MATERIALS AND METHODS

Humans

Six healthy subjects (skin types II and III; five Caucasians and one Asian; mean age 29 3 y) volunteered to take part in the study. Prior to experiments, volunteers were questioned to ensure that they did not take any medication and had no history of dermatologic diseases or other current medical problems.

Isolation of human SC proteins

Human SC proteins were obtained by tape stripping of skin with CuDerm D-Squame tape adhesive disks (22 mm diameter, CuDerm Corporation, Dallas, TX) and extraction using a sodium dodecyl sulfate (SDS)/-mercaptoethanol-based lysis solution. The D-Squame disks were chosen because its adhesive and the disks do not contain any traces of proteins (personal communication from Dr. Miller, CuDerm Corporation). Tape stripping was carried out as described (^{Thiele & Packer 1998}). Briefly, each disk was smoothly adhered on to the skin, equally flattened three times, and gently removed using moderate and even traction. After tape stripping, 10 disks were placed side by side in a 90 mm cell culture dish, SC side up, covered with 2 ml lysis buffer (2% SDS/0.5 mM Tris, pH 7.0/10% glycerol/5% -mercaptoethanol), and subjected to gentle agitation for 20 min at room temperature. SC extracts were collected, analyzed for protein content (DC Protein Assay, Bio-Rad, Hercules, CA), and stored at –80°C until used for further analysis.

Sequential tape stripping of forehead skin

Sequential tape stripping was used to collect specific layers of human forehead SC (^{Thiele & Packer 1998}). To avoid contamination by surface lipids, the forehead of each subject was cleaned with ethanol and the first tape stripping was discarded. Next, the forehead was divided into four quadrants and sequential tape stripping was performed in a similar fashion in each quadrant. Tape strippings 2–4 (hereafter referred to as "upper SC"), 9–11 ("intermediate SC"), and 14–16 ("lower SC") were collected from each quadrant and pooled into sterile cell culture plates so that each plate contained 12 strippings/disks. SC proteins were subsequently extracted as described.

Oxidative treatment of isolated human SC proteins in vitro

UVA treatment

Upper arm skin of volunteers (n = 3) was tape-stripped as described. D-Squame tape strippings (n = 10) were exposed for 30 min to UVA irradiation or sham-irradiated. UVA irradiations were performed using a UVA 700 illuminator from Waldmann (Villingen, Germany) at intensities of 42–44 mW per cm² and for 30 min (yielding a dose of approximately 770 kJ per m²).

Hypochlorite

This was obtained as a NaOCl solution from Aldrich (Milwaukee, WI). Hypochlorite was diluted in ddH₂O and incubated with human SC extracts at final concentrations of 25, 2.5, 0.25, 0.025, and 0 mM. Samples were incubated for 20 min at room temperature and subsequently analyzed for protein carbonyl formation as described.

Benzoyl peroxide

Benzoyl peroxide (Sigma, St Louis, MO) was dissolved in ethanol to yield a 50 mM stock solution, which was then further diluted in ddH₂O and incubated with human SC extracts at final concentrations between 0.01 and 1 mM. Benzoyl peroxide and control treated samples were incubated at room temperature for 15 min and subsequently assessed for the formation of protein carbonyls.

Isolation of human keratinocyte proteins

Human HaCaT keratinocytes were cultured in Dulbecco's modified Eagle's medium supplemented with 10% (vol/vol) fetal bovine serum, L-glutamine (2 mM), streptomycin (20 mg per liter), and penicillin (20,000 IU per liter). HaCaT cells were seeded on to 30 mm cell culture dishes. At 80% confluency, they were washed twice with phosphate-buffered saline. Cells were then lyzed directly on the cell culture plates with 1 ml Laemmli sample buffer containing 2% SDS/0.5 mM Tris, pH 7.0/10% glycerol/5% -mercaptoethanol and collected into Eppendorf tubes by gentle scraping with a pipette tip. Samples were then heated at 100°C for 5 min and stored at –20°C until ready for use.

