RESUMEN
Previous work demonstrated that lysine homopeptides adopt a polyproline II (PPII) structure. Lysine homopeptides with odd number of residues, especially with 11 residues (K11), were capable of inhibiting the growth of a broader spectrum of bacteria than those with an even number. Confocal studies also determined that K11 was able to localize exclusively in the bacterial membrane, leading to cell death. In this work, the mechanism of action of this peptide was further analyzed focused on examining the structural changes in bacterial membrane induced by K11, and in K11 itself when interacting with bacterial membrane lipids. Moreover, alanine and proline scans were performed for K11 to identify relevant positions in structure conformation and antibacterial activity. To do so, circular dichroism spectroscopy (CD) was conducted in saline phosphate buffer (PBS) and in lipidic vesicles, using large unilamellar vesicles (LUV), composed of 2-dimyristoyl-sn-glycero-3-phosphoglycerol (DMPG) or bacterial membrane lipid. Antimicrobial activity of K11 and their analogs was evaluated in Gram-positive and Gram-negative bacterial strains. The scanning electron microscopy (SEM) micrographs of Staphylococcus aureus ATCC 25923 exposed to the Lys homopeptide at MIC concentration showed blisters and bubbles formed on the bacterial surface, suggesting that K11 exerts its action by destabilizing the bacterial membrane. CD analysis revealed a remarkably enhanced PPII structure of K11 when replacing some of its central residues by proline in PBS. However, when such peptide analogs were confronted with either DMPG-LUV or membrane lipid extract-LUV, the tendency to form PPII structure was severely weakened. On the contrary, K11 peptide showed a remarkably enhanced PPII structure in the presence of DMPG-LUV. Antibacterial tests revealed that K11 was able to inhibit all tested bacteria with an MIC value of 5 µM, while proline and alanine analogs have a reduced activity on Listeria monocytogenes. Besides, the activity against Vibrio parahaemolyticus was affected in most of the alanine-substituted analogs. However, lysine substitutions by alanine or proline at position 7 did not alter the activity against all tested bacterial strains, suggesting that this position can be screened to find a substitute amino acid yielding a peptide with increased antibacterial activity. These results also indicate that the PPII secondary structure of K11 is stabilized by the interaction of the peptide with negatively charged phospholipids in the bacterial membrane, though not being the sole determinant for its antimicrobial activity.
Asunto(s)
Antibacterianos , Péptidos Catiónicos Antimicrobianos , Bacterias/crecimiento & desarrollo , Polilisina , Alanina/química , Antibacterianos/química , Antibacterianos/farmacología , Péptidos Catiónicos Antimicrobianos/química , Péptidos Catiónicos Antimicrobianos/farmacología , Polilisina/química , Polilisina/farmacología , Prolina/químicaRESUMEN
In this study, we report the effect of cholesterol content on the dynamic and structural properties of a dimyristoyl-phosphatidylcholine and distearoyl-phosphatidylcholine mixture in large unilamellar vesicles. The range of cholesterol concentrations studied varied around approximately 33.3mol%, where it has been postulated that an abrupt change in bilayer organization occurs. Steady-state fluorescence measurements demonstrated a typical behavior; at low temperatures in the main phase transition, the cholesterol concentration did not affect the gel phase, but at 37.5°C (phase coexistence) and in the liquid crystalline phase, the presence of cholesterol produced an increase in the fluorescence anisotropy of DPH and the generalized polarization of Laurdan. The greater effect was observed in the liquid crystalline phase, in which the bilayer became a mixture of fluid-like and liquid-ordered phases. The results obtained at approximately 33.3mol% of Cholesterol demonstrated that the Generalized Polarization of Laurdan, the DPH lifetime, the limiting anisotropy and the rotational correlation time, as well as the fluorescence quenching of DPH by TEMPO, are at maxima, while the fluorescence intensity of dehydroergosterol and the lipid solubility in TritonX-100 are at minima. These results correlate well with the hypothesis of domain segregation in the DMPC/DSPC/Cholesterol LUV system. In this context, we postulate that at 33.3mol% of Cho, the proportion of ordered domains reaches a maximum.
