RESUMO
Inorganic polyphosphate (polyP) is a linear polymer composed of several molecules of orthophosphate (Pi) linked by energy-rich phosphoanhydride bonds. In Pseudomonas aeruginosa, Pi is taken up by the ABC transporter Pst, encoded by an operon consisting of five genes. The first four genes encode proteins involved in the transport of Pi and the last gene of the operon, phoU, codes for a protein which exact function is unknown. We show here that the inactivation of phoU in P. aeruginosa enhanced Pi removal from the medium and polyP accumulation. The phoU mutant also accumulated high levels of the alarmone guanosine tetraphosphate (ppGpp), which in turn increased the buildup of polyP. In addition, phoU inactivation had several pleiotropic effects, such as reduced growth rate and yield and increased sensitivity to antibiotics and stresses. However, biofilm formation was not affected by the phoU mutation.
Assuntos
Proteínas de Bactérias/metabolismo , Técnicas de Inativação de Genes , Guanosina Tetrafosfato/metabolismo , Polifosfatos/metabolismo , Pseudomonas aeruginosa/enzimologia , Proteínas de Bactérias/genética , Óperon , Pseudomonas aeruginosa/genéticaRESUMO
The Danshen terpenoid cryptotanshinone (CPT) is gaining enormous interest in light of its various outstanding biological activities. Among those, CPT has been shown to interact with cell membranes and, for instance, to have antibacterial activity. Several works have shown that CPT alone, or in combination with other drugs, can effectively act as an antibiotic against various infectious bacteria. Some authors have related the mechanism underlying this action to CPT-membrane interaction. This work shows that CPT readily partitions into phosphatidylcholine membranes, but there is a limiting capacity of accommodation of ca. 1 mol CPT to 3 mol phospholipid. The addition of CPT to unilamellar liposomes composed of 1-palmitoyl-2-oleoylphosphatidylcholine (POPC) causes membrane permeabilization, as shown by fluorescent probe leakage. This process has been kinetically studied, as well as its modulation by incorporation of phosphatidylethanolamine or phosphatidylglycerol, as a model for pathogenic cell membranes. The thermotropic behavior of 1,2-dimyristoylphosphatidylcholine (DMPC) model membranes is weakly affected by CPT, but the terpenoid causes significant dehydration of the polar region of the bilayer and weak disordering of the acyl chain palisade, as observed in Fourier-transform infrared spectroscopy (FTIR) results. Small-angle X-ray scattering (SAXS) shows that CPT increases DMPC bilayer thickness, which could be due to localization near the phospholipid/water interface. Molecular dynamics (MD) simulations show that the lateral diffusion coefficient of the phospholipid increases with the presence of CPT. CPT extends from the polar head region to the center of the bilayer, being centered between the carbonyl groups and the unsaturated region of the POPC, where there is greater overlap. Interestingly, the free energy profiles of a water molecule crossing the lipid membrane show that the POPC membrane becomes more permeable in the presence of CPT. In summary, our results show that CPT perturbs the physicochemical properties of the phospholipid membrane and compromises its barrier function, which could be of relevance to explain part of its antimicrobial or anticancer activities.
RESUMO
Tamoxifen (TMX), commonly used in complementary therapy for breast cancer, also displays known effects on the structure and function of biological membranes. This work presents an experimental and simulation study on the permeabilization of model phospholipid membranes by TMX and its derivative 4-hydroxytamoxifen (HTMX). TMX induces rapid and extensive vesicle contents leakage in phosphatidylcholine (PC) liposomes, with the effect of HTMX being much weaker. Fitting of the leakage curves for TMX, yields two rate constants, corresponding to a fast and a slow process, whereas in the case of HTMX, only the slow process takes place. Interestingly, incorporation of phosphatidylglycerol (PG) or phosphatidylethanolamine (PE) protects PC membranes from TMXinduced permeabilization. Fourier-transform infrared spectroscopy (FTIR) shows that, in the presence of TMX there is a shift in the νCH2 band frequency, corresponding to an increase in gauche conformers, and a shift in the νC=O band frequency, indicating a dehydration of the polar region. A preferential association of TMX with PC, in mixed PC/PE systems, is observed by differential scanning calorimetry. Molecular dynamics (MD) simulations support the experimental results, and provide feasible explanations to the protecting effect of PG and PE. These findings add new information to explain the various mechanisms of the anticancer actions of TMX, not related to the estrogen receptor, and potential side effects of this drug.
