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 Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC surface films.

Bestrophinopathies are ocular diseases caused by mutations in the human bestrophin-1 (hBest1) - transmembrane Ca2+-activated chloride channel protein, mainly expressed in the retinal pigment epithelium (RPE) cells. hBest1 is also an important transporter for neurotransmitters such as glutamate (Glu) and γ-aminobutyric acid (GABA) in the nervous system. Recently, a new biological role of hBest1, related to its possible involvement in the pathology of brain diseases (Alzheimer's, Parkinson's disease) has been proposed. Here, we report the effects of Ca2+, Glu and GABA on hBest1 and composite hBest1/POPC (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine) Langmuir and Langmuir-Blodgett monolayers based on surface dynamics (π/A isotherms, hysteresis and compressibility), morphology (Brewster angle microscopy, BAM) and visualization of protein molecular organization (Atomic force microscopy, AFM). Ca2+ ions and neurotransmitters Glu and GABA affect hBest1 topology at the air/water interface altering its surface activity, size, orientation and organization. In contrast, no significant changes were detected on π/A isotherms and hysteresis of the composite hBest1/POPC films but their effects on structure, aggregation state and orientation hBest1 established by BAM and AFM differentiate. We found that the binary films of hBest1 and POPC are phase separated at the air/water interface, suggesting stronger lipid-lipid and protein-protein interactions than lipid-protein interactions that can significantly alter the molecular organization and activity of hBest1 in cell membranes. Our data shed light on structure, surface behavior and organization of hBest1 that define relationship structure-functional activity of hBest1 as transport channel.

Effects of Ca2+ ions on bestrophin-1 surface films.

Human bestrophin-1 (hBest1) is a transmembrane calcium-activated chloride channel protein - member of the bestrophin family of anion channels, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Mutations in the protein cause ocular diseases, named Bestrophinopathies. Here, we present the first Fourier transform infrared (FTIR) study of the secondary structure elements of hBest1, π/A isotherms and hysteresis, Brewster angle microscopy (BAM) and atomic force microscopy (AFM) visualization of the aggregation state of protein molecules dispersed as Langmuir and Langmuir-Blodgett films. The secondary structure of hBest1 consists predominantly of 310-helices (27.2%), α-helixes (16.3%), ß-turns and loops (32.2%). AFM images of hBest1 suggest approximate lateral dimensions of 100×160Å and 75Å height. Binding of calcium ions (Ca2+) induces conformational changes in the protein secondary structure leading to assembly of protein molecules and changes in molecular and macro-organization of hBest1 in monolayers. These data provide basic information needed in pursuit of molecular mechanisms underlying retinal and other pathologies linked to this protein.

Transepithelial resistance in human bestrophin-1 stably transfected Madin-Darby canine kidney cells.

Bestrophin-1 (Best1) is a transmembrane protein, found in the basolateral plasma membrane of retinal pigmented epithelial cells. The exact structure and functions of Best1 protein are still unclear. The protein is thought to be a regulator of ion channels, or an ion channel itself: it was shown to be permeable for chloride, thiocyanate, bicarbonate, glutamate and γ-aminobutyric acid (GABA). Mutations in the gene for Best1 are leading to best vitelliform macular dystrophy (BVMD) and are found in several other types of maculopathy. In order to obtain additional information about Best1 protein, we determined cell polarization of a stably transfected Madin-Darby canine kidney cell line II (MDCK II) cell line, expressing human Best1. We measured the transepithelial resistance of transfected and non-transfected MDCK cells by voltmeter EVOM, over 10 days at 24 hour intervals. The first few days (first-fourth day) both cell lines showed the same or similar values ​​of transmembrane resistance. As expected, on the fifth day the non-transfected cells showed maximum value of epithelial resistance, corresponding to the forming of monolayer. The transfected cells showed maximum value of transepithelial resistance on the ninth day of their cultivation. Phalloidin staining of actin demonstrated the difference in actin arrangements between transfected and non-transfected cells due to Best1. As a consequence of actin rearrangement, Best1 strongly affects the transepithelial resistance of polarizing stably transfected MDCK cells. Our results suggest that Best1 protein has an effect on transepithelial resistance and actin rearrangements of polarized stably transfected MDCK cells.

