NHE1 regulates the stratum corneum permeability barrier homeostasis. Microenvironment acidification assessed with fluorescence lifetime imaging

The outermost epidermal layer, the stratum corneum (SC), exhibits an acidic surface pH, whereas the pH at its base approaches neutrality. NHE1 is the only Na(+)/H(+) antiporter isoform in keratinocytes and epidermis, and has been shown to regulate intracellular pH. We now demonstrate a novel function for NHE1, as we find that it also controls acidification of extracellular "microdomains" in the SC that are essential for activation of pH-sensitive enzymes and the formation of the epidermal permeability barrier. NHE1 expression in epidermis is most pronounced in granular cell layers, and although the surface pH of NHE1 knockout mice is only slightly more alkaline than normal using conventional pH measurements, a more sensitive method, fluorescence lifetime imaging, demonstrates that the acidic intercellular domains at the surface and of the lower SC disappear in NHE1 -/- animals. Fluorescence lifetime imaging studies also reveal that SC acidification does not occur through a uniform gradient, but through the progressive accumulation of acidic microdomains. These findings not only visualize the spatial distribution of the SC pH gradient, but also demonstrate a role for NHE1 in the generation of acidic extracellular domains of the lower SC, thus providing the acidification of deep SC interstices necessary for lipid processing and barrier homeostasis.     

Direct visualization of lipid domains in human skin stratum corneum's lipid membranes: effect of pH and temperature

The main function of skin is to serve as a physical barrier between the body and the environment. This barrier capacity is in turn a function of the physical state and structural organization of the stratum corneum extracellular lipid matrix. This lipid matrix is essentially composed of very long chain saturated ceramides, cholesterol, and free fatty acids. Three unsolved key questions are i), whether the stratum corneum extracellular lipid matrix is constituted by a single gel phase or by coexisting crystalline (solid) domains; ii), whether a separate liquid crystalline phase is present; and iii), whether pH has a direct effect on the lipid matrix phase behavior. In this work the lateral structure of membranes composed of lipids extracted from human skin stratum corneum was studied in a broad temperature range (10 degrees C-90 degrees C) using different techniques such as differential scanning calorimetry, fluorescence spectroscopy, and two-photon excitation and laser scanning confocal fluorescence microscopy. Here we show that hydrated bilayers of human skin stratum corneum lipids express a giant sponge-like morphology with dimensions corresponding to the global three-dimensional morphology of the stratum corneum extracellular space. These structures can be directly visualized using the aforementioned fluorescence microscopy techniques. At skin physiological temperatures (28 degrees C-32 degrees C), the phase state of these hydrated bilayers correspond microscopically (radial resolution limit 300 nm) to a single gel phase at pH 7, coexistence of different gel phases between pH 5 and 6, and no fluid phase at any pH. This observation suggests that the local pH in the stratum corneum may control the physical properties of the extracellular lipid matrix by regulating membrane lateral structure and stability.     

Time-resolved fluorescence analysis of the recombinant photosystem II antenna complex CP29. Effects of zeaxanthin, pH and phosphorylation.

Nonradiative dissipation of excitation energy is the major photoprotective mechanism in plants. The formation of zeaxanthin in the antenna of photosystem II has been shown to correlate with the onset of nonphotochemical quenching in vivo. We have used recombinant CP29 protein, over-expressed in Escherichia coli and refolded in vitro with purified pigments, to obtain a protein indistinguishable from the native complex extracted from thylakoids, binding either violaxanthin or zeaxanthin together with lutein. These recombinant proteins and the native CP29 were used to measure steady-state chlorophyll fluorescence emission and fluorescence decay kinetics. We found that the presence of zeaxanthin bound to CP29 induces a approximately 35% decrease in fluorescence yield with respect to the control proteins (the native and zeaxanthin-free reconstituted proteins). Fluorescence decay kinetics showed that four components are always present but lifetimes (tau) as well as relative fluorescence quantum yields (rfqy) of the two long-lived components (tau3 and tau4) are modified by the presence of zeaxanthin. The most relevant changes are observed in the rfqy of tau3 and in the average lifetime ( approximately 2.4 ns with zeaxanthin and 3.2-3.4 ns in the control proteins). When studied in vitro, no significant effect of acidic pH (5.2-5.3) is observed on chlorophyll A fluorescence yield or kinetics. The data presented show that recombinant CP29 is able to bind zeaxanthin and this protein-bound zeaxanthin induces a significant quenching effect.     

