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1.
Molecules ; 26(13)2021 Jun 26.
Artigo em Inglês | MEDLINE | ID: mdl-34206777

RESUMO

Previous studies have revealed the numerous biological activities of the fruits of Illicium verum; however, the activities of its leaves and twigs have remained undiscovered. The study aimed to investigate the phytochemical components and antibacterial activity of the various extracts from the leaves and twigs of Illicium verum. The herbal extracts were prepared by supercritical CO2 extraction (SFE) and 95% ethanol extraction, followed by partition extraction based on solvent polarity. Analysis of antimicrobial activity was conducted through the usage of nine clinical antibiotic- resistant isolates, including Staphylococcus aureus, Pseudomonas aeruginosa and Acinetobacter baumannii. Among the tested samples, the SFE extracts exhibited broader and stronger antibacterial activities against the test strains, with a range of MIC between 0.1-4.0 mg/mL and MBC between 0.2-4.5 mg/mL. Observations made through scanning electron microscopy revealed potential mechanism of the antimicrobial activities involved disruption of membrane integrity of the test pathogens. Evaluation of the chemical composition by gas chromatography-mass spectrometry indicated the presence of anethole, anisyl aldehyde, anisyl acetone and anisyl alcohol within the SFE extracts, demonstrating significant correlations with the antibacterial activities observed. Therefore, the leaves and twigs of Illicium verum hold great potential in being developed as new natural antibacterial agents.


Assuntos
Antibacterianos/farmacologia , Anti-Infecciosos/farmacologia , Illicium/química , Extratos Vegetais/análise , Extratos Vegetais/farmacologia , Acinetobacter baumannii/efeitos dos fármacos , Acinetobacter baumannii/ultraestrutura , Antibacterianos/análise , Anti-Infecciosos/análise , Membrana Celular/efeitos dos fármacos , Membrana Celular/ultraestrutura , Sobrevivência Celular/efeitos dos fármacos , Cromatografia Gasosa , Espectrometria de Massas , Testes de Sensibilidade Microbiana , Microscopia Eletrônica de Varredura , Extratos Vegetais/química , Folhas de Planta/química , Pseudomonas aeruginosa/efeitos dos fármacos , Pseudomonas aeruginosa/ultraestrutura , Staphylococcus aureus/efeitos dos fármacos , Staphylococcus aureus/ultraestrutura
2.
ACS Appl Mater Interfaces ; 13(27): 31371-31378, 2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34196172

RESUMO

Owing to their remarkable infiltrative traits, glioblastomas develop unclear tumor margins toward the brain, hampering the complete resection. Since the remaining invasive cells tend to have resistance to therapeutics and cause recurrence around the surgical voids, this has been a major challenge for glioblastoma treatment. Thus, we design a cancer cell-sticky hydrogel (CSH) that interacts with the glioblastoma cells to impede their invasive motility by modifying the cell membrane with active thiol-enriched interfaces. Highly reactive thiols at the cell surface can make the infiltrated cancer cells adhere to the hydrogel, resulting in increased cell adhesion and decreased motility. Cotreatment with the CSH and chemical inhibitors of the major proinvasive molecules, focal adhesion kinase and hyaluronic acid synthase, maximized the invasion-inhibitory effect. In addition, a significant decrease in tumor mass was achieved via CSH implantation in mouse models. Overall, our results highlight the use of the CSH to inhibit the aggressive invasion as a novel therapeutic strategy against glioblastoma.


Assuntos
Neoplasias Encefálicas/patologia , Membrana Celular/efeitos dos fármacos , Glioblastoma/patologia , Hidrogéis/farmacologia , Adesão Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Movimento Celular/efeitos dos fármacos , Desenho de Fármacos , Proteína-Tirosina Quinases de Adesão Focal/metabolismo , Humanos , Hidrogéis/química , Invasividade Neoplásica , Compostos de Sulfidrila/química
3.
Int J Mol Sci ; 22(14)2021 Jul 14.
Artigo em Inglês | MEDLINE | ID: mdl-34299160

RESUMO

Infections with Candida spp. are commonly found in long-time denture wearers, and when under immunosuppression can lead to stomatitis. Imidazolium ionic liquids with an alkyl or alkyloxymethyl chain and a natural (1R,2S,5R)-(-)-menthol substituent possess high antifungal and antiadhesive properties towards C. albicans, C. parapsilosis, C. glabrata and C. krusei. We tested three compounds and found they disturbed fungal plasma membranes, with no significant hemolytic properties. In the smallest hemolytic concentrations, all compounds inhibited C. albicans biofilm formation on acrylic, and partially on porcelain and alloy dentures. Biofilm eradication may result from hyphae inhibition (for alkyl derivatives) or cell wall lysis and reduction of adhesins level (for alkyloxymethyl derivative). Thus, we propose the compounds presented herein as potential anti-fungal denture cleaners or denture fixatives, especially due to their low toxicity towards mammalian erythrocytes after short-term exposure.


