Multilayers at the surface of solutions of exogenous lung surfactant: Direct observation by neutron reflection

Pharmacy-grade exogenous lung surfactant preparations of bovine and porcine origin, dispersed in physiological electrolyte solution have been studied. The organization and dynamics at the air/water interface at physiological temperature was analysed by neutron reflection. The results show that a well-defined surface phase is formed, consisting of a multilayer structure of lipid/protein bilayers alternating with aqueous layers, with a repetition period of about 70 Å and correlation depths of 3 to > 25 bilayers, depending on electrolyte composition and time. The experimental surfactant concentration of 0.15% (w/w) is far below that used in therapeutic application of exogenous surfactants and it is therefore likely that similar multilayer structures are also formed at the alveolar surface in the clinical situation during surfactant substitution therapy. Lung surfactant preparations in dry form swell in aqueous solution towards a limit of about 60% (w/w) of water, forming a lamellar liquid-crystalline phase above about 34 °C, which disperses into lamellar bodies at higher water concentrations. The lamellar spacings in the surface multilayers at the air/water interface are smaller than those in the saturated limit even though they are in contact with much greater water concentrations. The surface multilayers are laterally disordered in a way that is consistent with fragments of Lα-phase lamellae. The near surface layers of the multilayer structure have a significant protein content (only SP-B and SP-C are present in the preparations). The results demonstrate that a multilayer structure can be formed in exogenous surfactant even at very low concentrations and indicate that multilayers need to be incorporated into present interpretations of in vitro studies of similar lung surfactant preparations, which are largely based on monolayer models.     

Structure of casein micelles studied by small-angle neutron scattering

The structure of casein micelles has been studied by small-angle neutron scattering and static light scattering. Alterations in structure upon variation of pH and scattering contrast, as well as after addition of chymosin, were investigated. The experimental data were analyzed by a model in which the casein micelle consists of spherical submicelles. This model gave good agreement with the data and gave an average micellar radius of about 100–120 nm and a submicellar radius of about 7 nm both with a polydispersity of about 40–50%. The contrast variation indicated that the scattering length density of the submicelles was largest at the center of the submicelles. The submicelles were found to be closely packed, the volume fraction varying slightly with pH. Upon addition of chymosin the submicellar structure remained unchanged within the experimental accuracy. Correspondence to: S. Hansen     

Effect of pH and aluminum on surface properties of barley roots as determined from water vapor adsorption

Water vapor adsorption isotherms were used for the estimation of surface areas and adsorption energy distribution functions of roots of barley grown at different pH levels and at a toxic Al level (10 mg·dm⁻³), induced at tillering and shooting stages of plants growth. Values of surface area as well as energy distributions were the same for the roots grown at all pH values studied: 2, 4 and 7 and not dependent on the age of the plants indicating that the protons do not alter the physicochemical build-up of the surface of roots. However, significant changes of the root surface properties under the influence of aluminum: increase of surface area, average adsorption energy and amount of highly energetic adsorption sites together with a decrease of low energetic sites were observed.     

Detection of embryonic stem cell lysate biomarkers by surface plasmon resonance with reduced nonspecific adsorption

Surface plasmon resonance imaging (SPRi) has emerged as a versatile biosensor to detect a wide range of biomolecular interactions with divergent potential applications. However, the use of this advanced-level technology for stem cell lysate study is still not much explored. Cell lysates are significant biological analytes used for disease diagnostics and proteomic studies, but their complex nature limits their use as an analyte for SPRi biosensors. Here, we review the problems associated with the use of SPRi for stem cell lysate study and examine the role of surface chemistry, running buffer, and blocking solution in order to minimize nonspecific adsorption (NSA). We detect the expression of Oct4, Sox2, Nanog, Rex1, and Lin28 biomarkers present in mouse embryonic stem cell (mESC) lysate against their corresponding antibodies immobilized on the sensor surface with reduced NSA. The current study shows that the conjunction of SPRi and microarray can be used as a label-free, high-throughput, and rapid technique for detection of biomarkers and their relative abundance in stem cell lysate study.     

