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1.
Results Probl Cell Differ ; 67: 233-250, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31435798

RESUMO

Morphology of Golgi apparatus changes frequently and diversely depending on various cellular conditions and these changes correlate with the balance between membrane inflow and outflow at the Golgi via vesicular transports. In a previous study, we introduced a semi-intact cell system suitable for the reconstitution of morphological changes that organelles undergo as well as the vesicular transport between them. Semi-intact cells are cells that have undergone plasma membrane permeabilization by the cholesterol-dependent pore-forming cytolysin, streptolysin O (SLO). Permeabilization enables the introduction of various molecules into the cells, as well as the substitution of the original cytosol with an exogenously made cytosol prepared from cells in various stages of cell cycle, differentiation, and disease progression. Coupled with a green fluorescent protein(GFP)-visualization technique, this cell-based system enables the analysis of the molecular mechanisms underlying biological processes that are highly dependent on the integrity of the intracellular architecture. In this chapter, we present a variety of reconstitution assays concerning biological reactions pertaining to the Golgi apparatus.


Assuntos
Permeabilidade da Membrana Celular , Complexo de Golgi/metabolismo , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citosol/química , Citosol/efeitos dos fármacos , Citosol/metabolismo , Complexo de Golgi/efeitos dos fármacos
2.
Life Sci ; 233: 116697, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31351968

RESUMO

AIMS: The present study investigated if berberine might induce Zrt-Irt-like protein 14 (ZIP14) and affect zinc redistribution to protect intestinal barrier in sepsis. MAIN METHODS: Rodent model of sepsis was induced by cecal ligation and puncture (CLP). Plasma endotoxin was assayed by LAL test and plasma zinc was measured by flame atomic spectrophotometer. Gut mucosal permeability was determined by plasma FITC-dextran. Zinc content and ZIP14 mRNA in gut mucosa were assayed by spectrophotometer and qRT-PCR, respectively. Tight junction integrity of Caco-2 was evaluated by transepithelial electrical resistance (TEER). Tight junction (TJ) protein expression was detected by Western blotting. KEY FINDINGS: Berberine and zinc gluconate pretreatment to CLP rats improved survival rate, reduced plasma endotoxin level, alleviated hypozincemia, increased zinc accumulation and ZIP14 mRNA expression in the intestinal mucosa. Berberine and zinc gluconate pretreatment decreased CLP-elicited intestinal hyperpermeability to FITC-dextran. These effects of berberine in vivo were abolished by AG1024. In vitro, lipopolysaccharide (LPS) repressed zinc transfer into Caco-2 cells exposed to zinc gluconate. Berberine and IGF-I treatment increased ZIP14 protein expression and promoted zinc transfer into Caco-2 cells exposed to zinc gluconate plus LPS. Berberine treatment induced TJ protein (claudin-1 and occludin) and raised TEER in LPS-treated Caco-2 cells. These effects of berberine in vitro were partially inhibited by ZIP14 siRNA. SIGNIFICANCE: The present study reveals that berberine induces ZIP14 expression and affects zinc re- distribution to protect intestinal barrier in sepsis, which is partially linked with the activation of IGF-I signaling.


Assuntos
Berberina/farmacologia , Proteínas de Transporte de Cátions/metabolismo , Coinfecção/prevenção & controle , Gluconatos/farmacologia , Mucosa Intestinal/efeitos dos fármacos , Sepse/prevenção & controle , Tirfostinas/farmacologia , Zinco/metabolismo , Animais , Células CACO-2 , Permeabilidade da Membrana Celular/efeitos dos fármacos , Coinfecção/metabolismo , Coinfecção/microbiologia , Humanos , Masculino , Substâncias Protetoras/farmacologia , Ratos , Ratos Wistar , Sepse/metabolismo , Sepse/microbiologia , Transdução de Sinais/efeitos dos fármacos
3.
Life Sci ; 230: 178-187, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31152810

RESUMO

AIMS: With the purpose of exploring combinatorial options that could enhance the bactericide efficacy of linezolid against Gram-negative bacteria, we assessed the extent of combination of nano-silver and linezolid. MAIN METHODS: In this study, we selected Escherichia coli MTCC 443 as a model to study the combinatorial effect of nano-silver and linezolid to combat efflux-mediated resistance in Gram-negative bacteria. The acting mechanism of nano-silver on E. coli MTCC 443 was investigated by evaluating interaction of nano-silver with bacterial membrane as well as bacterial surface charge, morphology, intracellular leakages and biological activities of membrane bound respiratory chain dehydrogenase and deoxyribonucleic acids (DNA) of the cells following treatment with nano-silver. KEY FINDINGS: The alternation of zeta potential due to the interaction of nano-silver towards bacterial membrane proteins was correlated with enhancement of membrane permeability, which allows the penetration of linezolid into the cells. In addition, the binding affinity of nano-silver towards bacterial membrane depressed biological activities of membrane bound respiratory chain dehydrogenases and DNA integrity. SIGNIFICANCE: Our findings suggested that nano-silver could not only obstruct the activities of efflux pumps, but also altered membrane integrity at the same time and thus increased the cytoplasmic concentration of the linezolid to the effective level.