Effect of hydrophilic polymers on the wettability, static and dynamic, of solid substrate covered by confluent monolayer of air-damaged SIRC cells.

The aim of this study was to evaluate the possible implementation of hydrophilic polymers as recovery agents in air-damaged corneal cells. The sessile bubble technique was implemented to measure the wetting properties of four selected polymers: hydroxyethyl cellulose (HEC), sodium chondroitin sulphate (SCS), hydroxypropyl-methylcellulose (HPMC) and poloxamer F127 (PO12), at equilibrium conditions and in the case of advancing and receding contact angle. For testing the wetting properties of the polymers, glass slides covered with a confluent monolayer of Statens Seruminstitut rabbit cornea (SIRC) cells were used. HEC showed best properties for a broad concentration range, as the polymer showed capability to maintain low values of the static (equilibrium) contact angle (average static contact angle - 36.07˚, compared to average static compact angles of HPMC - 38.44˚, PO12 - 38.92˚ and SCS - 37.85˚), i.e. better wettability. Sessile bubble technique provides quick, relatively simple and reliable approach for testing surface properties of the listed polymers. The nature of the surface damage produced by the exposition of SIRC cells was used as a plausible model of evaporative dry eye syndrome, and thus the results may have clinical implementation.

Interaction of Bestrophin-1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) in surface films.

Human bestrophin-1 (hBest1) is a transmembrane channel protein, predominantly expressed in the membrane of retinal pigment epithelium (RPE) cells. Although it is clear that hBest1's interactions with lipids are crucial for its function such studies were not performed as the protein was not purified. Here we describe an effective purification of hBest1 from Madin-Darby Canine Kidney (MDCK) cells via simple gel-filtration and affinity chromatographic steps, which makes possible to probe the protein interplay with lipids. The interaction of the purified hBest1 with 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) was studied in Langmuir monolayers. The surface pressure (π)-area (A) isotherms and compression/expansion isocycles of POPC monolayer were recorded in absence and presence of hBest1 in the subphase. The π(A) isotherms were analyzed in terms of surface compressional modulus and via two-dimensional virial equation of state. The dilatational rheological properties of the surface films and their surface potential were also measured. The morphology of the films was observed by Brewster angle microscopy. The inclusion of the protein in the film subphase does not lead to in-depth penetration of hBest1 but interaction takes place in the headgroup region of the monolayer. The hBest1/POPC interaction resulted in formation of more condensed films, which rheological properties and lateral structure differed significantly from the pure POPC monolayers. Our study sheds light on the still unclear question how hBest1 gets in touch with biomembrane phospholipids of eukaryotic cells that might be of key importance for the proper structure and function of RPE biomembranes.

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.

Surface chemistry study of the interactions of pharmaceutical ingredients with human meibum films.