RESUMO
The interaction of tamoxifen (TMX) and its metabolite 4-hydroxytamoxifen (HTMX) with a biomimetic membrane model system composed of 1,2-dielaidoylphosphatidylethanolamine (DEPE) has been studied using a biophysical approach. Incorporation of TMX into DEPE bilayers gives rise to a progressive broadening of the Lß/Lα phase transition and a downward temperature shift. The Lß/Lα phase transition presents multiple endotherms, indicating a lateral segregation of TMX/DEPE domains within the plane of the bilayer. TMX and HTMX also widen and shift the Lα to hexagonal-HII transition toward lower values, the phase diagrams showing that both compounds facilitate formation of the HII phase. TMX increases motional disorder of DEPE acyl chains in the Lß, Lα and HII phases, whereas the effect of HTMX is clearly different. In addition, neither TMX nor HTMX significantly perturb the hydration state of the polar headgroup region of DEPE. Molecular dynamics (MD) simulations indicate that these drugs do not affect membrane thickness, area per lipid, or the conformation of DEPE molecules. As a general rule, the interaction of HTMX with DEPE is qualitatively similar to TMX but less intense. However, a significant difference shown by MD is that HTMX is mainly placed around the center of each monolayer while TMX is located mainly at the center of the membrane, also having a greater tendency to cluster formation. These results are discussed to understand the modulation of phosphatidylethanolamine lipid polymorphism carried out by these drugs, which could be of relevance to explain their effects on enzyme activity or membrane permeabilization.
Assuntos
Antineoplásicos , Fosfatidiletanolaminas , Bicamadas Lipídicas , Transição de Fase , Tamoxifeno/farmacologiaRESUMO
The anticancer drug tamoxifen and its primary metabolite 4-hydroxytamoxifen tend to accumulate in membranes due to its strong hydrophobic character. Thus, in this work we have carried out a systematic study to investigate their effects on model phosphatidylcholine membranes. Tamoxifen and 4-hydroxytamoxifen affect the phase behaviour of phosphatidylcholine model membranes, giving rise to formation of drug/dipalmitoylphosphatidylcholine domains, which is more evident in the case of 4-hydroxytamoxifen. These drugs have differential effects on the polar and apolar regions of the phospholipid supporting a different location of both compounds within the bilayer. Both compounds induce contents leakage in fluid phosphatidylcholine unilamellar liposomes, the effect of 4-hydroxytamoxifen being negligible as compared to that of tamoxifen. Molecular dynamics confirmed the tendency of both drugs to form clusters, tamoxifen locating all along the bilayer, whereas 4-hydroxytamoxifen mostly locates near the lipid/water interface, which can explain the different effects of both drugs in fluid phosphatidylcholine membranes.
