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1.
Sensors (Basel) ; 23(22)2023 Nov 16.
Artigo em Inglês | MEDLINE | ID: mdl-38005599

RESUMO

Recently, security monitoring facilities have mainly adopted artificial intelligence (AI) technology to provide both increased security and improved performance. However, there are technical challenges in the pursuit of elevating system performance, automation, and security efficiency. In this paper, we proposed intelligent anomaly detection and classification based on deep learning (DL) using multi-modal fusion. To verify the method, we combined two DL-based schemes, such as (i) the 3D Convolutional AutoEncoder (3D-AE) for anomaly detection and (ii) the SlowFast neural network for anomaly classification. The 3D-AE can detect occurrence points of abnormal events and generate regions of interest (ROI) by the points. The SlowFast model can classify abnormal events using the ROI. These multi-modal approaches can complement weaknesses and leverage strengths in the existing security system. To enhance anomaly learning effectiveness, we also attempted to create a new dataset using the virtual environment in Grand Theft Auto 5 (GTA5). The dataset consists of 400 abnormal-state data and 78 normal-state data with clip sizes in the 8-20 s range. Virtual data collection can also supplement the original dataset, as replicating abnormal states in the real world is challenging. Consequently, the proposed method can achieve a classification accuracy of 85%, which is higher compared to the 77.5% accuracy achieved when only employing the single classification model. Furthermore, we validated the trained model with the GTA dataset by using a real-world assault class dataset, consisting of 1300 instances that we reproduced. As a result, 1100 data as the assault were classified and achieved 83.5% accuracy. This also shows that the proposed method can provide high performance in real-world environments.

2.
Int J Mol Sci ; 24(9)2023 Apr 30.
Artigo em Inglês | MEDLINE | ID: mdl-37175802

RESUMO

Platelet-derived growth factor type BB (PDGF-BB) regulates vascular smooth muscle cell (VSMC) migration and proliferation, which play critical roles in the development of vascular conditions. p90 ribosomal S6 kinase (p90RSK) can regulate various cellular processes through many different target substrates in several cell types, but the regulatory function of p90RSK on PDGF-BB-mediated cell migration and proliferation and subsequent vascular neointima formation has not yet been extensively examined. In this study, we investigated whether p90RSK inhibition protects VSMCs against PDGF-BB-induced cellular phenotypic changes and the molecular mechanisms underlying the effect of p90RSK inhibition on neointimal hyperplasia in vivo. Pretreatment of cultured primary rat VSMCs with FMK or BI-D1870, which are specific inhibitors of p90RSK, suppressed PDGF-BB-induced phenotypic changes, including migration, proliferation, and extracellular matrix accumulation, in VSMCs. Additionally, FMK and BI-D1870 repressed the PDGF-BB-induced upregulation of cyclin D1 and cyclin-dependent kinase-4 expression. Furthermore, p90RSK inhibition hindered the inhibitory effect of PDGF-BB on Cdk inhibitor p27 expression, indicating that p90RSK may induce VSMC proliferation by regulating the G0/G1 phase. Notably, treatment with FMK resulted in attenuation of neointima development in ligated carotid arteries in mice. The findings imply that p90RSK inhibition mitigates the phenotypic switch and neointimal hyperplasia induced by PDGF-BB.


Assuntos
Músculo Liso Vascular , Neointima , Ratos , Camundongos , Animais , Becaplermina/farmacologia , Becaplermina/metabolismo , Neointima/metabolismo , Hiperplasia/metabolismo , Músculo Liso Vascular/metabolismo , Proteínas Quinases S6 Ribossômicas 90-kDa/metabolismo , Proliferação de Células , Ratos Sprague-Dawley , Movimento Celular , Miócitos de Músculo Liso/metabolismo , Células Cultivadas , Proteínas Proto-Oncogênicas c-sis/farmacologia , Proteínas Proto-Oncogênicas c-sis/metabolismo
3.
Korean J Physiol Pharmacol ; 27(3): 231-240, 2023 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-37078297

RESUMO

Fabry disease is a lysosomal storage disorder characterized by the lysosomal accumulations of glycosphingolipids in a variety of cytotypes, which include endothelial cells. The disease is inherited and originates from an error in glycosphingolipid catabolism caused by insufficient α-galactosidase A activity, which causes uncontrolled progressive storage of intracellular globotriaosylceramide (Gb3) in the vasculature and extracellular accumulation of lyso-Gb3 (a deacetylated soluble form of Gb3). Necrosis can lead to inflammation, which exacerbates necrosis and creates a positive feedback loop that triggers necroinflammation. However, the role played by necroptosis, a form of programmed necrotic cell death, in the cell-to-cell inflammatory reaction between epithelial and endothelial cells is unclear. Thus, the present study was undertaken to determine whether lyso-Gb3 induces necroptosis and whether necroptosis inhibition protects endothelial dysfunction against lyso-Gb3 inflamed retinal pigment epithelial cells. We found lyso-Gb3 induced necroptosis of a retinal pigment epithelial cell line (ARPE-19) in an autophagy-dependent manner and that conditioned media (CM) from ARPE-19 cells treated with lyso-Gb3 induced the necroptosis, inflammation, and senescence of human umbilical vein endothelial cells. In addition, a pharmacological study showed CM from lyso-Gb3 treated ARPE-19 cells induced endothelial necroptosis, inflammation, and senescence were significantly inhibited by an autophagy inhibitor (3-MA) and by two necroptosis inhibitors (necrostatin and GSK-872), respectively. These results demonstrate lyso-Gb3 induces necroptosis via autophagy and suggest that lyso-Gb3 inflamed retinal pigment epithelial cells trigger endothelial dysfunction via the autophagy-dependent necroptosis pathway. This study suggests the involvement of a novel autophagy-dependent necroptosis pathway in the regulation of endothelial dysfunction in Fabry disease.

