摘要:

AbstractComparison of the protein expression patterns of proliferating normal primary human keratinocytes plated in serum‐free medium (SFKM), supplemented with epidermal growth factor (EGF) and bovine pituitary extract (BPE), and similar cultures induced to differentiate by the addition of Dulbecco's modified Eagle medium (DMEM), containing 10% fetal calf serum (FCS), revealed several known and unknown polypeptides that are abnormally regulated in the differentiated cells. Upregulated proteins included keratins (keratins 6, 10/11, 14 and 16), members of the S100 protein family psoriasin, MRP8, MRP14 and S100c), actin‐binding proteins (gelsolin and tropomyosin 9220), annexins (annexins IV and VIII), hsp28, the fatty acid binding protein 5 (FABP5), the squamous cell carcinoma (SCC) antigen, members of the 14‐3‐3 family, involucrin, E‐cadherin, cystatin A, desmoglein and integrins α2and β1, as well as several proteins of as yet unknown identity. The highest upregulated proteins corresponded to psoriasin (124.0 times), MRP8 (42.4 times), MRP14 (14.9 times), tropomyosin 9220 (11.5 times), involucrin (11.1 times), and FABP5 (9.1 times). FABP5, hsp28, and tropomyosin 9220 were also highly upregulated in quiescent keratinocytes indicating that their increased levels in the differentiated cell may be due to loss of proliferative activity. Highly downregulated proteins included PAI‐2, tropomyosins 9213, 9121 and 9122, keratin 5, calnexin, 14‐3‐3 beta and eta, nucleoside diphosphate kinase A, Rho GDIs, hsp60, hnRNPs H and C2, α‐enolase, eIF‐4D, thioredoxin, annexins III and V, moesin, nucleolar protein B23, GST π and PCNA/cyclin. Both the high expression of keratin 6 and 16 – which are markers for an alternative pathway of keratinocyte differentiation – as well as the extremely high upregulation of some members of the S100 protein family indicate that the cells have different

ISSN:0173-0835    

DOI:10.1002/elps.11501601356

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractPhagosomes are the organelles formedde novoin a variety of cells by the internalization of large particulate materials, including a wide range of pathogenic microorganisms. We present here a systematic approach that can be used to study the polypeptide composition of phagosomes/phagolysosomes and to yield analytical information on the characteristics of their proteins. A density shift approach was used to isolate pure preparations of phagosomes filled with low density latex beads from mouse J774 and human U937 macrophages. High resolution two‐dimensional (2‐D) gel electrophoresis was performed to generate a map of the overall [35S]methionine‐labeled protein profile of the isolated phagosomes. The resulting map showed the minimal presence of over 200 polypeptides, indicating the complexity of this organelle. Comigration experiments showed that several phagosome polypeptides, among them several known proteins, are shared by the two species. Extraction with Triton X‐114 and sodium carbonate was performed to distinguish between membrane and soluble proteins, and sensitivity to a panel of proteases was measured to identify proteins exposed on the cytoplasmic face of the phagosome membrane. The general value of the 2‐D gel approach in the mapping of organelle proteins is

ISSN:0173-0835    

DOI:10.1002/elps.11501601357

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

摘要:

AbstractWe present two procedures that can be used to map proteins in two‐dimensional gels if the cDNA is at hand. The first procedure, which is illustrated with the expression of cDNAs encoding the fatty acid binding protein 5 (FABP 5), psoriasin and stratifin, makes use of thein vitrotranscription/translation assay marketed by Promega. The procedure is simple and allows the mapping of the primary translation product in a very short time. The second method – which faithfully reproduces post‐translational modifications – is based on the expression of cDNAs transfected into COS‐1 cells using a eukaryotic expression plasmid. This procedure is illustrated with the expression of cDNAs encoding Ha‐ras p21 and rab 11, two small GTP‐binding proteins known to undergo complex

ISSN:0173-0835    

DOI:10.1002/elps.11501601358

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.11501601359

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.11501601360

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150160101

出版商:Wiley Subscription Services, Inc., A Wiley Company    

年代:1995

数据来源: WILEY