Assuntos
Fosfatidilcolinas , Tamoxifeno , Tamoxifeno/análogos & derivados , Tamoxifeno/metabolismo , Tamoxifeno/farmacologiaRESUMO
HYPOTHESIS: Pseudomonas aeruginosa dirhamnolipid (diRL) has been shown to form aggregates of different size and structure, under various conditions. Due to the presence of a carboxyl group in the molecule, it is expected that pH would strongly affect this aggregation behaviour. In addition, preliminary observations of temperature-induced changes in the states of aggregation of diRL supported the need of further investigation. EXPERIMENTS: A systematic experimental study, using differential scanning calorimetry (DSC), small-angle Xray diffraction (SAXD), and Fourier-transform infrared spectroscopy (FTIR), has been carried out to characterize pH and temperature driven changes in the aggregation behavior of diRL biosurfactant. Molecular dynamics (MD) simulations, supported by the experimental results, allowed depicting molecular details on formation of diRL membranes and other aggregated structures under various physicochemical conditions. FINDINGS: DiRL could adopt fairly organized multilayered structures (membranes) at low pH and temperature, which became highly disordered upon increasing either of these parameters. The effect of pH on the gauche/all-trans conformer ratio of the diRL acyl chains was not of significance, whereas temperature-induced effects were observed. For the first time it is described that diRL underwent an endothermic thermotropic transition with Tc = 34 °C as observed by DSC, at pH 4.5 (protonated diRL), but not at pH 7.4 (unprotonated diRL). FTIR confirmed these findings, showing a significant additional disordering of the all-trans acyl chains upon increasing temperature around that same value in the protonated form, an effect not observed for the dissociated form of the biosurfactant. In addition, at pH 7.4, changing temperature did not modify the hydration state of the polar moiety of diRL, whereas at pH 4.5 a significant decrease in the hydration state around 34 °C took place. SAXD data showed that protonated diRL formed multilayered structures at 20 °C, which converted into poorly correlated layers at 50 °C. MD simulations supported these findings, showing that the membrane-like structures formed by protonated diRL at 20 °C became unstable at higher temperatures, tending to form other structures, which could be micelles or other type of layered structures, whereas the negatively charged form of diRL organized in micelle-type aggregates in the whole range of temperature under study.
Assuntos
Simulação de Dinâmica Molecular , Tensoativos , Varredura Diferencial de Calorimetria , Glicolipídeos , Concentração de Íons de Hidrogênio , Espectroscopia de Infravermelho com Transformada de Fourier , TemperaturaRESUMO
Sophorolipids (SLs) constitute a group of unique biosurfactants (BS) in the light of their outstanding properties, among which their antimicrobial activities stand out. SLs can exist mainly in an acidic and a lactonic form, both of which display inhibitory activity. Given the amphipathic nature of SLs it is feasible that these antimicrobial actions are the result of the perturbation of the physicochemical properties of targeted membranes. Thus, in this work we have carried out a biophysical study to unveil the molecular details of the interaction of an acidic SL with a model phospholipid membrane made of 1,2-dipalmitoy-sn-glycero-3-phosphocholine (DPPC). Using differential scanning calorimetry it was found that SL altered the phase behaviour of DPPC at low molar fractions, producing fluid phase immiscibility with the result of formation of biosurfactant-enriched domains within the phospholipid bilayer. Fourier-transform infrared spectroscopy showed that SL interacted with DPPC increasing ordering of the phospholipid acyl chain palisade and hydration of the lipid/water interface. Small angle X-ray scattering showed that SL did not modify bilayer thickness in the biologically relevant Lα fluid phase. SL was found to induce contents leakage in 1-palmitoy-2-oleoy-sn-glycero-3-phosphocholine (POPC) unilamellar liposomes, at sublytic concentrations below the cmc. This SL-induced membrane permeabilization at concentrations below the onset for membrane solubilization can be the result of the formation of laterally segregated domains, which might contribute to provide a molecular basis for the reported antimicrobial actions of SLs.
Assuntos
Lecitinas , Fosfatidilcolinas , Varredura Diferencial de Calorimetria , Bicamadas Lipídicas , Membranas , Ácidos Oleicos , FosfolipídeosAssuntos
Enfermeiros Clínicos/organização & administração , Enfermeiros Clínicos/psicologia , Papel do Profissional de Enfermagem/psicologia , Higiene da Pele/enfermagem , Incontinência Fecal/enfermagem , Humanos , Satisfação no Emprego , Enfermeiros Clínicos/educação , Estomia/enfermagem , Higiene da Pele/métodos , Incontinência Urinária/enfermagem , Ferimentos e Lesões/enfermagemRESUMO
En nuestro sistema nacional de salud son incalculables los esfuerzos realizados en función de dotar a la Estomatología y dentro de ella a la Periodontología, de los medios más modernos y eficaces en las actividades preventivo curativas contra las enfermedades periodontales. Objetivo: determinar el comportamiento de las enfermedades periodontales en adolescentes de los consultorios 11, 51 y 64 del Área de Salud Norte perteneciente al Policlínico Docente Joaquín de Agüero y la Clínica Estomatológica Docente La Vigía. Método: se realizó un estudio descriptivo en adolescentes de once a quince años. La muestra quedó conformada por cuarenta adolescentes a los cuales se le realizó una encuesta de conocimientos sobre salud bucal. Resultados: predominó el sexo femenino, el nivel de información sobre salud bucal fue aceptable, predominaron los procesos inflamatorios. Conclusiones: los adolescentes que más concurrieron al estudio fueron los de 14 años. Más de la mitad de los adolescentes estudiados estaban afectados por la enfermedad periodontal.