Asunto(s)
Colesterol/metabolismo , Dimiristoilfosfatidilcolina/metabolismo , Membrana Dobles de Lípidos/metabolismo , Fosfatidilcolinas/metabolismo , Liposomas Unilamelares/metabolismo , Membrana Celular/metabolismo , Detergentes , Polarización de FluorescenciaRESUMEN
Research on biological influence of vanadium has gained major importance because it exerts potent toxic, mutagenic, and genotoxic effects on a wide variety of biological systems. However, hematological toxicity is one of the less studied effects. The lack of information on this issue prompted us to study the structural effects induced on the human erythrocyte membrane by vanadium (V). Sodium orthovanadate was incubated with intact erythrocytes, isolated unsealed human erythrocyte membranes (IUM) and molecular models of the erythrocyte membrane. The latter consisted of bilayers of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes located in the outer and inner monolayers of the human erythrocyte membrane, respectively. This report presents evidence in order that orthovanadate interacted with red cell membranes as follows: a) in scanning electron microscopy (SEM) studies it was observed that morphological changes on human erythrocytes were induced; b) fluorescence spectroscopy experiments in isolated unsealed human erythrocyte membranes (IUM) showed that an increase in the molecular dynamics and/or water content at the shallow depth of the lipids glycerol backbone at concentrations as low as 50µM was produced; c) X-ray diffraction studies showed that orthovanadate 0.25-1mM range induced increasing structural perturbation to DMPE; d) somewhat similar effects were observed by differential scanning calorimetry (DSC) with the exception of the fact that DMPC pretransition was shown to be affected; and e) fluorescence spectroscopy experiments performed in DMPC large unilamellar vesicles (LUV) showed that at very low concentrations induced changes in DPH fluorescence anisotropy at 18°C. Additional experiments were performed in mice cholinergic neuroblastoma SN56 cells; a statistically significant decrease of cell viability was observed on orthovanadate in low or moderate concentrations.
Asunto(s)
Eritrocitos/metabolismo , Neuroblastoma/metabolismo , Sodio/farmacología , Vanadatos/farmacología , Acetilcoenzima A/química , Animales , Anisotropía , Rastreo Diferencial de Calorimetría/métodos , Línea Celular Tumoral , Supervivencia Celular , Dimiristoilfosfatidilcolina/química , Eritrocitos/efectos de los fármacos , Humanos , Técnicas In Vitro , Lípidos/química , Ratones , Microscopía Electrónica de Rastreo/métodos , Fosfatidiletanolaminas/química , Espectrometría de Fluorescencia/métodos , Temperatura , Liposomas Unilamelares/química , Vanadio/farmacologíaRESUMEN
Zinc is an essential element for nutrition as well as for the proper development and function of brain cells, and its traces are present in a wide range of foods. It is a constituent of many enzyme systems and is an integral part of insulin and of the active site of intracellular enzymes. However, excessive accumulation of zinc or its release from the binding sites may become detrimental for neurons. With the aim to better understand the molecular mechanisms of the interaction of zinc ions with cell membranes, it was incubated with intact human erythrocytes, isolated unsealed human erythrocyte membranes (IUM), cholinergic murine neuroblastoma cells, and molecular models of cell membranes. These consisted in bilayers built-up of dimyristoylphosphatidylcholine (DMPC) and dimyristoylphosphatidylethanolamine (DMPE), phospholipid classes present in the outer and inner monolayers of most plasmatic cell membranes, particularly that of human erythrocytes, respectively. The capacity of zinc ions to perturb the bilayer structures of DMPC and DMPE was assessed by X-ray diffraction, DMPC large unilamellar vesicles (LUV) and IUM were studied by fluorescence spectroscopy, intact human erythrocytes were observed with scanning electron microscopy (SEM), and neuroblastoma cell morphology was observed under inverted microscope. This study presents evidence that 0.1mM Zn and higher concentrations affect cell membrane and molecular models.
