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  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 9-24 
    ISSN: 0091-7419
    Keywords: nascent chains ; co-translational modification ; glycosylation ; polypeptide folding ; covalent assembly ; heavy and light chains ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have investigated the in vivo co-translational covalent modification of nascent immunoglobulin heavy and light chains. Nascent polypeptides were separated from completed polypeptides by ion-exchange chromatography of solubilized ribosomes on QAE-Sephadex. First, we have demonstrated that MPC 11 nascent heavy chains are quantitatively glycosylated very soon after the asparaginyl acceptor site passes through the membrane into the cisterna of the rough endoplasmic reticulum. Nonglycosylated completed heavy chains of various classes cannot be glycosylated after release from the ribosome, due either to rapid intramolecular folding and/or intermolecular assembly, which cause the acceptor site to become unavailable for the glycosylation enzyme. Second, we have shown that the formation of the correct intrachain disulfide loop within the first light chain domain occurs rapidly and quantitatively as soon as the appropriate cysteine residues of the nascent light chain pass through the membrane into the cisterna of the endoplasmic reticulum. The intrachain disulfide loop in the second or constant region domain of the light chain is not formed on nascent chains, because one of the cysteine residues involved in this disulfide bond does not pass through the endoplasmic reticulum membrane prior to chain completion and release from the ribosome. Third, we have demonstrated that some of the initial covalent assembly (formation of interchain disulfide bonds) occurs on nascent heavy chains prior to their release from the ribosome. The results are consistent with the pathway of covalent assembly of the cell line, in that completed light chains are assembled onto nascent heavy chains in MPC 11 cells (IgG2b), where a heavy-light half molecule is the major initial covalent intermediate; and completed heavy chains are assembled onto nascent heavy chains in MOPC 21 cells (IgG1), where a heavy chain dimer is the major initial disulfide linked intermediate.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 269-281 
    ISSN: 0091-7419
    Keywords: fibronectin ; cell fractionation ; glial fibrillary acidic protein ; immunofluorescent labeling ; neuronal-glial cell interactions ; brain cell culture ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a basic approach to investigations of neuronal-glial interactions during both normal brain development and its pathogenesis, embryonic brain cell populations were fractionated into purified neuronal and glial components. Using separation procedures based on differential adhesion and cytotoxicity, the isolated neuronal and glial phenotypes could be identified by distinct morphological and biochemical characteristics, including the visualization of glial fibrillary acid protein (GFA) within glial cells in immunohistochemical assays with monospecific anti-GFA serum.When unfractionated cerebrum cells dissociated from 10-day chick or 14-day mouse embryos were plated as monolayers and cultured for 1-14 days, monospecific antiserum against fibronectin (LETS glycoprotein) was found to react with many, but not all, of the cells as revealed by indirect immunofluorescence microscopy. The isolated neuronal and glial components of these populations were used to determine whether the appearance of membrane-associated fibronectin was characteristic of one cell type or the other, or both, and if neuronal-glial cell interaction was required for its expression. It was found that the surfaces of glial cells, completely isolated from neurons, showed an intense fluorescent reaction to the anti-fibronectin serum. In contrast, the purified neuronal cultures showed no fluorescence with either the anti-GFA or anti-fibronectin sera. These results demonstrate fibronectin as a cell surface protein associated primarily with glial cells and independent of neuronal-glial cell interaction for its expression. Furthermore, the results indicate that the fibronectin observed on glial cell surfaces in these cultures is produced endogenously and is not due to the preferential binding of fibronectin present in the culture medium. The role of fibronectin as an adhesive molecule in neuronal-glial interactions is discussed.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 319-319 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Additional Material: 1 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 339-347 
    ISSN: 0091-7419
    Keywords: switch hypothesis ; cilia ; motility ; vanadate ; calcium ; dynein ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Lateral (L) cilia of freshwater mussel (Margaritana margaritifera and Elliptio complanatus) gills can be arrested in one of two unique positions. When treated with 12.5 mM CaCl2 and 10-5 M A23187 they arrest in a “hands up” position, ie, pointing frontally. When treated with approximately 10 mM vanadate (V) they arrest in a “hands down” position, ie, pointing abfrontally. L-cilia treated with 12.5 mM CaCl2 and 1 mM NaN3 also arrest in a “hands down” position; substitution of 20 mM KC1 and 1 mM NaN3 causes cilia to move rapidly and simultaneously to a “hands up” position.The observations suggest that there are two switching mechanisms for activation of active sliding in ciliary beat one at the end of the recovery stroke and the other at the end of the effective stroke; the first is inhibited by calcium and the second by vanadate or azide. This is consistent with a model of ciliary beating where microtubule doublet numbers 1, 2, 3, and 4 are active during the effective stroke while microtubule doublets numbers 6, 7, 8, and 9 are passive, and the converse occurs during the recovery stroke.
