A simple approach to examine early oral microbial biofilm formation and the effects of treatments.

BACKGROUND/AIMS: A simple in vivo approach to examine early dental plaque formation in the human mouth and to determine the effects of common dietary and oral hygiene procedures on biofilm formation is reported. METHODS: A custom designed device that fits securely behind the teeth of the mandibular arch provides a surface for microbial colonization. This device is prepared with denture acrylic and can be repeatedly used by the subject, exposing a large and constant surface area for microbial accumulation. RESULTS: Large numbers of oral bacteria colonized the device by 2 h; these increased significantly by 4 h (P < 0.05). Bacterial colonization increased significantly after rinsing with a sucrose solution (P < 0.05) but remained unaffected after rinsing with water, a commercially available fluoride mouthrinse without antimicrobial agents, or brushing with a fluoride dentifrice (P > 0.05). Rinsing with mouthrinses formulated with chlorhexidine, cetylpyridinium chloride or triclosan/copolymer significantly inhibited colonization (P < 0.05). A dose-dependent inhibition was noted with chlorhexidine rinses (P < 0.05). Brushing with a triclosan/copolymer dentifrice significantly inhibited microbial colonization compared with a control (P < 0.05). CONCLUSION: This simple approach was useful for examining the effects of common dietary and oral hygiene procedures. Significant biofilm inhibitory effects were noted with formulations that demonstrated efficacy in previous clinical studies.

Detection and quantification of apocrine secreted odor-binding protein on intact human axillary skin.

A proposed mechanism of axillary malodor formation is bacterial interaction with secreted odor carrier proteins leading to the release of volatile odor molecules. One primary volatile odor molecule, 3-methyl-2-hexenoic acid, is secreted into the apocrine glandular lumen bound to two carrier proteins known as apocrine secretion odor-binding proteins (ASOB1 and ASOB2). The objective of this study was to develop a biologic method to detect and quantify ASOB2 in vitro and on intact axillary skin. The proteins present in pure apocrine secretion were separated via SDS-polyacrylamide gel electrophoresis (PAGE), electro-blotted, and reacted with antibodies to detect ASOB2. The results of this study demonstrate that ASOB2 shares immunologically homologous epitopes with the human serum protein, apolipoprotein-D (apo-D). Axillary secretions and baseline microflora were collected from two groups of panelists 6 h after showering with a non-antibacterial soap. The extracts were fractionated by SDS-PAGE. ASOB2 was detected selectively by Western blot using a monoclonal mouse-antihuman apo-D antibody and quantified on human axillary skin using the presented methods. Axillary ASOB2 concentration varied among individuals (<0.1-4.1 microg cm(-2)) with significant differences (P < 0.05, anova) seen between those of Chinese descent and non-Chinese descent. Panelists of Chinese ancestry did not show significantly lower baseline microflora levels when compared to non-Chinese panelists.

Human cutaneous response to a mixed surfactant system: role of solution phenomena in controlling surfactant irritation.

Exposure of the skin to surfactant-based products can result in irritation. To control this effect researchers are probing mechanisms of surfactant action. In vitro studies show that mixing surfactants often results in less denaturation (swelling) of stratum corneum. We have explored the in vivo human irritation response (using a 21-day cumulative irritation test) to two of these surfactants-sodium lauryl sulfate (SLS) and (C12-C14) alkyl, 7-ethoxy sulfate (AEOS-7EO). Results demonstrate that addition of AEOS-7EO to a constant dose of SLS results in a significant reduction in erythema, hence producing a milder system. The reason for the synergism is unclear, but may related to experimentally determined alterations in the micellar solution properties of the SLS upon addition of AEOS-7EO.

Characterization of the L lambda phase in trehalose-stabilized dry membranes by solid-state NMR and X-ray diffraction.

