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1.
Int J Nanomedicine ; 15: 7651-7666, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-33116493

RESUMO

Introduction: Plants have always been a significant source of natural active components with biological properties. Celery seed oil (extracted from Apium graveolens) has several potential applications, but its therapeutic uses in the form of nanoemulsion formulation need to be investigated further in order to meet the demand in cancer treatment, and to alleviate the prevailing crisis arising from increased antimicrobial resistance. Methods: The therapeutic potential of celery seed oil was investigated through the formulation and testing of a nanoemulsion developed with Tween 80 (a non-ionic surfactant) and the utilization of an ultrasonication technique. Anticancer and apoptotic properties of the formulation were evaluated through MTT and Annexin V-FITC assays. The clonogenic assay aided in the identification of the antiproliferative properties of the formulation on oral squamous cell carcinoma. The antimicrobial study was supported by agar well diffusion assay, membrane integrity test and scanning electron microscopy. Results: Experiments identified relevant parameters, including optimal surfactant concentration and emulsification time. GC-MS analysis identified various components in the celery oil, but not their biological activities. A sonication time of 20 min resulted in a droplet diameter of 23.4 ± 1.80 nm. The IC50 concentration of the optimal nanoemulsion formulation against SAS cells was 1.4 µL/mL. At this concentration, cell proliferation was significantly reduced through inhibition of the anchorage-independent cell growth by disrupting colony formation and inducing cell death (apoptosis) of cancer cells. The nanoemulsion was also treated with a microbial suspension of S. aureus, and displayed antibacterial properties through lipid membrane fusion, causing cytoplasmic leakage as verified through agar well diffusion and membrane permeability assays. Scanning electron microscopy revealed complete distortion of the bacterial pathogen. Conclusion: The results in this study present celery as a possible constituent for cancer therapeutics and as a candidate for aggressive, yet safe cancer treatment. The celery-based nanoemulsion has the potential to act as a key alternative to standard antibiotic therapy.


Assuntos
Anti-Infecciosos/farmacologia , Antineoplásicos/farmacologia , Apium/química , Emulsões/química , Nanopartículas/química , Óleos Voláteis/farmacologia , Sonicação , Antineoplásicos/uso terapêutico , Apoptose/efeitos dos fármacos , Carcinoma de Células Escamosas/tratamento farmacológico , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Clonais , Composição de Medicamentos , Difusão Dinâmica da Luz , Cromatografia Gasosa-Espectrometria de Massas , Humanos , Testes de Sensibilidade Microbiana , Neoplasias Bucais/tratamento farmacológico , Nanopartículas/ultraestrutura , Óleos Voláteis/uso terapêutico , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura , Tensoativos/química
2.
J Med Microbiol ; 69(10): 1228-1234, 2020 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-32930657

RESUMO

Introduction. COVID-19 has rapidly emerged as a pandemic infection that has caused significant mortality and economic losses. Potential therapies and prophylaxis against COVID-19 are urgently needed to combat this novel infection. As a result of in vitro evidence suggesting zinc sulphate may be efficacious against COVID-19, our hospitals began using zinc sulphate as add-on therapy to hydroxychloroquine and azithromycin.Aim. To compare outcomes among hospitalized COVID-19 patients ordered to receive hydroxychloroquine and azithromycin plus zinc sulphate versus hydroxychloroquine and azithromycin alone.Methodology. This was a retrospective observational study. Data was collected from medical records for all patients with admission dates ranging from 2 March 2020 through to 11 April 2020. Initial clinical characteristics on presentation, medications given during the hospitalization, and hospital outcomes were recorded. The study included patients admitted to any of four acute care NYU Langone Health Hospitals in New York City. Patients included were admitted to the hospital with at least one positive COVID-19 test and had completed their hospitalization. Patients were excluded from the study if they were never admitted to the hospital or if there was an order for other investigational therapies for COVID-19.Results. Patients taking zinc sulphate in addition to hydroxychloroquine and azithromycin (n=411) and patients taking hydroxychloroquine and azithromycin alone (n=521) did not differ in age, race, sex, tobacco use or relevant comorbidities. The addition of zinc sulphate did not impact the length of hospitalization, duration of ventilation or intensive care unit (ICU) duration. In univariate analyses, zinc sulphate increased the frequency of patients being discharged home, and decreased the need for ventilation, admission to the ICU and mortality or transfer to hospice for patients who were never admitted to the ICU. After adjusting for the time at which zinc sulphate was added to our protocol, an increased frequency of being discharged home (OR 1.53, 95 % CI 1.12-2.09) and reduction in mortality or transfer to hospice among patients who did not require ICU level of care remained significant (OR 0.449, 95 % CI 0.271-0.744).Conclusion. This study provides the first in vivo evidence that zinc sulphate may play a role in therapeutic management for COVID-19.


