized. Using a direct and simple ambient desorption/ionization technique, i.e., easy ambient sonic-spray ionization mass spectrometry (EASI-MS), in a simpler, lower accuracy but robust single quadrupole mass spectrometer, the ANFO ion marker was directly detected from the surface of banknotes collected from ATM explosion theft.
ABSTRACT
A survey was carried out from 2008 to 2010 to determine the concentrations of 16 organochlorine pesticide residues (OPRs) from Tizayuca, Hidalgo, Mexico. Organochlorine residue determinations were made from milk fat, using chromatographic cleanup and analysis by gas chromatography with an electron capture detector. The OPR concentrations found were from below the detection limit (DL) to 0.91 ng g(-1) in 2008, DL to 0.38 ng g(-1) in 2009 and DL to 0.59 ng g(-1) in 2010. In general concentrations of organochlorine pesticides were higher in the wet season (3.37 ng g(-1) and 4.79 ng g(-1)) than the dry season (1.92 ng g(-1) and 2.71 ng g(-1)) for 2009 and 2010, due to control of pests in the pasture and sheds. According to Codex Alimentarius regulations, individual pesticides did not exceed the permissible limits, which for example were 10 µg kg(-)1 for alpha hexachlorocyclohexane (HCH) and endosulfan I, 20 µg kg(-1) for p,p'-DDT, and 6 µg kg(-1) for dieldrin, endrin and heptachlor. A reduction of organochlorine pesticide concentrations in cow's milk was noted, indicating that the Mexican government has achieved reduction or elimination of some organochlorine pesticides in response to global agreements on persistent organic pollutants.