4.
Molecules ; 27(9)2022 Apr 22.
Artigo em Inglês | MEDLINE | ID: mdl-35566046

RESUMO

Camphor tree (Cinnamomum camphora) is an ornamental plant that has been cultivated for a long time to obtain wood or camphor. Furthermore, its essential oil can be used as an alternative medicine and is an important source of perfume. Camphor obtained from camphor trees has long been used as a treatment for various symptoms such as inflammation, infection, congestion, muscle pain, and irritation in various regions. The purpose of this literature review is to provide knowledge of the well-established, wide, and extensive applications of camphor both in traditional and modern applications. Despite many studies focused on the essential oil of the camphor tree, there is a lack of systematic studies of its extraction or separation. Besides, various components of camphor are not fully understood, and further research is needed on the medicinal effects of individual components of C. camphor. The genus Cinnamomum has crucial economic value and theoretical significance. However, further systematic reviews and investigative studies based on existing research are needed to promote the modernization process of traditional applications of camphor. For proper use of the essential oil of C. camphora, it is imperative to consider its possible effects on humans and the environment.


Assuntos
Cinnamomum camphora , Óleos Voláteis , Perfumes , Cânfora , Humanos , Óleos Voláteis/farmacologia , Folhas de Planta
5.
FASEB J ; : fj201800011R, 2018 May 29.
Artigo em Inglês | MEDLINE | ID: mdl-29812969

RESUMO

The development of resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs) occurs by various mechanisms and appears to be almost inevitable, even in patients with lung cancer who initially respond well to EGFR-TKIs. Consequently, considerable efforts have been made to develop more effective EGFR-TKIs. Therefore, an understanding of the mechanisms behind TKI resistance is essential for improving EGFR-TKI therapeutic efficacy in non-small cell lung cancer (NSCLC) patients. In this study, we discovered that overexpression of antioxidant-responsive element (ARE)-containing Nrf2 target genes by increased transactivation of Nrf2 occurred because of an acquired Keap1 mutation in the gefitinib-resistant (GR) NSCLC cell line we established. These GR cells also acquired cross-resistance to the irreversible EGFR-TKIs, afatinib and osimertinib, and showed increased viability, invasiveness, proliferation, and tumorigenicity both in vitro and in vivo. These results were confirmed by the fact that inhibition of Nrf2 activity, either by treatment with brusatol or by inducing expression of exogenously introduced wild-type Keap1, suppressed tumor cell proliferation and tumorigenicity in vitro and in vivo. Our data suggest that disruption of the Keap1-Nrf2 pathway is one of the mechanisms by which EGFR-TKI resistance occurs, a fact that must be considered when treating patients with EGFR-TKI.-Park, S.-H., Kim, J. H., Ko, E., Kim, J.-Y., Park, M.-J., Kim, M. J., Seo, H., Li, S., Lee, J.-Y. Resistance to gefitinib and cross-resistance to irreversible EGFR-TKIs mediated by disruption of the Keap1-Nrf2 pathway in human lung cancer cells.

6.
Appl Microbiol Biotechnol ; 103(1): 211-223, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30343427

RESUMO

Lycopene is a red carotenoid pigment with strong antioxidant activity. Saccharomyces cerevisiae is considered a promising host to produce lycopene, but lycopene toxicity is one of the limiting factors for high-level production. In this study, we used heterologous lycopene biosynthesis genes crtE and crtI from Xanthophyllomyces dendrorhous and crtB from Pantoea agglomerans for lycopene production in S. cerevisiae. The crtE, crtB, and crtI genes were integrated into the genome of S. cerevisiae CEN.PK2-1C strain, while deleting DPP1 and LPP1 genes to inhibit a competing pathway producing farnesol. Lycopene production was further improved by inhibiting ergosterol production via downregulation of ERG9 expression and by deleting ROX1 or MOT3 genes encoding transcriptional repressors for mevalonate and sterol biosynthetic pathways. To further increase lycopene production, CrtE and CrtB mutants with improved activities were isolated by directed evolution, and subsequently, the mutated genes were randomly integrated into the engineered lycopene-producing strains via delta-integration. To relieve lycopene toxicity by increasing unsaturated fatty acid content in cell membranes, the OLE1 gene encoding stearoyl-CoA 9-desaturase was overexpressed. In combination with the overexpression of STB5 gene encoding a transcription factor involved in NADPH production, the final strain produced up to 41.8 mg/gDCW of lycopene, which is approximately 74.6-fold higher than that produced in the initial strain.


