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• W2014428796 abstract "Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21–30 and 75–92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85β subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-α and SSP 0107) that is induced by interferon-γ in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-γ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo. Aging of the human skin is a complex process that consists of chronological and extrinsic aging, the latter caused mainly by exposure to ultraviolet radiation (photoaging). Here we present studies in which we have used proteomic profiling technologies and two-dimensional (2D) PAGE database resources to identify proteins whose expression is deregulated in the epidermis of the elderly. Fresh punch biopsies from the forearm of 20 pairs of young and old donors (21–30 and 75–92 years old, respectively) were dissected to yield an epidermal fraction that consisted mainly of differentiated cells. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 18 h, lysed, and subjected to 2D PAGE (isoelectric focusing and non-equilibrium pH gradient electrophoresis) and phosphorimage autoradiography. Proteins were identified by matching the gels with the master 2D gel image of human keratinocytes (proteomics.cancer.dk). In selected cases 2D PAGE immunoblotting and/or mass spectrometry confirmed the identity. Quantitative analysis of 172 well focused and abundant polypeptides showed that the level of most proteins (148) remains unaffected by the aging process. Twenty-two proteins were consistently deregulated by a factor of 1.5 or more across the 20 sample pairs. Among these we identified a group of six polypeptides (Mx-A, manganese-superoxide dismutase, tryptophanyl-tRNA synthetase, the p85β subunit of phosphatidylinositol 3-kinase, and proteasomal proteins PA28-α and SSP 0107) that is induced by interferon-γ in primary human keratinocytes and that represents a specific protein signature for the effect of this cytokine. Changes in the expression of the eukaryotic initiation factor 5A, NM23 H2, cyclophilin A, HSP60, annexin I, and plasminogen activator inhibitor 2 were also observed. Two proteins exhibited irregular behavior from individual to individual. Besides arguing for a role of interferon-γ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo. Aging of the human skin is a complex process that comprises two components, chronological aging (replicative senescence) that is largely determined genetically and extrinsic aging, which is triggered by environmental factors, mainly exposure to UV radiation (photoaging) (Refs. 1.Jenkins G. Molecular mechanisms of skin ageing.Mech. Ageing Dev. 2002; 123: 801-810Google Scholar and 2.Wlaschek M. Tantcheva-Poor I. Naderi L. Ma W. Schneider L.A. Razi-Wolf Z. Schuller J. Scharffetter-Kochanek K. Solar UV irradiation and dermal photoaging.J. Photochem. Photobiol. 2001; 63: 41-51Google Scholar and references therein). UV radiation generates reactive oxygen species (ROS), 1The abbreviations used are: ROS, reactive oxygen species; 2D, two-dimensional; IEF, isoelectric focusing; NEPHGE, non-equilibrium pH gradient electrophoresis; IFN, interferon; TNF, tumor necrosis factor; SOD, superoxide dismutase; MALDI, matrix-assisted laser desorption ionization; TOF, time-of-flight; MS, mass spectrometry; eIF, eukaryotic initiation factor; PAI-2, plasminogen activator inhibitor 2; WRS, tryptophanyl-tRNA synthetase; HSP, heat shock protein; SSP, sample spot protein. which in the dermal compartment lead to the accumulation of disorganized elastic fibers and microfibrillar components as well as loss of interstitial collagen, the main component of the dermal connective tissue (Refs. 2.Wlaschek M. Tantcheva-Poor I. Naderi L. Ma W. Schneider L.A. Razi-Wolf Z. Schuller J. Scharffetter-Kochanek K. Solar UV irradiation and dermal photoaging.J. Photochem. Photobiol. 