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1.
Chemosphere ; 254: 126918, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32957302

RESUMO

The increasing application of various surfactants nowadays, may lead to the contamination of the natural environment and represent potential threat to terrestrial higher plants. In this article, the effect of 13 surfactants, with dodecyl alkyl chain and various aromatic (imidazolium, pyridinium, thiazolium) and aliphatic (guanidinium, ammonium, thiosemicarbazidium) polar heads, on germination, development and growth of wheat and cucumber was investigated. The study aimed to prove how changes in lipophilicity of surfactants and their various structural modifications (existence of the aliphatic or aromatic polar group, the introduction of oxygen and sulfur) influence toxicity towards investigated plants. The calculated lipophilic parameter (AlogP) is shown to be a useful parameter for predicting potential toxicity of the compound. The strategy of using surfactants with aliphatic polar heads instead of aromatic prove to be a promising strategy in reducing harmful effect, as well as the introduction of polar groups in the structure of cation. From all investigated compounds, surfactants with imidazolium polar head displayed the most harmful effect towards wheat and cucumber. The cucumber seeds were more sensitive to the addition of surfactants comparing to wheat. All obtained experimental results were additionally investigated using computational methods, simulating the transport of surfactants through a lipid bilayer. The influence of cation tendency to fit in lipid bilayer structure was correlated with toxicity. For the first time, it is concluded that cation ability to mimic the structure of bilayer have less harmful effect on plant development.


Assuntos
Cucumis sativus/efeitos dos fármacos , Imidazóis/toxicidade , Compostos de Piridínio/toxicidade , Tensoativos/toxicidade , Triticum/efeitos dos fármacos , Cátions , Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Cucumis sativus/crescimento & desenvolvimento , Germinação/efeitos dos fármacos , Imidazóis/química , Bicamadas Lipídicas/química , Simulação de Dinâmica Molecular , Compostos de Piridínio/química , Compostos de Piridínio/farmacologia , Plântula/efeitos dos fármacos , Plântula/crescimento & desenvolvimento , Sementes/efeitos dos fármacos , Sementes/crescimento & desenvolvimento , Relação Estrutura-Atividade , Tensoativos/química , Triticum/crescimento & desenvolvimento
2.
In Vivo ; 34(5): 3023-3026, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32871846

RESUMO

BACKGROUND/AIM: Coronavirus disease 2019 (COVID-19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). One drug that has attracted interest is the antiparasitic compound ivermectin, a macrocyclic lactone derived from the bacterium Streptomyces avermitilis. We carried out a docking study to determine if ivermectin might be able to attach to the SARS-CoV-2 spike receptor-binding domain bound with ACE2. MATERIALS AND METHODS: We used the program AutoDock Vina Extended to perform the docking study. RESULTS: Ivermectin docked in the region of leucine 91 of the spike and histidine 378 of the ACE2 receptor. The binding energy of ivermectin to the spike-ACE2 complex was -18 kcal/mol and binding constant was 5.8 e-08. CONCLUSION: The ivermectin docking we identified may interfere with the attachment of the spike to the human cell membrane. Clinical trials now underway should determine whether ivermectin is an effective treatment for SARS-Cov2 infection.


Assuntos
Betacoronavirus/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Ivermectina/química , Peptidil Dipeptidase A/química , Pneumonia Viral/tratamento farmacológico , Betacoronavirus/química , Betacoronavirus/patogenicidade , Sítios de Ligação/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Infecções por Coronavirus/virologia , Reposicionamento de Medicamentos , Histidina/química , Humanos , Ivermectina/uso terapêutico , Leucina/química , Simulação de Acoplamento Molecular , Pandemias , Peptidil Dipeptidase A/efeitos dos fármacos , Pneumonia Viral/virologia , Streptomyces/química
3.
Anticancer Res ; 40(9): 4857-4867, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32878773

RESUMO

BACKGROUND/AIM: Anticancer peptide PNC-27 binds to HDM-2 protein on cancer cell membranes inducing the formation of cytotoxic transmembrane pores. Herein, we investigated HDM-2 membrane expression and the effect of PNC-27 treatment on human non-stem cell acute myelogenous leukemia cell lines: U937, acute monocytic leukemia; OCI-AML3, acute myelomonocytic leukemia and HL60, acute promyelocytic leukemia. MATERIALS AND METHODS: We measured cell surface membrane expression of HDM-2 using flow cytometry. Cell viability was assessed using MTT assay while direct cytotoxicity was measured by lactate dehydrogenase (LDH) release and induction of apoptotic markers annexin V and caspase-3. RESULTS: HDM-2 is expressed at high levels in membranes of U937, OCI-AML3 and HL-60 cells. PNC-27 can bind to membrane HDM-2 to induce cell necrosis and LDH release within 4 h. CONCLUSION: Targeting membrane HDM-2 can be a potential strategy to treat leukemia. PNC-27 targeting membrane HDM-2 demonstrated significant anti-leukemia activity in a variety of leukemic cell lines.