Assuntos
Adesinas Bacterianas/metabolismo , Antifúngicos/farmacologia , Biofilmes/crescimento & desenvolvimento , Candida albicans/fisiologia , Imidazóis/química , Líquidos Iônicos/química , Mentol/farmacologia , Antipruriginosos/farmacologia , Biofilmes/efeitos dos fármacos , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos
4.
J Chem Phys ; 154(24): 245101, 2021 Jun 28.
Artigo em Inglês | MEDLINE | ID: mdl-34241335

RESUMO

Ethanol is highly effective against various enveloped viruses and can disable the virus by disintegrating the protective envelope surrounding it. The interactions between the coronavirus envelope (E) protein and its membrane environment play key roles in the stability and function of the viral envelope. By using molecular dynamics simulation, we explore the underlying mechanism of ethanol-induced disruption of a model coronavirus membrane and, in detail, interactions of the E-protein and lipids. We model the membrane bilayer as N-palmitoyl-sphingomyelin and 1-palmitoyl-2-oleoylphosphatidylcholine lipids and the coronavirus E-protein. The study reveals that ethanol causes an increase in the lateral area of the bilayer along with thinning of the bilayer membrane and orientational disordering of lipid tails. Ethanol resides at the head-tail region of the membrane and enhances bilayer permeability. We found an envelope-protein-mediated increase in the ordering of lipid tails. Our simulations also provide important insights into the orientation of the envelope protein in a model membrane environment. At ∼25 mol. % of ethanol in the surrounding ethanol-water phase, we observe disintegration of the lipid bilayer and dislocation of the E-protein from the membrane environment.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Coronavirus/metabolismo , Desinfetantes/farmacologia , Etanol/farmacologia , Proteínas do Envelope Viral/metabolismo , Coronavirus/fisiologia , Bicamadas Lipídicas/metabolismo , Conformação Molecular , Simulação de Dinâmica Molecular , Permeabilidade
5.
Int J Mol Sci ; 22(11)2021 Jun 04.
Artigo em Inglês | MEDLINE | ID: mdl-34200063

RESUMO

The modification of archaeal lipid bilayer properties by the insertion of apolar molecules in the lipid bilayer midplane has been proposed to support cell membrane adaptation to extreme environmental conditions of temperature and hydrostatic pressure. In this work, we characterize the insertion effects of the apolar polyisoprenoid squalane on the permeability and fluidity of archaeal model membrane bilayers, composed of lipid analogues. We have monitored large molecule and proton permeability and Laurdan generalized polarization from lipid vesicles as a function of temperature and hydrostatic pressure. Even at low concentration, squalane (1 mol%) is able to enhance solute permeation by increasing membrane fluidity, but at the same time, to decrease proton permeability of the lipid bilayer. The squalane physicochemical impact on membrane properties are congruent with a possible role of apolar intercalants on the adaptation of Archaea to extreme conditions. In addition, such intercalant might be used to cheaply create or modify chemically resistant liposomes (archeaosomes) for drug delivery.


Assuntos
Archaea/fisiologia , Membrana Celular/fisiologia , Bicamadas Lipídicas/metabolismo , Lipossomos/metabolismo , Fluidez de Membrana , Esqualeno/análogos & derivados , Archaea/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Esqualeno/farmacologia , Temperatura
6.
Ecotoxicol Environ Saf ; 221: 112431, 2021 Sep 15.
Artigo em Inglês | MEDLINE | ID: mdl-34146980

RESUMO

Detailed characterization of new Pseudomonas strains that degrade toxic pollutants is required and utterly necessary before their potential use in environmental microbiology and biotechnology applications. Therefore, phenol degradation by Pseudomonas putida KB3 under suboptimal temperatures, pH, and salinity was examined in this study. Parallelly, adaptive mechanisms of bacteria to stressful growth conditions concerning changes in cell membrane properties during phenol exposure as well as the expression level of genes encoding catechol 2,3-dioxygenase (xylE) and cyclopropane fatty acid synthase (cfaB) were determined. It was found that high salinity and the low temperature had the most significant effect on the growth of bacteria and the rate of phenol utilization. Degradation of phenol (300 mg L-1) proceeded 12-fold and seven-fold longer at 10 °C and 5% NaCl compared to the optimal conditions. The ability of bacteria to degrade phenol was coupled with a relatively high activity of catechol 2,3-dioxygenase. The only factor that inhibited enzyme activity by approximately 80% compared to the control sample was salinity. Fatty acid methyl ester (FAMEs) profiling, membrane permeability measurements, and hydrophobicity tests indicated severe alterations in bacteria membrane properties during phenol degradation in suboptimal growth conditions. The highest values of pH, salinity, and temperature led to a decrease in membrane permeability. FAME analysis showed fatty acid saturation indices and cyclopropane fatty acid participation at high temperature and salinity. Genetic data showed that suboptimal growth conditions primarily resulted in down-regulation of xylE and cfaB gene expression.


