Aminopeptidases of Human Skin — a Survey of Enzyme Activities in Extracts from Skin and Cultured Fibroblasts

Abstract A potent procoagulant synthesized by cultured human skin fibroblasts has been identified as tissue factor (factor III, thromboplastin), since it binds factor VII and is blocked by a specific antitissue factor antibody. Fibroblast tissue factor is, at least in part, a labile superficial, membrane-associated substance the synthesis of which is mediated by cell adhesion but is inhibited by actinomycin-D and puromycin. Tissue factor production is related to the shape change that occurs as the cells spread on the floor of the culture vessel, but tissue factor is not synonymous with the configuration on the cell surface responsible for cell adhesion. These observations suggest that cell membranes may play a significant role in hemostasis and thrombosis by virtue of their tissue factor content.

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Proteome Analysis of Cultured Fibroblasts from Type 1 Diabetic Patients and Normal Subjects

The Journal of Clinical Endocrinology & Metabolism ◽

10.1210/jc.2006-0274 ◽

2006 ◽

Vol 91 (9) ◽

pp. 3507-3514 ◽

Cited By ~ 13

Author(s):

Lucia Puricelli ◽

Elisabetta Iori ◽

Renato Millioni ◽

Giorgio Arrigoni ◽

Peter James ◽

...

Keyword(s):

Human Skin ◽

Proteome Analysis ◽

Normal Subjects ◽

Skin Fibroblasts ◽

Structural Proteins ◽

Diabetic Patients ◽

Human Skin Fibroblasts ◽

Cultured Fibroblasts ◽

Type 1 Diabetic Patients

Abstract Context: Protein profiling of diabetic tissues could provide useful biomarkers for early diagnosis, therapeutic targets, and disease response markers. Cultured fibroblasts are a useful in vitro model for proteome analysis and study of the molecular mechanisms involved in diabetes. Objective: The objective of the study was to isolate and characterize the proteins of cultured fibroblasts, obtained by skin biopsy, from long-term type 1 diabetic patients without complications and age- and sex-matched normal subjects as controls. Design: Proteins were separated by two-dimensional electrophoresis (2-DE), and the gel images were qualitatively and quantitatively analyzed. Protein identification was performed by matrix-assisted laser desorption/ionization mass spectrometry. Results: Reproducible protein maps of fibroblasts from diabetic and healthy subjects were obtained. A total of 125 protein spots were isolated and identified, among them 27 proteins not previously reported in published human fibroblast 2-DE maps, including 20 proteins never reported previously in the literature in human skin fibroblasts. Quantitative analyses revealed six protein spots differentially expressed in the fibroblasts from the diabetic vs. the control subjects (P < 0.05), representing glycolytic enzymes and structural proteins. An increase of triosephosphate I isomerase of two splice isoforms of pyruvate kinase and α-actinin 4 and a decrease of tubulin-β2 and splice isoform 2 of tropomyosin β-chain were detected. Conclusions: We generated 2-DE reference maps of the proteome of human skin fibroblasts from both normal and uncomplicated type 1 diabetic patients. Differences in glycolytic enzymes and structural proteins were found. The functional implications of the identified proteins are discussed.

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Biosynthesis and release of glycoproteins by human skin fibroblasts in culture

Biochemical Journal ◽

10.1042/bj1680091 ◽

1977 ◽

Vol 168 (1) ◽

pp. 91-103 ◽

Cited By ~ 21

Author(s):

Christopher H. J. Sear ◽

Michael E. Grant ◽

David S. Jackson

Keyword(s):

