Hexagonal phase with ordered acyl chains formed by a short chain asymmetric ceramide.

Ceramides constitute a group of lipids with usually high melting temperature that also favor negative curvature in membranes when mixed with other lipids. The short chain C10:0 ceramide is an asymmetric lipid which consists of an 18 carbon sphingosine base N-acylated with decanoic acid. According to high sensitivity differential scanning calorimetry, it shows a minor exothermic peak at 61°C and a main endothermic transition at 75°C. By small angle X-ray scattering and polarized light microscopy we found that, at temperatures below the main transition, the fully hydrated lipid dispersions are arranged in a tridimensional structure corresponding to an inverted hexagonal phase. Infrared spectroscopy and wide angle X-ray diffraction indicated that the acyl chains of ceramides exhibit a relatively high order in the hexagonal phase. As far as we know, this is the first report of a lipid hexagonal phase having highly ordered acyl chains. Molecular asymmetry due to the different length of the sphingosine and the N-acyl chains of C10:0 ceramide may explain why this novel phase is formed.

Surface interactions, thermodynamics and topography of binary monolayers of Insulin with dipalmitoylphosphatidylcholine and 1-palmitoyl-2-oleoylphosphatidylcholine at the air/water interface.

The molecular packing, thermodynamics and surface topography of binary Langmuir monolayers of Insulin and DPPC (dipalmitoylphosphatidylcholine) or POCP (1-palmitoyl-2-oleoylphosphatidylcholine) at the air/water interface on Zn(2+) containing solutions were studied. Miscibility and interactions were ascertained by the variation of surface pressure-mean molecular area isotherms, surface compressional modulus and surface (dipole) potential with the film composition. Brewster Angle Microscopy was used to visualize the surface topography of the monolayers. Below 20mN/m Insulin forms stable homogenous films with DPPC and POPC at all mole fractions studied (except for films with XINS=0.05 at 10mN/m where domain coexistence was observed). Above 20mN/m, a segregation process between mixed phases occurred in all monolayers without squeezing out of individual components. Under compression the films exhibit formation of a viscoelastic or kinetically trapped organization leading to considerable composition-dependent hysteresis under expansion that occurs with entropic-enthalpic compensation. The spontaneously unfavorable interactions of Insulin with DPPC are driven by favorable enthalpy that is overcome by unfavorable entropic ordering; in films with POPC both the enthalpic and entropic effects are unfavorable. The surface topography reveals domain coexistence at relatively high pressure showing a striped appearance. The interactions of Insulin with two major membrane phospholipids induces composition-dependent and long-range changes of the surface organization that ought to be considered in the context of the information-transducing capabilities of the hormone for cell functioning.

Rheological properties of regular insulin and aspart insulin Langmuir monolayers at the air/water interface: condensing effect of Zn2+ in the subphase.

The interfacial behavior of regular insulin (Reg-insulin) and aspart insulin (Asp-insulin) was critically affected by the presence of Zn(2+) in the subphase. This cation induced a condensed-like behavior in the compression isotherms, especially apparent for Reg-insulin films when observed by Brewster angle microscopy. Immediately after spreading, Reg-insulin, but not Asp-insulin, showed bright patches that moved in a gaseous-like state. Moreover, Zn(2+) caused marked variations of the surface electrostatics of both insulin monolayers and considerable hysteresis of their molecular organization. By oscillatory compression-expansion cycles, we observed in all cases the development of a dilatational response to the surface perturbation, and both monolayers exhibited well-defined shear moduli in the presence of Zn(2+), which was higher for Reg-insulin. Development of a shear modulus indicates behavior resembling a nominal solid, more apparent for Reg-insulin than for Asp-insulin, suggesting the presence of viscoelastic networks at the surface.

Controlled lateral packing of insulin monolayers influences neuron polarization in solid-supported cultures.