Protein carbonyl detection

As markers of protein oxidation, protein carbonyls were detected as described (^{Levine et al. 1994;Shacter et al. 1994}). Briefly, protein carbonyl groups in SC extracts and keratinocyte lysates were derivatized to 2,4-dinitrophenylhydrazone (DNP-hydrazone) by reaction with 2,4-dinitrophenylhydrazine (DNPH) solution containing 6% SDS/5 mM DNPH/5% trifluoroacetic acid for 15 min at room temperature. Following derivatization, the reaction was immediately neutralized using 2 M Tris base/30% glycerol. The samples were then electrophoresed on a 10% SDS–polyacrylamide gel and transferred on to polyvinylidene difluoride (PVDF) membrane (Amersham, Braunschweig, Germany). Blots were blocked in Tris-buffered saline containing 0.1% Tween and 5% nonfat dried milk and probed with anti-DNP antibody (OxyBlot Oxidized Protein Detection Kit, Oncor, Gaithersburg, MD) diluted 1:300 in Tris buffered saline-Tween (TBST)-milk. As a secondary antibody, anti-rabbit IgG horseradish peroxidase was used at 1:2000 dilution in TBST-milk and the blots were developed using Luminol (New England Biolabs, Beverly, MA). After developing, blots were stained with Coomassie Blue or Colloidal Gold Total Protein Stain (Bio-Rad) to ensure equal loading of proteins.

Identification of proteins using anti-keratin antibodies

Human SC protein extracts (4 g) were electrophoresed on 10% SDS–polyacrylamide gels and transferred to PVDF membrane using a semidry method. Membranes were blocked in Tris-buffered saline (200 mM Tris, 1.37 M NaCl, 0.01% Tween-20) containing 5% nonfat dried milk and probed using a 1:20 dilution of an anti-cytokeratin cocktail antibody CK22 (Biomeda, Foster City, CA), which recognizes human epidermal keratin polypeptides with molecular weights from 40 to 68 kDa. A secondary anti-mouse IgG horseradish peroxidase antibody (ICN Pharmaceuticals, Costa Mesa, CA) was used at 1:4000 dilution in TBST-milk buffer. To identify specific keratins further, monoclonal anti-cytokeratin 1, 10, and 11 antibodies (Progen, Heidelberg, Germany) was employed at 1:10 000 dilution and anti-mouse IgG horseradish peroxidase antibody was used at 1:4000 dilution. Blots were developed by Luminol. For estimation of protein size, a Multimark (Novex, San Diego, CA) and a 10 kDa protein standard (GIBCO, Eggenstein, Germany) were used.

Microsequencing

The same steps were carried out as described above for electrophoresis and immobilization of proteins. After transferring proteins on to a PVDF membrane, blots were briefly stained with Coomassie Blue and air-dried. Once dry, specific bands of interest were carefully excised from the membrane with sterile scalpels and stored at 4°C until sequenced. Sequencing of protein bands was performed by automated Edman degradation.

RESULTS

Isolation of human SC proteins

Protein separation of human SC protein extracts (4 g) by SDS–polyacrylamide gel electrophoresis (PAGE) revealed distinct protein bands in a range from 33 to 188 kDa when visualized by Gold stain (Figure 1a), and a complex of at least three major bands between 50 and 70 kDa when using Coomassie Blue stain (Figure 3a).

Figure 1.

Identification of keratins in human SC extracts. Extracts from human SC ("SC" 4 g protein) and cultured human keratinocytes (HaCaT cells, "K" 4 g protein) were electrophoresed by SDS–PAGE and visualized using a Colloidal Gold Total Protein Stain (a). Immunoblotting with anti-cytokeratin CK22 (b), anti-DNP (c), and monoclonal anti-cytokeratin 1, 10/11 (d) antibodies was carried out as described. (e) same immunoblot as left lane ("SC") in (d), developed with a shorter exposure time.

Full figure and legend (11K)

Figure 3.

Differential distribution of total versus oxidized levels of keratins in layers of human forehead SC. SC extracts from upper ("1"), intermediate ("2") and lower ("3") human forehead SC (4 g protein each) were electrophoresed by SDS–PAGE. Proteins were visualized by Coomassie Blue (a), by immunoblotting with monoclonal anti-cytokeratin 1, 10/11 antibody (b), or by anti-DNP antibodies (c), as described in Materials and Methods.

