RESUMEN
Endothelial dysfunction (ED) comes with age, even without overt vessel damage such as that which occurs in atherosclerosis and diabetic vasculopathy. We hypothesized that aging would affect the downstream signalling of the endothelial nitric oxide (NO) system in the vascular smooth muscle (VSM). With this in mind, resistance mesenteric arteries were isolated from 13-week (juvenile) and 40-week-old (aged) mice and tested under isometric conditions using wire myography. Acetylcholine (ACh)-induced relaxation was reduced in aged as compared to juvenile vessels. Pretreatment with L-NAME, which inhibits nitrix oxide synthases (NOS), decreased ACh-mediated vasorelaxation, whereby differences in vasorelaxation between groups disappeared. Endothelium-independent vasorelaxation by the NO donor sodium nitroprusside (SNP) was similar in both groups; however, SNP bolus application (10-6 mol L-1) as well as soluble guanylyl cyclase (sGC) activation by runcaciguat (10-6 mol L-1) caused faster responses in juvenile vessels. This was accompanied by higher cGMP concentrations and a stronger response to the PDE5 inhibitor sildenafil in juvenile vessels. Mesenteric arteries and aortas did not reveal apparent histological differences between groups (van Gieson staining). The mRNA expression of the α1 and α2 subunits of sGC was lower in aged animals, as was PDE5 mRNA expression. In conclusion, vasorelaxation is compromised at an early age in mice even in the absence of histopathological alterations. Vascular smooth muscle sGC is a key element in aged vessel dysfunction.
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Arterias Mesentéricas/fisiología , Guanilil Ciclasa Soluble/fisiología , Acetilcolina/farmacología , Factores de Edad , Animales , Aorta/metabolismo , GMP Cíclico/metabolismo , Células Endoteliales/metabolismo , Células Endoteliales/fisiología , Guanilato Ciclasa/metabolismo , Masculino , Arterias Mesentéricas/metabolismo , Ratones , Ratones Endogámicos C57BL , Músculo Liso Vascular/metabolismo , Músculo Liso Vascular/fisiología , NG-Nitroarginina Metil Éster/farmacología , Óxido Nítrico/metabolismo , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Óxido Nítrico Sintasa de Tipo III/metabolismo , Vasodilatación/efectos de los fármacos , Vasodilatadores/farmacologíaAsunto(s)
Diabetes Mellitus Tipo 2 , Receptor del Péptido 1 Similar al Glucagón , Hipoglucemiantes , Inhibidores del Cotransportador de Sodio-Glucosa 2 , Humanos , Inhibidores del Cotransportador de Sodio-Glucosa 2/uso terapéutico , Inhibidores del Cotransportador de Sodio-Glucosa 2/efectos adversos , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Diabetes Mellitus Tipo 2/epidemiología , Alemania/epidemiología , Receptor del Péptido 1 Similar al Glucagón/agonistas , Femenino , Hipoglucemiantes/uso terapéutico , Hipoglucemiantes/efectos adversos , Masculino , Persona de Mediana Edad , Anciano , Adulto , Factores Socioeconómicos , Agonistas Receptor de Péptidos Similares al GlucagónRESUMEN
In vertebrates, SLC22A13 is an evolutionarily conserved transport protein of the plasma membrane. In humans and rat, it is principally expressed in the kidney. The precise localization and physiological function are unknown. In the present study, immunohistochemistry revealed that expression of SLC22A13 is confined to the basolateral membrane of type A intercalated cells in rat kidney. Double-staining confirmed that SLC22A13 co-localizes with anion exchanger 1. LC-MS difference shading showed that heterologous expression of human and rat SLC22A13 in HEK (human embryonic kidney)-293 cells stimulates efflux of guanidinosuccinate, aspartate, glutamate and taurine. Time courses of uptake of [3H]aspartate and [3H]glutamate revealed that SLC22A13 counteracted endogenous uptake. By contrast, OAT2 (organic anion transporter 2), a bidirectional glutamate transporter, increased accumulation of [3H]glutamate. Thus SLC22A13 catalyses unidirectional efflux. Velocity of efflux of standard amino acids was measured by LC-MS/MS. Expression of SLC22A13 strongly stimulated efflux of aspartate, taurine and glutamate. When the intracellular concentrations of aspartate and taurine were increased by pre-incubation, velocities of efflux increased linearly. We propose that in type A intercalated cells, SLC22A13 compensates luminal exit of protons by mediating the basolateral expulsion of the anions aspartate and glutamate. In this context, unidirectional efflux is essential to avoid anion re-entering. Loss of SLC22A13 function could cause distal tubular acidosis.