Assuntos
Proteínas da Membrana Bacteriana Externa/efeitos dos fármacos , Nanopartículas Metálicas/uso terapêutico , Prata/farmacologia , Antibacterianos/farmacologia , Proteínas de Bactérias/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Farmacorresistência Bacteriana/efeitos dos fármacos , Farmacorresistência Bacteriana Múltipla , Escherichia coli/efeitos dos fármacos , Escherichia coli/metabolismo , Bactérias Gram-Negativas/metabolismo , Linezolida/metabolismo , Linezolida/farmacologia , Prata/metabolismo
4.
Eur J Med Chem ; 178: 30-38, 2019 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-31173969

RESUMO

Several studies that have identified agents that potentiate the antimicrobial activity of antibiotics, but there are limited insights into their structure-activity relationships (SAR). The SAR associated with select N-alkylaryl amide derivatives of ornithine was performed to establish those structural features that were associated with potentiation of the antimicrobial activity of clarithromycin against E. coli ATCC 25922. The data indicate that the N-propyl derivative was slightly more active in reducing the effective MIC of clarithromycin against E. coli ATCC 25922. In addition, the S-enantiomer of compound 9 was somewhat more potent than the R-enantiomer in potentiating clarithromycin activity. No significant enhancement in potentiation activity was observed with the conversion of these secondary amides to their N-methyl tertiary amides. Formation of the N-methyl or N,N-dimethyl derivatives of the primary amine of 9 was associated with the loss of potentiation activity. Conversion of this primary amine to a guanidine was also not associated with an increase in potentiation activity. Among the isomeric diamino pentamides, 15 potentiated the antibacterial activity of clarithromycin to the greatest extent. In addition to these amide derivatives, the desoxy derivatives 16 and 18 were the more potent potentiators within this triamine series. The relative location of the primary amines, as indicated by the relative differences in the potentiation observed with 16 compared to 14, appears to be a critical factor in determining potentiation activity. Cell-based membrane permeabilization and efflux inhibition studies in E. coli ATCC 25922 suggest that the potentiation of clarithromycin activity by 16 reflects its ability to inhibit efflux pump activity and to a lesser extent its actions as a permeabilizer of the outer leaflet of the outer cell membrane.


Assuntos
Amidas/farmacologia , Antibacterianos/farmacologia , Claritromicina/farmacologia , Escherichia coli/efeitos dos fármacos , Ornitina/farmacologia , Amidas/síntese química , Amidas/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sinergismo Farmacológico , Proteínas de Membrana Transportadoras/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Estrutura Molecular , Ornitina/análogos & derivados , Ornitina/síntese química , Relação Estrutura-Atividade
5.
Sci Data ; 6(1): 46, 2019 05 02.
Artigo em Inglês | MEDLINE | ID: mdl-31048742

RESUMO

The large amount of existing nanomaterials demands rapid and reliable methods for testing their potential toxicological effect on human health, preferably by means of relevant in vitro techniques in order to reduce testing on animals. Combining high throughput workflows with automated high content imaging techniques allows deriving much more information from cell-based assays than the typical readouts (i.e. one measurement per well) with optical plate-readers. We present here a dataset including data based on a maximum of 14 different read outs (including viable cell count, cell membrane permeability, apoptotic cell death, mitochondrial membrane potential and steatosis) of the human hepatoma HepaRG cell line treated with a large set of nanomaterials, coatings and supernatants at different concentrations. The database, given its size, can be utilized in the development of in silico hazard assessment and prediction tools or can be combined with toxicity results from other in vitro test systems.


Assuntos
Bases de Dados Factuais , Nanoestruturas/toxicidade , Apoptose/efeitos dos fármacos , Carcinoma Hepatocelular/patologia , Contagem de Células , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , Neoplasias Hepáticas/patologia , Potencial da Membrana Mitocondrial/efeitos dos fármacos
6.
AAPS PharmSciTech ; 20(5): 180, 2019 May 01.
Artigo em Inglês | MEDLINE | ID: mdl-31044335