Full figure and legend (15K)

Identification of human SC proteins

By immunoblotting of human SC proteins using an anti-cytokeratin antibody CK22, we have identified bands with molecular weights of approximately 54, 57, and 65 kDa, as well as a protein with molecular weight in the range of 110–120 kDa (Figure 1b). Human keratinocyte (HaCaT cells) lysates served as positive controls and when probed with anti-cytokeratin antibody CK22, showed signals in the range of 40–50 kDa (Figure 1b). To identify more specifically particular keratins, we employed monoclonal anti-cytokeratin 1, 10, and 11 antibodies and demonstrated that the keratin bands specified above in SC extracts correspond to keratins 1 (65 kDa) and 10 (54 and 57 kDa) (Figure 1e). At longer film exposure times, also the 110–120 kDa band showed a keratin 1, 10/11 positive signal (Figure 1d). As the molecular weights of individual keratins normally range between 40 and 70 kDa, the 110–120 kDa signal could refer to a keratin dimer, consisting, at least in part, of keratins 1, 10 and/or 11.

Identification of human keratin 10 by microsequencing

Proteins of 54 and 57 kDa were microsequenced and both identified as keratin 10, however, each matching best with two different known sequences of human keratin 10. Peptide sequences revealed by Edman protein sequencing were subjected to computer assisted database search (SwissProt). Keratin 10, homo sapiens, sequence from^{Zhou et al. (1988)}: 561 amino acids, 57,247 MW (calculated weight); matching sequence of the 54 kDa SC protein. The numbers given in front of and after the sequences represent the position of the amino acid in the respective keratin 10 sequences: (79) RGSYGSSSFGGSYGGSFGGG (98). Keratin 10, homo sapiens, sequence from^{Rieger & Franke (1988)}: 593 amino acids, 59,519 MW (calculated weight). Matching sequence of the 57 kDa SC protein: (37) SSSKGSLGGGFSSG (50).

Whereas the identification of human keratin 10 by microsequencing confirms the immunoreactivity of the respective bands, it remains unclear whether the two bands represent similar keratins or different fragments of the same protein. Remaining activities of proteases during the process of SC extraction may have led to the formation of two distinct protein bands.

Oxidants induce increase of keratin oxidation

Treatment of human SC proteins with UVA led to increased formation of protein carbonyls, in keratin 1 (65 kDa) and keratin 10 (54 and 57 kDa) (Figure 2a). Additionally, UVA treatment consistently induced an increased protein carbonyl signal in another band of 170–180 kDa. Treatment with hypochlorite (Figure 2b) or benzoyl peroxide (Figure 2c) led to a small increase in the carbonyl group immunoblot signal of keratins 1 (65 kDa) and 10 (54/57 kDa); and a more pronounced rise in immunoblot signal intensity in the 110–120 kDa keratin band.

Figure 2.

Treatment of human SC extracts with UVA, hypochlorite, and benzoyl peroxide increases levels of protein oxidation. (a) SC tape strippings were exposed (SC face up) for 30 min to UVA at intensities of 42–44 mW per cm² ("UVA") or sham irradiated ("contr."). (b, c) Equal amounts of human SC extracts (2.5 g protein) were treated with (b) either 250 M ("HOCl") or 0 M ("contr.") hypochlorite and (c) either 800 M ("BPO") or 0 M ("contr.") of benzoyl peroxide. Proteins were then separated by SDS–PAGE and immunoblotted using an anti-DNP antibody for detection of protein carbonyl. Results shown are representative of at least three independent experiments.

Full figure and legend (14K)

Evidence for an epidermal protein oxidation gradient

SC extracts contained many-fold higher levels of protein carbonyls than equivalent amounts of keratinocyte proteins (each 4 g; Figure 1c). To investigate further epidermal protein oxidation, we looked at different layers within the SC. In forehead SC of six volunteers, we found a remarkable gradient of keratin 10 (bands at 54 and 57 kDa) oxidation, with low levels of carbonyl groups in the lower SC, and dramatically increasing levels towards the outer layers (Figure 3c). This gradient was consistently detected in all six subjects (Figure 4a, b). In the case of the 54 and 57 kDa bands (keratin 10), however, both the protein stain (Figure 3a) and the keratin 1/10/11 immunostain (Figure 3b) indicated that the protein levels were comparatively constant in the three different SC layers, suggesting that the relative amount of carbonyl groups in keratin 10, and thus its level of oxidation, increases towards outer layers. Furthermore, the 110–120 kDa keratin containing protein was more pronounced in superficial SC layers (Figure 3b) and paralleled the carbonyl levels in the respective layers (Figure 4c).