Assuntos
Adaptação Fisiológica/genética , Fenol/metabolismo , Pseudomonas putida/genética , Pseudomonas putida/metabolismo , Biodegradação Ambiental , Catecol 2,3-Dioxigenase/genética , Membrana Celular/efeitos dos fármacos , Regulação Bacteriana da Expressão Gênica/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Metiltransferases/genética , Fenol/toxicidade , Pseudomonas putida/efeitos dos fármacos , Salinidade , Temperatura
7.
Int J Mol Sci ; 22(10)2021 May 14.
Artigo em Inglês | MEDLINE | ID: mdl-34069257

RESUMO

Candida albicans is an opportunistic pathogen that induces vulvovaginal candidiasis (VVC), among other diseases. In the vaginal environment, the source of carbon for C. albicans can be either lactic acid or its dissociated form, lactate. It has been shown that lactate, similar to the popular antifungal drug fluconazole (FLC), reduces the expression of the ERG11 gene and hence the amount of ergosterol in the plasma membrane. The Cdr1 transporter that effluxes xenobiotics from C. albicans cells, including FLC, is delocalized from the plasma membrane to a vacuole under the influence of lactate. Despite the overexpression of the CDR1 gene and the increased activity of Cdr1p, C. albicans is fourfold more sensitive to FLC in the presence of lactate than when glucose is the source of carbon. We propose synergistic effects of lactate and FLC in that they block Cdr1 activity by delocalization due to changes in the ergosterol content of the plasma membrane.


Assuntos
Antifúngicos/farmacologia , Candida albicans/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Fluconazol/farmacologia , Ácido Láctico/farmacologia , Candida albicans/genética , Candida albicans/metabolismo , Membrana Celular/metabolismo , Farmacorresistência Fúngica/efeitos dos fármacos , Farmacorresistência Fúngica/genética , Sinergismo Farmacológico , Ergosterol/metabolismo , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulação Fúngica da Expressão Gênica/efeitos dos fármacos , Glucose/metabolismo , Glucose/farmacologia , Ácido Láctico/metabolismo , Proteínas de Membrana Transportadoras/genética , Proteínas de Membrana Transportadoras/metabolismo , Testes de Sensibilidade Microbiana , Transporte Proteico/efeitos dos fármacos
8.
Nat Commun ; 12(1): 3444, 2021 06 08.
Artigo em Inglês | MEDLINE | ID: mdl-34103528

RESUMO

AKT is involved in a number of key cellular processes including cell proliferation, apoptosis and metabolism. Hyperactivation of AKT is associated with many pathological conditions, particularly cancers. Emerging evidence indicates that arginine methylation is involved in modulating AKT signaling pathway. However, whether and how arginine methylation directly regulates AKT kinase activity remain unknown. Here we report that protein arginine methyltransferase 5 (PRMT5), but not other PRMTs, promotes AKT activation by catalyzing symmetric dimethylation of AKT1 at arginine 391 (R391). Mechanistically, AKT1-R391 methylation cooperates with phosphatidylinositol 3,4,5 trisphosphate (PIP3) to relieve the pleckstrin homology (PH)-in conformation, leading to AKT1 membrane translocation and subsequent activation by phosphoinositide-dependent kinase-1 (PDK1) and the mechanistic target of rapamycin complex 2 (mTORC2). As a result, deficiency in AKT1-R391 methylation significantly suppresses AKT1 kinase activity and tumorigenesis. Lastly, we show that PRMT5 inhibitor synergizes with AKT inhibitor or chemotherapeutic drugs to enhance cell death. Altogether, our study suggests that R391 methylation is an important step for AKT activation and its oncogenic function.


