RESUMEN
A commitment to diversity, equity, inclusion, and belonging in medical education requires addressing both explicit and implicit biases based on sexual orientation, gender identity and expression, and sex characteristics and the intersectionality with other identities. Heterosexism and heteronormative attitudes contribute to health and healthcare disparities for lesbian, gay, bisexual, transgender and queer or questioning, intersex, asexual individuals. Student, trainee, and faculty competencies in medical education curricula regarding the care of lesbian, gay, bisexual, transgender and queer or questioning, intersex, asexual patients and those who are gender nonconforming or born with differences of sex development allow for better understanding and belonging within the clinical learning environment of lesbian, gay, bisexual, transgender and queer/questioning, intersex, asexual learners and educators. The Association of Professors of Gynecology and Obstetrics issued a call to action to achieve a future free from racism and bias through inclusivity in obstetrics and gynecology education and healthcare, which led to the creation of the Association of Professors of Gynecology and Obstetrics Diversity, Equity, and Inclusion Guidelines Task Force. The task force initially addressed racism, racial- and ethnicity-based bias, and discrimination in medical education and additionally identified other groups that are subject to bias and discrimination, including sexual orientation, gender identity and expression, and sex characteristic identities, persons with disabilities, and individuals with various religious and spiritual practices. In this scholarly perspective, the authors expand on previously developed guidelines to address sexual orientation, gender identity and expression, and sex characteristics bias, heterosexism, and heteronormative attitudes in obstetrics and gynecology educational products, materials, and clinical learning environments to improve access and equitable care to vulnerable individuals of the lesbian, gay, bisexual, transgender and queer or questioning, intersex, asexual community.
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Educación Médica , Identidad de Género , Ginecología , Conducta Sexual , Minorías Sexuales y de Género , Humanos , Femenino , Masculino , Ginecología/educación , Obstetricia/educación , Curriculum , Sexismo , RacismoRESUMEN
Racism and bias contribute to healthcare disparities at a patient and population health level and also contribute to the stagnation or even regression of progress toward equitable representation in the workforce and in healthcare leadership. Medical education and healthcare systems have expended tremendous efforts over the past several years to address these inequities. However, systemic racism continues to impact health outcomes and the future physician workforce. The Association of Professors of Gynecology and Obstetrics called for action to achieve a future free from racism in obstetrics and gynecology education and healthcare. As a result of this call to action, the Diversity, Equity, and Inclusion Guidelines Task Force was created. The mission of the Task Force was to support educators in their efforts to identify and create educational materials that augment antiracist educational goals and prepare, recruit, and retain a talented and diverse workforce. In this Special Report, the authors share these guidelines that describe best practices and set new standards to increase diversity, foster inclusivity, address systemic racism, and eliminate bias in obstetrics and gynecology educational products, materials, and environments.
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Educación Médica , Ginecología , Obstetricia , Racismo , Humanos , Racismo/prevención & control , Ginecología/educación , Obstetricia/educación , Disparidades en Atención de SaludRESUMEN
Human DJ-1 is a cytoprotective protein whose absence causes Parkinson's disease and is also associated with other diseases. DJ-1 has an established role as a redox-regulated protein that defends against oxidative stress and mitochondrial dysfunction. Multiple studies have suggested that DJ-1 is also a protein/nucleic acid deglycase that plays a key role in the repair of glycation damage caused by methylglyoxal (MG), a reactive α-keto aldehyde formed by central metabolism. Contradictory reports suggest that DJ-1 is a glyoxalase but not a deglycase and does not play a major role in glycation defense. Resolving this issue is important for understanding how DJ-1 protects cells against insults that can cause disease. We find that DJ-1 reduces levels of reversible adducts of MG with guanine and cysteine in vitro. The steady-state kinetics of DJ-1 acting on reversible hemithioacetal substrates are fitted adequately with a computational kinetic model that requires only a DJ-1 glyoxalase activity, supporting the conclusion that deglycation is an apparent rather than a true activity of DJ-1. Sensitive and quantitative isotope-dilution mass spectrometry shows that DJ-1 modestly reduces the levels of some irreversible guanine and lysine glycation products in primary and cultured neuronal cell lines and whole mouse brain, consistent with a small but measurable effect on total neuronal glycation burden. However, DJ-1 does not improve cultured cell viability in exogenous MG. In total, our results suggest that DJ-1 is not a deglycase and has only a minor role in protecting neurons against methylglyoxal toxicity.
