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UVA-Induced Modification of Catalase Charge Properties in the Epidermis Is Correlated with the Skin Phototype  Vittoria Maresca, Enrica Flori, Stefania.

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Presentation on theme: "UVA-Induced Modification of Catalase Charge Properties in the Epidermis Is Correlated with the Skin Phototype  Vittoria Maresca, Enrica Flori, Stefania."— Presentation transcript:

1 UVA-Induced Modification of Catalase Charge Properties in the Epidermis Is Correlated with the Skin Phototype  Vittoria Maresca, Enrica Flori, Stefania Briganti, Emanuela Camera, Muriel Cario-André, Alain Taïeb, Mauro Picardo  Journal of Investigative Dermatology  Volume 126, Issue 1, Pages (January 2006) DOI: /sj.jid Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 UVA is more effective than UVB in inducing modifications of enzymatic antioxidant activities in reconstructed epidermis and the phototype of melanocytes has a role in the photoprotection. Reconstructed epidermis was irradiated with UVA or UVB. The epidermis was homogenated and enzymatic activities were determined on the supernatants by spectrophotometrical techniques. REK=reconstructed epidermis with only keratinocytes; REML=reconstructed epidermis with melanocytes and keratinocytes from low phototype subjects; REMH=reconstructed epidermis with melanocytes and keratinocytes from high phototype subjects; CTR=not-irradiated samples; UVA=reconstructed epidermis irradiated with 8J/cm2 UVA; UVB=reconstructed epidermis irradiated with 0.15J/cm2 UVB. Each value is the mean±SD of three determinations in duplicate. *P<0.05 in not-irradiated REML with respect to not-irradiated REMH; *P<0.05 in UV-treated reconstructed epidermis versus not-irradiated ones; **P<0.005 in UV-treated reconstructed epidermis versus not-irradiated ones; ***P<0.001 in UV-treated reconstructed epidermis versus non-irradiated ones. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 After UVA low phototype melanocytes in reconstructed epidermis influence charge properties of Cat. Reconstructed epidermis, REK, REML, and REMH were irradiated with UVA or UVB. Thereafter, samples were processed for zymography. One unit of Cat activity was loaded in each electrophoretic lane and the run was performed on undenaturing native acrylamide PAGE, as described in Materials and methods. The spots in the gel represent tetrameric Cat in its native three-dimensional folding from differently treated and untreated samples. The “x” symbol identifies the center of each Cat spot. REK=reconstructed epidermis with only keratinocytes; REML=reconstructed epidermis with melanocytes and keratinocytes from low phototype subjects; REMH=reconstructed epidermis with melanocytes and keratinocytes from high phototype subjects; CTR=non-irradiated samples; UVA=reconstructed epidermis irradiated with 8J/cm2 UVA; UVB=reconstructed epidermis irradiated with 0.15J/cm2 UVB. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Irradiation of U937 cells was performed through CDM or DM. Synthetic melanins, CDM or DM, were put onto a filter, allowing the cells to be separated from the synthetic pigment by an air space. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Cysteinyl-DOPA-melanin but not DOPA-melanin acts as a photosensitizing agent in the induction of charge modification of Cat after UVA and His is able to counteract the phenomenon. U937 cells were irradiated with UVA (20J/cm2) or UVB (0.45J/cm2) through CDM (4μg/ml) or DM (4μg/ml) in the presence or absence of His (30mm) in the cell suspension (see Figure 3). Thereafter, cells were processed for zymographic analysis. (a) CDM acts as a photosensitizing agent in the induction of charge modification of Cat in U937 after UVA, and His, a quencher of singlet oxygen, is able to counteract the phenomenon. CTR=non-irradiated cells; CDM=non-irradiated cells with CDM put onto the filter; UVA=cells irradiated with UVA; CDM UVA=cells irradiated with UVA through CDM; His=non-irradiated cells in the presence of His in the cell suspension; His UVA=cells irradiated with UVA in the presence of His in the cell suspension; His CDM UVA=cells irradiated with UVA through CDM in the presence of His in the cell suspension. (b) UVA irradiation through DM does not modify charge properties of Cat in U937. CTR=non-irradiated cells; DM=non-irradiated cells with DM put onto the filter; UVA=cells irradiated with UVA; DM UVA=cells irradiated with UVA through DM. (c) UVB irradiation through CDM does not modify the charge properties of Cat. CTR=non-irradiated cells; CDM=non-irradiated cells with CDM put onto the filter; UVB=cells irradiated with UVB; CDM UVB=cells irradiated with UVB through CDM. (d) UVB irradiation through DM does not modify the charge properties of Cat. CTR=non-irradiated cells; DM=non-irradiated cells with DM put onto the filter; UVB=cells irradiated with UVB; DM UVB=cells irradiated with UVB through DM. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Cat charge alteration, after UVA through CDM, does not seem to be correlated with the introduction of carbonyl groups in the amino-acid residues. The irradiation procedure, with 20J/cm2 UVA through 4μg/ml CDM, was performed as reported in Figure 3. CTR (lanes 1 and 2): non-irradiated cells; UVA (lanes 3 and 4): cells irradiated with UVA; CDM (lanes 5 and 6): not-irradiated cells with CDM put onto the filter; CDM-UVA (lanes 7 and 8): cells irradiated with UVA through CDM. (a) Western blot analysis of oxidized proteins from U937 samples, after reaction with a specific antibody that recognizes oxidatively introduced carbonyl groups (aldehydes and ketones) at lysine, arginine, proline, or threonine residues, after derivatization with DNPH (see Materials and Methods): even number lanes ( ) represent samples subjected to the derivatization reaction with DNPH; odd number lanes ( ) represent negative control of the derivatization reaction (see Materials and Methods). (b) Western blot analysis of immunoprecipitated Cat from U937 samples, after reaction with an antibody that recognizes oxidatively introduced carbonyl groups after derivatization with DNPH. (c) Western blot analysis of immunoprecipitated Cat from U937 samples, after reaction with an antibody that recognizes the enzyme itself (see Materials and Methods). Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 UVA even through CDM modifies Trp residue oxidative status in Cat. The irradiation procedure, with 20J/cm2 UVA through CDM (4μg/ml), was performed as reported in Figure 3. After irradiation with UVA or UVA through CDM, Cat was immunoprecipitated from total cellular lysate, and Cat antibody-conjugated immunocomplexes, associated with GammaBind™ G Sepharose™ beads (Amersham Pharmacia Biotech, Sweden), were analyzed for the detection of 5-OH-Trp by spectrofluorimetric analysis. After UVA irradiation, a decrease of the fluorescence excitation of the Trp (282nm) was observed, associated with a parallel increase of 5-OH-Trp fluorescence (276nm). This effect was amplified after irradiation through CDM. CTR: immunoprecipitated Cat from not-irradiated U937 cells; UVA: immunoprecipitated Cat from cells irradiated with UVA; CDM: immunoprecipitated Cat from not-irradiated cells with CDM put onto the filter; CDM-UVA: immunoprecipitated Cat from cells irradiated with UVA through CDM; 5-OH-Trp: standard of oxidized Trp. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 UVA even through CDM modifies Met residue oxidative status in Cat. The irradiation procedure with 20J/cm2 UVA through CDM (4μg/ml), was performed as reported in Figure 3. After irradiation with UVA or UVA through CDM, Cat was immunoprecipitated from total cellular lysate, and Cat antibody-conjugated immunocomplexes, associated with GammaBind™ G Sepharose™ beads (Amersham Pharmacia Biotech, Sweden), were analyzed for the detection of Met-SO by micro-Raman spectroscopy. Met-SO leads to the well-assigned SO stretch at 1,025cm−1. After UVA irradiation, the spectrum presented the expected stretch, more intense after irradiation with UVA through CDM (see arrows). (a) CTR: immunoprecipitated Cat from non-irradiated U937 cells; UVA: immunoprecipitated Cat from cells irradiated with UVA; Met-SO: standard of oxidized Met. (b) CDM: immunoprecipitated Cat from non-irradiated cells with CDM put onto the filter; CDM-UVA: immunoprecipitated Cat from cells irradiated with UVA through CDM; Met-SO: standard of oxidized Met. Journal of Investigative Dermatology  , DOI: ( /sj.jid ) Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

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