RESUMO
Measurements of ammonia with inexpensive and reliable sensors are necessary to obtain information about e.g., ammonia emissions. The concentration information is needed for mitigation technologies and documentation of existing technologies in agriculture. A flow-based fluorescence sensor to measure ammonia gas was developed. The automated sensor is robust, flexible and made from inexpensive components. Ammonia is transferred to water in a miniaturized scrubber with high transfer efficiency (>99%) and reacts with o-phthalaldehyde and sulfite (pH 11) to form a fluorescent adduct, which is detected with a photodiode. Laboratory calibrations with standard gas show good linearity over a dynamic range from 0.03 to 14 ppm, and the detection limit of the analyzer based on three-times the standard deviation of blank noise was approximately 10 ppb. The sampling frequency is 0.1 to 10 s, which can easily be changed through serial commands along with UV LED current and filter length. Parallel measurements with a cavity ring-down spectroscopy analyzer in a pig house show good agreement (R2 = 0.99). The fluorescence sensor has the potential to provide ammonia gas measurements in an agricultural environment with high time resolution and linearity over a broad range of concentrations.
Assuntos
Amônia , Gado , Agricultura , Animais , Análise Espectral , SuínosRESUMO
Nanozymes, nanoparticles that mimic the natural activity of enzymes, are intriguing academically and are important in the context of the Origin of Life. However, current nanozymes offer mimicry of a narrow range of mammalian enzymes, near-exclusively performing redox reactions. We present an unexpected discovery of non-proteinaceous enzymes based on metals, metal oxides, 1D/2D-materials, and non-metallic nanomaterials. The specific novelty of these findings lies in the identification of nanozymes with apparent mimicry of diverse mammalian enzymes, including unique pan-glycosidases. Further novelty lies in the identification of the substrate scope for the lead candidates, specifically in the context of bioconversion of glucuronides, that is, human metabolites and privileged prodrugs in the field of enzyme-prodrug therapies. Lastly, nanozymes are employed for conversion of glucuronide prodrugs into marketed anti-inflammatory and antibacterial agents, as well as "nanozyme prodrug therapy" to mediate antibacterial measures.
Assuntos
Nanoestruturas/química , Pró-Fármacos/química , Catálise , HumanosRESUMO
The innate immune response is essential for combating infectious disease. Macrophages and other cells respond to infection by releasing cytokines, such as interleukin-1ß (IL-1ß), which in turn activate a well-described, myeloid-differentiation factor 88 (MYD88)-mediated, nuclear factor-κB (NF-κB)-dependent transcriptional pathway that results in inflammatory-cell activation and recruitment. Endothelial cells, which usually serve as a barrier to the movement of inflammatory cells out of the blood and into tissue, are also critical mediators of the inflammatory response. Paradoxically, the cytokines vital to a successful immune defence also have disruptive effects on endothelial cell-cell interactions and can trigger degradation of barrier function and dissociation of tissue architecture. The mechanism of this barrier dissolution and its relationship to the canonical NF-κB pathway remain poorly defined. Here we show that the direct, immediate and disruptive effects of IL-1ß on endothelial stability in a human in vitro cell model are NF-κB independent and are instead the result of signalling through the small GTPase ADP-ribosylation factor 6 (ARF6) and its activator ARF nucleotide binding site opener (ARNO; also known as CYTH2). Moreover, we show that ARNO binds directly to the adaptor protein MYD88, and thus propose MYD88-ARNO-ARF6 as a proximal IL-1ß signalling pathway distinct from that mediated by NF-κB. Finally, we show that SecinH3, an inhibitor of ARF guanine nucleotide-exchange factors such as ARNO, enhances vascular stability and significantly improves outcomes in animal models of inflammatory arthritis and acute inflammation.