SummaryBackground Melasma is a common acquired symmetrical hypermelanosis characterized by irregular light to dark brown macules and patches on sun-exposed areas of the skin. Its histopathological characteristics are not fully understood. Objectives To characterize the histopathological features of facial melasma skin in comparison with adjacent normal skin. Methods Biopsies were taken from both melasma lesional skin and adjacent perilesional normal skin in 56 Korean women with melasma. The sections were stained using haematoxylin and eosin, Fontana±Masson, diastase-resistant periodic acid-Schiff, Masson trichrome and Verhoeff±van Gieson stains, and immunostaining for melanocytes. Data on the changes in number of melanocytes and melanin contents of the epidermis were analysed by a computer-assisted image analysis program. The ultrastructure of the skin was also examined. Results The amount of melanin was signi®cantly increased in all epidermal layers in melasma skin. The staining intensity and number of epidermal melanocytes increased in melasma lesions. Lesional skin showed more prominent solar elastosis compared with normal skin. Melanosomes increased in number and were more widely dispersed in the keratinocytes of the lesional skin. Lesional melanocytes had many more mitochondria, Golgi apparatus, rough endoplasmic reticulum and ribosomes in their cytoplasm. A dihydroxyphenylalanine reaction was apparent in the cisternae and vesicles of the trans-Golgi network in melanocytes from lesional skin. Conclusions Melasma is characterized by epidermal hyperpigmentation, possibly caused both by an increased number of melanocytes and by an increased activity of melanogenic enzymes overlying dermal changes caused by solar radiation.

The temperature-dependent conformational change of poly(N-isopropylacrylamide) (PNIPAM) brushes in D 2 O was investigated as a function of the molecular weight (M) at a high grafting density with neutron reflection. PNIPAM chains with three different M values were grafted at the same high surface density from a gold surface by atom transfer radical polymerization. A significant change in the segment concentration profile was observed for all three samples as the temperature passed through the lower critical solution temperature (ϳ30°C), in contrast to previous results obtained for samples with much lower surface density. Somewhat surprisingly, the fractional change in the first moment of the segment concentration profile (͗z͘) from 20 to 41°C was weaker with increasing M. This is contrary to the trend for systems involving only van der Waals (VDW) interactions, in which higher M chains experience larger conformational changes with change in solvent quality. Indeed, the M dependence of the first moment of the segment concentration profile for the grafted PNIPAM chains at 20°C was much weaker than has been reported previously for dense brushes involving only VDW interactions under good solvent conditions. At 20°C, the form of the segment concentration profile varied systematically with M. A single-layer profile resulted for the highest M, but the profiles became more bilayer in character with decreasing M. At 41°C, the profiles for all three samples were adequately described by a single dense layer with a smooth transition region to bulk D 2 O. The weak dependence of ͗z͘ on M at 20°C and the trend from a bilayer profile at lower M to a single-layer profile at higher M appear to be related. These results are interpreted in terms of concentration-dependent segment-segment interactions that result in a weak attraction for high segment densities at 20°C.

The structure and orientation of adsorbed myoglobin as directed by metal-histidine complexation at the liquid-film interface was studied as a function of time using neutron and X-ray reflectivity (NR and XR, respectively). In this system, adsorption is due to the interaction between iminodiacetate (IDA)-chelated divalent metal ions Ni(II) and Cu(II) and histidine moieties at the outer surface of the protein. Adsorption was examined under conditions of constant area per lipid molecule at an initial pressure of 40 mN/m. Adsorption occurred over a time period of about 15 h, allowing detailed characterization of the layer structure throughout the process. The layer thickness and the in-plane averaged segment volume fraction were obtained at roughly 40 min intervals by NR. The binding constant of histidine with Cu(II)-IDA is known to be about four times greater than that of histidine with Ni(II)-IDA. The difference in interaction energy led to significant differences in the structure of the adsorbed layer. For Cu(II)-IDA, the thickness of the adsorbed layer at low protein coverage was < or = 20 A and the thickness increased almost linearly with increasing coverage to 42 A. For Ni(II)-IDA, the thickness at low coverage was approximately 38 A and increased gradually with coverage to 47 A. The in-plane averaged segment volume fraction of the adsorbed layer independently confirmed a thinner layer at low coverage for Cu(II)-IDA. These structural differences at the early stages are discussed in terms of either different preferred orientations for isolated chains in the two cases or more extensive conformational changes upon adsorption in the case of Cu(II)-IDA. Subphase dilution experiments provided additional insight, indicating that the adsorbed layer was not in equilibrium with the bulk solution even at low coverages for both IDA-chelated metal ions. We conclude that the weight of the evidence favors the interpretation based on more extensive conformational changes upon adsorption to Cu(II)-IDA.