Solid-state nuclear magnetic resonance (NMR) spectroscopy and X-ray powder diffraction were used to investigate the mechanism of trehalose (TRE) stabilization of lipid bilayers. Calorimetric investigation of dry TRE-stabilized bilayers reveals a first-order phase transition (L kappa----L lambda) at temperatures similar to the L beta'----(P beta')----L alpha transition of hydrated lipid bilayers. X-ray diffraction studies show that dry mixtures of TRE and 1,2-dipalmitoyl-sn-phosphatidylcholine (DPPC) have a lamellar structure with excess crystalline TRE being present. The L kappa phase shows typical gel-phase X-ray diffraction patterns. In contrast, the L lambda-phase diffraction patterns indicate disordered hydrocarbon chains. 2H NMR of specifically 2H chain-labeled DPPC confirmed that the acyl chains are disordered in the L lambda phase over their entire lengths. 2H spectra of the choline headgroup show hindered molecular motions as compared to dry DPPC alone, and 13C spectra of the sn-2-carbonyl show rigid lattice powder patterns indicating very little motion at the headgroup and interfacial regions. Thus, the sugar interacts extensively with the hydrophilic regions of the lipid, from the choline and the phosphate moieties in the headgroup to the glycerol and carbonyls in the interfacial region. We postulate that the sugar and the lipid form an extensive hydrogen-bonded network with the sugar acting as a spacer to expand the distance between lipids in the bilayer. The fluidity of the hydrophobic region in the L lambda phase together with the bilayer stabilization at the headgroup contributes to membrane viability in anhydrobiotic organisms.

Interactions of metal ions with phosphatidylserine bilayer membranes: effect of hydrocarbon chain unsaturation.

A combination of surface monolayer, scanning calorimetry, 31P NMR, and spin-label ESR techniques has been used to monitor the interactions of monovalent (NH4+, Na+, and Li+) and divalent (Ca2+) cations with phosphatidylserines (PS) differing in their levels of chain unsaturation. Comparisons are made between the disaturated dimyristoyl-, dipalmitoyl-, and dihexadecyl-PS (DMPS, DPPS, and DHPS), saturated cis-monounsaturated palmitoyloleoyl-PS (POPS) (and bovine brain PS), di-trans-monounsaturated dielaidoyl-PS (DEPS), and di-cis-monounsaturated dioleoyl-PS (DOPS). Na+ and NH4+ cations interact weakly with all PS monolayers and bilayers without significant changes in molecular conformation, chain packing, or headgroup dynamics and without dependence on chain composition. In contrast, considering these structural and dynamic parameters, Li+ shows a gradation in its interaction with PS (DMPS greater than POPS approximately bovine brain PS greater than DOPS), suggesting that Li+-PS interactions depend on the interfacial properties of the PS molecules (e.g., surface area). Finally, Ca2+ interacts strongly with all PS monolayers and bilayers, without obvious chain selectivity. Thus, ion binding to PS depends not only on the properties of the cation (Na+ vs Li+ vs Ca2+) but also on the molecular details of the PS membrane surface.

Quantitative similarity of zinc and calcium binding to heparin in excess salt solution.

Zn2+ binding to the anticoagulant heparin was examined using a dye spectrophotometric method, with added NaCl concentrations of 0.005, 0.0075, 0.01, 0.02 and 0.04 mol/l. The results are shown as Scatchard plots and demonstrate the entropy-driven anticooperativity of Zn2+ binding to heparin. From these Scatchard plots, intrinsic binding constants are determined and are compared to our earlier data for Mg2+ and Ca2+ binding to heparin at similar ionic strengths (J. Mattai and J.C.T. Kwak, Biochim. Biophys. Acta 677 (1981) 303), and to Manning's two-variable theory (G.S. Manning, Q. Rev. Biophys. 2 (1978) 179) for a generalized system of polyelectrolyte + divalent cations + univalent cations. While Mg2+ binding to heparin is purely electrostatic (delocalized or territorial), Zn2+ and Ca2+ binding is much stronger and more specific. Binding constants for these two cations are identical, suggesting similar mechanisms for Zn2+ and Ca2+ binding to heparin.

Gel phase polymorphism in ether-linked dihexadecylphosphatidylcholine bilayers.