Assuntos
Azitromicina/uso terapêutico , Infecções por Coronavirus/tratamento farmacológico , Hidroxicloroquina/uso terapêutico , Pneumonia Viral/tratamento farmacológico , Sulfato de Zinco/uso terapêutico , Betacoronavirus/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Quimioterapia Combinada , Hospitalização , Humanos , Ionóforos/uso terapêutico , Tempo de Internação , Pandemias , Estudos Retrospectivos
3.
Pharm Res ; 37(10): 194, 2020 Sep 11.
Artigo em Inglês | MEDLINE | ID: mdl-32918191

RESUMO

PURPOSE: We characterized three canine P-gp (cP-gp) deficient MDCKII cell lines. Their relevance for identifying efflux transporter substrates and predicting limitation of brain penetration were evaluated. In addition, we discuss how compound selection can be done in drug discovery by using these cell systems. METHOD: hMDR1, hBCRP-transfected, and non-transfected MDCKII ZFN cells (all with knock-down of endogenous cP-gp) were used for measuring permeability and efflux ratios for substrates. The compounds were also tested in MDR1_Caco-2 and BCRP_Caco-2, each with a double knock-out of BCRP/MRP2 or MDR1/MRP2 transporters respectively. Efflux results were compared between the MDCK and Caco-2 models. Furthermore, in vitro MDR1_ZFN efflux data were correlated with in vivo unbound drug brain-to-plasma partition coefficient (Kp,uu). RESULTS: MDR1 and BCRP substrates are correctly classified and robust transporter affinities with control substrates are shown. Cell passage mildly influenced mRNA levels of transfected transporters, but the transporter activity was proven stable for several years. The MDCK and Caco-2 models were in high consensus classifying same efflux substrates. Approx. 80% of enlisted substances were correctly predicted with the MDR1_ZFN model for brain penetration. CONCLUSION: cP-gp deficient MDCKII ZFN models are reliable tools to identify MDR1 and BCRP substrates and useful for predicting efflux liability for brain penetration.


Assuntos
Membro 1 da Subfamília B de Cassetes de Ligação de ATP/deficiência , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/metabolismo , Avaliação Pré-Clínica de Medicamentos/métodos , Proteínas de Neoplasias/metabolismo , Farmacocinética , Subfamília B de Transportador de Cassetes de Ligação de ATP/antagonistas & inibidores , Subfamília B de Transportador de Cassetes de Ligação de ATP/genética , Subfamília B de Transportador de Cassetes de Ligação de ATP/metabolismo , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/antagonistas & inibidores , Membro 2 da Subfamília G de Transportadores de Cassetes de Ligação de ATP/genética , Animais , Barreira Hematoencefálica/metabolismo , Encéfalo/metabolismo , Células CACO-2 , Permeabilidade da Membrana Celular , Dibenzocicloeptenos/farmacologia , Dicetopiperazinas/farmacologia , Cães , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Madin Darby de Rim Canino , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Prazosina/farmacocinética , Quinidina/farmacocinética , Quinolinas/farmacologia , Especificidade por Substrato , Transfecção
4.
J Vis Exp ; (162)2020 08 06.
Artigo em Inglês | MEDLINE | ID: mdl-32831302

RESUMO

Neurite outgrowth assay and neurotoxicity assessment are two major studies that can be performed using the presented method herein. This protocol provides reliable analysis of neuronal morphology together with quantitative measurements of modifications on neurite length and synaptic protein localization and abundance upon treatment with small molecule compounds. In addition to the application of the presented method in neurite outgrowth studies, neurotoxicity assessment can be performed to assess, distinguish and rank commercial chemical compounds based on their potential developmental neurotoxicity effect. Even though cell lines are nowadays widely used in compound screening assays in neuroscience, they often differ genetically and phenotypically from their tissue origin. Primary cells, on the other hand, maintain important markers and functions observed in vivo. Therefore, due to the translation potential and physiological relevance that these cells could offer neurite outgrowth assay and neurotoxicity assessment can considerably benefit from using human neural progenitor cells (hNPCs) as the primary human cell model. The presented method herein can be utilized to screen for the ability of compounds to induce neurite outgrowth and neurotoxicity by taking advantage of the human neural progenitor cell-derived neurons, a cell model closely representing human biology."


Assuntos
Bioensaio/métodos , Células-Tronco Neurais/patologia , Crescimento Neuronal , Neurônios/patologia , Neurotoxinas/toxicidade , Animais , Diferenciação Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Separação Celular , Sobrevivência Celular/efeitos dos fármacos , Células Cultivadas , Epigênese Genética/efeitos dos fármacos , Fluorescência , Congelamento , Humanos , Células-Tronco Neurais/efeitos dos fármacos , Crescimento Neuronal/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Software , Coloração e Rotulagem
5.
Arch Biochem Biophys ; 692: 108517, 2020 10 15.
Artigo em Inglês | MEDLINE | ID: mdl-32738196

RESUMO

The relationship between prion propagation and the generation of neurotoxic species and clinical onset remains unclear. Several converging lines of evidence suggest that interactions with lipids promote various precursors to form aggregation-prone states that are involved in amyloid fibrils. Here, we compared the cytotoxicities of different soluble isolated oligomeric constructs from murine full-length PrP and from the restricted helical H2H3 domain with their effects on lipid vesicles. The helical H2H3 domain is suggested to be the minimal region of PrP involved in the oligomerization process. The discrete PrP oligomers of both the full-length sequence and the H2H3 domain have de novo ß-sheeted structure when interacting with the membrane. They were shown to permeabilize synthetic negatively charged vesicles in a dose-dependent manner. Restricting the polymerization domain of the full-length PrP to the H2H3 helices strongly diminished the ability of the corresponding oligomers to associate with the lipid vesicles. Furthermore, the membrane impairment mechanism occurs differently for the full-length PrP oligomers and the H2H3 helices, as shown by dye-release and black lipid membrane experiments. The membrane damage caused by the full-length PrP oligomers is correlated to their neuronal toxicity at submicromolar concentrations, as shown by cell culture assays. Although oligomers of synthetic H2H3 could compromise in vitro cell homeostasis, they followed a membrane-disruptive pattern that was different from the full-length oligomers, as revealed by the role of PrPC in cell viability assays.