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Proteínas Ativadoras de GTPase/metabolismo , Fator 88 de Diferenciação Mieloide/metabolismo , Receptores de Interleucina/metabolismo , Fator 6 de Ribosilação do ADP , Adjuvantes Imunológicos/farmacologia , Animais , Artrite/patologia , Caderinas/metabolismo , Permeabilidade Capilar/efeitos dos fármacos , Linhagem Celular , Células Endoteliais/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Humanos , Interleucina-1beta/farmacologia , NF-kappa B/metabolismo , Inibidores de Proteínas Quinases/farmacologia , Transporte Proteico/efeitos dos fármacos , Purinas/farmacologia , Transdução de Sinais , Tiofenos/farmacologiaRESUMO
The vascular endothelium responds to infection by destabilizing endothelial cell-cell junctions to allow fluid and cells to pass into peripheral tissues, facilitating clearance of infection and tissue repair. During sepsis, endotoxin and other proinflammatory molecules induce excessive vascular leak, which can cause organ dysfunction, shock, and death. Current therapies for sepsis are limited to antibiotics and supportive care, which are often insufficient to reduce morbidity and prevent mortality. Previous attempts at blocking inflammatory cytokine responses in humans proved ineffective at reducing the pathologies associated with sepsis, highlighting the need for a new therapeutic strategy. The small GTPase ARF6 is activated by a MyD88-ARNO interaction to induce vascular leak through disruption of endothelial adherens junctions. In this study, we show that the MyD88-ARNO-ARF6-signaling axis is responsible for LPS-induced endothelial permeability and is a destabilizing convergence point used by multiple inflammatory cues. We also show that blocking ARF6 with a peptide construct of its N terminus is sufficient to reduce vascular leak and enhance survival during endotoxic shock, without inhibiting the host cytokine response. Our data highlight the therapeutic potential of blocking ARF6 and reducing vascular leak for the treatment of inflammatory conditions, such as endotoxemia.
Assuntos
Fatores de Ribosilação do ADP/imunologia , Junções Aderentes/imunologia , Permeabilidade Capilar/imunologia , Células Endoteliais/imunologia , Choque Séptico/imunologia , Transdução de Sinais/imunologia , Fator 6 de Ribosilação do ADP , Junções Aderentes/patologia , Animais , Permeabilidade Capilar/efeitos dos fármacos , Células Cultivadas , Células Endoteliais/patologia , Feminino , Proteínas Ativadoras de GTPase/imunologia , Humanos , Lipopolissacarídeos/toxicidade , Masculino , Camundongos , Fator 88 de Diferenciação Mieloide/imunologia , Choque Séptico/induzido quimicamente , Choque Séptico/patologia , Transdução de Sinais/efeitos dos fármacosRESUMO
This is a retrospective study to investigate whether motivational interviewing increases weight loss among obese or overweight women prior to fertility treatment. Women with body mass index (BMI) > 30 kg/m(2) approaching the Fertility Clinic, Regional Hospital Skive, were given advice about diet and physical activity with the purpose of weight loss. In addition, they were asked if they wanted to receive motivational interviewing. Among other data, age, height and weight were obtained. Main outcomes were weight loss measured in kg and decrease in BMI. We studied 187 women: 110 received sessions of motivational interviewing (intervention group, n = 110), 64 received motivational support by phone or e-mail only and 13 women did not wish any motivational support (control group, n = 77). The mean weight loss and decrease in BMI was greater in the intervention group compared with the control group (9.3 kg versus 7.3 kg, difference p = 0.01, 3.3 kg/m(2) versus 2.6 kg/m(2), difference p = 0.02). The mean period of intervention was comparable in the two groups, 7.9 month and 7.3 month, respectively, (difference non significant: NS). The study indicates that motivational interviewing may be a valuable tool in weight loss programs for obese and overweight women prior to fertility treatment.
