RESUMEN

Staphylococcus aureus is a bacterium responsible for resistance to multiple drugs and the efflux system is widely studied among the resistance mechanisms developed by this species. The present study evaluates the inhibition of the MepA efflux pump by thiadiazine-derived compounds. For this purpose, thiadiazine-derived compounds (IJ-14 to IJ-20) were tested against S. aureus K2068 strains. Microdilution tests were initially conducted to assess the Minimum Inhibitory Concentration (MIC) of the compounds and their efflux pump inhibition activity. In addition, fluorimetry tests were performed using BrEt emission and tests were conducted to inhibit the expression of the mepA gene. This involved comparing the bacterial gene expression with the antibiotic alone to the gene expression after combining compounds (IJ-17 and IJ-20) with the antibiotic. Furthermore, membrane permeability assessment tests and in silico molecular docking tests were performed. It was observed that the IJ17 and IJ20 compounds exhibited direct activity against the tested strain. The IJ17 compound produced significant results in the gene inhibition tests, which was also evidenced through the membrane permeability alteration test. These findings suggest that thiadiazine-derived compounds have promising effects against one of the main resistance mechanisms, with the IJ17 compound presenting observable mechanisms of action.

Asunto(s)

Antibacterianos , Proteínas Bacterianas , Permeabilidad de la Membrana Celular , Pruebas de Sensibilidad Microbiana , Simulación del Acoplamiento Molecular , Staphylococcus aureus , Staphylococcus aureus/efectos de los fármacos , Staphylococcus aureus/genética , Antibacterianos/farmacología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Tiazinas/farmacología , Proteínas de Transporte de Membrana/metabolismo , Proteínas de Transporte de Membrana/genética

RESUMEN

Botulinum neurotoxins (BoNTs) are valuable tools to unveil molecular mechanisms of exocytosis in neuronal and non-neuronal cells due to their peptidase activity on exocytic isoforms of SNARE proteins. They are produced by Clostridia as single-chain polypeptides that are proteolytically cleaved into light, catalytic domains covalently linked via disulfide bonds to heavy, targeting domains. This format of two subunits linked by disulfide bonds is required for the full neurotoxicity of BoNTs. We have generated a recombinant version of BoNT/B that consists of the light chain of the toxin fused to the protein transduction domain of the human immunodeficiency virus-1 (TAT peptide) and a hexahistidine tag. His6-TAT-BoNT/B-LC, expressed in Escherichia coli and purified by affinity chromatography, penetrated membranes and exhibited strong enzymatic activity, as evidenced by cleavage of the SNARE synaptobrevin from rat brain synaptosomes and human sperm cells. Proteolytic attack of synaptobrevin hindered exocytosis triggered by a calcium ionophore in the latter. The novel tool reported herein disrupts the function of a SNARE protein within minutes in cells that may or may not express the receptors for the BoNT/B heavy chain, and without the need for transient transfection or permeabilization.

Asunto(s)

Toxinas Botulínicas Tipo A , Exocitosis , Animales , Humanos , Ratas , Toxinas Botulínicas Tipo A/metabolismo , Toxinas Botulínicas Tipo A/genética , Toxinas Botulínicas Tipo A/aislamiento & purificación , Proteínas SNARE/metabolismo , Proteínas SNARE/genética , Masculino , Sinaptosomas/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/aislamiento & purificación , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Toxinas Botulínicas/metabolismo , Toxinas Botulínicas/genética , Toxinas Botulínicas/química , Toxinas Botulínicas/aislamiento & purificación

RESUMEN

Doxazosin and carvedilol have been evaluated as an alternative treatment against chronic liver lesions and for their possible role during the regeneration of damage caused by liver fibrosis in a hamster model. However, these drugs have been reported to induce morphological changes in hepatocytes, affecting the recovery of liver parenchyma. The effects of these α/𝛽 adrenoblockers on the viability of hepatocytes are unknown. Herein, we demonstrate the protective effect of curcumin against the possible side effects of doxazosin and carvedilol, drugs with proven antifibrotic activity. After pretreatment with 1 µM curcumin for 1 h, HepG2 cells were exposed to 0.1-25 µM doxazosin or carvedilol for 24, 48, and 72 h. Cell viability was assessed using the MTT assay and SYTOX green staining. Morphological changes were detected using the hematoxylin and eosin (H&E) staining and scanning electron microscopy (SEM). An expression of apoptotic and oxidative stress markers was analyzed using reverse transcription-quantitative PCR (RT-qPCR). The results indicate that doxazosin decreases cell viability in a time- and dose-dependent manner, whereas carvedilol increases cell proliferation; however, curcumin increases or maintains cell viability. SEM and H&E staining provided evidence that doxazosin and carvedilol induced morphological changes in HepG2 cells, and curcumin protected against these effects, maintaining the morphology in 90% of treated cells. Furthermore, curcumin positively regulated the expression of Nrf2, HO-1, and SOD1 mRNAs in cells treated with 0.1 and 0.5 µM doxazosin. Moreover, the Bcl-2/Bax ratio was higher in cells that were treated with curcumin before doxazosin or carvedilol. The present study demonstrates that curcumin controls doxazosin- and carvedilol-induced cytotoxicity and morphological changes in HepG2 cells possibly by overexpression of Nrf2.