Assuntos
Permeabilidade da Membrana Celular , Membrana Celular/metabolismo , Neurônios/metabolismo , Proteínas da Gravidez/metabolismo , Multimerização Proteica , Animais , Membrana Celular/genética , Camundongos , Camundongos Knockout , Proteínas da Gravidez/genética , Domínios Proteicos , Estrutura Secundária de Proteína
6.
PLoS Pathog ; 16(8): e1008766, 2020 08.
Artigo em Inglês | MEDLINE | ID: mdl-32857822

RESUMO

Pathogens commonly disrupt the intestinal epithelial barrier; however, how the epithelial immune system senses the loss of intestinal barrier as a danger signal to activate self-defense is unclear. Through an unbiased approach in the model nematode Caenorhabditis elegans, we found that the EGL-44/TEAD transcription factor and its transcriptional activator YAP-1/YAP (Yes-associated protein) were activated when the intestinal barrier was disrupted by infections with the pathogenic bacterium Pseudomonas aeruginosa PA14. Gene Ontology enrichment analysis of the genes containing the TEAD-binding sites revealed that "innate immune response" and "defense response to Gram-negative bacterium" were two top significantly overrepresented terms. Genetic inactivation of yap-1 and egl-44 significantly reduced the survival rate and promoted bacterial accumulation in worms after bacterial infections. Furthermore, we found that disturbance of the E-cadherin-based adherens junction triggered the nuclear translocation and activation of YAP-1/YAP in the gut of worms. Although YAP is a major downstream effector of the Hippo signaling, our study revealed that the activation of YAP-1/YAP was independent of the Hippo pathway during disruption of intestinal barrier. After screening 10 serine/threonine phosphatases, we identified that PP2A phosphatase was involved in the activation of YAP-1/YAP after intestinal barrier loss induced by bacterial infections. Additionally, our study demonstrated that the function of YAP was evolutionarily conserved in mice. Our study highlights how the intestinal epithelium recognizes the loss of the epithelial barrier as a danger signal to deploy defenses against pathogens, uncovering an immune surveillance program in the intestinal epithelium.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Proteínas de Caenorhabditis elegans/metabolismo , Permeabilidade da Membrana Celular , Células Epiteliais/imunologia , Microbioma Gastrointestinal/imunologia , Salmonelose Animal/imunologia , Salmonella typhimurium/imunologia , Proteínas Adaptadoras de Transdução de Sinal/genética , Animais , Caenorhabditis elegans , Proteínas de Caenorhabditis elegans/genética , Células Epiteliais/metabolismo , Células Epiteliais/microbiologia , Células Epiteliais/patologia , Camundongos , Salmonelose Animal/metabolismo , Salmonelose Animal/microbiologia , Salmonelose Animal/patologia , Transdução de Sinais
7.
PLoS One ; 15(8): e0237505, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32790727

RESUMO

Increased intestinal permeability (IP) and inflammation are both linked with functionality of the intestinal barrier and in particular enterocytes. Currently, almost all assessment methods of the intestinal barrier function are invasive. The present study aimed to quantify selected proteins as novel biomarkers in excreta of broiler chickens to facilitate non-invasive assessment of gut barrier function using enzyme-linked immunosorbent assays (ELISA). It was further hypothesised that probiotics as feed additives may counteract gut barrier dysfunction. A 3 × 2 factorial arrangement of treatments was used with the main factors being gut barrier dysfunction models (control, rye-based diet, and dexamethasone-DEX) with and without probiotic supplementation (a three-strain Bacillus) using 72 male Ross 308 day-old chickens. Each of the 6 experimental treatments was replicated 12 times. On d 21 of age, fluorescein isothiocyanate dextran (FITC-d) uptake into serum was examined to test IP. Fresh excreta samples were collected on d 20. The biomarkers included alpha-1 antitrypsin (A1AT), intestinal fatty acid binding protein (I-FABP), lipocalin-2 (LCN2), fibronectin (FN), intestinal alkaline phosphatase (IAP), ovotransferrin (OVT) and superoxide dismutase [Cu-Zn] (SOD1). Only DEX increased (P<0.001) FITC-d passage to the blood on d 21 of age, indicating a greater IP. The excreta concentrations of A1AT, I-FABP and SOD1 were unaltered by the experimental treatments. DEX increased (P<0.05) FN concentration in excreta compared with control birds. Conversely, inclusion of rye in the diet reduced (P<0.05) FN but increased (P<0.001) OVT in excreta. Independently, DEX decreased IAP (P<0.05) in excreta compared with control and rye-fed birds. The excreta concentration of LCN2 tended (P = 0.086) to increase in birds injected by DEX. There was no demonstrable effect of probiotic addition on any of the studied parameters. Among the tested biomarkers, FN, IAP, and LCN2 revealed promise as biomarkers of intestinal barrier function quantified by ELISA kits.