Assuntos
Arginina/metabolismo , Carcinogênese/metabolismo , Carcinogênese/patologia , Proteína-Arginina N-Metiltransferases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Animais , Antineoplásicos/farmacologia , Biocatálise/efeitos dos fármacos , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Proliferação de Células/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Células HEK293 , Humanos , Metilação/efeitos dos fármacos , Camundongos Nus , Mutação/genética , Ligação Proteica/efeitos dos fármacos , Inibidores de Proteínas Quinases/farmacologia , Proteína-Arginina N-Metiltransferases/deficiência , Proteínas Proto-Oncogênicas c-akt/química , Piruvato Desidrogenase Quinase de Transferência de Acetil/metabolismo , RNA Interferente Pequeno/metabolismo , Transdução de Sinais/efeitos dos fármacos
9.
Nat Commun ; 12(1): 3675, 2021 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-34135326

RESUMO

Gangliosides in the outer leaflet of the plasma membrane of eukaryotic cells are essential for many cellular functions and pathogenic interactions. How gangliosides are dynamically organized and how they respond to ligand binding is poorly understood. Using fluorescence anisotropy imaging of synthetic, fluorescently labeled GM1 gangliosides incorporated into the plasma membrane of living cells, we found that GM1 with a fully saturated C16:0 acyl chain, but not with unsaturated C16:1 acyl chain, is actively clustered into nanodomains, which depends on membrane cholesterol, phosphatidylserine and actin. The binding of cholera toxin B-subunit (CTxB) leads to enlarged membrane domains for both C16:0 and C16:1, owing to binding of multiple GM1 under a toxin, and clustering of CTxB. The structure of the ceramide acyl chain still affects these domains, as co-clustering with the glycosylphosphatidylinositol (GPI)-anchored protein CD59 occurs only when GM1 contains the fully saturated C16:0 acyl chain, and not C16:1. Thus, different ceramide species of GM1 gangliosides dictate their assembly into nanodomains and affect nanodomain structure and function, which likely underlies many endogenous cellular processes.


Assuntos
Membrana Celular/química , Ceramidas/química , Actinas/química , Antígenos CD59/química , Membrana Celular/efeitos dos fármacos , Toxina da Cólera/química , Toxina da Cólera/farmacologia , Colesterol/química , Gangliosídeo G(M1)/química , Glicoesfingolipídeos/química , Glicosilfosfatidilinositóis/química , Modelos Biológicos , Simulação de Dinâmica Molecular , Fosfatidilserinas/química
10.
Int J Mol Sci ; 22(9)2021 May 07.
Artigo em Inglês | MEDLINE | ID: mdl-34067102

RESUMO

Significant antibacterial properties of non-thermal plasma (NTP) have converted this technology into a promising alternative to the widespread use of antibiotics in assisted reproduction. As substantial data available on the specific in vitro effects of NTP on male reproductive cells are currently missing, this study was designed to investigate selected quality parameters of human spermatozoa (n = 51) exposed to diffuse coplanar surface barrier discharge NTP for 0 s, 15 s, 30 s, 60 s and 90 s. Sperm motility characteristics, membrane integrity, mitochondrial activity, production of reactive oxygen species (ROS), DNA fragmentation and lipid peroxidation (LPO) were investigated immediately following exposure to NTP and 2 h post-NTP treatment. Exposure to NTP with a power input of 40 W for 15 s or 30 s was found to have no negative effects on the sperm structure or function. However, a prolonged NTP treatment impaired all the sperm quality markers in a time- and dose-dependent manner. The most likely mechanism of action of high NTP doses may be connected to ROS overproduction, leading to plasma membrane destabilization, LPO, mitochondrial failure and a subsequent loss of motility as well as DNA integrity. As such, our findings indicate that appropriate plasma exposure conditions need to be carefully selected in order to preserve the sperm vitality, should NTP be used in the practical management of bacteriospermia in the future.


Assuntos
Gases em Plasma/farmacologia , Espermatozoides/fisiologia , Adulto , Apoptose/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Fragmentação do DNA/efeitos dos fármacos , Humanos , Peroxidação de Lipídeos/efeitos dos fármacos , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Motilidade Espermática/efeitos dos fármacos , Espermatozoides/efeitos dos fármacos , Superóxidos/metabolismo , Fatores de Tempo
11.
Nat Commun ; 12(1): 3405, 2021 06 07.
Artigo em Inglês | MEDLINE | ID: mdl-34099708

RESUMO

Single-cell multiparameter measurement has been increasingly recognized as a key technology toward systematic understandings of complex molecular and cellular functions in biological systems. Despite extensive efforts in analytical techniques, it is still generally challenging for existing methods to decipher a large number of phenotypes in a single living cell. Herein we devise a multiplexed Raman probe panel with sharp and mutually resolvable Raman peaks to simultaneously quantify cell surface proteins, endocytosis activities, and metabolic dynamics of an individual live cell. When coupling it to whole-cell spontaneous Raman micro-spectroscopy, we demonstrate the utility of this technique in 14-plexed live-cell profiling and phenotyping under various drug perturbations. In particular, single-cell multiparameter measurement enables powerful clustering, correlation, and network analysis with biological insights. This profiling platform is compatible with live-cell cytometry, of low instrument complexity and capable of highly multiplexed measurement in a robust and straightforward manner, thereby contributing a valuable tool for both basic single-cell biology and translation applications such as high-content cell sorting and drug discovery.