Assuntos
Antineoplásicos/farmacologia , Leucemia Mieloide/patologia , Proteínas Proto-Oncogênicas c-mdm2/metabolismo , Proteína Supressora de Tumor p53/farmacologia , Antineoplásicos/metabolismo , Linhagem Celular Tumoral , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Sobrevivência Celular/efeitos dos fármacos , Relação Dose-Resposta a Droga , Humanos , L-Lactato Desidrogenase/metabolismo , Leucemia Mieloide/metabolismo , Necrose , Proteína Supressora de Tumor p53/metabolismo
4.
Artigo em Inglês | MEDLINE | ID: mdl-32687406

RESUMO

Severe acute respiratory syndrome coronavirus (SARS-CoV), an enveloped virus with a positive-sense single-stranded RNA genome, facilitates the host cell entry through intricate interactions with proteins and lipids of the cell membrane. The detailed molecular mechanism involves binding to the host cell receptor and fusion at the plasma membrane or after being trafficked to late endosomes under favorable environmental conditions. A crucial event in the process is the proteolytic cleavage of the viral spike protein by the host's endogenous proteases that releases the fusion peptide enabling fusion with the host cellular membrane system. The present review details the mechanism of viral fusion with the host and highlights the therapeutic options that prevent SARS-CoV-2 entry in humans.


Assuntos
Betacoronavirus/metabolismo , Membrana Celular/metabolismo , Infecções por Coronavirus/metabolismo , Infecções por Coronavirus/prevenção & controle , Pandemias/prevenção & controle , Pneumonia Viral/metabolismo , Pneumonia Viral/prevenção & controle , Proteínas Virais de Fusão/metabolismo , Sequência de Aminoácidos , Animais , Betacoronavirus/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/virologia , Humanos , Peptidil Dipeptidase A/metabolismo , Inibidores de Proteases/farmacologia , Inibidores de Proteases/uso terapêutico , Ligação Proteica/efeitos dos fármacos , Ligação Proteica/fisiologia , Glicoproteína da Espícula de Coronavírus/antagonistas & inibidores , Glicoproteína da Espícula de Coronavírus/metabolismo , Proteínas Virais de Fusão/efeitos dos fármacos
5.
PLoS One ; 15(7): e0236373, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32702063

RESUMO

The diagnosis of patients with malignancies relies on the results of a clinical cytological examination. To enhance the diagnostic qualities of cytological examinations, it is important to have a detailed analysis of the cell's characteristics. There is, therefore, a need for developing a new auxiliary method for cytological diagnosis. In this study, we focused on studying the charge of the cell membrane surface of fixed cells, which is one of important cell's characteristics. Although fixed cells lose membrane potential which is observed in living cells owing to ion dynamics, we hypothesized that fixed cells still have a cell membrane surface charge due to cell membrane components and structure. We used 5 cell lines in this study (ARO, C32TG, RT4, TK, UM-UC-14). After fixation with CytoRich Red, we measured the cell membrane surface charge of fixed cells in solution using zeta potential measurements and fixed cells on glass slides, visualizing it using antibody-labeled beads and positively-charged beads. Furthermore, we measured the cell membrane surface charge of fixed cells under different conditions, such as different solution of fixative, ion concentration, pH, and pepsin treatments. The zeta potential measurements and visualization using the beads indicated that the cell membrane surface of fixed cells was negatively charged, and also that the charge varied among fixed cells. The charge state was affected by the different treatments. Moreover, the number of cell-bound beads was small in interphase, anaphase, and apoptotic cells. We concluded that the negative cell membrane surface charge was influenced by the three-dimensional structure of proteins as well as the different types of amino acids and lipids on the cell membrane. Thus, cell surface charge visualization can be applied as a new auxiliary method for clinical cytological diagnosis. This is the first systematic report of the cell membrane surface charge of fixed cells.