Asunto(s)

Carvedilol/toxicidad , Curcumina/farmacología , Doxazosina/toxicidad , Estrés Oxidativo/efectos de los fármacos , Apoptosis/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Proliferación Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Expresión Génica/efectos de los fármacos , Células Hep G2 , Hepatocitos/efectos de los fármacos , Hepatocitos/metabolismo , Hepatocitos/ultraestructura , Humanos , Factor 2 Relacionado con NF-E2/genética , Factor 2 Relacionado con NF-E2/metabolismo , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo

RESUMEN

Fructooligosaccharides (FOSs)-fructose-based oligosaccharides-are typical prebiotics with health-promoting effects in humans and animals. The trisaccharide 1-kestotriose is the most attractive inulin-type FOS. We previously reported a recombinant sucrose:sucrose 1-fructosyltransferase (1-SST, EC 2.4.1.99) from Schedonorus arundinaceus (Sa) that efficiently converts sucrose into 1-kestotriose. In this study, Pichia pastoris PGFT6x-308 constitutively expressing nine copies of the Sa1-SST gene displayed fructosyltransferase activity in undisrupted biomass (49.8 U/ml) and culture supernatant (120.7 U/ml) in fed-batch fermentation (72 hr) with sugarcane molasses. Toluene permeabilization increased 2.3-fold the Sa1-SSTrec activity of whole cells entrapped in calcium-alginate beads. The reaction with refined or raw sugar (600 g/l) yielded 1-kestotriose and 1,1-kestotetraose in a ratio of 8:2 with their sum representing above 55% (wt/wt) of total carbohydrates. The FOSs yield decreased to 45% (wt/wt) when sugarcane syrup and molasses were used as cheaper sucrose sources. The beads retained 80% residual Sa1-SSTrec activity after a 30-day batchwise operation with refined cane sugar at 30°C and pH 5.5. The immobilized biocatalyst is attractive for the continuous production of short-chain FOSs, most particularly 1-kestotriose.

Asunto(s)

Hexosiltransferasas/metabolismo , Oligosacáridos/metabolismo , Pichia/metabolismo , Alginatos/química , Carbohidratos/análisis , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células Inmovilizadas , Fermentación , Hexosiltransferasas/genética , Humanos , Microbiología Industrial , Inulina/metabolismo , Melaza , Pichia/genética , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Saccharomycetales , Sacarosa , Tolueno/farmacología , Trisacáridos/biosíntesis

RESUMEN

The development of multidrug-resistant bacteria has revealed the need for new antimicrobial compounds. Cannabis sativa preparations have a long history of medical applications, including the treatment of infectious diseases. This review collects the information about the activity of C. sativa extracts and its main components (cannabinoids and terpenes) against pathogenic bacteria and fungus, to assess its potential using as antimicrobial agents.

Asunto(s)

Antibacterianos/farmacología , Infecciones Bacterianas/tratamiento farmacológico , Cannabis/química , Extractos Vegetales/farmacología , Animales , Antibacterianos/química , Antibacterianos/uso terapéutico , Cannabinoides/farmacología , Cannabinoides/uso terapéutico , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Farmacorresistencia Bacteriana Múltiple , Sinergismo Farmacológico , Humanos , Pruebas de Sensibilidad Microbiana , Extractos Vegetales/química , Extractos Vegetales/uso terapéutico , Terpenos/farmacología , Terpenos/uso terapéutico

RESUMEN

Cell-penetrating peptides (CPPs) comprise a class of short polypeptides that possess the ability to selectively interact with the cytoplasmic membrane of certain cell types, translocate across plasma membranes and accumulate in the cell cytoplasm, organelles (e.g., the nucleus and mitochondria) and other subcellular compartments. CPPs are either of natural origin or de novo designed and synthesized from segments and patches of larger proteins or designed by algorithms. With such intrinsic properties, along with membrane permeation, translocation and cellular uptake properties, CPPs can intracellularly convey diverse substances and nanomaterials, such as hydrophilic organic compounds and drugs, macromolecules (nucleic acids and proteins), nanoparticles (nanocrystals and polyplexes), metals and radionuclides, which can be covalently attached via CPP N- and C-terminals or through preparation of CPP complexes. A cumulative number of studies on animal toxins, primarily isolated from the venom of arthropods and snakes, have revealed the cell-penetrating activities of venom peptides and toxins, which can be harnessed for application in biomedicine and pharmaceutical biotechnology. In this review, I aimed to collate examples of peptides from animal venoms and toxic secretions that possess the ability to penetrate diverse types of cells. These venom CPPs have been chemically or structurally modified to enhance cell selectivity, bioavailability and a range of target applications. Herein, examples are listed and discussed, including cysteine-stabilized and linear, α-helical peptides, with cationic and amphipathic character, from the venom of insects (e.g., melittin, anoplin, mastoparans), arachnids (latarcin, lycosin, chlorotoxin, maurocalcine/imperatoxin homologs and wasabi receptor toxin), fish (pardaxins), amphibian (bombesin) and snakes (crotamine and cathelicidins).