RESUMO

The process of liposome fusion with cellular membrane plays key role in delivering encapsulated drug molecule into the cell. This process becomes very important for molecules having low permeability as they fail to reach the site of action located inside the cell. Ciprofloxacin (CIP), a broad-spectrum BCS class IV antibiotic, has poor permeability. In the present work, CIP-loaded liposomes were prepared using solvent evaporation method and optimized by 32 factorial design approach. The optimized batch of CIP-loaded liposomes was characterized for size, entrapment efficiency, zeta potential, FTIR, and microbial susceptibility study on Staphylococcus aureus (gram-positive bacteria) and Escherichia coli (gram-negative bacteria). Confocal microscopy was used to study the fusogenicity process of CIP-loaded liposomes with bacterial cells. Additionally, the kinetics of fusogenicity process was studied using SAXS for the first time. Surprisingly, the rate of fusion of CIP-loaded liposomes with cell wall of S. aureus was twice when compared to the cell wall of E. coli. It is believed that the current work can act as a roadmap in selection of proper excipients while developing formulations which would expedite the fusogenicity and may execute pharmacological activity of poorly penetrable drug molecules at lower dose.


Assuntos
Antibacterianos/administração & dosagem , Antibacterianos/farmacologia , Bactérias/efeitos dos fármacos , Ciprofloxacino/administração & dosagem , Ciprofloxacino/farmacologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Composição de Medicamentos , Sistemas de Liberação de Medicamentos , Escherichia coli/efeitos dos fármacos , Lipossomos , Staphylococcus aureus/efeitos dos fármacos
7.
Int J Nanomedicine ; 14: 2781-2795, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31114195

RESUMO

Purpose: Amoxicillin is a commonly used antibiotic, although degraded by the acidic pH of the stomach. This is an important limitation for the treatment of Helicobacter pylori infections. The purpose of this work was to encapsulate amoxicillin in lipid nanoparticles, increasing the retention time at the site of infection (gastric mucosa), while protecting the drug from the harsh conditions of the stomach lumen. Materials and methods: The nanoparticles were produced by the double emulsion technique and optimized by a three-level Box-Behnken design. Tween 80 and linolenic acid were used as potential therapeutic adjuvants and dioleoylphosphatidylethanolamine as a targeting agent to Helicobacter pylori. Nanoparticles were characterized regarding their physico-chemical features, their storage stability, and their usability for oral administration (assessment of in vitro release, in vitro cell viability, permeability, and interaction with mucins). Results: The nanoparticles were stable for at least 6 months at 4°C. In vitro release studies revealed a high resistance to harsh conditions, including acidic pH and physiologic temperature. The nanoparticles have a low cytotoxicity effect in both fibroblasts and gastric cell lines, and they have the potential to be retained at the gastric mucosa. Conclusion: Overall, the designed formulations present suitable physico-chemical features for being henceforward used by oral administration to treat Helicobacter pylori infections.


Assuntos
Amoxicilina/administração & dosagem , Amoxicilina/uso terapêutico , Infecções por Helicobacter/tratamento farmacológico , Lipídeos/química , Nanopartículas/química , Amoxicilina/farmacologia , Animais , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Sobrevivência Celular , Quitosana/química , Portadores de Fármacos/química , Liberação Controlada de Fármacos , Helicobacter pylori/efeitos dos fármacos , Humanos , Camundongos , Mucinas/metabolismo , Nanopartículas/ultraestrutura , Tamanho da Partícula
8.
Carbohydr Polym ; 217: 58-68, 2019 Aug 01.
Artigo em Inglês | MEDLINE | ID: mdl-31079685

RESUMO

The fruit of Akebia trifoliata var. australis can be consumed as food. However, the peel of this fruit is typically regarded as waste. The application of such waste can create opportunities to produce new and valuable by-products. Herein, we have shown that citric acid extracted pectin (CEP) from Akebia trifoliata var. australis peel has good water solubility and high galacturonic units, which helps reduce AgNO3 into Ag nanoparticles (CEP-AgNPs) through a one-step, eco-friendly process. The resulting CEP-AgNPs showed sustained release of Ag+ and remarkable antibacterial activity. Subsequently, the CEP-AgNPs were processed into a CEP-Ag sponge with excellent water absorption and prolonged water retention properties. The CEP-Ag sponge could support the cell adhesion and proliferation. Most importantly, the sponge effectively facilitated a moist environment with bacterial disinfection capability which accelerated the healing of infected wounds. Thus, CEP-Ag sponge, a sustainable and high value by-product, was obtained from food waste.