Assuntos
Separação Celular/métodos , Microscopia Intravital/métodos , Microscopia Óptica não Linear/métodos , Análise de Célula Única/métodos , Animais , Células COS , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Chlorocebus aethiops , Descoberta de Drogas/métodos , Endocitose/efeitos dos fármacos , Células HeLa , Humanos , Proteínas de Membrana/metabolismo , Estudo de Prova de Conceito
12.
Aging (Albany NY) ; 13(9): 12817-12832, 2021 05 11.
Artigo em Inglês | MEDLINE | ID: mdl-33972461

RESUMO

Aging is one of the significant risk factors for Alzheimer's disease (AD). Therefore, this study aimed to propose a new hypothesis "membrane aging" as a critical pathogenesis of AD. The concept of "membrane aging" was reviewed, and the possible mechanisms of membrane aging as the primary culprit of AD were clarified. To further prove this hypothesis, a hydroxyurea-induced "membrane aging" model was established in vitro and in vivo. First, neuronal aging was validated by immunocytochemistry with age-related markers, and membrane aging phenotypes were confirmed. The alterations of membrane fluidity within APP/PS1 mice were re-proved by intracerebroventricular injection of hydroxyurea. Decreased membrane fluidity was found in vitro and in vivo, accompanied by increased total cholesterol concentration in neurons but decreased cholesterol levels within membrane fractions. The Aß level increased considerably after hydroxyurea treatment both in vitro and in vivo. DHA co-treatment ameliorated membrane aging phenotypes and Aß aggregation. The study revealed the AMP-activated protein kinase/acetyl CoA carboxylase/carnitine palmitoyl transferase 1 pathway involved in membrane aging processes. These results strongly supported the idea that membrane aging was a pathogenesis of AD and might serve as a new therapeutic target for AD.


Assuntos
Envelhecimento/patologia , Doença de Alzheimer/patologia , Membrana Celular/patologia , Fluidez de Membrana/efeitos dos fármacos , Neurônios/patologia , Doença de Alzheimer/induzido quimicamente , Doença de Alzheimer/genética , Peptídeos beta-Amiloides/metabolismo , Animais , Animais Recém-Nascidos , Encéfalo/citologia , Encéfalo/patologia , Membrana Celular/efeitos dos fármacos , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Hidroxiureia/administração & dosagem , Hidroxiureia/toxicidade , Injeções Intraventriculares , Masculino , Camundongos , Camundongos Transgênicos , Neurônios/citologia , Neurônios/efeitos dos fármacos , Cultura Primária de Células , Ratos
13.
Methods Mol Biol ; 2274: 391-441, 2021.
Artigo em Inglês | MEDLINE | ID: mdl-34050488

RESUMO

Single-molecule imaging (SMI) is a powerful method to measure the dynamics of membrane proteins on the cell membrane. The single-molecule tracking (SMT) analysis provides information about the diffusion dynamics, the oligomer size distribution, and the particle density change. The affinity and on/off-rate of a protein-protein interaction can be estimated from the dual-color SMI analysis. However, it is difficult for trainees to determine quantitative information from the SMI movies. The present protocol guides the detailed workflows to measure the drug-activated dynamics of a G protein-coupled receptor (GPCR) and metabotropic glutamate receptor 3 (mGluR3), by using the total internal reflection fluorescence microscopy (TIRFM). This tutorial guidance comprises an open-source software, named smDynamicsAnalyzer, with which one can easily analyze the SMT dataset by just following the workflows after building a designated folder structure ( https://github.com/masataka-yanagawa/IgorPro8-smDynamicsAnalyzer ).