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Estrés Oxidativo , Piruvaldehído , Animales , Glicosilación , Guanina , Humanos , Ratones , Neuronas/metabolismo , Proteína Desglicasa DJ-1/metabolismo , Piruvaldehído/química , Piruvaldehído/metabolismoRESUMEN
The mammalian pyruvate dehydrogenase complex (PDC) is a mitochondrial multienzyme complex that connects glycolysis to the tricarboxylic acid cycle by catalyzing pyruvate oxidation to produce acetyl-CoA, NADH, and CO2. This reaction is required to aerobically utilize glucose, a preferred metabolic fuel, and is composed of three core enzymes: pyruvate dehydrogenase (E1), dihydrolipoyl transacetylase (E2), and dihydrolipoyl dehydrogenase (E3). The pyruvate-dehydrogenase-specific kinase (PDK) and pyruvate-dehydrogenase-specific phosphatase (PDP) are considered the main control mechanism of mammalian PDC activity. However, PDK and PDP activity are allosterically regulated by several effectors fully overlapping PDC substrates and products. This collection of positive and negative feedback mechanisms confounds simple predictions of relative PDC flux, especially when all effectors are dynamically modulated during metabolic states that exist in physiologically realistic conditions, such as exercise. Here, we provide, to our knowledge, the first globally fitted, pH-dependent kinetic model of the PDC accounting for the PDC core reaction because it is regulated by PDK, PDP, metal binding equilibria, and numerous allosteric effectors. The model was used to compute PDH regulatory complex flux as a function of previously determined metabolic conditions used to simulate exercise and demonstrates increased flux with exercise. Our model reveals that PDC flux in physiological conditions is primarily inhibited by product inhibition (â¼60%), mostly NADH inhibition (â¼30-50%), rather than phosphorylation cycle inhibition (â¼40%), but the degree to which depends on the metabolic state and PDC tissue source.
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Condicionamiento Físico Animal , Proteínas Serina-Treonina Quinasas , Complejo Piruvato Deshidrogenasa , Animales , Fosforilación , Piruvato Deshidrogenasa (Lipoamida)-Fosfatasa/metabolismo , Piruvato Deshidrogenasa Quinasa Acetil-Transferidora , Complejo Piruvato Deshidrogenasa/metabolismoRESUMEN
Succinate dehydrogenase (SDH) is an inner mitochondrial membrane protein complex that links the Krebs cycle to the electron transport system. It can produce significant amounts of superoxide ([Formula: see text]) and hydrogen peroxide (H2O2); however, the precise mechanisms are unknown. This fact hinders the development of next-generation antioxidant therapies targeting mitochondria. To help address this problem, we developed a computational model to analyze and identify the kinetic mechanism of [Formula: see text] and H2O2 production by SDH. Our model includes the major redox centers in the complex, namely FAD, three iron-sulfur clusters, and a transiently bound semiquinone. Oxidation state transitions involve a one- or two-electron redox reaction, each being thermodynamically constrained. Model parameters were simultaneously fit to many data sets using a variety of succinate oxidation and free radical production data. In the absence of respiratory chain inhibitors, model analysis revealed the 3Fe-4S iron-sulfur cluster as the primary [Formula: see text] source. However, when the quinone reductase site is inhibited or the quinone pool is highly reduced, [Formula: see text] is generated primarily by the FAD. In addition, H2O2 production is only significant when the enzyme is fully reduced, and fumarate is absent. Our simulations also reveal that the redox state of the quinone pool is the primary determinant of free radical production by SDH. In this study, we showed the importance of analyzing enzyme kinetics and associated side reactions in a consistent, quantitative, and biophysically detailed manner using a diverse set of experimental data to interpret and explain experimental observations from a unified perspective.
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Modelos Biológicos , Especies Reactivas de Oxígeno/metabolismo , Succinato Deshidrogenasa/metabolismo , Algoritmos , Animales , Cobayas , CinéticaRESUMEN
Mammalian carnitine acetyltransferase (CrAT) is a mitochondrial enzyme that catalyzes the reversible transfer of an acetyl group from acetyl-CoA to carnitine. CrAT knockout studies have shown that this enzyme is critical to sustain metabolic flexibility, or the ability to switch between different fuel types, an underlying theme of the metabolic syndrome. These recent physiological findings imply that CrAT dysfunction, or its catalytic impairment, may lead to disease. To gain insight into the CrAT kinetic mechanism, we conducted stopped-flow experiments in various enzyme substrate/product conditions and analyzed full progress curves by global fitting. Simultaneous mixing of both substrates with CrAT produced relatively fast kinetics that follows an ordered bi bi mechanism. A great preference for ordered binding is supported by stopped-flow double mixing experiments such that premixed CrAT with acetyl-CoA or CoA demonstrated a biphasic decrease in initial rate that produces about a 100-fold attenuation in catalysis. Double mixing experiments also revealed that the CrAT initial rate is inhibited by 50% in approximately 8 s by either acetyl-CoA or CoA premixing. Analysis of available CrAT structures support a substrate conformational change between acetyl-CoA/CoA binary versus ternary complexes. Additional viscosity-based kinetic experiments yielded strong evidence that product release is the rate limiting step in the CrAT-catalyzed reaction.
