RESUMO
Both clinical and experimental data suggest that podocyte injury is involved in the onset and progression of diabetic kidney disease (DKD). Although the mechanisms underlying the development of podocyte loss are not completely understood, critical structural proteins such as podocin play a major role in podocyte survival and function. We have reported that the protein tyrosine phosphatase SHP-1 expression increased in podocytes of diabetic mice and glomeruli of patients with diabetes. However, the in vivo contribution of SHP-1 in podocytes is unknown. Conditional podocyte-specific SHP-1-deficient mice (Podo-SHP-1-/-) were generated to evaluate the impact of SHP-1 deletion at four weeks of age (early) prior to the onset of diabetes and after 20 weeks (late) of diabetes (DM; Ins2+/C96Y) on kidney function (albuminuria and glomerular filtration rate) and kidney pathology. Ablation of the SHP-1 gene specifically in podocytes prevented and even reversed the elevated albumin/creatinine ratio, glomerular filtration rate progression, mesangial cell expansion, glomerular hypertrophy, glomerular basement membrane thickening and podocyte foot process effacement induced by diabetes. Moreover, podocyte-specific deletion of SHP-1 at an early and late stage prevented diabetes-induced expression of collagen IV, fibronectin, transforming growth factor-ß, transforming protein RhoA, and serine/threonine kinase ROCK1, whereas it restored nephrin, podocin and cation channel TRPC6 expression. Mass spectrometry analysis revealed that SHP-1 reduced SUMO2 post-translational modification of podocin while podocyte-specific deletion of SHP-1 preserved slit diaphragm protein complexes in the diabetic context. Thus, our data uncovered a new role of SHP-1 in the regulation of cytoskeleton dynamics and slit diaphragm protein expression/stability, and its inhibition preserved podocyte function preventing DKD progression.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Animais , Camundongos , Diabetes Mellitus Experimental/induzido quimicamente , Nefropatias Diabéticas/genética , Nefropatias Diabéticas/prevenção & controle , Nefropatias Diabéticas/metabolismo , Podócitos/patologia , Proteína Tirosina Fosfatase não Receptora Tipo 6/genética , Proteína Tirosina Fosfatase não Receptora Tipo 6/metabolismo , Quinases Associadas a rho/metabolismo , SumoilaçãoRESUMO
Polymyxin antibiotics are often used as a last-line defense to treat life-threatening Gram-negative pathogens. However, polymyxin-induced kidney toxicity is a dose-limiting factor of paramount importance and can lead to suboptimal treatment. To elucidate the mechanism and develop effective strategies to overcome polymyxin toxicity, we employed a whole-genome CRISPR screen in human kidney tubular HK-2 cells and identified 86 significant genes that upon knock-out rescued polymyxin-induced toxicity. Specifically, we discovered that knockout of the inwardly rectifying potassium channels Kir4.2 and Kir5.1 (encoded by KCNJ15 and KCNJ16, respectively) rescued polymyxin-induced toxicity in HK-2 cells. Furthermore, we found that polymyxins induced cell depolarization via Kir4.2 and Kir5.1 and a significant cellular uptake of polymyxins was evident. All-atom molecular dynamics simulations revealed that polymyxin B1 spontaneously bound to Kir4.2, thereby increasing opening of the channel, resulting in a potassium influx, and changes of the membrane potential. Consistent with these findings, small molecule inhibitors (BaCl2 and VU0134992) of Kir potassium channels reduced polymyxin-induced toxicity in cell culture and mouse explant kidney tissue. Our findings provide critical mechanistic information that will help attenuate polymyxin-induced nephrotoxicity in patients and facilitate the design of novel, safer polymyxins.