A zymogel enhances the self-cleaning abilities of the skin of the pilot whale (Globicephala melas)

Enzyme activity in the stratum corneum of the pilot whale Globicephala melas was investigated employing colorimetric enzyme screening assays combined with NATIVE PAGE, size exclusion chromatography (SEC) and histochemical staining procedures. Applying different substrates, several enzymes were detected. The histochemical demonstration of some selected hydrolytic enzymes enriched in the stratum corneum showed high extracellular accumulation. As demonstrated by size exclusion chromatography, high molar mass aggregates were built up from a glycoprotein-rich 20-30-kD fraction. Using NATIVE PAGE experiments under non-reducing conditions, a selection of five degrading enzymes was recovered within the above-reported aggregates. Activity of extracellular aggregate-attached enzymes in the superficial layer of the stratum corneum exhibited no remarkable decrease potentially resulting from self-degradation. We thus conclude that due to their enclosure within the microenvironment of aggregates, a zymogel is formed and autolysis of the stratum corneum is reduced. With respect to the skin surface, the zymogel with hydrolytic activities covering major parts of it enhances the self-cleaning abilities of the skin of the pilot whale based on physical pre-requisites by hydrolyzing adhesive glycoconjugates of settling biofouling organisms considered as primary steps in fouling.     

Frequency domain measurements of the fluorescence lifetime of ribonuclease T1.

Using multifrequency phase/modulation fluorometry, we have studied the fluorescence decay of the single tryptophan residue of ribonuclease T1 (RNase T1). At neutral pH (7.4) we find that the decay is a double exponential (tau 1 = 3.74 ns, tau 2 = 1.06 ns, f1 = 0.945), in agreement with results from pulsed fluorometry. At pH 5.5 the decay is well described by a single decay time (tau = 3.8 ns). Alternatively, we have fitted the frequency domain data by a distribution of lifetimes. Temperature dependence studies were performed. If analyzed via a double exponential model, the activation energy for the inverse of the short lifetime component (at pH 7.4) is found to be 3.6 kcal/mol, as compared with a value of 1.0 kcal/mol for the activation energy of the inverse of the long lifetime component. If analyzed via the distribution model, the width of the distribution is found to increase at higher temperature. We have also repeated, using lifetime measurements, the temperature dependence of the acrylamide quenching of the fluorescence of RNase T1 at pH 5.5. We find an activation energy of 8 kcal/mol for acrylamide quenching, in agreement with our earlier report.     

Permeability parameters of the toad isolated stratum corneum.

A technique for isolating the stratum corneum from the subjacent layers of the epithelium was developed which permits studying the stratum corneum as an isolated membrane mounted between half-chambers. The method basically consists of an osmotic shock induced by immersing a piece of skin in distilled water at 50 degrees C for 2 min. When the membrane is bathed on each surface by NaCl-Ringer's solution, its electrical resistance is 14.1 +/- 1.3 omega cm2 (n=10). This value is about 1/100 of the whole skin resistance in the presence of the same solution. The hydraulic filtration coefficient (Lp) measured by a hydrostatic pressure method, with identical solutions on each side of the membrane, is 8.8 X 10(-5) +/- 1.5 X 10(-5) cm sec-1 atm-1 (n=10) in distilled water and 9.2 X 10(-5) +/- 1.4 X 10(-5) cm sec-1 atm-1 (n=10) in NaCl-Ringer's solution. These values are not statistically different and are within the range of 1/80 to 1/120 of the whole skin Lp. The stratum corneum shows an amphoteric character when studied by KCl diffusion potentials at different pH'S. The membrane presents an isoelectric pH of 4.6 +/- 0.3 (n=10). Above the isoelectric pH the potassium transport number is higher than the chloride transport number; below it, the reverse situation is valid. Divalent cations (Ca++ or Cu++) reduce membrane ionic discrimination when the membrane is negatively charged and are ineffective when the membrane fixed charges are protonated at low pH.     