Human Skin ◽

De Novo ◽

Polyacrylamide Gel Electrophoresis ◽

Sodium Dodecyl ◽

Gel Filtration ◽

Skin Fibroblasts ◽

Human Skin Fibroblasts ◽

Cultured Fibroblasts ◽

Cscl Density Gradient ◽

Before And After

1. Confluent human skin fibroblasts maintained in a chemically defined medium incorporate l-[1-3H]fucose in a linear manner with time into non-diffusible macromolecules for up to 48h. Chromatographic analysis demonstrated that virtually all the macromolecule-associated3H was present as [3H]fucose. 2. Equilibrium CsCl-density-gradient centrifugation established that [3H]fucose-labelled macromolecules released into the medium were predominantly glycoproteins. Confirmation of this finding was provided by molecular-size analyses of the [3H]fucose-labelled material before and after trypsin digestion. 3. The [3H]fucose-labelled glycoproteins released into fibroblast culture medium were analysed by gel-filtration chromatography and sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. These techniques demonstrated that the major fucosylated glycoprotein had an apparent mol.wt. of 230000–250000; several minor labelled species were also detected. 4. Dual-labelling experiments with [3H]fucose and14C-labelled amino acids indicated that the major fucosylated glycoprotein was synthesized de novo by cultured fibroblasts. The non-collagenous nature of this glycoprotein was established by three independent methods. 5. Gel-filtration analysis before and after reduction with dithiothreitol showed that the major glycoprotein occurs as a disulphide-bonded dimer when analysed under denaturing conditions. Further experiments demonstrated that this glycoprotein was the predominant labelled species released into the medium when fibroblasts were incubated with [35S]cysteine. 6. The relationship between the major fucosylated glycoprotein and a glycoprotein, or group of glycoproteins, variously known as fibronectin, LETS protein, cell-surface protein etc., is discussed.

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Inhibition of Enzyme Activities and the Antiwrinkle Effect of Polyphenol Isolated from the Persimmon Leaf (Diospyros kaki folium) on Human Skin

Dermatologic Surgery ◽

10.1111/j.1524-4725.2005.31730 ◽

2006 ◽

Vol 31 ◽

pp. 848-855 ◽

Cited By ~ 32

Author(s):

Bong-Jeun An ◽

Jae-Hoon Kwak ◽

Jung-Mi Park ◽

Jin-Young Lee ◽

Tae-Soon Park ◽

...

Keyword(s):

Human Skin ◽

Enzyme Activities ◽

Diospyros Kaki ◽

Persimmon Leaf

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METABOLISM OF ANDROGENS IN VITRO BY HUMAN FACIAL AND AXILLARY SKIN

Journal of Endocrinology ◽

10.1677/joe.0.0590475 ◽

1973 ◽

Vol 59 (3) ◽

pp. 475-486 ◽

Cited By ~ 41

Author(s):

J. B. HAY ◽

M. B. HODGINS

Keyword(s):

Human Skin ◽

Enzyme Activities ◽

Hydroxysteroid Dehydrogenase ◽

Facial Skin ◽

Steroid Hydroxylase ◽

Skin Formation

SUMMARY Human skin from forehead, cheek and axilla was incubated in vitro with [7α-3H]dehydroepiandrosterone (DHA), [7α-3H]DHA sulphate, [7α-3H]-androstenedione and [7α-3H]testosterone. The following enzyme activities were detected: 3β-hydroxysteroid dehydrogenase Δ4-5 isomerase, 17β-hydroxysteroid dehydrogenase, 3β-hydroxysteroid dehydrogenase, 3α-hydroxysteroid dehydrogenase, 5α-reductase, 5β-reductase, sulphotransferase, sulphatase, steroid hydroxylase. 5α-Reduced steroids were the major metabolites. All four substrates were converted to 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol. In axillary skin, conversion of 17-oxosteroids to 17β-hydroxysteroids was favoured, 5α-dihydrotestosterone and 5α-androstane-3α,17β-diol being major metabolites. In facial skin, formation of 17-oxosteroids predominated with little accumulation of 5α-dihydrotestosterone or 5α-androstane-3α,17β-diol. 5α-Androstane-3β,17β-diol was a metabolite of DHA, androstenedione and testosterone but was found in lower amounts than 5α-androstane-3α,17β-diol. Similarly conversions to epiandrosterone were much lower than to androsterone in all the skin specimens. It was concluded that the differences in accumulation of 5α-dihydrotestosterone were determined by the differences in 17β-oxidoreduction rather than differences in 5α-reductase, the activity of which was high in all skin specimens.

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