2001; 63: 41-51Google Scholar, 3.Glogau R.G. Physiologic and structural changes associated with aging skin.Dermatol. Clin. 1997; 15: 555-559Google Scholar, 4.Gilchrest B.A. A review of skin aging and its medical therapy.Br. J. Dermatol. 1996; 135: 867-875Google Scholar, 5.Pascuali-Ronchetti I. Baccarani-Contri M. Elastic fibers during development and aging.Microsc. Res. Tech. 1997; 38: 428-435Google Scholar and references therein). Currently there is mounting evidence indicating that the aging process of cells and organs is associated with increased oxidative stress (6.Sohal R.S. Role of oxidative stress and protein oxidation in the aging process.Free Radic. Biol. Med. 2002; 33: 37-44Google Scholar) as well as alterations in apoptosis (Ref. 7.Higami Y. Schimokawa I. Apoptosis in the aging process.Cell Tissue Res. 2000; 301: 125-132Google Scholar and references therein), a homeostatic mechanism that is exacerbated by increased production of ROS (8.Curtin J.F. Donovan M. Cotter T.G. Regulation and measurement of oxidative stress in apoptosis.J. Immunol. Methods. 2002; 265: 49-72Google Scholar, 9.Chandra J. Samali A. Orrenius S. Triggering and modulation of apoptosis by oxidative stress.Free Radic. Biol. Med. 2000; 29: 323-333Google Scholar, 10.Clutton S. The importance of oxidative stress in apoptosis.Br. Med. Bull. 1997; 53: 662-668Google Scholar). Increased production of ROS, decline of the autoxidant cellular defenses to cope with oxidative stress, and the accumulation of mitochondrial DNA mutations and oxidized proteins are among the events that may play an important role in the aging process (Refs. 6.Sohal R.S. Role of oxidative stress and protein oxidation in the aging process.Free Radic. Biol. Med. 2002; 33: 37-44Google Scholar, 7.Higami Y. Schimokawa I. Apoptosis in the aging process.Cell Tissue Res. 2000; 301: 125-132Google Scholar, 11.Lenaz G. Bovina C. D’Aurelio M. Fato R. Formiggini G. Genova M.L. Giuliano G. Pich M.M. Paolucci U. Castelli G.P. Ventura B. Role of mitochondria in oxidative stress and aging.Ann. N. Y. Acad. Sci. 2002; 959: 199-213Google Scholar, and 12.Martindale J.L. Holbrook N.J. Cellular response to oxidative stress: signaling for suicide and survival.J. Cell. Physiol. 2002; 192: 1-15Google Scholar and references therein). Identification of the molecular components that underlie these events is an area of priority in aging research today. While some of the components and pathways may play a common role in the aging process of various organs, others may be specific as tissues are differentiated to exert a defined function and are exposed to different environmental conditions. Gene expression profiling techniques such as two-dimensional (2D) PAGE and DNA microarrays have been used to reveal genes and proteins that may be associated with senescence and longevity, particularly in cultured fibroblasts (13.Celis J.E. Bravo R. Synthesis of the nuclear protein cyclin in growing, senescent and morphologically transformed human skin fibroblasts.FEBS Lett. 1984; 165: 21-25Google Scholar, 14.Toda T. Satoh M. Sugimoto M. Goto M. Furuichi Y. Kimura N. A comparative analysis of the proteins between the fibroblasts from Werner’s syndrome patients and age-matched normal individuals using two-dimensional gel electrophoresis.Mech. Ageing Dev. 1998; 100: 133-143Google Scholar, 15.Toda T. Kaji K. Kimura N. TMIG-2DPAGE: a new concept of two-dimensional gel protein database for research on aging.Electrophoresis. 1998; 19: 344-348Google Scholar, 16.Dierick J.F. Pascal T. Chainiaux F. Eliaers F. Remacle J. Larsen P.M. Roepstorff P. Toussaint O. Transcriptome and proteome analysis in human senescent fibroblasts and fibroblasts undergoing premature senescence induced by repeated sublethal stresses.Ann. N. Y. Acad. Sci. 2000; 908: 302-305Google Scholar, 17.Kondo T. Sakaguchi M. Namba M. Two-dimensional gel electrophoretic studies on the cellular aging: accumulation of α-2-macroglobulin in human fibroblasts with aging.Exp. Gerontol. 