Human stratum corneum lipid organization as observed by atomic force microscopy on Langmuir-Blodgett films

The barrier function of skin ultimately depends on the physical state and structural organisation of the stratum corneum extracellular lipid matrix. Ceramides, cholesterol and a broad distribution of saturated long-chain free fatty acids dominate the stratum corneum lipid composition. Additionally, smaller amounts of cholesterol sulfate and cholesteryl oleate may be present. A key feature determining skin barrier capacity is thought to be whether or not different lipid domains coexist laterally in the stratum corneum extracellular lipid matrix. In this study, the overall tendency for lipid domain formation in different mixtures of extracted human stratum corneum ceramides, cholesterol, free fatty acids, cholesterol sulfate and cholesteryl oleate were studied using atomic force microscopy (AFM) on Langmuir-Blodgett (LB) films on mica. It is shown that the saturated long-chain free fatty acid distribution of human stratum corneum prevents hydrocarbon chain segregation. Further, LB-films of human stratum corneum ceramides express a pattern of connected elongated domains with a granular domain interface. The dominating effect of both cholesterol and cholesterol sulfate is that of increased ceramide domain dispersion. This effect is counteracted by the presence of free fatty acids, which preferentially mix with ceramides and not with cholesterol. Cholesteryl oleate does not mix with other skin lipid components, supporting the hypothesis of an extra-endogenous origin. In the system composed of endogenous human ceramides and cholesterol plus 15 wt% stratum corneum distributed free fatty acids, i.e., the system mimicking most closely the lipid composition of the stratum corneum extracellular space, LB-films on mica express lateral domain formation.     

Determination of rotational correlation times from deconvoluted fluorescence anisotropy decay curves. Demonstration with 6,7-dimethyl-8-ribityllumazine and lumazine protein from Photobacterium leiognathi as fluorescent indicators

The experimental and analytical protocols required for obtaining rotational correlation times of biological macromolecules from fluorescence anisotropy decay measurements are described. As an example, the lumazine protein from Photobacterium leiognathi was used. This stable protein (Mr 21 200) contains the noncovalently bound, natural fluorescent marker 6,7-dimethyl-8-ribityllumazine, which has in the bound state a long fluorescence lifetime (tau = 14 ns). Shortening of the fluorescence lifetime to 2.6 ns at room temperature was achieved by addition of the collisional fluorescence quencher potassium iodide. The shortening of tau had virtually no effect on the rotational correlation time of the lumazine protein (phi = 9.4 ns, 19 degrees C). The ability to measure biexponential anisotropy decay was tested by the addition of Photobacterium luciferase (Mr 80 000), which forms an equilibrium complex with lumazine protein. Under the experimental conditions used (2 degrees C) the biexponential anisotropy decay can best be described with correlation times of 20 and 60 ns, representing the uncomplexed and luciferase-associated lumazine proteins, respectively. The unbound 6,7-dimethyl-8-ribityllumazine itself (tau = 9 ns) was used as a model compound for determining correlation times in the picosecond time range. In the latter case rigorous deconvolution from the excitation profile was required to recover the correlation time, which was shorter (100-200 ps) than the measured laser excitation pulse width (500 ps).     

Determination of local conformation of proteins from 1H-NMR spectroscopy data

A method is proposed to determine conformations of amino acid residues of the protein and effective correlation time tau c from cross-peak intensities in two-dimensional nuclear Overhauser enhancement (NOESY) spectra. The method consists in fitting complete relaxation matrix of dipeptide unit protons to experimental cross-peak intensities by varying phi, psi, chi torsional angles and tau c. To verify the method, NOESY spectra of basic pancreatic trypsin inhibitor (BPTI) were theoretically generated at mixing times tau m = 25-300 ms and tau c = 4 ns and used for local structure determination. The method works well with optimum for measurement of NOE intensities tau m 100-200 ms. As a result, the backbone phi, psi torsion angles were unambiguously determined at tau m = 100 ms for all but Gly residues of BPTI, and chi 1 angles were determined for the majority of side chains. The obtained dipeptide unit conformations are very close to the BPTI crystallographic structure: root mean square deviation (RMSD) of interproton distances within dipeptide units, on the average, is 0.08 A (maximal deviation 0.44 A), and RMSD of phi and psi angles are 18 and 9 degrees, respectively (maximal deviations are 44 and 22 degrees).     