Assuntos
Licopeno/metabolismo , Microrganismos Geneticamente Modificados , NADP/biossíntese , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Basidiomycota/genética , Membrana Celular/metabolismo , Evolução Molecular Direcionada , Farneseno Álcool/metabolismo , Farnesil-Difosfato Farnesiltransferase/genética , Farnesil-Difosfato Farnesiltransferase/metabolismo , Ácidos Graxos Insaturados/metabolismo , Regulação Fúngica da Expressão Gênica , Pantoea/genética , Engenharia de Proteínas , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Proteínas Repressoras/genética , Proteínas Repressoras/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Estearoil-CoA Dessaturase/genética , Estearoil-CoA Dessaturase/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo
7.
Artigo em Inglês | MEDLINE | ID: mdl-28137806

RESUMO

SCY-078 (MK-3118) is a novel, semisynthetic derivative of enfumafungin and represents the first compound of the triterpene class of antifungals. SCY-078 exhibits potent inhibition of ß-(1,3)-d-glucan synthesis, an essential cell wall component of many pathogenic fungi, including Candida spp. and Aspergillus spp. SCY-078 is currently in phase 2 clinical development for the treatment of invasive fungal diseases. In vitro disposition studies to assess solubility, intestinal permeability, and metabolic stability were predictive of good oral bioavailability. Preclinical pharmacokinetic studies were consistent with once-daily administration to humans. After intravenous delivery, plasma clearance in rodents and dogs was low, representing <15% and <25% of hepatic blood flow, respectively. The terminal elimination-phase half-life was 5.5 to 8.7 h in rodents, and it was ∼9.3 h in dogs. The volume of distribution at steady-state was high (4.7 to 5.3 liters/kg), a finding suggestive of extensive tissue distribution. Exposure of SCY-078 in kidney tissue, a target organ for invasive fungal disease such as candidiasis, exceeded plasma by 20- to 25-fold for the area under the concentration-time curve from 0 h to infinity (AUC0-∞) and Cmax SCY-078 achieved efficacy endpoints following oral delivery across multiple murine models of disseminated candidiasis. The pharmacokinetic/pharmacodynamic indices Cmax/MIC and AUC/MIC correlated with outcome. Target therapeutic exposure, expressed as the plasma AUC0-24, was comparable across models, with an upper value of 11.2 µg·h/ml (15.4 µM·h); the corresponding mean value for free drug AUC/MIC was ∼0.75. Overall, these results demonstrate that SCY-078 has the oral and intravenous (i.v.) pharmacokinetic properties and potency in murine infection models of disseminated candidiasis to support further investigation as a novel i.v. and oral treatment for invasive fungal diseases.


Assuntos
Antifúngicos/farmacocinética , Candida albicans/efeitos dos fármacos , Candidíase/tratamento farmacológico , Ácidos Carboxílicos/farmacocinética , Glucanos/antagonistas & inibidores , Fenantrenos/farmacocinética , Animais , Antifúngicos/sangue , Antifúngicos/síntese química , Área Sob a Curva , Disponibilidade Biológica , Células CACO-2 , Candida albicans/crescimento & desenvolvimento , Candida albicans/metabolismo , Candidíase/sangue , Candidíase/microbiologia , Ácidos Carboxílicos/sangue , Ácidos Carboxílicos/síntese química , Parede Celular/efeitos dos fármacos , Parede Celular/metabolismo , Modelos Animais de Doenças , Cães , Esquema de Medicação , Cálculos da Dosagem de Medicamento , Feminino , Glucanos/biossíntese , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Microssomos Hepáticos/efeitos dos fármacos , Microssomos Hepáticos/metabolismo , Fenantrenos/sangue , Fenantrenos/síntese química , Ratos
8.
Artigo em Inglês | MEDLINE | ID: mdl-28416547

RESUMO

Tenofovir disoproxil fumarate (TDF), a prodrug of tenofovir, has oral bioavailability (25%) limited by intestinal transport (P-glycoprotein), and intestinal degradation (carboxylesterase). However, the influence of luminal pancreatic enzymes is not fully understood. Physiologically based pharmacokinetic (PBPK) modeling has utility for estimating drug exposure from in vitro data. This study aimed to develop a PBPK model that included luminal enzyme activity to inform dose reduction strategies. TDF and tenofovir stability in porcine pancrelipase concentrations was assessed (0, 0.48, 4.8, 48, and 480 U/ml of lipase; 1 mM TDF; 37°C; 0 to 30 min). Samples were analyzed using mass spectrometry. TDF stability and permeation data allowed calculation of absorption rates within a human PBPK model to predict plasma exposure following 6 days of once-daily dosing with 300 mg of TDF. Regional absorption of drug was simulated across gut segments. TDF was degraded by pancrelipase (half-lives of 0.07 and 0.62 h using 480 and 48 U/ml, respectively). Previously reported maximum concentration (Cmax; 335 ng/ml), time to Cmax (Tmax; 2.4 h), area under the concentration-time curve from 0 to 24 h (AUC0-24; 3,045 ng · h/ml), and concentration at 24 h (C24; 48.3 ng/ml) were all within a 0.5-fold difference from the simulated Cmax (238 ng/ml), Tmax (3 h), AUC0-24 (3,036 ng · h/ml), and C24 (42.7 ng/ml). Simulated TDF absorption was higher in duodenum and jejunum than in ileum (p<0.05). These data support that TDF absorption is limited by the action of intestinal lipases. Our results suggest that bioavailability may be improved by protection of drug from intestinal transporters and enzymes, for example, by coadministration of enzyme-inhibiting agents or nanoformulation strategies.