Assuntos
Ração Animal/análise , Biomarcadores/análise , Dieta/veterinária , Fezes/química , Microbioma Gastrointestinal/efeitos dos fármacos , Intestinos/patologia , Probióticos/farmacologia , Animais , Permeabilidade da Membrana Celular , Galinhas , Suplementos Nutricionais , Intestinos/efeitos dos fármacos , Masculino
8.
Int J Nanomedicine ; 15: 4049-4062, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606663

RESUMO

Background: Calcium carbonate (CC) nanoparticles have broad biomedical utilizations, owing to their multiple intrinsic merits. However, bare CC nanoparticles do not allow for the development of multifunctional devices suitable for advanced drug delivery in cancer therapy. Methods: Phospholipid-modified phospholipid-CC hybrid nanoparticles were prepared in our study using a combination of vapor-diffusion and solvent-diffusion methods to offer optimized pharmaceutical capabilities. Results: Considering that particle size is a critical parameter that plays an important role in both in vitro and in vivo behaviors of nanoparticles, we here for the first time a present detailed protocol for the size-controlled preparation of hybrid nanoparticles, as well as analysis of the in vitro/in vivo behaviors of differently sized hybrid nanoparticles. Conclusion: Our results might significantly advance the application of this promising material in more varied fields.


Assuntos
Carbonato de Cálcio/química , Nanopartículas/química , Tamanho da Partícula , Fosfolipídeos/química , Animais , Linhagem Celular , Permeabilidade da Membrana Celular , Sistemas de Liberação de Medicamentos/métodos , Endocitose , Feminino , Humanos , Lisossomos/metabolismo , Camundongos Endogâmicos BALB C , Nanopartículas/administração & dosagem , Nanopartículas/ultraestrutura , Coelhos , Esferoides Celulares/metabolismo , Eletricidade Estática , Temperatura , Fatores de Tempo , Distribuição Tecidual
9.
PLoS One ; 15(7): e0235737, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32726322

RESUMO

The primary aim of in vitro testing of chemicals delivered via the percutaneous route is to predict the absorption that would ensue if exposure occurred in live animals. While there is mounting evidence that in vitro diffusion studies in mammalian skin can provide valid information regarding likely in vivo absorption, little is known whether such a correlation exists between in vitro diffusion testing and in vivo blood levels in amphibians. The current study used previously-reported in vitro absorption data for caffeine, benzoic acid, and ibuprofen across isolated skin from the cane toad (Rhinella marina) to produce a series of linear mixed-effect models of the absorption parameters flux and permeability coefficient (Kp). Models investigated the relative impacts of animal weight, physicochemical characteristics of the applied chemical (logP or molecular weight), and site of application. The top models were then used to predict the flux, Kp and serum concentrations of the same three model chemicals. Finally, the absorption of these chemicals was determined in live cane toads, and results compared to the model predictions. LogP and site of application were included in all top models. In vivo absorption rates were lower than predicted for all chemicals, however, the models provided reasonable predictions of serum concentration, with factors of difference (FOD) ranging from 2.5-10.5. Ibuprofen, the chemical with the highest relative lipophilicity, had the poorest predictive performance, consistently having the highest FOD for all predictions. This report presents the first models of percutaneous absorption in an amphibian. These models provide a basic method to establish the approximate in vivo absorption of hydrophilic and moderately-lipophilic chemicals through frog skin, and could therefore be used to predict absorption when formulating such chemicals for treatment of disease in frogs, or for risk-assessments regarding chemical pollutants in frog habitats.


Assuntos
Ácido Benzoico/metabolismo , Bufo marinus/metabolismo , Cafeína/metabolismo , Ibuprofeno/metabolismo , Modelos Biológicos , Absorção Cutânea/fisiologia , Pele/metabolismo , Animais , Permeabilidade da Membrana Celular , Difusão , Técnicas In Vitro , Masculino , Absorção Cutânea/efeitos dos fármacos
10.
Int J Nanomedicine ; 15: 4573-4589, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32606693