Assuntos
Antibacterianos/uso terapêutico , Bandagens , Nanopartículas Metálicas/uso terapêutico , Pectinas/química , Resíduos Sólidos , Cicatrização/efeitos dos fármacos , Animais , Antibacterianos/química , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Permeabilidade da Membrana Celular/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , Feminino , Fibroblastos/efeitos dos fármacos , Frutas/química , Química Verde/métodos , Humanos , Nanopartículas Metálicas/química , Nanopartículas Metálicas/toxicidade , Staphylococcus aureus Resistente à Meticilina/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Pectinas/isolamento & purificação , Pectinas/toxicidade , Ranunculales/química , Ratos Sprague-Dawley , Prata/química , Prata/uso terapêutico , Prata/toxicidade , Nitrato de Prata/química , Pele/patologia , Solubilidade , Água/química , Molhabilidade
9.
Invest Ophthalmol Vis Sci ; 60(6): 1943-1952, 2019 05 01.
Artigo em Inglês | MEDLINE | ID: mdl-31050722

RESUMO

Purpose: Ocular angiogenesis, including retinopathy of prematurity, diabetic retinopathy, and exudative age-related macular degeneration, are closely related to oxidative stress. Many reports have shown that the cellular protective mechanism against oxidative stress and inflammatory response has nuclear factor-erythroid 2-related factor-2 (Nrf2) activity. The aim of this study was to investigate the effectiveness and mechanism of Nrf2 activation in treating the ocular diseases with abnormal vessels. Methods: The effects of Nrf2 activators, bardoxolone methyl (BARD) and RS9, were evaluated against vascular endothelial growth factor (VEGF)-induced cell migration in human retinal microvascular endothelial cells (HRMECs). We measured the expression of the Nrf2 target genes, Ho-1 and Nqo-1 mRNA, in mouse retinas after a single injection of BARD and RS9. The effects and mechanisms of RS9 against retinal angiogenesis were evaluated using an oxygen-induced retinopathy (OIR) model in mice. Moreover, the effect of RS9 against choroidal neovascularization (CNV) was evaluated in a laser-induced CNV monkey model. Results: Both BARD and RS9 decreased VEGF-induced cell migration, and significantly increased Ho-1 mRNA expression; however, only RS9 significantly increased Nqo-1 mRNA. RS9 decreased retinal neovascularization through suppressing VEGF expression and increasing Nrf2, HO-1, platelet-derived growth factor receptor (PDGFR)-ß, and tight junction proteins in OIR murine retinas. Furthermore, RS9 showed a tendency toward decreasing CNV lesions, and improved vascular leakage in a CNV monkey model. Conclusions: These data indicate that a Nrf2 activator might be a candidate for treatment of ocular diseases characterized by pathophysiological angiogenesis and hyperpermeability.


Assuntos
Neovascularização de Coroide/tratamento farmacológico , Fator 2 Relacionado a NF-E2/metabolismo , Ácido Oleanólico/análogos & derivados , Neovascularização Retiniana/tratamento farmacológico , Vasos Retinianos/metabolismo , Triterpenos/farmacologia , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Neovascularização de Coroide/metabolismo , Neovascularização de Coroide/patologia , Modelos Animais de Doenças , Células Endoteliais/efeitos dos fármacos , Células Endoteliais/metabolismo , Células Endoteliais/patologia , Angiofluoresceinografia , Fundo de Olho , Humanos , Immunoblotting , Macaca fascicularis , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fator 2 Relacionado a NF-E2/efeitos dos fármacos , Ácido Oleanólico/farmacologia , Neovascularização Retiniana/metabolismo , Neovascularização Retiniana/patologia , Vasos Retinianos/patologia , Tomografia de Coerência Óptica
10.
Molecules ; 24(7)2019 Mar 27.
Artigo em Inglês | MEDLINE | ID: mdl-30934783

RESUMO

Plant essential oils (EOs) are one of the most relevant natural products due to their biological, medicinal, and nutritional properties. The promising biological effects of many plants EOs encourage researchers to study their biochemical properties to be used as possible natural alternatives for commercial pesticides and not only as herbal medicines. The current research has been conducted to study the microbicide effect of Solidago canadensis L. EO to control some common plant diseases caused by several postharvest phytopathogenic fungi (Monilinia fructicola, Botrytis cinerea, Aspergillus niger, and Penicillium expansum) in comparison with Azoxystrobin as a large spectrum fungicide. The antibacterial activity has been carried out against some phytopathogenic bacteria (Bacillus megaterium and Clavibacter michiganensis (G+ve) and Xanthomonas campestris, Pseudomonas fluorescens, and Pseudomonas syringae pv. phaseolicola (G-ve)) compared to the synthetic antibiotic Tetracycline. Minimum inhibitory concentration was carried out to determine the lowest effective EO dose using a 96-well microplate. The cell membrane permeability was also evaluated by measuring the electric conductivity (EC) to examine the possible mechanisms of action of S. canadensis EO. Chemical characterization of EO has been carried out using gas chromatography and mass spectrometry (GC-MS). Thirty-two identified components in S. canadensis EO presented 97.7% of total compounds in EO. The principal compounds were identified as germacrene D (34.9%), limonene (12.5%), α-pinene (11.6%), ß-elemene (7.1%), and bornyl acetate (6.3%). In addition, S. canadensis EO demonstrated promising in vitro antimicrobial activities against the majority of tested phytopathogens at all tested concentrations.