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Carnitina O-Acetiltransferasa/química , Acetilcoenzima A/química , Acetilcoenzima A/metabolismo , Animales , Carnitina/química , Carnitina/metabolismo , Carnitina O-Acetiltransferasa/metabolismo , Catálisis , Dominio Catalítico , Coenzima A/química , Coenzima A/metabolismo , Columbidae , Cristalografía por Rayos X , Cinética , Ratones , Unión ProteicaRESUMEN
High capacity and low capacity running rats, HCR and LCR respectively, have been bred to represent two extremes of running endurance and have recently demonstrated disparities in fuel usage during transient aerobic exercise. HCR rats can maintain fatty acid (FA) utilization throughout the course of transient aerobic exercise whereas LCR rats rely predominantly on glucose utilization. We hypothesized that the difference between HCR and LCR fuel utilization could be explained by a difference in mitochondrial density. To test this hypothesis and to investigate mechanisms of fuel selection, we used a constraint-based kinetic analysis of whole-body metabolism to analyze transient exercise data from these rats. Our model analysis used a thermodynamically constrained kinetic framework that accounts for glycolysis, the TCA cycle, and mitochondrial FA transport and oxidation. The model can effectively match the observed relative rates of oxidation of glucose versus FA, as a function of ATP demand. In searching for the minimal differences required to explain metabolic function in HCR versus LCR rats, it was determined that the whole-body metabolic phenotype of LCR, compared to the HCR, could be explained by a ~50% reduction in total mitochondrial activity with an additional 5-fold reduction in mitochondrial FA transport activity. Finally, we postulate that over sustained periods of exercise that LCR can partly overcome the initial deficit in FA catabolic activity by upregulating FA transport and/or oxidation processes.
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Simulación por Computador , Condicionamiento Físico Animal , Carrera/fisiología , Adenosina Trifosfatasas/metabolismo , Adenosina Trifosfato/metabolismo , Algoritmos , Animales , Dióxido de Carbono/metabolismo , Ácidos Grasos/metabolismo , Glucosa/metabolismo , Mitocondrias/metabolismo , Modelos Estadísticos , Músculo Esquelético/fisiología , Oxidación-Reducción , Oxígeno/metabolismo , Proteómica , Ratas , TermodinámicaRESUMEN
Proline utilization A from Escherichia coli (EcPutA) is a multifunctional flavoenzyme that oxidizes proline to glutamate through proline dehydrogenase (PRODH) and Δ1-pyrroline-5-carboxylate dehydrogenase (P5CDH) activities, while also switching roles as a DNA-bound transcriptional repressor and a membrane-bound catabolic enzyme. This phenomenon, termed functional switching, occurs through a redox-mediated mechanism in which flavin reduction triggers a conformational change that increases EcPutA membrane binding affinity. Structural studies have shown that reduction of the FAD cofactor causes the ribityl moiety to undergo a crankshaft motion, indicating that the orientation of the ribityl chain is a key element of PutA functional switching. Here, we test the role of a conserved histidine that bridges from the FAD pyrophosphate to the backbone amide of a conserved leucine residue in the PRODH active site. An EcPutA mutant (H487A) was characterized by steady-state and rapid-reaction kinetics, and cell-based reporter gene experiments. The catalytic activity of H487A is severely diminished (>50-fold) with membrane vesicles as the electron acceptor, and H487A exhibits impaired lipid binding and in vivo transcriptional repressor activity. Rapid-reaction kinetic experiments demonstrate that H487A is 3-fold slower than wild-type EcPutA in a conformational change step following reduction of the FAD cofactor. Furthermore, the reduction potential (Em) of H487A is â¼40 mV more positive than that of wild-type EcPutA, and H487A has an attenuated ability to catalyze the reverse PRODH chemical step of reoxidation by P5C. In this process, significant red semiquinone forms in contrast to the same reaction with wild-type EcPutA, in which facile two-electron reoxidation occurs without the formation of a measurable amount of semiquinone. These results indicate that His487 is critically important for the proline/P5C chemical step, conformational change kinetics, and functional switching in EcPutA.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Biocatálisis , Secuencia Conservada , Histidina/análisis , Histidina/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Enzimas Multifuncionales/química , Enzimas Multifuncionales/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Escherichia coli/química , Escherichia coli/genética , Escherichia coli/metabolismo , Cinética , Proteínas de la Membrana/genética , Modelos Moleculares , Enzimas Multifuncionales/genética , Prolina Oxidasa/química , Prolina Oxidasa/genética , Prolina Oxidasa/metabolismo , Alineación de SecuenciaRESUMEN