Assuntos
Fármacos Anti-HIV/farmacologia , Fármacos Anti-HIV/farmacocinética , Tenofovir/farmacologia , Tenofovir/farmacocinética , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Adolescente , Adulto , Carboxilesterase/metabolismo , Infecções por HIV/metabolismo , Humanos , Lipase/metabolismo , Masculino , Pessoa de Meia-Idade , Pancrelipase/metabolismo , Adulto Jovem
9.
Biochem Biophys Res Commun ; 493(1): 671-676, 2017 11 04.
Artigo em Inglês | MEDLINE | ID: mdl-28864415

RESUMO

Streptococcus pneumoniae is an important human pathogen responsible for more than 2 million deaths annually worldwide. The airway epithelium acts as the first-line of defense against pneumococcal infections by regulating acute inflammation against invading pneumococcus. Despite the intact adaptive immunity, failure in early defense due to loss of pattern recognition receptors (PRRs) and/or acute phase proteins (APPs) results in detrimental damage and death. C-reactive protein (CRP), the first found APP, is a member of the pentraxin family of proteins and an important soluble PRR for pneumococcus. CRP and another short pentraxin, serum amyloid P, are critical for acute defense against pneumococcal infection. However, the role of the long pentraxin PTX3 in regulating pneumococcal infections is unknown. In this study, PTX3 expression was upregulated by pneumococcus in epithelial cells and in lungs of mice. In addition, PTX3 potentiated pneumococcal inflammation; overexpression of PTX3 enhanced pneumococcus-induced cytokine expression, whereas knock-down of PTX3 with siPTX3 inhibited the cytokine expression. Furthermore, PTX3 deficiency indeed ameliorated acute inflammation and protected mice against death following pneumococcal infection. Pneumococcal toxin pneumolysin was responsible for PTX3 expression and upregulated PTX3 expression via JNK MAPK signaling. These data implicate PTX3 as a novel therapeutic target for the control of acute inflammation by pneumococcus.


Assuntos
Proteína C-Reativa/imunologia , Citocinas/imunologia , Mediadores da Inflamação/imunologia , Sistema de Sinalização das MAP Quinases/imunologia , Proteínas do Tecido Nervoso/imunologia , Pneumonia Pneumocócica/imunologia , Mucosa Respiratória/imunologia , Células A549 , Animais , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Knockout , Pneumonia Pneumocócica/patologia , Mucosa Respiratória/patologia
10.
Am J Physiol Cell Physiol ; 310(6): C414-22, 2016 Mar 15.
Artigo em Inglês | MEDLINE | ID: mdl-26739495

RESUMO

Membrane contact sites (MCS) are critical junctions that form between the endoplasmic reticulum (ER) and membranes of various organelles, including the plasma membrane (PM). Signaling complexes, including mediators of Ca(2+) signaling, are assembled within MCS, such as the ER/PM junction. This is most evident in polarized epithelial cells, such as pancreatic cells. Core Ca(2+) signaling proteins cluster at the apical pole, the site of inositol 1,4,5-trisphosphate-mediated Ca(2+) release and Orai1/transient receptor potential canonical-mediated store-dependent Ca(2+) entry. Recent advances have characterized the proteins that tether the membranes at MCS and the role of these proteins in modulating physiological and pathological intracellular signaling. This review discusses recent advances in the characterization of Ca(2+) signaling at ER/PM junctions and the relation of these junctions to physiological and pathological Ca(2+) signaling in pancreatic acini.


Assuntos
Membrana Celular/metabolismo , Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Pâncreas/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio/metabolismo , Sinalização do Cálcio/fisiologia
11.
Biochem Biophys Res Commun ; 470(4): 907-12, 2016 Feb 19.
Artigo em Inglês | MEDLINE | ID: mdl-26820527

RESUMO

The intermediate conductance calcium-activated potassium channel (KCa3.1) mediates proliferation of many cell types including fibroblasts, and is a molecular target for intervention in various cell proliferative diseases. Our previous study showed that reduction of KCa3.1 channel expression by lyso-globotriaosylceramide (lyso-Gb3) inhibits differentiation into myofibroblasts and collagen synthesis, which might lead to development of ascending thoracic aortic aneurysm secondary to Fabry disease. However, how lyso-Gb3 downregulates KCa3.1 channel expression is unknown. Therefore, we aimed to investigate the underlying mechanisms of lyso-Gb3-mediated KCa3.1 channel downregulation, focusing on the cAMP signaling pathway. We found that lyso-Gb3 increased the intracellular cAMP concentration by upregulation of adenylyl cyclase 6 and inhibited ERK 1/2 phosphorylation through the protein kinase A (PKA) pathway, leading to the inhibition of KCa3.1 channel synthesis, not the exchange protein directly activated by cAMP (Epac) pathway. Moreover, lyso-Gb3 suppressed expression of class II phosphatidylinositol 3-kinase C2ß (PI3KC2ß) by PKA activation, which reduces the production of phosphatidylinositol 3-phosphate [PI(3)P], and the reduced membrane surface expression of KCa3.1 channel was recovered by increasing the intracellular levels of PI(3)P. Consequently, our findings that lyso-Gb3 inhibited both KCa3.1 channel synthesis and surface expression by increasing intracellular cAMP, and controlled surface expression through changes in PI3KC2ß-mediated PI(3)P production, suggest that modulation of PKA and PI3KC2ß activity to control of KCa3.1 channel expression can be an alternative important target to attenuate ascending thoracic aortic aneurysms in Fabry disease.