Fluorescence anisotropy studies of molecularly imprinted polymers.

A molecularly imprinted polymer (MIP) is a biomimetic material that can be used as a biochemical sensing element. We studied the steady-state and time-resolved fluorescence and fluorescence anisotropy of anthracene-imprinted polyurethane. We compared MIPs with imprinted analytes present, MIPs with the imprinted analytes extracted, MIPs with rebound analytes, non-imprinted control polymers (non-MIPs) and non-MIPs bound with analytes to understand MIP's binding behaviour. MIPs and non-MIPs had similar steady-state fluorescence anisotropy in the range 0.11-0.24. Anthracene rebound in MIPs and non-MIPs had a fluorescence lifetime of tau = 0.64 ns and a rotational correlation time of phi(F) = 1.2-1.5 ns, both of which were shorter than that of MIPs with imprinted analytes present (tau = 2.03 ns and phi(F) = 2.7 ns). The steady-state anisotropy of polymer solutions increased exponentially with polymerization time and might be used to characterize the polymerization extent in situ.     

Local temperature rises influence in vivo electroporation pore development: a numerical stratum corneum lipid phase transition model

Electroporation is an approach used to enhance transdermal transport of large molecules in which the skin is exposed to a series of electric pulses. Electroporation temporarily destabilizes the structure of the outer skin layer, the stratum corneum, by creating microscopic pores through which agents, ordinarily unable to pass into the skin, are able to pass through this outer barrier. Long duration electroporation pulses can cause localized temperature rises, which result in thermotropic phase transitions within the lipid bilayer matrix of the stratum corneum. This paper focuses on electroporation pore development resulting from localized Joule heating. This study presents a theoretical model of electroporation, which incorporates stratum corneum lipid melting with electrical and thermal energy equations. A transient finite volume model is developed representing electroporation of in vivo human skin, in which stratum corneum lipid phase transitions are modeled as a series of melting processes. The results confirm that applied voltage to the skin results in high current densities within the less resistive regions of the stratum corneum. The model captures highly localized Joule heating within the stratum corneum and subsequent temperature rises, which propagate radially outward. Electroporation pore development resulting from the decrease in resistance associated with lipid melting is captured by the lipid phase transition model. As the effective pore radius grows, current density and subsequent Joule heating values decrease.     

Conformational transitions in the calcium adenosinetriphosphatase studied by time-resolved fluorescence resonance energy transfer

We have used time-resolved fluorescence to study proposed conformational transitions in the Ca-ATPase in skeletal sarcoplasmic reticulum (SR). Resonance energy transfer was used to measure distances between the binding sites of 5-[[2-[(iodoacetyl)amino]ethyl]amino]naphthalene-1-sulfonic acid (IAEDANS) and fluorescein 5-isothiocyanate (FITC) as a function of conditions proposed to affect the enzyme's conformation. When 1.0 +/- 0.15 IAEDANS is bound per Ca-ATPase, most (76 +/- 4%) of the probes have an excited-state lifetime (tau) of 18.6 +/- 0.5 ns, and the remainder have a lifetime of 2.5 +/- 0.9 ns. When FITC is bound to a specific site on each IAEDANS-labeled enzyme, most of the long-lifetime component is quenched into two short-lifetime components, indicating energy transfer that corresponds to two donor-acceptor distances. About one-third of the quenched population has a lifetime tau = 11.1 +/- 2.5 ns, corresponding to a transfer efficiency E = 0.40 +/- 0.07 and a donor-acceptor distance R1 = 52 +/- 3 A. The remaining two-thirds exhibit lifetimes in the range of 1.2-4.2 ns, corresponding to a second distance 31 A less than or equal to R2 less than or equal to 40 A. Addition of Ca2+ (in the micromolar to millimolar range), or vanadate (to produce a phosphoenzyme analogue), had no effect on the donor-acceptor distances. Addition of decavanadate results in the quenching of IAEDANS fluorescence but has no effect on the energy-transfer distance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Proteolytic modification of acidic and basic keratins during terminal differentiation of mouse and human epidermis