The structure and properties of the ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) have been examined as a function of hydration. By differential scanning calorimetry, DHPC exhibits an endothermic (chain melting) transition with the transition temperature (limiting value, 44.2 degrees C) and enthalpy (limiting value, delta H = 8.0 kcal/mol) being hydration dependent. For hydration values greater than 30 wt % water, DHPC exhibits a pretransition at approximately 36 degrees C (delta H = 1.1 kcal/mol) and a subtransition at approximately 5 degrees C (delta H = 0.2 kcal/mol). By X-ray diffraction, at 22 degrees C DHPC exhibits a normal bilayer gel structure with the bilayer periodicity increasing from 58.0 to 62.5 A over the hydration range 9.5-25.4% water. At 30-32% water, two coexisting gel phases are observed with d = 63-64 A and d = 44-45 A; at higher hydration, only the latter phase is present, reaching a limiting d = 47.0 A at 37.5% water. Two different gel phases clearly exist at low and high hydrations. Electron density profiles at low hydration (9.5-25.4%) show a bilayer thickness dp-p = 46 A, whereas at greater than 32% water the bilayer thickness is markedly reduced, dp-p = 30 A. These and other structural parameters indicate a hydration-dependent gel----gel structural transition between a normal bilayer (two chains per polar group) and the chain-interdigitated bilayer (four chains per polar group) arrangement described previously for DHPC [Ruocco, M. J., Siminovitch, D. J., & Griffin, R. G. (1985) Biochemistry 24, 2406-2411].(ABSTRACT TRUNCATED AT 250 WORDS)

Bilayer interactions of ether- and ester-linked phospholipids: dihexadecyl- and dipalmitoylphosphatidylcholines.

Mixed phospholipid systems of ether-linked 1,2-dihexadecylphosphatidylcholine (DHPC) and ester-linked 1,2-dipalmitoylphosphatidylcholine (DPPC) have been studied by differential scanning calorimetry and X-ray diffraction. At maximum hydration (60 wt % water), DHPC shows three reversible transitions: a main (chain melting) transition, TM = 44.2 degrees C; a pretransition, TP = 36.2 degrees C; and a subtransition, TS = 5.5 degrees C. DPPC shows two reversible transitions: TM = 41.3 degrees C and TP = 36.5 degrees C. TM decreases linearly from 44.2 to 41.3 degrees C as DPPC is incorporated into DHPC bilayers; TP exhibits eutectic behavior, decreasing sharply to reach 23.3 degrees C at 40.4 mol % DPPC and then increasing over the range 40-100 mol % DPPC; TS remains constant at 4-5 degrees C and is not observed at greater than 20 mol % DPPC. At 50 degrees C, X-ray diffraction shows a liquid-crystalline bilayer L alpha phase at all DHPC:DPPC mole ratios. At 22 degrees C, DHPC shows an interdigitated bilayer gel L beta phase (bilayer periodicity d = 47.0 A) into which approximately 30 mol % DPPC can be incorporated. Above 30 mol % DPPC, a noninterdigitated gel L beta' phase (d = 64-66 A) is observed. Thus, at T greater than TM, DHPC and DPPC are miscible in all proportions in an L alpha bilayer phase. In contrast, a composition-dependent gel----gel transition between interdigitated and noninterdigitated bilayers is observed at T less than TP, and this leads to eutectic behavior of the DHPC/DPPC system.

Mixed-chain phosphatidylcholine bilayers: structure and properties.

Calorimetric and X-ray diffraction data are reported for two series of saturated mixed-chain phosphatidylcholines (PCs), 18:0/n:0-PC and n:0/18:0-PC, where the sn-1 and sn-2 fatty acyl chains on the glycerol backbone are systematically varied by two methylene groups from 18:0 to 10:0 (n = 18, 16, 14, 12, or 10). Fully hydrated PCs were annealed at -4 degrees C and their multilamellar dispersions characterized by differential scanning calorimetry and X-ray diffraction. All mixed-chain PCs form low-temperature "crystalline" bilayer phases following low-temperature incubation, except 18:0/10:0-PC. The subtransition temperature (Ts) shifts toward the main (chain melting) transition temperature (Tm) as the sn-1 or sn-2 fatty acyl chain is reduced in length; for the shorter chain PCs (18:0/12:0-PC, 12:0/18:0-PC, and 10:0/18:0-PC), Ts is 1-2 degrees C greater than Tm, and the subtransition enthalpy (delta Hs) is much greater than for the longer acyl chain PCs. Tm decreases with acyl chain length for both series of PCs except 18:0/10:0-PC, while for the positional isomers, n:0/18:0-PC and 18:0/n:0-PC, Tm is higher for the isomer with the longer acyl chain in the sn-2 position of the glycerol backbone. The conversion from the crystalline bilayer Lc phase to the liquid-crystalline L alpha phase with melted hydrocarbon chains occurs through a series of phase changes which are chain length dependent. For example, 18:0/18:0-PC undergoes the phase changes Lc----L beta'----P beta'----L alpha, while the shorter chain PC, 10:0/18:0-PC, is directly transformed from the Lc phase to the L alpha phase. However, normalized enthalpy and entropy data suggest that the overall thermodynamic change, Lc----L alpha, is essentially chain length independent. On cooling, the conversion to the Lc phases occurs via bilayer gel phases, L beta', for the longer chain PCs or through triple-chain interdigitated bilayer gel phases, L beta, for the shorter chain PC 18:0/12:0-PC and possibly 10:0/18:0-PC. Molecular models indicate that the bilayer gel phases for the more asymmetric PC series, 18:0/n:0-PC, must undergo progressive interdigitation with chain length reduction to maintain maximum chain-chain interaction. The L beta phase of 18:0/10:0-PC is the most stable structure for this PC below Tm. The formation and stability of the triple-chain structures can be rationalized from molecular models.