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Ácido Aspártico/metabolismo , Células Epiteliales/metabolismo , Ácido Glutámico/metabolismo , Túbulos Renales Colectores/metabolismo , Transportadores de Anión Orgánico/biosíntesis , Animales , Catálisis , Regulación de la Expresión Génica , Células HEK293 , Humanos , Transportadores de Anión Orgánico/genética , Transporte de Proteínas/fisiología , RatasRESUMEN
Mutations in GDAP1 lead to recessively or dominantly inherited peripheral neuropathies (Charcot-Marie-Tooth disease, CMT), indicating that GDAP1 is essential for the viability of cells in the peripheral nervous system. GDAP1 contains domains characteristic of glutathione-S-transferases (GSTs), is located in the outer mitochondrial membrane and induces fragmentation of mitochondria. We found GDAP1 upregulated in neuronal HT22 cells selected for resistance against oxidative stress. GDAP1 over-expression protected against oxidative stress caused by depletion of the intracellular antioxidant glutathione (GHS) and against effectors of GHS depletion that affect the mitochondrial membrane integrity like truncated BH3-interacting domain death agonist and 12/15-lipoxygenase. Gdap1 knockdown, in contrast, increased the susceptibility of motor neuron-like NSC34 cells against GHS depletion. Over-expression of wild-type GDAP1, but not of GDAP1 with recessively inherited mutations that cause disease and reduce fission activity, increased the total cellular GHS content and the mitochondrial membrane potential up to a level where it apparently limits mitochondrial respiration, leading to reduced mitochondrial Ca(2+) uptake and superoxide production. Fibroblasts from autosomal-recessive CMT4A patients had reduced GDAP1 levels, reduced GHS concentration and a reduced mitochondrial membrane potential. Thus, our results suggest that the potential GST GDAP1 is implicated in the control of the cellular GHS content and mitochondrial activity, suggesting an involvement of oxidative stress in the pathogenesis of CMT4A.
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Enfermedad de Charcot-Marie-Tooth/metabolismo , Glutatión/metabolismo , Potencial de la Membrana Mitocondrial , Proteínas del Tejido Nervioso/metabolismo , Línea Celular , Enfermedad de Charcot-Marie-Tooth/genética , Humanos , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/genética , Estrés OxidativoRESUMEN
Patients with idiopathic pulmonary fibrosis show a strongly upregulated expression of chemokine CXCL14, whose target is still unknown. Screening of CXCL14 in a panel of human G protein-coupled receptors (GPCRs) revealed its potent and selective activation of the orphan MAS-related GPCR X2 (MRGPRX2). This receptor is expressed on mast cells and - like CXCL14 - upregulated in bronchial inflammation. CXCL14 induces robust activation of MRGPRX2 and its putative mouse ortholog MRGPRB2 in G protein-dependent and ß-arrestin recruitment assays that is blocked by a selective MRGPRX2/B2 antagonist. Truncation combined with mutagenesis and computational studies identified the pharmacophoric sequence of CXCL14 and its presumed interaction with the receptor. Intriguingly, C-terminal domain sequences of CXCL14 consisting of 4 to 11 amino acids display similar or increased potency and efficacy compared to the full CXCL14 sequence (77 amino acids). These results provide a rational basis for the future development of potential idiopathic pulmonary fibrosis therapies.