RESUMO

Background: Therapeutic efficiency of ceragenins against cancers may be limited by lack of their hemocompatibility when high concentrations of molecules are required to reach a desired result. Synergistic effects observed upon administration of anticancer agents and metal nanoparticles may provide an opportunity to limit toxicity of immobilized ceragenins on the surface of metal nanoparticles and to improve their therapeutic efficiency at the same time. The aim of present work is to investigate the anticancer activities and hemocompatibility of nanoformulations consisting of ceragenin CSA-131 united with aminosilane-modified iron oxide-based magnetic nanoparticles (MNP) and prepared by 1) covalent bonding (MNP@CSA-131) or 2) by combining CSA-131 with MNP in 1:1 ratio (CSA-131 + MNP). Possible synergistic interactions between CSA-131 and magnetic nanoparticles were also quantified. Methods: MNP@CSA-131 and CSA-131+MNP were tested in vitro against selected lung and colon cancer cells using colorimetric, fluorimetric and flow cytometry methods. Results: Performed analysis demonstrates that MNP-based nanosystems significantly improve the killing efficiency of tested ceragenin, decreasing the viability of extra 1.37±4.72% to 76.07±15.30% cancer cells when compared to free CSA-131. Quantification of synergistic effects indicates the favorable interactions between CSA-131 and magnetic nanoparticles (CI < 1 for all tested doses), revealing at the same time a reduction in effective doses of ceragenin from 1.17 ± 0.61 to 34.57 ± 12.78 times when combined with MNP. We demonstrate that both MNP@CSA-131 and CSA-131+MNP induce significantly apoptosis of cancer cells and prevent the division of colon cancer cells even at relatively low doses of the active compound (10 µg/mL). Importantly, combining CSA-131 with MNP decreases the hemolytic activity of free ceragenin 4.72 to 7.88 times, which indicates a considerable improvement of hemotoxicity profile. Conclusion: Comparative analyses have revealed that both developed CSA-containing nanoformulations due to the utility of synergistic interactions between MNP and CSA-131, which are effective against lung and colon cancer cells. This indicates the new directions in preparation of MNP-based therapeutics, which are relatively easy to synthetize, cost-effective and safe when intravenously administrated.


Assuntos
Compostos Férricos/uso terapêutico , Nanopartículas de Magnetita/uso terapêutico , Esteroides/farmacologia , Antineoplásicos/administração & dosagem , Antineoplásicos/farmacologia , Apoptose/efeitos dos fármacos , Morte Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Neoplasias do Colo/patologia , Sinergismo Farmacológico , Humanos , Neoplasias Pulmonares/patologia , Nanopartículas de Magnetita/administração & dosagem , Nanopartículas de Magnetita/ultraestrutura , Teste de Materiais
11.
Life Sci ; 258: 118136, 2020 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-32726662

RESUMO

The endothelium is a critical regulator of vascular homeostasis, controlling vascular tone and permeability as well as interactions of leukocytes and platelets with blood vessel walls. Consequently, endothelial dysfunction featuring inflammation and reduced vasodilation are considered central to cardiovascular disease (CVD) pathogenesis and have become a therapeutic area of focus. Type II endothelial cell (EC) activation by stress-related stimuli such as tumor necrosis factor-α (TNF-α) initiates the nuclear factor-κB (NF-κB) signaling pathway, a master regulator of inflammatory responses. Because dysregulated NF-κB signaling has been tightly linked to several CVDs, EC-specific inhibition of NF-κB represents an attractive pharmacological strategy. As accumulating evidence highlights the clinical benefits of tea catechin for multiple diseases including CVDs, we sought to determine whether the tea catechin epigallocatechin gallate (EGCG) that displays antioxidative, anti-inflammatory, hypolipidemic, anti-thrombogenic, and anti-hypertensive properties offers protection against CVDs by suppressing the canonical NF-κB pathway. Our findings indicate that EGCG downregulates multiple components of the TNF-α-induced NF-κB signaling pathway and thereby reduces the consequent increase in inflammatory gene transcription and protein expression. Furthermore, EGCG blocked type II EC activation, evidenced by diminished EC leakage and monocyte adhesion in EGCG-treated cells. In summary, our study advances knowledge of EGCG's anti-inflammatory effects on the NF-κB pathway and hence its benefits on endothelial health, supporting its therapeutic potential for CVDs.


Assuntos
Catequina/análogos & derivados , Vasos Coronários/patologia , Células Endoteliais/patologia , Inflamação/tratamento farmacológico , Catequina/farmacologia , Catequina/uso terapêutico , Adesão Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Humanos , Inflamação/genética , Inflamação/patologia , Mediadores da Inflamação/metabolismo , Monócitos/efeitos dos fármacos , Monócitos/patologia , NF-kappa B/metabolismo , Fator de Necrose Tumoral alfa/metabolismo
12.
Mol Cell ; 79(1): 68-83.e7, 2020 07 02.
Artigo em Inglês | MEDLINE | ID: mdl-32533918

RESUMO

BAX is a pro-apoptotic protein that transforms from a cytosolic monomer into a toxic oligomer that permeabilizes the mitochondrial outer membrane. How BAX monomers assemble into a higher-order conformation, and the structural determinants essential to membrane permeabilization, remain a mechanistic mystery. A key hurdle has been the inability to generate a homogeneous BAX oligomer (BAXO) for analysis. Here, we report the production and characterization of a full-length BAXO that recapitulates physiologic BAX activation. Multidisciplinary studies revealed striking conformational consequences of oligomerization and insight into the macromolecular structure of oligomeric BAX. Importantly, BAXO enabled the assignment of specific roles to particular residues and α helices that mediate individual steps of the BAX activation pathway, including unexpected functionalities of BAX α6 and α9 in driving membrane disruption. Our results provide the first glimpse of a full-length and functional BAXO, revealing structural requirements for the elusive execution phase of mitochondrial apoptosis.