Regulation of bilayer stability in Clostridium butyricum: studies on the polymorphic phase behavior of the ether lipids.

Three of the major phospholipids of the cell membrane of Clostridium butyricum are phosphatidylethanolamine (PE), plasmenylethanolamine (PlaE), and the glycerol acetal of plasmenylethanolamine. When cultured in the absence of biotin in media supplemented with a cis-unsaturated fatty acid, the cellular lipids become highly enriched with the fed fatty acid. Under these conditions, the ratio of the glycerol acetal of PlaE to the sum of PE plus PlaE increases markedly over that seen in cells containing mixtures of saturated and unsaturated fatty acids [Johnston, N.C., & Goldfine, H. (1985) Biochim. Biophys. Acta 813, 10-18]. We have studied the polymorphic phase behavior of the phospholipids from C. butyricum grown on oleic acid using differential scanning calorimetry, 31P nuclear magnetic resonance, and X-ray diffraction. The mixed PE plus PlaE fraction undergoes a transition from the gel to liquid-crystalline state at -1.9 degrees C and a lamellar to reversed hexagonal (L----H) transition at or near 0 degrees C. The glycerol acetal of PlaE melts at 16.1 degrees C, and as predicted from lipid packing theory, the lamellar phase is stabilized, up to 50 degrees C. Addition of the oleate-enriched glycerol acetal of PlaE to dioleoylphosphatidylethanolamine, or the PE plus PlaE fraction from oleate-grown cells, stabilized the lamellar arrangement of the mixtures. A ratio of glycerol acetal of PlaE to total PE (PE plus PlaE) of 0.5, which is close to that found in cells grown on palmitic plus oleic acid, 0.6-0.7, did not produce a lamellar phase at 37 degrees C when the lipids enriched with oleic acid were tested,(ABSTRACT TRUNCATED AT 250 WORDS)

Interaction of cholera toxin with ganglioside GM1 receptors in supported lipid monolayers.

Lipid monolayers formed at the air-water interface containing the ganglioside GM1 in egg yolk phosphatidylcholine have been transferred according to the Langmuir-Blodgett technique to glass cover slips coated with octadecyl- or hexadecyltrichlorosilane and carbon-coated electron microscope grids. Monolayer transfer has been demonstrated with fluorescence microscopy, by the transfer of a fluorescent phospholipid analogue, N-(7-nitro-2,1,3-benzoxadiazol-4-yl)phosphatidylethanolamine or Lucifer yellow labeled GM1 (LY-GM1), incorporated into the lipid monolayer. Incubation of supported monolayers with solutions of fluorescein-labeled cholera toxin (FITC cholera toxin) resulted in specific binding of the toxin to monolayers containing GM1, as revealed by fluorescence microscopy. Lateral diffusion coefficients were measured for both the receptor (LY-GM1) [(3.9 +/- 2.1) X 10(-8) cm2/s] and the receptor-ligand complex (GM1-FITC cholera toxin) [(8.9 +/- 3.2) X 10(-9) cm2/s] according to the technique of fluorescence recovery after photobleaching. In separate studies, GM1-containing monolayers transferred to electron microscope grids were incubated with solutions containing unlabeled cholera toxin, followed by negative staining with uranyl acetate. Electron microscopy revealed patches of stained cholera toxin molecules (diameter approximately 70 A) in crystalline, two-dimensional hexagonal arrays. Optical diffraction and image reconstruction showed the arrangement of the cholera toxin molecules in a planar hexagonal cell, a = 81 A. These initial reconstructions give structural information to a resolution of approximately 30 A and indicate a doughnut-shaped molecule with a central aqueous channel.

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes.

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.

The kinetics of formation and structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine.