Assuntos
Membrana Celular/metabolismo , Classe II de Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Glicolipídeos/administração & dosagem , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/biossíntese , Fosfatos de Fosfatidilinositol/metabolismo , Esfingolipídeos/administração & dosagem , Animais , Relação Dose-Resposta a Droga , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/efeitos dos fármacos , Camundongos , Células NIH 3T3
12.
Appl Microbiol Biotechnol ; 100(17): 7591-8, 2016 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-27225475

RESUMO

Subcellular compartmentalization of the biosynthetic enzymes is one of the limiting factors for isobutanol production in Saccharomyces cerevisiae. Previously, it has been shown that mitochondrial compartmentalization of the biosynthetic pathway through re-locating cytosolic Ehrlich pathway enzymes into the mitochondria can increase isobutanol production. In this study, we improved mitochondrial isobutanol production by increasing mitochondrial pool of pyruvate, a key substrate for isobutanol production. Mitochondrial isobutanol biosynthetic pathway was introduced into bat1Δald6Δlpd1Δ strain, where genes involved in competing pathways were deleted, and MPC1, MPC2, and MPC3 genes encoding the subunits of mitochondrial pyruvate carrier (MPC) hetero-oligomeric complex were overexpressed with different combinations. Overexpression of Mpc1 and Mpc3 forming high-affinity MPCOX was more effective in improving isobutanol production than overexpression of Mpc1 and Mpc2 forming low-affinity MPCFERM. The final engineered strain overexpressing MPCOX produced 330.9 mg/L isobutanol from 20 g/L glucose, exhibiting about 22-fold increase in production compared to wild type.


Assuntos
Butanóis/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Mitocôndrias/metabolismo , Ácido Pirúvico/metabolismo , Saccharomyces cerevisiae/metabolismo , Proteínas de Transporte de Ânions/genética , Proteínas de Transporte de Ânions/metabolismo , Transporte Biológico/fisiologia , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas de Transporte da Membrana Mitocondrial , Transportadores de Ácidos Monocarboxílicos , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Complexo Piruvato Desidrogenase/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo
13.
Adv Exp Med Biol ; 898: 47-66, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27161224

RESUMO

The Ca(2+) second messenger is initiated at ER/PM junctions and propagates into the cell interior to convey the receptor information. The signal is maintained by Ca(2+) influx across the plasma membrane through the Orai and TRPC channels. These Ca(2+) influx channels form complexes at ER/PM junctions with the ER Ca(2+) sensor STIM1, which activates the channels. The function of STIM1 is modulated by other STIM isoforms like STIM1L, STIM2 and STIM2.1/STIM2ß and by SARAF, which mediates the Ca(2+)-dependent inhibition of Orai channels. The ER/PM junctions are formed at membrane contact sites by tethering proteins that generate several types of ER/PM junctions, such as PI(4,5)P2-poor and PI(4,5)P2-rich domains. This chapter discusses several properties of the TRPC channels, the Orai channels and the STIMs, their key interacting proteins and how interaction of the STIMs with the channels gates their activity. The chapter closes by highlighting open questions and potential future directions in this field.


Assuntos
Retículo Endoplasmático/metabolismo , Proteínas de Membrana/metabolismo , Proteínas de Neoplasias/metabolismo , Canais de Cátion TRPC/metabolismo , Cálcio/metabolismo , Membrana Celular/metabolismo , Humanos , Ativação do Canal Iônico , Molécula 1 de Interação Estromal
14.
Biochem Biophys Res Commun ; 468(4): 883-8, 2015 Dec 25.
Artigo em Inglês | MEDLINE | ID: mdl-26592662

RESUMO

Fabry disease is an X-linked lysosomal storage disorder that is caused by a deficiency of α-galactosidase A. The disease ultimately manifests as multiple organ dysfunctions owing to excessive accumulation of globotriaosylceramide (Gb3). Among the several complications of Fabry disease, ascending thoracic aortic aneurysm is relatively common, which is classically associated with connective tissue disorders characterized by abnormal defects or deficiencies in structural proteins such as collagen and elastin. Although an elevated Gb3 level is regarded as a prerequisite for the manifestations of Fabry disease, only this excess accumulation cannot explain the pathophysiology of these complications. Recently, an increased plasma level of lyso-Gb3 was suggested as a new biomarker in Fabry disease. Therefore, the aim of this study was to assess the effects of lyso-Gb3 on the pathogenesis of thoracic ascending aortic aneurysms in Fabry disease, with a particular focus on the responses related to aortic remodeling by fibroblasts. We found that lyso-Gb3 inhibited the growth of fibroblasts, as well as their differentiation into myofibroblasts, and collagen expression. Moreover, all of these compromised responses could be attributed to the effects of lyso-Gb3 on downregulation of KCa3.1 channel expression, and these impairments could be rescued when activating the KCa3.1 channel or increasing intracellular Ca(2+) concentration. This study provides new evidence that lyso-Gb3 inhibits the differentiation into myofibroblasts and collagen synthesis of fibroblasts owing to decreased Ca(2+) levels by KCa3.1 channel dysfunction. These findings suggest that the KCa3.1 channel can serve as a new target to attenuate and prevent development of ascending thoracic aortic aneurysm in Fabry disease.