Mammalian E3 is an essential mitochondrial enzyme responsible for catalyzing the terminal reaction in the oxidative catabolism of several metabolites. E3 is a key regulator of metabolic fuel selection as a component of the pyruvate dehydrogenase complex (PDHc). E3 regulates PDHc activity by altering the affinity of pyruvate dehydrogenase kinase, an inhibitor of the enzyme complex, through changes in reduction and acetylation state of lipoamide moieties set by the NAD(+)/NADH ratio. Thus, an accurate kinetic model of E3 is needed to predict overall mammalian PDHc activity. Here, we have combined numerous literature data sets and new equilibrium spectroscopic experiments with a multitude of independently collected forward and reverse steady-state kinetic assays using pig heart E3. The latter kinetic assays demonstrate a pH-dependent transition of NAD(+) activation to inhibition, shown here, to our knowledge, for the first time in a single consistent data set. Experimental data were analyzed to yield a thermodynamically constrained four-redox-state model of E3 that simulates pH-dependent activation/inhibition and active site redox states for various conditions. The developed model was used to determine substrate/product conditions that give maximal E3 rates and show that, due to non-Michaelis-Menten behavior, the maximal flux is different compared with the classically defined kcat.
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Dihidrolipoamida Deshidrogenasa/química , Proteínas Mitocondriales/química , Proteínas Musculares/química , Miocardio/enzimología , NAD/química , Animales , Dihidrolipoamida Deshidrogenasa/metabolismo , Activación Enzimática , Concentración de Iones de Hidrógeno , Cinética , Proteínas Mitocondriales/metabolismo , Proteínas Musculares/metabolismo , NAD/metabolismo , PorcinosRESUMEN
PutA (proline utilization A) is a large bifunctional flavoenzyme with proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate dehydrogenase (P5CDH) domains that catalyze the oxidation of l-proline to l-glutamate in two successive reactions. In the PRODH active site, proline undergoes a two-electron oxidation to Δ(1)-pyrroline-5-carboxlylate, and the FAD cofactor is reduced. In the P5CDH active site, l-glutamate-γ-semialdehyde (the hydrolyzed form of Δ(1)-pyrroline-5-carboxylate) undergoes a two-electron oxidation in which a hydride is transferred to NAD(+)-producing NADH and glutamate. Here we report the first kinetic model for the overall PRODH-P5CDH reaction of a PutA enzyme. Global analysis of steady-state and transient kinetic data for the PRODH, P5CDH, and coupled PRODH-P5CDH reactions was used to test various models describing the conversion of proline to glutamate by Escherichia coli PutA. The coupled PRODH-P5CDH activity of PutA is best described by a mechanism in which the intermediate is not released into the bulk medium, i.e., substrate channeling. Unexpectedly, single-turnover kinetic experiments of the coupled PRODH-P5CDH reaction revealed that the rate of NADH formation is 20-fold slower than the steady-state turnover number for the overall reaction, implying that catalytic cycling speeds up throughput. We show that the limiting rate constant observed for NADH formation in the first turnover increases by almost 40-fold after multiple turnovers, achieving half of the steady-state value after 15 turnovers. These results suggest that EcPutA achieves an activated channeling state during the approach to steady state and is thus a new example of a hysteretic enzyme. Potential underlying causes of activation of channeling are discussed.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Escherichia coli/enzimología , Proteínas de la Membrana/química , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/química , Prolina Oxidasa/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Cinética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , NAD/química , NAD/genética , NAD/metabolismo , Oxidación-Reducción , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/genética , Oxidorreductasas actuantes sobre Donantes de Grupo CH-NH/metabolismo , Prolina Oxidasa/genética , Prolina Oxidasa/metabolismoRESUMEN