2001; 36: 487-495Google Scholar, 18.Benvenuti S. Cramer R. Quinn C.C. Bruce J. Zvelebil M. Corless S. Bond J. Yang A. Hockfield S. Burlingame A.L. Waterfield M.D. Jat P.S. Differential proteome analysis or replicative senescence in rat embryo fibroblasts.Mol. Cell Proteomics. 2002; 1: 280-292Google Scholar). Similar studies at the tissue and organ level, however, have proven difficult due to the heterogeneous nature of the specimens. To date, tissue profiling of the aging process has been confined mainly to DNA microarray analysis of mouse and human muscle (19.Lee C.-K. Klopp R.G. Weindruch R. Prolla T.A. Gene expression profile of aging and its retardation by caloric restriction.Science. 1999; 285: 1390-1393Google Scholar, 20.Welle S. Bhatt K. Thornton C.A. High-abundance mRNAs in human muscle: comparison between young and old.J. Appl. Physiol. 2000; 89: 297-304Google Scholar, 21.Weindruch R. Kayo T. Lee C.K. Prolla T.A. Gene expression profiling of aging using DNA microarrays.Mech. Ageing Dev. 2002; 123: 177-193Google Scholar) and mouse brain (22.Lee C.K. Weindruch R. Prolla T.A. Gene-expression profile of the ageing brain in mice.Nat. Genet. 2000; 25: 294-297Google Scholar, 23.Jiang C.H. Tsien J.Z. Schultz P.G. Hu Y. The effects of aging on gene expression in the hypothalamus and cortex of mice.Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 1930-1934Google Scholar) and liver (24.Han E. Hilsenbeck S.G. Richardson A. Nelson J.F. cDNA expression arrays reveal incomplete reversal of age-related changes in gene expression by caloric restriction.Mech. Ageing Dev. 2000; 115: 157-174Google Scholar, 25.Dozmorov I. Bartke A. Miller R.A. Array-based expression analysis of mouse liver genes: effect of age and of the longevity mutant Prop1df.J. Gerontol. A Biol. Sci. Med. Sci. 2001; 56: B72-B80Google Scholar). In general, these studies have shown that the expression of only a small fraction of the genes analyzed changed during the aging process. A comparison between the effect of senescence in the muscle of mice and men showed that of 70 homologous genes studied by oligonucleotide microarrays only 17 showed similar age-related changes (26.Welle S. Brooks A. Thornton C.A. Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.Physiol. Genomics. 2001; 5: 67-73Google Scholar). Interestingly there was no evidence indicating that human muscle from old individuals exhibited deregulated expression of stress response genes as observed in the old murine muscle indicating important differences among species (26.Welle S. Brooks A. Thornton C.A. Senescence-related changes in gene expression in muscle: similarities and differences between mice and men.Physiol. Genomics. 2001; 5: 67-73Google Scholar). Our laboratory has for many years applied proteomic technologies to the study of human epidermal biopsies in health and disease, and we have gathered a substantial amount of information on keratinocyte proteins under various physiological conditions (Refs. 27.Celis J.E. Madsen P. Rasmussen H.H. Leffers H. Honore B. Gesser B. Dejgaard K. Olsen E. Magnusson N. Kiil J. Celis A. Lauridsen J.B. Basse B. Ratz G.P. Andersen A.H. Walbum E. Brandstrup B. Pedersen P.S. Brandt N.J. Puype M. Van Damme J. Vandekerckhove J. A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.Electrophoresis. 1991; 12: 802-872Google Scholar, 28.Celis J.E. Rasmussen H.H. Gromov P. Olsen E. Madsen P. Leffers H. Honore B. Dejgaard K. Vorum H. Kristensen B. Østergaard M. Hauns⊘ A. Jensen N.A. Celis A. Basse B. Lauridsen J.B. Ratz G.P. Andersen A.H. Walbum E. Kjærgaard I. Andersen I. Puype M. Van Damme J. Vandekerckhove J. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.Electrophoresis. 