    Additional Material: 6 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 391-399 
    ISSN: 0091-7419
    Keywords: cold-insoluble globulin ; carbohydrate structure ; human plasma ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cold-insoluble globulin (CIg) is a member of a group of circulating and cell-associated, high-molecular-weight glycoproteins termed fibronectins. CIg was isolated from human plasma by affinity chromatography on gelatin-Sepharose. SDS-polyacrylamide gel electrophoresis of the purified glycoprotein gave a double band that migrated near myosin. The CIg glycopeptides were released by pronase digestion and isolated by chromatography on Sephadex G-50. Affinity chromatography of the major G-50 peak on Con A-Sepharose resulted in two fractions: one-third of the glycopeptides were unbound and two-thirds were weakly bound (WB). Sugar composition analysis of the unbound glycopeptides by GLC of the trimethylsilyl methyl glycosides gave the following molar ratios: sialic acid, 2.5; galactose, 3.0; N-acetylglucosamine, 4.9; and mannose, 3.0. Sugar composition analysis of the WB glycopeptides gave the following molar ratios: sialic acid, 1.7; galactose, 2.0; N-acetylglucosamine, 4.1; and mannose, 3.0. The WB CIg glycopeptides cochromatographed on Sephadex G-50 with WB transferrin glycopeptides giving an estimated molecular weight of 2,800. After degradation with neuraminidase alone or sequentially with β-galactosidase the CIg and transferrin glycopeptides again cochromatographed. Methylation linkage analysis of the intact and the partially degraded glycopeptides indicated that the carbohydrate structure of the major human CIg glycopeptide resembles that of the major glycopeptide from transferrin.
    Additional Material: 5 Ill.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 401-427 
    ISSN: 0091-7419
    Keywords: fibronectin ; glycosaminoglycans ; proteoglycans ; adhesion ; substrate-attached material cytoskeleton ; immunofluorescence ; heparan sulfate ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: When normal or SV40-transformed Balb/c 3T3 cells are treated with the Ca++-specific chelator EGTA, they round up and pull away from their footpad adhesion sites to the serum-coated tissue culture substrate, as shown by scanning electron microscope studies. Elastic membranous retraction fibers break upon culture agitation, leaving adhesion sites as substrate-attached material (SAM) (Cells leave “footprints” of substrate adhesion sites during movement by a very similar process.) SAM contains 1-2% of the cell's total protein and phospholipid content and 5-10% of its glucosamine-radiolabeled polysaccharide, most of which is glycosaminoglycan (GAG). By one- and two-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis, there is considerable enrichment in SAM for specific GAGs; for the glycoprotein fibronectin; and for the cytoskeletal proteins actin, myosin, and the subunit protein of the 10 nm-diameter filaments. Fibrillar fibronectin of cellular origin and substratum bound fibronectin of serum origin (cold-insoluble globulin, CIg) have been visualized by immunofluorescence microscopy. The GAG composition in SAM has been examined under different cellular growth and attachment conditions. Heparan sulfate content correlates with glycopeptide content (derived from glycoprotein). Newly attaching cells deposit SAM with principally heparan sulfate and fibronectin and little of the other GAGs. Hyaluronate and chrondroitin proteoglycans are coordinately deposited in SAM as cells begin spreading and movement over the substrate. Cells attaching to serum-coated or CIg coated substrates deposited SAM with identical compositions. The proteoglycan nature of the GAGs in SAM has been examined as well as the ability of proteoglycans to form two classes of reversibly dissociable “supramolecular complexes” - one class with heparan sulfate and glycopeptide-containing material and the second with hyaluronate-chondroitin complexes. Enzymatic digestion of “intact” SAM with trypsin or testicular hyaluronidase indicates that (1) only a small portion of long-term radiolabeled fibronectin and cytoskeletal protein is bound to the substrate via hyaluronate or chondroitin classes of GAG; (2) most of the fibronectin, cytoskeletal protein and heparan sulfate coordinately resist solubilization; and (3) newly synthesized fibronectin, which is metabolically labile in SAM, is linked to SAM by hyaluronate- and/or chondroitin-dependent binding. All of our studies indicate that heparan sulfate is a direct mediator of adhesion of cells to the substrate, possibly by binding to both cell-surface fibronectin and substrate-bound CIg in the serum coating; hyaluronate-chondroitin complexes in SAM appear to be most important in motility of cells by binding and labilizing fibronectin at the periphery of footpad adhesions, with subsequent cytoskeletal disorganization.
    Additional Material: 12 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 445-449 
    ISSN: 0091-7419
    Keywords: subcellular fractionation ; brown adipose tissue ; plasma membranes ; microsomes ; Metrizamide ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The present study proposes a technique, using Metrizamide, which permits the preparation of brown adipose tissue plasma membranes from the crude mitochondria as well as from the crude microsome fraction. These plasma membranes have high relative specific activities of their marker enzyme, 5′-nucleotidase (15 ± 3 and 14 ± 2 respectively) and, particularly those originating in the crude microsomes, are relatively free of mitochondria contamination. This study also shows the influence of the mode of cell disruption on microsome integrity. When cell disruption was achieved by grinding in liquid nitrogen the purified microsome NADPH cytochrome c reductase specific activity was found to be 3.5 times greater than that of microsomes obtained after homogenization of the tissue.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 429-444 
    ISSN: 0091-7419
    Keywords: (H++K+)-ATPase ; transport ATPase ; proton transport ; phospholipids ; phospholipase A2 ; CD spectrum ; gastric ATPase ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The phospholipid and fatty acid composition and role of phospholipids in enzyme and transport function of gastric (H++K+)-ATPase vesicles was studied using phospholipase A2 (bee venom). The composition (%) was phosphatidylcholine (PC) 33%; sphingomyelin (sph) 25%; phosphatidylethanolamine (PE) 22%; phosphatidylserine (PS) 11%; and phosphatidylinositol (PI) 8%. The fatty acid composition showed a high degree of unsaturation. In both fresh and lyophilized preparations, even with prolonged incubation, only 50% of phospholipids were hydrolyzed, but the amount of PE and PS disappearing was increased following lyophilization. There was a marked decrease in K+-ATPase activity (75%) but essentially no loss of the associated K+ p-nitrophenyl phosphatase was found. ATPase activity could be largely restored by various phospholipids (PE 〉 PC 〉 PS). There was also an increase in Mg2+-ATPase activity, partially reversed in fresh preparations by the addition of phospholipids (PE 〉 PS 〉 PC). Proton transport activity of the preparation was rapidly inhibited, initially due to a large increase in the HC1 permeability of the preparation. Associated with these enzymatic and functional changes, the ATP-induced conformational changes, as indicated by circular dichroism spectra were inhibited.