Asunto(s)
Membrana Celular/efectos de los fármacos , Eritrocitos/efectos de los fármacos , Membrana Dobles de Lípidos/química , Zinc/farmacología , Animales , Línea Celular Tumoral , Supervivencia Celular/efectos de los fármacos , Dimiristoilfosfatidilcolina/química , Eritrocitos/ultraestructura , Humanos , Ratones , Microscopía Electrónica de Rastreo , Fosfatidiletanolaminas/química , Espectrometría de Fluorescencia , Difracción de Rayos XRESUMEN
A simple method useful for the joint evaluation of substrate partitioning and kinetic parameters for reactions catalyzed by enzymes entrapped in reverse micelles is proposed. The method is applied to the hydrolysis of 2-naphthyl acetate (2-NA) catalyzed by lipase in sodium 1,4-bis(2-ethylhexyl) sulfosuccinate (AOT)/buffer/heptane reverse micellar solutions. In the presence of micelles, the relationship between the initial reaction rate and the analytical concentration of 2-NA was dependent on AOT concentration at a constant W ([water]/[AOT]) value. The dependence of the initial reaction rate profiles with [AOT] was analyzed according with the method proposed to obtain the partition constant of 2-NA between the micelles and the external solvent, Kp. A value of Kp = 2.7 L mol(-1) was obtained irrespective of the water content of the micelles (W from 5 to 20). The catalytic rate constant kcat in the micellar solutions was independent of [AOT] but slightly decreased with an increase in W from 2 x 10(-6) mol g(-1) s(-1) at W = 5 to 1.2 x 10(-6) mol g(-1) s(-1) at W = 20. The apparent Michaelis constant determined in terms of the analytical concentration of 2-NA increased with [AOT] at a given W and moderately decreased with W at a fixed [AOT]. The increase with [AOT] is accounted for by considering the partitioning of the substrate. After correction for the partitioning of 2-NA values of (Km)corr were obtained as 3.9 x 10(-3) mol L(-1) (W = 5), 4.6 x 10(-3) mol L(-1) (W = 10), 2.3 x 10(-3) mol L(-1) (W = 15), and 1.7 x 10(-3) mol L(-1) (W = 20). The rate parameters in the aqueous phase in the absence of micelles, were obtained as (kcat)aq = 7.9 x 10(-6) mol g(-1) s(-1) and (Km)aq = 2.5 x 10(-3) mol L(-1). In order to compare the efficiency of the enzyme in the micellar solution with that in aqueous phase, the values of (Km)corr were in turn corrected to take into account differences in the substrate activity, obtaining so a set of (Km)*corr values. The efficiency of the enzyme in the micellar solution, defined as the ratio, kcat/(Km)*corr, was found to be higher than in the aqueous phase, even at high water contents (W = 20). This higher efficiency is due to a significant decrease in (Km)*corr values.