Assuntos
Membrana Celular/metabolismo , Microscopia de Fluorescência/métodos , Preparações Farmacêuticas/administração & dosagem , Receptores Acoplados a Proteínas G/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Imagem Individual de Molécula/métodos , Software , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ligação Proteica , Fluxo de Trabalho
14.
Int J Food Microbiol ; 348: 109224, 2021 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-33965694

RESUMO

ε-Poly-l-lysine (ε-PL) is a natural antimicrobial poly-cationic peptide widely applied as a natural preservative in the food industry, whereas its application in preventing postharvest loss of fruit was largely absent. This study investigated the antifungal activity of ε-PL and determined the possible mechanisms involved. The in vivo results indicated that 500 mg L-1 exogenous ε-PL treatment significantly inhibited black spot rot in apple, jujube, and tomato. The lesion diameter inhibition rate was range from 20.11% to 29.09% by 500 mg L-1 ε-PL treatment. ε-PL exerts antifungal activity against A. alternata in vitro, the half-inhibition concentration is 160.1 mg L-1. ε-PL induced morphology and ultrastructure change on the pathogen, which resulted in the inhibition of A. alternata. This was accomplished by disturbing pathogen membrane integrity and functionality. The fluorometric assay confirmed that ε-PL induced endogenous reactive oxygen species formation and accumulation in A. alternata and the elicited severe lipid peroxidation that caused membrane lesions. Further, ε-PL treatment enhanced the expression of genes involved in antioxidant metabolism and pathogenesis-related responses in apple fruit. These findings illustrated that ε-PL exhibits multifaceted antifungal activity by the direct effect on the pathogen as well as induce host defense responses. ε-PL may be conducive as a promising alternative for Alternaria rot management.


Assuntos
Alternaria/efeitos dos fármacos , Antifúngicos/farmacologia , Membrana Celular/efeitos dos fármacos , Frutas/microbiologia , Polilisina/farmacologia , Antioxidantes/metabolismo , Lycopersicon esculentum/microbiologia , Malus/microbiologia , Testes de Sensibilidade Microbiana , Ziziphus/microbiologia
15.
J Biosci Bioeng ; 132(1): 71-80, 2021 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-33895082

RESUMO

Cryopreservation is important for enabling long-term cell preservation. However, physical damage due to ice crystal formation and membrane permeation by dimethyl sulfoxide (DMSO) severely affects cryopreserved cell viability. To ensure cell survival and functional maintenance after cryopreservation, it is important to protect the cell membrane, the most vulnerable cell component, from freeze-thaw damage. This study aimed to create a glycolipid derivative having a positive interaction with the cell membrane and cytoprotective effects. As a result, we synthesized a novel trehalose derivative, oleyl-trehalose (Oleyl-Treh), composed of trehalose and oleyl groups. Its use led to increased viable cell counts when used with DMSO in a non-cytotoxic concentration range (1.6 nM-16 µM). Oleyl-Treh significantly improved viability and liver-specific functions of hepatocytes after cryopreservation, including albumin secretion, ethoxyresorufin-O-deethylase activity (an indicator of cytochrome P450 family 1 subfamily A member 1 activity), and ammonia metabolism. Oleyl-Treh could localize trehalose to the cell membrane; furthermore, the oleyl group affected cell membrane fluidity and exerted cryoprotective effects. This novel cryoprotective agent, which shows a positive interaction with the cell membrane, provides a unique approach toward cell protection during cryopreservation.


Assuntos
Membrana Celular/efeitos dos fármacos , Criopreservação/métodos , Crioprotetores/química , Crioprotetores/farmacologia , Glicolipídeos/química , Trealose/química , Trealose/farmacologia , Animais , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Humanos
16.
Int J Mol Sci ; 22(6)2021 Mar 11.
Artigo em Inglês | MEDLINE | ID: mdl-33799744

RESUMO

Lasioglossin III (LL-III) is a cationic antimicrobial peptide derived from the venom of the eusocial bee Lasioglossum laticeps. LL-III is extremely toxic to both Gram-positive and Gram-negative bacteria, and it exhibits antifungal as well as antitumor activity. Moreover, it shows low hemolytic activity, and it has almost no toxic effects on eukaryotic cells. However, the molecular basis of the LL-III mechanism of action is still unclear. In this study, we characterized by means of calorimetric (DSC) and spectroscopic (CD, fluorescence) techniques its interaction with liposomes composed of a mixture of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) and 1-palmitoyl-2-oleoyl-sn-glycero-3-rac-phosphoglycerol (POPG) lipids as a model of the negatively charged membrane of pathogens. For comparison, the interaction of LL-III with the uncharged POPC liposomes was also studied. Our data showed that LL-III preferentially interacted with anionic lipids in the POPC/POPG liposomes and induces the formation of lipid domains. Furthermore, the leakage experiments showed that the peptide could permeabilize the membrane. Interestingly, our DSC results showed that the peptide-membrane interaction occurs in a non-disruptive manner, indicating an intracellular targeting mode of action for this peptide. Consistent with this hypothesis, our gel-retardation assay experiments showed that LL-III could interact with plasmid DNA, suggesting a possible intracellular target.


Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Abelhas/metabolismo , Membrana Celular/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Proteínas Citotóxicas Formadoras de Poros/farmacologia , Animais , Peptídeos Catiônicos Antimicrobianos/análise , Calorimetria/métodos , Membrana Celular/química , Permeabilidade da Membrana Celular/efeitos dos fármacos , Bicamadas Lipídicas/química , Lipossomos/química , Fosfatidilcolinas/química , Fosfatidilgliceróis/química , Proteínas Citotóxicas Formadoras de Poros/análise , Espectrofotometria/métodos
17.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33804887

RESUMO

Ultrashort cationic lipopeptides (USCLs) and gemini cationic surfactants are classes of potent antimicrobials. Our recent study has shown that the branching and shortening of the fatty acids chains with the simultaneous addition of a hydrophobic N-terminal amino acid in USCLs result in compounds with enhanced selectivity. Here, this approach was introduced into arginine-rich gemini cationic surfactants. l-cystine diamide and l-lysine amide linkers were used as spacers. Antimicrobial activity against planktonic and biofilm cultures of ESKAPE (Enterococcus faecium, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Enterobacter spp.) strains and Candida sp. as well as hemolytic and cytotoxic activities were examined. Moreover, antimicrobial activity in the presence of human serum and the ability to form micelles were evaluated. Membrane permeabilization study, serum stability assay, and molecular dynamics were performed. Generally, critical aggregation concentration was linearly correlated with hydrophobicity. Gemini surfactants were more active than the parent USCLs, and they turned out to be selective antimicrobial agents with relatively low hemolytic and cytotoxic activities. Geminis with the l-cystine diamide spacer seem to be less cytotoxic than their l-lysine amide counterparts, but they exhibited lower antibiofilm and antimicrobial activities in serum. In some cases, geminis with branched fatty acid chains and N-terminal hydrophobic amino acid resides exhibited enhanced selectivity to pathogens over human cells.


Assuntos
Peptídeos Catiônicos Antimicrobianos/síntese química , Biofilmes/efeitos dos fármacos , Lipoproteínas/síntese química , Tensoativos/síntese química , Motivos de Aminoácidos , Peptídeos Catiônicos Antimicrobianos/farmacologia , Arginina/química , Candida/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Cistina/química , Enterobacteriaceae/efeitos dos fármacos , Ácidos Graxos/química , Hemólise , Interações Hidrofóbicas e Hidrofílicas , Lipoproteínas/farmacologia , Lisina/química , Micelas , Tensoativos/farmacologia
18.
Int J Mol Sci ; 22(7)2021 Mar 24.
Artigo em Inglês | MEDLINE | ID: mdl-33805017

RESUMO

Identification of novel agents for bladder cancer treatment is highly desirable due to the high incidence of tumor recurrence and the risk of progression to muscle-invasive disease. The key feature of the cholesterol-dependent toxin listeriolysin O mutant (LLO Y406A) is its preferential activity at pH 5.7, which could be exploited either directly for selective targeting of cancer cells or the release of accumulated therapeutics from acidic endosomes. Therefore, our goal was to compare the cytotoxic effect of LLO Y406A on cancer cells (RT4) and normal urothelial cells (NPU), and to identify which cell membranes are the primary target of LLO Y406A by viability assays, life-cell imaging, fluorescence, and electron microscopy. LLO Y406A decreased viability, altered cell morphology, provoked membrane blebbing, and induced apoptosis in RT4 cells, while it did not affect NPU cells. LLO Y406A did not cause endosomal escape in RT4 cells, while the plasma membrane of RT4 cells was revealed as the primary target of LLO Y406A. It has been concluded that LLO Y406A has the ability to selectively eliminate cancer urothelial cells through pore-forming activity at the plasma membrane, without cytotoxic effects on normal urothelial cells. This promising selective activity merits further testing as an anti-cancer agent.