Assuntos
Canais de Potássio Corretores do Fluxo de Internalização , Animais , Humanos , Rim/metabolismo , Potenciais da Membrana , Camundongos , Polimixinas/metabolismo , Polimixinas/toxicidade , Potássio/metabolismo , Canais de Potássio Corretores do Fluxo de Internalização/genética , Canais de Potássio Corretores do Fluxo de Internalização/metabolismoRESUMO
Congenital heart disease (CHD) is the most common birth defect and brings with it significant mortality and morbidity. The application of exome and genome sequencing has greatly improved the rate of genetic diagnosis for CHD but the cause in the majority of cases remains uncertain. It is clear that genetics, as well as environmental influences, play roles in the aetiology of CHD. Here we address both these aspects of causation with respect to the Notch signalling pathway. In our CHD cohort, variants in core Notch pathway genes account for 20% of those that cause disease, a rate that did not increase with the inclusion of genes of the broader Notch pathway and its regulators. This is reinforced by case-control burden analysis where variants in Notch pathway genes are enriched in CHD patients. This enrichment is due to variation in NOTCH1. Functional analysis of some novel missense NOTCH1 and DLL4 variants in cultured cells demonstrate reduced signalling activity, allowing variant reclassification. Although loss-of-function variants in DLL4 are known to cause Adams-Oliver syndrome, this is the first report of a hypomorphic DLL4 allele as a cause of isolated CHD. Finally, we demonstrate a gene-environment interaction in mouse embryos between Notch1 heterozygosity and low oxygen- or anti-arrhythmic drug-induced gestational hypoxia, resulting in an increased incidence of heart defects. This implies that exposure to environmental insults such as hypoxia could explain variable expressivity and penetrance of observed CHD in families carrying Notch pathway variants.
Assuntos
Interação Gene-Ambiente , Predisposição Genética para Doença , Genômica/métodos , Cardiopatias Congênitas/patologia , Mutação , Receptor Notch1/genética , Animais , Estudos de Casos e Controles , Feminino , Cardiopatias Congênitas/etiologia , Cardiopatias Congênitas/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Sequenciamento do ExomaRESUMO
Congenital heart disease (CHD) is the most common type of birth defect. In recent years, research has focussed on identifying the genetic causes of CHD. However, only a minority of CHD cases can be attributed to single gene mutations. In addition, studies have identified different environmental stressors that promote CHD, but the additive effect of genetic susceptibility and environmental factors is poorly understood. In this context, we have investigated the effects of short-term gestational hypoxia on mouse embryos genetically predisposed to heart defects. Exposure of mouse embryos heterozygous for Tbx1 or Fgfr1/Fgfr2 to hypoxia in utero increased the incidence and severity of heart defects while Nkx2-5+/- embryos died within 2 days of hypoxic exposure. We identified the molecular consequences of the interaction between Nkx2-5 and short-term gestational hypoxia, which suggest that reduced Nkx2-5 expression and a prolonged hypoxia-inducible factor 1α response together precipitate embryo death. Our study provides insight into the causes of embryo loss and variable penetrance of monogenic CHD, and raises the possibility that cases of foetal death and CHD in humans could be caused by similar gene-environment interactions.
Assuntos
Interação Gene-Ambiente , Cardiopatias Congênitas/genética , Coração/embriologia , Proteína Homeobox Nkx-2.5/genética , Proteínas de Homeodomínio/genética , Subunidade alfa do Fator 1 Induzível por Hipóxia/genética , Animais , Apoptose , Proliferação de Células , Embrião de Mamíferos/metabolismo , Feminino , Predisposição Genética para Doença , Coração/diagnóstico por imagem , Heterozigoto , Proteína Homeobox Nkx-2.5/fisiologia , Hipóxia , Subunidade alfa do Fator 1 Induzível por Hipóxia/fisiologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxigênio/metabolismo , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/genética , Proteínas com Domínio T/genética , Fatores de TempoRESUMO
BACKGROUND: Congenital malformations can be manifested as combinations of phenotypes that co-occur more often than expected by chance. In many such cases, it has proved difficult to identify a genetic cause. We sought the genetic cause of cardiac, vertebral, and renal defects, among others, in unrelated patients. METHODS: We used genomic sequencing to identify potentially pathogenic gene variants in families in which a person had multiple congenital malformations. We tested the function of the variant by using assays of in vitro enzyme activity and by quantifying metabolites in patient plasma. We engineered mouse models with similar variants using the CRISPR (clustered regularly interspaced short palindromic repeats)-Cas9 system. RESULTS: Variants were identified in two genes that encode enzymes of the kynurenine pathway, 3-hydroxyanthranilic acid 3,4-dioxygenase (HAAO) and kynureninase (KYNU). Three patients carried homozygous variants predicting loss-of-function changes in the HAAO or KYNU proteins (HAAO p.D162*, HAAO p.W186*, or KYNU p.V57Efs*21). Another patient carried heterozygous KYNU variants (p.Y156* and p.F349Kfs*4). The mutant enzymes had greatly reduced activity in vitro. Nicotinamide adenine dinucleotide (NAD) is synthesized de novo from tryptophan through the kynurenine pathway. The patients had reduced levels of circulating NAD. Defects similar to those in the patients developed in the embryos of Haao-null or Kynu-null mice owing to NAD deficiency. In null mice, the prevention of NAD deficiency during gestation averted defects. CONCLUSIONS: Disruption of NAD synthesis caused a deficiency of NAD and congenital malformations in humans and mice. Niacin supplementation during gestation prevented the malformations in mice. (Funded by the National Health and Medical Research Council of Australia and others.).