PURPOSE: To perform a surface chemistry study of the interactions between the benzalkonium chloride (BAC)-preserved eyedrops Travatan, the SofZia-preserved TravatanZ, and the Polyquad-preserved DuoTrav, and tear film (TF) constituents. The interactions of TF compounds with the individual preservatives, BAC, SofZia, and Polyquad, were also examined. METHODS: Langmuir surface balance measurements were used to examine the interactions between the pharmaceuticals and films of human meibum and rabbit corneal cell lipid extracts. Surface pressure-area isocycles were used to assess the sample's capability to compress and spread during dynamic area changes. The dilatational rheologic properties of human meibum films, pure and in the presence of preservatives, were probed by stress-relaxation studies. Lipid film morphology was monitored by Brewster angle microscopy. The viability of SofZia- and Polyquad-treated Statens Seruminstitut Rabbit Cornea (SIRC) cell cultures was also evaluated. RESULTS: The interactions between BAC-preserved eyedrops and lipids resulted in impaired lipid spread, formation of discontinuous nonuniform surface layers, and increased surface pressure-area hysteresis during compression/expansion. In contrast, TravatanZ, DuoTrav, and the individual preservatives SofZia and Polyquad proved to be safe to the lipid film structure and isothermal reversibility. The stress-relaxation experiments revealed that the viscoelastic properties of meibomian film are impaired by BAC, and remain unaffected by SofZia and Polyquad. SIRC cells' viability and capability to form confluent cellular monolayer were also maintained after exposure to SofZia and Polyquad. CONCLUSIONS: Surface chemistry studies present criteria for preclinical in vitro molecular scale characterization of the interactions between eyedrop compounds and TF constituents.

Surface chemistry study of the interactions of benzalkonium chloride with films of meibum, corneal cells lipids, and whole tears.

PURPOSE: To perform a surface chemistry study of the interactions between benzalkonium chloride (BAC), a common preservative used in ophthalmic formulations, and tear film (TF) constituents. METHODS: The interactions between BAC and human tears, meibum, and rabbit corneal cell lipid extracts at the air-water interface were examined in vitro during controlled compression-expansion of the film area by a Langmuir surface balance, surface potential measurements, and pendant drop-axisymmetric drop shape analysis (PD-ADSA). Surface pressure-area isotherms and isocycles were used to assess the sample's lateral elasticity and capability of compressing and spreading during dynamic area changes. Lipid film morphology was monitored by Brewster angle microscopy. The viability of BAC-treated Statens Seruminstitut rabbit cornea (SIRC) cell cultures was also examined. The BAC concentration was kept within the clinical range of 0.001% to 0.02%. RESULTS: In the Langmuir balance and PD-ADSA experiments, the interactions between BAC and lipids or tears resulted in (1) impaired lipid spread and formation of discontinuous nonuniform surface layers, (2) increased surface pressure-area hysteresis during compression and expansion, and (3) displacement of the lipids by BAC from the surface. A decrease (>50%) in SIRC cell viability was observed. The effects occurred within seconds after BAC exposure, and their magnitude increased with BAC concentration. CONCLUSIONS: The surface chemistry approach used in this study provided molecular-scale insights into the detrimental effect of BAC on TF, which well explain the TF instability and corneal epithelial barrier dysfunction after exposure to BAC in the in vivo human eye.

Surface properties and behavior of lipid extracts from plasma membranes of cells cultured as monolayer and in tissue-like conditions.

The differences in the surface active properties of native lipids extracted from plasma membranes of cells cultured as a monolayer and in three-dimensional (3D) matrix were investigated. This experimental model was chosen because most of the current knowledge on cellular physiological processes is based on studies performed with conventional monolayer two-dimensional (2D) cell cultures, where cells are forced to adjust to unnaturally rigid surfaces that differ significantly from the natural matrix surrounding cells in living organisms. Differences between monolayer and 3D cells were observed in the lipid composition of plasma membranes and especially in the level of the two major microdomain-forming lipids--sphingomyelin (SM) and cholesterol, which were significantly elevated in 3D cells. The obtained results showed that culturing of cells in in vivo-like environment affected the surface active properties of plasma membrane lipids at interfaces which might influence certain membrane-associated interface processes. The detected differences in the lipid levels in 2D and 3D cell extracts affected significantly the behavior of the model lipid monolayers at the air-water interface (Langmuir monolayers) which resulted in different values of the monolayer equilibrium (gamma(eq)) and dynamic (gamma(max), gamma(min)) surface tension and surface potential. Compensation of the SM content in extracts of 2D cell cultures up to a level close to the one measured in 3D cells approximated the monolayer properties to the values observed for 3D cells. These results implied that the interactions between the cells and the surrounding medium affected the level of plasma membrane SM and other lipids, which had a strong impact on the surface properties of lipid monolayers, such as gamma(eq), gamma(max), and gamma(min), the compression/decompression curve shape, the hysteresis area during cycling of the monolayers, etc. We suggest that the elevated content of SM observed in plasma membranes of 3D fibroblasts could be responsible for an increased rigidity and possibly reduced permeability of cells cultured in 3D environment. The current results provide useful information that should be taken into account in the interpretation of the membrane physico-chemical properties of cells cultured under different conditions.