    Additional Material: 8 Ill.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 477-483 
    ISSN: 0091-7419
    Keywords: virus transformation ; membrane fluidity ; plasma membrane ; filipin ; cholesterol ; spin label ; lectin agglutination ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Polyene antibiotics such as filipin selectively inhibit wheat germ agglutinin-induced agglutination of transformed and malignant cells compared to normal cells (Hatten ME, Burger MM: Biochemistry 18:739, 1979). Since filipin binds specifically to cholesterol, we measured cholesterol levels in 3T3 cells and SV101-3T3 cells. SV101-3T3 cells contained 50-100% more cholesterol per cell than 3T3 cells. Both cell types were starved for cholesterol by growth in lipid-depleted medium plus 25-hydroxycholesterol. The cholesterol level of SV101-3T3 cells decreased by 30-50%, while the level in 3T3 cells remained constant. Filipin-stained SV101-3T3 cells revealed bright patches of filipin under fluorescence microscopy. These patches were absent in 3T3 cells and in SV101-3T3 and 3T3 cells starved for cholesterol. We selectively labeled plasma membranes of these cells with a spin label analog of phosphatidylcholine. The spin label indicated differences in plasma membrane fluidity that may be related to the different cholesterol levels in 3T3 and SV101-3T3 cells.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 485-492 
    ISSN: 0091-7419
    Keywords: carcinoma ; cell surface ; ganglioside ; hepatoma ; metastatis ; sialic acid ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In previous investigations, we correlated levels of sialic acid, gangliosides, and ganglioside glycosyltransferases with tumorigenesis over a 24-week continuum of growth of hepatocellular neoplasms of the rat induced by the carcinogen N-2-fluorenylacetamide. However, metastatic tumors developed only rarely and were not analyzed. To investigate surface changes associated with metastasis, well-differentiated and poorly differentiated hepatocellular carcinomas were transplanted to syngeneic recipient rats. From those, several metastatic and nonmetastatic isolates were obtained and compared. Both total and ganglioside sialic acid amounts in transplantable hepatomas were elevated above control liver values but were significantly lower for metastatic lines than for nonmetastatic lines. The nonmetastatic lines were characterized by ganglioside patterns depleted in the precursor ganglioside GM3 (sialic acid-galactose-glucose-ceramide) and elevated in the products of the monosialoganglioside pathway. In contrast, metastatic isolates exhibited a restoration of GM3 and nearer normal amounts of other gangliosides. The findings point to differences in sialic acid-containing glycolipids, comparing metastatic and nonmetastatic hepatocellular carcinomas, and further extend the concept that ganglioside alterations do not cause tumorigenesis but are the end result of a cascade of events which apparently continue beyond the onset of metastasis.
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  • 11
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 493-502 
    ISSN: 0091-7419
    Keywords: cell surface ; plasma membrane ; glycoproteins ; affinity chromatography ; lectins ; Novikoff hepatocellular carcinoma ; neuraminidase ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Novikoff hepatocellular carcinoma cells were radioiodinated by a cell surface-specific method using lactoperoxid ase/125I. The iodinated proteins were solubilized in 0.5% Nonidet P-40 and subjected to affinity chromatography on Sepharose-conjugated lectins (Ricinus communis agglutinins I or II, soybean agglutinin, concanavalin A, or wheat germ agglutinin) and analyzed by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Almost all the iodinated proteins bound to one or more of the Sepharose-conjugated lectins, presumptive evidence that these peptides are glycosylated. Lectin affinity chromatography resolved defined subsets of iodinated glycoproteins and suggested that certain glycoproteins could be fractionated on the basis of heterogeneity of their heterosaccharide moieties. Incubation of the iodinated cells with neuraminidase resulted in increased binding of iodinated proteins to Sepharose-conjugated Ricinus communis agglutinins I and II and soybean agglutinin and decreased binding to Sepharose-conjugated wheat germ agglutinin. Binding of iodinated proteins to concanavalin A was unaffected by neuraminidase treatment of the cells. These studies demonstrate the utility of lectins for the multicomponent analysis of plasma membrane proteins.