Difference in the hydration water mobility around F-actin and myosin subfragment-1 studied by quasielastic neutron scattering

Hydration water is essential for a protein to perform its biological function properly. In this study, the dynamics of hydration water around F-actin and myosin subfragment-1 (S1), which are the partner proteins playing a major role in various cellular functions related to cell motility including muscle contraction, was characterized by incoherent quasielastic neutron scattering (QENS). The QENS measurements on the D₂O- and H₂O-solution samples of F-actin and S1 provided the spectra of hydration water, from which the translational diffusion coefficient (D_T), the residence time (τ_T), and the rotational correlation time (τ_R) were evaluated. The D_T value of the hydration water of S1 was found to be much smaller than that of the hydration water of F-actin while the τ_T values were similar between S1 and F-actin. On the other hand, the τ_R values of the hydration water of S1 was found to be larger than that of the hydration water of F-actin. It was also found that the D_T and τ_R values of the hydration water of F-actin are similar to those of bulk water. These results suggest a significant difference in mobility of the hydration water between S1 and F-actin: S1 has the typical hydration water, the mobility of which is reduced compared with that of bulk water, while F-actin has the unique hydration water, the mobility of which is close to that of bulk water rather than the typical hydration water around proteins.     

Potato water content impact on soil moisture measurement by neutron meter

A field study was conducted to examine the effect of the water content of potato tubers on the neutron count ratios of a neutron probe in an irrigated potato field. At the 20 cm depth, where tubers are concentrated, the neutron count ratios within the potato row (CW) were significantly higher than the neutron count ratios between the rows (CB). In contrast, the measured soil water contents within the rows (SW) at this depth, tended to be less than those measured between the rows (SB). At 45 cm depth, results were similar in a dry but not a wet year. The SW values, in contrast, were less than SB. Neither (CW-CB) or (SW-SB) were significant at 75 cm depth and values of (CW-CB) and (SW-SB) were similar. This study provides qualitative information regarding the influence of a potato tuber water content on neutron measurements, resulting in an overestimations of soil moisture in a potato field. Further studies should be conducted to develop relationships for corrections.Contribution no 3879215Contribution no 3879215     

The aggregation behavior of zinc-free insulin studied by small-angle neutron scattering

The aggregation behavior of zinc-free insulin has been studied by small-angle neutron scattering as a function of pH and ionic strength of the solution. The pair distance distribution functions for the 12 samples have been obtained by indirect Fourier transformation. The results show that the diameter of the aggregates is 40 Å at pH 11 and 10 mM NaCl, independent of the protein concentration. The largest diameter of about 120 Å is found for pH 8, 100 mM NaCl, and a protein concentration of 10 mg/ml. Estimates of the pair distance distribution functions, free of inter-particle correlation effects, were obtained by an indirect Fourier transformation, omitting the data at small scattering vectors, which are influenced by these effects. By this procedure the weight-averaged molecular mass and the average radius of gyration were determined. These parameters vary from 1.3 times the monomer mass and 14 Å, to 6.8 times the monomer mass and 31 Å, respectively. The mass distribution between the oligomers was determined by a model based on the crystal structure of zinc-free insulin. The results from this model and the Fourier transformations have been compared to an equilibrium model recently introduced by Kadima et al. (1993). The neutron scattering results agree well with the predictions of this model except that broader mass distributions are suggested by neutron scattering. Correspondence to: J. Skov Pedersen     

Protein adsorption and displacement at lipid layers determined by total internal reflection fluorescence (TIRF)

In many drug delivery systems such as liposomes, the adsorption of interstitial proteins upon administration can have a huge effect on the elimination, release, and stability of the delivery system. For example, it is assumed that PEGylated liposomes prevent the adsorption of opsonins and thereby prolong the circulation time in vivo, and EMEA guidelines recommend that more than 80% of the protein antigen is adsorbed in the formulation of adjuvant systems. However, few methods exist to elucidate this protein adsorption. The present study indicates that total internal reflection fluorescence (TIRF) is a possible method to examine the adsorption and exchange of proteins at lipid surfaces. In the TIRF set-up, a lipid layer can be formed [exemplified with dimethyldioctadecylammonium bromide (DDA) and D-(+)-trehalose 6,6’-dibehenate (TDB)] whereafter protein (i.e., ovalbumin or an antigen, Ag85B-ESAT-6) is adsorbed, and these proteins can subsequently be displaced by the abundant interstitial protein (i.e., serum albumin).     

The effects of surface adsorption on the thermal stability of proteins

The effect of surface adsorption on the structure and stability of proteins is a matter of increasing interest in biotechnology. Therefore, we have examined the effect of adsorption to silica on the thermal stability of 7 proteins employing differential scanning calorimetry (DSC) and front surface fluorescence (FSF) spectroscopy. In general, it was found that surface adsorption decreased the thermal stability of the bound protein. Using lysozyme for further studies, DSC, FSF, and FTIR spectroscopies, as well as enzymatic activity measurements, were used to explore the effect of decreasing surface apolarity on stability. It was observed that increasing surface apolarity produced decreasing stability and increasing structural alteration of the adsorbed protein.     