Neurons are highly polarized cells, composed of one axon and several branching dendrites. One important issue in neurobiology is to understand the molecular factors that determine the neuron to develop polarized structures. A particularly early event, in neurons still lacking a discernible axon, is the segregation of IGF-1 (Insulin like Growth Factor-1) receptors in one neurite. This receptor can be activated by insulin in bulk, but, it is not known if changes of insulin organization as a monomolecular film may affect neuron polarization. To this end, in this work we developed solid-supported Langmuir-Blodgett films of insulin with different surface packing density. Hyppocampal pyramidal neurons, in early stage of differentiation, were cultured onto those substrates and polarization was studied after 24 h by confocal microscopy. Also we used surface reflection interference contrast microscopy and confocal microscopy to study attachment patterns and morphology of growth cones. We observed that insulin films packed at 14 mN/m induced polarization in a similar manner to high insulin concentration in bulk, but insulin packed at 44 mN/m did not induce polarization. Our results provide novel evidence that the neuron polarization through IGF-1 receptor activation can be selectively modulated by the lateral packing of insulin organized as a monomolecular surface for cell growth.

A preliminary investigation into the use of denture adhesives combined with dietary advice to improve diets in complete denture wearers.

OBJECTIVES: To investigate how nutritional advice and denture adhesives may be associated with eating healthier foods. METHODS: 35 edentulous subjects (13 males and 22 females, mean age 73.9 years (55-84 years)), wearing complete dentures more than one year old, completed validated questionnaires analysing saturated fat, protein, Vitamin C, the number of servings of fruit/vegetables. In addition subjects completed the NDNS and OHIP Edent questionnaires. At baseline, nutritional information and the use of denture adhesive was provided. Subjects returned after 30 consecutive days and the questionnaires were repeated. A Wilcoxon signed rank test was used to test the effect of the denture adhesive on diet and on quality of life measures. RESULTS: The subjects increased mean intake from 2.2 portions of fruit/vegetables a day to 3.6. Fat and saturated fats were reduced from 23.2g to 11.3g and Vitamin C intake increased by 34.4mg. All were statistically significant (p<0.0001). There was a statistically significant improvement over the 30-day treatment period in subjects' ability as measured by using OHIP Edent scores to bite (p=0.017) and chew a range of foods (p=0.007). CONCLUSION: Within the confines of the study, use of simple dietary advice and denture adhesives improved diet. CLINICAL SIGNIFICANCE: The results of this pilot study suggest that denture fixatives may improve dietary behaviour of complete denture wearers.

A 3-day randomised clinical study investigating the efficacy of two toothpastes, designed to occlude dentine tubules, for the treatment of dentine hypersensitivity.

OBJECTIVES: A product comparison study to compare the short term clinical efficacy of a strontium acetate/silica toothpaste with an arginine/calcium carbonate paste for pain reduction in dentine hypersensitivity. METHODS: The study was examiner blind of two arm parallel design. Eighty healthy adult subjects from general dental practice with ≥2 sensitive teeth but otherwise good oral health, were enrolled and randomised to 1 of 2 toothpaste treatments, schedule provided by the sponsor. Almost equal numbers received each treatment. Tooth sensitivity was measured in three ways; evaporative (Schiff score; Visual Analogue Scale) and tactile stimuli (Yeaple probe), prior to and immediately after subjects' self application of a single pea sized dose of toothpaste, and following subsequent twice daily brushing for three days with the paste. RESULTS: All 80 subjects completed the study. Results confirm that for both treatments, pain was reduced immediately and relief was sustained after 3 days use. For all 3 measures, benefit was similar between the two pastes, with no statistical or clinical difference demonstrated, apart from response to evaporative stimulus at 3 days, where Schiff scores were significantly lower in the arginine group, p=0.02. CONCLUSIONS: It can be concluded that both desensitising, occluding toothpastes provided reduction of pain from dentine hypersensitivity on a short term basis: toothpastes appearing to be clinically similarly effective both after a single subject dab on application and post twice daily brushing for three days. National Research Ethics Service register number 09/H020/57.

Synthesis, benzodiazepine receptor binding and molecular modelling of isochromeno[4,3-c]pyrazol-5(1H)-one derivatives.