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  • 12
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 503-515 
    ISSN: 0091-7419
    Keywords: glycoproteins ; two-dimensional electrophoresis ; differentiation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The present work examined the expression of cell surface glycoprotein antigens in cultured human cell lines. The set of glycoproteins studied was defined by their immunoreactivity with antiserum developed to Triton-solubilized extracts of placental brush border membranes. Studies were performed using cell lines of trophoblastic (BeWo, JEG-3) and nontrophoblastic (Chang liver cells) origin, as well as diploid fibroblast cell lines (WI-38, GM-38).Antiplacental brush border antiserum reacts with at least 19 distinct antigens present in placental membrane preparations, each of which can be resolved and identified in two-dimensional electrophoresis. The subunit molecular weight and isoelectric point for all components were defined by their positions in the two-dimensional matrix. Thirteen of these could be detected among the five cell lines examined by lactoperoxidase-catalyzed cell surface iodination. One of these 13 antigens has been identified as the placental isoenzyme of alkaline phosphatase (PAP). The expression of this component is limited to choriocarcinonia cells and Chang liver cells and it is not present in diploid fibroblasts. Under normal circumstances expression of PAP is unique to the differentiated placenta but has been frequently demonstrated in both trophoblastic and nontrophoblastic neoplasms.Two other antigens are variably expressed among the different cell types examined in the present study and their presence or absence was independent of the trophoblastic, epithelial nontrophoblastic, or fibroblastic origin of the cells.Ten surface antigens were expressed in all five cell lines. Six of these had previously been found common to membranes from three adult differentiated tissues, including liver and kidney, as well as placenta (Wada et al, J Supramol Struc 10(3):287-305, 1979). The presence of this set of antigens in cultured cells as well extends the possibility that these are ubiquitously expressed on human cell surfaces. Two other antigens observed in all cultured cells had been found in both placental and either kidney or liver membranes and may represent common functions shared by many tissues which are also necessary for growth in vitro. The two remaining placental antigens seen in all cultured cells have previously been shown to be absent in adult tissues. Their presence in cultured cells but not in the membranes of resting differentiated tissues may signify the expression of glycoproteins characteristic of trophoblasts in all cells adapted to growth in culture.
    Additional Material: 3 Ill.
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  • 13
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 517-528 
    ISSN: 0091-7419
    Keywords: cell variants ; electron microscopy ; malignant melanoma ; melanin ; metastasis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Murine melanoma line B16-F1, which shows some specificity for metastatic organ colonization of lung but rarely metastasizes to ovary, was used to select variant cell lines with increased preference for experimental ovary metastasis. Ovary-colonizing melanoma cell lines were sequentially selected in syngeneic C57BL/6 mice by repeated intravenous administration and surgical recovery of ovarian melanoma tumors for tissue culture. After ten selections for experimental ovary metastasis, line B16-010 was established which formed experimental metastatic ovary tumors in almost every test animal. In tissue culture B16-010 cells grew in circular in circular colonies with rounded, smooth cell peripheries compared to B16-F1 cells which were flatter, grew in irregular patterns, and exhibited long cellular projections. Ovary-selected B16 lines contained less melainin pigment (B16-010 〈 B16-05 〈 B16-01 ≅ B16-F1) compared to the parental melanoma line. Together with previous cloning and selection data, these results are consistent with the preexistence of highly malignant cells in the parental tumor population that possess the ability to metastasize to specific organs.
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  • 14
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 529-538 
    ISSN: 0091-7419
    Keywords: 3T3 cells ; transformed cells ; restriction point ; labile proteins ; growth factors ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Both serum factors and protein synthesis are required for normal cell growth. Swiss 3T3 cells require the serum growth factors insulin and EGF (epidermal growth factor) during the initial part of the G1 period, until they pass a restriction point about 2 h before the initiation of DNA synthesis. Concentration of cycloheximide that inhibit protein synthesis by as much as 70% dramatically lengthen the cell cycle before the restriction point, while the cell cycle after the restriction point remains nearly constant. These results are consistent with a model in which labile proteins are required for transit of cells past the serum-sensitive restriction point. The relation of these findings to the growth control of transformed cells is discussed.
    Additional Material: 6 Ill.
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  • 15
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 539-546 
    ISSN: 0091-7419
    Keywords: SV40 transformation ; tumorigenicity ; anchorage independence ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A syndrome of in vitro properties correlates with the tumorigenicity of SV40-transformed rodent cells. These properties are plasminogen activator production, loss of large actin cables, and anchorage-independent growth. An established rat fibroblast line, its SV40 transformant, several T-antigen negative revertants, and a spontaneous retransformant isolated form one of the revertants were analyzed in vivo for their tumorigenicity and in vitro for the syndrome. The two transformed lines were highly tumorigenic, and had clearly abnormal in vitro properties. The parental rat line was weakly tumorigenic in nude mice and demonstrated a slightly transformed response in the in vitro assays. The revertants were completely nontumorigenic. Expression of the in vitro syndrome was not uniform for all revertants; however, most cell lines maintained the correlation of the syndrome and tumorigenicity.
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  • 16
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 151-164 
    ISSN: 0091-7419
    Keywords: membrane glycoproteins ; posttranslational modifications ; intracellular transport ; secretion ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The early steps in the biosynthesis of glycoproteins associated with the plasma membranes of rat hepatoma tissue culture cells has been analyzed. By measuring the effect of tunicamycin on the incorporation of [3H] mannose and [3H] fucose into cell glycoproteins, it was determined that an interval of about 1 h was required to transfer the glycoprotein from the site of mannosylation to the site of fucosylation. This result was corroborated by an analysis of the time required for the appearance of either mannose or fucose-labeled glycoproteins at the cell surface. The separation of membrane glycoproteins by a two-dimensional gel system allowed the visualization of the modifications leading to both size and charge heterogeneity of these proteins. By following the changes in electrophoretic mobility introduced into membrane glycoproteins during a chase period after a pulse labeling, the time course of these molecular alterations could be estimated. Several glycoproteins have apparently higher rates of synthesis than the bulk of membrane-associated glycoproteins. Most of these glycoproteins were released within 2 h after biosynthesis from the intracellular membrane fraction and appear after 3 h in the medium. In addition to the glycoproteins that contain both mannose and fucose and that show a high degree of charge heterogeneity, there are other membrane-bound species that are not noticeably modified by the in corporation of fucose or sialic acids. These glycoproteins could represent constituents limited to the internal membrane system of the HTC cell.