Assuntos
Apoptose , Mitocôndrias/patologia , Membranas Mitocondriais/metabolismo , Multimerização Proteica , Proteína X Associada a bcl-2/química , Proteína X Associada a bcl-2/metabolismo , Animais , Transporte Biológico , Permeabilidade da Membrana Celular , Citosol/metabolismo , Humanos , Camundongos , Mitocôndrias/metabolismo , Modelos Moleculares , Conformação Proteica , Proteínas Proto-Oncogênicas c-fos
13.
Food Chem ; 328: 127174, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32492604

RESUMO

This study investigated the effect of lysosomal iron involvement in the mechanism of mitochondrial apoptosis on bovine muscle protein degradation during postmortem aging. Six crossbred cattle were studied to evaluate intracellular reactive oxygen species (ROS), antioxidant enzyme activity, lysosomal membrane stability, mitochondrial dysfunction-induced apoptosis, desmin and troponin-T degradation in both control and iron chelator desferrioxamine (DFO) groups. Results showed that lysosomal iron induced ROS accumulation and lysosomal membrane destabilization by decreasing the antioxidant enzyme activity (P < 0.05). Subsequently, lysosomal dysfunction mediated by iron increased mitochondrial membrane permeability and decreased mitochondrial membrane potential, thereby enhancing Bid and cytochrome c release and caspase-9/-3 activation (P < 0.05). Ultimately, lysosomal iron mediated lysosomal-mitochondrial apoptosis increased the postmortem bovine muscle desmin and troponin-T degradation (P < 0.05). The results indicated that lysosomal iron contributes to postmortem meat tenderization through the lysosomal-mitochondrial dysfunction-induced apoptosis pathway.


Assuntos
Ferro/metabolismo , Lisossomos/metabolismo , Proteínas de Carne/metabolismo , Mitocôndrias Musculares/patologia , Membranas Mitocondriais/patologia , Animais , Apoptose/efeitos dos fármacos , Autopsia , Bovinos , Permeabilidade da Membrana Celular , Citocromos c/metabolismo , Masculino , Mitocôndrias/metabolismo , Mitocôndrias Musculares/metabolismo , Membranas Mitocondriais/metabolismo , Proteínas Musculares/metabolismo , Proteólise , Espécies Reativas de Oxigênio/metabolismo
14.
Nat Commun ; 11(1): 2992, 2020 06 12.
Artigo em Inglês | MEDLINE | ID: mdl-32532974

RESUMO

Activated protein C (APC) is a plasma serine protease with antithrombotic and cytoprotective functions. Based on the hypothesis that specific inhibition of APC's anticoagulant but not its cytoprotective activity can be beneficial for hemophilia therapy, 2 types of inhibitory monoclonal antibodies (mAbs) are tested: A type I active-site binding mAb and a type II mAb binding to an exosite on APC (required for anticoagulant activity) as shown by X-ray crystallography. Both mAbs increase thrombin generation and promote plasma clotting. Type I blocks all APC activities, whereas type II preserves APC's cytoprotective function. In normal monkeys, type I causes many adverse effects including animal death. In contrast, type II is well-tolerated in normal monkeys and shows both acute and prophylactic dose-dependent efficacy in hemophilic monkeys. Our data show that the type II mAb can specifically inhibit APC's anticoagulant function without compromising its cytoprotective function and offers superior therapeutic opportunities for hemophilia.


Assuntos
Anticorpos Monoclonais/farmacologia , Hemofilia A/prevenção & controle , Fragmentos Fab das Imunoglobulinas/imunologia , Inibidor da Proteína C/farmacologia , Proteína C/antagonistas & inibidores , Animais , Anticorpos Monoclonais/classificação , Anticorpos Monoclonais/imunologia , Tempo de Sangramento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Cultivadas , Cristalografia por Raios X , Hemofilia A/sangue , Hemorragia/prevenção & controle , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Células Endoteliais da Veia Umbilical Humana/fisiologia , Humanos , Fragmentos Fab das Imunoglobulinas/metabolismo , Macaca fascicularis , Masculino , Proteína C/química , Proteína C/imunologia , Proteína C/metabolismo , Inibidor da Proteína C/sangue , Inibidor da Proteína C/farmacocinética
15.
PLoS One ; 15(6): e0220598, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32579545

RESUMO

Roof greening is an important national policy for maintaining the hydrological balance in China; however, plant growth is limited by drought stress. This study aims to identify strong drought resistant plant species for roof greening from ten common species: Paeonia lactiflora, Hemerocallis dumortieri, Meehania urticifolia, Iris lactea var. chinensis, Hylotelephium erythrostictum, Sedum lineare, Iris germanica, Cosmos bipinnata, Hosta plantaginea, and Dianthus barbatus. By controlling the soil relative water content (RWC), we designed three treatments: moderate drought stress (40±2% < RWC < 45±2%), severe drought stress (RWC < 30±2%) and well-watered control (RWC > 75±2%). After the seedlings were provided different levels of water, their membrane permeability (MP), chlorophyll concentration (Chl), and superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) activity were measured. Finally, the membership function method was used to assess the drought resistance of these species. The results showed that C. bipinnata and M. urticifolia were not suitable for moderate or severe drought stress and did not survive. The other species presented variations in physiological and biochemical parameters. The MP of He. dumortieri, I. lactea and Ho. plantaginea showed minor changes between the well-watered control and drought stress. Most of the species showed reduced SOD activity under moderate drought stress but increased activity under severe stress. All of the plant species showed decreases in the protective enzymes POD and APX with increasing drought stress. The membership function method was applied to calculate the plant species' drought resistance, and the following order of priority of the roof-greening plant species was suggested: He. dumortieri > I. germanica > I. lactea > D. barbatus > Hy. erythrostictum > S. lineare > Ho. plantaginea > P. lactiflora.