Assuntos
3-Hidroxiantranilato 3,4-Dioxigenase/genética , Anormalidades Congênitas/genética , Suplementos Nutricionais , Hidrolases/genética , NAD/deficiência , Niacina/uso terapêutico , 3-Hidroxiantranilato 3,4-Dioxigenase/metabolismo , Canal Anal/anormalidades , Animais , Anormalidades Congênitas/prevenção & controle , Modelos Animais de Doenças , Esôfago/anormalidades , Feminino , Cardiopatias Congênitas/genética , Cardiopatias Congênitas/prevenção & controle , Humanos , Hidrolases/metabolismo , Rim/anormalidades , Deformidades Congênitas dos Membros/genética , Deformidades Congênitas dos Membros/prevenção & controle , Masculino , Camundongos , Camundongos Knockout , Mutação , NAD/biossíntese , NAD/genética , Análise de Sequência de DNA , Coluna Vertebral/anormalidades , Traqueia/anormalidadesRESUMO
OBJECTIVE: Preeclampsia is a hypertensive disorder of pregnancy associated with proteinuria detected by 24-hour urine collection (≥0.3 g/24 h) or protein/creatinine ratio (≥30 mg/mmol). The albumin/creatinine ratio (ACR) is used outside pregnancy to detect abnormal amounts of albumin in the urine, but there is little data on its value in pregnancy. Our objective was to determine the diagnostic threshold for ACR to detect significant proteinuria in women investigated for preeclampsia. METHODS: A prospective observational study involving 99 hypertensive women (≥140/90 mm Hg) over 20 weeks gestation who were hospitalized at 2 Canadian tertiary centres. A 24-hour urine collection and a morning urine sample were collected. The optimal ACR threshold was determined by a receiver operating characteristic (ROC) curve using the 24-hour collection as the reference test; sensitivity and specificity analyses were performed. Maternal and perinatal characteristics were extracted from medical records. RESULTS: Of the 87 women who had completed urine collection, 74 (85%) had an initial diagnosis of preeclampsia and 63 (72%) had significant proteinuria confirmed by 24-hour collection. The area under the morning ROC curve was 0.92 (95% CI 0.86-0.98) and the optimal threshold obtained for the ACR was 9 mg/mmol, with a sensitivity and specificity of 84% (95% CI 73-92) and 88% (95% CI 68-97), respectively. CONCLUSION: Our results suggest that an ACR threshold of 9 mg/mmol on a morning urine sample can be used to detect significant proteinuria of preeclampsia in hospitalized hypertensive women.
RESUMO
Congenital heart disease (CHD) is an enigma. It is the most common human birth defect and yet, even with the application of modern genetic and genomic technologies, only a minority of cases can be explained genetically. This is because environmental stressors also cause CHD. Here we propose a plausible non-genetic mechanism for induction of CHD by environmental stressors. We show that exposure of mouse embryos to short-term gestational hypoxia induces the most common types of heart defect. This is mediated by the rapid induction of the unfolded protein response (UPR), which profoundly reduces FGF signaling in cardiac progenitor cells of the second heart field. Thus, UPR activation during human pregnancy might be a common cause of CHD. Our findings have far-reaching consequences because the UPR is activated by a myriad of environmental or pathophysiological conditions. Ultimately, our discovery could lead to preventative strategies to reduce the incidence of human CHD.