The adsorption of myoglobin to Langmuir monolayers of a metal-chelating lipid in crystalline phase was studied using neutron and X-ray reflectivity (NR and XR) and grazing incidence X-ray diffraction (GIXD). In this system, adsorption is due to the interaction between chelated divalent copper or nickel ions and the histidine moieties at the outer surface of the protein. The binding interaction of histidine with the Ni-IDA complex is known to be much weaker than that with Cu-IDA. Adsorption was examined under conditions of constant surface area with an initial pressure of 40 mN/m. After approximately 12 h little further change in reflectivity was detected, although the surface pressure continued to slowly increase. For chelated Cu2+ ions, the adsorbed layer structure in the final state was examined for bulk myoglobin concentrations of 0.10 and 10 microM. For the case of 10 microM, the final layer thickness was approximately 43 A. This corresponds well to the two thicker dimensions of myoglobin in the native state (44 A x 44 A x 25 A) and so is consistent with an end-on orientation for this disk-shaped protein at high packing density. However, the final average volume fraction of amino acid segments in the layer was 0.55, which is substantially greater than the value of 0.44 calculated for a completed monolayer from the crystal structure. This suggests an alternative interpretation based on denaturation. GIXD was used to follow the effect of protein binding on the crystalline packing of the lipids and to check for crystallinity within the layer of adsorbed myoglobin. Despite the strong adsorption of myoglobin, very little change was observed in the structure of the DSIDA film. There was no direct evidence in the XR or GIXD for peptide insertion into the lipid tail region. Also, no evidence for in-plane crystallinity within the adsorbed layer of myoglobin was observed. For 0.1 microM bulk myoglobin concentration, the average segment volume fraction was only 0.13 and the layer thickness was < or = 25 A. Adsorption of myoglobin to DSIDA-loaded with Ni2+ was examined at bulk concentrations of 10 and 50 microM. At 10 microM myoglobin, the adsorbed amount was comparable to that obtained for adsorption to Cu2+-loaded DSIDA monolayers at 0.1 M. But interestingly, the adsorbed layer thickness was 38 A, substantially greater than that obtained at low coverage with Cu-IDA. This indicates that either there are different preferred orientations for isolated myoglobin molecules adsorbed to Cu-IDA and Ni-IDA monolayer films or else myoglobin denatures to a different extent in the two cases. Either interpretation can be explained by the very different binding energies for individual interactions in the two cases. At 50 microM myoglobin, the thickness and segement volume fraction in the adsorbed layer for Ni-IDA were comparable to the values obtained with Cu-IDA at 10 microM myoglobin.

Silane adhesion promoters are commonly used to improve the adhesion, durability, and corrosion resistance of polymer-oxide interfaces. The current study investigates a model interface consisting of the natural oxide of 〈100〉 Si and an epoxy cured from diglycidyl ether of bisphenol A (DGEBA) and triethylenetetraamine (TETA). The thickness of (3-glycidoxypropyl)trimethoxysilane (GPS) films placed between the two materials provided the structural variable. Five surface treatments were investigated: a bare interface, a rough monolayer film, a smooth monolayer film, a 5 nm thick film, and a 10 nm thick film. Previous neutron reflection experiments revealed large extension ratios (>2) when the 5 and 10 nm thick GPS films were exposed to deuterated nitrobenzene vapor. Despite the larger extension ratio for the 5 nm thick film, the epoxy/Si fracture energy (Gc) was equal to that of the 10 nm thick film under ambient conditions. Even the smooth monolayer exhibited the same Gc. Only when the monolayer included a significant number of agglomerates did the Gc drop to levels closer to that of the bare interface. When immersed in water at room temperature for 1 week, the threshold energy release rate (Gth) was nearly equal to Gc for the smooth monolayer, 5 nm thick film, and 10 nm thick film. While the Gth for all three films decreased with increasing water temperature, the Gth of the smooth monolayer decreased more rapidly. The bare interface was similarly sensitive to temperature; however, the Gth of the rough monolayer did not change significantly as the temperature was raised. Despite the influence of pH on hydrolysis, the Gth was insensitive to the pH of the water for all surface treatments.