Assuntos
Secas , Fenômenos Fisiológicos Vegetais , Plântula/fisiologia , Estresse Fisiológico , Ascorbato Peroxidases/metabolismo , Permeabilidade da Membrana Celular , Clorofila/metabolismo , Peroxidases/metabolismo , Plântula/metabolismo , Solo/química , Superóxido Dismutase/metabolismo , Análise de Sobrevida , Água/análise
16.
Life Sci ; 256: 117884, 2020 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-32502546

RESUMO

AIMS: Endothelial barrier dysfunction is associated with multiple diseases, and barrier repair may be a possible therapeutic target. Yes-associated protein and its pathway have been implicated in organ repair after injury. However, the mechanisms underlying barrier repair and any role YAP plays in the process are unclear. This study aimed to explore the role and mechanism of YAP in the repair of endothelial cell permeability after TNF-α-induced injury. MAIN METHODS: A trans-endothelial electrical resistance assay was performed to investigate changes in endothelial cell permeability. Lentivirus packaging by calcium phosphate transfection was used to construct endothelial cell lines with knocked down or overexpressed YAP. Western blotting, immunofluorescence, CO-IP, and real-time PCR were used to detect related protein and gene expression. KEY FINDINGS: YAP is involved in the repair process of TNF-α-induced endothelial cell permeability injury; its overexpression promotes repair of endothelial cell permeability, and knockdown weakens repair ability. Moreover, YAP may promote repair by down-regulating STAT3 activity, thereby inhibiting VEGF expression. SIGNIFICANCE: Elucidating the role of YAP in endothelial cell permeability repair process after injury might reveal mechanisms of endothelial barrier repair and provide therapeutic targets for treatment of vascular hyper-permeability disease.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Células Endoteliais da Veia Umbilical Humana/metabolismo , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator A de Crescimento do Endotélio Vascular/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Humanos , Ligação Proteica/efeitos dos fármacos , Transdução de Sinais/efeitos dos fármacos , Fator de Necrose Tumoral alfa/farmacologia
17.
Biochim Biophys Acta Bioenerg ; 1861(10): 148251, 2020 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-32598881

RESUMO

Saponins are a diverse group of secondary plant metabolites, some of which display hemolytic toxicity due to plasma membrane permeabilization. This feature is employed in biological applications for transferring hydrophilic molecules through cell membranes. Widely used commercial saponins include digitonin and saponins from soap tree bark, both of which constitute complex mixtures of little definition. We assessed the permeabilization power of pure saponins towards cellular membranes in an effort to detect novel properties and to improve existing applications. In a respirometric assay, we characterized half-maximal permeabilization of the plasma membrane for different metabolites, of the mitochondrial outer membrane for cytochrome C and the full solubilization of mitochondrial inner membrane protein complexes. Beyond the complete list as repository for the field, we highlight several findings with direct applicability. First, we identified and validated α-chaconine as alternative permeabilization agent in respirometric assays of cultured cells and isolated synaptosomes, superior to digitonin in its tolerability for mitochondria. Second, we identified glycyrrhizic acid to form exceptionally small pores impermeable for adenosine diphosphate. Third, in a concentration dependent manner, tomatine proved to be able to selectively permeabilize the mitochondrial outer, but not inner membrane, allowing for novel states in which to determine cytochrome C oxidase activity. In summary, we provide a list of the permeabilization properties of 18 pure saponins. The identification of two saponins, namely tomatine and chaconine, with direct usability in improved or novel cell biological applications within this small subgroup demonstrates the tremendous potential for further functional screening of pure saponins.


Assuntos
Metabolismo/efeitos dos fármacos , Saponinas/farmacologia , Animais , Calorimetria , Permeabilidade da Membrana Celular/efeitos dos fármacos , Complexo IV da Cadeia de Transporte de Elétrons/metabolismo , Células HEK293 , Humanos , Camundongos
18.
Nat Commun ; 11(1): 3184, 2020 06 23.
Artigo em Inglês | MEDLINE | ID: mdl-32576824

RESUMO

Peptide antibiotics are an abundant and synthetically tractable source of molecular diversity, but they are often cationic and can be cytotoxic, nephrotoxic and/or ototoxic, which has limited their clinical development. Here we report structure-guided optimization of an amphipathic peptide, arenicin-3, originally isolated from the marine lugworm Arenicola marina. The peptide induces bacterial membrane permeability and ATP release, with serial passaging resulting in a mutation in mlaC, a phospholipid transport gene. Structure-based design led to AA139, an antibiotic with broad-spectrum in vitro activity against multidrug-resistant and extensively drug-resistant bacteria, including ESBL, carbapenem- and colistin-resistant clinical isolates. The antibiotic induces a 3-4 log reduction in bacterial burden in mouse models of peritonitis, pneumonia and urinary tract infection. Cytotoxicity and haemolysis of the progenitor peptide is ameliorated with AA139, and the 'no observable adverse effect level' (NOAEL) dose in mice is ~10-fold greater than the dose generally required for efficacy in the infection models.