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Quimiocinas , Fibrosis Pulmonar Idiopática , Animales , Humanos , Ratones , Aminoácidos , Bioensayo , Quimiocinas CXC , Fibrosis Pulmonar Idiopática/genética , Proteínas del Tejido Nervioso/genética , Receptores Acoplados a Proteínas G/genética , Receptores de NeuropéptidoRESUMEN
TOX3 is a nuclear protein containing a high mobility group (HMG)-box domain, which regulates Ca(2+)-dependent transcription in neurons through interaction with the cAMP-response-element-binding protein (CREB). TOX3 appears to be associated with breast cancer susceptibility and was previously shown to be expressed downstream of a cytoprotective cascade together with CITED1, a transcriptional regulator that does not bind directly to DNA. In the present study we show that TOX3 is predominantly expressed in the brain, forms homodimers and interacts with CITED1. TOX3 overexpression protects neuronal cells from cell death caused by endoplasmic reticulum stress or BAX overexpression through the induction of anti-apoptotic transcripts and repression of pro-apoptotic transcripts, which correlates with enhanced transcription involving isolated estrogen-responsive elements and estrogen-responsive promoters. However, both functions cannot be inhibited with the anti-estrogen fulvestrant and are only attenuated by mutation of estrogen-responsive elements. TOX3 also interacts with native CREB and induces the CREB-responsive BCL-2 promoter, which can be inhibited by coexpression of CITED1. Coexpression of CREB, by contrast, abolishes TOX3-mediated transcription from the estrogen-responsive complement C3 promoter. Our results suggest that TOX3 can enhance transcriptional activation from different cytoprotective promoters and that this is dependent on the predominance of either phosphorylated CREB or CITED1 within the transcriptionally active complex.
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Proteína de Unión a Elemento de Respuesta al AMP Cíclico/metabolismo , Neuronas/citología , Neuronas/metabolismo , Proteínas Nucleares/metabolismo , Receptores de Progesterona/metabolismo , Factores de Transcripción/metabolismo , Activación Transcripcional , Animales , Proteínas Reguladoras de la Apoptosis , Células COS , Supervivencia Celular , Chlorocebus aethiops , Proteína de Unión a Elemento de Respuesta al AMP Cíclico/genética , Regulación de la Expresión Génica , Células HEK293 , Proteínas del Grupo de Alta Movilidad , Humanos , Proteínas Nucleares/genética , Regiones Promotoras Genéticas , Unión Proteica , Receptores de Progesterona/genética , Transactivadores , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Arrhythmogenic right ventricular cardiomyopathy (ARVC) is an inherited cardiomyopathy primarily of the right ventricle characterized through fibrofatty replacement of cardiomyocytes. The genetic etiology in ARVC patients is most commonly caused by dominant inheritance and high genetic heterogeneity. Though histological examinations of ARVC-affected human myocardium reveals fibrolipomatous replacement, the molecular mechanisms leading to loss of cardiomyocytes are largely unknown. We therefore analyzed the transcriptomes of six ARVC hearts and compared our findings to six nonfailing donor hearts (NF). To characterize the ARVC-specific transcriptome, we compared our findings to samples from seven patients with idiopathic dilated cardiomyopathy (DCM). The myocardial DCM and ARVC samples were prepared from hearts explanted during an orthotopic heart transplantation representing myocardium from end-stage heart failure patients (NYHA IV). From each heart, left (LV) and right ventricular (RV) myocardial samples were analyzed by Affymetrix HG-U133 Plus 2.0 arrays, adding up to six sample groups. Unsupervised cluster analyses of the groups revealed a clear separation of NF and cardiomyopathy samples. However, in contrast to the other samples, the analyses revealed no distinct expression pattern in LV and RV of myocardial ARVC samples. We further identified differentially expressed transcripts using t-tests and found transcripts separating diseased and NF ventricular myocardium. Of note, in failing myocardium only ~15-16% of the genes are commonly regulated compared with NF samples. In addition both cardiomyopathies are clearly distinct on the transcriptome level. Comparison of the expression patterns between the failing RV and LV using a paired t-test revealed a lack of major differences between LV and RV gene expression in ARVC hearts. Our study is the first analysis of specific ARVC-related RV and LV gene expression patterns in terminal failing human hearts.
