Recently, we synthesized a new fluorescent thiol reagent, N-(7-dimethylamino-4-methylcoumarinyl)maleimide (DACM) which is nonfluorescent by itself but will react readily with-SH groups to form highly fluorescent addition products. By the use of this reagent, we studied the localization and concentration of-SH groups and S-S linkages in the human epidermis. The distribution of-SH groups in living layers was abundant in cytoplasm but not in nuclei. The fluorescence was concentrated on the cell membrane or intercellular spaces (MIC parts) and was increased at the spino-granular junction. In the horny layer, the fluorescence of the MIC parts appeared brilliantly in the lower layers and decreased gradually. On the other hand, the fluorescence of cytoplasm in keratinized cells in the stratum corneum was faint. The localization of S-S linkages was not a characteristic of the living layers, but appeared abruptly at the junction of living and horny layers. The fluorescence was localized to the MIC parts and disappeared
A new fluorogenic maleimide DACM, i.e., N(7-dimethyl amino-4-methyl coumarinyl) maleimide, does not fluoresce by itself. It is specifically combined with --SH groups and becomes fluorescent (lambda ex: 400 nm, lambda em: 485 nm). At this range of excitation the frozen skin section has no native fluorescence because the emission maximum of DACM does not overlap with any of aromatic residues of proteins such as tryptophane. S--S groups can be demonstrated with DACM by inhibiting native --SH and then reducing SS to --SH. --Sh was generally abundant at the bulb region. --SH of hair cortex was more concentrated at keratogenous zone. Further up in the follicle, --SH of hair cortex was gradually decreased, although at the region of isthmus, --SH reaction of the hair cortex was still moderately strong. Outer root sheath had --SH from the upper bulb to the surface of the epidermis. One the other hand, no S--S was demonstrable at the bulb region. At the level of keratogenous zone, however, S--S linkages of hair cortex and inner root sheath began to appear and further up in the follicle S--S linkages were increased gradually. Outer root sheath had no S--S linkages by DACM staining up until it is keratinized along the hair canal in the upper follicle. The concentration of --SH and S--S thus seemed to be reciprocal; --SH is found in non-keratinized tissues, whereas S--S is abundant in keratinized areas. These findings are at some variance with conventional data, which suggest, for example, that --SH groups disappear suddenly above the keratogenous zone.
New factors controlling the reaction of pemphigus antibody and the pemphigus antigens were defined by studying the action of antibody on section prepared from human skin. The pemphigus antigenicity was protected by adding dithiothreitol and CaCl2 in buffer. Blocking studies of the -SH groups by sulfhydryl reagents rapidly decreased the reactivity of antigens. In addition pretreatment calcium chelating agents also decreased this reactivity.
The enzyme activities of normal-looking skin and blister fluid from a patient with recessive dystrophic epidermolysis bullosa (RDEB) were measured. Of the hydrolytic enzymes measured, both collagenase and neutral protease activities were considerably increased in the skin and blister fluid samples compared with values found in normal control skin and in blister fluid from a patient with a burn. In addition, skin from a healthy person cultured with RDEB blister fluid showed dermal-epidermal separation. These findings suggest that collagenase and neutral protease may be involved in the formation of blisters in RDEB.
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