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  • 17
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 299-304 
    ISSN: 0091-7419
    Keywords: mycoplasma ; cytochalasin B ; actin-like protein ; cytoskeleton ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Studies are presented on the effect of cytochalasin B (CB) on the growth of five Mycoplasma species, three Acholeplasma species, and one Spiroplasma species. The three gliding mycoplasma species (M) gallisepticum, M pneumoniae and M pulmonis are the only mycoplasmas inhibited by CB. These are the only prolaryotes reported to be inhibited by CB. This suggested that these three mycoplasmas might have some sort of cytoskeletal structure. A protein fraction has been isolated from M gallisepticum which polymerizes in 0.6 M KC1 and depolymerizes when KC1 is removed. This fraction contains a major 58,000-dalton protein, a 46,000-dalton protein, and a minor 87,000-dalton protein.
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  • 18
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 305-320 
    ISSN: 0091-7419
    Keywords: tobacco mosaic virus ; structure ; RNA-binding site ; assembly ; protein-nucleic acid interactions ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A review of the structural studies of tobacco mosaic virus (TMV) is given. TMV is essentially a flat helical microcrystal with 16 1/3 subunits per turn. A single strand of RNA runs along the helix and is deeply embedded in the protein. The virus particles form oriented gels from which high-resolution X-ray fiber diffraction data can be obtained. This may be interpreted by the use of six heavy-atom derivatives to give an electron density map at 0.4 nm resolution from which the RNA configuration and the form of the inner part of the protein subunit may be determined. In addition, the protein subunits form a stable 17-fold two-layered disk which is involved in virus assembly and which crystallizes. By the use of noncrystallographic symmetry and a single heavy-atom derivative, it has been possible to solve the structure of the double disk to 0.28 nm resolution. In this structure one sees that an important structural role is played by four alpha-helices, one of which (the LR helix) appears to form the main binding site for the RNA. The main components of the binding site appear to be hydrophobic interactions with the bases, hydrogen bonds between aspartate groups and the sugars, and arginine salt bridges to the phosphate groups. The binding site is between two turns of the virus helix or between the turns of the double disk. In the disk, the region proximal to the RNA binding site is in a random coil until the RNA binds, whereupon the 24 residues involved build a well-defined structure, thereby encapsulating the RNA.
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  • 19
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 321-334 
    ISSN: 0091-7419
    Keywords: denervated sarcolemma ; nonsynaptic acetylcholine receptors ; 125I-α-bungarotoxin ; ferritin-α-bungarotoxin ; electron microscopy ; freeze-fracture ; freeze-etching ; autoradiography ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The nonsynaptic sarcolemma of denervated skeletal muscle of rat shows an abundance of ∼15 nm intramembranous particles on the P face. These particles are either singly distributed or are in clusters, and they are essentially lacking from the comparable freeze-fractures of the innervated sarcolemma. Autoradiographic studies using 125I-α-bungarotoxin (BGT) on 1 μ-thick sections, and freeze-etch studies using ferritin-α-BGT conjugates on membrane fractions, show that the distribution of the label corresponds to the distribution of the 15-nm particles in the nonsynaptic sarcolemma. On the basis of these results and existing physiologic and biochemical data, it is suggested that the 15-nm intramembranous particles are components of the α-BGT binding sites, ie, acetylcholine (Ach) receptors, in the nonsynaptic sarcolemma of denervated muscle and that the two types of distributions represent two spatial manifestations of Ach receptor molecules. The significance of these findings in relation to synapse formation in denervated muscle is discussed.
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  • 20
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 369-384 
    ISSN: 0091-7419
    Keywords: phagocytosis ; actin ; myosin ; macrophages ; immunofluorescence ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Contractile proteins are thought to play a causative role in motile processes such as phagocytosis. In order to investigate their role in phagocytosis further, simultaneous immunofluorescence localization of F-actin and myosin was carried out in resident mouse peritoneal macrophages after phagocytosis of opsonized zymosan particles. Both actin and myosin appeared to concentrate rapidly at sites of particle phagocytosis. The observed concentration of both proteins at such sites preceded ultimate particle engulfment. Cytochalasin B, a drug which was shown to block pseudopod extensions around the particle, did not prevent the concentration of the two contractile proteins at cell-particle binding sites. This result ruled out path-length effects as an explanation for the observed concentration of actin and myosin at phagocytic sites. Kinetic analysis showed that actin rapidly concentrates at particle-cell binding sites within minutes (or less) of contact with cell surface. The two proteins are present throughout the engulfment phase until and after ingestion is complete. Finally, at later times the particles become clustered over the cell nucleus and the particle-associated actin-myosin seen earlier is no longer evident.
    Additional Material: 9 Ill.