Assuntos
Anti-Infecciosos/química , Anti-Infecciosos/farmacologia , Óleos Voláteis/química , Óleos Voláteis/farmacologia , Compostos Fitoquímicos/química , Extratos Vegetais/química , Extratos Vegetais/farmacologia , Solidago/química , Antifúngicos/química , Antifúngicos/farmacologia , Bactérias/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Fungos/efeitos dos fármacos , Cromatografia Gasosa-Espectrometria de Massas , Testes de Sensibilidade Microbiana
11.
Toxicon ; 165: 110-115, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-31029638

RESUMO

BACKGROUND: Cytotoxin 1 (CTX1) purified from Naja atra Cantor venom could inhibit cancer cell proliferation, but the mechanism is not clear. This study aimed to investigate the mechanism by which leukemia cells are killed by CTX1. MATERIALS AND METHODS: HL-60 and KG1a cells were treated with CTX1 and the cell death was detected. RESULTS: The viability of HL-60 and KG1a cells decreased in a dose- and time-dependent manner after treatment with CTX1. CTX1 mainly induced late apoptosis and necrosis. The cell death induced by CTX1 could be rescued by specific necroptosis inhibitor Nec-1 but not by caspase inhibitor Z-VAD-fmk in HL-60 cells. In addition, CTX1 increased lysosome membrane permeability (LMP) and release of cathepsin B. CONCLUSION: CTX1 could induce necroptosis in leukemia cells, and it is related to LMP increase and cathepsin release. CTX1 could be a promising anti-cancer drug for leukemia therapy.


Assuntos
Antineoplásicos/farmacologia , Citotoxinas/farmacologia , Venenos Elapídicos/farmacologia , Necrose/induzido quimicamente , Animais , Antineoplásicos/isolamento & purificação , Apoptose/efeitos dos fármacos , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Citotoxinas/isolamento & purificação , Venenos Elapídicos/química , Células HL-60 , Humanos , Leucemia , Naja naja
12.
Food Microbiol ; 82: 504-514, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31027812

RESUMO

As a result of the rapidly growing human population, reducing post-harvest crop losses of cereals due to microbial pests has major importance. Plant defensins have the potential to fulfil these demands, being highly specific and efficient antimicrobial agents. Hence, this study aimed to extract and characterise a peptide from cowpea seeds and investigate its antifungal performance. After extraction and partial purification, N-terminal sequencing was used to identify the primary peptide in the extract as cowpea-thionin II. Antifungal activity in vitro was found against Fusarium culmorum (MIC = 50 µg/mL), but Aspergillus niger and Penecillium expansum showed an MIC > 500 µg/mL. The extract was resistant against heat treatment (100 °C, 15 min) but lost its antifungal activity in presence of cations (Na+, K+, Ca2+ and Mg2+, respectively). Membrane permeabilization of fungal hyphae was evident at 25 µg/mL, while induction of oxidative stress only had minor contribution to the antifungal performance. The extract did not induce haemolysis at all concentrations tested (up to 200 µg/mL). Finally, it was successfully used to protect stored wheat grains from fungal spoilage (determined via ergosterol content) when applied at 100 µg/mL. In conclusion, the defensin Cp-thionin II showed the potential for future application as food bio-preservative.


Assuntos
Antifúngicos/farmacologia , Conservantes de Alimentos/farmacologia , Fungos/efeitos dos fármacos , Proteínas de Plantas/farmacologia , Tioninas/farmacologia , Vigna/química , Antifúngicos/química , Antifúngicos/isolamento & purificação , Cátions , Permeabilidade da Membrana Celular/efeitos dos fármacos , Defensinas/química , Defensinas/isolamento & purificação , Defensinas/farmacologia , Grão Comestível/microbiologia , Ergosterol/análise , Ergosterol/metabolismo , Microbiologia de Alimentos , Conservantes de Alimentos/química , Conservantes de Alimentos/isolamento & purificação , Fungos/metabolismo , Fungos/fisiologia , Temperatura Alta , Hifas/efeitos dos fármacos , Hifas/metabolismo , Hifas/fisiologia , Testes de Sensibilidade Microbiana , Proteínas de Plantas/química , Proteínas de Plantas/isolamento & purificação , Estabilidade Proteica , Sementes/química , Tioninas/química , Tioninas/isolamento & purificação
13.
Nat Commun ; 10(1): 1541, 2019 04 04.
Artigo em Inglês | MEDLINE | ID: mdl-30948723

RESUMO

Protein aggregation is a complex process resulting in the formation of heterogeneous mixtures of aggregate populations that are closely linked to neurodegenerative conditions, such as Alzheimer's disease. Here, we find that soluble aggregates formed at different stages of the aggregation process of amyloid beta (Aß42) induce the disruption of lipid bilayers and an inflammatory response to different extents. Further, by using gradient ultracentrifugation assay, we show that the smaller aggregates are those most potent at inducing membrane permeability and most effectively inhibited by antibodies binding to the C-terminal region of Aß42. By contrast, we find that the larger soluble aggregates are those most effective at causing an inflammatory response in microglia cells and more effectively inhibited by antibodies targeting the N-terminal region of Aß42. These findings suggest that different toxic mechanisms driven by different soluble aggregated species of Aß42 may contribute to the onset and progression of Alzheimer's disease.