A series of isochromeno[4,3-c]pyrazole-5(1H)-one derivatives 7b-h were prepared and tested at 10 µM for their ability to displace specific [(3)H]flunitrazepam from bovine brain membranes. The substitution pattern of the above derivatives was shown to influence the receptor affinity. The most active compound of the series was 7e, showing a 54% inhibition of [(3)H]flunitrazepam binding. Compounds 7a-d,i were compared with the known isomers chromeno[4,3-c]pyrazole-4(1H)-ones 14a-d,i, showing that the isochromene/chromene isomerism influences the activity.

Rheological properties of a two phase lipid monolayer at the air/water interface: effect of the composition of the mixture.

Many biologically relevant monolayers show coexistence of discrete domains of a long-range ordered condensed phase dispersed in a continuous, disordered, liquid-expanded phase. In this work, we determined the viscous and elastic components of the compressibility modulus and the shear viscosity of monolayers exhibiting phase coexistence with the aim at elucidating the contribution of each phase to the observed monolayer mechanical properties. To this purpose, mixed monolayers with different proportions of distearoylphosphatidylcholine (DSPC) and dimyristoylphosphatidylcholine (DMPC) were prepared and their rheological properties were analyzed. The relationship between the phase diagram of the mixture at 10 mN m(-1) and the rheological properties was studied. We found that the monolayer shear viscosity is highly dependent on the presence of domains and on the domain density. In turn, the monolayer compressibility is only influenced by the presence of domains for high domain densities. For monolayers that look homogeneous on the micrometer scale (DSPC amount lower that 23 mol %), all the analyzed rheological properties remain similar to those observed for pure DMPC monolayers, indicating that in this proportion range the DSPC molecules contribute as DMPC to the surface rheology in spite of having hydrocarbon chains four carbons longer.

Clinical study investigating abrasive effects of three toothpastes and water in an in situ model.

OBJECTIVES: This in situ study compared the abrasive effect of repeated brushings (over 10 days) of a low relative abrasive dentine (RDA) toothpaste with moderate and high relative abrasive dentine (RDA) toothpastes, on human dentine in situ. MATERIALS AND METHODS: The study design was single centre, single blind, randomized, split mouth, two period, four-treatment cross-over, in situ study in 20 healthy subjects. Subjects wore bi-lateral lower buccal appliances each fitted with four dentine sections with treatment applied with a power toothbrush, during each 10 day study period. Samples were measured at baseline, day 5 and day 10 by contact profilometry, and baseline and day 10 with non-contact profilometry. RESULTS: Nineteen subjects were included in the efficacy analysis. Results as measured by contact and non-contact profilometry from brushing with the moderate RDA paste and high RDA paste showed significantly (p<0.0001) more abrasion to dentine than brushing with the low RDA paste or water after 10 days. Dentine loss following tooth brushing with the low RDA paste was not significantly different from brushing with water, after 10 days. CONCLUSIONS: The methodology successfully showed clear differentiation between the amount of dentine lost following toothbrushing with the low RDA paste compared to the moderate or high RDA pastes. Dentine loss following brushing with the low RDA paste showed a comparable degree of abrasion to brushing with water.

Micron-scale phase segregation in lipid monolayers induced by myelin basic protein in the presence of a cholesterol analog.

It was previously shown that myelin basic protein (MBP) can induce phase segregation in whole myelin monolayers and myelin lipid films, which leads to the accumulation of proteins into a separate phase, segregated from a cholesterol-enriched lipid phase. In this work we investigated some factors regulating the phase segregation induced by MBP using fluorescent microscopy of monolayers formed with binary and ternary lipid mixtures of dihydrocholesterol (a less-oxidable cholesterol analog) and phospholipids. The influence of the addition of salts to the subphase and of varying the lipid composition was analyzed. Our results show that MBP can induce a dihydrocholesterol-dependent segregation of phases that can be further regulated by the electrolyte concentration in the subphase and the composition (type and proportion) of non-sterol lipids. In this way, changes of the lipid composition of the film or the ionic strength in the aqueous media modify the local surface density of MBP and the properties (phase state and composition) of the protein environment.

Development of an in situ methodology for the clinical evaluation of dentine hypersensitivity occlusion ingredients.