Assuntos
Cálcio/metabolismo , Colágeno/biossíntese , Fibroblastos/citologia , Fibroblastos/fisiologia , Glicolipídeos/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Esfingolipídeos/metabolismo , Animais , Diferenciação Celular/efeitos dos fármacos , Diferenciação Celular/fisiologia , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/fisiologia , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/fisiologia , Fibroblastos/efeitos dos fármacos , Regulação da Expressão Gênica/efeitos dos fármacos , Regulação da Expressão Gênica/fisiologia , Glicolipídeos/administração & dosagem , Camundongos , Células NIH 3T3 , Esfingolipídeos/administração & dosagem
15.
Arterioscler Thromb Vasc Biol ; 34(1): 81-9, 2014 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-24158513

RESUMO

OBJECTIVE: Globotriaosylceramide (Gb3) induces KCa3.1 downregulation in Fabry disease (FD). We investigated whether Gb3 induces KCa3.1 endocytosis and degradation. APPROACH AND RESULTS: KCa3.1, especially plasma membrane-localized KCa3.1, was downregulated in both Gb3-treated mouse aortic endothelial cells (MAECs) and human umbilical vein endothelial cells. Gb3-induced KCa3.1 downregulation was prevented by lysosomal inhibitors but not by a proteosomal inhibitor. Endoplasmic reticulum stress-inducing agents did not induce KCa3.1 downregulation. Gb3 upregulated the protein levels of early endosome antigen 1 and lysosomal-associated membrane protein 2 in MAECs. Compared with MAECs from age-matched wild-type mice, those from aged α-galactosidase A (Gla)-knockout mice, an animal model of FD, showed downregulated KCa3.1 expression and upregulated early endosome antigen 1 and lysosomal-associated membrane protein 2 expression. In contrast, no significant difference was found in early endosome antigen 1 and lysosomal-associated membrane protein 2 expression between young Gla-knockout and wild-type MAECs. In aged Gla-knockout MAECs, clathrin was translocated close to the cell border and clathrin knockdown recovered KCa3.1 expression. Rab5, an effector of early endosome antigen 1, was upregulated, and Rab5 knockdown restored KCa3.1 expression, the current, and endothelium-dependent relaxation. CONCLUSIONS: -Gb3 accelerates the endocytosis and lysosomal degradation of endothelial KCa3.1 via a clathrin-dependent process, leading to endothelial dysfunction in FD.


Assuntos
Células Endoteliais/enzimologia , Endotélio Vascular/enzimologia , Doença de Fabry/enzimologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Lisossomos/metabolismo , Triexosilceramidas/metabolismo , Animais , Células Cultivadas , Clatrina/genética , Clatrina/metabolismo , Modelos Animais de Doenças , Endocitose , Endotélio Vascular/fisiopatologia , Doença de Fabry/genética , Doença de Fabry/fisiopatologia , Células Endoteliais da Veia Umbilical Humana/enzimologia , Humanos , Proteína 2 de Membrana Associada ao Lisossomo/metabolismo , Camundongos , Camundongos Knockout , Transporte Proteico , Proteólise , Interferência de RNA , Transfecção , Vasodilatação , Proteínas de Transporte Vesicular/metabolismo , alfa-Galactosidase/genética , alfa-Galactosidase/metabolismo , Proteínas rab5 de Ligação ao GTP/genética , Proteínas rab5 de Ligação ao GTP/metabolismo
16.
Pflugers Arch ; 466(8): 1487-99, 2014 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-24240699

RESUMO

A central function of epithelia is the control of the volume and electrolyte composition of bodily fluids through vectorial transport of electrolytes and the obligatory H2O. In exocrine glands, fluid and electrolyte secretion is carried out by both acinar and duct cells, with the portion of fluid secreted by each cell type varying among glands. All acinar cells secrete isotonic, plasma-like fluid, while the duct determines the final electrolyte composition of the fluid by absorbing most of the Cl(-) and secreting HCO3 (-). The key transporters mediating acinar fluid and electrolyte secretion are the basolateral Na(+)/K(+) /2Cl(-) cotransporter, the luminal Ca(2+)-activated Cl(-) channel ANO1 and basolateral and luminal Ca(2+)-activated K(+) channels. Ductal fluid and HCO3 (-) secretion are mediated by the basolateral membrane Na(+)-HCO3 (-) cotransporter NBCe1-B and the luminal membrane Cl(-)/HCO3 (-) exchanger slc26a6 and the Cl(-) channel CFTR. The function of the transporters is regulated by multiple inputs, which in the duct include major regulation by the WNK/SPAK pathway that inhibit secretion and the IRBIT/PP1 pathway that antagonize the effects of the WNK/SPAK pathway to both stimulate and coordinate the secretion. The function of these regulatory pathways in secretory glands acinar cells is yet to be examined. An important concept in biology is synergism among signaling pathways to generate the final physiological response that ensures regulation with high fidelity and guards against cell toxicity. While synergism is observed in all epithelial functions, the molecular mechanism mediating the synergism is not known. Recent work reveals a central role for IRBIT as a third messenger that integrates and synergizes the function of the Ca(2+) and cAMP signaling pathways in activation of epithelial fluid and electrolyte secretion. These concepts are discussed in this review using secretion by the pancreatic and salivary gland ducts as model systems.