Assuntos
Linhagem Celular/ultraestrutura , Membrana Celular/ultraestrutura , Células Cultivadas/ultraestrutura , Citodiagnóstico , Anáfase/efeitos dos fármacos , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Divisão Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Células Cultivadas/efeitos dos fármacos , Fixadores/farmacologia , Humanos , Potenciais da Membrana/efeitos dos fármacos , Pepsina A/farmacologia , Propriedades de Superfície
6.
PLoS One ; 15(7): e0233252, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32701962

RESUMO

Phthiocerol dimycocerosates (PDIMs) are a class of mycobacterial lipids that promote virulence in Mycobacterium tuberculosis and Mycobacterium marinum. It has recently been shown that PDIMs work in concert with the M. tuberculosis Type VII secretion system ESX-1 to permeabilize the phagosomal membranes of infected macrophages. As the zebrafish-M. marinum model of infection has revealed the critical role of PDIM at the host-pathogen interface, we set to determine if PDIMs contributed to phagosomal permeabilization in M. marinum. Using an ΔmmpL7 mutant defective in PDIM transport, we find the PDIM-ESX-1 interaction to be conserved in an M. marinum macrophage infection model. However, we find PDIM and ESX-1 mutants differ in their degree of defect, with the PDIM mutant retaining more membrane damaging activity. Using an in vitro hemolysis assay-a common surrogate for cytolytic activity, we find that PDIM and ESX-1 differ in their contributions: the ESX-1 mutant loses hemolytic activity while PDIM retains it. Our observations confirm the involvement of PDIMs in phagosomal permeabilization in M. marinum infection and suggest that PDIM enhances the membrane disrupting activity of pathogenic mycobacteria and indicates that the role they play in damaging phagosomal and red blood cell membranes may differ.


Assuntos
Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Lipídeos/farmacologia , Macrófagos/citologia , Mycobacterium marinum/metabolismo , Fagossomos/efeitos dos fármacos , Linhagem Celular , Humanos , Macrófagos/efeitos dos fármacos , Mycobacterium marinum/fisiologia , Permeabilidade/efeitos dos fármacos , Fagossomos/metabolismo
7.
Gene ; 759: 144987, 2020 Oct 30.
Artigo em Inglês | MEDLINE | ID: mdl-32712065

RESUMO

BACKGROUND: The immune response is influenced by the administration of omega-3 polyunsaturated fatty acids (PUFA). Multiple sclerosis (MS) and its animal model, experimental autoimmune encephalomyelitis (EAE) are affected by PUFA. The combination of evening primrose/hemp seed oil (EPO/HSO) has essential fatty acids (EFAs) for human optimal health due to the favorable ratio of omega-6/omega-3 and antioxidantal properties. The study was conducted to evaluate the effects of EPO/HSO on improving the membrane fatty acids composition of spleen and blood cells and immunologic factors in compared to rapamycin (RAPA) in the EAE model. METHODS AND MATERIALS: Chronic-EAE was induced by induction of MOG in C57BL/6J mice (female, age: 6-8 weeks, weight 18-21). Mice were assigned to 5 groups (6/group) to evaluate the therapeutic effects of EPO/HSO supplement in comparison with rapamycin: A group; EPO/HSO + RAPA, B group; RAPA, C group; EPO/HSO. Results were compared to two control groups (EAE and naive). The fatty acid profile of the spleen and blood cell membrane was evaluated. Real-time-polymerase chain reaction was used for the evaluate the genes expression levels of interleukin (IL) -4, IL-5, and IL-13 in lymphocytes. Also, IL-4 of serum was evaluated by enzyme-linked immunosorbent assay (ELISA). RESULTS: Our findings indicated that EPO/HSO therapy significantly increased the percentage of essential fatty acids in cell membrane of the spleen and blood. The relative expression of IL-4, IL-5, and IL-13 genes in lymphocytes and serum level of IL-4 was significantly increased in the HSO/EPO treated group versus other groups. CONCLUSION: These results point to potential therapeutic effects on the repair of the structure of cell membranes and suppression of inflammation by EPO/HSO in EAE.


Assuntos
Antioxidantes/uso terapêutico , Encefalomielite Autoimune Experimental/tratamento farmacológico , Ácidos Graxos Essenciais/metabolismo , Fatores Imunológicos/uso terapêutico , Interleucinas/metabolismo , Óleos Vegetais/uso terapêutico , Sirolimo/uso terapêutico , Animais , Antioxidantes/administração & dosagem , Antioxidantes/farmacologia , Cannabis/química , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Suplementos Nutricionais , Combinação de Medicamentos , Feminino , Fatores Imunológicos/administração & dosagem , Fatores Imunológicos/farmacologia , Lipídeos de Membrana/metabolismo , Camundongos , Camundongos Endogâmicos C57BL , Óleos Vegetais/administração & dosagem , Primula/química , Sirolimo/administração & dosagem
8.
Proc Natl Acad Sci U S A ; 117(26): 14978-14986, 2020 06 30.
Artigo em Inglês | MEDLINE | ID: mdl-32554490