Dihydrolipoamide dehydrogenase is a flavoenzyme that reversibly catalyzes the oxidation of reduced lipoyl substrates with the reduction of NAD(+) to NADH. In vivo, the dihydrolipoamide dehydrogenase component (E3) is associated with the pyruvate, α-ketoglutarate, and glycine dehydrogenase complexes. The pyruvate dehydrogenase (PDH) complex connects the glycolytic flux to the tricarboxylic acid cycle and is central to the regulation of primary metabolism. Regulation of PDH via regulation of the E3 component by the NAD(+)/NADH ratio represents one of the important physiological control mechanisms of PDH activity. Furthermore, previous experiments with the isolated E3 component have demonstrated the importance of pH in dictating NAD(+)/NADH ratio effects on enzymatic activity. Here, we show that a three-state mechanism that represents the major redox states of the enzyme and includes a detailed representation of the active-site chemistry constrained by both equilibrium and thermodynamic loop constraints can be used to model regulatory NAD(+)/NADH ratio and pH effects demonstrated in progress-curve and initial-velocity data sets from rat, human, Escherichia coli, and spinach enzymes. Global fitting of the model provides stable predictions to the steady-state distributions of enzyme redox states as a function of lipoamide/dihydrolipoamide, NAD(+)/NADH, and pH. These distributions were calculated using physiological NAD(+)/NADH ratios representative of the diverse organismal sources of E3 analyzed in this study. This mechanistically detailed, thermodynamically constrained, pH-dependent model of E3 provides a stable platform on which to accurately model multicomponent enzyme complexes that implement E3 from a variety of organisms.
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Proteínas Bacterianas/química , Dihidrolipoamida Deshidrogenasa/química , Proteínas de Plantas/química , Animales , Escherichia coli/enzimología , Humanos , Concentración de Iones de Hidrógeno , Cinética , NAD/química , Oxidación-Reducción , Ratas , Especificidad de la Especie , Spinacia oleracea/enzimologíaRESUMEN
Circadian rhythm (CR) dysregulation negatively impacts health and contributes to mental disorders. The role of melatonin, a hormone intricately linked to CR, is still a subject of active study. The enzyme arylalkylamine N-acetyltransferase (AANAT) is responsible for melatonin synthesis, and it is a potential target for disorders that involve abnormally high melatonin levels, such as seasonal affective disorder (SAD). Current AANAT inhibitors suffer from poor cell permeability, selectivity, and/or potency. To address the latter, we have employed an X-ray crystal-based model to guide the modification of a previously described AANAT inhibitor, containing a rhodanine-indolinone core. We made various structural modifications to the core structure, including testing the importance of a carboxylic acid group thought to bind in the CoA site, and we evaluated these changes using MD simulations in conjunction with enzymatic assay data. Additionally, we tested three AANAT inhibitors in a zebrafish locomotion model to determine their effects inâ vivo. Key discoveries were that potency could be modestly improved by replacing a 5-carbon alkyl chain with rings and that the central rhodanine ring could be replaced by other heterocycles and maintain potency.
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Melatonina , Rodanina , Animales , Humanos , Melatonina/metabolismo , Acetiltransferasas , Rodanina/farmacología , Pez Cebra , N-Acetiltransferasa de Arilalquilamina/metabolismoRESUMEN
The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli catalyzes the oxidation of proline to glutamate in two reaction steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. Here, the kinetic mechanism of PRODH in PutA is studied by stopped-flow kinetics to determine microscopic rate constants for the proline:ubiquinone oxidoreductase mechanism. Stopped-flow data for proline reduction of the flavin cofactor (reductive half-reaction) and oxidation of reduced flavin by CoQ(1) (oxidative half-reaction) were best-fit by a double exponential from which maximum observable rate constants and apparent equilibrium dissociation constants were determined. Flavin semiquinone was not observed in the reductive or oxidative reactions. Microscopic rate constants for steps in the reductive and oxidative half-reactions were obtained by globally fitting the stopped-flow data to a simulated mechanism that includes a chemical step followed by an isomerization event. A microscopic rate constant of 27.5 s(-1) was determined for proline reduction of the flavin cofactor followed by an isomerization step of 2.2 s(-1). The isomerization step is proposed to report on a previously identified flavin-dependent conformational change [Zhang, W. et al. (2007) Biochemistry 46, 483-491] that is important for PutA functional switching but is not kinetically relevant to the in vitro mechanism. Using CoQ(1), a soluble analogue of ubiquinone, a rate constant of 5.4 s(-1) was obtained for the oxidation of flavin, thus indicating that this oxidative step is rate-limiting for k(cat) during catalytic turnover. Steady-state kinetic constants calculated from the microscopic rate constants agree with the experimental k(cat) and k(cat)/K(m) parameters.