Assuntos
Infertilidade/terapia , Entrevista Motivacional/métodos , Obesidade/terapia , Sobrepeso/terapia , Complicações na Gravidez/prevenção & controle , Cuidado Pré-Natal/métodos , Técnicas de Reprodução Assistida , Adulto , Estudos de Casos e Controles , Feminino , Humanos , Infertilidade/complicações , Obesidade/complicações , Sobrepeso/complicações , Gravidez , Estudos Retrospectivos , Programas de Redução de PesoRESUMO
Formation of the vascular system within organs requires the balanced action of numerous positive and negative factors secreted by stromal and epithelial cells. Here, we used a genetic approach to determine the role of SLITs in regulating the growth and organization of blood vessels in the mammary gland. We demonstrate that vascularization of the gland is not affected by loss of Slit expression in the epithelial compartment. Instead, we identify a stromal source of SLIT, mural cells encircling blood vessels, and show that loss of Slit in the stroma leads to elevated blood vessel density and complexity. We examine candidate SLIT receptors, Robo1 and Robo4, and find that increased vessel angiogenesis is phenocopied by loss of endothelial-specific Robo4, as long as it is combined with the presence of an angiogenic stimulus such as preneoplasia or pregnancy. In contrast, loss of Robo1 does not affect blood vessel growth. The enhanced growth of blood vessels in Robo4(-/-) endothelium is due to activation of vascular endothelial growth factor (VEGF)-R2 signaling through the Src and FAK kinases. Thus, our studies present a genetic dissection of SLIT/ROBO signaling during organ development. We identify a stromal, rather than epithelial, source of SLITs that inhibits blood vessel growth by signaling through endothelial ROBO4 to down-regulate VEGF/VEGFR2 signaling.
Assuntos
Glândulas Mamárias Animais/irrigação sanguínea , Glândulas Mamárias Animais/metabolismo , Neovascularização Fisiológica , Proteínas do Tecido Nervoso/metabolismo , Receptores Imunológicos/metabolismo , Transdução de Sinais , Fator A de Crescimento do Endotélio Vascular/metabolismo , Animais , Peptídeos e Proteínas de Sinalização Intercelular/deficiência , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Proteínas de Membrana/deficiência , Proteínas de Membrana/metabolismo , Camundongos , Camundongos Knockout , Proteínas do Tecido Nervoso/deficiência , Receptores de Superfície Celular , Receptores Imunológicos/deficiência , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Proteínas RoundaboutRESUMO
Preventing or effectively treating metastatic uveal melanoma (UM) is critical because it occurs in about half of patients and confers a very poor prognosis. There is emerging evidence that hepatocyte growth factor (HGF) and insulin-like growth factor 1 (IGF-1) promote metastasis and contribute to the striking metastatic hepatotropism observed in UM metastasis. However, the molecular mechanisms by which HGF and IGF-1 promote UM liver metastasis have not been elucidated. ASAP1, which acts as an effector for the small GTPase ARF6, is highly expressed in the subset of uveal melanomas most likely to metastasize. Here, we found that HGF and IGF-1 hyperactivate ARF6, leading to its interaction with ASAP1, which then acts as an effector to induce nuclear localization and transcriptional activity of NFAT1. Inhibition of any component of this pathway impairs cellular invasiveness. Additionally, knocking down ASAP1 or inhibiting NFAT signaling reduces metastasis in a xenograft mouse model of UM. The discovery of this signaling pathway represents not only an advancement in our understanding of the biology of uveal melanoma metastasis but also identifies a novel pathway that could be targeted to treat or prevent metastatic uveal melanoma.
Assuntos
Melanoma , Neoplasias Uveais , Humanos , Animais , Camundongos , Fator de Crescimento de Hepatócito/genética , Fator de Crescimento de Hepatócito/metabolismo , Fator de Crescimento Insulin-Like I/genética , Fator de Crescimento Insulin-Like I/metabolismo , Melanoma/patologia , Neoplasias Uveais/metabolismo , Modelos Animais de Doenças , Proteínas Adaptadoras de Transdução de Sinal/metabolismoRESUMO
Breakdown of the blood-central nervous system barrier (BCNSB) is a hallmark of many neuroinflammatory disorders, such as multiple sclerosis (MS). Using a mouse model of MS, experimental autoimmune encephalomyelitis (EAE), we show that endothelial-to-mesenchymal transition (EndoMT) occurs in the CNS before the onset of clinical symptoms and plays a major role in the breakdown of BCNSB function. EndoMT can be induced by an IL-1ß-stimulated signaling pathway in which activation of the small GTPase ADP ribosylation factor 6 (ARF6) leads to crosstalk with the activin receptor-like kinase (ALK)-SMAD1/5 pathway. Inhibiting the activation of ARF6 both prevents and reverses EndoMT, stabilizes BCNSB function, reduces demyelination, and attenuates symptoms even after the establishment of severe EAE, without immunocompromising the host. Pan-inhibition of ALKs also reduces disease severity in the EAE model. Therefore, multiple components of the IL-1ß-ARF6-ALK-SMAD1/5 pathway could be targeted for the treatment of a variety of neuroinflammatory disorders.