RESUMO

AB5 bacterial toxins and polyomaviruses induce membrane curvature as a mechanism to facilitate their entry into host cells. How membrane bending is accomplished is not yet fully understood but has been linked to the simultaneous binding of the pentameric B subunit to multiple copies of glycosphingolipid receptors. Here, we probe the toxin membrane binding and internalization mechanisms by using a combination of superresolution and polarized localization microscopy. We show that cholera toxin subunit B (CTxB) can induce membrane curvature only when bound to multiple copies of its glycosphingolipid receptor, GM1, and the ceramide structure of GM1 is likely not a determinant of this activity as assessed in model membranes. A mutant CTxB capable of binding only a single GM1 fails to generate curvature either in model membranes or in cells, and clustering the mutant CTxB-single-GM1 complexes by antibody cross-linking does not rescue the membrane curvature phenotype. We conclude that both the multiplicity and specific geometry of GM1 binding sites are necessary for the induction of membrane curvature. We expect this to be a general rule of membrane behavior for all AB5 toxins and polyomaviruses that bind glycosphingolipids to invade host cells.


Assuntos
Membrana Celular/química , Membrana Celular/efeitos dos fármacos , Toxina da Cólera/farmacologia , Receptores de Superfície Celular/metabolismo , Animais , Células COS , Membrana Celular/metabolismo , Chlorocebus aethiops , Receptores de Superfície Celular/genética
9.
Eur J Pharmacol ; 882: 173237, 2020 Sep 05.
Artigo em Inglês | MEDLINE | ID: mdl-32525005

RESUMO

Pirfenidone (PFD), a pyridone compound, is well recognized as an antifibrotic agent tailored for the treatment of idiopathic pulmonary fibrosis. Recently, through its anti-inflammatory and anti-oxidant effects, PFD based clinical trial has also been launched for the treatment of coronavirus disease (COVID-19). To what extent this drug can perturb membrane ion currents remains largely unknown. Herein, the exposure to PFD was observed to depress the amplitude of hyperpolarization-activated cation current (Ih) in combination with a considerable slowing in the activation time of the current in pituitary GH3 cells. In the continued presence of ivabradine or zatebradine, subsequent application of PFD decreased Ih amplitude further. The presence of PFD resulted in a leftward shift in Ih activation curve without changes in the gating charge. The addition of this compound also led to a reduction in area of voltage-dependent hysteresis evoked by long-lasting inverted triangular (downsloping and upsloping) ramp pulse. Neither the amplitude of M-type nor erg-mediated K+ current was altered by its presence. In whole-cell potential recordings, addition of PFD reduced the firing frequency, and this effect was accompanied by the depression in the amplitude of sag voltage elicited by hyperpolarizing current stimulus. Overall, this study highlights evidence that PFD is capable of perturbing specific ionic currents, revealing a potential additional impact on functional activities of different excitable cells.


Assuntos
Membrana Celular/efeitos dos fármacos , Infecções por Coronavirus/tratamento farmacológico , Pneumonia Viral/tratamento farmacológico , Piridonas/farmacologia , Animais , Betacoronavirus/metabolismo , Cátions/metabolismo , Linhagem Celular Tumoral , Membrana Celular/metabolismo , Infecções por Coronavirus/virologia , Humanos , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Transporte de Íons/efeitos dos fármacos , Potenciais da Membrana/efeitos dos fármacos , Pandemias , Pneumonia Viral/virologia , Potássio/metabolismo , Piridonas/uso terapêutico , Ratos , Sódio/metabolismo
10.
Nat Commun ; 11(1): 2694, 2020 06 01.
Artigo em Inglês | MEDLINE | ID: mdl-32483155

RESUMO

Toxin complex (Tc) toxins are virulence factors of pathogenic bacteria. Tcs are composed of three subunits: TcA, TcB and TcC. TcA facilitates receptor-toxin interaction and membrane permeation, TcB and TcC form a toxin-encapsulating cocoon. While the mechanisms of holotoxin assembly and pore formation have been described, little is known about receptor binding of TcAs. Here, we identify heparins/heparan sulfates and Lewis antigens as receptors for different TcAs from insect and human pathogens. Glycan array screening reveals that all tested TcAs bind negatively charged heparins. Cryo-EM structures of Morganella morganii TcdA4 and Xenorhabdus nematophila XptA1 reveal that heparins/heparan sulfates unexpectedly bind to different regions of the shell domain, including receptor-binding domains. In addition, Photorhabdus luminescens TcdA1 binds to Lewis antigens with micromolar affinity. Here, the glycan interacts with the receptor-binding domain D of the toxin. Our results suggest a glycan dependent association mechanism of Tc toxins on the host cell surface.