Assuntos
Líquidos Corporais/metabolismo , Eletrólitos/metabolismo , Epitélio/metabolismo , Transdução de Sinais/fisiologia , Animais , Humanos , Ductos Pancreáticos/metabolismo , Ductos Salivares/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia
17.
Gastroenterology ; 145(1): 232-241, 2013 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-23542070

RESUMO

BACKGROUND & AIMS: The cyclic adenosine monophosphate (cAMP) and Ca(2+) signaling pathways synergize to regulate many physiological functions. However, little is known about the mechanisms by which these pathways interact. We investigated the synergy between these signaling pathways in mouse pancreatic and salivary gland ducts. METHODS: We created mice with disruptions in genes encoding the solute carrier family 26, member 6 (Slc26a6(-/-) mice) and inositol 1,4,5-triphosphate (InsP3) receptor-binding protein released with InsP3 (Irbit(-/-)) mice. We investigated fluid secretion by sealed pancreatic ducts and the function of Slc26a6 and the cystic fibrosis transmembrane conductance regulator (CFTR) in HeLa cells and in ducts isolated from mouse pancreatic and salivary glands. Slc26a6 activity was assayed by measuring intracellular pH, and CFTR activity was assayed by measuring Cl(-) current. Protein interactions were determined by immunoprecipitation analyses. RESULTS: Irbit mediated the synergistic activation of CFTR and Slc26a6 by Ca(2+) and cAMP. In resting cells, Irbit was sequestered by InsP3 receptors (IP3Rs) in the endoplasmic reticulum. Stimulation of Gs-coupled receptors led to phosphorylation of IP3Rs, which increased their affinity for InsP3 and reduced their affinity for Irbit. Subsequent weak stimulation of Gq-coupled receptors, which led to production of low levels of IP3, caused dissociation of Irbit from IP3Rs and allowed translocation of Irbit to CFTR and Slc26a6 in the plasma membrane. These processes stimulated epithelial secretion of electrolytes and fluid. These pathways were not observed in pancreatic and salivary glands from Irbit(-/-) or Slc26a6(-/-) mice, or in salivary gland ducts expressing mutant forms of IP3Rs that could not undergo protein kinase A-mediated phosphorylation. CONCLUSIONS: Irbit promotes synergy between the Ca(2+) and cAMP signaling pathways in cultured cells and in pancreatic and salivary ducts from mice. Defects in this pathway could be involved in cystic fibrosis, pancreatitis, or Sjögren syndrome.


Assuntos
Adenosil-Homocisteinase/fisiologia , Cálcio/metabolismo , AMP Cíclico/fisiologia , Transdução de Sinais/fisiologia , Animais , Antiporters/metabolismo , Transporte Biológico , Proteínas Quinases Dependentes de AMP Cíclico/fisiologia , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Epitélio/metabolismo , Inositol 1,4,5-Trifosfato/biossíntese , Receptores de Inositol 1,4,5-Trifosfato/fisiologia , Camundongos , Ductos Pancreáticos/metabolismo , Fosforilação , Ductos Salivares/metabolismo , Transportadores de Sulfato
18.
Appl Microbiol Biotechnol ; 98(21): 9139-47, 2014 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-25280745

RESUMO

Saccharomyces cerevisiae naturally produces small amounts of isobutanol and 3-methyl-1-butanol via Ehrlich pathway from the catabolism of valine and leucine, respectively. In this study, we engineered CEN.PK2-1C, a leucine auxotrophic strain having a LEU2 gene mutation, for the production of isobutanol and 3-methyl-1-butanol. First, ALD6 encoding aldehyde dehydrogenase and BAT1 involved in valine synthesis were deleted to eliminate competing pathways. We also increased transcription of endogenous genes in the valine and leucine biosynthetic pathways by expressing Leu3Δ601, a constitutively active form of Leu3 transcriptional activator. For the production of isobutanol, genes involved in isobutanol production (ILV2, ILV3, ILV5, ARO10, and ADH2) were additionally overexpressed in ald6Δbat1Δ strain expressing LEU3Δ601, resulting in 376.9 mg/L isobutanol production from 100 g/L glucose. To increase 3-methyl-1-butanol production, leucine biosynthetic genes were additionally overexpressed in the final isobutanol-production strain. The resulting strain overexpressing LEU2 and LEU4 (D578Y) , a feedback inhibition-insensitive mutant of LEU4, showed a 34-fold increase in 3-methyl-1-butanol synthesis compared with CEN.PK2-1C control strain, producing 765.7 mg/L 3-methyl-1-butanol.