Evaluation of different carbon sources for growth and biosurfactant production by Pseudomonas fluorescens isolated from wastewaters.

The indigenous strain Pseudomonas fluorescens, isolated from industrial wastewater, was able to produce glycolipid biosurfactants from a variety of carbon sources, including hydrophilic compounds, hydrocarbons, mineral oils, and vegetable oils. Hexadecane, mineral oils, vegetable oils, and glycerol were preferred carbon sources for growth and biosurfactant production by the strain. Biosurfactant production was detected by measuring the surface and interfacial tension, rhamnose concentration and emulsifying activity. The surface tension of supernatants varied from 28.4 mN m(-1) with phenanthrene to 49.6 mN m(-1) with naphthalene and heptane as carbon sources. The interfacial tension has changed in a narrow interval between 6.4 and 7.6 mN m(-1). The emulsifying activity was determined to be highest in media with vegetable oils as substrates. The biosurfactant production on insoluble carbon sources contributed to a significant increase of cell hydrophobicity and correlated with an increased growth of the strain on these substrates. Based on these results, a mechanism of biosurfactant-enhanced interfacial uptake of hydrophobic substrates could be proposed as predominant for the strain. With hexadecane as a carbon source, the pH value of 7.0-7.2 and temperature of (28 +/- 2) degrees C were optimum for growth and biosurfactant production by P. fluorescens cells. The increased specific protein and biosurfactant release during growth of the strain on hexadecane in the presence of NaCl at contents up to 2% could be due to increased cell permeability. The capability of P. fluorescens strain HW-6 to adapt its own metabolism to use different nutrients as energy sources and to keep up relatively high biosurfactant levels in the medium during the stationary phase is a promising feature for its possible application in biological treatments.

Influence of surfactant protein C on the interfacial behavior of phosphatidylethanolamine monolayers.

In the current work we study with monolayer tensiometry and Brewster angle microscopy (BAM) the surface properties of Dipalmitoleoylphosphatidylethanolamine (DPoPE) films at the air/water interface in presence and absence of specific surfactant protein C (SP-C). DPoPE is used, as it readily forms both lamellar (L(alpha)) and non-lamellar inverted hexagonal (H(II)) phases and appears as a suitable model phospholipid for probing the interfacial properties of distinct lipid phases. At pure air/water interface L(alpha) shows faster adsorption and better surface disintegration than H(II) phase. The interaction of DPoPE molecules with SP-C (predeposited at the interface) results in equalizing of the interfacial disintegration of the both phases (reaching approximately the same equilibrium surface tension) although the adsorption kinetics of the lamellar phase remains much faster. Monolayer compression/decompression cycling revealed that the effect of SP-C on dynamic surface tensions (gamma (max) and gamma (min)) of mixed films is remarkably different for the two phases. If gamma (max) for L(alpha) decreased from the first to the third cycle, the opposite effect is registered for H(II) where gamma (max) increases during cycling. Also the significant decrease of gamma (min) for L(alpha) in SP-C presence is not observed for H(II) phase. BAM studies reveal the formation of more uniform and homogeneously packed DPoPE monolayers in the presence of SP-C.

Characterization of bacterial isolates from industrial wastewater according to probable modes of hexadecane uptake.