Assuntos
Peptídeos beta-Amiloides/toxicidade , Bicamadas Lipídicas/metabolismo , Agregação Patológica de Proteínas , Peptídeos beta-Amiloides/metabolismo , Animais , Permeabilidade da Membrana Celular/efeitos dos fármacos , Camundongos , Microglia/efeitos dos fármacos , Microglia/metabolismo , Ultracentrifugação
14.
J Microbiol ; 57(4): 288-297, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30929229

RESUMO

The detailed antibacterial mechanism of cordycepin efficacy against food-borne germs remains ambiguous. In this study, the antibacterial activity and action mechanism of cordycepin were assessed. The results showed that cordycepin effectively inhibited the growth of seven bacterial pathogens including both Gram-positive and Gram-negative bacterial pathogens; the minimum inhibitory concentrations (MIC) were 2.5 and 1.25 mg/ml against Escherichia coli and Bacillus subtilis, respectively. Scanning electron microscope and transmission electron microscope examination confirmed that cordycepin caused obvious damages in the cytoplasmatic membranes of both E. coli and B. subtilis. Outer membrane permeability assessment indicated the loss of barrier function and the leakage of cytoplasmic contents. Propidium iodide and carboxyfluorescein diacetate double staining approach coupled with flow cytometry analysis indicated that the integrity of cell membrane was severely damaged during a short time, while the intracellular enzyme system still remained active. This clearly suggested that membrane damage was one of the reasons for cordycepin efficacy against bacteria. Additionally, results from circular dichroism and fluorescence analysis indicated cordycepin could insert to genome DNA base and double strand, which disordered the structure of genomic DNA. Basis on these results, the mode of bactericidal action of cordycepin against E. coli and B. subtilis was found to be a dual mechanism, disrupting bacterial cell membranes and binding to bacterial genomic DNA to interfere in cellular functions, ultimately leading to cell death.


Assuntos
Antibacterianos/farmacologia , Bacillus subtilis/efeitos dos fármacos , Desoxiadenosinas/farmacologia , Escherichia coli/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Testes de Sensibilidade Microbiana
15.
Microbes Environ ; 34(2): 155-160, 2019 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-30905896

RESUMO

Aphids have a mutualistic relationship with the bacterial endosymbiont Buchnera aphidicola. We previously reported seven cysteine-rich peptides in the pea aphid Acyrthosiphon pisum and named them Bacteriocyte-specific Cysteine-Rich (BCR) peptides; these peptides are exclusively expressed in bacteriocytes, special aphid cells that harbor symbionts. Similar symbiotic organ-specific cysteine-rich peptides identified in the root nodules of leguminous plants are named Nodule-specific Cysteine-Rich (NCR) peptides. NCR peptides target rhizobia in the nodules and are essential for symbiotic nitrogen fixation. A BacA (membrane protein) mutant of Sinorhizobium is sensitive to NCR peptides and is unable to establish symbiosis. Based on the structural and expressional similarities between BCR peptides and NCR peptides, we hypothesized that aphid BCR peptides exhibit antimicrobial activity, similar to some NCR peptides. We herein synthesized BCR peptides and investigated their antimicrobial activities and effects on the bacterial membrane of Escherichia coli. The peptides BCR1, BCR3, BCR5, and BCR8 exhibited antimicrobial activities with increased membrane permeability. An sbmA mutant of E. coli, a homolog of bacA of S. meliloti, was more sensitive to BCR peptides than the wild type. Our results suggest that BCR peptides have properties that may be required to control the endosymbiont, similar to NCR peptides in legumes.


Assuntos
Anti-Infecciosos/farmacologia , Afídeos/metabolismo , Cisteína/química , Proteínas de Insetos/farmacologia , Peptídeos/farmacologia , Animais , Anti-Infecciosos/síntese química , Anti-Infecciosos/química , Afídeos/microbiologia , Buchnera/fisiologia , Permeabilidade da Membrana Celular/efeitos dos fármacos , Permeabilidade da Membrana Celular/genética , Escherichia coli/citologia , Escherichia coli/efeitos dos fármacos , Escherichia coli/genética , Proteínas de Insetos/síntese química , Proteínas de Insetos/química , Mutação , Peptídeos/síntese química , Peptídeos/química , Sinorhizobium meliloti/efeitos dos fármacos , Sinorhizobium meliloti/genética , Simbiose
16.
Methods Mol Biol ; 1943: 313-322, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-30838625