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  • 21
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 491-504 
    ISSN: 0091-7419
    Keywords: oocyte protein transport ; receptor solubilization ; phosvitin receptor ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Phosvitin (PV), a subunit of a female-specific protein, vitellogenin, binds to oocyte membranes with a KD of 10-6 M. Binding reaches equilibrium within 30 min after incubation at 25°C. Bound 125I-PV dissociates from the membrane with a t1/2 of 13 h when incubated in buffer. However, when 125I-PV-labeled membranes are incubated in buffer containing 10-5 M unlabeled PV, 50% of the initially bound 125I-PV dissociates from the membrane within 10 min. These results support the conclusion that PV binds to a membrane-associated receptor.Solubilization studies show that Triton X-100 solubilizes up to 45% of the total membrane-bound 125I-PV. Gel-exculsion chromatography of the solubilized material yields a 500,000 dalton 125I-PV-containing complex separated from free 125I-PV. The 500,000 dalton complex completely dissociates to yield free 125I-PV when incubated with excess unlabeled PV. However, when incubated with (1) no addition, (2) IgG, or (3) serum albumin, the extent of dissociation is significantly reduced and is consistent with that which would be predicted on the basis of the observed dissociation rate in the absence of unlabeled PV.These results suggest that bound 125I-PV can only be displaced by unlabeled PV. These results also indicate that the 500,000 dalton species is a solubilized PV-receptor complex and that it is possible to solubilize the PV-receptor in an active form.
    Additional Material: 6 Ill.
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  • 22
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 505-516 
    ISSN: 0091-7419
    Keywords: fibronectin ; cold-insoluble globulin ; carbohydrate content ; proteoglycan ; proteolytic cleavage ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Human amniotic fluid fibronectin and plasma fibronectin (cold-incoluble globulin) are indistinguishable both immunologically and by amino acid composition. Cyanogen bromide and tryptic peptides also suggest substantial structural homology. However, carbohydrate analysis has demonstrated additional saccharides in fibronectin and an overall increase in carbohydrate content relative to coldinsoluble globulin. Furthermore, limited proteolytic cleavage of the two proteins indicates differences in primary structure or in conformation. Using affinity-purified antibodies to cold-insoluble globulin, a glucosamine-labeled pronaseresistant component, probably proteoglycan, was found to coprecipitate with fibronectin, suggesting an association between these two macromolecules in the connective tissue matrix.
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  • 23
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 24
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 1-30 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 25
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 31-78 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 26
    ISSN: 0091-7419
    Keywords: protein conformation ; phospholipids ; diglycerides ; lipid fluidity ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In order to gain direct evidence for lipid-dependent protein conformation in membrane, effects of modification of lipid composition on mobility of spin-labeled cysteine residues were investigated in the plasma membrane of the yeast Saccharomyces cerevisiae. Conversion of the bulk of phospholipids to diglycerides by treatment of the membrane with phospholipase C substantially enhanced spectral anisotropy. However, alteration of the viscosity of the lipid-bilayer by enriching the membrane with palmitelaidic or oleic acid had no effect on mobility of spin-labeled cysteine residues. These observations indicate that while the spin-labeled residues are not in direct contact with the lipid core of the membrane, there are lipid-protein interactions to the extent that removal of polar portion of the bulk of phospholipids induces conformational changes in proteins, which in turn restrict mobility of these residues. It is concluded that conformation of membrane proteins depends on lipid structure and that phospholipids have a role in preserving the native conformation of proteins.
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  • 27
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 307-327 
    ISSN: 0091-7419
    Keywords: fibroblasts ; plasma membranes ; contact inhibition ; growth control ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Addition of a suspension of a surface membrane enriched fraction prepared from confluent 3T3 cells to sparse 3T3 cells in culture results in a concentration dependent and saturable decrease in the rate of DNA synthesis. The inhibition of cell growth by membranes resembles the inhibition of cell growth observed at confluent cell densities by a number of criteria: (1) In both cases the cells are arrested in the G1 protion of the cell cycle; (2) the inhibition by membranes or by high local cell density can to a large extent be compensated for by raising the serum concentration or by addition of fibroblast growth factor plus dexamethasone. Membranes prepared from sparse cultures inhibit less well than membranes from confluent cultures in a manner which suggests that binding of membranes to cells is not by itself sufficient to cause inhibition of cell growth. The inhibitory activity has a subcellular distribution similar to phosphodiesterase (a plasma membrane marker) and appears to reside in one or more intrinsic membrane components. Maximally, membranes can arrest about 40% of the cell population in each cell cycle. Plasma membranes obtained from sparse 3T3 cells are less inhibitory than membranes obtained from confluent cells. This suggests either that the inhibitory component(s) in the plasma membrane responsible for growth inhibition may be in part induced by high cell density, or that this component(s) may be lost from these membranes during purification.
    Additional Material: 10 Ill.
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  • 28
    ISSN: 0091-7419
    Keywords: ANS fluorescence ; membrane hydration ; cholesterol ; phospholipid-cholesterol interaction ; infrared spectra ; red cell membranes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The addition of bacteriophage T5 to anaerobic, fermenting cells of Escherichia coli B or K-12 in the presence of 8-anilino-1-naphthalene sulfonate (ANS), N-phenylnaphthyl-1-amine (NPN), or dansyl ethylamine causes the fluorescence of these probes to rise in two steps, the first occurring immediately upon addition, the second delayed by 6 min. The conditions necessary for observing this phenomenon are defined (cell density, probe concentration, substrate, absence of an electron acceptor, multiplicity of infection, growth, and harvesting conditions).The magnitudes of the first and second steps in fluorescence are dependent upon the multiplicity of infection; the timing of the steps is not. The first step correlates with a breakdown in the potassium or rubidium permeability barrier of the cell, and it occurs either aerobically or anaerobically, with fermentable or nonfermentable substrates. The second step occurs only with cells that are without an available electron acceptor, are fermenting, and which have a functional membrane-bound, Ca2+-Mg2+-dependent adenosine triphosphatase (ATPase). The results are consistent with disturbance of energization of the cell membrane by the membrane-bound ATPase at the time of the second step in fluorescence. No change in the intracellular level of adenosine 5′-triphosphate (ATP) was seen, whereas the extracellular level increased sharply, starting 3-6 min after phage addition. The quantity of ATP found in the medium by 30 min after infection amounted to about four times the amount present inside the cells at the time of infection. The quantity and rate of efflux of ATP was similar under aerobic and anaerobic conditions.