Bacterial isolates from industrial wastewater were characterized according to probable modes of hexadecane uptake based on data for cell surface hydrophobicity, emulsifying activity, glycoside content and surface tension of cell-free culture medium. The results obtained suggested that both modes of biosurfactant-enhanced hexadecane uptake by bacterial strains take place, direct uptake and alkane transfer. The increase in cell surface hydrophobicity and glycoside production by the strains suggested the existence of biosurfactant-enhanced interfacial uptake of the alkane. Such mechanism is probably predominant for three isolates, Staphylococcus sp. HW-2, Streptococcus sp. HW-9 and Bacillus sp. HW-4. Secreted biosurfactants enhanced mainly alkane emulsification for most hydrophobic isolate Arthrobacter sp. HW-8, and micellar transfer for most hydrophilic isolate Streptococcus sp. HW-5. For other strains (67%) both mechanisms of biosurfactant-enhanced hexadecane uptake probably take place in similar degree, interfacial uptake and alkane emulsification. The results obtained could contribute to clarifying the natural relationships between the members of water ecosystem studied as well as will reveal potential producers of surface active compounds.

Effects of poly (ethylene glycol) chains conformational transition on the properties of mixed DMPC/DMPE-PEG thin liquid films and monolayers.

Foam thin liquid films (TLF) and monolayers at the air-water interface formed by DMPC mixed with DMPE-bonded poly (ethylene glycol)s (DMPE-PEG(550), DMPE-PEG(2000) and DMPE-PEG(5000)) were obtained. The influence of both (i) PEG chain size (evaluated in terms of Mw) and mushroom-to-brush conformational transition and (ii) of the liposome/micelle ratio in the film-forming dispersions, on the interfacial properties of mixed DMPC/DMPE-PEG films was compared. Foam film studies demonstrated that DMPE-PEG addition to foam TLFs caused (i) delayed kinetics of film thinning and black spot expansion and (ii) film stabilization. At the mushroom-to-brush transition, due to steric repulsion increased DMPE-PEG films thickness reached 25 nm while pure DMPC films were only 8 nm thick Newton black films. It was possible to differentiate DMPE-PEG(2000/5000) from DMPE-PEG(550) by the ability to change foam TLF formation mechanism, which could be of great importance for "stealth" liposome design. Monolayer studies showed improved formation kinetics and equilibrium surface tension decrease for DMPE-PEG monolayers compared with DMPC pure films. SEM observations revealed "smoothing" and "sealing" of the defects in the solid-supported layer surface by DMPE-PEGs adsorption, which could explain DMPE-PEGs ability to stabilize TLFs and to decrease monolayer surface tension. All effects in monolayers, foam TLFs and solid-supported layers increased with the increase of PEG Mw and DMPE-PEG concentration. However, at the critical DMPE-PEG concentration (where mushroom-to-brush conformational transition occurred) maximal magnitude of the effects was reached, which only slightly changed at further DMPE-PEG content and micelle/liposome ratio increase.

Production and properties of biosurfactants from a newly isolated Pseudomonas fluorescens HW-6 growing on hexadecane.

The newly isolated from industrial wastewater Pseudomonas fluorescens strain HW-6 produced glycolipid biosurfactants at high concentrations (1.4-2.0 g l(-1)) when grown on hexadecane as a sole carbon source. Biosurfactants decreased the surface tension of the air/ water interface by 35 mN m(-1) and possessed a low critical micelle concentration value of 20 mg l(-1), which indicated high surface activity. They efficiently emulsified aromatic hydrocarbons, kerosene, n-paraffins and mineral oils. Biosurfactant production contributed to a significant increase in cell hydrophobicity correlated with an increased growth of the strain on hexadecane. The results suggested that the newly isolated strain of Ps. fluorescens and produced glycolipid biosurfactants with effective surface and emulsifying properties are very promising and could find application for bioremediation of hydrocarbon-polluted sites.

Thin liquid films and monolayers of DMPC mixed with PEG and phospholipid linked PEG.