Assuntos
Encefalomielite Autoimune Experimental , Proteínas Monoméricas de Ligação ao GTP , Esclerose Múltipla , Receptores de Ativinas/metabolismo , Animais , Sistema Nervoso Central/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Monoméricas de Ligação ao GTP/metabolismo , Doenças Neuroinflamatórias , Receptores Proteína Tirosina Quinases/metabolismo , Transdução de SinaisRESUMO
In eutherian mammals, embryonic growth and survival is dependent on the formation of the placenta, an organ that facilitates the efficient exchange of oxygen, nutrients, and metabolic waste between the maternal and fetal blood supplies. Key to the placenta's function is the formation of its vascular labyrinth, a series of finely branched vessels whose molecular ontogeny remains largely undefined. In this report, we demonstrate that HOXA13 plays an essential role in labyrinth vessel formation. In the absence of HOXA13 function, placental endothelial cell morphology is altered, causing a loss in vessel wall integrity, edema of the embryonic blood vessels, and mid-gestational lethality. Microarray analysis of wild-type and mutant placentas revealed significant changes in endothelial gene expression profiles. Notably, pro-vascular genes, including Tie2 and Foxf1, exhibited reduced expression in the mutant endothelia, which also exhibited elevated expression of genes normally expressed in lymphatic or sinusoidal endothelia. ChIP analysis of HOXA13-DNA complexes in the placenta confirmed that HOXA13 binds the Tie2 and Foxf1 promoters in vivo. In vitro, HOXA13 binds sequences present in the Tie2 and Foxf1 promoters with high affinity (K(d) = 27-42 nM) and HOXA13 can use these bound promoter regions to direct gene expression. Taken together, these findings demonstrate that HOXA13 directly regulates Tie2 and Foxf1 in the placental labyrinth endothelia, providing a functional explanation for the mid-gestational lethality exhibited by Hoxa13 mutant embryos as well as a novel transcriptional program necessary for the specification of the labyrinth vascular endothelia.
Assuntos
Endotélio Vascular/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/metabolismo , Placenta/irrigação sanguínea , Animais , Sequência de Bases , Endotélio Vascular/embriologia , Endotélio Vascular/crescimento & desenvolvimento , Fatores de Transcrição Forkhead/genética , Genes Reporter , Proteínas de Homeodomínio/genética , Homozigoto , Técnicas In Vitro , Camundongos , Camundongos Knockout , Dados de Sequência Molecular , Neovascularização Fisiológica , Análise de Sequência com Séries de Oligonucleotídeos , Placenta/embriologia , Placenta/metabolismo , Regiões Promotoras Genéticas , Receptor TIE-2/genética , Trofoblastos/fisiologiaRESUMO
INTRODUCTION: Modifiable lifestyle behaviors represent a central target for public health interventions. This study investigates the association between adherence to 4 modifiable lifestyle recommendations and all-cause, cancer, or cardiovascular disease mortality. METHODS: Investigators used data from the Danish Diet, Cancer and Health cohort (1993-2013; N=54,276). Lifestyle recommendations included smoking (never smoking), diet (adherence to 6 national food-based dietary guidelines), alcohol consumption (≤7 units per week for women and ≤14 units per week for men), and physical activity (≥30 minutes per day of moderate-to-vigorous leisure-time physical activity). Pseudo-values were used to estimate the adjusted risk differences and 95% CIs for all-cause, cancer, or cardiovascular disease mortality. Data were analyzed in 2019-2020. RESULTS: A total of 8,860 participants died during a median follow-up of 17.0 years. Adherence to all modifiable lifestyle recommendations was associated with an 18.46% (95% CI= -20.52%, -16.41%) lower absolute risk of all-cause mortality than no adherence. Never smokers had a 13.19% (95% CI= -13.95%, -12.44%) lower risk, those adhering to dietary guidelines (diet score ≥5) had a 7.52% (95% CI= -8.89%, -6.14%) lower risk, and those adhering to recommended levels of alcohol (2.11%, 95% CI= -2.75%, -1.48%) and physical activity (1.58%, 95% CI= -2.20%, -1.00%) had a lower risk than those who did not adhere. Stronger associations were observed in men than in women and in older than in middle-aged participants. CONCLUSIONS: Findings suggest that adherence to modifiable lifestyle recommendations is associated with a lower risk of mortality from all causes, cancer, and cardiovascular disease, underlining the importance of supporting adherence to national guidelines for lifestyle recommendations.