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Proteínas Bacterianas/química , Proteínas de Escherichia coli/química , Proteínas de la Membrana/química , Prolina Oxidasa/química , Prolina/química , Multimerización de Proteína , 1-Pirrolina-5-Carboxilato Deshidrogenasa/química , 1-Pirrolina-5-Carboxilato Deshidrogenasa/fisiología , Anaerobiosis , Proteínas Bacterianas/fisiología , Bradyrhizobium/enzimología , Catálisis , Cristalografía por Rayos X , Escherichia coli/enzimología , Proteínas de Escherichia coli/fisiología , Flavina-Adenina Dinucleótido/química , Ácido Glutámico/química , Homeostasis/fisiología , Isomerismo , Cinética , Proteínas de la Membrana/fisiología , Oxidación-Reducción , Prolina Oxidasa/fisiología , Multimerización de Proteína/fisiología , Estructura Terciaria de ProteínaRESUMEN
Exposure of insulin-producing cells to elevated levels of the free fatty acid (FFA) palmitate results in the loss of ß-cell function and induction of apoptosis. The induction of endoplasmic reticulum (ER) stress is one mechanism proposed to be responsible for the loss of ß-cell viability in response to palmitate treatment; however, the pathways responsible for the induction of ER stress by palmitate have yet to be determined. Protein palmitoylation is a major posttranslational modification that regulates protein localization, stability, and activity. Defects in, or dysregulation of, protein palmitoylation could be one mechanism by which palmitate may induce ER stress in ß-cells. The purpose of this study was to evaluate the hypothesis that palmitate-induced ER stress and ß-cell toxicity are mediated by excess or aberrant protein palmitoylation. In a concentration-dependent fashion, palmitate treatment of RINm5F cells results in a loss of viability. Similar to palmitate, stearate also induces a concentration-related loss of RINm5F cell viability, while the monounsaturated fatty acids, such as palmoleate and oleate, are not toxic to RINm5F cells. 2-Bromopalmitate (2BrP), a classical inhibitor of protein palmitoylation that has been extensively used as an inhibitor of G protein-coupled receptor signaling, attenuates palmitate-induced RINm5F cell death in a concentration-dependent manner. The protective effects of 2BrP are associated with the inhibition of [(3)H]palmitate incorporation into RINm5F cell protein. Furthermore, 2BrP does not inhibit, but appears to enhance, the oxidation of palmitate. The induction of ER stress in response to palmitate treatment and the activation of caspase activity are attenuated by 2BrP. Consistent with protective effects on insulinoma cells, 2BrP also attenuates the inhibitory actions of prolonged palmitate treatment on insulin secretion by isolated rat islets. These studies support a role for aberrant protein palmitoylation as a mechanism by which palmitate enhances ER stress activation and causes the loss of insulinoma cell viability.
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Retículo Endoplásmico/fisiología , Ácidos Grasos no Esterificados/toxicidad , Células Secretoras de Insulina/fisiología , Lipoilación/fisiología , Animales , Western Blotting , Carnitina O-Palmitoiltransferasa/antagonistas & inhibidores , Caspasas/metabolismo , Muerte Celular/fisiología , Separación Celular , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Ceramidas/metabolismo , Glucosa/farmacología , Insulina/metabolismo , Secreción de Insulina , Masculino , Palmitatos/farmacología , Reacción en Cadena de la Polimerasa , Procesamiento Proteico-Postraduccional/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The multifunctional proline utilization A (PutA) flavoenzyme from Escherichia coli performs the oxidation of proline to glutamate in two catalytic steps using separate proline dehydrogenase (PRODH) and Δ(1)-pyrroline-5-carboxylate (P5C) dehydrogenase domains. In the first reaction, the oxidation of proline is coupled to the reduction of ubiquinone (CoQ) by the PRODH domain, which has a ß(8)α(8)-barrel structure that is conserved in bacterial and eukaryotic PRODH enzymes. The structural requirements of the benzoquinone moiety were examined by steady-state kinetics using CoQ analogs. PutA displayed activity with all the analogs tested; the highest k(cat)/K(m) was obtained with CoQ(2). The kinetic mechanism of the PRODH reaction was investigated use a variety of steady-state approaches. Initial velocity patterns measured using proline and CoQ(1), combined with dead-end and product inhibition studies, suggested a two-site ping-pong mechanism for PutA. The kinetic parameters for PutA were not strongly influenced by solvent viscosity suggesting that diffusive steps do not significantly limit the overall reaction rate. In summary, the kinetic data reported here, along with analysis of the crystal structure data for the PRODH domain, suggest that the proline:ubiquinone oxidoreductase reaction of PutA occurs via a rapid equilibrium ping-pong mechanism with proline and ubiquinone binding at two distinct sites.