Asunto(s)
Displasia Ventricular Derecha Arritmogénica/genética , Miocardio/metabolismo , Transcriptoma , Adolescente , Adulto , Anciano , Displasia Ventricular Derecha Arritmogénica/metabolismo , Displasia Ventricular Derecha Arritmogénica/patología , Estudios de Casos y Controles , Análisis por Conglomerados , Estudios de Cohortes , Femenino , Perfilación de la Expresión Génica , Estudios de Asociación Genética , Insuficiencia Cardíaca/genética , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/patología , Humanos , Masculino , Análisis por Micromatrices , Persona de Mediana Edad , Miocardio/patología , Transcriptoma/genética , Adulto JovenRESUMEN
OAT (organic anion transporter) 2 [human gene symbol SLC22A7 (SLC is solute carrier)] is a member of the SLC22 family of transport proteins. In the rat, the principal site of expression of OAT2 is the sinusoidal membrane domain of hepatocytes. The particular physiological function of OAT2 in liver has been unresolved so far. In the present paper, we have used the strategy of LC (liquid chromatography)-MS difference shading to search for specific and cross-species substrates of OAT2. Heterologous expression of human and rat OAT2 in HEK (human embryonic kidney)-293 cells stimulated accumulation of the zwitterion trigonelline; subsequently, orotic acid was identified as an excellent and specific substrate of OAT2 from the rat (clearance=106 µl·min⻹·mg of protein⻹) and human (46 µl·min⻹·mg of protein⻹). The force driving uptake of orotic acid was identified as glutamate antiport. Efficient transport of glutamate by OAT2 was directly demonstrated by uptake of [³H]glutamate. However, because of high intracellular glutamate, OAT2 operates as glutamate efflux transporter. Thus expression of OAT2 markedly increased the release of glutamate (measured by LC-MS) from cells, even without extracellular exchange substrate. Orotic acid strongly trans-stimulated efflux of glutamate. We thus propose that OAT2 physiologically functions as glutamate efflux transporter. OAT2 mRNA was detected, after laser capture microdissection of rat liver slices, equally in periportal and pericentral regions; previous reports of hepatic release of glutamate into blood can now be explained by OAT2 activity. A specific OAT2 inhibitor could, by lowering plasma glutamate and thus promoting brain-to-blood efflux of glutamate, alleviate glutamate exotoxicity in acute brain conditions.
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Ácido Glutámico/metabolismo , Transportadores de Anión Orgánico Sodio-Independiente/metabolismo , Ácido Orótico/metabolismo , Alcaloides/metabolismo , Animales , Transporte Biológico Activo/genética , Dominio Catalítico/genética , Línea Celular Transformada , Células HEK293 , Humanos , Transportadores de Anión Orgánico Sodio-Independiente/genética , Ratas , Especificidad por Sustrato/genéticaRESUMEN
Short-term glycemic variability is associated with the risk of hypoglycemia and hyperglycemia in people living with type 1 diabetes and can potentially affect clinical outcomes. Continuous glucose monitoring (CGM) is of increasing importance to evaluate glycemic variability in greater detail. Specific metrics for assessing glycemic variability were proposed, such as the SD of mean glucose level and associated coefficient of variation, and time in target glucose range to guide study designs, therapy and allow people with diabetes more transparency in interpreting their own CGM data. Randomized controlled trials (RCT) and real-world evidence provide complementary information about the efficacy/effectiveness and safety of interventions. Insulin glargine 300 U/mL (Gla-300) has a longer lasting and less variable action than insulin glargine U100 (Gla-100) with a lower risk of hypoglycemia. While insulin degludec U100 (iDeg-100) was associated with lower glucose values but more time below range in one randomized study compared with Gla-300, Gla-300 was associated with a higher per cent time in range, but also above the therapeutic range. However, a real-world study did not find differences during the day between Gla-300 and iDeg-100. The upcoming InRange RCT is the first head-to-head comparison of Gla-300 with iDeg-100 using CGM in an international population using CGM metrics as the primary endpoint. The non-interventional COMET-T real-world study will determine the real-world effectiveness of Gla-300 using CGM metrics and cover a broad spectrum of clinical practice decisions irrespective of the prior basal insulin.