1995; 16: 2177-2240Google Scholar, 29.Celis J.E. Østergaard M. Jensen N.A. Gromova I.I. Rasmussen H.H. Gromov P. Human and mouse proteomic databases: a novel resources in the proteome universe.FEBS Lett. 1998; 430: 64-72Google Scholar; proteomics.cancer.dk). Here we present a quantitative analysis of the proteome expression profiles of fresh human epidermal biopsies obtained from the forearm of young and old donors. Besides arguing for a role of IFN-γ in the aging process, the biological activities associated with the deregulated proteins support the contention that aging is linked with increased oxidative stress that could lead to apoptosis in vivo (Refs. 6.Sohal R.S. Role of oxidative stress and protein oxidation in the aging process.Free Radic. Biol. Med. 2002; 33: 37-44Google Scholar and 7.Higami Y. Schimokawa I. Apoptosis in the aging process.Cell Tissue Res. 2000; 301: 125-132Google Scholar and references therein). Skin punch biopsies were obtained from Rigshospitalet, Copenhagen. Biopsies were taken from both forearms of normal Danish individuals of different ages (from 21 to 92 years old), placed on ice, and immediately transported to the Department of Medical Biochemistry, Aarhus University. The forearm was selected to minimize the effect of solar irradiation. Fresh skin biopsies were dissected with the aid of a scalpel to yield enriched epidermis. One- to two-mm3 epidermal pieces were labeled with [35S]methionine for 14 h in 0.1 ml of Dulbecco's modified Eagle's medium containing 1% dialyzed fetal calf serum and 100 μCi of radioactivity (Amersham Biosciences, catalog number SJ204). Following labeling, the tissues were dissolved in 0.3 ml of lysis solution (30.O’Farrell P.Z. Goodman H.M. O’Farrell P.H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell. 1977; 12: 1133-1141Google Scholar) and kept at −20 °C until use. Whole protein lysates were then subjected to both IEF and NEPHGE 2D PAGE as described previously (31.Celis J.E. Ratz G. Basse B. Lauridsen J.B. Celis A. Gromov P. Celis J.E. Carter N. Hunter T. Shotton D. Simons K. Small J.V. Cell Biology. A Laboratory Handbook. 4. Academic Press, New York1998: 375-385Google Scholar). Several gels were run from each sample. Proteins were visualized using autoradiography and/or phosphorimaging. Primary cultures of normal human keratinocytes were prepared and grown as described previously (32.Madsen P. Rasmussen H.H. Leffers H. Honore B. Celis J.E. Molecular cloning and expression of a novel keratinocyte protein (psoriasis-associated fatty acid-binding protein [PA-FABP]) that is highly up-regulated in psoriatic skin and that shares similarity to fatty acid-binding proteins.J. Investig. Dermatol. 1992; 99: 299-305Abstract Full Text PDF Google Scholar). Cells were labeled for 14 h in Dulbecco's modified Eagle's medium lacking methionine and containing 1% dialyzed fetal calf serum, 500 μCi/ml [35S]methionine, and 50 units/ml recombinant cytokines: IFN-γ, IFN-α, or TNF-α. After labeling, the medium was aspirated, and the cells were resuspended in lysis solution for 2D PAGE (see above and Ref. 30.O’Farrell P.Z. Goodman H.M. O’Farrell P.H. High resolution two-dimensional electrophoresis of basic as well as acidic proteins.Cell. 1977; 12: 1133-1141Google Scholar). Samples were kept at −20 °C until use. Control cells were labeled under the same conditions except that no cytokine was added. Phosphorimage autoradiographs were obtained with the aid of the Molecular Imager from Bio-Rad and were quantitated using the Multi-Analyst 1.0.1 software (manually driven) from the same company. Only gels depicting well focused spots and limited amount of protein remaining at the origin were selected for quantitation. The levels of actin (IEF) and of annexin II, which migrated both in IEF and NEPHGE gels, were used as reference to normalize protein levels in both gel types (33.Celis J.E. Rasmussen H.H. Olsen E. Madsen P. Leffers H. Honore B. Dejgaard K. Gromov P. Vorum H. Vassilev A. Baskin Y. Liu X. Celis A. Basse B. Lauridsen J.B. Ratz G.P. Andersen A.H. Walbum E. Kjærgaard I. Andersen I. Puype M. Van Damme J. Vandekerckhove J. The human keratinocyte two-dimensional gel protein database (update 1994): towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.Electrophoresis. 