Asunto(s)

Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Celular/efectos de los fármacos , Péptidos de Penetración Celular/farmacología , Ponzoñas/metabolismo , Animales , Membrana Celular/metabolismo , Péptidos de Penetración Celular/aislamiento & purificación , Péptidos de Penetración Celular/metabolismo , Portadores de Fármacos , Humanos

RESUMEN

BACKGROUND: Antimicrobial resistance poses substantial risks to human health. Thus, there is an urgent need for novel antimicrobial agents, including alternative compounds, such as peptides derived from bacterial toxin-antitoxin (TA) systems. ParELC3 is a synthetic peptide derived from the ParE toxin reported to be a good inhibitor of bacterial topoisomerases and is therefore a potential antibacterial agent. However, ParELC3 is inactive against bacteria due to its inability to cross the bacterial membranes. To circumvent this limitation we prepared and used rhamnolipid-based liposomes to carry and facilitate the passage of ParELC3 through the bacterial membrane to reach its intracellular target - the topoisomerases. METHODS AND RESULTS: Small unilamellar liposome vesicles were prepared by sonication from three formulations that included 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine and cholesterol. ParELC3 was loaded with high efficiency into the liposomes. Characterization by DLS and TEM revealed the appropriate size, zeta potential, polydispersity index, and morphology. In vitro microbiological experiments showed that ParELC3 loaded-liposomes are more efficient (29 to 11 µmol·L-1) compared to the free peptide (>100 µmol·L-1) at inhibiting the growth of standard E. coli and S. aureus strains. RL liposomes showed high hemolytic activity but when prepared with POPC and Chol this activity had a significant reduction. Independently of the formulation, the vesicles had no detectable cytotoxicity to HepG2 cells, even at the highest concentrations tested (1.3 mmol·L-1 and 50 µmol·L-1 for rhamnolipid and ParELC3, respectively). CONCLUSION: The present findings suggest the potential use of rhamnolipid-based liposomes as nanocarrier systems to enhance the bioactivity of peptides.

Asunto(s)

Antibacterianos/farmacología , Portadores de Fármacos/química , Glucolípidos/química , Nanopartículas/química , Péptidos/farmacología , Sistemas Toxina-Antitoxina , Secuencia de Aminoácidos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Liberación de Fármacos , Dispersión Dinámica de Luz , Escherichia coli/efectos de los fármacos , Hemólisis/efectos de los fármacos , Células Hep G2 , Humanos , Hidrodinámica , Liposomas , Pruebas de Sensibilidad Microbiana , Péptidos/química , Sonicación , Staphylococcus aureus/efectos de los fármacos

RESUMEN

Tepary bean (Phaseolus acutifolius) lectins exhibit differential in vitro and in vivo biological effects, but their gastrointestinal interactions and digestion have not yet been assessed. This work aimed to evaluate the changes of a recombinant Tepary bean lectin (rTBL-1) through an in vitro and ex vivo gastrointestinal process. A polyclonal antibody was developed to selectively detect rTBL-1 by Western blot (WB) and immunohistochemical analysis. Everted gut sac viability was confirmed until 60 min, where protein bioaccessibility, apparent permeability coefficient, and efflux ratio showed rTBL-1 partial digestion and absorption. Immunoblot assays suggested rTBL-1 internalization, since the lectin was detected in the digestible fraction. The immunohistochemical assay detected rTBL-1 presence at the apical side of the small intestine, potentially due to the interaction with the intestinal cell membrane. The in silico interactions between rTBL-1 and some saccharides or derivatives showed high binding affinity to sialic acid (-6.70 kcal/mol) and N-acetylglucosamine (-6.10 kcal/mol). The ultra-high-performance liquid chromatography-electron spray ionization-quantitative time-of-flight coupled to mass spectrometry (UHPLC-ESI-QTOF/MS) analysis showed rTBL-1 presence in the gastric content and the non-digestible fraction after intestinal simulation conditions. The results indicated that rTBL-1 partially resisted the digestive conditions and interacted with the intestinal membrane, whereas its digestion allowed the absorption or internalization of the protein or the derivative peptides. Further purification of digestion samples should be conducted to identify intact rTBL-1 protein and digested peptides to assess their physiological effects.