Assuntos
Cardiopatias Congênitas/etiologia , Cardiopatias Congênitas/patologia , Estresse Fisiológico , Resposta a Proteínas não Dobradas , Animais , Apoptose/efeitos dos fármacos , Diferenciação Celular/efeitos dos fármacos , Hipóxia Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Embrião de Mamíferos/efeitos dos fármacos , Embrião de Mamíferos/patologia , Feminino , Fatores de Crescimento de Fibroblastos/metabolismo , Camundongos Endogâmicos C57BL , Oxigênio/farmacologia , Fenótipo , Gravidez , Biossíntese de Proteínas/efeitos dos fármacos , Receptor Tipo 1 de Fator de Crescimento de Fibroblastos/metabolismo , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/efeitos dos fármacos , Resposta a Proteínas não Dobradas/efeitos dos fármacosRESUMO
Much has been written on the successful lesbian, gay, bisexual, transgender, and intersex movement in South Africa, and the resulting institutionalization of sexual minority rights. Comparatively less has been written about the forms of activism undertaken specifically by Black lesbians that are not oriented toward legal change. In this article, I assert the need to examine public demonstrations of mourning as an act of Black lesbian resistance to violence in South Africa. Based on in-depth interviews with members of Free Gender, a Black lesbian organization, I argue that members' conceptualizations of mourning as providing community support force a reconsideration of what it means to be human. In order to grasp the decolonial potential of Free Gender's activism, I draw on Sylvia Wynter's argument that a singular Western bourgeois conception of human has come to dominate globally.
Assuntos
Pesar , Homofobia/etnologia , Homossexualidade Feminina/etnologia , Política , Minorias Sexuais e de Gênero/psicologia , Violência/etnologia , Adulto , Feminino , Humanos , Pesquisa Qualitativa , África do Sul/etnologiaRESUMO
BACKGROUND: Excessive gestational weight gain (GWG) is associated with adverse pregnancy outcomes. In non-pregnant populations, low leptin levels stimulate positive energy balance. In pregnancy, both the placenta and adipose tissue contribute to circulating leptin levels. We tested whether maternal leptin levels are associated with subsequent GWG and whether this association varies depending on stage of pregnancy and on maternal body mass index (BMI). METHODS: This prospective cohort study included 675 pregnant women followed from 1(st) trimester until delivery. We collected anthropometric measurements, blood samples at 1(st) and 2(nd) trimester, and clinical data until delivery. Maternal leptin was measured by ELISA (Luminex technology). We classified women by BMI measured at 1(st) trimester: BMI < 25 kg/m(2) = normal weight; 25 ≤ BMI < 30 kg/m(2) = overweight; and BMI ≥ 30 kg/m(2) = obese. RESULTS: Women gained a mean of 6.7 ± 3.0 kg between 1(st) and 2(nd) trimester (mid pregnancy GWG) and 5.6 ± 2.5 kg between 2(nd) and the end of 3(rd) trimester (late pregnancy GWG). Higher 1(st) trimester leptin levels were associated with lower mid pregnancy GWG, but the association was no longer significant after adjusting for % body fat (%BF; ß = 0.38 kg per log-leptin; SE = 0.52; P = 0.46). Higher 2(nd) trimester leptin levels were associated with greater late pregnancy GWG and this association remained significant after adjustment for BMI (ß = 2.35; SE = 0.41; P < 0.0001) or %BF (ß = 2.01; SE = 0.42; P < 0.0001). In BMI stratified analyses, higher 2(nd) trimester leptin levels were associated with greater late pregnancy GWG in normal weight women (ß = 1.33; SE = 0.42; P =0.002), and this association was stronger in overweight women (ß = 2.85; SE = 0.94; P = 0.003--P for interaction = 0.05). CONCLUSIONS: Our results suggest that leptin may regulate weight gain differentially at 1(st) versus 2(nd) trimester of pregnancy: at 2(nd) trimester, higher leptin levels were associated with greater subsequent weight gain--the opposite of its physiologic regulation in non-pregnancy--and this association was stronger in overweight women. We suspect the existence of a feed-forward signal from leptin in second half of pregnancy, stimulating a positive energy balance and leading to greater weight gain.