Assuntos
Antineoplásicos/toxicidade , Toxinas Bacterianas/toxicidade , Membrana Celular/efeitos dos fármacos , Proteínas de Choque Térmico/toxicidade , Proteínas Hemolisinas/toxicidade , Neoplasias da Bexiga Urinária/metabolismo , Urotélio/efeitos dos fármacos , Animais , Toxinas Bacterianas/genética , Cálcio/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Células Cultivadas , Endossomos/efeitos dos fármacos , Endossomos/metabolismo , Proteínas de Choque Térmico/genética , Proteínas Hemolisinas/genética , Humanos , Mutação , Suínos , Urotélio/metabolismo
19.
Int J Mol Sci ; 22(6)2021 Mar 18.
Artigo em Inglês | MEDLINE | ID: mdl-33803867

RESUMO

Transient receptor potential channels-vanilloid receptor 1 (TRPV1) regulates thermotaxis in sperm-oriented motility. We investigated the role of membrane cholesterol (Chol) on TRPV1-mediated human sperm migration. Semen samples were obtained from five normozoospemic healthy volunteers. Sperm membrane Chol content, quantified by liquid chromatography-mass spectrometry, was modified by incubating cells with 2-hydroxypropyl-ß-cyclodextrin (CD) or the complex between CD and Chol (CD:Chol). The effect on sperm migration on a 10 µM capsaicin gradient (CPS), a TRPV1 agonist, was then investigated. Motility parameters were evaluated by Sperm Class Analyser. Intracellular calcium concentration and acrosome reaction were measured by staining with calcium orange and FITC-conjugated anti-CD46 antibody, respectively. TRPV1-Chol interaction was modelled by computational molecular-modelling (MM). CD and CD:Chol, respectively, reduced and increased membrane Chol content in a dose-dependent manner, resulting in a dose-dependent increase and reduction of sperm migration in a CPS gradient. MM confirmed a specific interaction of Chol with a TRPV1 domain that appeared precluded to the Chol epimer epicholesterol (Epi-Chol). Accordingly, CD:Epi-Chol was significantly less efficient than CD:Chol, in reducing sperm migration under CPS gradient. Chol inhibits TRPV1-mediated sperm function by directly interacting with a consensus sequence of the receptor.


Assuntos
Colesterol/metabolismo , Motilidade Espermática , Espermatozoides/metabolismo , Canais de Cátion TRPV/metabolismo , Adulto , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Ciclodextrinas/farmacologia , Humanos , Masculino , Modelos Moleculares , Canais de Cátion TRPV/química
20.
Int J Mol Sci ; 22(8)2021 Apr 15.
Artigo em Inglês | MEDLINE | ID: mdl-33920953

RESUMO

TMEM16A is a Ca2+-activated Cl- channel that controls broad cellular processes ranging from mucus secretion to signal transduction and neuronal excitability. Recent studies have reported that membrane phospholipid phosphatidylinositol 4,5-bisphosphate (PI(4,5)P2) is an important cofactor that allosterically regulates TMEM16A channel activity. However, the detailed regulatory actions of PIP2 in splice variants of TMEM16A remain unclear. Here, we demonstrated that the attenuation of membrane phosphoinositide levels selectively inhibited the current amplitude of the TMEM16A(ac) isoform by decreasing the slow, but not instantaneous, Cl- currents, which are independent of the membrane potential and specific to PI(4,5)P2 depletion. The attenuation of endogenous PI(4,5)P2 levels by the activation of Danio rerio voltage-sensitive phosphatase (Dr-VSP) decreased the Cl- currents of TMEM16A(ac) but not the TMEM16A(a) isoform, which was abolished by the co-expression of PIP 5-kinase type-1γ (PIPKIγ). Using the rapamycin-inducible dimerization of exogenous phosphoinositide phosphatases, we further revealed that the stimulatory effects of phosphoinositide on TMEM16A(ac) channels were similar in various membrane potentials and specific to PI(4,5)P2, not PI4P and PI(3,4,5)P3. Finally, we also confirmed that PI(4,5)P2 resynthesis is essential for TMEM16A(ac) recovery from Dr-VSP-induced current inhibition. Our data demonstrate that membrane PI(4,5)P2 selectively modulates the gating of the TMEM16A(ac) channel in an agonistic manner, which leads to the upregulation of TMEM16A(ac) functions in physiological conditions.


Assuntos
Processamento Alternativo/genética , Anoctamina-1/genética , Cálcio/metabolismo , Membrana Celular/metabolismo , Fosfatidilinositol 4,5-Difosfato/metabolismo , Processamento Alternativo/efeitos dos fármacos , Sequência de Aminoácidos , Animais , Anoctamina-1/química , Anoctamina-1/metabolismo , Membrana Celular/efeitos dos fármacos , Células HEK293 , Humanos , Ativação do Canal Iônico/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Camundongos , Monoéster Fosfórico Hidrolases/metabolismo , Receptor Muscarínico M1/metabolismo , Sirolimo/farmacologia , Peixe-Zebra , Proteínas de Peixe-Zebra/metabolismo
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