Asunto(s)

Permeabilidad de la Membrana Celular , Absorción Intestinal , Mucosa Intestinal/metabolismo , Lectinas/metabolismo , Phaseolus/genética , Proteínas Recombinantes/metabolismo , Metabolismo de los Hidratos de Carbono , Carbohidratos/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular , Cromatografía Líquida de Alta Presión , Inmunohistoquímica , Absorción Intestinal/efectos de los fármacos , Mucosa Intestinal/efectos de los fármacos , Lectinas/química , Lectinas/genética , Ligandos , Modelos Moleculares , Unión Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/farmacología , Espectrometría de Masa por Ionización de Electrospray , Espectrometría de Masa por Láser de Matriz Asistida de Ionización Desorción , Relación Estructura-Actividad

RESUMEN

Three-dimensional skin models, also named 3D skin models, human skin equivalents (HSEs), or Human Epidermal Equivalents (HEEs), have been increasingly used for chemical assessments in terms of efficacy and safety. Considering this, we developed an HEE model using immortalized HaCaT cells, aiming to overcome the limitation of primary tissue source. Our 3D model (HaCaT-HEE) exhibited important markers of cell differentiation (CK10, CK14, involucrin, and filaggrin), although the stratum corneum was shown to be modest. Besides, the model showed a good prediction potential considering membrane permeability, sensitivity, specificity, and accuracy in distinguishing irritant and corrosive effects after exposure to selected chemicals recommended by the OECD protocols. We also validated the formazan determination for the MTT method using High-Performance Liquid Chromatography (HPLC). For that, we considered carry over, linearity, reproducibility/robustness, accuracy, precision, selectivity, and matrix effect, according to the Food and Drug Administration (FDA) guideline. Based on our results, we can conclude that our model has an acceptable predictive value for the safety evaluation of compounds after skin exposure, with the great advantage of being constructed using immortalized cells.

Asunto(s)

Cáusticos/toxicidad , Irritantes/toxicidad , Queratinocitos/efectos de los fármacos , Pruebas de Irritación de la Piel/métodos , Alternativas a las Pruebas en Animales , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Epidermis , Proteínas Filagrina , Humanos

RESUMEN

To determine and compare the antimicrobial effect of Hibiscus sabdariffa calyx extracts, six types of commercial mouthwashes, and chlorhexidine on Streptococcus mutans, Streptococcus sanguinis, Capnocytophaga gingivalis, and Staphylococcus aureus. Two varieties of H. sabdariffa cultivated in Mexico were used. Aqueous, methanolic, ethanolic, acetonic, and ethyl acetate extracts were obtained from H. sabdariffa calyces. Six different types of mouthwash (Astringosol®, Colgate plax-ice-infinity®, Crest pro-health®, Dental max®, Equate®, and Listerine zero®) and chlorhexidine (0.12%) were purchased at a pharmacy. The antimicrobial activity of the H. sabdariffa calyx extracts, mouthwashes, and chlorhexidine was determined by the agar disc diffusion technique. The minimum inhibitory concentration (MIC) and the minimum bactericidal concentration (MBC) of all solutions were determined by the broth dilution method and the pour plate technique, respectively. Also, the effect of H. sabdariffa extracts and chlorhexidine on permeability of the bacterial membrane was determined by the violet crystal assay. All H. sabdariffa calyx extracts and chlorhexidine showed antibacterial activity against all oral pathogenic bacteria. The mouthwashes showed lower antibacterial effect than H. sabdariffa extracts and chlorhexidine. Dental max showed no antibacterial effect. The MICs and MBCs, respectively, for H. sabdariffa extracts were between 5-20 and 10-20 mg/mL; and for chlorhexidine, between 3-4 and 3-5 mg/mL. For the Listerine®, the MIC and MBC values were between 20-25 and 25-33 mg/mL, respectively. The results of the crystal violet test indicate that H. sabdariffa calyx extracts and chlorhexidine alter the permeability of the bacterial membrane. All H. sabdariffa extracts and chlorhexidine showed significantly greater antimicrobial effect than mouthwashes. This is the first report in which the antimicrobial effect of the H. sabdariffa calyx extracts, mouthwashes, and chlorhexidine is compared.

Asunto(s)

Clorhexidina/farmacología , Hibiscus/química , Antisépticos Bucales/farmacología , Extractos Vegetales/farmacología , Capnocytophaga/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , México , Pruebas de Sensibilidad Microbiana , Staphylococcus aureus/efectos de los fármacos , Streptococcus/efectos de los fármacos