Root surface area measurements based on adsorption and desorption of nitrite

A method, based on the negative adsorption of NO₂ ^-, has been developed to determine surface area of roots. Young roots of 3–4 year old plants ofAcacia catechu, Eucalyptus camaldulensis andLeucaena leucocephala were up-rooted and cut into 18 cylindrical pieces. Each root piece was immersed individually for 10 sec in 0.05M, 0.10M and 0.15M aqueous NaNO₂ solution and the excess solution on the root surface allowed to drain off. It was then transferred to conical flask containing distilled water and shaken for 15 min for desorption of nitrite. A known quantity of this aliquot was reacted with 1% acidic sulphanilamide and 0.02% NED HCl. A pink colour developed, and its optical density was read at 540 nm. A positive linear correlation was noted between colour density and root surface area. The respective correlation coefficientvalues for 0.05M, 0.10M and 0.15M NaNO₂ solutions were 0.973, 0.963 and 0.964 forAcacia catechu, 0.933, 0.903 and 0.898 forEucalyptus camaldulensis and 0.968, 0.976 and 0.972 forLeucaena leucocephala (significant atp<0.001). The method was successfully adopted to determine the root surface area of seedlings ofAlbizia lebbek, A. procera, Acacia auriculiformis, A. nilotica, Dalbergia latifolia andD. sissoo.     

Effects of salt concentration on association of the amyloid protofilaments of hen egg white lysozyme studied by time-resolved neutron scattering

Various proteins have been shown to form various aggregated structures including the filamentous aggregates known as amyloid fibrils depending on the solution conditions. Hen egg white lysozyme (HEWL) is one of the proteins that form the amyloid fibrils. To gain insight into the mechanism of this polymorphism of the aggregated structures, we employed a model system consisting of HEWL, pure water, and ethanol, and investigated the kinetic process of the fibril formation in various salt concentrations with time-resolved neutron scattering. It was shown that by addition of NaCl in a range between 0.3 mM and 1.0 mM to HEWL solution in 90% ethanol, gelation occurred, and this gelation proceeded through a two-step process: the lateral association of the protofilaments, followed by the cross-linking of these fibrils formed. Both the structures of the fibrils and the rate of the gelation depended on NaCl concentration. The average structures of the fibrils formed at 1.0 mM NaCl were characterized by the radius of gyration of their cross-section (45.9(+/-0.4)A) and the number of the protofilaments within the fibril (4.10(+/-0.12)), corresponding to the mature amyloid fibrils. A range of intermediate structures was formed below 1 mM NaCl. Above 2 mM NaCl, precipitation occurred because of the formation of amorphous aggregates. Here the branch point to the formation of the mature amyloid fibrils or to the amorphous aggregates was after the formation of the protofilaments. Sensitivity of the aggregated structures to salt concentration suggests that electrostatic interaction plays an essential role in the formation of these structures. The structural diversity both in the fibrils and the aggregated structures of the fibrils can be interpreted in terms of the difference in the degree of the electrostatic shielding at different salt concentrations.     

Effect of helper lipids on the interaction of DNA with cationic lipid monolayers studied by specular neutron reflection

The interaction of DNA with monolayers of the cationic lipid dimethyldioctadecylammonium bromide, with/without 50 mol % of a neutral "helper" lipid, either dioleoylphosphatidylethanolamine or cholesterol, has been studied using specular neutron reflection, surface pressure-area isotherms, and Brewster angle microscopy. The amount of DNA bound to the lipid head groups has been comprehensively quantified in the range of 8-39 vol% of DNA with respect to the monolayer composition (monolayers composed of dimethyldioctadecylammonium bromide binding the most DNA and monolayers containing dioleoylphosphatidylethanolamine binding the least) and surface pressure (DNA binding being greatest at highest surface pressures). Surprisingly, regardless of these variables, the thickness of the DNA-containing layer remained approximately constant between 18 and 25 ?. This systematic study is the first direct quantification of the binding of DNA with two different helper-lipid-containing multicomponent monolayers, an important step toward understanding interaction parameters in more realistic models of gene delivery systems.     