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Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Proteínas Bacterianas/antagonistas & inhibidores , Escherichia coli/enzimología , Proteínas de Escherichia coli/antagonistas & inhibidores , Cinética , Proteínas de la Membrana/antagonistas & inhibidores , Modelos Moleculares , Prolina/metabolismo , Estructura Terciaria de Proteína , Quinonas/metabolismo , Quinonas/farmacología , Solventes , Espectrometría de Fluorescencia , Especificidad por Sustrato , Triptófano/química , ViscosidadRESUMEN
Objective This study aimed to identify factors associated with meeting the Obstetric Care Consensus (OCC) guidelines for nulliparous, term, singleton, and vertex (NTSV) cesarean births. Materials and methods This was a retrospective case control study of women with NTSV cesarean births between January 2014 and December 2017 at single tertiary care center. Demographics and clinical characteristics were compared between women with NTSV cesarean births which did or did not meet OCC guidelines. A multivariable logistic regression model was used to evaluate the effect of each variable on the odds of meeting OCC guidelines. Results There were 1,834 women with NTSV cesarean births of which 744 (40.6%) met OCC guidelines for delivery and 1,090 (59.4%) did not. After controlling for confounding factors, the odds of meeting OCC guidelines were increased for in-house providers managing with residents (adjusted odds ratio [aOR] = 2.03, 95% confidence interval [CI]: 1.44-2.87) and without residents (aOR = 1.66, 95% CI: 1.30-2.12), compared with non-in-house providers managing without residents. There was no significant difference in the odds of meeting OCC guidelines for in-house providers managing with or without residents (aOR = 1.23, 95% CI: 0.84-1.79). Conclusion After adjusting for confounding factors, in-house provider coverage, regardless of resident involvement, is associated with increased odds of NTSV cesarean births meeting OCC guidelines. Key Points Frequency of adherence to OCC guidelines for NTSV cesarean births was 40.6%.Neither patient demographics nor comorbidities was associated with the odds of meeting OCC guidelines.In-house providers are associated with increased odds of meeting OCC guidelines.
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High serum levels of insulin-like growth factor-I (IGF-I) are reported to be a risk factor for several common cancers, and recent cross-sectional data suggest a possible additional association of IGF-I with cervical neoplasia. To prospectively assess whether circulating IGF-I levels influence the natural history of oncogenic human papillomavirus (HPV), the viral cause of cervical cancer, we conducted a pilot investigation of 137 women who underwent semiannual type-specific HPV DNA PCR testing and cervical cytology. Total IGF-I and IGF binding protein-3 (IGFBP-3), the most abundant IGFBP in circulation, were measured using baseline serum specimens. Having a high IGF-I/IGFBP-3 ratio was associated with increased persistence of oncogenic HPV infection [that is, a lower rate of clearance; adjusted hazard ratio (AHR), 0.14; 95% confidence interval (95% CI), 0.04-0.57], whereas IGFBP-3 was inversely associated with both the incident detection of oncogenic HPV (AHR, 0.35; 95% CI, 0.13-0.93) and the incidence of oncogenic HPV-positive cervical neoplasia (that is, squamous intraepithelial lesions at risk of progression; AHR, 0.07; 95% CI, 0.01-0.66). These prospective data provide initial evidence that the IGF axis may influence the natural history of oncogenic HPV.
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Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/metabolismo , Factor I del Crecimiento Similar a la Insulina/metabolismo , Infecciones por Papillomavirus/epidemiología , Neoplasias del Cuello Uterino/epidemiología , Adulto , Estudios de Casos y Controles , Estudios de Cohortes , ADN Viral/genética , ADN Viral/metabolismo , Femenino , Seropositividad para VIH , Humanos , Proteína 3 de Unión a Factor de Crecimiento Similar a la Insulina/genética , Factor I del Crecimiento Similar a la Insulina/genética , Papillomaviridae/genética , Papillomaviridae/aislamiento & purificación , Infecciones por Papillomavirus/metabolismo , Infecciones por Papillomavirus/virología , Proyectos Piloto , Estudios Prospectivos , Neoplasias del Cuello Uterino/metabolismo , Neoplasias del Cuello Uterino/virologíaRESUMEN
OBJECTIVE: Despite the low mortality and morbidity of major obstetric and gynecologic surgeries (including hysterectomy and cesarean delivery), women undergoing these procedures occasionally suffer from intractable postoperative suprapubic and groin pain. We present seven patients whose intractable pain lasted longer than 6 months and was not due to gynecologic disease or other obvious pathology. METHODS: Neuromas of the ilioinguinal, iliohypogastric, and/or genitofemoral nerves were suspected clinically and confirmed intraoperatively. RESULTS: After neuroma resection, all patients reported complete and durable pain relief. CONCLUSION: Intractable pain after obstetric or gynecologic surgery can be due to neuroma formation, and resection is therapeutic. We suggest an algorithm for the management of women with chronic intractable suprapubic or groin pain after major obstetric and gynecologic surgery. LEVEL OF EVIDENCE: II-3.