Figure 4.

Oxidation gradients of keratins in untreated human forehead SC. SC proteins were extracted from sequential tape strippings of forehead skin from human volunteers (n = 6) and separated by SDS–PAGE. Protein bands were identified by using keratin antibodies and by Edman sequencing. Levels of protein oxidation were determined by immunoblotting and quantitated by densitometry analysis. Highest levels in the upper SC were assigned to 100%; values in other layers represent levels relative to those in the upper SC in percentage. Data are means (n = 4–6) and standard deviations. "upper SC": tape strippings 1–3; "med. SC": tape strippings 8–10; "lower SC": tape strippings 13–15. (A) 54 kDa band; human keratin 10 (microsequenced), (B) 57 kDa band; keratin 10 (microsequenced), (C) 110–120 kDa band recognized by both keratin mix and keratin 1, 10, and 11 antibodies as shown in Figure 1(b, d).

Full figure and legend (8K)

DISCUSSION

By analyzing the levels of protein carbonyls in human SC keratins using an immunoblotting technique, we provide direct evidence that: (i) human SC keratins are susceptible to carbonyl formation by model oxidants and UVA radiation; (ii) one of the oxidizable keratins was identified as keratin 10; and (iii) keratin 10 oxidation increases dramatically from the inner to the outer SC in healthy forehead skin.

Previously, we have demonstrated that vitamin E is the major lipophilic anti-oxidant of the human SC, and that it is highly susceptible to depletion by ozone (^{Thiele et al. 1997b}), as well as by UVB and UVA irradiation (^{Thiele et al. 1998a}). In this study, incubation of SC proteins with the chemical oxidants hypochlorite and benzoyl peroxide, as well as UVA treatment induced a significant increase in carbonyl levels of SC keratins (Figure 2), one of which was identified as keratin 10 by immunoblotting (Figure 1e) and microsequencing of corresponding SDS–PAGE protein bands. Another SC protein, recognized by immunoblotting using a monoclonal antibody against keratins 1, 10 and 11, 110–120 kDa, was shown to be susceptible to carbonyl formation upon the same oxidant challenges. In analogy to the high degree of saturation of fatty acids in the SC (^{Wertz & Downing 1991}), the amount of disulfide cross-links in human SC is known to be many-fold higher than in lower epidermal layers (^{Broekaert et al. 1982}). Similarly, we found that keratins in human SC contain dramatically more carbonyl groups than the keratins present in keratinocytes (Figure 1c), indicating that the baseline levels of keratin oxidation are considerably higher in the SC as compared with lower epidermal layers. By using sequential tape strippings, a steep gradient with lowest levels of carbonyl groups in keratins from lower layers and highest in the upper layers was found (Figure 3c and Figure 4). Importantly, this protein oxidation gradient is inversely correlated with the gradients of the anti-oxidant vitamin E (^{Thiele et al. 1998a}), and free thiols (^{Broekaert et al. 1982}) in human SC. There is in vitro and in vivo evidence from other biologic systems that protein oxidation can be counteracted by anti-oxidants such as vitamin E (^{Ibrahim et al. 1997}) and thiols (^{Yan et al. 1996}). Furthermore, the oxygen partial pressure (pO₂), a rate-limiting factor for the formation of reactive oxygen intermediates in skin (^{Fuchs & Thiele 1998}), decreases gradually from outer to inner SC layers (^{Grossmann & Luebbers 1981;Hatcher & Plachy 1993}). Besides oxygen, the percutaneous penetration of most molecules, among them noxious, oxidizing xenobiotics, leads to a gradient within the SC with highest concentrations in the outer layers (^{Rougier et al. 1990}). The inverse correlation with SC anti-oxidant levels on the one hand and the positive correlation with the levels of oxygen and oxidizing xenobiotics within the SC on the other, may account for the protein oxidation gradient in SC keratins.