Keratins from the living cell layers of human and neonatal mouse epidermis (prekeratins) have been compared to those from the stratum corneum (SC keratins). Human and mouse epidermis contained four prekeratins, two of each keratin subfamily: type II basic (pI 6.5-8.5; human 68 kDa, 60.5 kDa and mouse 67 kDa, 60 kDa) and type I acidic (pI 4.7-5.7; human 57 kDa, 51 kDa and mouse 58 kDa, 53 kDa,). While all four were present in equal amounts in adult human epidermis, two (67 kDa basic, 58 kDa acidic) were more prominent in neonatal mouse epidermis. Preliminary results with cell fractions (basal, spinous and granular) indicated that quantitative differences were a function of morphology, basal cells containing the smaller member of each subfamily and granular cells the larger. Mouse stratum corneum extracts contained four keratins (three in human): type II neutral-acidic (pI 5.7-6.7; human 65 kDa and mouse 64 kDa, 62 kDa) and type I acidic (pI 4.9-5.4; human 57.5 kDa, 55 kDa and mouse 58.5 kDa, 57.5 kDa). In both species, one-dimensional and two-dimensional peptide mapping (with V8 protease and trypsin respectively) indicated that while all four prekeratins were distinct gene products, similarities existed in the type II basic and the type I acidic keratin subfamilies. A strong homology also existed between type II SC keratins and the larger basic (type II) prekeratin (human 68 kDa and mouse 67 kDa) and between type I SC keratins and the larger acidic (type I) prekeratin (human 57 kDa and mouse 58 kDa). These results indicate a precursor-product relationship within each keratin subfamily, between SC keratins and the prekeratins abundant in the adjacent granular layer. This differentiation-related keratin processing was similar in mouse and human epidermis, and may represent a widespread phenomenon amongst keratinising epithelia.     

The Grainyhead-like epithelial transactivator Get-1/Grhl3 regulates epidermal terminal differentiation and interacts functionally with LMO4

Defective permeability barrier is an important feature of many skin diseases and causes mortality in premature infants. To investigate the control of barrier formation, we characterized the epidermally expressed Grainyhead-like epithelial transactivator (Get-1)/Grhl3, a conserved mammalian homologue of Grainyhead, which plays important roles in cuticle development in Drosophila. Get-1 interacts with the LIM-only protein LMO4, which is co-expressed in the developing mammalian epidermis. The epidermis of Get-1(-/-) mice showed a severe barrier function defect associated with impaired differentiation of the epidermis, including defects of the stratum corneum, extracellular lipid composition and cell adhesion in the granular layer. The Get-1 mutation affects multiple genes linked to terminal differentiation and barrier function, including most genes of the epidermal differentiation complex. Get-1 therefore directly or indirectly regulates a broad array of epidermal differentiation genes encoding structural proteins, lipid metabolizing enzymes and cell adhesion molecules. Although deletion of the LMO4 gene had no overt consequences for epidermal development, the epidermal terminal differentiation defect in mice deleted for both Get-1 and LMO4 is much more severe than in Get-1(-/-) mice with striking impairment of stratum corneum formation. These findings indicate that the Get-1 and LMO4 genes interact functionally to regulate epidermal terminal differentiation.     