Asunto(s)
Lipasa/metabolismo , Ácidos Naftalenoacéticos/metabolismo , Rhizopus/enzimología , Tampones (Química) , Catálisis , Ácido Dioctil Sulfosuccínico/química , Heptanos/química , Hidrólisis , Cinética , Lipasa/química , Micelas , Especificidad por Sustrato , Agua/químicaRESUMEN
Cholesterol is known to affect the activity of membrane-bound enzymes, including Na(+)/K(+)-ATPase. To gain insight into the mechanism of cholesterol's effect, we have used various hydrophobic fluorescent probes which insert into different regions of the membrane bilayer and report on the degree of hydration of their environment. Specifially, we have measured the generalized polarization of Laurdan and the lifetime of DPH and derivatives of DPH inserted into membranes from pig kidneys enriched in Na(+)/K(+)-ATPase. Spectral measurements were also carried out on these membranes after modification of their cholesterol content. The generalized polarization of Laurdan increased with increasing cholesterol, showing an abrupt modification at the native cholesterol content. The fluorescence lifetimes of DPH and the DPH derivatives were analyzed using a distribution model. The center value of these lifetime distributions and their widths also changed with increasing cholesterol. One DPH derivative, DPH-PC, showed a minimum value for the lifetime center at the native cholesterol concentration, whereas the other derivatives showed a maximum value for the lifetime center at that cholesterol concentration. DPH-PC is known to sense the protein-lipid interface, whereas the other derivatives sense the bulk lipid phase. These data suggest that hydration at the protein-lipid interface is maximal at the native cholesterol concentration as is the enzymatic activity. Hydration at the protein-lipid interface is therefore proposed to be required for activity. These results are in agreement with current models of membrane dynamics and thermodynamics of protein function.
Asunto(s)
Colesterol/química , Riñón/enzimología , ATPasa Intercambiadora de Sodio-Potasio/química , Animales , Membrana Celular/enzimología , Colesterol/metabolismo , Difenilhexatrieno/química , Activación Enzimática , Polarización de Fluorescencia , Membrana Dobles de Lípidos/química , Liposomas/química , Lípidos de la Membrana/química , Modelos Químicos , Fosfatidilcolinas/química , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Espectrometría de Fluorescencia , Porcinos , Agua/químicaRESUMEN
Using patch-clamp and fluorescence techniques we found that ethanol (10-200 mM) potentiated strychnine-sensitive glycine receptors without having detectable effects on lipid order parameters in mouse spinal cord neurons. Hepthanol (0.01-1 mM), in contrast, did not affect the glycine current, but it altered the core and surface of spinal neuron membranes as detected by changes in 1,6-diphenyl-1,3,5-hexatriene (DPH) and Laurdan fluorescence parameters. These findings support the idea that ethanol affects these membrane proteins without changing lipid fluidity.
Asunto(s)
Etanol/farmacología , Lípidos de la Membrana/metabolismo , Neuronas/efectos de los fármacos , Receptores de Glicina/efectos de los fármacos , Animales , Células Cultivadas , Canales de Cloruro/efectos de los fármacos , Canales de Cloruro/fisiología , Femenino , Glicina/metabolismo , Fluidez de la Membrana , Ratones , Ratones Endogámicos C57BL , Neuronas/fisiología , Receptores de Glicina/fisiología , Médula Espinal/efectos de los fármacos , Médula Espinal/fisiologíaRESUMEN
PIP: The study of perinatal mortality in Mexico is of considerable importance due to the natality rate presented by the country as a result of inefficient development. Women in reproductive age, very young or very late, contribute with high risk pregnancies. Classification of medical causes of perinatal mortality continues to be a problem because of inexact records and the multiplicity of circumstances that may provoke the same effects. The World Health Organization groups the causes of perinatal mortality as follows: congenital malformations; obstetric causes; and other causes, including chronic or acute illness of the mother, illnesses of pregnancy and childbirth. Perinatal mortality expresses the obstetric risks of the fetus and the newlyborn, that is of that period of life that precedes the beginning of the viability of the fetus. Good care at childbirth, prevention of toxemias, of arytoblastosis, the use of adequate procedures of resuscitation, and incubators are all effective factors in the prevention of deaths. Socioeconomic conditions, particularly those related to nutrition, physical status and education of the pregnant women also have an important role in the reduction of perinatal mortality causes. Even through these preventive aspects have been intensified in the whole Mexican territory, there continues to be a great difference in its implementation in rural zones where disperse human settlements exist with scarce communication. Fetal mortality in Mexico has declined markedly over the 1930-1975 period. Perinatal mortality also has declined, from 51-190/1000 in 1930 to 10-39/1000 liveborn in 1975.^ieng