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  • 29
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 30
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 397-404 
    ISSN: 0091-7419
    Keywords: electron microscopy ; hybrid ribosome ; ribosome structure ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Small 40S Artemia salina and large 50S Escherichia coli ribosomal subunits can be assembled into 73S hybrid monosomes active in model assays for protein synthesis. The reciprocal combination-small 30S E coli and large 60S A salina-fails to form hybrids. The 73S hybrid particles strongly resemble homologous 70S E coli and 80S A salina monosomes. The morphologic differences between the corresponding eukaryotic and prokaryotic ribosomal particles, established by electron microscopy, do not significantly affect the assembly and mutual orientation of 40S A salina and 50S E coli subunits in the heterologous monosome. The fact that the structure of the interface, the supposed site of protein synthesis, is preserved in the active hybrid implies that retention or loss of biologic activity of hybrid ribosomes is determined by the extent of conformational changes in the interface.
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  • 31
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 377-395 
    ISSN: 0091-7419
    Keywords: wet replicas ; microsurface spreading ; DNA ; subunits ; superbeads ; supercoils ; fibers ; chromosome bridges ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Superpacking of chromatin and the surface features of metaphase chromosomes have been studied by SiO replication of wet, unstained, and unfixed specimens in an exceedingly thin (≤ 1 nm) aqueous layer, keeping them wet. Hydrophilic Formvar substrates allow controlled thinning of the aqueous layer covering the wet specimens. Whole mounts of chromatin and chromosomes were prepared by applying a microsurface spreading method to swollen nuclei and mitotic cells at metaphase.The highest level of nucleosome folding of the inactive chromatin in chicken erythrocytes and rat liver nuclei is basically a second-order superhelical organization (width 150-200 nm, pitch distance 50-150 nm) of the elementary nucleosome filament. In unfavorable environments (as determined by ionic agents, fixative, and dehydrating agents) this superstructure collapses into chains of superbeads and beads. Formalin (10%) apparently attacks at discrete sites of chromatin, which are then separated into superbeads. The latter consist of 4-6 nucleosomes and seemingly correspond to successive turns of an original solenoidal coil (width 30-35 nm), which forms the superhelical organization. When this organization is unfolded, eg, in 1-2 mM EDTA, DNAse-sensitive filaments (diameter 1.7 nm) are seen to be wrapped around the nucleosomes.The wet chromosomes in each metaphase spread are held to each other by smooth microtubular fibers, 20-30 nm in diameter. Before they enter into a chromsome, these fibers branch into 9-13 protofilaments, each 5 nm wide. The chromosome surface contains a dense distribution of subunits about 10-25 nm in diameter. This size distribution corresponds to that of nucleosomes and their superbeads. Distinct from this beaded chromosome surface are several smooth, 23-30-nm-diameter fibers, which are longitudinal at the centromere and seem to continue into the chromatid structure. The surface replicas of dried chromosomes do not show these features, which are revealed only in wet chromosomes.
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  • 32
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 1-7 
    ISSN: 0091-7419
    Keywords: erythrocyte membranes ; LCAT deficiency ; electron spin resonance ; spin label ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The membrane fluidity of erythrocytes from patients with Lecithin: cholesterol acyltransferase (LCT) deficiency was studied by means of electron spin resonance. The temperature dependence of the separation of the outer extrema of the spectra of 2-(3-carboxy-propyl)-4,4-dimethyl, 2-tridecyl-3-oxazolidinyloxyl spin probe was monitored for normal, presumed carrier and clinically affected subjects. The temperature profile of controls was significantly different form that of the presumed carriers and the clinically affected individuals. The results show that the compositional abnormalities previously noted in erythrocyte membranes from patients with LCAT deficiency are associated with alterations in the physicochemical state of the membrane. An investigation of the spectral lineshapes below 10°C allowed a distinction to be made at the membarne level between clinically affected subjects and clinically normal heterozygous carriers. Alterations in the temperature dependence of electron spin resonance parameters may provide a sensitive index of red cell membrane alterations in pathological states of generalized membrane involvement.
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  • 33
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 25-31 
    ISSN: 0091-7419
    Keywords: simian virus 40 ; flow cytometry ; DNA synthesis induction ; transformation ; human diploid cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Simian virus 40 (SV40) is capable of inducing cellular DNA synthesis in permissive and nonpermissive cells. Utilizing flow cytometry, we analyzed the DNA content changes in two diploid human cell strains and two monkey cell lines. The osteogenesis imperfects (OI) human skin fibroblasts were induced into DNA synthesis, and within one to two cell generations, a polyploid cell population was produced. With WI-38 phase II cells, a similar pattern of increased cycling of cells into DNA synthesis was observed; however, the majority (∼60%) of the cells were blocked in the G2 + M phase of the cell cycle. At later time intervals, an increase in the G1 population was demonstrated. The two monkey cell lines responded to SV40 virus with an accumulation of cells in the G2 + M phase of the cell cycle. Thus, two diploid human cell strains exhibited different cell cycle kinetics early after infection with SV40 virus. The one strain (WI-38) behaved similarly to the two monkey cell lines studied. The other strain (OI) responded similarly to nonpermissive (transformin) cells infected with SV40 virus.