RESUMEN

The currents of α7 nicotinic acetylcholine receptors activated by acetylcholine (ACh) are brief. The channel has high permeability to calcium relative to monovalent cations and shows inward rectification. It has been previously noted that in the presence of positive allosteric modulators (PAMs), currents through the channels of α7 receptors differ from normal α7 currents both in sensitivity to specific channel blockers and their current-voltage (I-V) relationships, no longer showing inward rectification. Linear I-V functions are often associated with channels lacking calcium permeability, so we measured the I-V functions of α7 receptors activated by ACh when PAMs were bound to the allosteric binding site in the transmembrane domain. Currents were recorded in chloride-free Ringer's solution with low or high concentrations of extracellular calcium to determine the magnitude of the reversal potential shift in the two conditions as well as the I-V relationships. ACh-evoked currents potentiated by the allosteric agonist-PAMs (ago-PAMs) (3aR,4S,9bS)-4-(4-bromophenyl)-3a,4,5,9b-tetrahydro-3H-cyclopenta[c]quinoline-8-sulfonamide (GAT107) and 3-(3,4-difluorophenyl)-N-(1-(6-(4-(pyridin-2-yl)piperazin-1-yl)pyrazin-2-yl)ethyl)propenamide (B-973B) showed reduced inward rectification and calcium-dependent reversal potential shifts decreased by 80%, and 50%, respectively, compared with currents activated by ACh alone, indicative of reduced calcium permeability. Currents potentiated by 3a,4,5,9b-tetrahydro-4-(1-naphthalenyl)-3H-cyclopentan[c]quinoline-8-sulfonamide were also linear and showed no calcium-dependent reversal potential shifts. The ago-PAMs GAT-107 and B-973B stimulated increases in intracellular calcium in stably transfected HEK293 cells. However, these calcium signals were delayed relative to channel activation produced by these agents and were insensitive to the channel blocker mecamylamine. Our results indicate that, although allosterically activated α7 nicotinic ACh receptor may affect intracellular calcium levels, such effects are not likely due to large channel-dependent calcium influx. SIGNIFICANCE STATEMENT: Positive allosteric modulators (PAMs) of α7 nicotinic acetylcholine receptor can increase channel activation by two or more orders of magnitude, raising the concern that, due to the relatively high calcium permeability of α7 receptors activated by acetylcholine alone, such efficacious PAMs may have cytotoxic side effects. We show that PAMs alter the ion conduction pathway and, in general, reduce the calcium permeability of the channels. This supports the hypothesis that α7 effects on intracellular calcium may be independent of channel-mediated calcium influx.

Asunto(s)

Calcio/metabolismo , Receptor Nicotínico de Acetilcolina alfa 7/metabolismo , Acetilcolina/metabolismo , Regulación Alostérica/efectos de los fármacos , Sitio Alostérico , Animales , Permeabilidad de la Membrana Celular/efectos de los fármacos , Células HEK293 , Humanos , Oocitos , Técnicas de Placa-Clamp , Fenilpropionatos/farmacología , Piperazinas/farmacología , Quinolinas/farmacología , Sulfonamidas/farmacología , Xenopus laevis , Receptor Nicotínico de Acetilcolina alfa 7/agonistas

RESUMEN

Brazil is the biggest producer of sweet oranges and the main exporter of concentrated orange juice in the world. Among the diseases that affect citriculture, Asiatic citrus canker, caused by the bacterial pathogen Xanthomonas citri, represents one of the most significant threats. The current Brazilian legislation regulating the control of citrus canker no longer requires the eradication of affected trees in states where the incidence of the disease is high. Instead, control involves disease control measures, including periodic preventative spraying of copper compounds. The long-term use of copper for plant disease control has raised concerns about environmental accumulation and toxicity, as well as the selective pressure it exerts leading to the emergence of copper-resistant X. citri strains. Here, we evaluated hexyl gallate (G6) as an alternative to copper compounds for citrus plant protection. G6 was able to protect citrus nursery trees against X. citri infection. Thirty days after inoculation, the trees treated with G6 developed 0.5 lesions/cm2 leaf area compared with the 2.84 lesions/cm2 observed in the untreated control trees. Also, G6 did not interfere with germination and root development of tomato, lettuce, and arugula, which is consistent with our previous data showing that G6 is safe for tissue culture cell lines. Membrane permeability tests showed that the primary target of G6 is the bacterial outer membrane. Finally, we could not isolate spontaneous X. citri mutants resistant to G6 nor induce resistance to G6 after long-term exposures to increasing concentrations of the compound, which suggests that G6 may have multiple cellular targets. This study demonstrated that G6 is a promising candidate for the development and use in citrus canker management.

Asunto(s)

Citrus sinensis/microbiología , Enfermedades de las Plantas/prevención & control , Xanthomonas/efectos de los fármacos , Brasil , Permeabilidad de la Membrana Celular/efectos de los fármacos , Farmacorresistencia Bacteriana , Germinación/efectos de los fármacos , Pruebas de Sensibilidad Microbiana , Enfermedades de las Plantas/microbiología , Hojas de la Planta/microbiología , Raíces de Plantas/efectos de los fármacos , Raíces de Plantas/crecimiento & desarrollo , Xanthomonas/fisiología

RESUMEN

Candida albicans is a major cause of human infections, ranging from relatively simple to treat skin and mucosal diseases to systemic life-threatening invasive candidiasis. Fungal infections treatment faces three major challenges: the limited number of therapeutic options, the toxicity of the available drugs, and the rise of antifungal resistance. In this study, we demonstrate the antifungal activity and mechanism of action of peptides ToAP2 and NDBP-5.7 against planktonic cells and biofilms of C. albicans. Both peptides were active against C. albicans cells; however, ToAP2 was more active and produced more pronounced effects on fungal cells. Both peptides affected C. albicans membrane permeability and produced changes in fungal cell morphology, such as deformations in the cell wall and disruption of ultracellular organization. Both peptides showed synergism with amphotericin B, while ToAP2 also presents a synergic effect with fluconazole. Besides, ToAP2 (6.25 µM.) was able to inhibit filamentation after 24 h of treatment and was active against both the early phase and mature biofilms of C. albicans. Finally, ToAP2 was protective in a Galleria mellonella model of infection. Altogether these results point to the therapeutic potential of ToAP2 and other antimicrobial peptides in the development of new therapies for C. albicans infections.