Molecular motions and hydration of purple membranes and disk membranes studied by neutron scattering

Fast stochastic equilibrium fluctuations (time scale: 10^–10–10^–13 seconds) in purple membranes (PM) and in disk membranes (DM) have been measured with quasielastic incoherent neutron scattering. The comparison of predominantly stochastic motions occurring in purple membranes and in disk membranes revealed qualitatively similar dynamical behaviour. Models of internal motions within restricted volumes have been shown to be useful to fit the spectra from both samples. From fits using these models we found “amplitudes” 15 to 20% larger for motions in DM samples compared to PM samples. This indicates a higher internal flexibility of the DM. Because the dynamical behaviour is very sensitive to the hydration of the protein-lipid complex, we also performed neutron diffraction experiments to determine lamellar spacings as a measure of level of hydration and as a function of temperature. From these studies the interaction of solvent molecules with the surface of the protein-lipid complex appears to be qualitatively similar for both types of membranes. Received: 12 February 1998 / Revised version: 18 March 1998 / Accepted: 27 March 1998     

Moisture adsorption capacity and surface area as deduced from vapour pressure isotherms in relation to hygroscopic water of soils

Six calcareous and alluvial soil profiles differing in their texture, CaCO₃ and salinity were chosen from west and middle Nile Delta for the present study. The 1^st and 2^nd profiles from Borg El-Arab area were sandy loam in texture and > 30% CaCO₃, while the 3^rd and 4^th profiles (from Nubaria area) were sandy clay loam and < 30% CaCO₃. The 2^nd and 4^th profiles were taken from cultivated area with maize. The 5^th profile from Epshan area was non-saline clay alluvial soil and the 6^th from El-Khamsen was saline clay alluvial soil. The relation between soil moisture content (W%) and water vapour pressure (P/P _o) was established for the mentioned soils. Data showed that the specific surface area (S) values were 34–53 and 44–60 m²/g for calcareous soils of Borg El-Arab and Nubaria areas, 206–219 and 206–249 m²/g for non-saline and saline clay alluvial soils of Epshan and El-Khamsen areas, respectively. The corresponding values of the external specific surface area (S _e) were 16–21, 14–22, 72–86 and 92–112 m²/g. Submitting W _m+W _me as an adsorption boundary of moisture films (W _c) (where W _m is mono-adsorbed layer of water vapour on soil particles and W _me is the external mono-adsorbed layer), the maximum water adsorption capacity (W _a) was found to be W _c + W _me or W _m + 2W _me. It was ranged from 1.88 to 2.70%, 1.97 to 2.95%, 9.70–10.70% and 10.80 to 13.12% while the maximum hygroscopic water (M _H) values were 2.43–3.78%, 2.91–4.65%, 16–17% and 18.30–21.9% for the studied soil profiles respectively. The residual moisture content (θ _r) at pF 7 and P/P _o = 0 was ranged from 0.0005–0.0010%, 0.0007–0.0019% and 0.0043–0.0048% in Borg El-Arab, Nubaria and Epshan soil profiles, respectively. The inter-relations between the surface area and the hygroscopic moisture parameters of the soils under investigation were as follows Calcareous soils; W _m = 0.40 M _H, W _c = 0.55 M _H, W _a = 0.70 M _H, S = 14 M _H Non-saline soil; W _m = 0.35 M _H, W _c = 0.49 M _H, W _a = 0.63 M _H, S = 13 M _H Saline soil; W _m = 031 M _H, W _c = 0.45 M _H, W _a = 0.59 M _H, S = 12 M _H These relations give possibility to deduce the soil moisture adsorption capacities and specific surface area via maximum hygroscopic water, which can be obtained through the experimental determination of water vapor adsorption isotherms.     

Interaction of proteins with lipid monolayers at the air-solution interface studied by reflection spectroscopy

The interaction of a fluorescein-labelled insulin and of cytochrome C with the air-solution interface and with lipid monolayers at the air-solution interface has been studied by measuring the change in surface pressure at constant area and by reflection spectroscopy. Chromophores at the interface only give rise to enhanced light reflection without contribution to the signal from chromophores in the bulk. The accumulation of labelled insulin at the solution surface is very weak as concluded from the shape of the spectrum and reflection intensity. No interaction with a monolayer of dipalmitoyl-phosphatidylcholine at initial surface pressure of 5mN/m was detected. In contrast, the interaction with monolayers of dioctadecyl-dimethyl-ammonium bromide at initial surface pressures between 5 and 40 mN/m is much stronger, leading to a remarkable increase of surface pressure at constant area and strong reflection signal. The technique was also used to detect cytochrome C at the air-solution interface.     