Assuntos
Doenças Cardiovasculares , Estilo de Vida , Idoso , Doenças Cardiovasculares/prevenção & controle , Estudos de Coortes , Dinamarca/epidemiologia , Exercício Físico , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Mortalidade , Fatores de RiscoRESUMO
Melanoma has an unusual capacity to spread in early-stage disease, prompting aggressive clinical intervention in very thin primary tumors. Despite these proactive efforts, patients with low-risk, low-stage disease can still develop metastasis, indicating the presence of permissive cues for distant spread. Here, we show that constitutive activation of the small GTPase ARF6 (ARF6Q67L) is sufficient to accelerate metastasis in mice with BRAFV600E/Cdkn2aNULL melanoma at a similar incidence and severity to Pten loss, a major driver of PI3K activation and melanoma metastasis. ARF6Q67L promoted spontaneous metastasis from significantly smaller primary tumors than PTENNULL, implying an enhanced ability of ARF6-GTP to drive distant spread. ARF6 activation increased lung colonization from circulating melanoma cells, suggesting that the prometastatic function of ARF6 extends to late steps in metastasis. Unexpectedly, ARF6Q67L tumors showed upregulation of Pik3r1 expression, which encodes the p85 regulatory subunit of PI3K. Tumor cells expressing ARF6Q67L displayed increased PI3K protein levels and activity, enhanced PI3K distribution to cellular protrusions, and increased AKT activation in invadopodia. ARF6 is necessary and sufficient for activation of both PI3K and AKT, and PI3K and AKT are necessary for ARF6-mediated invasion. We provide evidence for aberrant ARF6 activation in human melanoma samples, which is associated with reduced survival. Our work reveals a previously unknown ARF6-PI3K-AKT proinvasive pathway, it demonstrates a critical role for ARF6 in multiple steps of the metastatic cascade, and it illuminates how melanoma cells can acquire an early metastatic phenotype in patients. SIGNIFICANCE: These findings reveal a prometastatic role for ARF6 independent of tumor growth, which may help explain how melanoma spreads distantly from thin, early-stage primary tumors.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/79/11/2892/F1.large.jpg.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Melanoma/patologia , Fosfatidilinositol 3-Quinases/metabolismo , Neoplasias Cutâneas/patologia , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Animais , Inibidor p16 de Quinase Dependente de Ciclina/genética , Guanosina Trifosfato/metabolismo , Humanos , Neoplasias Pulmonares/secundário , Melanoma/metabolismo , Melanoma Experimental/metabolismo , Melanoma Experimental/patologia , Camundongos Mutantes , Camundongos SCID , Metástase Neoplásica , PTEN Fosfo-Hidrolase/genética , Proteínas Proto-Oncogênicas B-raf/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Neoplasias Cutâneas/metabolismoRESUMO
Macromolecular (pro)drugs hold much promise as broad-spectrum antiviral agents as either microbicides or carriers for intracellular delivery of antiviral drugs. Intriguing opportunity exists in combining the two modes of antiviral activity in the same polymer structure such that the same polymer acts as a microbicide and also serves to deliver the conjugated drug (ribavirin) into the cells. We explore this opportunity in detail and focus on the polymer backbone as a decisive constituent of such formulations. Fourteen polyanions (polycarboxylates, polyphosphates and polyphosphonates, and polysulfonates) were analyzed for blood pro/anti coagulation effects, albumin binding and albumin aggregation, inhibitory activity on polymerases, cytotoxicity, and anti-inflammatory activity in stimulated macrophages. Ribavirin containing monomers were designed to accommodate the synthesis of macromolecular prodrugs with disulfide-exchange triggered drug release. Kinetics of drug release was fast in all cases however enhanced hydrophobicity of the polymer significantly slowed release of ribavirin. Results of this study present a comprehensive view on polyanions as backbone for macromolecular prodrugs of ribavirin as broad-spectrum antiviral agents.