Assuntos
Toxinas Bacterianas/toxicidade , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Polissacarídeos/metabolismo , Animais , Toxinas Bacterianas/química , Toxinas Bacterianas/farmacocinética , Sítios de Ligação , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células HEK293 , Heparina/química , Heparina/metabolismo , Humanos , Insetos/microbiologia , Antígenos CD15/química , Antígenos CD15/metabolismo , Modelos Moleculares , Simulação de Acoplamento Molecular , Morganella morganii/patogenicidade , Photorhabdus/patogenicidade , Polissacarídeos/química , Xenorhabdus/patogenicidade
11.
Parasitol Res ; 119(8): 2703-2711, 2020 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-32537718

RESUMO

It is known that the current treatment for toxoplasmosis causes side effects. Thus, it is essential to develop new therapies with reduced adverse effects while concurrently maintaining broad coverage and prophylactic therapy. Melatonin is a hormone that participates in the circadian cycle in vertebrates and has antioxidant, immunomodulatory, and antitumoral functions. In addition, it has been shown that melatonin can modulate immune responses and parasitic development during infection by Trypanosoma cruzi and Leishmania spp. Furthermore, studies indicate that melatonin increases the number of lymphocytes in rats infected by Toxoplasma gondii. However, there is no information on the possible effects of melatonin in T. gondii-infected host cells in vitro. This study analyzed the effects of melatonin treatment in the monkey kidney cell epithelial cell line, LLC-MK2, after infection with T. gondii. LLC-MK2 cells were infected and treated/not treated with melatonin, and the infection index was then quantified. Melatonin treatment did not alter host cell viability and was able to reduce parasite proliferation in LLC-MK2 cells at 24 and 48 h and at 6 days. Analysis by scanning electron microscopy confirmed reduction of parasite proliferation and alterations of tachyzoite shapes. Transmission electron microscopy images showed parasites with ruptured plasma membranes and cytoplasmic leakage. After treatment, parasites showed positive staining for apoptotic-like cell death. These results suggest that the use of melatonin as the lead compound for the synthesis of new compounds may constitute an alternative treatment for toxoplasmosis.


Assuntos
Coccidiostáticos/farmacologia , Melatonina/farmacologia , Toxoplasma/efeitos dos fármacos , Toxoplasma/crescimento & desenvolvimento , Animais , Morte Celular/efeitos dos fármacos , Linhagem Celular , Membrana Celular/efeitos dos fármacos , Células Epiteliais/parasitologia , Haplorrinos , Estágios do Ciclo de Vida/efeitos dos fármacos
12.
J Environ Sci Health B ; 55(8): 719-725, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32538258

RESUMO

The industrialization of the agricultural sector has significantly increased the use of chemicals such as pesticides. Therefore, exposure to them is unavoidable, which makes it necessary to assess their safety for humans at actual exposure doses. This paper aims to determine toxicity of three types of pesticides toward human immune cells (HL-60 and U-937): glyphosate (GLY), deltamethrin (DEL), and chlorothalonil (CHL). Cell viability, membrane integrity, inflammation induction, and antioxidant activity were evaluated to determine differences in cellular response to the tested plant protection agents. In experimental models, all tested substances caused increased mortality of cells after only 24 h. Cell membrane damage was recorded under DEL and CHL influences. The largest disintegration of the cell membrane was due to the action of 100 µg/mL DEL for U-937 and CHL at 1 µg/mL for HL-60. GLY at a concentration of 3,600 µg/mL caused significant peroxidation of U-937 cells' lipids. CHL-induced inflammation in both types of cells tested. DEL and GLY also induced antioxidant activity in cells. These results lead to the conclusion that the tested pesticides act cytotoxically to the cells of the human immune system in doses to which both farmers and consumers are exposed.


Assuntos
Glicina/análogos & derivados , Sistema Imunitário/efeitos dos fármacos , Nitrilos/toxicidade , Praguicidas/toxicidade , Piretrinas/toxicidade , Agricultura , Antioxidantes/metabolismo , Membrana Celular/efeitos dos fármacos , Membrana Celular/imunologia , Sobrevivência Celular/efeitos dos fármacos , Fazendeiros , Glicina/toxicidade , Células HL-60 , Humanos , Sistema Imunitário/citologia , Peroxidação de Lipídeos/efeitos dos fármacos , Exposição Ocupacional , Testes de Toxicidade
13.
PLoS One ; 15(5): e0233854, 2020.
Artigo em Inglês | MEDLINE | ID: mdl-32470006

