Gene/Protein Disease Symptom Drug Enzyme Compound
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Query: EC:3.4.21.4 (trypsin)
 42,187 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

Basal keratinocytes of human epidermis strongly express the cell surface glycoprotein beta(1)-integrin, and putatively harbour epidermal stem cells. Selective sorting and culturing of keratinocyte stem cells forms the basis for studies on the role of these cells as targets for therapeutic intervention and gene therapy. Here we have studied variables which affect cell surface labelling for beta(1)-integrin, flow sorting and subsequent culturing of beta(1)-integrin-positive and beta(1)-integrin-negative keratinocytes. Keratinocytes were derived from small human skin punch biopsies (3 or 4 mm in diameter), and we tested a number of variables such as choice of proteolytic enzyme for cell isolation, cell concentration, fixation, storage of fixed cell suspensions and labelling conditions. In contrast to thermolysin treatment for cell isolation, trypsin treatment left most cell surface beta(1)-integrin molecules intact. Ethanol and paraformaldehyde fixation interfered with beta(1)-integrin detection, and unfixed cells gave the best results. Optimisation of all the individual steps resulted in a labelling protocol for reproducible staining and sorting of the cells. Sorted cells were seeded in 96-well plates (300 cells/well) and colonies were obtained in more than 50% of the wells with beta(1)-integrin-positive keratinocytes. In plates with beta(1)-integrin-negative cells, only 10% of the wells contained keratinocyte colonies. Flow sorted keratinocytes obtained by trypsin formed numerous colonies in cell culture experiments. In cell suspensions obtained with thermolysin, only sparse colonies were formed. We conclude that our methodology permits the use of small human tissue samples for cell labelling and sorting, while preserving the clonogenic potential.
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PMID:Immunofluorescent surface labelling, flow sorting and culturing of putative epidermal stem cells derived from small skin punch biopsies. 1216 32

We previously isolated AF20, a murine monoclonal antibody that recognizes a cell surface glycoprotein of approximately 90-110 kDa. The AF20 antigen is specifically expressed in human hepatoma and colon cancer cell lines, and thus could serve as a cancer biomarker. To uncover the molecular identity of the AF20 antigen, a combination of ion-exchange chromatography, immunoprecipitation, and SDS-polyacrylamide gel electrophoresis was employed to purify the AF20 antigen followed by trypsin digestion and mass spectrometry. Surprisingly, three host proteins were thus purified from human hepatoma and colon cancer cell lines: transferrin receptor 1 (TFR1), heat shock protein 90 (HSP90), and Na+/K+ ATPase or Mg++ ATPase. Co-immunoprecipitation followed by Western blot analysis confirmed interaction among the three proteins. However, only the cDNA encoding TFR1 conferred strong cell surface staining by the AF20 antibody following its transient transfection into a cell line lacking endogenous AF20. In support of the molecular identity of AF20 as TFR1, diferric but not iron-free transferrin could prevent AF20 antigen-antibody interaction during immunoprecipitation. Moreover, very similar patterns of AF20 and TFR1 overexpression was documented in colon cancer tissues. In conclusion, AF20 is glycosylated TFR1. This finding could explain the molecular structure of AF20, its cell surface localization, as well as overexpression in cancer cells. Glycosylated TFR1 should serve as a usefulness target for anti-cancer therapy, or a vehicle for delivery of anti-tumor drugs with high affinity and specificity. The biological significance of the complex formation between TFR1, HSP90, and/or transporting ATPase warrants further investigation.
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PMID:Identification of Tumor Antigen AF20 as Glycosylated Transferrin Receptor 1 in Complex with Heat Shock Protein 90 and/or Transporting ATPase. 2780 97


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