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Cesárea , Histerectomía , Neuroma/cirugía , Dolor Postoperatorio/cirugía , Neoplasias del Sistema Nervioso Periférico/cirugía , Algoritmos , Femenino , Ingle/inervación , Humanos , Neuroma/diagnóstico , Dolor Postoperatorio/etiología , Neoplasias del Sistema Nervioso Periférico/diagnóstico , Complicaciones Posoperatorias/cirugía , EmbarazoRESUMEN
OBJECTIVE: To characterize ovarian failure and prolonged amenorrhea from other causes in women who are both human immunodeficiency virus (HIV) seropositive and seronegative. METHODS: This was a cohort study nested in the Women's Interagency HIV Study, a multicenter U.S. study of HIV infection in women. Prolonged amenorrhea was defined as no vaginal bleeding for at least 1 year. A serum follicle stimulating hormone more than 25 milli-International Units/mL and prolonged amenorrhea were used to define ovarian failure. Logistic regressions, chi2, and t tests were performed to estimate relationships between HIV-infection and cofactors with both ovarian failure and amenorrhea from other causes. RESULTS: Results were available for 1,431 women (1,139 HIV seropositive and 292 seronegative). More than one half of the HIV positive women with prolonged amenorrhea of at least 1 year did not have ovarian failure. When adjusted for age, HIV seropositive women were about three times more likely than seronegative women to have prolonged amenorrhea without ovarian failure. Body mass index, serum albumin, and parity were all negatively associated with ovarian failure in HIV seropositive women. CONCLUSION: HIV serostatus is associated with prolonged amenorrhea. It is difficult to ascertain whether the cause of prolonged amenorrhea is ovarian in HIV-infected women without additional testing. LEVEL OF EVIDENCE: II-2.
Asunto(s)
Amenorrea/complicaciones , Seropositividad para VIH/complicaciones , Insuficiencia Ovárica Primaria/complicaciones , Adolescente , Adulto , Estudios de Cohortes , Femenino , Seronegatividad para VIH , Humanos , Persona de Mediana Edad , Factores de RiesgoRESUMEN
OBJECTIVE: To estimate the risk of and risk factors for progression among human immunodeficiency virus (HIV)-seropositive women with abnormal cervical cytology but negative colposcopy. METHODS: In a prospective cohort study, 391 HIV-seropositive and 103 seronegative women with cervical cytology read as atypical squamous cells (ASC) or low-grade squamous intraepithelial lesion (LSIL) but negative colposcopy were followed up for a mean of 4.0 years with cytology at 6-month intervals. Colposcopy was prescribed for any epithelial abnormality. RESULTS: Progression to CIN2, CIN3, high-grade SIL/severe dysplasia, or cancer occurred in 47 (12%) HIV-seropositive women and 4 (4%) HIV-seronegative women (P = .02). Progression to CIN1 was seen in an additional 12 HIV-seropositive women and 1 seronegative woman. In multivariate analysis, high-risk but not low-risk HPV detection (hazard ratio [HR] 2.46-95% confidence interval [CI] 1.18-5.12, P = .02 for high risk, HR 1.41, 95% CI 0.62-3.21, P = .42 for low risk), satisfactory colposcopy (HR 2.01, 95% CI 1.11-3.65, P = .02), and non-Hispanic African-American ethnicity (HR 5.08, 95% CI 1.72-14.98, P = .003) were the only factors associated with progression, while HIV serostatus was marginally significant (HR 2.53, 95% CI 0.85-7.50, P = .09). CONCLUSION: Human immunodeficiency virus-seropositive women with negative colposcopy after borderline cytology face a higher risk of progression than seronegative women, but the absolute risk is low and becomes nonsignificant after controlling for HPV risk type, ethnicity, and colposcopic findings. Observation is appropriate. LEVEL OF EVIDENCE: II-2.