In our national system of health are incalculable the efforts carried out in function of endowing Odontology, and inside her to Periodontology, of the most modern and effective means in the preventive curative activities against the periodontal diseases. Objective: to determine the behavior of the periodontal diseases in adolescents from the doctors´ office 11, 51 and 64 of the North Health Area belonging to the Teaching Polyclinic "Joaquín de Agüero" and the Teaching Odontology Clinic "La Vigía". Method: a descriptive study in adolescents from eleven to fifteen years was performed. The sample was constituted by forty adolescents to whom a survey of knowledge on oral health was carried out. Results: the feminine sex prevailed, the level of information on oral health was acceptable, and the inflammatory processes predominated. Conclusions: adolescents that most concurred to the study were those of 14 years. More than the half of the studied adolescents was affected by the periodontal disease.
Assuntos
Humanos , Adolescente , Inflamação , Saúde Bucal , Doenças PeriodontaisRESUMO
Fundamento: La caries dental se describe como una secuencia de procesos de destrucción localizada en los tejidos duros del diente que evoluciona en forma progresiva e irreversible. La misma comienza en la superficie y luego avanza en profundidad. Objetivo: Determinar el comportamiento de algunos factores de riesgo y caries dental en adolescentes de 12 a 15 años. Método: Se realizó una investigación en servicios de salud de tipo descriptivo en los consultorios 11, 51 y 64 del área de salud Norte, perteneciente al Policlínico Comunitario «Joaquín de Agüero¼ y la Clínica Estomatológica Docente «La Vigía¼, del municipio Camagüey, desde octubre de 2005 a septiembre del 2006. La muestra estuvo constituida por 40 adolescentes, a los cuales se les aplicó un cuestionario para establecer el nivel de información sobre salud bucal, se realizó la historia clínica individual y en un formulario se recogieron datos tales como, experiencia anterior de caries dental, índice de higiene bucal, dieta cariogénica, apiñamiento dentario, pH salival y prematuridad al nacer. Resultados: La mayoría poseía una buena información sobre salud bucal, sin embargo, hubo predominio de los afectados por caries dental, higiene bucal deficiente, dieta cariogénica, donde un elevado por ciento de ellos tenían valores de pH ácido y neutro y más de la mitad de los prematuros estaban afectados por caries dental, en tanto el mayor por ciento clasificó en alto y moderado riesgo a caries dental. Conclusiones: Más de la ¾ parte de la muestra clasificó en alto y moderado riesgo a caries dental.
Background: Dental caries is described like a located destruction processes sequence in the hard tissues of the tooth that evolves in progressive and irreversible form. It begins in the surface and then advances in depth. Objective: To determine the behavior of some risk factors and dental caries in adolescents from 12 to 15 years. Method: An investigation of descriptive type in the doctor's offices 11, 51 and 64 of the North health area was performed, belonging to «Joaquin de Agüero¼ Community Hospital and «La Vigía¼ Educational Odontology Clinic, of Camagüey municipality, from October 2005 to September 2006. The sample was constituted by 40 adolescents, to which a questionnaire to establish the level of information on oral health was applied, the individual medical history was carried out and in a form, data were collected such as, previous experience of dental caries, oral hygiene rate, cariogenic diet, crowding, salivary pH and prematurity upon being born. Results: The majority possessed a good information on oral health, nevertheless, there was predominance of the affected ones by dental caries, deficient oral hygiene, cariogenic diet, where a raised percent of them had acid and neutral pH values and more than the half of the premature were affected by dental caries, in the meanwhile the major percent classified in high and moderate risk to dental caries. Conclusions: More than the ¾ part of the sample classified in high and moderate risk to dental caries.