摘要:

AbstractIn this study both a Ligand‐dependent treatment [concanavalin A (Con A)] and a ligand‐independent treatment [high‐voltage pulsed galvanic stimulation (HVPGS)]have been used to initiate lymphocyte activation via a transmembrane signaling process. Our results show that both treatments cause the exposure of two different hormone [insulin and interIeukin‐2 (IL‐2)] receptors within the first 5 min of stimulation. When either insulin or IL‐2 is present in the culture medium, the stimulated lymphocytes undergo the following responses: (1) increased free intra‐cellular Ca2+activity; (2) aggregation of insulin or IL‐2 receptors into patch/cap structures; (3) tyrosine‐kinase‐specific phosphorylation of a 32‐kd membrane protein; and finally (4) induction of DNA synthesis.Further analysis indicates that hormone receptor capping is inhibited by (1) cytochalasin D, suggesting the involvement of microfilaments; (2) sodium azide, indicating a requirement for ATP production; and (3) W‐5, W‐7, and W‐12 drugs, implying a need for Ca2 +/calmodulin activity. Treatment with these metabolic or cytoskeletal inhibitors also prevents both the tyrosine‐kinase‐specific protein phosphorylation and DNA synthesis which normally follow hormone receptor capping. Double immunofluorescence staining shows that actomyosin, Ca2+/calmodulin, and myosin light‐chain kinase are all closely associated with the insulin and IL‐2 receptor cap structures.These findings strongly suggest that an actomyosin‐mediated contractile system (regulated by Ca2+, calmodulin, and myosin light‐chain kinase in an energy‐dependent manner) is required not only for the collection of insulin and IL‐2 receptors into patch and cap structures but also for the subsequent activation of tyrosine kinase and the initiation of DNA synthesis. We, therefore, propose that the exposure and subsequent patching/capping of at least one hormone receptor are required for th

ISSN:0730-2312    

DOI:10.1002/jcb.240370202

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

年代:1988

数据来源: WILEY

摘要:

AbstractRous sarcoma virus‐trans formed baby hamster kidney fibroblasts (RSV/B4‐BHK) adhere to a fibronectin‐coated substratum by means of dot‐like adhesion sites called podosomes in view of their shape and function as cellular feet (Tarone et al.:Exp Cell Res159:141, 1985). Podosomes concentrate tyrosine‐phosphorylated proteins, including pp60v‐src, and appear in many cells transformed by oncogenes coding for tyrosine kinases. In this paper we used orthovanadate, an inhibitor of phosphotyrosine phosphatases, in order to increase the cellular concentration of phosphotyrosine and to study whether this treatment induced the cytoskeleton remodeling leading to the formation of podosomes. Indeed, orthovanadate (10–100 μM) induced in a time‐and dose‐dependent manner the redistribution of F‐actin and the formation of podosomes in BHK cells. Cytoskeleton remodeling occurred along with a marked increase of tyrosine phosphorylatcd proteins. The vanadate effect on the cytoskeletal phenotype was enhanced by the simultaneous treatment of cells with a phorbol ester. Under the latter conditions almost all BHK cells showed podosomes. The vanadate effect was reversible insofar as podosomes and tyrosine‐phosphorylated proteins disappeared. Then, vanadate treatment of normal cells induced the cascade of events leading to the cytoskeletal changes typical of transformation and suggested that the transformed cytoskeletal phenotype may he primarily induced by the tyrosine phosphorylation of unknown target(s) operated

ISSN:0730-2312    

DOI:10.1002/jcb.240370203

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

年代:1988

数据来源: WILEY

摘要:

AbstractIt has been shown that cAMP‐dependent phosphorylation of a soluble sperm protein is important for the initiation of flagellar motion. The suggestion has been made that this motility initiation protein, named axokinin, is the major 56,000‐dalton phos‐phoprotein present in both dog sperm and in other cells containing axokinin‐like activity. Since the regulatory subunit of a type II cAMP‐dependent protein kinase is a ubiquitous cAMP‐dependent phosphoprotein of similar subunit molecular weight as reported for axokinin, we have addressed the question of how many soluble 56,000‐dalton cAMP‐dependent phosphoproteins are present in mammalian sperm. We report that in bovine sperm cytosol, the ratio of the type I to type II cAMP‐dependent protein kinase is approximately 1:1. The type II regulatory subunit is related to the non‐neural form of the enzyme and undergoes a phosphorylation‐dependent electrophoretic mobility shift. The apparent subunit molecular weights of the phospho and dephospho forms are 56,000 and 54,000 daltons, respectively. When bovine sperm cytosol or detergent extracts are phosphorylated in the presence of catalytic subunits, two major proteins are phosphorylated and have subunit molecular weights of 56,000 and 40,000 daltons. If, however, the type II regulator subunit (RII) is quantitatively removed from these extracts using cither immobilized cAMP or an anti‐RII monoclonal affinity column, the ability to phosphorylate the 56,000‐ but not 40,000‐dalton polypeptide is lost. These data suggest that the major 56,000 dalton cAMP‐dependent phosphoprotein present in bovine sperm is the regulatory subunit of a type II cAMP‐dependent protein kinase and not the motil