Assuntos
Leptina/sangue , Segundo Trimestre da Gravidez/sangue , Aumento de Peso/fisiologia , Adulto , Índice de Massa Corporal , Feminino , Humanos , Sobrepeso/sangue , Gravidez , Complicações na Gravidez/sangue , Primeiro Trimestre da Gravidez/sangue , Estudos Prospectivos , Fatores de RiscoRESUMO
CITED2 is a transcriptional co-factor with important roles in many organs of the developing mammalian embryo. Complete deletion of this gene causes severe malformation of the placenta, and results in significantly reduced embryonic growth and death from E14.5. The placenta is a complex organ originating from cells derived from three lineages: the maternal decidua, the trophectoderm, and the extra-embryonic mesoderm. Cited2 is expressed in many of these cell types, but its exact role in the formation of the placenta is unknown. Here we use a conditional deletion approach to remove Cited2 from overlapping subsets of trophectoderm and extra-embryonic mesoderm. We find that Cited2 in sinusoidal trophoblast giant cells and syncytiotrophoblasts is likely to have a non-cell autonomous role in patterning of the pericytes associated with the embryonic capillaries. This function is likely to be mediated by PDGF signaling. Furthermore, we also identify that loss of Cited2 in syncytiotrophoblasts results in the subcellular mislocalization of one of the major lactate transporters in the placenta, SLC16A3 (MCT4). We hypothesize that the embryonic growth retardation observed in Cited2 null embryos is due in part to a disorganized embryonic capillary network, and in part due to abnormalities of the nutrient transport functions of the feto-maternal interface.
Assuntos
Padronização Corporal , Capilares/embriologia , Placenta/irrigação sanguínea , Placenta/embriologia , Circulação Placentária/genética , Proteínas Repressoras/genética , Transativadores/genética , Trofoblastos/enzimologia , Actinas/biossíntese , Animais , Proteínas de Transporte/metabolismo , Desenvolvimento Embrionário , Feminino , Deleção de Genes , Camundongos , Camundongos Endogâmicos C57BL , Transportadores de Ácidos Monocarboxílicos/biossíntese , Proteínas Musculares/biossíntese , Neovascularização Fisiológica/genética , Neovascularização Fisiológica/fisiologia , Pericitos/citologia , Pericitos/fisiologia , Molécula-1 de Adesão Celular Endotelial a Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Fator de Crescimento Derivado de Plaquetas/metabolismo , Gravidez , Proteínas Proto-Oncogênicas c-sis/biossíntese , Receptor beta de Fator de Crescimento Derivado de Plaquetas/biossíntese , Simportadores/biossínteseRESUMO
A series of redox-labeled L-tyrosinamide (L-Tym) derivatives was prepared and the nature of the functional group and the chain length of the spacer were systematically varied in a step-by-step affinity optimization process of the tracer for the L-Tym aptamer. The choice of the labeling position on L-Tym proved to be crucial for the molecular recognition event, which could be monitored by cyclic voltammetry and is based on the different diffusion rates of free and bound targets in solution. From this screening approach an efficient electroactive tracer emerged. Comparable dissociation constants Kd were obtained for the unlabeled and labeled targets in direct or competitive binding assays. The enantiomeric tracer was prepared and its enantioselective recognition by the corresponding anti-D-Tym aptamer was demonstrated. The access to both enantiomeric tracer molecules opens the door for the development of one-pot determination of the enantiomeric excess when using different labels with well-separated redox potentials for each enantiomer.