Asunto(s)

Antifúngicos/farmacología , Biopelículas/efectos de los fármacos , Candida albicans/efectos de los fármacos , Candidiasis/tratamiento farmacológico , Proteínas Citotóxicas Formadoras de Poros/farmacología , Anfotericina B/farmacología , Anfotericina B/uso terapéutico , Animales , Antifúngicos/uso terapéutico , Candidiasis/microbiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Pared Celular/efectos de los fármacos , Modelos Animales de Enfermedad , Farmacorresistencia Fúngica , Sinergismo Farmacológico , Quimioterapia Combinada/métodos , Fluconazol/farmacología , Fluconazol/uso terapéutico , Humanos , Pruebas de Sensibilidad Microbiana , Mariposas Nocturnas , Proteínas Citotóxicas Formadoras de Poros/uso terapéutico

RESUMEN

Starting with cholesterol homeostasis, the first part of the review addresses various aspects of cholesterol metabolism in neuronal and glial cells and the mutual crosstalk between the two cell types, particularly the transport of cholesterol from its site of synthesis to its target loci in neuronal cells, discussing the multiple mechanistic aspects and transporter systems involved. Statins are next analyzed from the point of view of their chemical structure and its impingement on their pharmacological properties and permeability through cell membranes and the blood-brain barrier in particular. The following section then discusses the transcriptional effects of statins and the changes they induce in brain cell genes associated with a variety of processes, including cell growth, signaling and trafficking, uptake and synthesis of cholesterol. We review the effects of statins at the cellular level, analyzing their impact on the cholesterol composition of the nerve and glial cell plasmalemma, neurotransmitter receptor mobilization, myelination, dendritic arborization of neurons, synaptic vesicle release, and cell viability. Finally, the role of statins in disease is exemplified by Alzheimer and Parkinson diseases and some forms of epilepsy, both in animal models and in the human form of these pathologies.

Asunto(s)

Encéfalo/metabolismo , Colesterol/metabolismo , Inhibidores de Hidroximetilglutaril-CoA Reductasas/química , Neuronas/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/patología , Encéfalo/patología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Homeostasis/genética , Humanos , Inhibidores de Hidroximetilglutaril-CoA Reductasas/uso terapéutico , Neuroglía/metabolismo , Enfermedad de Parkinson/tratamiento farmacológico , Enfermedad de Parkinson/patología , Vesículas Sinápticas/efectos de los fármacos , Vesículas Sinápticas/genética

RESUMEN

Artepillin C is the main compound present in propolis from Baccharis dracunculifolia, whose antitumor activity has been the focus of many studies. Herein, we shall investigate the Artepillin C mechanisms of action against cells derived from the oropharyngeal carcinoma (HEp-2). Cytotoxicity tests revealed that the concentrations of Artepillin C required to reduce cell viability by 50% (CC50) are dependent on the incubation time, decreasing from 40.7 × 10-5 mol/L to 15.7 × 10-5 mol/L and 9.05 × 10-5 mol/L considering 12, 24 and 48 h, respectively. Hydrophobic interactions on neutral species of Artepillin C induce aggregation over the HEp-2 plasma membrane, given the acid conditions of the cellular culture. Indeed, Langmuir monolayers mimicking cellular membranes of tumor cells revealed Artepillin C affinity to interact with 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) containing 20 mol% of 1,2-dipalmitoyl-sn-glychero-3-phosphoserine (DPPS), leading aggregation on giant unilamellar vesicles (GUVs) at pH 3.2. Moreover, leakage experiments on GUVs have shown that the presence of DPPS enhances the efflux of the fluorescent probe signaling the membrane permeabilization, which is the origin of the necrotic pathway triggered in HEp-2 cells, as observed by flow cytometry assays.

Asunto(s)

Antineoplásicos/farmacología , Membrana Celular/efectos de los fármacos , Fenilpropionatos/química , Antineoplásicos/química , Antineoplásicos/metabolismo , Línea Celular Tumoral , Membrana Celular/química , Membrana Celular/metabolismo , Permeabilidad de la Membrana Celular/efectos de los fármacos , Supervivencia Celular/efectos de los fármacos , Humanos , Concentración de Iones de Hidrógeno , Interacciones Hidrofóbicas e Hidrofílicas , Microscopía Confocal , Fenilpropionatos/metabolismo , Fenilpropionatos/farmacología , Liposomas Unilamelares/química , Liposomas Unilamelares/metabolismo