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Diabetes Mellitus Tipo 1 , Hipoglucemia , Glucemia , Diabetes Mellitus Tipo 1/tratamiento farmacológico , Humanos , Hipoglucemia/inducido químicamente , Hipoglucemia/prevención & control , Hipoglucemiantes/efectos adversos , Insulina Glargina/efectos adversos , Ensayos Clínicos Controlados Aleatorios como AsuntoRESUMEN
Förster resonance energy transfer within a protein-protein complex has previously been invoked to explain emission spectral modulation observed in several bioluminescence systems. Here we present a spatial structure of a complex of the Ca(2+)-regulated photoprotein clytin with its green-fluorescent protein (cgGFP) from the jellyfish Clytia gregaria, and show that it accounts for the bioluminescence properties of this system in vitro. We adopted an indirect approach of combining x-ray crystallography determined structures of the separate proteins, NMR spectroscopy, computational docking, and mutagenesis. Heteronuclear NMR spectroscopy using variously (15)N,(13)C,(2)H-enriched proteins enabled assignment of backbone resonances of more than 94% of the residues of both proteins. In a mixture of the two proteins at millimolar concentrations, complexation was inferred from perturbations of certain (1)H-(15)N HSQC-resonances, which could be mapped to those residues involved at the interaction site. A docking computation using HADDOCK was employed constrained by the sites of interaction, to deduce an overall spatial structure of the complex. Contacts within the clytin-cgGFP complex and electrostatic complementarity of interaction surfaces argued for a weak protein-protein complex. A weak affinity was also observed by isothermal titration calorimetry (K(D) = 0.9 mM). Mutation of clytin residues located at the interaction site reduced the degree of protein-protein association concomitant with a loss of effectiveness of cgGFP in color-shifting the bioluminescence. It is suggested that this clytin-cgGFP structure corresponds to the transient complex previously postulated to account for the energy transfer effect of GFP in the bioluminescence of aequorin or Renilla luciferase.
Asunto(s)
Simulación por Computador , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/química , Proteínas Luminiscentes/química , Modelos Moleculares , Complejos Multiproteicos/química , Sustitución de Aminoácidos , Animales , Cnidarios/química , Cnidarios/genética , Cristalografía por Rayos X , Proteínas Fluorescentes Verdes/genética , Proteínas Luminiscentes/genética , Complejos Multiproteicos/genética , Mutación Missense , Resonancia Magnética Nuclear Biomolecular , Estructura Cuaternaria de ProteínaRESUMEN
The bioluminescent systems of many marine organisms are comprised of two proteins--the Ca(2+)-regulated photoprotein and green-fluorescent protein (GFP). This work reports the cloning of the full-size cDNA encoding GFP (cgreGFP) from jellyfish Clytia gregaria, its expression and properties of the recombinant protein. The overall degree of identity between the amino acid sequence of the novel cgreGFP and the sequence of GFP (avGFP) from Aequorea victoria is 42% (similarity--64%) despite these GFPs originating from jellyfish that both belong to the same class, Hydrozoa. However although the degree of identity is low, three residues, Ser-Tyr-Gly, which form the chromophore are identical in both GFPs. The cgreGFP displayed two absorption peaks at 278 and 485 nm, and the fluorescence maximum at 500 nm. The fluorescence quantum yield was determined to be 0.86, the brightness to be 54 mM(-1) cm(-1). For the first time we have also demonstrated an efficient radiationless energy transfer in vitro between clytin and cgreGFP in solution at micromolar concentrations. The cgreGFP may be a useful intracellular fluorescent marker, as it was able to be expressed in mammalian cells.