Assuntos
Butanóis/metabolismo , Engenharia Metabólica , Redes e Vias Metabólicas/genética , Pentanóis/metabolismo , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Deleção de Genes , Expressão Gênica , Saccharomyces cerevisiae/enzimologia
19.
Physiology (Bethesda) ; 27(5): 291-9, 2012 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-23026752

RESUMO

Fluid and electrolyte homeostasis is a fundamental physiological function required for survival and is associated with a plethora of diseases when aberrant. Systemic fluid and electrolyte composition is regulated by the kidney, and all secretory epithelia generate biological fluids with defined electrolyte composition by vectorial transport of ions and the obligatory water. A major regulatory pathway that immerged in the last several years is regulation of ion transporters by the WNK/SPAK kinases and IRBIT/PP1 pathways. The IRBIT/PP1 pathway functions to reverse the effects of the WNK/SPAK kinases pathway, as was demonstrated for NBCe1-B and CFTR. Since many transporters involved in fluid and electrolyte homeostasis are affected by PP1 and/or calcineurin, it is possible that WNK/SPAK and IRBIT/PP1 form a common regulatory pathway to tune the activity of fluid and electrolyte transport in response to physiological demands.


Assuntos
Peptídeos e Proteínas de Sinalização Intracelular/fisiologia , Transporte de Íons/fisiologia , Lectinas Tipo C/fisiologia , Proteínas de Membrana/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Receptores de Neuropeptídeo Y/fisiologia , Transdução de Sinais/fisiologia , Células Epiteliais/fisiologia , Humanos , Antígenos de Histocompatibilidade Menor , Proteína Quinase 1 Deficiente de Lisina WNK , Equilíbrio Hidroeletrolítico/fisiologia
20.
Pharmacol Res ; 66(1): 51-9, 2012 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-22414869

RESUMO

Modafinil has been used as a psychostimulant for the treatment of narcolepsy. However, its primary mechanism of action remains elusive. Therefore, we examined the effects of modafinil on K(Ca)3.1 channels and vascular smooth muscle contraction. K(Ca)3.1 currents and channel activity were measured using a voltage-clamp technique and inside-out patches in mouse embryonic fibroblast cell line, NIH-3T3 fibroblasts. Intracellular adenosine 3',5'-cyclic monophosphate (cAMP) concentration was measured, and the phosphorylation of K(Ca)3.1 channel protein was examined using western blotting in NIH-3T3 fibroblasts and/or primary cultured mouse aortic smooth muscle cells (SMCs). Muscle contractions were recorded from mouse aorta and rat pulmonary artery by using a myograph developed in-house. Modafinil was found to inhibit K(Ca)3.1 currents in a concentration-dependent manner, and the half-maximal inhibition (IC(50)) of modafinil for the current inhibition was 6.8 ± 0.7 nM. The protein kinase A (PKA) activator forskolin also inhibited K(Ca)3.1 currents. The inhibitory effects of modafinil and forskolin on K(Ca)3.1 currents were blocked by the PKA inhibitors PKI(14-22) or H-89. In addition, modafinil relaxed blood vessels (mouse aorta and rat pulmonary artery) in a concentration-dependent manner. Modafinil increased cAMP concentrations in NIH-3T3 fibroblasts or primary cultured mouse aortic SMCs and phosphorylated K(Ca)3.1 channel protein in NIH-3T3 fibroblasts. However, open probability and single-channel current amplitudes of K(Ca)3.1 channels were not changed by modafinil. From these results, we conclude that modafinil inhibits K(Ca)3.1 channels and vascular smooth muscle contraction by cAMP-dependent phosphorylation, suggesting that modafinil can be used as a cAMP-dependent K(Ca)3.1 channel blocker and vasodilator.


Assuntos
Compostos Benzidrílicos/farmacologia , AMP Cíclico/metabolismo , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/antagonistas & inibidores , Músculo Liso Vascular/efeitos dos fármacos , Miócitos de Músculo Liso/efeitos dos fármacos , Bloqueadores dos Canais de Potássio/farmacologia , Sistemas do Segundo Mensageiro/efeitos dos fármacos , Vasoconstrição/efeitos dos fármacos , Vasodilatadores/farmacologia , Animais , Proteínas de Transporte/farmacologia , Colforsina/farmacologia , Proteínas Quinases Dependentes de AMP Cíclico/antagonistas & inibidores , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Relação Dose-Resposta a Droga , Ativação Enzimática , Ativadores de Enzimas/farmacologia , Humanos , Iloprosta/farmacologia , Canais de Potássio Ativados por Cálcio de Condutância Intermediária/metabolismo , Isoquinolinas/farmacologia , Masculino , Potenciais da Membrana , Camundongos , Modafinila , Músculo Liso Vascular/metabolismo , Miócitos de Músculo Liso/metabolismo , Células NIH 3T3 , Técnicas de Patch-Clamp , Fragmentos de Peptídeos/farmacologia , Fosforilação , Inibidores de Proteínas Quinases/farmacologia , Ratos , Sulfonamidas/farmacologia , Fatores de Tempo , Regulação para Cima
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