Assuntos
Anti-Inflamatórios/administração & dosagem , Antivirais/administração & dosagem , Polímeros/administração & dosagem , Pró-Fármacos/administração & dosagem , Ribavirina/administração & dosagem , Animais , Anti-Inflamatórios/química , Antivirais/química , Coagulação Sanguínea/efeitos dos fármacos , DNA Polimerase Dirigida por DNA/genética , Liberação Controlada de Fármacos , Humanos , Camundongos , Polímeros/química , Pró-Fármacos/química , Células RAW 264.7 , Ribavirina/química , Resultado do TratamentoRESUMO
Most differentiated cells convert glucose to pyruvate in the cytosol through glycolysis, followed by pyruvate oxidation in the mitochondria. These processes are linked by the mitochondrial pyruvate carrier (MPC), which is required for efficient mitochondrial pyruvate uptake. In contrast, proliferative cells, including many cancer and stem cells, perform glycolysis robustly but limit fractional mitochondrial pyruvate oxidation. We sought to understand the role this transition from glycolysis to pyruvate oxidation plays in stem cell maintenance and differentiation. Loss of the MPC in Lgr5-EGFP-positive stem cells, or treatment of intestinal organoids with an MPC inhibitor, increases proliferation and expands the stem cell compartment. Similarly, genetic deletion of the MPC in Drosophila intestinal stem cells also increases proliferation, whereas MPC overexpression suppresses stem cell proliferation. These data demonstrate that limiting mitochondrial pyruvate metabolism is necessary and sufficient to maintain the proliferation of intestinal stem cells.
Assuntos
Proliferação de Células , Drosophila melanogaster/metabolismo , Glicólise , Mucosa Intestinal/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Células-Tronco/metabolismo , Acrilatos/farmacologia , Animais , Proteínas de Transporte de Ânions/antagonistas & inibidores , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Diferenciação Celular , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Genótipo , Humanos , Intestinos/citologia , Intestinos/efeitos dos fármacos , Ácido Láctico/metabolismo , Camundongos Knockout , Mitocôndrias/efeitos dos fármacos , Proteínas de Transporte da Membrana Mitocondrial/antagonistas & inibidores , Proteínas de Transporte da Membrana Mitocondrial/genética , Proteínas de Transporte da Membrana Mitocondrial/metabolismo , Proteínas Mitocondriais/metabolismo , Transportadores de Ácidos Monocarboxílicos , Fenótipo , Interferência de RNA , Receptores Acoplados a Proteínas G/genética , Receptores Acoplados a Proteínas G/metabolismo , Transdução de Sinais , Células-Tronco/efeitos dos fármacos , Fatores de Tempo , Técnicas de Cultura de Tecidos , TransfecçãoRESUMO
The devastating sequelae of diabetes mellitus include microvascular permeability, which results in retinopathy. Despite clinical and scientific advances, there remains a need for new approaches to treat retinopathy. Here, we have presented a possible treatment strategy, whereby targeting the small GTPase ARF6 alters VEGFR2 trafficking and reverses signs of pathology in 4 animal models that represent features of diabetic retinopathy and in a fifth model of ocular pathological angiogenesis. Specifically, we determined that the same signaling pathway utilizes distinct GEFs to sequentially activate ARF6, and these GEFs exert distinct but complementary effects on VEGFR2 trafficking and signal transduction. ARF6 activation was independently regulated by 2 different ARF GEFs - ARNO and GEP100. Interaction between VEGFR2 and ARNO activated ARF6 and stimulated VEGFR2 internalization, whereas a VEGFR2 interaction with GEP100 activated ARF6 to promote VEGFR2 recycling via coreceptor binding. Intervening in either pathway inhibited VEGFR2 signal output. Finally, using a combination of in vitro, cellular, genetic, and pharmacologic techniques, we demonstrated that ARF6 is pivotal in VEGFR2 trafficking and that targeting ARF6-mediated VEGFR2 trafficking has potential as a therapeutic approach for retinal vascular diseases such as diabetic retinopathy.