Intramolecular mobility of human major histocompatibility complex protein. A spin-label study

Membranes of human splenocytes were hydrolyzed by papain and extracellular portions of class I and class II HLA antigen molecules were isolated by monoclonal antibodies fixed on Sepharose 4B. The isolated proteins were spin-labeled by TEMPO-dichlorotriazine and the values of rotational correlation times (tau) of labeled proteins were found using dependencies of ESR spectra parameters vs viscosity at constant temperature. The tau-values were equal to 8 ns for class I molecules and 14 ns for class II molecules. These values are 2-3 times lower than predicted for a rigid ellipsoid with mol wt. 50 kDa (about 20 ns). This fact suggests the existence of flexibility of HLA molecules which seems to be important for their biological activity. In this respect extracellular portions of HLA antigen molecules resemble flexible Fc fragments (tau = 12 ns) and differ from rigid Fab fragments (tau = 20 ns) of immunoglobulins G. The values of tau of spin-labeled proteins adsorbed from membrane hydrolysates on IgG-column was equal to 6.5 ns. The proteins adsorbed on lentil lectin column (after isolation of HLA proteins) have the tau-values equal to 9 ns.     

Measurement of the transit time for cells through the epidermis and stratum corneum of the mouse and guinea-pig

A new approach to determine the transit time through the epidermis is presented, involving a gentle washing of the skin surface to collect the loosely attached surface corneocytes. This, it is believed, will be less likely to stimulate the system than tape-stripping or scraping. Radioactively labelled thymidine and iododeoxyuridine have been used to label cells in the basal layer and various labelled amino acids (glycine, cystine and methionine) have been used to label the metabolically viable cell layers (up to and including the granular layer). The resulting changes in surface radioactivity levels have been interpreted to provide a basal to surface transit time of 8-9.5 days for hairless and haired mouse epidermis and about 13.5 days for guinea-pigs. The basal to granular layer transit time, which probably includes some basal layer residence time, is about 4.5 days in the mouse and 8 days in the guinea-pig. The granular to surface time in mice is about 5 days. The results also suggest that when nuclear and cytoplasmic organelles are degraded in the granular layer, material is released that can diffuse rapidly through the stratum corneum to the surface. Some of this can be shown by chromatography to be thymidine. Hence, the stratum corneum is previous to molecules such as nucleosides. This rapid diffusion outwards through the skin can also be detected shortly after injecting [125I]-iododeoxyuridine.     

Fluorescence spectroscopic studies to characterize the internal environment of tetraethyl-orthosilicate derived sol-gel bulk and thin films with aging

Characterization of the internal environment of a sol-gel matrix is an important area of investigation in optical biosensors. In the present study, different sol-gel compositions were prepared by varying the water (H2O) to tetraethyl-orthosilicate (TEOS) ratio (R) from 1 to 16 and the changes in the internal environment of the sol-gel both in bulk and thin films as a function of aging (storage) were investigated using fluorescence spectroscopy. We focussed on the fluorescence characteristics , viz. emission and excited state lifetime of Hoechst 33258 (H258), a bisbenzimidazole derivative, which was used as fluorescence probe entrapped in the TEOS derived sol-gel bulk and thin films. These sols were prepared at a low pH (approximately 2.0) and the thin films were coated by dip coating technique at withdrawal speeds of 1 cm/min and 0.1cm/min. Usually, uniform thin films were obtained at a high speed (1 cm/min) and partially cracked film at a low speed (0.1 cm/min) as observed by fluorescence microscope. These observations did not change during aging. On the contrary, three months long observations on steady-state fluorescence emission measurements on H258 depicted a blue shift from 535 nm to 508 nm at R = 1 in the sol-gel bulk, whereas at higher ratios this was not prominent. At all ratios, dual emission bands were observed in thin films. This may be due to faster sol-gel to xerogel transition during aging depending on the ratio (R). Analysis of the excited state decay profiles of H258 revealed a double exponential fitting having a short (tau1) and a long (tau2) component in both fresh and during aging, in the sol-gel bulk and thin films, indicating heterogeneity in the internal environment. The value of tau1 increased from 0.4 ns to 1.2 ns whereas tau2 attained a value from 3.0 ns to 3.6 ns at R = 1 upon aging in the sol-gel bulk. The corresponding values of tau1 and tau2 in thin films were 0.3 ns and 3.5 ns, respectively. The values of these decay components in thin films did not alter much due to storage, but their relative contributions showed more systematic changes in the thin films. The observed changes could be correlated to rigidification in the bulk depending on the ratio (R). This process was very slow at R > or = 4. The heterogeneity in the internal environment of bulk and thin films upon aging appeared to be different as revealed from analysis of excited-state lifetime. Thus, the bisbenzimidazole derivative H258 appears to be very useful probe for characterizing the internal environment of both the sol-gel bulk and thin films.     