Asunto(s)
Proteínas de Peces/química , Proteínas Fluorescentes Verdes/química , Escifozoos/metabolismo , Secuencia de Aminoácidos , Animales , Células CHO , Cricetinae , Cricetulus , Proteínas de Peces/genética , Proteínas de Peces/metabolismo , Transferencia Resonante de Energía de Fluorescencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Proteínas Luminiscentes/química , Datos de Secuencia Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Alineación de Secuencia , Espectrometría de FluorescenciaRESUMEN
Kidney fibrosis presents a hallmark of chronic kidney disease. With ever-increasing patient numbers and limited treatment options available, novel strategies for therapeutic intervention in kidney disease are warranted. Fibrosis commonly results from a wound healing response to repeated or chronic tissue damage, irrespective of the underlying etiology, and can occur in virtually any solid organ or tissue. In order to identify targets relevant for kidney fibrosis, we aimed to employ CRISPR screening in primary human kidney fibroblasts. We demonstrate that CRISPR technology can be applied in primary kidney fibroblasts and can furthermore be used to conduct arrayed CRISPR screening using a high-content imaging readout in a whole genome-wide manner. Hits coming out of this screen were validated using orthogonal approaches and present starting points for validation of novel targets relevant to kidney disease.
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Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Fibrosis/genética , Enfermedades Renales/genética , Riñón/efectos de los fármacos , Fibroblastos/metabolismo , Fibrosis/tratamiento farmacológico , Fibrosis/patología , Genoma Humano/genética , Secuenciación de Nucleótidos de Alto Rendimiento/tendencias , Humanos , Riñón/patología , Enfermedades Renales/tratamiento farmacológico , Enfermedades Renales/patología , Terapia Molecular Dirigida/tendenciasRESUMEN
Cardiomyocytes derived from pluripotent embryonic stem cells (ESC) have the advantage of providing a source for standardized cell cultures. However, little is known on the regulation of the genome during differentiation of ESC to cardiomyocytes. Here, we characterize the transcriptome of the mouse ESC line CM7/1 during differentiation into beating cardiomyocytes and compare the gene expression profiles with those from primary adult murine cardiomyocytes and left ventricular myocardium. We observe that the cardiac gene expression pattern of fully differentiated CM7/1-ESC is highly similar to adult primary cardiomyocytes and murine myocardium, respectively. This finding is underlined by demonstrating pharmacological effects of catecholamines and endothelin-1 on ESC-derived cardiomyocytes. Furthermore, we monitor the temporal changes in gene expression pattern during ESC differentiation with a special focus on transcription factors involved in cardiomyocyte differentiation. Thus, CM7/1-ESC-derived cardiomyocytes are a promising new tool for functional studies of cardiomyocytes in vitro and for the analysis of the transcription factor network regulating pluripotency and differentiation to cardiomyocytes.
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Células Madre Embrionarias/metabolismo , Perfilación de la Expresión Génica , Miocardio/metabolismo , Recombinación Genética , Factores de Transcripción/genética , Animales , Diferenciación Celular , Línea Celular , Células Madre Embrionarias/citología , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
In addition to its function as carnitine transporter, novel organic cation transporter type 2 (OCTN2; human gene symbol SLC22A5) is widely recognized as a transporter of drugs. This notion is based on several reports of direct measurement of drug accumulation. However, a rigorous, comparative, and comprehensive analysis of transport efficiency of OCTN2 has not been available so far. In the present study, OCTN2 orthologs from human, rat, and chicken were expressed in 293 cells using an inducible expression system. Uptake of trans-4-[4-(dimethylamino)styryl]-1-methylpyridinium iodide (ASP(+)), cephaloridine, ergothioneine, gabapentin, mildronate, pyrilamine, quinidine, spironolactone, tetraethylammonium, verapamil, and vigabatrin was determined by liquid chromatography/mass spectrometry. For reference, uptake of carnitine was measured in parallel. Our results indicate that OCTN2-mediated uptake of drugs was not significantly different from zero or, with tetraethylammonium and ergothioneine, was minute relative to carnitine. The carnitine congener mildronate, by contrast, was transported very efficiently. Thus, OCTN2 is not a general drug transporter but a highly specific carrier for carnitine and closely related molecules. Transport parameters (cellular accumulation, transporter affinity, sodium dependence) were similar for mildronate and carnitine. Efficiency of transport of mildronate was even higher than that of carnitine. Hence, our results establish that OCTN2 is a key target of the cardioprotective agent mildronate because it controls, as integral protein of the plasma membrane, cellular entry of mildronate and enables efficient access to intracellular targets. The highest levels of human OCTN2 mRNA were detected by real-time reverse transcription-polymerase chain reaction in kidney, ileum, breast, small intestine, skeletal muscle, and ovary but also in some heart and central nervous system tissues.