RESUMO

Alpha-toxin (Hla) is a major virulence factor of Staphylococcus aureus (S. aureus) and plays an important role in S. aureus-induced pneumonia. It binds as a monomer to the cell surface of eukaryotic host cells and forms heptameric transmembrane pores. Sensitivities toward the toxin of various types of potential host cells have been shown to vary substantially, and the reasons for these differences are unclear. We used three human model airway epithelial cell lines (16HBE14o-, S9, A549) to correlate cell sensitivity (measured as rate of paracellular gap formation in the cell layers) with Hla monomer binding, presence of the potential Hla receptors ADAM10 or α5ß1 integrin, presence of the toxin-stabilizing factor caveolin-1 as well as plasma membrane lipid composition (phosphatidylserine/choline, sphingomyelin). The abundance of ADAM10 correlated best with gap formation or cell sensitivities, respectively, when the three cell types were compared. Caveolin-1 or α5ß1 integrin did not correlate with toxin sensitivity. The relative abundance of sphingomyelin in plasma membranes may also be used as a proxi for cellular sensitivity against alpha-toxin as sphingomyelin abundances correlated well with the intensities of alpha-toxin mediated gap formation in the cell layers.


Assuntos
Toxinas Bacterianas/metabolismo , Toxinas Bacterianas/toxicidade , Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Proteínas Hemolisinas/metabolismo , Proteínas Hemolisinas/toxicidade , Interações Hospedeiro-Patógeno , Sistema Respiratório/patologia , Células A549 , Caveolina 1/metabolismo , Membrana Celular/efeitos dos fármacos , Tamanho Celular , Células Epiteliais/efeitos dos fármacos , Interações Hospedeiro-Patógeno/efeitos dos fármacos , Humanos , Modelos Biológicos , Fosfolipídeos/metabolismo , Ligação Proteica , Receptores de Superfície Celular/metabolismo
15.
Int J Food Microbiol ; 328: 108663, 2020 Sep 02.
Artigo em Inglês | MEDLINE | ID: mdl-32454366

RESUMO

ε-Polylysine (ε-PL) is a natural and highly effective cationic antimicrobial, of which antibacterial activity is limited in food matrix because of ε-PL's charged amino groups that form complexes with food polyanions. Whey protein-ε-PL complexes delivery system was found to be able to solve the problem and keep the antibacterial activity. This study investigated the antibacterial activity of the complexes and its mechanism against Escherichia coli. The minimal inhibitory concentration of ε-PL was in the range 11.72-25.00 g/mL for the complexes containing different amount of ε-PL and was similar to that of free ε-PL. The results of scanning electron microscopy showed that the complexes could destroy the structure of E. coli cell membrane surface, leaving holes on the surface of the bacteria, leading to the death of the bacteria. The molecular dynamics simulation results showed that the mechanism of the antibacterial activity of the complexes was as follows: under electrostatic interaction, the complexes captured the phospholipid molecules of the bacterial membrane through the hydrogen bonds between the positively charged amino groups of ε-PL and the oxygen atom of the phosphate head groups of the membrane, which could create holes on the surface of the bacteria and lead to the death of the bacteria. The results of activity on real food systems showed that the complexes kept the number of E. coli within 5.8 log10 CFU/g after 7 d storage in sauced duck products, while the positive control (ε-PL) was 6.5 log10 CFU/g and negative control (sterile water) was 7.8 log10 CFU/g. Overall, this study confirmed the antibacterial activity of the complexes and provided fundamental knowledge of its antibacterial activity mechanism.


Assuntos
Antibacterianos/farmacologia , Peptídeos Catiônicos Antimicrobianos/farmacologia , Escherichia coli/efeitos dos fármacos , Polilisina/farmacologia , Proteínas do Soro do Leite/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Patos , Microbiologia de Alimentos , Ligação de Hidrogênio/efeitos dos fármacos , Testes de Sensibilidade Microbiana , Simulação de Dinâmica Molecular , Proteínas do Soro do Leite/metabolismo
16.
Proc Natl Acad Sci U S A ; 117(24): 13757-13766, 2020 06 16.
Artigo em Inglês | MEDLINE | ID: mdl-32467161

RESUMO

Inhaled anesthetics are a chemically diverse collection of hydrophobic molecules that robustly activate TWIK-related K+ channels (TREK-1) and reversibly induce loss of consciousness. For 100 y, anesthetics were speculated to target cellular membranes, yet no plausible mechanism emerged to explain a membrane effect on ion channels. Here we show that inhaled anesthetics (chloroform and isoflurane) activate TREK-1 through disruption of phospholipase D2 (PLD2) localization to lipid rafts and subsequent production of signaling lipid phosphatidic acid (PA). Catalytically dead PLD2 robustly blocks anesthetic TREK-1 currents in whole-cell patch-clamp recordings. Localization of PLD2 renders the TRAAK channel sensitive, a channel that is otherwise anesthetic insensitive. General anesthetics, such as chloroform, isoflurane, diethyl ether, xenon, and propofol, disrupt lipid rafts and activate PLD2. In the whole brain of flies, anesthesia disrupts rafts and PLDnull flies resist anesthesia. Our results establish a membrane-mediated target of inhaled anesthesia and suggest PA helps set thresholds of anesthetic sensitivity in vivo.