In this work thin liquid films (TLFs) and monolayers at the air/water interface formed by dimyristoylphosphatidylcholine (DMPC) and by DMPC mixed with poly ethylene glycols (PEGs) and dimyristoylphosphatidylethanolamine (DMPE) linked PEGs were studied. Film forming dispersions were composed of two types of particles: liposomes and micelles. TLFs stability, threshold concentration C(t) (i.e., the minimum one for stable film formation), and hydrodynamic behavior were measured. At equivalent conditions, DMPC films were Newton black films (real bilayers), while DMPE-PEGs films were much thicker with free water between the monolayers. DMPE-PEG addition to DMPC films caused both C(t) decrease (depending on PEG moiety length and Mw) and change of TLF formation mechanism. TLFs' hydrodynamic behavior also strongly depended on DMPE-PEG content and Mw. It was observed that thinning of the DMPC and DMPE-PEGs films continued to different film types and thickness, being much thicker for the latter films. Addition of free PEGs (PEG-200/6000) did not alter TLF type or stability, but changed TLF thinning time, confirming that free PEGs with Mw<8000 could not penetrate in the membrane and alter "near-membrane" water layer viscosity. Monolayer studies showed improved formation kinetics of both adsorbed and spread films, decrease of surface tension (equilibrium and dynamic), and of film compression/decompression histeresis area in DMPE-PEGs monolayers compared with DMPC pure films. Our study shows that combining the models of phospholipid TLFs and monolayers provide the opportunity to investigate the properties of membrane surface and to clarify some mechanisms of its interactions with membrane-active agents.

Model study of interactions of high-molecular dextran sulfate with lipid monolayers and foam films.

The interaction of high-molecular dextran sulfate (DS-5000) with dimyristoylphosphatidylcholine (DMPC) monolayers and foam films (FF) at the air-water interface in the presence of Ca2+ and Na+ ions was studied. DS-5000 was added in monolayer films (MF) and in FF as monomer molecules and in liposomal form. When added in liposomal form in FF, DS-5000 decreased the stability of DMPC common black films (CBF), and no formation of Newton black films (NBF) was observed. However, when included as monomer molecules in FF, DS-5000 caused film thinning, and drastically decreased the expansion rate of the black spots and transition of thick films to NBF, thus avoiding formation of CBF. The above effects were observed in both gel and liquid-crystalline phase states of DMPC in the presence of Ca2+ ions only, and not in the presence of Na+ ions. We postulate that the interaction of DMPC with DS-5000 in the plane of FF is mediated by Ca2+ bridges and results in dehydration of the DMPC polar heads. The interaction between DMPC and DS-5000 in monolayers resulted in slower adsorption and spreading of DMPC molecules at the interface, lower monolayer surface pressure, and penetration of DS-5000 molecules to DMPC monolayers when surface lipid density was higher than 50 A2 per DMPC molecule. The applicability of the FF model for studying the interactions of phospholipids with polysaccharides at interfaces surrounded by bulk solution, and for modeling such interactions in biological systems, e.g. LDL adhesion to the arterial walls, aggregation and fusion of liposomes, etc., is discussed.

Rhamnolipid biosurfactants produced by Renibacterium salmoninarum 27BN during growth on n-hexadecane.

A new strain Renibacterium salmoninarum 27BN was isolated for its capacity to utilize n-hexadecane as sole substrate. Growth on n-hexadecane was accompanied with the production of glycolipid surface active substances detected by surface pressure lowering and emulsifying activity. Glycolipid detection by thin layer chromatography and infrared spectra analyses showed for the first time that Renibacterium salmoninarum 27BN secretes the two rhamnolipids RLL and RRLL typical for Pseudomonas aeruginosa. Growth of Renibacterium salmoninarum 27BN on n-hexadecane depended on the bioavailability of the substrate and the secreted rhamnolipids appeared to be efficient in increasing hexadecane availability for the cells.