We propose that the protein oxidation gradient with increased levels towards outer SC layers may have important implications for the process of desquamation. Because proteins in corneodesmosomes play a crucial part in SC cell cohesion, and specific proteases have been identified in human SC (^{Egelrud 1993;Brysk et al. 1994;Suzuki et al. 1996}), proteolysis is generally believed to be a key event in desquamation. As the SC consists of enucleated, "dead" cells, however, it is still unclear, how the onset of desquamation in the upper SC is regulated (^{Egelrud et al. 1996}). Many common proteases degrade oxidized proteins more rapidly than unoxidized forms (^{Davies et al. 1987;Stadtman 1992}). Thus, in addition to regulation by other factors, the higher levels of protein oxidation detected in the upper SC may account for an increased susceptibility of keratins and other macromolecules to be degraded by SC proteases, leading to desquamation in the superficial SC layers. Oxidative protein damage not only targets enzyme substrates, but can also affect activities of enzymes (^{Davies et al. 1987}), which may be as well targets of oxidative stress in the SC. Trypsin-like SC proteases, such as desquamin, however, appear to be extremely resistant to chemical and thermal degradation (^{Brysk et al. 1994}), and hence, might be also resistant to the increased oxidative challenges in the upper SC.

Whereas protein oxidation increases proteolytic susceptibility up to a protein-specific degree, further damage actually causes a decrease in proteolytic susceptibility and leads to cross-linking and aggregation (^{Grune et al. 1997;Sommerburg et al. 1997}). Furthermore, protein-bound carbonyl groups are believed to be involved in intra- and intermolecular cross-linking (^{Smith et al. 1996}). Although it is well accepted that cross-linking of SC keratins serves to improve the physical stability of the keratin network, very little is known about the biochemical details (^{Pang et al. 1993}). The introduction of carbonyl groups into SC keratins is likely to have implications for keratin cross-linking, because protein cross-linking and aggregation is not limited to disulfide cross-linking, but also other forms of covalent cross-links such as the formation of an intermolecular Schiff base by reaction of a carbonyl group from one protein with an amino group from another. Possibly, transglutaminase, which catalyzes the formation of an amide bond between the -carbonyl group of glutamine and the -amino group of lysine and plays an important part in the formation of the cornified envelope (^{Greenberg et al. 1991}), is involved in this dimerization. Notably, eye lens proteins were shown to be far more susceptible to transglutaminase catalyzed reactions when preincubated with reactive oxygen species (^{Brossa et al. 1990}). Finally, an additional functional aspect that should be considered is that SC proteins, while being oxidized, may act as macromolecular anti-oxidants, preventing oxidative damage in subjacent epidermal layers.

In esophageal epithelium, the presence of disulfide-stabilized keratin dimers was demonstrated to occur with epithelial differentiation and to be most prominent in the most superficial epidermal layers; these proteins could only be visualized under nonreducing SDS–PAGE condition (^{Pang et al. 1993}). Similarly, we observed an approx. 110–120 kDa protein in human forehead SC with increasing levels towards the outer SC (Figure 3b). Its reactivity with a keratin 1, 10 and 11 antibody and its high molecular weight, exceeding the molecular weights of known epidermal keratins, suggest that it might be a keratin dimer. As this 110–120 kDa band did not disappear under reducing conditions in the SDS–PAGE, however it was concluded that non-disulfide-cross-links, such as Schiff bases could be involved in the keratin cross-linking.

In conclusion, our findings demonstrate that the introduction of carbonyl groups into human SC keratins is inducible by oxidants, and that the levels of protein oxidation increase towards outer SC layers. These findings may contribute to a better understanding of the complex biochemical processes of keratinization and desquamation.