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Retinopatia Diabética/metabolismo , Transdução de Sinais , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/genética , Linhagem Celular , Retinopatia Diabética/genética , Retinopatia Diabética/patologia , Proteínas Ativadoras de GTPase/genética , Proteínas Ativadoras de GTPase/metabolismo , Humanos , Transporte Proteico , Receptor 2 de Fatores de Crescimento do Endotélio Vascular/genéticaRESUMO
Activating mutations in Gαq proteins, which form the α subunit of certain heterotrimeric G proteins, drive uveal melanoma oncogenesis by triggering multiple downstream signaling pathways, including PLC/PKC, Rho/Rac, and YAP. Here we show that the small GTPase ARF6 acts as a proximal node of oncogenic Gαq signaling to induce all of these downstream pathways as well as ß-catenin signaling. ARF6 activates these diverse pathways through a common mechanism: the trafficking of GNAQ and ß-catenin from the plasma membrane to cytoplasmic vesicles and the nucleus, respectively. Blocking ARF6 with a small-molecule inhibitor reduces uveal melanoma cell proliferation and tumorigenesis in a mouse model, confirming the functional relevance of this pathway and suggesting a therapeutic strategy for Gα-mediated diseases.
Assuntos
Fatores de Ribosilação do ADP/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/genética , Melanoma/tratamento farmacológico , Bibliotecas de Moléculas Pequenas/administração & dosagem , Neoplasias Uveais/tratamento farmacológico , beta Catenina/metabolismo , Fator 6 de Ribosilação do ADP , Fatores de Ribosilação do ADP/antagonistas & inibidores , Fatores de Ribosilação do ADP/genética , Animais , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Citoplasma/metabolismo , Subunidades alfa Gq-G11 de Proteínas de Ligação ao GTP/metabolismo , Humanos , Melanoma/genética , Melanoma/metabolismo , Camundongos , Transplante de Neoplasias , Transporte Proteico/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Bibliotecas de Moléculas Pequenas/farmacologia , Neoplasias Uveais/genética , Neoplasias Uveais/metabolismoRESUMO
A hallmark of vascular smooth muscle cells (VSMCs) is their dynamic ability to assemble and disassemble contractile proteins into sarcomeric units depending upon their phenotypic state. This phenotypic plasticity plays an important role during vascular development and in obstructive vascular disease. Previously, we showed that the Elastin gene product, tropoelastin, activates myofibrillar organization of VSMCs. Recently, others have suggested that elastin does not have a direct signaling role but rather binds to and alters the interactions of other matrix proteins with their cognate receptors or disrupts the binding of growth factors and cytokines. In contrast, we provide evidence that tropoelastin directly regulates contractile organization of VSMCs. First, we show that a discrete domain within tropoelastin, VGVAPG, induces myofibrillogenesis in a time- and dose-dependent fashion. We confirm specificity using a closely related control peptide that fails to stimulate actin stress fiber formation. Second, the activity of VGVAPG is not affected by the presence or absence of other serum or matrix components. Third, both the elastin hexapeptide and tropoelastin stimulate actin polymerization through a common pertussis toxin-sensitive G protein pathway that activates RhoA-GTPase and results in the conversion of G to F actin. Collectively, these data support a model whereby the elastin gene product, signaling through the VGVAPG domain, directly induces VSMC myofibrillogenesis.