OBJECTIVE: The aim of these clinical studies was to evaluate an in situ dentine tubule occlusion model, and to determine the occluding effect from novel occluding agents on patent dentine tubules compared to a positive control (8% strontium acetate--Sensodyne Mint) and negative control (a non-occluding agent) after four days of brushing treatment. METHODS: These two in situ clinical studies were of single-center, randomized, crossover, single-blind design. Healthy participants wore two lower intra-oral appliances retaining four dentine samples for four treatment days for each period of the study. Samples were power-brushed each day with the test product. Assessment utilized surface topological analysis with a replica-based methodology under scanning electron microscopy (SEM). RESULTS: Both clinical trials demonstrated that the positive control (8% strontium acetate) occluded dentine tubules significantly better (p = 0.0007; p < 0.0009) than the negative controls in the two studies, respectively. The experimental occluding agents demonstrated varying degrees of success for occluding effect compared to the controls. CONCLUSION: The methodology clearly demonstrates that this in situ clinical model can robustly and reproducibly detect the dentine tubular occlusive effects of positive and negative controls in the treatment of dentine hypersensitivity brushed on the dentine surface. Using this methodology, new occlusion agents for the relief of dentine hypersensitivity can be assessed for occlusive effects on dentine.

The influence of domain crowding on the lateral diffusion of ceramide-enriched domains in a sphingomyelin monolayer.

In this paper, we analyze the Brownian motion of ceramide-enriched condensed domains immersed in a fluid sphingomyelin-enriched monolayer at the air-water interface. The diffusion coefficient of the domains is determined under different molecular packings and domain arrays, and the monolayer viscosity is calculated. With this approach, the effect of domain crowding and of intrinsic monolayer viscosity can be split. We found that, for mixed monolayers of palmitoylated sphingomyelin and ceramide, the monolayer viscosity depends on the lateral pressure and on the domain-domain distance. In a 9:1 proportion of sphingomyelin:ceramide, the viscosity is about 4x10(-9) N s m(-1) below 15 mN m(-1) (continuum sphingomyelin phase in a liquid expanded state) and increases at higher lateral pressures (continuum phase in a condensed state). The viscosity change with pressure is caused by both an increase of intrinsic viscosity and an increase in domain crowding. At very high domain crowding, the monolayer viscosity increases because the domains are held in place by steric hindrance generated by the other condensed domains of the array. These are short-range forces, effective when the domains are close together. As the domain array is relaxed and the domain-domain distance increases, these forces become negligible, and repulsive dipolar interactions appear to acquire importance. For the lipid mixture analyzed, the dipolar repulsion is more noticeable on subphases of 0.15 M NaCl than on pure water, revealing the influence of surface electrostatics.

Externally applied electric fields on immiscible lipid monolayers: repulsion between condensed domains precludes domain migration.

Lipid and protein molecules anisotropically oriented at a hydrocarbon-aqueous interface configure a dynamic array of self-organized molecular dipoles. Electrostatic fields applied to lipid monolayers have been shown to induce in-plane migration of domains or phase separation in a homogeneous system. In this work, we have investigated the effect of externally applied electrostatic fields on different lipid monolayers exhibiting surface immiscibility. In the monolayers studied, lipids in the condensed state segregate in discontinuous round-shaped domains, with the lipid in the liquid-expanded state forming the continuous phase. The use of fluorescent probes with selective phase partitioning allows analyzing by epifluorescence microscopy the migrations of the domains under the influence of inhomogeneous electric fields applied to the surface. Our observations indicate that a positive potential applied to an electrode placed over the monolayer promotes a repulsion of the domains until a steady state is reached, indicating the presence of a force opposed to the externally applied electric force. The experimental results were modeled by considering that the opposing force is generated by the dipole-dipole repulsion between the domains.

Protein-mediated surface structuring in biomembranes

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Protein-mediated surface structuring in biomembranes.

