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1.
Tumor promoter and fibronectin induce actin stress fibers and focal adhesion sites in spreading human erythroleukemia (HEL) cells 总被引:2,自引:0,他引:2
The effects of plasma fibronectin (pFn) and the tumor promoter 12-0-tetradecanoyl-phorbol-13-acetate (TPA) on adhesion and cytoskeletal organization of human erythroleukemia (HEL) cells were studied. HEL cells, that normally grow in suspension, attached rapidly on pFn-coated growth substratum and some cells showed spreading. Upon exposure to TPA most of the cells adhered and showed some degree of spreading also when plated on plastic. The spread cells showed mostly peripheral accumulations of F-actin in addition to actin fibers seen in some of the cells. When the cells were plated in the presence of TPA on pFn or on pFn-fragments, containing the cell binding site, all the cells adhered rapidly, spread extensively, organized prominent F-actin stress fibers and typical ventral plaques of vinculin and alpha-actinin. Both proteins were revealed also in the suspended cells by Western blot analysis. When plated on substratum coated with other pFn-fragments or laminin, the HEL cells did not adhere or spread. Both adhesion on pFn as well as formation of stress fibers in the presence of TPA could be prevented by the synthetic peptide Arg-Gly-Asp-Ser (RGDS). HEL cells were also able to organize typical ventral fibrillar arrays of Fn. Immunostaining and metabolic labeling experiments showed that the cells did not contain or synthesize Fn, indicating that the plaques were formed from exogenous pFn by the cells. The results suggest that Fn and TPA synergistically induce the organization of the actomyosin system in HEL cells by promoting the formation of prominent actin stress fibers and focal adhesion sites. 相似文献
2.
The properties of the nucleotides tightly bound with mitochondrial F1-ATPase were examined. One of three bound nucleotide molecules is localized at the site with Kd approximately 10(-7) M and released with koff approximately 0.1 s-1. The second nucleotide molecule is bound with the enzyme with Kd approximately 10(-8) M and koff for its dissociation is 3 X 10(-4) s-1. The third is never released even in the presence of 1 mM ATP or ADP. The last two nucleotides are believed to be bound at the noncatalytic sites of F1-ATPase. Pyrophosphate promotes liberation of two releasable nucleotide molecules, decreasing the affinity of the enzyme to AD(T)P. From the results obtained it follows that the only suitable criterion for localization of the nucleotide at the F1-ATPase catalytic site is the high rate (koff greater than or equal to 0.1 s-1) of its spontaneous release. 相似文献
3.
Role of the polar head group stereoconfiguration in the cation-induced aggregation of dimyristoylphosphatidylglycerol vesicles 总被引:2,自引:0,他引:2
Cation-induced aggregation of small unilamellar vesicles of 1,2-dimyristoyl-sn-glycero-3-phosphatidyl-sn-1'-glycerol (1'-DMPG), the corresponding 3' stereoisomer (3'-DMPG), and their 1:1 mixture was studied as a function of the concentration of different mono- and divalent cations. The order of efficiency, Na+ greater than Li+ greater than K+ greater than Cs+, of the monovalent cations to induce the aggregation of DMPG vesicles is the same for both stereoisomers and their mixture. However, significant differences in the Na+-induced aggregation of 1'-DMPG and 3'-DMPG were evident. The threshold concentration of aggregation by Na+ was 0.35 M for 3'-DMPG, 0.55 M for 1'-DMPG, and 0.50 M for the mixed liposomes. Such difference in the aggregation of DMPG stereoisomers was not observed for the other mono- and divalent cations. The higher affinity of 3'-DMPG for Na+ is suggested to be due to a slightly different favored conformation of the head group glycerol moiety. Aggregation of the stereoisomers by 1 M NaCl was identical, indicating that the differences in the affinity of 1'-DMPG and 3'-DMPG for sodium can be overcome by very high ionic strength. Inclusion of 20 mol % cholesterol in vesicles enhanced the aggregation of 1'-DMPG and decreased the aggregation of 3'-DMPG by Na+ and thus abolished the difference between the two stereoisomers. 相似文献
4.
I Virtanen K Heikinheimo M Hormia T Kivel? L Laitinen L E Thornell 《The International journal of developmental biology》1989,33(1):55-61
Intermediate filaments are found in most nucleated cells as part of their cytoskeleton. Intermediate filaments are formed by different proteins in cells of major tissues types. Therefore, antibodies against intermediate filaments can be used in tissue typing, in the analysis of cell lineages during development and in the elucidation of the origin of unknown tumors. 相似文献
5.