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Transporte Biológico/fisiología , Carnitina/metabolismo , Proteínas de Transporte de Membrana/metabolismo , Metilhidrazinas/metabolismo , Proteínas de Transporte de Catión Orgánico/metabolismo , Animales , Pollos , Clonación de Organismos , Femenino , Humanos , Reacción en Cadena de la Polimerasa , Ratas , Miembro 5 de la Familia 22 de Transportadores de SolutosRESUMEN
BACKGROUND: The aim of the current study was to determine whether the time to insulin therapy initiation in patients with type 2 diabetes in primary care in Germany has changed in recent years. METHODS: Longitudinal data from general practices in Germany (Disease Analyzer) were analyzed. These data included information of 7128 patients (age: 68.5 [SD: 11.5] years; 54.4% male) receiving incident insulin therapy in 2010/2011 and 8216 patients (age: 69.1 [SD: 11.9] years; 54.9% male) receiving incident insulin therapy in 2016/2017. Changes in time to insulin initiation in the practices and the last HbA1c value before the start of insulin therapy were analyzed, stratified by index date. To analyze the impact of covariables on the time to insulin initiation, a multivariate regression analysis was performed, adjusted for age, sex, diabetologist care, and HbA1c as independent variables. RESULTS: Median time from T2D diagnosis to insulin therapy in the Disease Analyzer database increased from 1717 days in 2010/2011 to 1917 days in 2016/2017 (P < .001). The proportion of patients with a HbA1c value ≥9% before insulin initiation was high in both groups (2010/2011: 33.0%, 2016/2017: 34.2%, P = .347). The time to insulin initiation in DPP-4i patients was 112 days longer, and in SGLT2 patients 346 days longer than in patients treated with sulfonylurea. CONCLUSIONS: The present analysis confirms an increasing delay of the insulin therapy initiation as a consequence of the more frequent use of newer oral antidiabetics. However, the rather moderate increase of time to insulin might display insufficient long-term glycemic control using these agents. Still, more than one-third of patients receive insulin only when HbA1c levels exceed 9%.
Asunto(s)
Glucemia/análisis , Diabetes Mellitus Tipo 2/tratamiento farmacológico , Hipoglucemiantes/uso terapéutico , Insulina/uso terapéutico , Pautas de la Práctica en Medicina , Anciano , Bases de Datos Factuales , Femenino , Alemania , Hemoglobina Glucada/análisis , Humanos , Estudios Longitudinales , Masculino , Persona de Mediana Edad , Atención Primaria de Salud , Tiempo de TratamientoRESUMEN
Ischemia/reperfusion injury holds a key position in many pathological conditions such as acute kidney injury and in the transition to chronic stages of renal damage. We hypothesized that besides a reported disproportional activation of vasoconstrictor response, hypoxia/reoxygenation (H/R) adversely affects endothelial dilatory systems and impairs relaxation in renal arteries. Rat renal interlobar arteries were studied under isometric conditions. Hypoxia was induced by application of 95% N2, 5% CO2 for 60 min to the bath solution, followed by a 10 min period of reoxygenation (95% O2, 5% CO2). The effect of H/R on relaxation was assessed using various inhibitors of endothelial dilatory systems. mRNA expression of phosphodiesterase 5 (PDE5), NADPH oxidases (NOX), and nitric oxide synthase (NOS) isoforms were determined using qRT-PCR; cGMP was assayed with direct cGMP ELISA. Acetylcholine induced relaxation was impaired after H/R. Inhibition of the NOS isoforms with L-NAME, and cyclooxygenases (COXs) by indomethacin did not abolish the H/R effect. Moreover, blocking the calcium activated potassium channels KCa3.1 and KCa2.1, the main mediators of the endothelium-derived hyperpolarizing factor, with TRAM34 and UCL1684, respectively, showed similar effects in H/R and control. Arterial stiffness did not differ comparing H/R with controls, indicating no impact of H/R