Shigeru Kusuda, Cui Chang-Yi, Masae Takahashi, Tadashi Tezuka

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Presentation transcript:

Localization of Sphingomyelinase in Lesional Skin of Atopic Dermatitis Patients Shigeru Kusuda, Cui Chang-Yi, Masae Takahashi, Tadashi Tezuka Journal of Investigative Dermatology Volume 111, Issue 5, Pages 733-738 (November 1998) DOI: 10.1046/j.1523-1747.1998.00370.x Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Anti-SM Ab cross-reacts with a single 58 kDa epidermal protein and a 70 kDa human placental SMase. (A) The extracts of normal epidermis obtained from suction blisters were separated by SDS-PAGE, transferred onto Immobilon, and reacted with preimmune serum (lane 1) or with anti-SM Ab (lane 2). The proteins extracted from the epidermis were stained with Coomassie brilliant blue R (lane 3). Lane 4 shows the following molecular weight markers (from the top): bovine serum albumin (67 kDa), ovalbumin (43 kDa), carbonic anhydrase (30 kDa), soybean trypsin inhibitor (20.1 kDa), and lactalbumin (14.4 kDa). (B) The commercially available human placental SMase was separated by SDS and transferred onto Immobilon and reacted with preimmune serum (lane 1) or with anti-SM Ab (lane 2), and stained with Coomassie brilliant blue R (lane 3). Lane 4 shows the molecular weight markers. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 A single spot reacts with anti-SM Ab in two-dimensional immunoblotting analysis of epidermal extracts. (a) Immunoblot; (b) Coomassie blue stains. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 A 58 kDa epidermal protein-rich fraction is obtained by immunoprecipitation. Thirty micrograms of SF was applied to lane 1, and the supernatants after immunoprecipitation of 30 μg of SF and 15 μl of either anti-SM Ab or preimmune serum were applied to lanes 2 and 3, respectively. To lanes 4 and 5, 5.2 and 3.0 μg of solubilized immunoprecipitates were applied, respectively. (A) Coomassie brilliant blue R staining, (B) immunoblotting. In (A) and (B), the materials analyzed on the gels were as follows; lane 1, SF; lane 2, SF, anti-SM Ab, and PAS; lane 3, SF, preimmune serum, and PAS; lane 4, acetic acid-soluble fraction from the pellet after incubation of SF, anti-SM Ab, and PAS; lane 5, acetic acid-soluble fraction from the pellet after incubation of SF, preimmune serum, and PAS. The 58 kDa protein was partially purified, as shown in part (B), lane 4, indicated by an arrow. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 The 58 kDa protein was detected immunohistochemically in the stratum granulosum cells. Frozen sections of normal human epidermis (A and C) or a chronic dermatitis lesion (lichenification) of an AD patient (B) were reacted with either anti-SM Ab, diluted 1:2000 (A and B), or preimmune rabbit serum, diluted 1:2000 (C), and visualized by the labeled streptavidin biotin method. In (A), there were three to four lines of dots in the stratum granulosum and the innermost cell layers of the stratum corneum in normal epidermis. In contrast, in (B), there were many dots at the periphery of the cells in the upper part of the stratum spinosum, stratum granulosum, and stratum corneum, and weakly reacted product was seen in the cytoplasm of the lower part of stratum spinosum cells. In (C), there was no positive reaction. b, basal cell layer; s, stratum spinosum; g, stratum granulosum; c, stratum corneum. Arrowheads indicate positive reaction products. Scale bars: 40 μm. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Gold particles in the intercellular domains by immunoelectron-microscopy. Gold particles were seen in the small vesicles in the stratum granulosum cells and in the intercellular domain of the innermost three cell layers of the stratum corneum. Scale bar: 1 μm. Insertion: higher magnification; gold particles were seen in the small vesicles, some of which were fused with the cell membrane. The arrows indicate the boundary of the cell, the arrowheads indicate the membrane of the small vesicles in the stratum granulosum, and the open arrowheads indicate the intercellular materials. G, keratohyalin granules; C, stratum corneum. Scale bar: 1 μm. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 6 Gold particles in the spinous cells and in the stratum corneum cells of AD patients. Gold particles were seen in the intercellular domain of cornified cells from the innermost to the middle part of the stratum corneum cell layers (A), and also in the cytoplasm and the intercellular domain of the spinous cells (B) in the lesional skin of AD patients. Scale bar: 1 μm. Arrows indicate desmosomes and arrowheads indicate melanosomes. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 7 The amount of the 58 kDa protein in the lesional skin of AD was slightly increased. The nonlesional skin of the inner surface of the upper arm of AD patients, normal skins, and the lesional skins of various parts of the body of AD patients were punch-biopsied. The total amount of the 58 kDa protein was slightly increased in the lesional skin of AD patients. The difference is significant (p < 0.05, nonpaired student’s t test). 1, the normal control (n = 10); 2, the nonlesional skins of AD patients (n = 10); 3, the lesional skins of AD patients (n = 7, three foreheads, one cheek, two necks, and one breast). Error bars: SE. Journal of Investigative Dermatology 1998 111, 733-738DOI: (10.1046/j.1523-1747.1998.00370.x) Copyright © 1998 The Society for Investigative Dermatology, Inc Terms and Conditions