摘要:

AbstractTwo‐dimensional gel electrophoresis was used to study the regulation of cytoskeletal protein synthesis during growth activation and development of the differentiated phenotype. We demonstrated a correlation between the state of organization and the expression of the respective cytoskeletal protein by showing that depolymerization of microtubules leads to a rapid decrease in new tubulin synthesis. We found that the synthesis of vimentin in both fibroblasts and epithelial cells correlates with extensive cell spreading on the substrate, while cytokeratin synthesis is maximal when cell to cell contacts are abundant. The analysis of cytoskeletal elements, involved directly in the formation of cell contacts, revealed that the level of vinculin synthesis is dependent on the extent of adherent type of cell contacts formed. Moreover, we found that the transient disappearance of vinculin from adhesion plaques of quiescent fibroblasts in response to serum factors was followed by an induction of vinculin mRNA and protein synthesis. The morphological changes associated with establishment of the differentiated phenotype were also found to include changes in the expression of the cytoskeletal‐extracellular matrix complex. This was demonstrated in several differentiating systems: in 3T3 preadipocytes which change their shape from a fibroblastic to a spherical shape when stimulated to differentiate with adipogenic medium, we observed a decrease in mRNA levels and in the synthesis of fibronectin, β‐integrin, and the microfilament proteins, vinculin, α‐actinin, tropomyosin and actin. The culturing of these cells on a certain extracellular matrix prevented the morphological changes occurring in the presence of adipogenic medium and blocked the shifts in cytoskeletal‐ and differentiation‐related gene expression. Similar changes in the organization and expression of cytoskeletal proteins were identified during maturation of primary ovarian granulosa cell cultures, stimulated with gonadotropic hormones to form highly steroidogenic cells. The cell rounding and aggregation occurring during this process were associated with a decreased synthesis of vinculin, α‐actinin, actin and the nonmuscle tropomyosins. The physiological relevance of these changes was suggested by the observation that the level of tropomyosin mRNA was lower in follicles of animals at late stages of granulosa cell maturation when compared to earlier stages. The expression of tissue‐specific and cytoskeletal proteins was also determined in primary cultures of liver hepatocytes, maintained under conditions either favorable for growth or for expression of liver‐specific functions. When DNA synthesis was elevated, cytoskeletal protein synthesis was high and that of liver‐specific proteins was low. In contrast, when the expression of liver specific genes was maintained at high level, cytoskeletal gene expression and DNA synthesis were inhibited, as in the adult liver hepatocytes. Taken together, these results suggest that: (i) there is a close correlation between the mode of organization and expression of cytoskeletal proteins, (ii) such changes induced by the environment in the extracellular and intracellular matrices are programmed events occurring during growth activati

ISSN:0173-0835    

DOI:10.1002/elps.1150110302

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

年代:1990

数据来源: WILEY

摘要:

AbstractDifferent applications of two‐dimensional gel electrophoresis and the research strategies that this methodology allows, with examples drawn from our own work on thyroid and liver cells, are describe

ISSN:0173-0835    

DOI:10.1002/elps.1150110303

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

年代:1990

数据来源: WILEY

摘要:

AbstractTwo‐dimensional polyacrylamide gel electrophoresis (2‐D PAGE) was used to identify and analyse subsets of proteins in cells and tissues. The combination of 2‐D PAGE and [125I]concanavalin A overlay revealed an extraordinary complexity and diversity in the glycoprotein profiles of different cell types. However, the glycoproteins are not expressed idiosyncratically. Rather, their expression is closely linked to the state of differentiation of a particular cell type. Such glycoproteins can therefore be used to generate antibodies specific for differentiated cells. 2‐D PAGE analyses of cellular glycoproteins also revealed a major common glycoprotein of 100 kDa. This was localised to the lumen of the endoplasmic reticulum and is referred to as endoplasmin. The combinat on of 2‐D PAGE with electroblotting and45Ca overlay revealed that endoplasmin and several other luminal endoplasmic reticulum proteins (reticuloplasmins) are high capacity, low affinity calcium binding proteins which could function as calcium storage proteins in the endoplasmic reticulum. One of these called calreticulin is also found in the sarcoplasmic reticulum. 2‐D PAGE and45Ca overlay has been used to demonstrate the presence of a calcium‐binding protein (CP22/sorcin) in the cytosol of rodent multidrug resistant cells. Analyses of murine serum by 2‐D PAGE revealed the presence of a novel stress protein serum amyloid P component. These studies illustrate the value of 2‐D PAGE when used in combination with detection methods which select specific subsets of proteins such

ISSN:0173-0835    

DOI:10.1002/elps.1150110304

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

年代:1990

数据来源: WILEY

摘要:

AbstractInfection of tissue culture cells with certain viruses results in the shutoff of host cell protein synthesis. We have examined virally infected cell lysates using two‐dimensional gel electrophoresis and immunoblotting to ascertain whether initiation factor protein modifications are correlated with translational repression. Moderate increases in eukaryotic initiation factor (eIF)‐2α phosphorylation are detected in reovirus‐ and adenovirus‐infected cells, as reported previously (Samuelet al., 1984; O'Malleyet al., 1989). Neither vesicular stomatitis virus, vaccinia virus, frog virus III, rhinovirus, nor encephalomyocarditis virus caused significantly increased 2α phosphorylation. There were no reproducible, significant changes in eIF‐4A, eIF‐4B, or eIF‐2β in cells infected by any of these viruses. The cleavage of eIF‐4F subunit p220, such as has been previously demonstrated to occur in poliovirus (Etchisonet al., 1982) and rhinovirus (Etchison and Fout, 1985), was not detected in any of the other virus i