RESUMEN

Sporotrichosis is a chronic and subacute mycosis causing epidemiological outbreaks involving sick cats and humans in southeastern Brazil. The systemic disease prevails in cats, and in humans, the symptoms are restricted to skin in immunocompetent individuals. Under these conditions, the prolonged treatment of animals and cases of recurrence justify the discovery of new treatments for sporotrichosis. This work addresses the antifungal activity of silver salts of Keggin-type heteropolyacid salts (Ag-HPA salts) such as Ag3[PW12O40], Ag6[SiW10V2O40], Ag4[SiW12O40] and Ag3[PMo12O40] and interactions with the antifungal drugs itraconazole (ITC), terbinafine (TBF) and amphotericin B (AMB) on the yeast and mycelia forms of Sporothrix spp. Sporothrix spp. yeast cells were susceptible to Ag-HPA salts at minimum inhibitory concentration (MIC) values ranging from 8 to 128 µg/mL. Interactions between Ag3[PW12O40] and Ag3[PMo12O40] with itraconazole and amphotericin B resulted in higher antifungal activity with a reduction in growth and melanization. Treated cells showed changes in cell membrane integrity, vacuolization, cytoplasm disorder, and membrane detachment. Promising antifungal activity for treating sporotrichosis was observed for the Ag-HPA salts Ag3[PMo12O40] and Ag3[PW12O40], which have a low cost, high yield and activity at low concentrations. However, further evaluation of in vivo tests is still required.

Asunto(s)

Antifúngicos/farmacología , Plata/farmacología , Sporothrix/efectos de los fármacos , Compuestos de Tungsteno/farmacología , Antifúngicos/química , Permeabilidad de la Membrana Celular/efectos de los fármacos , Sinergismo Farmacológico , Pruebas de Sensibilidad Microbiana , Micelio/efectos de los fármacos , Micelio/crecimiento & desarrollo , Micelio/metabolismo , Pigmentación/efectos de los fármacos , Sales (Química)/química , Sales (Química)/farmacología , Plata/química , Sporothrix/crecimiento & desarrollo , Sporothrix/metabolismo , Sporothrix/ultraestructura , Compuestos de Tungsteno/química

RESUMEN

The physicochemical characteristics of nanostructured suspensions are important prerequisites for the success of new drug development. This work aimed to develop nanometric systems containing Cymbopogon densiflorus leaf essential oil and to evaluate their antimicrobial activity. The essential oil was isolated by hydrodistillation from leaves and analyzed by GC-MS. The main constituents were found to be trans-p-mentha-2,8-dien-1-ol, cis-p-mentha-2,8-dien-1-ol, trans-p-mentha-1(7),8-dien-2-ol, cis-piperitol, and cis-p-mentha-1(7),8-dien-2-ol. In silico prediction analysis suggested that this oil possesses antimicrobial potential and the main mechanism of action might be the peptidoglycan glycosyltransferase inhibition. Nanoemulsions were prepared by the phase inversion method, and liposomes were made by the film hydration method. Qualitative evaluation of the antimicrobial activity was performed by the diffusion disk assay with 24 microorganisms; all of them were found to be sensitive to the essential oil. Subsequently, this property was quantified by the serial microdilution technique, where the nanoformulations demonstrated improved activity in comparison with the free oil. Bactericidal action was tested by the propidium iodide method, which revealed that free essential oil and nanoemulsion increased cytoplasmic membrane permeability, while no difference was observed between negative control and liposome. These results were confirmed by images obtained using transmission electron microscopy. This study has shown an optimization in the antimicrobial activity of C. densiflorus essential oil by a nanoemulsion and a liposomal formulation of the active substances.

Asunto(s)

Antiinfecciosos/farmacología , Cymbopogon/química , Nanoestructuras , Aceites Volátiles/farmacología , Hojas de la Planta/química , Animales , Antiinfecciosos/química , Bacterias/efectos de los fármacos , Línea Celular , Permeabilidad de la Membrana Celular/efectos de los fármacos , Ensayos de Selección de Medicamentos Antitumorales , Hongos/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Concentración de Iones de Hidrógeno , Ratones , Pruebas de Sensibilidad Microbiana , Microscopía Electrónica de Transmisión , Aceites Volátiles/química , Tamaño de la Partícula , Relación Estructura-Actividad Cuantitativa

RESUMEN

In recent years, hopanoids, a group of pentacyclic compounds found in bacterial membranes, are in the spotlight since it was proposed that they induce order in lipid membranes in a similar way cholesterol do in eukaryotes, despite their structural differences. We studied here whether diplopterol (an abundant hopanoid) promoted similar effects on model membranes as sterols do. We analyzed the compaction, dynamics, phase segregation, permeability and compressibility of model membranes containing diplopterol, and compared with those containing sterols from animals, plants and fungi. We also tested the effect that the incubation with diplopterol had on hopanoid-lacking bacteria. Our results show that diplopterol induces phase segregation, increases lipid compaction, and decreases permeability on phospholipid membranes, while retaining membrane fluidity and compressibility. Furthermore, the exposition to this hopanoid decreases the permeability of the opportunistic pathogen Pseudomonas aeruginosa and increases the resistance to antibiotics. All effects promoted by diplopterol were similar to those generated by the sterols. Our observations add information on the functional significance of hopanoids as molecules that play an important role in membrane organization and dynamics in model membranes and in a bacterial system.