1994; 15: 1349-1458Google Scholar). The levels of selected proteins were quantitated in 20 pairs of young and old individuals. The average means and S.D. were determined. Groups in which the average means differed by a factor 1.5 or more were compared by using the heteroscedastic t test and evaluated for several proteins with the nonparametric Wilcoxon-Mann-Whitney test. Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (Refs. 27.Celis J.E. Madsen P. Rasmussen H.H. Leffers H. Honore B. Gesser B. Dejgaard K. Olsen E. Magnusson N. Kiil J. Celis A. Lauridsen J.B. Basse B. Ratz G.P. Andersen A.H. Walbum E. Brandstrup B. Pedersen P.S. Brandt N.J. Puype M. Van Damme J. Vandekerckhove J. A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.Electrophoresis. 1991; 12: 802-872Google Scholar, 28.Celis J.E. Rasmussen H.H. Gromov P. Olsen E. Madsen P. Leffers H. Honore B. Dejgaard K. Vorum H. Kristensen B. Østergaard M. Hauns⊘ A. Jensen N.A. Celis A. Basse B. Lauridsen J.B. Ratz G.P. Andersen A.H. Walbum E. Kjærgaard I. Andersen I. Puype M. Van Damme J. Vandekerckhove J. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.Electrophoresis. 1995; 16: 2177-2240Google Scholar, 29.Celis J.E. Østergaard M. Jensen N.A. Gromova I.I. Rasmussen H.H. Gromov P. Human and mouse proteomic databases: a novel resources in the proteome universe.FEBS Lett. 1998; 430: 64-72Google Scholar; proteomics.cancer.dk). One or a combination of procedures that included Edman degradation (34.Rasmussen H.H. van Damme J. Puype M. Gesser B. Celis J.E. Vandekerckhove J. Microsequences of 145 proteins recorded in the two-dimensional gel protein database of normal human epidermal keratinocytes.Electrophoresis. 1992; 13: 960-969Google Scholar), mass spectrometry (35.Rasmussen H.H. Mortz E. Mann M. Roepstorff P. Celis J.E. Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.Electrophoresis. 1994; 15: 406-416Google Scholar), and 2D PAGE Western immunoblotting (36.Celis J.E. Gromov P. High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.EXS (Basel). 2000; 88: 55-67Google Scholar) has identified proteins in the database. Mass spectrometry and/or 2D PAGE Western immunoblotting confirmed the identity of selected proteins. In short, for mass spectrometry, protein spots were cut out from the dry gel with the aid of the corresponding x-ray film and were prepared as described previously (35.Rasmussen H.H. Mortz E. Mann M. Roepstorff P. Celis J.E. Identification of transformation sensitive proteins recorded in human two-dimensional gel protein databases by mass spectrometric peptide mapping alone and in combination with microsequencing.Electrophoresis. 1994; 15: 406-416Google Scholar, 37.Jensen O.N. Wilm M. Shevchenko A. Mann M. Sample preparation methods for mass spectrometric peptide mapping directly from 2-DE gels.Methods Mol. Biol. 1999; 112: 513-530Google Scholar, 38.Celis J.E. Kruhoffer M. Gromova I. Frederiksen C. Ostergaard M. Thykjaer T. Gromov P. Yu J. Palsdottir H. Magnusson N. Orntoft T.F. Gene expression profiling: monitoring transcription and translation products using DNA microarrays and proteomics.FEBS Lett. 2000; 480: 2-16Google Scholar). All MALDI-MS measurements were performed using a Bruker Reflex III MALDI-TOF-MS (Bruker Daltonik GmbH). Prior to peptide mass fingerprint analysis, the instrument was calibrated using the known masses of a mixture of synthetic peptides spotted on the target disc closer to the sample. Peptide masses were searched using the Expasy algorithm (www.expasy.ch/tools/). 2D PAGE Western immunoblotting was performed as described previously (36.Celis J.E. Gromov P. High-resolution two-dimensional gel electrophoresis and protein identification using western blotting and ECL detection.EXS (Basel). 