Study of the Mechanism of the Antimicrobial Activity of Novel Water Soluble Ammonium Quaternary Benzanthrone on Model Membranes.

The increasing resistance of many pathogens to most of the common antimicrobials requires the development of new substances with more effective antimicrobial properties. In the present work, we investigated the mechanism of the antimicrobial activity of novel water soluble ammonium quaternary benzanthrone (Compound B) on model membranes, composed of dipalmitoylphosphatidylcholine, 1-palmitoyl-2-oleoylphosphatidylcholine, dipalmitoylphosphatidylglycerol, 1-palmitoyl-2-oleoylphosphatidylglycerol, and dipalmitoylphosphatidylethanolamine (DPPE). The lipids were chosen to represent a model of a bacterial membrane. The changes in surface pressure of the model membranes, before and after the addition of Compound B, were studied by the Langmuir's monolayer method, and the compressional modulus for each monolayer was determined. In addition, the surface morphology of the lipid monolayers before and after injection of Compound B was monitored by Brewster Angle Microscopy. The results showed that Compound B penetrated all the monolayers studied. The most noticeable effects were found with the negatively charged phosphatidylglycerols and with DPPE leading to the conclusion that the electrostatic interactions between the compound and the lipid head groups and the possible formation of hydrogen bonds between the amino group of the ethanolamine and the keto groups in the structure of Compound B are of great importance. In addition, the penetration ability of the benzoquinone with all phospholipids studied was stable even at higher values of the surface pressure, i.e. thicker monolayers, due to the hydrophobic interaction, which plays also an important role for the antimicrobial activity of Compound B.

Rapid biophysical analyses of gastric aspirates from risk newborns for lung maturity assessment after corticosteroid therapy.

BACKGROUND: One of the main causes for the higher mortality among risk newborn children (including preterm infants) is neonatal respiratory distress syndrome (NRDS), which develops as a result of primary deficiency or secondary inactivation of alveolar surfactant (AS). Therefore, fast and early diagnostics of risk newborns lung maturity is crucial for their prompt therapy. MATERIALS AND METHODS: Gastric aspirates (GA) were collected from 77 infants divided into three groups: a control of 38 healthy full-term infants; 16 prematurely newborns with NRDS, and 23 prematurely born infants after in vitro fertilization and corticosteroid therapy (CST). Surface parameters: equilibrium (γeq), maximal (γmax) and minimal (γmin) surface tension, and the shape of hysteresis curves of GA monolayers were measured by axisymmetric drop shape analysis (ADSA) of a pending drop. In addition, the morphology of GA monolayers was studied by Brewster angle microscopy (BAM). RESULTS: Our results showed that only γmin values were reliable and were significantly lower in full-term infants, as compared to the risk neonates. The results obtained were proved by the shape of hysteresis curves of GA surface active films. BAM images of GA monolayers from NRDS group showed impaired surface morphology due to the surfactant insufficiency, as compared to the control group. Corticosteroid therapy improved both GA surface characteristics and monolayer morphology. CONCLUSIONS: GAs analyses by ADSA and BAM are fast and informative approaches for lung maturity assessment. In addition, the corticosteroid therapy applied improved all GAs surface parameters due to AS maturation.

Effects of Leucin-Enkephalins on Surface Characteristics and Morphology of Model Membranes Composed of Raft-Forming Lipids.

During the last decades opioid peptides, like enkephalins (Tyr-Gly-Gly-Phe-Met/Leu) are subject to extensive studies due to their antinociceptive action in organism. According to the membrane catalysis theory, in order to adopt a proper conformation for binding to their receptors, opioid peptides interact with the lipid phase of the membrane receptor surrounding. With this regard, the aim of the present work was to study the effects of synthetic leucine-enkephalin and leucine-enkephalinamide on surface characteristics and morphology of lipid monolayers, composed of 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, sphingomyelin, and cholesterol alone and with their mixtures. The lipids were chosen to represent a model of a membrane raft, since it is known that G-protein-coupled receptors, including opioid receptors, are located preferably in membrane rafts. By using Langmuir's monolayer method, the change in surface pressure of the model membranes before and after the addition of the synthetic enkephalins was studied, and the compressional moduli of the lipids and lipid-peptides monolayers were determined. In addition, by Brewster angle microscopy, the surface morphology of the lipid monolayers alone and after the injection of both enkephalins was monitored. Our results showed that both leucine-enkephalins affected the lipid monolayers surface characteristics, and led to an increase in surface density of the mixed surface lipids/enkephalins films at loose lipid packing. This effect was more pronounced for the enkephalinamide, suggesting a different mechanism of interaction for the amidated enkephalin with the lipid phase, as compared to leucine-enkephalin.

Biochemical and biophysical investigation of surfactant in neonatal gastric aspirate at birth.