Assuntos
Elastina/química , Elastina/metabolismo , Músculo Liso Vascular/citologia , Miócitos de Músculo Liso/metabolismo , Tropoelastina/química , Actinas/química , Animais , Western Blotting , Linhagem Celular , Movimento Celular , Quimiotaxia , AMP Cíclico/metabolismo , Citoplasma/metabolismo , Densitometria , Relação Dose-Resposta a Droga , Técnica Indireta de Fluorescência para Anticorpo , Peptídeos e Proteínas de Sinalização Intracelular , Camundongos , Camundongos Transgênicos , Microscopia Eletrônica , Microscopia de Fluorescência , Peptídeos/química , Toxina Pertussis/farmacologia , Fenótipo , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Terciária de Proteína , Fatores de Tempo , Vinculina/química , Quinases Associadas a rhoRESUMO
A generic, highly selective, and robust capillary electrophoresis (CE) method was developed for separation of a racemic mixture of three available glitazone compounds (also known as thiazolidinediones) in active pharmaceutical ingredients (API) and tablets. The method separated the R and S enantiomers of balaglitazone, pioglitazone and rosiglitazone, and showed that the samples contained an equal (50:50) quantity of the enantiomers as a mixture. After a simple extraction of samples with acetonitrile:water (80:20), separation was performed using a combination of two cyclodextrins: sulfobuthylether-beta-cyclodextrin (SB-beta-CD) and dimethyl-beta-cyclodextrin (DM-beta-CD) in the electrolyte at pH 8.0. The method showed a very good specificity, and all separations were achieved with a resolution (Rs) over 3.0. The developed CE method was then validated. The Rs for the separations were 3.5 for balaglitazone enantiomers, 3.5 for pioglitazone enantiomers, and 3.7 for rosiglitazone. The squared correlation coefficients (r2) were found to be 0.999 for all three compounds. The range of the CE method (injection volume was approximately 4 nl) was demonstrated to be from 1.0 to 2.4 ng. The R.S.D. in the repeatability study was found to be less than 0.5 for area/area ratio (and 3.0% for area) for all three compounds. The R.S.D. in the intermediate precision study was found to be less than 0.7 for area/area ratio (and 4.5% for area) for all three compounds. Generally, the method showed good robustness. Resolution between the enantiomers peak was maintained acceptable throughout the small variations around the pH value of the buffer, different capillary, CE instrument and electrolytes ion strength capacity, but changes in concentration of cyclodextrins and acetonitrile showed significant effects on separations and affected the resolution. The validation results showed that the CE method was suitable for separation of the racemic mixtures of the three glitazone drugs. The CE method was then applied for routine test during the drug and formulation development work of balaglitazone. Due to the achieved results from this work, it is the authors' belief that this method can easily separate other glitazone racemic mixtures.
Assuntos
Hipoglicemiantes/isolamento & purificação , Quinazolinas/química , Tiazolidinedionas/química , Tiazolidinedionas/isolamento & purificação , Ciclodextrinas/química , Eletrólitos/química , Eletroforese Capilar , Hipoglicemiantes/química , Indicadores e Reagentes , Pioglitazona , Reprodutibilidade dos Testes , Rosiglitazona , Soluções , Estereoisomerismo , ComprimidosRESUMO
Many highly productive marine ecosystems exhibit nitrogen limitation or co-limitation. This article is a status review of research into the exchange of nitrogen between the atmosphere and these ecosystems with a particular focus on reactive nitrogen compounds. A summary of research conducted over the past ten years is presented and a perspective given as to remaining uncertainities and research needs. Looking toward development of coastal management modeling tools, we illustrate the processes that need to be resolved in order to accurately simulate the flux from the atmosphere and provide guidance on the required resolution of such models.