RESUMO

A better understanding of the molecular basis of polycation-mediated impairment of mitochondrial bioenergetics might improve the design and synthesis of more efficient and safer polymeric transfectants. Here we utilize the phosphorylation control protocol for studying the effect of polycations on mitochondrial respiration in intact mammalian cells using Oxygraph-2k (OROBOROS). The protocol offers an opportunity to comprehensively monitor mitochondrial respiration through consecutive additions of various cell membrane permeable compounds that alter mitochondrial respiration, thus providing useful information on different states of mitochondrial respiration. Furthermore, we demonstrate how to analyze the data obtained with the phosphorylation control protocol and how to calculate the respiratory flux ratios, which can be used as indicators of respiratory functionality and mitochondrial health.


Assuntos
Mitocôndrias/efeitos dos fármacos , Poliaminas/toxicidade , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Respiração Celular/efeitos dos fármacos , Humanos , Mitocôndrias/metabolismo , Fosforilação Oxidativa/efeitos dos fármacos , Consumo de Oxigênio/efeitos dos fármacos , Espectrofotometria/instrumentação , Espectrofotometria/métodos , Transfecção/métodos
17.
Microsc Microanal ; 25(1): 135-150, 2019 02.
Artigo em Inglês | MEDLINE | ID: mdl-30869575

RESUMO

Possible multidrug-resistant (MDR) mechanisms of four resistant strains of Escherichia coli to a model ß-lactam, ampicillin, were investigated using contact angle measurements of wettability, crystal violet assays of permeability, biofilm formation, fluorescence imaging, and nanoscale analyses of dimensions, adherence, and roughness. Upon exposure to ampicillin, one of the resistant strains, E. coli A5, changed its phenotype from elliptical to spherical, maintained its roughness and biofilm formation abilities, decreased its length and surface area, maintained its cell wall integrity, increased its hydrophobicity, and decreased its nanoscale adhesion to a model surface of silicon nitride. Such modifications are suggested to allow these cells to conserve energy during metabolic dormancy. In comparison, resistant strains E. coli D4, A9, and H5 elongated their cells, increased their roughness, increased their nanoscale adhesion forces, became more hydrophilic, and increased their biofilm formation upon exposure to ampicillin. These results suggest that these strains resisted ampicillin through biofilm formation that possibly introduces diffusion limitations to antibiotics. Investigations of how MDR bacterial cells modify their surfaces in response to antibiotics can guide research efforts aimed at designing more effective antibiotics and new treatment strategies for MDR bacterial infections.


Assuntos
Antibacterianos/farmacologia , Farmacorresistência Bacteriana Múltipla/efeitos dos fármacos , Escherichia coli/efeitos dos fármacos , beta-Lactamas/farmacologia , Ampicilina/farmacologia , Aderência Bacteriana/efeitos dos fármacos , Biofilmes/efeitos dos fármacos , Biofilmes/crescimento & desenvolvimento , Permeabilidade da Membrana Celular/efeitos dos fármacos , Parede Celular/efeitos dos fármacos , Escherichia coli/citologia , Escherichia coli/crescimento & desenvolvimento , Escherichia coli/metabolismo , Gelatina , Interações Hidrofóbicas e Hidrofílicas/efeitos dos fármacos , Imobilização , Testes de Sensibilidade Microbiana , Fenótipo , Propriedades de Superfície/efeitos dos fármacos , Molhabilidade/efeitos dos fármacos
18.
Nat Chem ; 11(4): 342-350, 2019 04.
Artigo em Inglês | MEDLINE | ID: mdl-30903037

RESUMO

The architecture and bioactivity of natural products frequently serve as embarkation points for the exploration of biologically relevant chemical space. Total synthesis followed by derivative synthesis has historically enabled a deeper understanding of structure-activity relationships. However, synthetic strategies towards a natural product are not always guided by hypotheses regarding the structural features required for bioactivity. Here, we report an approach to natural product total synthesis that we term 'pharmacophore-directed retrosynthesis'. A hypothesized, pharmacophore of a natural product is selected as an early synthetic target and this dictates the retrosynthetic analysis. In an ideal application, sequential increases in the structural complexity of this minimal structure enable development of a structure-activity relationship profile throughout the course of the total synthesis effort. This approach enables the identification of simpler congeners retaining bioactivity at a much earlier stage of a synthetic effort, as demonstrated here for the spongiane diterpenoid, gracilin A, leading to simplified derivatives with potent neuroprotective and immunosuppressive activity.