Assuntos
Antibacterianos/química , Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Peptídeos Catiônicos Antimicrobianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Animais , Carbapenêmicos/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Colistina/farmacologia , Modelos Animais de Doenças , Descoberta de Drogas , Feminino , Proteínas de Helminto/química , Proteínas de Helminto/farmacologia , Humanos , Masculino , Camundongos , Testes de Sensibilidade Microbiana , Peritonite/tratamento farmacológico , Peritonite/microbiologia , Pneumonia/tratamento farmacológico , Pneumonia/microbiologia , Infecções Urinárias/tratamento farmacológico , Infecções Urinárias/microbiologia
19.
PLoS One ; 15(6): e0232549, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32484808

RESUMO

Prodigiosin is an important secondary metabolite produced by Serratia marcescens. It can help strains resist stresses from other microorganisms and environmental factors to achieve self-preservation. Prodigiosin is also a promising secondary metabolite due to its pharmacological characteristics. However, pigmentless S. marcescens mutants always emerge after prolonged starvation, which might be a way for the bacteria to adapt to starvation conditions, but it could be a major problem in the industrial application of S. marcescens. To identify the molecular mechanisms of loss of prodigiosin production, two mutants were isolated after 16 days of prolonged incubation of wild-type (WT) S. marcescens 1912768R; one mutant (named 1912768WR) exhibited reduced production of prodigiosin, and a second mutant (named 1912768W) was totally defective. Comparative genomic analysis revealed that the two mutants had either mutations or deletions in rpoS. Knockout of rpoS in S. marcescens 1912768R had pleiotropic effects. Complementation of rpoS in the ΔrpoS mutant further confirmed that RpoS was a positive regulator of prodigiosin production and that its regulatory role in prodigiosin biosynthesis was opposite that in Serratia sp. ATCC 39006, which had a different type of pig cluster; further, rpoS from Serratia sp. ATCC 39006 and other strains complemented the prodigiosin defect of the ΔrpoS mutant, suggesting that the pig promoters are more important than the genes in the regulation of prodigiosin production. Deletion of rpoS strongly impaired the resistance of S. marcescens to stresses but increased membrane permeability for nutritional competence; competition assays in rich and minimum media showed that the ΔrpoS mutant outcompeted its isogenic WT strain. All these data support the idea that RpoS is pleiotropic and that the loss of prodigiosin biosynthesis in S. marcescens 1912768R during prolonged incubation is due to a mutation in rpoS, which appears to be a self-preservation and nutritional competence (SPANC) trade-off.


Assuntos
Proteínas de Bactérias/metabolismo , Biofilmes/crescimento & desenvolvimento , Enzimas/metabolismo , Prodigiosina/biossíntese , Serratia marcescens/fisiologia , Sideróforos/biossíntese , Fator sigma/metabolismo , Proteínas de Bactérias/genética , Permeabilidade da Membrana Celular/fisiologia , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes , Movimento/fisiologia , Regiões Promotoras Genéticas , Fator sigma/genética , Estresse Fisiológico
20.
Nat Immunol ; 21(7): 746-755, 2020 07.
Artigo em Inglês | MEDLINE | ID: mdl-32514064

RESUMO

Plasma membranes of animal cells are enriched for cholesterol. Cholesterol-dependent cytolysins (CDCs) are pore-forming toxins secreted by bacteria that target membrane cholesterol for their effector function. Phagocytes are essential for clearance of CDC-producing bacteria; however, the mechanisms by which these cells evade the deleterious effects of CDCs are largely unknown. Here, we report that interferon (IFN) signals convey resistance to CDC-induced pores on macrophages and neutrophils. We traced IFN-mediated resistance to CDCs to the rapid modulation of a specific pool of cholesterol in the plasma membrane of macrophages without changes to total cholesterol levels. Resistance to CDC-induced pore formation requires the production of the oxysterol 25-hydroxycholesterol (25HC), inhibition of cholesterol synthesis and redistribution of cholesterol to an esterified cholesterol pool. Accordingly, blocking the ability of IFN to reprogram cholesterol metabolism abrogates cellular protection and renders mice more susceptible to CDC-induced tissue damage. These studies illuminate targeted regulation of membrane cholesterol content as a host defense strategy.


Assuntos
Infecções Bacterianas/imunologia , Toxinas Bacterianas/imunologia , Hidroxicolesteróis/metabolismo , Interferons/isolamento & purificação , Fagócitos/imunologia , Estreptolisinas/imunologia , Animais , Bactérias/imunologia , Bactérias/metabolismo , Proteínas de Bactérias/administração & dosagem , Proteínas de Bactérias/imunologia , Proteínas de Bactérias/metabolismo , Toxinas Bacterianas/metabolismo , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular/imunologia , Células Cultivadas , Modelos Animais de Doenças , Suscetibilidade a Doenças/imunologia , Feminino , Interações entre Hospedeiro e Microrganismos/imunologia , Humanos , Microscopia Intravital , Masculino , Camundongos , Camundongos Transgênicos , Fagócitos/citologia , Fagócitos/metabolismo , Cultura Primária de Células , Esteroide Hidroxilases/genética , Esteroide Hidroxilases/metabolismo , Estreptolisinas/administração & dosagem , Estreptolisinas/metabolismo
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