Asunto(s)

Permeabilidad de la Membrana Celular/fisiología , Membrana Celular/química , Triterpenos/metabolismo , Membrana Celular/fisiología , Permeabilidad de la Membrana Celular/efectos de los fármacos , Membrana Dobles de Lípidos/química , Lípidos de la Membrana/química , Lípidos de la Membrana/fisiología , Membranas/química , Membranas/fisiología , Modelos Biológicos , Permeabilidad , Fosfolípidos/química , Fosfolípidos/fisiología , Pseudomonadaceae/metabolismo , Esteroles/química , Triterpenos/farmacología

RESUMEN

Xanthomonas campestris pv.campestris (Xcc) is the causative agent of black rot, a disease that causes serious damage to plants from Brassicaceae family. However, there are no chemicals or biological agent commercially registered for the control of this disease. Thus, this study aimed to evaluate the antimicrobial activity and chemical composition of Lippia gracilis essential oils (EOs) on Xcc aiming its use as effective biological control. We also investigated the effect of EOs on the integrity of the bacterial cytoplasmic membrane. Chemical analysis by GC/MS showed that the major compounds of the seven EOs of L. gracilis are thymol or carvacrol. The seven genotypes showed inhibition of bacterial growth with MIC from 700 µg.ml-1 to 1000 µg.ml-1, with the genotype LGRA-106 (rich in Thymol) with higher antimicrobial activity. The MIC for thymol and carvacrol were 250 µg.ml-1. After exposure to LGRA-106 EO (2×, 1×, 1/2×, 1/4×, and 1/8 x MIC for 5 min, it was observed a decreased cell viability and increased pI fluorescence, which indicates damage to the cytoplasmic cell membrane. This study demonstrates that L. gracilis EOs have antimicrobial activity and have a potential to be used in the control of black rot. Furthermore this antimicrobial activity is due, at least in part, to bacterial cytoplasmic membrane damage.

Asunto(s)

Antibacterianos/farmacología , Lippia/química , Aceites Volátiles/farmacología , Xanthomonas campestris/efectos de los fármacos , Permeabilidad de la Membrana Celular/efectos de los fármacos , Citoplasma/efectos de los fármacos , Cromatografía de Gases y Espectrometría de Masas , Pruebas de Sensibilidad Microbiana

RESUMEN

Phytophthora parasitica is an important oomycete that causes disease in a variety of plants, dimethomorph fungicides being specific for oomycetes. The aim of this study was to use RNA-seq to rapidly discover the mechanism by which dimethomorph acts in the treatment of P. parasitica. We found that the expression of 832 genes changed significantly after the dimethomorph treatment, including 365 up-regulated genes and 467 down-regulated genes. According to the Gene Ontology (GO) enrichment analysis, pathway enrichment and verification test results, the following conclusions are obtained: (i) the treatment of P. parasitica with dimethomorph causes changes in the expression levels of genes associated with the cell wall and cell wall synthesis; (ii) dimethomorph treatment results in reduced permeability of the cell membrane and changes in the expression of certain transport-related proteins; (iii) dimethomorph treatment increased reactive oxygen species and reduced the expression of genes related to the control of oxidative stress.

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Phytophthora parasitica es un importante oomiceto que origina enfermedades en una variedad de plantas; el fungicida dimetomorf es específico contra oomicetos. El objetivo de este estudio fue utilizar la tecnología de RNA-seq para descubrir rápidamente el mecanismo por el que el dimetomorf actúa en el tratamiento de P. parasitica. Descubrimos que la expresión de 832 genes se modificaba significativamente tras el tratamiento con dimetomorf, incluyendo 365 genes que son sobrerregulados y 467 genes que son subrregulados. El análisis de enriquecimiento de ontología de genes (GO), análisis de enriquecimiento de las vías y pruebas de verificación permitieron extraer las conclusiones siguientes: 1) el tratamiento de P. parasitica con dimetomorf origina cambios en los niveles de expresión de los genes relacionados con la pared celular y su síntesis; 2) el tratamiento con dimetomorf origina una reducción de la permeabilidad de la membrana celular, así como cambios en la expresión de ciertas proteínas relacionadas con el transporte, y 3) el tratamiento con dimetomorf incrementó las especies reactivas del oxígeno y redujo la expresión de los genes relacionados con el control del estrés oxidativo.

Asunto(s)

Phytophthora/efectos de los fármacos , ARN Mensajero/biosíntesis , Morfolinas/farmacología , Fungicidas Industriales/farmacología , RNA-Seq , Phytophthora/genética , Enfermedades de las Plantas/parasitología , ARN Mensajero/genética , Proteínas Portadoras/biosíntesis , Proteínas Portadoras/genética , Permeabilidad de la Membrana Celular/efectos de los fármacos , Permeabilidad de la Membrana Celular/genética , Pared Celular/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Alineación de Secuencia , Especies Reactivas de Oxígeno , Estrés Oxidativo/genética , beta-Glucanos/análisis , Reacción en Cadena en Tiempo Real de la Polimerasa , Ontología de Genes