2000; 88: 55-67Google Scholar). Eight-μm cryostat sections from frozen human skin were placed on round coverslips, washed three times with phosphate-buffered saline, and treated for 5 min with methanol at −20 °C (39.Celis J.E. Celis P. Ostergaard M. Basse B. Lauridsen J.B. Ratz G. Rasmussen H.H. Orntoft T.F. Hein B. Wolf H. Celis A. Proteomics and immunohistochemistry define some of the steps involved in the squamous differentiation of the bladder transitional epithelium: a novel strategy for identifying metaplastic lesions.Cancer Res. 1999; 59: 3003-3009Google Scholar). Coverslips were washed several times with phosphate-buffered saline, covered with 20 μl of the primary antibody, and incubated for 60 min at 37 °C in a humidified box. Following incubation, the coverslips were washed several times with phosphate-buffered saline, covered with 20 μl of rhodamine-conjugated secondary antibody (dilution, 1:50), and incubated for 60 min at 37 °C in a humidified box. Coverslips were washed extensively with phosphate-buffered saline, washed once with distilled water, and covered with DAKO mounting medium. Samples were observed using a Leica photomicroscope equipped with epifluorescence and phase contrast optics. To monitor the structural disorganization and fragmentation of the elastic fiber meshwork, which is a hallmark of skin aging (1.Jenkins G. Molecular mechanisms of skin ageing.Mech. Ageing Dev. 2002; 123: 801-810Google Scholar, 2.Wlaschek M. Tantcheva-Poor I. Naderi L. Ma W. Schneider L.A. Razi-Wolf Z. Schuller J. Scharffetter-Kochanek K. Solar UV irradiation and dermal photoaging.J. Photochem. Photobiol. 2001; 63: 41-51Google Scholar), we performed immunohistochemistry of skin biopsies using a monoclonal antibody prepared in our laboratory (monoclonal antibody b9) that specifically decorates these fibers (40.Palsdottir H. Preparation and Characterization of Monoclonal Antibodies against Urines from Bladder Cancer Patients with Invasive Disease. Institute for Molecular and Structural Biology, Aarhus Universitet, Aarhus, Denmark2000Google Scholar). As shown in Fig. 1, the forearm skin from old individuals displayed disorganization of the elastic fiber meshwork as well as loss of anchoring of the fibers oriented perpendicularly to the dermal/epidermal junction (Fig. 1b). In young and middle-aged individuals, the latter formed arcades that projected toward the junction (Fig. 1a). Immunostaining of similar skin sections with a collagen IV antibody (DAKO AS), which decorates the basement membrane, revealed flattening of the dermal/epidermal junction, a feature that is characteristic of aging skin (Fig. 1, compare d with c (young)). Representative 2D gel phosphorimages of human epidermal proteins resolved in IEF and NEPHGE gels are shown in Fig. 2. Samples showed little contamination with connective tissue as judged by the low levels of expression of vimentin, a protein that is expressed by dermal fibroblasts (41.Celis J.E. Celis P. Palsdottir H. Østergaard M. Gromov P. Primdahl H. Orntoft T.F. Wolf H. Celis A. Gromova I. Proteomic strategies to reveal tumor heterogeneity among urothelial papillomas.Mol. Cell. Proteomics. 2002; 1: 269-279Google Scholar). A total of 172 well focused and abundant proteins were selected for quantitation (Table I). These are indicated with their name and/or SSP number in Fig. 2, A and B, and are listed in Table I together with their apparent Mr and pI values. Proteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (Fig. 3; proteomics.cancer.dk) (27.Celis J.E. Madsen P. Rasmussen H.H. Leffers H. Honore B. Gesser B. Dejgaard K. Olsen E. Magnusson N. Kiil J. Celis A. Lauridsen J.B. Basse B. Ratz G.P. Andersen A.H. Walbum E. Brandstrup B. Pedersen P.S. Brandt N.J. Puype M. Van Damme J. Vandekerckhove J. A comprehensive two-dimensional gel protein database of noncultured unfractionated normal human epidermal keratinocytes: towards an integrated approach to the study of cell proliferation, differentiation and skin diseases.Electrophoresis. 