on passive vessel properties. Moreover, superoxide was not responsible for the observed H/R effect. Remarkably, H/R attenuated the endothelium-independent relaxation by sodium nitroprusside, suggesting endothelium-independent mechanisms of H/R action. Investigating the signaling downstream of NO revealed significantly decreased cGMP and impaired relaxation during PDE5 inhibition with sildenafil after H/R. Inhibition of PKG, the target of cGMP, did not normalize SNP-induced relaxation following H/R. However, the soluble guanylyl cyclase (sGC) inhibitor ODQ abolished the H/R effect on relaxation. The mRNA expressions of the endothelial and the inducible NOS were reduced. NOX and PDE5 mRNA were similarly expressed in H/R and control. Our results provide new evidence that impaired renal artery relaxation after H/R is due to a dysregulation of sGC leading to decreased cGMP levels. The presented mechanism might contribute to an insufficient renal reperfusion after ischemia and should be considered in its pathophysiology.
RESUMEN
Recently, we have identified the ergothioneine (ET) transporter ETT (gene symbol SLC22A4). Much interest in human ETT has been generated by case-control studies that suggest an association of polymorphisms in the SLC22A4 gene with susceptibility to chronic inflammatory diseases. ETT was originally designated a multispecific novel organic cation transporter (OCTN1). Here we reinvestigated, based on stably transfected 293 cells and with ET as reference substrate, uptake of quinidine, verapamil, and pyrilamine. ETT from human robustly catalyzed transport of ET (68micfrol/(minmgprotein)), but no transport of organic cations was discernible. With ET as substrate, ETT was relatively resistant to inhibition by selected drugs; the most potent inhibitor was verapamil (K(i)=11micromol/l). The natural compound hercynine and antithyroid drug methimazole are related in structure to ET. However, efficiency of ETT-mediated transport of methimazole (K(i)=7.5mmol/l) was 130-fold lower, and transport of hercynine (K(i)=1.4mmol/l) was 25-fold lower than transport of ET. ETT from mouse, upon expression in 293 cells, catalyzed high affinity, sodium-driven uptake of ET very similar to ETT from human. Additional real-time PCR experiments based on 16 human tissues revealed ETT mRNA levels considerably lower than in bone marrow. Our experiments establish that ETT is highly specific for its physiological substrate ergothioneine. ETT is not a cationic drug transporter, and it does not have high affinity for organic cation inhibitors. Detection of ETT mRNA or protein can therefore be utilized as a specific molecular marker of intracellular ET activity.
Asunto(s)
Betaína/análogos & derivados , Ergotioneína/metabolismo , Histidina/análogos & derivados , Metimazol/metabolismo , Proteínas de Transporte de Catión Orgánico/fisiología , Betaína/metabolismo , Transporte Biológico , Células Cultivadas , Histidina/metabolismo , Humanos , Proteínas de Transporte de Catión Orgánico/genética , ARN Mensajero/análisis , Especificidad por Sustrato , SimportadoresAsunto(s)
Diabetes Mellitus Tipo 1 , Diabetes Mellitus Tipo 1/terapia , Humanos , Hipoglucemiantes , InsulinaRESUMEN
A method for high-throughput cloning and analysis of short hairpin RNAs (shRNAs) is described. Using this approach, 464 shRNAs against 116 different genes were screened for knockdown efficacy, enabling rapid identification of effective shRNAs against 74 genes. Statistical analysis of the effects of various criteria on the activity of the shRNAs confirmed that some of the rules thought to govern small interfering RNA (siRNA) activity also apply to shRNAs. These include moderate GC content, absence of internal hairpins, and asymmetric thermal stability. However, the authors did not find strong support for position specific rules. In addition, analysis of the data suggests that not all genes are equally susceptible to RNA interference (RNAi).