ISSN:0730-2312    

DOI:10.1002/jcb.240370204

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

年代:1988

数据来源: WILEY

摘要:

AbstractAn automated, video‐driven system was used to measure approximately 30 parameters of cell motion and accompanying changes in shape. This “Dynamic Morphology System” is based upon the Expertvision Motion Analysis System and is driven by a SUN computer. With the aid of this system, amoebic movement and shape changes were compared for vegetative wild‐typeDictyostelium discoideumamoebae and a motility mutant, Mo‐1. The measured parameters included speed, angle change, bearing, length, width, roundness, boundary flow, and curvature; and cell behavior was visualized monitoring amoebic tracks, difference pictures, and a newly developed ring expansion plot. Wild‐type cells remained elongated, moved continuously and retained polarity throughout migration. In contrast, Mo‐1 did not translocate, was round rather than elongated, formed bulges rather than elongated pseudopods, and exhibited no polarity. In contrast to the anterior f‐action distribution in wild‐type cells, f‐actin in Mo‐1 was distributed evenly as a shell just under the entire plasma membrane, a distribution consistent with the lack of polar

ISSN:0730-2312    

DOI:10.1002/jcb.240370205

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

年代:1988

数据来源: WILEY

摘要:

AbstractRecent interest has focused on fucosylated epitopes expressed on human neoplasms. The plant lectinUlex europusagglutinin, Type I (UEA) binds fucosylated oligosac‐charides, while UEA‐reactive substances have a tissue distribution similar to carci‐noembryonic antigen (CEA). We sought to determine if UEA reacted with CEA in extracts of fresh primary and metastatic colorectal carcinomas and paired normal tissues. The extracts were electrophoretically transferred to nitrocellulose membranes after the proteins were separated by SDS‐PAGE in 10% polyacrylamide gels. The transfer membranes were then stained with peroxidase‐conjugated UEA (UEA‐P) or antibody to CEA (CEA‐P). UEA‐P reacted with a 170‐190‐kDa band in extracts of 22 of 30 primary tumors, 10 of 12 metastases, but only 1 of 5 villous adenomas. UEA‐P generally did not react with normal colon or liver extracts. UEA‐P also did not bind to 170–190‐kDa molecules in Western transfers of a breast carcinoma metastatic to bowel and a focal nodular hyperplasia of liver. CEA‐P displayed similar reactivity and detected CEA in a tumor extract negative for UEA. Fucose blocked binding of UEA‐P to Western transfers of tumor extracts. CEA‐P reacted with a 170–190‐kDa substance in tumor extracts eluted with fucose from a column of immobilized UEA. Thus, UEA reacts with fucosylated oligosaccharides on most, but not all, species of CEA and may be a useful adju

ISSN:0730-2312    

DOI:10.1002/jcb.240370206

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

年代:1988

数据来源: WILEY

摘要:

AbstractPrevious studies from this and other laboratories have shown that variants of tumor cell lines can be selected for resistance to the lytic action of natural killer (NK) cells. One of these (K562‐Clone I), when made resistant to the toxic effects of Concanavalin A (Con A‐R1), regained its sensitivity to NK. Comparing the plasma membranes of Clone I and Con A‐R1, we observed (1) a very similar electrophoretic pattern of membrane glycoproteins identified by binding to the lectins Con A, WGA, PNA, and SBA; (2) an increase in binding ofUlex europaeuslectin to a group of glycoproteins from Con A‐R1 that were sensitive to treatment with fucosidase and N‐glycanase and that had a diffuse mobility ranging in apparent molecular weight from 30 to 200 kDa; and (3) a marked decrease in binding of an antibody reactive with the lactoneofucopentaose III antigen (Lewis x). This constellation of changes is an unusual pattern to follow Con A resistance and may point to a pathway of glyco‐sylation that a leukemic cell might use to modify its recognition by the N

ISSN:0730-2312    

DOI:10.1002/jcb.240370207

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

年代:1988

数据来源: WILEY

摘要:

AbstractMurine squamous carcinoma cells (KLN205) grown in a medium supplemented with the retinoid, 13‐cis retinoic acid (RA), had dose‐dependent, selective increases in the expression of certain lectin receptors, which correlated with a dramatic decrease in the ability to form pulmonary colonies (P−.0003) (Couch MJ, Pauli BU, Weinstein RS, Coon JS: JNCI, 78:971 −977, 1987). These findings suggest a possible relationship between the RA‐induced glycoconjugate alterations and the decreased experimental metastatic behavior. We further define the mechanism of RA's action. The finding that RA treatment (5 × 10−6M, 5 × 10−7M) did not perturb the cell cycle of KLN205 cells provides further proof that the decreased metastatic behavior is not attributable to any inhibition in the rate of growth or to alterations in the cell cycle. Furthermore, since stable subpopulations with variable lectin binding could not be detected, the mechanism of RA's action does not appear to be due to selection of variant tumor‐cell subpopulations. Finally, in a scries of experiments designed to determine the reversibility of the RA treatment, the RA‐induced decrease in metastatic behavior reverted back to a more metastatic state in the same time frame (3 days) as the reversion of the RA‐induced changes in cell‐surface glycoconjugate expression. This reversion provides further evidence for a close relationship between the RA‐induced modulation of tumor cell‐surface glycoconjugate expression and the decreased metastatic behavior; it suggests that transient, reversible modulation of the tumor cell surface may play a role in dete

ISSN:0730-2312    

DOI:10.1002/jcb.240370208

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

年代:1988

数据来源: WILEY

摘要:

AbstractRecent studies have desmonstrated that Rous sarcoma virus‐transformed baby hamster kidney (RS‐BHK) cells express twofold higher levels of those N‐linked oligosac‐charides that contain the sequence [GlcNAc‐β(1,6)Man (1,6)] compared to nontrans‐formed parental BHK cells (Pierce and Arango,J. Biol. Chem. 261, 10772 [1986]). We have investigated in RS‐BHK and BHK cells the activity of UDP‐GlcNAc:α‐D‐mannoside β(l,6)N‐acetylglucosaminyltransferase V, the enzyme that begins the synthesis of the sequence that is increased in the RS‐BHK cells. We have measured GnT V activity using UDP‐ [3H]‐ GlcNAc and a synthetic oligosaccharide acceptor, GlcNAcβ(1,2)Man α(1,6)Manβ‐O‐ (Ch2)8COOCH3, separating the radioactive product by a newly devised reverse‐phase chromatographic technique. Assayed under optimal conditions, the specific activity of GnT V is about fourfold higher in RS‐BHK sonicates than in BHK sonicates, suggesting that this increase in activity may be the primary mechanism that causes the increase in [GlcNAcβ(1,6) Man] sequences in the RS‐BHK cells. The apparent Km, values of the enzymes in RS‐BHK and BHK cell sonicates for UDP‐GIcNAc and the synthetic acceptor are similar, as are the pH optima. These results suggest that the increase in GnT V‐specific activity in RS‐BHK cells is not caused by the presence in these cells o

ISSN:0730-2312    

DOI:10.1002/jcb.240370209

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

年代:1988

数据来源: WILEY

摘要:

AbstractThe internalization of surface‐bound diphtheria toxin (DT) in BS‐C‐1 cells correlated with its appearance in intracellular endosomal vesicles; essentially no toxin appeared within secondary lysosomal vesicles. In contrast, internalized epidermal growth factor (EGF) was localized within both endosomal and lysosomal vesicles. Upon prein‐cubation of cells with leupeptin, a lysosomal protease inhibitor, a threefold increase in the accumulation of EGF into lysosomes was observed. Under identical conditions, essentially all of the diphtheria toxin remained within endosomes (less than 2% of the intracellular diphtheria toxin accumulated in the lysosomaJ fraction), indicating that the inability to detect diphtheria toxin in lysosomes was not due to its rapid turnover within this vesicle. Following internalization of EGF or DT, up to 40% of the lgand appeared in the medium as TCA‐soluble radioactivity. EGF degradation was partially leupeptin‐sensitive and markedly NH4Cl‐sensitive, indicating lysosomal degradation. In contrast, DT A‐fragment degradation was resistant to these inhibitors, while B‐fragment showed only partial sensitivity. These data suggest that the bulk of endocytosed diphtheria toxin is localized within endosomes and degraded by a pathway essentially indepen

ISSN:0730-2312    

DOI:10.1002/jcb.240370210

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

年代:1988

数据来源: WILEY

摘要:

AbstractCloned cDNAs were used in hybridization analyses to assess hormonal responsiveness of two similarly regulated genes in livers of late‐term fetal rats. Transcription of the tyrosine aminotransferase gene and of gene 33 (Lee et al.:J Biol Chem260:16433–16438, 1985) is enhanced by glucocorticoids and by each of the usually antagonistic hormonal agents, insulin and cAMP, in adult liver, and that of both genes is developmentally activated at or just prior to birth. The mRNA of gene 33 was found to be significantly increased by each of the hormonal regulators in livers of fetuses treated in utero. Expression of the nearly silent aminotransferase gene in fetal liver was appreciably increased by cAMP but was refractory to control by either glucocorticoids or insulin; capacity of this gene to respond to insulin was not realized until several days postpartum. The data indicate specificity in the developmental acquisition of the capacity of individual genes to respond to hormonal regulat

ISSN:0730-2312    

DOI:10.1002/jcb.240370211

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

年代:1988

数据来源: WILEY