References

1. Berlett, BS, Stadtman, ER: Protein oxidation in aging, disease, and oxidative stress. J Biol Chem 1997 272:20313–20316,  | Article | PubMed | ISI | ChemPort |
2. Broekaert, D, Cooreman, K, Coucke, P, Nsabumukunzi, S, Reyniers, P, Kluyskens, P, Gillis, E: A quantitative histochemical study of sulphydryl and disulphide content during normal epidermal keratinization. Histochem J 1982 14:573–584,  | Article | PubMed | ISI | ChemPort |
3. Brossa, O, Seccia, M, Gravela, E: Increased susceptibility to transglutaminase of eye lens proteins exposed to activated oxygen species produced in the glucose-glucose oxidase reaction. Free Radic Res Commun 1990 11:223–229,  | PubMed | ISI | ChemPort |
4. Brysk, MM, Bell, T, Brysk, H, Selvanayagam, P, Rajaraman, S: Enzymic activity of desquamin. Exp Cell Res 1994 214:22–26,  | Article | PubMed | ISI | ChemPort |
5. Davies, KJA: Protein damage and degradation by oxygen radicals: I. General Aspects. J Biol Chem 1987 262:9895–9901,  | PubMed | ISI | ChemPort |
6. Davies, KJA, Lin, SW, Pacifici, RE: Protein damage and degradation by oxygen radicals: Degradation of denatured protein. J Biol Chem 1987 262:9914–9920,  | PubMed | ISI | ChemPort |
7. Dean, RT, Fu, S, Stocker, R, Davies, MJ: Biochemistry and pathology of radical-mediated protein oxidation. Biochem J 1997 324:1–18,  | PubMed | ISI | ChemPort |
8. Egelrud, T: Purification and preliminary characterization of stratum corneum chymotryptic enzyme: a proteinase that may be involved in desquamation. J Invest Dermatol 1993 101:200–204,  | Article | PubMed | ISI | ChemPort |
9. Egelrud, T, Lundstroem, A, Sondell, B: Stratum corneum cell cohesion and desquamation in maintenance of the skin barrier. In: Marzulli, FN Maibach, HI (eds). Dermatotoxicology 1996 Washington: Taylor & Francis, pp 19–27,
10. Elias, PM: Stratum corneum architecture, metabolic activity and interactivity with subjacent cell layers. Exp Dermatol 1996 5:191–201,  | PubMed | ChemPort |
11. Esterbauer, H, Schaur, RJ, Zollner, H: Chemistry and biochemistry of 4-hydroxynonenal, malonaldehyde and related aldehydes. Free Radic Biol Med 1991 11:81–128,  | Article | PubMed | ISI | ChemPort |
12. Fuchs, J: Oxidative Injury in Dermatopathology 1992 Berlin: Springer-Verlag,
13. Fuchs, J, Thiele, J: The role of oxygen in cutaneous photodynamic therapy. Free Radic Biol Med 1998 24:835–847,  | Article | PubMed | ISI | ChemPort |
14. Greenberg, CS, Birckbichler, PJ, Rice, RH: Transglutaminases: multifunctional cross-linking enzymes that stabilize tissues. FASEB J 1991 5:3071–3077,  | PubMed | ISI | ChemPort |
15. Grossmann, U, Luebbers, DW: Analysis of epidermal oxygen supply by simulation of oxygen partial pressure fields under varying conditions. Crit Care Med 1981 9:734–735,  | PubMed | ISI | ChemPort |
16. Grune, T, Reinheckel, T, Davies, KJ: Degradation of oxidized proteins in mammalian cells. FASEB J 1997 11:526–534,  | PubMed | ISI | ChemPort |
17. Hatcher, ME, Plachy, WZ: Dioxygen diffusion in the stratum corneum an EPR spin label study. Biochim Biophys Acta 1993 1149:73–78,  | PubMed | ISI | ChemPort |
18. Ibrahim, W, Lee, U, Yeh, C, Szabo, J, Bruckner, G, Chow, C: Oxidative stress and antioxidant status in mouse liver: effects of dietary lipid, vitamin E and iron. J Nutr 1997 127:1401–1406,  | PubMed | ISI | ChemPort |
19. Levine, RL, Williams, JA, Stadtman, EA, Shacter, E: Carbonyl assays for determination of oxidatively modified proteins. Methods Enzymol 1994 233:346–357,  | PubMed | ISI | ChemPort |
20. Oliver, CN, Ahn, B-W, Moerman, EJ, Goldstein, S, Stadtman, ER: Age-related changes in oxidized proteins. J Biol Chem 1987 262:5488,  | PubMed | ISI | ChemPort |
21. Pang, Y-YS, Schermer, A, Yu, J, Sun, T-T: Suprabasal change and subsequent formation of disulfide-stabilized homo- and heterodimers of keratins during esophageal epithelial differentiation. J Cell Sci 1993 104:727–740,  | PubMed | ISI |