Assuntos
Anestésicos Inalatórios/administração & dosagem , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/genética , Membrana Celular/metabolismo , Clorofórmio/administração & dosagem , Drosophila/efeitos dos fármacos , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Isoflurano/administração & dosagem , Ácidos Fosfatídicos/metabolismo , Fosfolipase D/genética , Fosfolipase D/metabolismo , Canais de Potássio/genética , Canais de Potássio/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Canais de Potássio de Domínios Poros em Tandem/metabolismo
17.
Artigo em Inglês | MEDLINE | ID: mdl-32348180

RESUMO

The ubiquitous calpains, calpain-1 and -2, play important roles in Ca2+-dependent membrane repair. Mechanically active tissues like skeletal muscle are particularly reliant on mechanisms to repair and remodel membrane injury, such as those caused by eccentric damage. We demonstrate that calpain-1 and -2 are master effectors of Ca2+-dependent repair of mechanical plasma membrane scrape injuries, although they are dispensable for repair/removal of small wounds caused by pore-forming agents. Using CRISPR gene-edited human embryonic kidney 293 (HEK293) cell lines, we established that loss of both calpains-1 and -2 (CAPNS1-/-) virtually ablates Ca2+-dependent repair of mechanical scrape injuries but does not affect injury or recovery from perforation by streptolysin-O or saponin. In contrast, cells with targeted knockout of either calpain-1 (CAPN1-/-) or -2 (CAPN2-/-) show near-normal repair of mechanical injuries, inferring that both calpain-1 and calpain-2 are equally capable of conducting the cascade of proteolytic cleavage events to reseal a membrane injury, including that of the known membrane repair agent dysferlin. A severe muscular dystrophy in a murine model with skeletal muscle knockout of Capns1 highlights vital roles for calpain-1 and/or -2 for health and viability of skeletal muscles not compensated for by calpain-3 (CAPN3). We propose that the dystrophic phenotype relates to loss of maintenance of plasma membrane/cytoskeletal networks by calpains-1 and -2 in response to directed and dysfunctional Ca2+-signaling, pathways hyperstimulated in the context of membrane injury. With CAPN1 variants associated with spastic paraplegia, a severe dystrophy observed with muscle-specific loss of calpain-1 and -2 activity identifies CAPN2 and CAPNS1 as plausible candidate neuromuscular disease genes.


Assuntos
Calpaína/deficiência , Membrana Celular/enzimologia , Músculo Esquelético/enzimologia , Distrofia Muscular do Cíngulo dos Membros/enzimologia , Distrofia Muscular Animal/enzimologia , Animais , Proteínas de Bactérias/farmacologia , Sinalização do Cálcio , Calpaína/genética , Membrana Celular/efeitos dos fármacos , Membrana Celular/patologia , Modelos Animais de Doenças , Disferlina/deficiência , Disferlina/genética , Feminino , Células HEK293 , Humanos , Masculino , Camundongos Knockout , Músculo Esquelético/patologia , Distrofia Muscular do Cíngulo dos Membros/genética , Distrofia Muscular do Cíngulo dos Membros/patologia , Distrofia Muscular Animal/genética , Distrofia Muscular Animal/patologia , Saponinas/farmacologia , Índice de Gravidade de Doença , Estreptolisinas/farmacologia
18.
J Appl Microbiol ; 129(3): 541-553, 2020 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-32271977

RESUMO

AIM: This study investigated the in vitro mechanism of action of a commercial citrus EO, Brazilian orange terpenes (BOT), on an enterotoxigenic Escherichia coli (ETEC) isolated from pig gut and on Lactobacillus rhamnosus. METHODS AND RESULTS: Firstly, bacteria were exposed sequentially to BOT every 3 h (three times) at sub-minimal inhibitory concentrations and results showed that sequential exposure to BOT provoked a higher reduction of bacteria viability than a single exposure and the reduction of ETEC viability was higher compared to that of L. rhamnosus. Then, evaluation of the BOT effects on the cell membrane permeability and integrity, indicated that BOT increased the membrane permeability and caused disruptive effects on the integrity of bacterial cells as reflected by an increase of the relative electric conductivity and the release of essential cell constituents. Interestingly, BOT effects were more pronounced on the ETEC than on L. rhamnosus. This was ratified by scanning electron microscopy, which showed more noticeable morphological damages and disturbances on ETEC cells than on the L. rhamnosus cells. Limonene was detected as the major compound in BOT by polar/nonpolar GC-MS (78·65%/79·38%). CONCLUSIONS: Results revealed that the probable mechanism of the selective antibacterial action of the citrus EO, BOT, can be described as altering more remarkable the permeability and integrity of the cytoplasmic membrane as well as the external structure of ETEC cells than L. rhamnosus cells. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides information about the mechanism of antibacterial action displayed by a citrus EO, a by-product of the citrus processing industry, as a natural alternative to antibiotics used in pig production sector to combat pathogens such as ETECs.