Assuntos
Acetatos/química , Diterpenos/química , Furanos/química , Imunossupressores/química , Fármacos Neuroprotetores/química , Acetatos/síntese química , Acetatos/farmacologia , Linhagem Celular Tumoral , Permeabilidade da Membrana Celular/efeitos dos fármacos , Cristalografia por Raios X , Reação de Cicloadição , Diterpenos/síntese química , Diterpenos/farmacologia , Desenho de Drogas , Furanos/síntese química , Furanos/farmacologia , Humanos , Imunossupressores/síntese química , Imunossupressores/farmacologia , Membranas Mitocondriais/metabolismo , Conformação Molecular , Neurônios/citologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/síntese química , Fármacos Neuroprotetores/farmacologia , Espécies Reativas de Oxigênio/metabolismo , Relação Estrutura-Atividade
19.
Nat Commun ; 10(1): 1390, 2019 03 27.
Artigo em Inglês | MEDLINE | ID: mdl-30918252

RESUMO

The AB5 toxins cholera toxin (CT) from Vibrio cholerae and heat-labile enterotoxin (LT) from enterotoxigenic Escherichia coli are notorious for their roles in diarrheal disease, but their effect on other intestinal bacteria remains unexplored. Another foodborne pathogen, Campylobacter jejuni, can mimic the GM1 ganglioside receptor of CT and LT. Here we demonstrate that the toxin B-subunits (CTB and LTB) inhibit C. jejuni growth by binding to GM1-mimicking lipooligosaccharides and increasing permeability of the cell membrane. Furthermore, incubation of CTB or LTB with a C. jejuni isolate capable of altering its lipooligosaccharide structure selects for variants lacking the GM1 mimic. Examining the chicken GI tract with immunofluorescence microscopy demonstrates that GM1 reactive structures are abundant on epithelial cells and commensal bacteria, further emphasizing the relevance of this mimicry. Exposure of chickens to CTB or LTB causes shifts in the gut microbial composition, providing evidence for new toxin functions in bacterial gut competition.


Assuntos
Toxinas Bacterianas/farmacologia , Campylobacter jejuni/efeitos dos fármacos , Permeabilidade da Membrana Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Enterotoxinas/farmacologia , Proteínas de Escherichia coli/farmacologia , Microbioma Gastrointestinal/efeitos dos fármacos , Mucosa Intestinal/metabolismo , Lipopolissacarídeos/metabolismo , Animais , Bactérias/efeitos dos fármacos , Bactérias/metabolismo , Campylobacter jejuni/metabolismo , Galinhas , Gangliosídeo G(M1)/metabolismo , Glicoconjugados/metabolismo , Mucosa Intestinal/patologia , Microscopia de Fluorescência , Receptores de Superfície Celular/efeitos dos fármacos , Receptores de Superfície Celular/metabolismo
20.
Mol Cells ; 42(3): 262-269, 2019 Mar 31.
Artigo em Inglês | MEDLINE | ID: mdl-30841024

RESUMO

The porcine myeloid antimicrobial peptide (PMAP), one of the cathelicidin family members, contains small cationic peptides with amphipathic properties. We used a putative lysozyme originated from the bacteriophage P22 (P22 lysozyme) as a fusion partner, which was connected to the N-terminus of the PMAP36 peptide, to markedly increase the expression levels of recombinant PMAP36. The PMAP36-P22 lysozyme fusion protein with high solubility was produced in Escherichia coli. The final purified yield was approximately 1.8 mg/L. The purified PMAP36-P22 lysozyme fusion protein exhibited antimicrobial activity against both Gram-negative and Gram-positive bacteria (Staphylococcus aureus, Salmonella enterica serovar Typhimurium, Pseudomonas aeruginosa, and Bacillus subtilis). Furthermore, we estimated its hemolytic activity against pig erythrocytes as 6% at the high concentration (128 µM) of the PMAP36-P22 lysozyme fusion protein. Compared with the PMAP36 peptide (12%), our fusion protein exhibited half of the hemolytic activity. Overall, our recombinant PMAP36-P22 lysozyme fusion protein sustained the antimicrobial activity with the lower hemolytic activity associated with the synthetic PMAP36 peptide. This study suggests that the PMAP36-P22 lysozyme fusion system could be a crucial addition to the plethora of novel antimicrobials.


Assuntos
Peptídeos Catiônicos Antimicrobianos/metabolismo , Muramidase/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Proteínas Recombinantes de Fusão/farmacologia , Animais , Anti-Infecciosos/farmacologia , Peptídeos Catiônicos Antimicrobianos/química , Bactérias/efeitos dos fármacos , Bactérias/ultraestrutura , Permeabilidade da Membrana Celular/efeitos dos fármacos , Feminino , Hemólise/efeitos dos fármacos , Fatores Imunológicos/farmacologia , Camundongos Endogâmicos C57BL , Testes de Sensibilidade Microbiana , Muramidase/química , Plasmídeos/metabolismo , Estrutura Secundária de Proteína , Proteínas Recombinantes de Fusão/química , Suínos
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