22. Rieger, M, Franke, WW: Identification of an orthologous mammalian cytokeratin gene. High degree of intron sequence conservation during evolution of human cytokeratin 10. J Mol Biol 1988 204:841–856,  | Article | PubMed | ISI | ChemPort |
23. Rougier, A, Rallis, M, Krien, P, Lotte, C: In vivo percutaneous absorption: a key role for stratum corneum/vehicle partitioning. Arch Dermatol Res 1990 282:498–505,  | Article | PubMed | ISI | ChemPort |
24. Scharffetter-Kochanek, K: Photoaging of the connective tissue of skin: Its prevention and therapy. Adv Pharmacol 1997 38:639–655,  | PubMed | ChemPort |
25. Shacter, E, Williams, JA, Lim, M, Levine, RL: Differential susceptibility of plasma proteins to oxidative modification: examination by western blot immunoassay. Free Radic Biol Med 1994 17:429–437,  | Article | PubMed | ISI | ChemPort |
26. Smith, MA, Perry, G, Richey, PL, Sayre, LM, Anderson, VE, Beal, MF, Kowall, N: Oxidative damage in Alzheimer's. Nature 1996 382:120–121,  | Article | PubMed | ISI | ChemPort |
27. Sommerburg, O, Ullrich, O, von Sitte, N, Zglinicki, D, Siems, W, Grune, T: Dose- and wavelength-dependent oxidation of crystallins by uv light-selective recognition and degradation by the 20s proteasome. Free Radic Biol Med 1997 24:1369–1374,  | ISI |
28. Stadtman, ER: Protein oxidation and aging. Science 1992 257:1220–1224,  | PubMed | ISI | ChemPort |
29. Suzuki, Y, Koyama, J, Moro, O, Horii, I, Kikuchi, K, Tanida, M, Tagami, H: The role of two endogenous proteases of the stratum corneum in degradation of desmoglein-1 and their reduced activity in the skin of ichthyotic patients. Br J Dermatol 1996 134:460–464,  | Article | PubMed | ISI | ChemPort |
30. Thiele, JJ, Packer, L: Non-Invasive Measurement of -tocopherol gradients in human stratum corneum by HPLC analysis of sequential tape strippings. Methods Enzymol 1998 300:413–419,  | ISI |
31. Thiele, JJ, Podda, M, Packer, L: Tropospheric ozone: an emerging environmental stress to skin. Biol Chem 1997a 378:1299–1305,  | ISI | ChemPort |
32. Thiele, JJ, Traber, MG, Polefka, TG, Cross, CE, Packer, LP: Ozone exposure depletes vitamin E and induces lipid peroxidation in murine stratum corneum. J Invest Dermatol 1997b 108:753–757,  | Article | PubMed | ISI | ChemPort |
33. Thiele, JJ, Traber, MG, Tsang, KG, Cross, CE, Packer, L: In vivo exposure to ozone depletes vitamins C and E and induces lipid peroxidation in epidermal layers of murine skin. Free Radic Biol Med 1997c 23:385–391,  | Article | ISI | ChemPort |
34. Thiele, JJ, Traber, MG, Packer, L: Depletion of human stratum corneum vitamin E. An early and sensitive in vivo marker of UV-induced photooxidation. J Invest Dermatol 1998a 110:756–761,  | Article | PubMed | ISI | ChemPort |
35. Thiele, JJ, Traber, MG, Re, R, Espuno, N, Yan, L-J, Cross, CE, Packer, L: Macromolecular carbonyls in human stratum corneum: a biomarker for environmental oxidant exposure? FEBS Lett 1998b 422:403–406,  | Article | ISI | ChemPort |
36. Wertz, PW, Downing, DT: Epidermal lipids. In: Goldsmith, LA (eds). Physiology, Biochemistry, and Molecular Biology of the Skin 1991 New York: Oxford University Press, pp 205–236,
37. Yan, LJ, Traber, M, Kobuchi, H, Matsugo, S, Tritschler, H, Packer, L: Efficacy of hypochlorous acid scavengers in the prevention of protein carbonyl formation. Arch Biochem Biophys 1996 327:330–334,  | Article | PubMed | ISI | ChemPort |
38. Zhou, XM, Idler, WW, Steven, AC, Roop, DR, Steinert, PM: The complete sequence of the human intermediate filament chain keratin 10. Subdomainal divisions and model for folding of end domain sequences. J Biol Chem 1988 263:15584–15589,  | PubMed | ISI | ChemPort |

Acknowledgments

We thank Dr. Michael Kiess (GBF, Braunschweig, Germany) for protein microsequencing and helpful discussions. This study was supported by the Deutsche Forschungsgemeinschaft, SFB 503, Project B1.