Using the method of homogenization to calculate the effective diffusivity of the stratum corneum with permeable corneocytes

The stratum corneum is the outermost layer of the skin, which acts as a barrier membrane against the penetration of molecules into and out of the body. It has a biphasic structure consisting of keratinized cells (corneocytes) that are embedded in a lipid matrix. The macroscopic transport properties of the stratum corneum are functions of its microstructure and the transport properties of the corneocytes and the lipid matrix, and are of considerable interest in the context of transdermal drug delivery and quantifying exposure to toxins, as well as for determining the relation of skin disorders to disruption of the stratum corneum barrier. Due to the complexity of the tissue and the difference in length scales involved in its microstructure, a direct analysis of the mass transport properties of the stratum corneum is not feasible. In this study, we undertake an approach where the macroscopic diffusion tensor of the stratum corneum is obtained through homogenization using the method of asymptotic expansions. The biphasic structure of the stratum corneum is fully accounted for by allowing the corneocytes to be permeable and considering the partitioning between the corneocytes and the lipid phases. By systematically exploring the effect of permeable corneocytes on the macroscopic transport properties of the stratum corneum, we show that solute properties such as lipophilicity and relative permeabilities in the two phases have large effects on its transdermal diffusion behavior.     

Marked difference between self-aggregations of first and fourth repeat peptides on tau microtubule-binding domain in acidic solution

The heparin-induced self-aggregation behaviours of four repeat peptides (R1-R4) in an acidic solution (pH = 4.5) were investigated by fluorescence and circular dichroism (CD) measurements and compared with those in a neutral solution (pH = 7.5). In contrast with the self-aggregation-resistive behaviours of the R1 and R4 repeat peptides in the neutral solution, the R4 peptide formed a filament similarly to the R2 and R3 peptides in the acidic solution, whereas the R1 peptide still showed resistive behaviour for filament formation. This is the first report on the markedly different self-aggregation behaviours of the first and fourth repeat peptides on tau microtubule-binding domain.     

Organization of skin stratum corneum extracellular lamellae: diffraction evidence for asymmetric distribution of cholesterol

Lipid suspensions containing 2:1:1 skin ceramides:palmitic acid:cholesterol, similar to the lipid composition found in the extracellular matrix of skin stratum corneum, were analyzed by X-ray diffraction methods. These suspensions gave a sharp wide-angle reflection at 4.1 A, indicating tight hydrocarbon chain packing that would function as a water barrier, and low-angle lamellar diffraction with a repeat period near 130 A, similar to that previously recorded from intact stratum corneum. The lamellar repeat increased from 121 A at pH 6 to 133 A at pH 8.5, allowing phase angles of the lamellar data to be obtained by a sampling theorem "swelling" analysis. Electron density profiles showed that each repeating unit contained two asymmetric bilayers, with a fluid space on one side of the bilayer that increased with increasing pH, due to electrostatic repulsion between bilayers because of ionization of the palmitic acid. Profiles obtained from lamellae with cholesterol sulfate partially substituted for cholesterol showed large density increases on that same side of the bilayer, indicating that cholesterol is asymmetrically distributed in each bilayer. A molecular model was developed postulating that this asymmetry is due to the exclusion of cholesterol from lipid monolayers containing the ester-linked unsaturated (linoleic) hydrocarbon chain of skin ceramide 1. This model can explain the altered organization of extracellular lamellae in epidermal cysts (P. W. Wertz, D. C. Swartzendruber, K. C. Madison, D. T. Downing. 1987. J. Invest. Dermatol. 89:419-425) where the ester-linked chains have a higher percentage of saturated fatty acids than found in normal epidermis.