A combination of differential scanning calorimetry (DSC) and X-ray diffraction have been used to study the kinetics of formation and the structure of the low-temperature phase of 1-stearoyl-lysophosphatidylcholine (18:0-lysoPC). For water contents greater than 40 weight %, DSC shows a sharp endothermic transition at 27 degrees C (delta H = 6.75 kcal/mol) corresponding to a low-temperature phase----micelle transition. This sharp transition is not reversible, but is regenerated in a time and temperature-dependent manner. For example, with incubation at 0 degrees C the maximum transition enthalpy (delta H = 6.75 kcal/mol) is generated in about 45 min after an initial slow nucleation process of approx. 20 min. The kinetics of formation of the low-temperature phase is accelerated at lower temperatures and may be related to the disruption of 18:0-lysoPC micelles by ice crystal formation. X-ray diffraction patterns of 18:0-lysoPC recorded at 10 degrees C over the hydration range 20-80% are characteristic of a lamellar gel phase with tilted hydrocarbon chains with the bilayer repeat distance increasing from 47.6 A at 20% hydration to a maximum of 59.4 A at 39% hydration. At this maximum hydration, approx. 19 molecules of water are bound per molecule of 18:0-lysoPC. Electron density profiles show a phosphate-phosphate distance of 30 A, indicating an interdigitated lamellar gel phase for 18:0-lysoPC at all hydration values. The angle of chain tilt is calculated to be between 20 and 30 degrees. For water contents greater than 40%, this interdigitated lamellar phase converts to the micellar phase at 27 degrees C in a kinetically fast process, while the reverse (micelle----interdigitated bilayer) transition is a kinetically slower process (see also Wu, W. and Huang, C. (1983) Biochemistry 22, 5068-5073).

2H and 31P NMR study of the interaction of general anesthetics with phosphatidylcholine membranes.

The effect of the general anesthetics alpha-chloralose and chloral hydrate as well as the nonanesthetic beta-chloralose on the order of phosphatidylcholine and phosphatidylcholine-cholesterol liposomes has been examined by 2H nuclear magnetic resonance. Chloral hydrate interacts with the hydrophilic head-group region, causing a change in the torsion angle of the C alpha-C beta bond. The membrane interior is also disordered by the presence of this agent. alpha-Chloralose, on the other hand, disorders only the central position of the membrane. beta-Chloralose produces little significant change in bilayer order.

The displacement of phenothiazines from phospholipid binding sites by cholesterol.

The interaction of the phenothiazine type drug, methochlorpromazine, with phosphatidylcholine membranes has been investigated by using this tranquilizer in a deuterium labeled form. Two distinct binding sites were found, with exchange between them being fast on the 2H NMR time scale. Cholesterol preferentially displaces the chlorpromazine from the more hydrophobic of these sites, making possible an explanation of the modulation of the effects of amphipathic agents by cholesterol. In addition, the phenomenon of displacement of membrane active agents by sterols may explain discrepancies between membrane/water partition coefficients as measured by centrifugation and hygroscopic desorption.

A dye spectrophotometric method for binding studies of Zn2+ and Mn2+ by biopolyelectrolytes.

A dual-wavelength dye spectrophotometric method is reported for measuring zinc and manganese activities using the dyes tetramethylmurexide (TMMX) and murexide (MX) respectively. The method is applied to the measurement of the activities of these metal ions in solutions of the polyelectrolyte dextransulfate with added sodium chloride. Polyion concentrations, Cp (expressed as moles sulfate ion litre ) of 0.001 and 0.002 are studied at total ionic strengths 0.005, 0.0075, 0.01, 0.02 and 0.03 mole/1. Divalent metal ion concentrations are varied between 0 and 1.0 Cp. The results for the metal ion activities are expressed in the form of binding isotherms, theta2 versus C2/Cp (theta2 = C2b/Cp; C2b = bound divalent metal ion concentration) and Scatchard plots, K2 versus theta2/(C2-C2b), at different ionic strengths. The experimental data are correlated with the "two-variable theory" eveloped for these mixed counterion systems by Manning. This comparison shows that the observed decrease in theta2 and K2 with ionic strength at fixed C2 and Cp is generally well predicted by the two-variable theory. Both Zn and Mn bind to the same extent to dextransulfate. This observation, and the reasonable agreement of the data with the "two-variable theory" may be interpreted as indicating a delocalized form of binding of these metal ions to the polymer.