BACKGROUND: The optimal approach to detection of surfactant deficiency in the prematurely born infants at birth remains unclear and the decision to apply exogenous surfactant is based mainly on the development of clinical and radiological signs of neonatal respiratory distress syndrome (NRDS). OBJECTIVES: We studied the biochemical and biophysical properties of gastric aspirates (GA) from prematurely born infants with NRDS and healthy full term infants with an aim to find an approachable method for assessment of surfactant maturity at birth. MATERIAL AND METHODS: Forty-seven newborn infants divided into two groups were enrolled in the study. The first group comprised 34 healthy infants born at term (after 37 weeks of gestation). The second group included 13 premature infants (aged from 26 to 32 weeks of gestation) developing clinical signs of NRDS for which they were treated by assisted ventilation and exogenous surfactant. A biochemical analysis of the protein and lipid content of GA collected at birth was performed. The fatty acid composition of the GA samples was determined by Gas Chromatography-Mass Selective Detector (GS-MSD) analysis. The surface characteristics (equilibrium, maximal and minimal surface tension values) of the GA samples were measured by using the pending drop method. Data were compared between the groups by using Student's t test or Mann-Whitney analysis. Values were considered significantly different if the p value was = 0.05. RESULTS: The mean phospholipids' concentration in GA of the premature infants was lower (295.7 µg / ml vs. 374.5 µg / ml) than in the term infants and the mean protein content was less in GA of the premature babies than the term newborns (574.5 µg / ml vs. 641.5 µg / ml). The measurement of dynamic surface characteristics of GA showed significantly higher mean values of the minimal surface tension (γmin) in the premature infants - 20.5 m / Nm compared to the term babies - 12.3 mN/m (p < 0,01). There was no difference between the equilibrium surface tensions (38 mN/m vs. 38 mN/m) of both groups; The mean values of maximal surface tension (?max) in GA did not differ significantly between the groups (50.1 mN/m vs. 48.5 mN/m). CONCLUSION: Our findings revealed lower phospholipids' and protein concentrations in the GA at birth from premature infants as compared to the healthy term infants. The dynamic surface characteristics of GA had significant differences between the two groups, the minimal surface tension being the most important parameter for evaluation of surfactant maturity. It could be used in the clinical practice for fast surfactant's assessment in the premature infants in regard to administration of exogenous surfactant.

Introdução: A melhor abordagem para detecção de deficiência de surfactante em recém-nascidos prematuros no nascimento permanece pouco clara e a decisão de aplicar surfactante exógeno é baseada principalmente no desenvolvimento de sinais clínicos e radiológicos da síndrome de angústia respiratória neonatal (NRDS). Objetivos: Estudamos as propriedades bioquímicas e biofísicas do aspirado gástrico (GA) de crianças nascidas prematuramente com NRDS e saudáveis nascidos a termo, com o objetivo de encontrar um método acessível para avaliação da maturidade do surfactante no nascimento. Material e Métodos: Quarenta e sete recém-nascidos, divididos em dois grupos foram incluídos no estudo. O primeiro grupo composto por 34 crianças saudáveis nascidas a termo (após 37 semanas de gestação). O segundo grupo incluiu 13 recém-nascidos prematuros (idade entre 26 - 32 semanas de gestação) desenvolveram sinais clínicos de NRDS para os quais foram tratados por ventilação assistida e surfactante exógeno. A análise bioquímica da proteína e lipídio da GA coletado no nascimento foi realizado. A composição de ácidos graxos das amostras GA foi determinada pelo detector de cromatografia de massa seletiva (GS-MSD) análise. As características da superfície (de equilíbrio, os valores máximo e mínimo de tensão de superfície) das amostras de AG foram medidos utilizando o método de gota pendente. Os dados foram comparados entre os grupos, utilizando o teste t de Student ou teste de Mann-Whitney análise. Os valores foram considerados significativamente diferentes se o valor de p ≤ 0,05. Resultados: A concentração média de fosfolipídeos em GA dos prematuros foi menor (295,7 g / ml vs 374,5 mg / ml) do que em crianças a termo e do conteúdo médio de proteína foi menor no GA dos bebês prematuros que o termo recém-nascidos (574,5 ng / ml vs 641,5 ug / ml). A medição das características de superfície dinâmicas da GA mostraram significativamente maiores valores médios da tensão superficial mínima (γmin) nos bebés prematuros - 20,5 m / Nm em comparação com as crianças a termo - 12,3 mN / m (p < 0,01). Não houve diferença entre as tensões superficiais de equilíbrio (38 mN / m contra 38 mN / m) de ambos os grupos, os valores médios de tensão de superfície máxima (γmax) em GA não diferiram significativamente entre os grupos (50,1 mN / m vs 48,5 mN / m). Conclusão: Os achados revelaram fosfolipídios inferiores e concentrações de proteína na IG ao nascimento de bebês prematuros em comparação com os lactentes saudáveis a longo prazo. As características da superfície dinâmicas da GA apresentaram diferenças significativas entre os dois grupos, a tensão de superfície mínima sendo o parâmetro mais importante para a avaliação da maturidade surfactante. Ele pode ser utilizado na prática clínica para a avaliação rápida de surfactante em recém-nascidos prematuros com respeito à administração de surfactante.

Comparative study of the interaction of synthetic methionine-enkephalin and its amidated derivate with monolayers of zwitterionic and negatively charged phospholipids.

Using Langmuir's monolayer technique, the surface behavior and the interaction of the synthetic neuropeptide methionine-enkephalin (Met-enk) and its amidated derivate (Met-enk-NH(2)) with monolayers of the zwitterionic dimyristoylphosphatidylcholine (DMPC) and the negatively charged dimyristoylphosphatidylglycerol (DMPG) were studied. The surface tension (γ, mN/m) of DMPG and DMPC monolayers as a function of time (after injection of the peptide under the interface) was detected. The decrease in γ values showed that there was a strong penetration effect of both types of Met-enk molecules into the monolayers, being significantly stronger for the amidated derivate, Met-enk-NH(2). We suggest that the interaction between the neuropeptides and DMPC was predominantly determined by peptides amphiphilicity, while the electrostatic forces play significant role for the insertion of the cationic Met-enk-NH(2) in DMPG monolayers, especially at high packing densities. Our results demonstrate the potential of lipid monolayers formed in Langmuir's trough to be successfully used as an elegant and simple membrane models to study lipid-peptide interactions at the air/water interface.