Effect of halothane on lung carcinoma cells A 549.

The halogenated hydrocarbons, such as halothane, are widely used as anesthetics in clinical practice; however their application is often accompanied with metabolic, cardiovascular and respiratory complications. One of the possible factors for this negative outcome might be the severe toxicity of these agents. In this paper, we investigate in vitro effects of halothane on human lung carcinoma A 549 cells, namely on their cytotoxicity, adhesive properties and metabolic activity. The cytotoxicity response of lung carcinoma A 549 cells to halothane was determined by lactate dehydrogenase (LDH) assay (for cytotoxicity), by detachment assay after adhesion to type IV collagen (for cell adhesive properties) and by surface tension measurements of culture medium (for cell metabolic activity). Regarding the cytotoxicity, the determined maximal non-toxic concentration of halothane on A 549 cells, given here as volume percentages (vol.%) was 0.7 vol.% expressed as aqueous concentration in the culture medium. Direct measurement of the actual halothane concentration in the culture medium showed that 0.7 vol.% corresponds to 1.05 mM and 5.25 aqueous-phase minimum alveolar concentration (MAC). Concentrations equal or higher than 1.4 vol.% (2.1 mM; 10.5 MAC) of halothane provoked complete detachment (cell death), or reduction of initial adhesion to collagen IV in half of the cell population. Surfactant production of A 549 cells, registered up to 48 h after halothane treatment, was inhibited by halothane concentrations as low as 0.6 vol.% (0.9 mM; 4.5 MAC). Our results demonstrate that sub toxic halothane concentrations of 0.6 vol.% inhibits surfactant production; concentrations in the range 0.8-1.4 vol.% induce membrane damages and concentrations equal and higher than 1.4 vol.%--cell death of approximately 50% of the cells.

Formation of monolayers and bilayer foam films from lamellar, inverted hexagonal and cubic lipid phases.

This study revealed large distinctions between the lamellar and non-lamellar liquid crystalline lipid phases in their spreading at the air/water interface and propensity to form bilayer foam films. Comparative measurements were made for the lamellar L(alpha), the inverted hexagonal H(II) and the bicontinuous cubic Pn3m phases of the phospholipid dipalmitoleoylphosphatidylethanolamine (DPoPE). With regard to monolayer formation, followed as the decrease of surface tension with time, the best spreading (lowest surface tension) was observed for the L(alpha) phase, and poorest spreading (highest surface tension) was recorded for the H(II) phase. The cubic Pn3m phase of DPoPE, induced by temperature cycling, retained an intermediate position between the L(alpha) and H(II) phases. According to their ability to lower surface tension and disintegrate at the air/water interface, the three phases thus order as L(alpha)>Pn3m>H(II). Clearly expressed threshold (minimum) bulk lipid concentrations, C(t), required for formation of stable foam bilayers from these phases, were determined and their values were found to correlate well with the bulk lipid phase behaviour. The C(t) values for L(alpha) and H(II) substantially increase with the temperature. Their Arrhenius plots, ln C(t) versus 1/ T, are linear and intersect at approximately 36-37 degrees C, coinciding with the onset of the bulk L(alpha)-->H(II) phase transition, as determined by differential scanning calorimetry. However, the C(t) value for the Pn3m phase, equal to 30 micro g/mL, was found to be constant over the whole range investigated between 20 degrees C and 50 degrees C. The horizontal C(t) versus T plot for the Pn3m phase crosses the respective plot for the L(alpha) phase at the temperature bounding from below the hysteretic loop of the L(alpha)<-->H(II) transition (approximately 26 degrees C), thus providing a certain insight about the thermodynamic stability of the Pn3m phase relative to the L(alpha) phase. The established strong effect of the particular lipid phase on the formation of monolayers and stable black foam films should be of importance in various in vitro and in vivo systems, where lipid structures are in contact with interfaces and disintegrate there to different extents.