Assuntos
Aptâmeros de Nucleotídeos/química , Oligonucleotídeos/química , Tirosina/análogos & derivados , Ligação Competitiva , Difusão , Eletroquímica , Eletroforese Capilar , Oxirredução , Estereoisomerismo , Tirosina/químicaRESUMO
Purpose: Breath-hold (BH) technique can mitigate target motion, minimize target margins, reduce normal tissue doses, and lower the effect of interplay effects with intensity-modulated proton therapy (IMPT). This study presents dosimetric comparisons between BH and nonbreath-hold (non-BH) IMPT plans and investigates the reproducibility of BH plans using frequent quality assurance (QA) computed tomography scans (CT). Methods and Materials: Data from 77 consecutive patients with liver (n = 32), mediastinal/lung (n = 21), nonliver upper abdomen (n = 20), and malignancies in the gastroesophageal junction (n = 4), that were treated with a BH spirometry system (SDX) were evaluated. All patients underwent both BH CT and 4-dimensional CT simulations. Clinically acceptable BH and non-BH plans were generated on each scan, and dose-volume histograms of the 2 plans were compared. Reproducibility of the BH plans for 30 consecutive patients was assessed using 1 to 3 QA CTs per patient and variations in dose-volume histograms for deformed target and organs at risk (OARs) volumes were compared with the initial CT plan. Results: Use of BH scans reduced initial and boost target volumes to 72% ± 20% and 70% ± 17% of non-BH volumes, respectively. Additionally, mean dose to liver, stomach, kidney, esophagus, heart, and lung V20 were each reduced to 71% to 79% with the BH technique. Similarly, small and large bowels, heart, and spinal cord maximum doses were each lowered to 68% to 84%. Analysis of 62 QA CT scans demonstrated that mean target and OAR doses using BH scans were reproducible to within 5% of their nominal plan values. Conclusions: The BH technique reduces the irradiated volume, leading to clinically significant reductions in OAR doses. By mitigating tumor motion, the BH technique leads to reproducible target coverage and OAR doses. Its use can reduce motion-related uncertainties that are normally associated with the treatment of thoracic and abdominal tumors and, therefore, optimize IMPT delivery.
RESUMO
Msx1, a member of the Msx gene family, encodes a homeodomain transcription factor and plays critical roles during mouse development in numerous organs. By homologous recombination, we generated a new Msx1 allele (Msx1(CreERT2) ) in which the CreERT2 fusion protein is produced in place of the endogenous Msx1 protein. Using different reporter mouse strains and appropriate tamoxifen treatments, we show that, in mice bearing the Msx1(CrERT2) allele, CreERT2 is capable to induce loxP genomic recombination specifically in Msx1-expressing cells and that this can be obtained during embryonic development as well as after birth. These results show that this new mouse line can be used for lineage tracing of Msx1-expressing cells and their descendants and, combined with Cre-inducible Msx null alleles, for the analysis of Msx1 and/or Msx2 functions in the Msx1-expressing organs, in a time-dependant manner.
Assuntos
Alelos , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Introdução de Genes/métodos , Fator de Transcrição MSX1/genética , Tamoxifeno/farmacologia , Animais , Clonagem Molecular , Embrião de Mamíferos/efeitos dos fármacos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/imunologia , Proteínas de Fluorescência Verde/metabolismo , Heterozigoto , Fator de Transcrição MSX1/metabolismo , Camundongos , Camundongos TransgênicosRESUMO
Oxime bond formation has been applied to the preparation of oligonucleotides labeled with electrochemical ferrocene and viologen labels. Aminooxy functionalized ferrocene and viologen derivatives were prepared by a straightforward route and efficiently conjugated with aldehyde containing oligonucleotides either at 3' or 5' end. Both labels were found to not disturb the recognition properties of the oligonucleotide. The versatility of the method was further demonstrated by preparing bi-functionalized conjugates with a disulfide at 3' end and an electrochemical label at 5' end.