The lipids and proteins of biomembranes exhibit highly dissimilar conformations, geometrical shapes, amphipathicity, and thermodynamic properties which constrain their two-dimensional molecular packing, electrostatics, and interaction preferences. This causes inevitable development of large local tensions that frequently relax into phase or compositional immiscibility along lateral and transverse planes of the membrane. On the other hand, these effects constitute the very codes that mediate molecular and structural changes determining and controlling the possibilities for enzymatic activity, apposition and recombination in biomembranes. The presence of proteins constitutes a major perturbing factor for the membrane sculpturing both in terms of its surface topography and dynamics. We will focus on some results from our group within this context and summarize some recent evidence for the active involvement of extrinsic (myelin basic protein), integral (Folch-Lees proteolipid protein) and amphitropic (c-Fos and c-Jun) proteins, as well as a membrane-active amphitropic phosphohydrolytic enzyme (neutral sphingomyelinase), in the process of lateral segregation and dynamics of phase domains, sculpturing of the surface topography, and the bi-directional modulation of the membrane biochemical reactivity.

Properties of galactocerebroside layers transferred to glassy carbon electrodes: effect of an applied electric field.

Galactocerebroside films deposited onto glassy carbon electrodes have been previously studied through the electrochemical response of a redox couple present in solution. Those experiments indicated that the film is inhomogeneous and that there are lipid-free places. In this work, we present experimental results indicating that those bare regions are formed when the electrode is introduced in an aqueous solution, and that the size and/or amount of uncovered domains increase when negative potentials are applied to the film. The experimental techniques employed for these findings are epifluorescence microscopy and ellipsometry.

The Folch-Lees proteolipid induces phase coexistence and transverse reorganization of lateral domains in myelin monolayers.

Solvent solubilized myelin membranes spread as monomolecular layers at the air-water interface show a heterogeneous pattern at all surface pressures. In order to asses the role of myelin protein and lipid components in the surface structuring we compared the topography, as seen by Brewster angle microscopy (BAM) and epifluorescence microscopy, of monolayers made from mixtures containing all myelin lipids (except gangliosides) and variable proportions of Folch-Lees proteolipid protein (PLP, the major protein component of myelin). The presence of the single PLP, in the absence of the other myelin proteins, can reproduce the surface pattern of the whole myelin extract films in a concentration-dependant manner. Moreover, a threshold mole fraction of PLP is necessary to induce the lipid-protein component reorganization leading to the appearance of a rigid (gray) phase, acting as a surface skeleton, at low surface pressures and of fractal clusters at high surface pressures. The average size of those clusters is also dependent on the PLP content in the monolayer and on the time elapsed from the moment of film spreading, as they apparently result from an irreversible lateral aggregation process. The transverse rearrangement of the monolayer occurring under compression was different in films with the highest and lowest PLP mole fractions tested.

Synthesis and antimicrobial evaluation of new phenoxyacetamide derivatives.

New N-(5-methylisoxazol-3-yl)-2 or 3 or 4-(phenoxyacetamido)benzamides 6a-t were synthesized and tested for their in vitro antimicrobial activity against gram positive (Staphylococcus aureus ATCC 25923) and gram negative (Escherichia coli ATCC 25922 and Pseudomonas aeruginosa ATCC 27853) bacteria as well as fungi (Candida albicans ATCC 10231, Candida tropicalis ATCC 13803 and Cryptococcus neoformans ATCC 90112). Compounds 6 were devoid of antibacterial as well as antifungal activities at maximum tested concentrations of 50 micrograms/ml for bacteria and 100 micrograms/ml for yeast.

The amphiphilic character of glycogenin.

This study describes for the first time the amphiphilicity of the protein moiety of proteoglycogen. Glycogenin but not proteoglycogen associates to phospholipid vesicles and forms by itself stable Gibbs and Langmuir monolayers at the air-buffer interface. The adsorption free energy (-6.7 kcal/mol) and the glycogenin collapse pressure (47 mN/m) are indicative of its high surface activity which can thermodynamically drive and retain the protein at the membrane interface to a maximum equilibrium adsorption surface pressure of 21 mN/m. The marked surface activity of glycogenin is further enhanced by its thermodynamically favorable penetration into zwitterionic and anionic phospholipids with a high cut-off surface pressure point above 30 mN/m. The strong association to phospholipid vesicles and the marked surface activity of glycogenin correspond to a high amphiphilic character which supports its spontaneous association to membrane interfaces, in which the de novo biosynthesis of glycogen was proposed to initiate.