I. Virtanen 《Histochemistry and cell biology》1990,94(4):397-401
Summary Fluorochrome-coupled Helix pomatia agglutinin (HPA), but not other lectin-conjugates with the same nominal specificity, bound specifically to the Golgi apparatus in cultured human fibroblasts, revealing a cytoplasmic juxtanuclear reticular structure. Unlike other Golgi-binding lectins the HPA-conjugates did not bind to the cell surface membrane or pericellular matrix. Experiments with 35S-methionine-labeled cells showed that HPA recognized two glycoproteins of Mr 170000 and 400000 among the secreted products of fibroblasts and two major cellular glycoproteins of Mr 40000 and Mr 180000 in Triton X-100 extracts of the cells. The two cellular HPA-binding polypeptides were also found in cells depleted of secretory products and in cells pulselabeled shortly with 35S-methionine and then chased with methionine containing medium up to 12 h. These findings suggest that the two cellular glycoproteins recognized by HPA are retained in the Golgi apparatus and are therefore not precursors of secretory proteins. The results suggest that there are two endogenous, Golgi apparatus-specific glycoproteins in cultured human fibroblasts with terminal non-reducing O-glycosidic N-acetyl galactosaminyl residues. 相似文献
6.
M Hormia A L Kariniemi L Laitinen I Virtanen 《The journal of histochemistry and cytochemistry》1988,36(10):1231-1237
Dolichos biflorus agglutinin (DBA) binds to N-acetyl-D-galactosamine (GalNAc) residues in glycoconjugates and agglutinates erythrocytes carrying blood group antigen A. In cryostat sections of various tissues from blood group-specified humans, fluorochrome-coupled DBA bound preferentially to fusiform connective tissue cells and to certain epithelial cells. The connective tissue cells were identified as mast cells by their typical metachromasia in consecutive staining with toluidine blue. Double labeling with DBA and conjugated avidin revealed two distinct populations of mast cells. In several tissues the DBA-reactive cells likewise displayed uniform avidin reactivity. In intestinal mucosa, however, morphologically distinct DBA-binding mast cells were found, which were labeled with the avidin conjugates only in specially fixed paraffin sections. DBA did not bind to vascular endothelial cells, which could be identified by double staining with antibodies to factor VIII-related antigen. Labeling with Helix pomatia agglutinin (HPA), another blood group A-reactive lectin, resulted in distinct blood group-dependent fluorescence of the endothelia. Sophora japonica agglutinin (SJA), a blood group B-reactive lectin, labeled vascular endothelial cells in tissues from blood group A, AB, and B donors. HPA and SJA reacted with small mast cells in the gastrointestinal mucosa but failed to label large mast cells in any of the tissues. These results indicate that the blood group reactivity of lectins, as determined by erythroagglutination, is not necessarily consistent with their reactivity with blood group determinants in tissue sections. Moreover, DBA conjugates appear to be a reliable probe for detection of mast cells in various human connective tissues. 相似文献
7.
Using immunoblotting and immunofluorescent microscopy, we showed the presence in Xenopus laevis oocytes of two prosomal proteins (27 and 31-33 kDa) and studied their distribution during oogenesis. In the ooplasm, both proteins are detected in prosomal clusters of various size. During previtellogenesis, prosomal proteins are diffusely distributed in the nucleoplasm and form evenly distributed clusters in the cytoplasm. During oocyte growth, prosomal proteins disappear from the nucleus and form animal-vegetal and cortical gradients in the cytoplasm. In the course of oocyte maturation prosomal clusters become smaller. After artificial activation of the egg, the dorso-ventral gradient of distribution of prosomal proteins is observed. Double immunohistochemical labeling revealed morphological association between prosomal clusters and fibril-like structures of the oocyte containing actin and myosin. The latter are then replaced by diffusely distributed actin and myosin. Thus, correlation is observed between localization of the acto-myosin complex of the oocyte and that of prosomal proteins. 相似文献
8.