1991; 12: 802-872Google Scholar, 28.Celis J.E. Rasmussen H.H. Gromov P. Olsen E. Madsen P. Leffers H. Honore B. Dejgaard K. Vorum H. Kristensen B. Østergaard M. Hauns⊘ A. Jensen N.A. Celis A. Basse B. Lauridsen J.B. Ratz G.P. Andersen A.H. Walbum E. Kjærgaard I. Andersen I. Puype M. Van Damme J. Vandekerckhove J. The human keratinocyte two-dimensional gel protein database (update 1995): mapping components of signal transduction pathways.Electrophoresis. 1995; 16: 2177-2240Google Scholar, 29.Celis J.E. Østergaard M. Jensen N.A. Gromova I.I. Rasmussen H.H. Gromov P. Human and mouse proteomic databases: a novel resources in the proteome universe.FEBS Lett. 1998; 430: 64-72Google Scholar). In selected cases, mass spectrometry and 2D PAGE Western immunoblotting further confirmed their identity. Known proteins listed in Table I are categorized according to the following functional groups: (i) energy metabolism; (ii) protein synthesis, folding, and degradation; (iii) cytoskeleton; (iv) RNA metabolism; (v) calcium-modulated metabolism; (vi) cell proliferation and differentiation; and (vii) others.Table ILevels of [35S]methionine-labeled proteins in normal human skin biopsies obtained from young (21–30-year-old) and old (75–92-year-old) individualsProteinaProteins were identified by matching the gels with the master image of the human keratinocyte 2D PAGE database (proteomics.cancer.dk). In selected cases the identity was verified by mass spectrometry and/or 2D PAGE immunoblotting.Mr × 10−3bProteins in each functional category are listed in order of increasing Mr.pISSPcSSP number from the human keratinocyte 2D PAGE database (proteomics.cancer.dk). I, IEF gel; N, NEPHGE gel.Spot volume (×102)dProtein spots were quantitated as described under “Experimental Procedures.” The column shows the average means and S.D. Proteins changing by a factor of 1.5 or more are highlighted in bold.Ratio old/youngep values were determined for groups in which the average means changed by a factor of 1.5 or more.YoungOldEnergy metabolism 1. Hydrogen-transporting ATP synthetase13.94.19010 (I)6.2 ± 1.57.9 ± 1.71.2 2. Triose-phosphate isomerase28.37.35106 (N)15 ± 6.313 ± 4.00.9 3. Phosphoglyceromutase28.67.01107 (I)12 ± 3.910 ± 3.40.8 4. Phosphoglyceromutase30.47.25203 (N)24 ± 9.721 ± 7.70.9 5. Aldose reductase37.56.53327 (I)5.6 ± 0.94.7 ± 0.70.8 6. Glyceraldehyde-3-phosphate dehydrogenase38.48.91206 (N)167 ± 57166 ± 411.0 7. Isocytrate dehydrogenase41.27.41320 (I)4.4 ± 0.95.2 ± 0.71.2 8. Fructose 1,6-bisphosphate aldolase43.48.81302 (N)37 ± 1132 ± 7.50.8 9. Phosphoglycerate kinase43.68.23308 (N)54 ± 2466 ± 301.2 10. Citrate synthetase44.58.13302 (N)12 ± 3.015 ± 2.81.1 11. α-Enolase46.87.41325 (I)72 ± 2182 ± 171.1 12. α-Enolase48.77.45406 (N)152 ± 52168 ± 341.1 13. Aldehyde dehydrogenase (mitochondrial)51.96.33303 (I)4.1 ± 1.25.3 ± 1.11.2 14. Vacuolar ATPase68.95.36515 (I)7.8 ± 3.37.1 ± 3.00.9Protein synthesis, folding, and degradation 15. Ubiquitin9.67.80013 (I)33 ± 1342 ± 111.3 16. Cystatin11.25.36011 (I)5.5 ± 2.05.7 ± 1.51.0 17. Ribosomal P-protein14.44.09005 (I)5.2 ± 1.16.0 ± 1.41.1 18. eIF-5A16.04.98016 (I)6.9 ± 4.916 ± 4.02.3 (p < 0.001) 19. eIF-5A, variant16.04.68010 (I)5.3 ÷ 21fThe division sign indicates values that were highly variable.1.6 ÷ 11fThe division sign indicates values that were highly variable.Variable, not determined 20. Cyclophilin A17.38.32003 (N)26 ± 8.941 ± 111.6 (p < 0.001) 21. Cyclophilin A" @default.
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• W2014428796 creator A5073018306 @default.
• W2014428796 date "2003-02-01" @default.
• W2014428796 modified "2023-10-16" @default.
• W2014428796 title "Protein Profiling of the Human Epidermis from the Elderly Reveals Up-regulation of a Signature of Interferon-γ-induced Polypeptides That Includes Manganese-superoxide Dismutase and the p85β Subunit of Phosphatidylinositol 3-Kinase" @default.
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