ISSN:0173-0835    

DOI:10.1002/elps.1150110305

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

年代:1990

数据来源: WILEY

摘要:

AbstractA technique is described for the analysis of degradation rates of individual intracellular proteins, based on pulse‐chase‐labeling of cells using radioactive amino acids [35S]methionine, two‐dimensional polyacrylamide gel electrophoresis, fluorography and scanning of the fluorograms by a computerized video densitomter. As compared to scintillation counting of individual protein spots resolved by two‐dimensional gel electrophoresis, this method allows a rapid and precise determination of the degradation rates of individual intracellular proteins. In the present study, degradation rates of individual intracellular proteins of normal human skin fibroblasts and skin fibroblasts from patients with Duchenne muscular dystrophy were compared. Rates of degradation for proteins PIIa, PIIb and PIIc recently described as cell‐type‐specific proteins were significantly enhanced (p>0.01) in fibroblast cultures of Duchenne muscular dystr

ISSN:0173-0835    

DOI:10.1002/elps.1150110306

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

年代:1990

数据来源: WILEY

摘要:

AbstractTumor necrosis factor (TNF) synergistically enhanced the antiproliferative activity of interferon‐gamma (IFN‐γ) in both TNF‐sensitive and TNF‐resistant variants of the cervical carcinoma line, ME‐180. TNF alone had no apparent effect on the levels of synthesis of individual proteins in either of these variant cell lines as assessed by computerized two‐dimensional gel analysis of cell lysates using the PDQUEST system. However, IFN‐γ enhanced the levels of 18 polypeptides and suppressed the levels of 10 polypeptides in both cell lines. When used in combination in both cell lines, TNF and IFN‐γ induced the synthesis of 10 polypeptides that were not induced by either agent alone. These synergistically induced polypeptides may be crucial to the mechanism of the synergistic antiproliferative action of TNF and IFN

ISSN:0173-0835    

DOI:10.1002/elps.1150110307

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

年代:1990

数据来源: WILEY

摘要:

AbstractA two‐dimensional (2‐D) gel database of proteins from noncultured total normal human epidermal keratinocytes has been established. A total of 1449 [35S]methionine labelled proteins (1112 isoelectric focusing, 337 nonequilibrium pH gradient electrophoresis) were resolved and recorded using computer assisted (PDQ‐SCAN and PDQUEST software) 2‐D gel electrophoresis. By matching the protein patterns of total keratinocytes and transformed human amnion cells (master database; Celiset al., Leukemia1988,2, 561–602) as well as by 2‐D immunoblotting and microsequencing of keratinocyte proteins, it was possible to identify 72 polypeptides in the keratinocyte database. The database also includes data on polypeptides that are synthesized at a higher level by keratinocytes enriched in basal cells, and on six secreted proteins which are produced, albeit at a reduced rate, by normal keratinocytes and that are strongly up‐regulated in psoriatic epidermis (Celiset al., FEBS Lette

ISSN:0173-0835    

DOI:10.1002/elps.1150110308

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

年代:1990

数据来源: WILEY

摘要:

AbstractAn improved method of high‐resolution two‐dimensional gel electrophoresis has been used to study the patterns of protein synthesis in wing imaginal discs of late instar larvae ofDrosophila melanogaster. A total of one thousand and twenty five labelled polypeptides (787 acidic and 238 basic) have so far been separated and catalogued. For convenience, all these polypeptides have been numbered and their position fixed by its molecular weight and relative mobility. They are indicated on a reference protein map for further stud

ISSN:0173-0835    

DOI:10.1002/elps.1150110309

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

年代:1990

数据来源: WILEY

摘要:

AbstractTwo‐dimensional (2‐D) gel electrophoresis methods for separating complex mixtures of proteins have not changed fundamentally since their original description in the late 1970′s. Nevertheless, 2‐D gel resolution has improved substantially as a result of a series of incremental modifications. One of these was the development of the „giantgel”︁ format, using gels measuring at least 30 x 30 cm to provide the highest resolution 2‐D gel system available. As originally described, this procedure has several important limitations: it requires custom‐built equipment; it is expensive in terms of time, reagents, film and support matrices; and it generates gels which are difficult to manupulate, particularly for silver staining. This report describes modifications in the giant gel procedure to permit use of a commercially available gel apparatus and to obtain gaint gels of improved mechanical strength suitable for silver staining. The resolution of giant gels is compared with that obtained using two systems currently being marketed for use by laboratories performing large numbers of 2‐D gel analyses. The smaller format gels resolved fewer proteins, by 30–40%, compared with the giant gels. This difference in resolving power suggests that giant gels will continue to be useful in

ISSN:0173-0835    

DOI:10.1002/elps.1150110310

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

年代:1990

数据来源: WILEY

ISSN:0173-0835    

DOI:10.1002/elps.1150110311

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

年代:1990

数据来源: WILEY