Phospholipase A2 assay using an intramolecularly quenched pyrene-labeled phospholipid analog as a substrate 总被引:1,自引:0,他引:1
A phospholipid analog 1-palmitoyl-2-6(pyren-1-yl)hexanoyl-sn-glycero-3-phospho-N- (trinitrophenyl)aminoethanol (PPHTE) in which pyrene fluorescence is intramolecularly quenched by the trinitrophenyl group was used as a substrate for pancreatic phospholipase A2. Upon phospholipase A2 catalyzed hydrolysis of this molecule pyrene monomer fluorescence emission intensity increased as a result of the transfer of the pyrene fatty acid to the aqueous phase. Optimal conditions for phospholipase A2 hydrolysis of PPHTE were similar to those observed earlier for other pyrenephospholipids (T. Thuren, J. A. Virtanen, R. Verger, and P. K. J. Kinnunen (1987) Biochim. Biophys. Acta 917, 411-417). Although differential scanning calorimetry revealed no thermal phase transitions for PPHTE between +5 and +60 degrees C the Arrhenius plot of the enzymatic hydrolysis of the lipid showed a discontinuity at 30 degrees C. The molecular origin of this discontinuity remains at present unknown. To study the effects of dimyristoylphosphatidylcholine (DMPC) phase transition at 23.9 degrees C on phospholipase A2 reaction PPHTE was mixed with DMPC in a molar ratio of 1:200 in small unilamellar vesicles. The hydrolysis of DMPC-PPHTE vesicles was measured by following the increase in pyrene monomer fluorescence emission due to phospholipase A2 action on PPHTE. Below the phase transition of DMPC the enzymatic reaction exhibited a hyperbolic behavior. At the transition as well as at slightly higher temperatures a lag period was observed. The longest lag period was approximately 20 min. Above 26 degrees C no lag time could be observed. However, the reaction rates were slower than below the phase transition temperature.(ABSTRACT TRUNCATED AT 250 WORDS) 相似文献
9.
Summary Conditions were used where the action of porcine pancreatic phospholipase A2 on phospholipids can be followed in the absence of added calcium and the catalytic activity is supported by the calcium brought with the nanomolar enzyme. Therefore, alterations in the enzyme velocity resulting from the presence of spermine or spermidine could be specifically studied using 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (PPHPC) and 1-palmitoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphoglycerol (PPHPG) as substrates. Both spermine and spermidine activated the hydrolysis of PPHPG fourfold at polyamine/phospholipid molar ratios of approximately 11 and 121, respectively. Double-reciprocal plots of enzyme activityvs. PPHPG concentration revealed the enhancement to be due to increased apparentV
max while the apparentK
m
was slightly increased. In the presence of 4mm CaCl2 inhibition by polyamines of PPHPG hydrolysis by phospholipase A2 was observed. Using synthetic diamines we could further demonstrate that two primary amino groups are required for the activation. In the absence of exogenous CaCl2 polyamines inhibited the hydrolysis of PPHPC by phospholipase A2. The presence of 4mm CaCl2 reversed this inhibition and a twofold activation was observed at 10 m spermine. The results obtained indicate that the activation of PLA2 by spermine and spermidine is produced at the level of the substrate, PPHPG. This implies the formation of complexes of phosphatidylglycerol and polyamines with defined stoichiometries. 相似文献
10.
Reorganization of intermediate filament cytoskeleton in induced metanephric mesenchyme cells is independent of tubule morphogenesis 总被引:5,自引:0,他引:5
The metanephric mesenchyme becomes converted into epithelial tubules if cultured in transfilter contact with an inductor tissue. The expression of intermediate filaments (IFs), used as cell-type-specific markers has been studied in this model system for differentiation and organogenesis. In immunofluorescence microscopy of frozen sections, the undifferentiated cells of isolated metanephric mesenchymes uniformly showed IFs of vimentin type only. Also, when cultured as a monolayer, cells from the uninduced mesenchymes showed only vimentin filaments. In frozen sections of transfilter explants, epithelial tubules apparently negative for vimentin could be seen after 3 days in culture, but expression of cytokeratin could not be demonstrated in the developing tubules until the fourth day of culture. Sections of explants cultured further showed tubule cells with distinct fibrillar cytokeratin positivity. The appearance of cytokeratin in the explants was also demonstrated with immunoblotting experiments, using two different cytokeratin antibodies. Expression of IFs was further examined in monolayer cultures of metanephric mesenchymes which had been initially exposed to a short transfilter induction pulse. In these experiments, cytokeratin-positive cells could be demonstrated after a total of 4 days in culture. Double immunofluorescence experiments showed varying amounts of vimentin in the cytokeratin-positive cells: after 4 days in culture, most cytokeratin-positive cells still showed vimentin-positivity although often in a nonfibrillar form. During further culture, gradual disappearance of vimentin-specific fluorescence was observed in cytokeratin-positive cells. The results suggest that the vimentin-positive metanephric mesenchyme cells lose their fibrillar vimentin organization upon induction that leads to kidney tubule formation. This change may be essential for the transformation from an undifferentiated mesenchymal cell into a specialized epithelial cell. Cytokeratin filaments, regarded as a marker for epithelial cells, seem to appear simultaneously with or soon after the change in vimentin organization. These changes in IF expression also occur in monolayer cultures of mesenchyme cells initially exposed to a short transfilter induction pulse. This suggests that epithelial differentiation, as revealed by the emergence of cytokeratin positivity, may occur even in the absence of a clear morphological differentiation and three-dimensional organization of the cells. 相似文献