Assuntos
Oligonucleotídeos/química , Oximas/química , Técnicas Eletroquímicas/métodos , Compostos Ferrosos/química , Metalocenos , Oligonucleotídeos/síntese química , Oximas/síntese química , Viologênios/químicaRESUMO
The Ras/mitogen-activated protein kinase (MAPK) pathway regulates a variety of cellular processes by activating specific transcriptional and translational programs. Ras/MAPK signaling promotes mRNA translation and protein synthesis, but the exact molecular mechanisms underlying this regulation remain poorly understood. Increasing evidence suggests that the mammalian target of rapamycin (mTOR) plays an essential role in this process. Here, we show that Raptor, an essential scaffolding protein of the mTOR complex 1 (mTORC1), becomes phosphorylated on proline-directed sites following activation of the Ras/MAPK pathway. We found that ERK1 and ERK2 interact with Raptor in cells and mediate its phosphorylation in vivo and in vitro. Using mass spectrometry and phosphospecific antibodies, we found three proline-directed residues within Raptor, Ser(8), Ser(696), and Ser(863), which are directly phosphorylated by ERK1/2. Expression of phosphorylation-deficient alleles of Raptor revealed that phosphorylation of these sites by ERK1/2 normally promotes mTORC1 activity and signaling to downstream substrates, such as 4E-BP1. Our data provide a novel regulatory mechanism by which mitogenic and oncogenic activation of the Ras/MAPK pathway promotes mTOR signaling.
Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Proteínas/metabolismo , Proteínas ras/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/química , Sequência de Aminoácidos , Sítios de Ligação , Linhagem Celular , Proliferação de Células , Humanos , Sistema de Sinalização das MAP Quinases , Alvo Mecanístico do Complexo 1 de Rapamicina , Dados de Sequência Molecular , Complexos Multiproteicos , Fosforilação , Prolina/metabolismo , Proteína Regulatória Associada a mTOR , Serina-Treonina Quinases TORRESUMO
A new electrochemical methodology is reported for monitoring in homogeneous solution the enantiospecific binding of a small chiral analyte to an aptamer. The principle relies on the difference of diffusion rates between the targeted molecule and the aptamer/target complex, and thus on the ability to more easily electrochemically detect the former over the latter in a homogeneous solution. This electrochemical detection strategy is significant because, in contrast to the common laborious and time-consuming heterogeneous binding approaches, it is based on a simple and fast homogeneous binding assay which does not call for an aptamer conformational change upon ligand binding. The methodology is here exemplified with the specific chiral recognition of trace amounts of l- or d-tyrosinamide by a 49-mer d- or l-deoxyribooligonucleotide receptor. Detection as low as 0.1% of the minor enantiomer in a nonracemic mixture can be achieved in a very short analysis time (<1 min). The assay finally combines numerous attractive features including simplicity, rapidity, low cost, flexibility, low volume samples (few microliters), and homogeneous format.
Assuntos
Aptâmeros de Nucleotídeos/metabolismo , Eletroquímica/métodos , Tirosina/análogos & derivados , Aptâmeros de Nucleotídeos/química , Aptâmeros de Nucleotídeos/genética , Sequência de Bases , Modelos Moleculares , Conformação de Ácido Nucleico , Estereoisomerismo , Especificidade por Substrato , Fatores de Tempo , Tirosina/química , Tirosina/metabolismoRESUMO
Background: Diabetic kidney disease (DKD) remains the leading cause of end stage kidney disease worldwide. Despite significant advances in kidney care, there is a need to improve noninvasive techniques to predict the progression of kidney disease better for patients with diabetes. After injury, podocytes are shed in urine and may be used as a biologic tool. We previously reported that SHP-1 is upregulated in the kidney of diabetic mice, leading to podocyte dysfunction and loss. Our objective was to evaluate the expression levels of SHP-1 in urinary podocytes and kidney tissues of patients with diabetes. Methods: In this prospective study, patients with and without diabetes were recruited for the quantification of SHP-1 in kidney tissues, urinary podocytes, and peripheral blood monocytes. Immunochemistry and mass spectrometry techniques were applied for kidney tissues. Urinary podocytes were counted, and expression of SHP-1 and podocyte markers were measured by quantitative PCR. Results: A total of 66 participants (diabetic n=48, nondiabetic n=18) were included in the analyses. Diabetes was associated with increased SHP-1 expression in kidney tissues (P=0.03). Nephrin and podocin mRNA was not significantly increased in urinary podocytes from patients with diabetes compared with those without diabetes, whereas levels of SHP-1 mRNA expression significantly correlated with HbA1c and estimated glomerular filtration rate (eGFR). Additionally, follow-up (up to 2 years post recruitment) evaluation indicated that SHP-1 mRNA expression continued to increase with eGFR decline. Conclusions: Levels of SHP-1 in urinary podocytes may serve as an additional marker of glomerular disease progression in this population.
Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Podócitos , Diabetes Mellitus Experimental/complicações , Nefropatias Diabéticas/etiologia , Rim/metabolismo , Podócitos/metabolismo , Estudos Prospectivos , HumanosRESUMO
The stereoselective convergent total syntheses of both enantiomers of the tetrahydroisoquinoline (THIQ) alkaloid crispine A are described. The THIQ precursors (-)-6 (90:10 dr) and (-)-11 (85:15 dr) were prepared from the alkylation-reduction sequence of a common α-amino nitrile (+)-4 derivative that has been conveniently prepared by anodic cyanation. Elaboration of the pyrrolidine ring of the title compound was cleanly achieved by two efficient ring closures methods involving (a) the displacement of a halogen atom and (b) the formation of a cyclic iminium cation to afford (-)-crispine A in 90% and 85% yields, respectively. A crystallization of enantioenriched (-)-crispine A (90:10 er) with 1 equiv of (-)-DBTA afforded the tartrate salt (-)-14 (≥98:2 dr) in 81% yield. The absolute S configuration of (-)-crispine A was simply deduced from examination of the X-ray data of tartrate salt (-)-14. Likewise, the natural (+)-crispine A was prepared in seven workup steps in an overall 30% yield, and reciprocal crystallization with (+)-DBTA afforded the enantiomeric tartrate salt (+)-14 in a ≥98:2 dr. Both enantiomers of crispine A were liberated from their respective DBTA salts in ≥98:2 er's which were determined by proton and carbon NMR spectroscopy, utilizing (R)-(+)-tert-butylphenylphosphinothioic acid (+)-15 as chiral solvating agent.
Assuntos
Isoquinolinas/química , Isoquinolinas/síntese química , Eletroquímica , Estrutura Molecular , EstereoisomerismoRESUMO
BACKGROUND: Measurement of proteinuria in women with hypertensive disorders of pregnancy is of major importance in the diagnosis and management of preeclampsia. Urinary protein/creatinine ratio, which is commonly used to detect kidney damage in preeclampsia, suffers from important analytical limitations, including poor harmonization of results between laboratories. Adoption of albuminuria could help reduce interlaboratory bias, since methods used to quantify it are better harmonized. METHODS: A total of 27 urinary samples collected from hypertensive women evaluated for preeclampsia were sent to four different clinical laboratories in Canada. Urinary proteins and albumin as well as urinary creatinine were measured in duplicates in one batch to calculate protein/creatinine (PCR) and albumin/creatinine (ACR) ratio. Statistical analyses were done to evaluate interlaboratory variability of urinary proteins and urinary albumin. RESULTS: Interlaboratory bias for urinary proteins ranged from 64.7% at low concentration to 3.9% at higher concentrations. In contrast, urinary albumin interlaboratory bias ranged from 29.2% to 4% from low to high concentrations. Coefficient of variation for urinary proteins reached a maximum of 91.5% in lower concentration while urinary albumin highest value was 42.7%. When looking at PCR and ACR ratio, eight samples had PCR measurement range that contained the diagnostic threshold used to detect kidney damage in HDP (30 mg/mmol), while only four samples had ACR ratio measurement range that contained the diagnostic threshold used outside of pregnancy in Canada (2 mg/mmol). CONCLUSION: Interlaboratory bias was lower for urinary albumin measurement compared to urinary proteins in hypertensive women evaluated for preeclampsia. Better harmonization with the use of albumin instead of protein measurement would reduce instances where results of different laboratories lead to opposite diagnosis of kidney damage in pregnancy.