A different way of teaching the reflection of light: Using the clock model

The goal of this study is to present and evaluate a different way of teaching concepts related to the reflection of light (incident beam, reflecting beam, angle of incidence, angle of reflection, surface, regular reflection, and diffuse reflection). The participants were 42 sixth-grade students (between the ages of 11 and 12) attending a public middle school in the eastern region of Turkey. Students were given the opportunity to learn concepts related to reflection while performing experiments and making observations on their own with a mirror and clock model created by the teacher. The study enabled students to learn about the subject of reflection in a meaningful way, showing a full grasp of the concepts based on both interviews and students worksheets. The implications of these results could help teachers to support students’ learning of concepts related to light based on the exhibited benefits of using a clock model.     

Comparative adsorption of natural lung surfactant, extracted phospholipids, and artificial phospholipid mixtures to the air-water interface

Adsorption to the air-water interface of natural lung surfactant obtained by bovine lung lavage is compared and contrasted with the adsorption of mixtures of synthetic phospholipids and of extracted mixed lung lipids containing minimal protein. Surface pressure-time (pi-t) adsorption isotherms are measured at 35 degrees C for the surfactant mixtures as a function of the presence or absence of divalent metal cations (Ca2+ and Mg2+) and of heating to 45 degrees C or 90 degrees C. The effect of aqueous dispersion technique (sonication or mechanical vortexing) on the adsorption process is also studied for the extracted or synthetic phospholipid mixtures. The results imply that the protein component is necessary for the optimal adsorption of natural lung surfactant. However, by taking advantage of different methods available for phospholipid dispersion in an aqueous phase in vitro, it is possible to formulate dispersions of extracted lung phospholipids containing of order 1% protein which adsorb as well as the complete surfactant system. These results suggest that protein concentrations in surfactant mixtures can be minimized for applications such as exogenous lung surfactant replacement for the neonatal Respiratory Distress Syndrome (RDS). However, for situations which may involve alterations in endogenous surfactant function such as in lung injury, effects involving pulmonary surfactant protein and protein-lipid interactions may be of functional significance.     

Targeted amplification of delivery to cell surface receptors by dendrimer self-assembly

Nanometer-scale architectures assembled on cell surface receptors from smaller macromolecular constituents generated a large amplification of fluorescence. A targeted dendrimer was synthesized from a cystamine-core G4 PAMAM dendrimer, and contained an anti-BrE3 monoclonal antibody as the targeting group, several fluorophores and an average of 12 aldehyde moieties as complementary bio-orthogonal reactive sites for the covalent assembly. A cargo dendrimer, derived from a PAMAM G4 dendrimer, contained several fluorophores as the cargo for delivery and five hydrazine moieties as complimentary bio-orthogonal reactive sites. The system is designed to be flexible and allow for facile incorporation of a variety of targeting ligands.     

Correlation between the synthetic activity of lymphocytes and the intensity of neutron radiation near the Earth surface

The synthetic activity of lymphocytes from rat and ground squirrel was assessed for correlation with the rate of neutron radiation about the Earth surface. In the periods 1999–2002 for rats and 2002–2003 for ground squirrels, significant positive correlation was obtained with the neutron flux incident on Earth and the overall “terrestrial neutron field” but not with the neutron efflux from Earth. No correlation was observed in 2005–2006 when the solar activity and the variation in neutron count were minimal. It is concluded that low-energy neutrons may be bioeffective in periods of substantial variation in flux intensity.     

Changes in specific area and energy of root surface of cereal plants in Al-solution cultures. Water vapor adsorption studies

Surface areas and energetic properties of the shooting stage roots of rye (Secale L.), triticale (Triticale), barley (Hordeum L.) and four wheat (Triticum L.) varieties were estimated from experimental water vapor adsorption data. Roots stressed during 10 days at pH 4 with aluminium concentrations ranging from 0 to 40 mg dm^–3 were studied. Roots grown continuously at pH 7 were taken as controls. The surface properties of the roots grown at pH 4 without Al addition were apparently the same as those of the control roots. With the increase of the concentration of the aluminium treatment the surface area of the roots increased for all of the plants, beginning at 5 mg Al dm^–3 for barley, at 10 mg Al dm^–3for wheat and triticale, and at 40 mg Al dm^–3 for rye. The average water vapor adsorption energy of the root surface decreased in general with the increase of Al stress concentration for all plants but triticale, for which this increased. The sensitive cereal varieties seem to have greater amount of high energy adsorption centers (more polar surface) than the resistant ones (lower surface polarity), however more data is needed to justify this hypothesis. For Al-sensitive roots, fraction of high energy adsorption sites decreased and fraction of low energy sites increased under the Al stress. Smaller changes in adsorption energy sites were noted for roots of Al-resistant plants.