Assuntos
Antibacterianos/farmacologia , Citrus/química , Escherichia coli Enterotoxigênica/efeitos dos fármacos , Lactobacillus rhamnosus/efeitos dos fármacos , Óleos Voláteis/farmacologia , Animais , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Escherichia coli Enterotoxigênica/isolamento & purificação , Testes de Sensibilidade Microbiana , Óleos Voláteis/química , Especificidade da Espécie , Suínos , Terpenos/farmacologia
19.
Plant Physiol Biochem ; 151: 429-437, 2020 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-32289636

RESUMO

Potassium (K+) has been reported to alleviate ammonium (NH4+) toxicity in rice through some underlying mechanisms, but it still not clear. In addition, K+ is an important cation for activation of plasma membrane (PM) H+-ATPase activity. Here, we hypothesized that K+ alleviated NH4+ toxicity by mediating PM H+-ATPase function in rice root. In this study, rice plants were cultivated in hydroponic solution with various concentrations of K+ and NH4+. By concurrently supplying K+ with NH4+ or re-supplying K+ after NH4+ toxicity, we found that high K+ concentration reduced the NH4+ uptake rate, enhanced the H+ extrusion rate by the roots, and alleviated rice NH4+ toxicity. The gene expression levels of PM H+-ATPase members (OsA1, 3, 7, 8, and 9) were upregulated by application of increasing concentrations of K+ under NH4+ toxicity. The PM H+-ATPase activity and protein expression in rice roots were also enhanced. Furthermore, the enhancement of PM H+-ATPase activity by a specific stimulator (fusicoccin) rescued rice seedlings from NH4+ toxicity. Taken together, these results indicate that K+ can alleviate NH4+ toxicity, possibly by activating PM H+-ATPase to extrude more H+ and inhibit NH4+ uptake by root. Our results may enhance understanding of the strategy of applying K+ fertilizer to mitigate crop NH4+ toxicity in agriculture.


Assuntos
Compostos de Amônio , Oryza , Potássio , ATPases Translocadoras de Prótons , Compostos de Amônio/metabolismo , Compostos de Amônio/toxicidade , Membrana Celular/efeitos dos fármacos , Membrana Celular/enzimologia , Ativação Enzimática/efeitos dos fármacos , Oryza/efeitos dos fármacos , Oryza/enzimologia , Raízes de Plantas/efeitos dos fármacos , Raízes de Plantas/enzimologia , Potássio/farmacologia , ATPases Translocadoras de Prótons/metabolismo
20.
J Microbiol Biotechnol ; 30(3): 382-390, 2020 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-32238758

RESUMO

Periplanetasin-4 is an antimicrobial peptide with 13 amino acids identified in cockroaches. It has been reported to induce fungal cell death by apoptosis and membrane-targeted action. Analogs were designed by substituting arginine residues to modify the electrostatic and hydrophobic interactions accordingly and explore the effect of periplanetasin-4 through the increase of net charge and the decrease of hydrophobicity. The analogs showed lower activity than periplanetasin-4 against gram-positive and gram-negative bacteria. Similar to periplanetasin-4, the analogs exhibited slight hemolytic activity against human erythrocytes. Membrane studies, including determination of changes in membrane potential and permeability, and fluidity assays, revealed that the analogs disrupt less membrane integrity compared to periplanetasin-4. Likewise, when the analogs were treated to the artificial membrane model, the passage of molecules bigger than FD4 was difficult. In conclusion, arginine substitution could not maintain the membrane disruption ability of periplanetasin-4. The results indicated that the attenuation of hydrophobic interactions with the plasma membrane caused a reduction in the accumulation of the analogs on the membrane before the formation of electrostatic interactions. Our findings will assist in the further development of antimicrobial peptides for clinical use.


Assuntos
Membrana Celular/efeitos dos fármacos , Antibacterianos/farmacologia , Eritrócitos/efeitos dos fármacos , Bactérias Gram-Negativas/efeitos dos fármacos , Bactérias Gram-Positivas/efeitos dos fármacos , Humanos , Interações Hidrofóbicas e Hidrofílicas
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