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  • 34
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 33-49 
    ISSN: 0091-7419
    Keywords: cell enucleation ; cell reconstruction ; nuclear control of tumorigenicity ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The techniques of somatic cell hybridization have provided a valuable means of studying mechanisms of regulation of mammalian cell differentiation and transformation. Most previous studies have indicated that fusions between tumorigenic and nontumorigenic cells result in hybrid cells that are usually tumorigenic. In recent years it has been demonstrated that the phenotypic expression of tumorigenicity is at least partially due to the extensive chromosome loss that occurs in most interspecific and some intraspecific hybrid cells. In the present study we have utilized enucleation techniques that permit cells to be divided into nuclear (karyoplast) and cytoplasmic (cytoplast) cell fragments. Even though these nuclear and cytoplasmic fragments are metabolically stable for short periods of time, in our hands they ultimately degenerate. Viable cells can be reconstructed by PEG-induced fusion of karyoplasts to cytoplasts. Since reconstructed cells apparently do not segregate chromosomes, they may provide a clearer understanding of the interactions between the nucleus and the cytoplasm in the control of the expression of tumorigenicity. We have reconstructed cells using karyoplasts from the tumorigenic Y-1 cell line and cytoplasts from a nontumorigenic cell line, A-MT-BU-A1. In addition we have reconstructed cells containing Y-1 cytoplasts and A-MT-BU-A1 karyoplasts. The reconstructed cells porduced were assayed for tumorigenicity by their ability to grow in soft agar and in nude mice. The results of these experiments indicate that the reconstructed cells containing a tumorigenic nucleus and a nontumorigenic cytoplasm ultimately are tumorigenic and conversely the reconstructed cells containing a nontumorigenic nucleus and a tumorigenic cytoplasm are nontumorigenic. These experiments support the concept that with these cell lines the nucleus (karyoplast) is sufficient to control the phenotypic expression of tumorigenicity.
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  • 35
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 61-67 
    ISSN: 0091-7419
    Keywords: lectin ; glycosaminoglycan ; extracellular material ; cell matrix ; cellular interactions ; myoblast development ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Embryonic chick muscle contains two developmentally regulated lectins, which may be involved in cell interactions. These endogenous lectins are assayed as agglutinins of appropriate test erythrocytes. One of these, called lectin-2, interacts with specific glycosaminoglycans, especially heparin and dermatan sulfate. Lectin-2 is present at constant levels in both chick fibroblast and chick muscle cells throughout 14 days of culture but is released into the medium of cultured embryonic muscle after 7-8 days of culture, soon after myoblast fusion. Lectin-2 interacts strongly with a component of substrate-attached material in embryonic muscle cultures which is extractable from the culture dishes with alkali after the cells have been removed with ethylediaminetetraacetic acid. The active component in the substrate-attached material appears to be a glycosaminoglycan that is a more potent inhibitor of lectin-2 agglutination activity than any of the known glycosaminoglycans that we have tested. The active material is degraded by chondroitinase ABC but not by chondrotinase AC, hyaluronidase, or proteolytic enzymes and thus appears to be similar to dermatan sulfate. The results of these studies raise the possibility that lectin-2 functions by interacting with glycosaminoglycans, either associated with the cell surface or with the extracellular matrix.
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  • 36
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 79-93 
    ISSN: 0091-7419
    Keywords: Phorbol esters ; retinoids ; vasopressin ; mitogens ; uridine uptake ; deoxyglucose uptake ; ion fluxes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: 12-O-Tetradecanoyl-phorbol-13-acetate (TPA), in the absence of serum, acts synergistically with a range of polypeptide growth factors to stimulate DNA synthesis in quiescent Swiss 3T3 cells. These growth factors include epidermal growth factor (EGF), insulin, and the peptide produced by BHK cells transformed by SV-40 virus (fibroblast-derived growth factor, FDGF). Retinoids also show mitogenic synergism with TPA or polypeptide growth factors. The spectrum of mitogenic synergisms displayed by TPA are similar to those of vasopressin, a pituitary peptide. However, TPA and vasopressin do not synergistically interact to stimulate DNA synthesis in quiescent 3T3 cells. This suggests that TPA and vasopressin act via an identical biochemical pathway. Several lines of evidence suggest rapid postreceptor convergence of the mitogenic mechanisms of action of the hormone and the tumor promotor. Thus, vasopressin and TPA both inhibit EGF binding to cellular receptors. Furthermore, TPA and vasopressin induce a similar array of early events in quiescent cells - most strikingly, identical stimulation of Rb+ influx. Stimulation of ion flux is suggested as the possible convergence point of the pathway by which TPA and vasopressin act as mitogens.
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  • 37
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 38
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 13-30 
    ISSN: 0091-7419
    Keywords: hepatoma ; cell culture ; tight junctions ; gap junctions ; freeze-fracture ; dexamethasone ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Freeze-fracture and thin-section methods were used to study tight junction formation between confluent H4-II-E hepatoma cells that were plated in monolayer culture in media with and without dexamethasone, a synthetic glucocorticoid. Three presumptive stages in the genesis of tight junctions were suggested by these studies: (1) “formation zones” (smooth P-fracture face ridges deficient in intramembranous particles), apparently matched across a partially reduced extracellular space, develop between adjacent cells; (2) linear strands and aggregates of 9-11 nm particles collect along the ridges of the formation zones. The extracellular space was always reduced when these structures were found matched with pits in gentle E-face depressions; (3) the linear arrays of particles on the ridges associate within the membranes to form the fibrils characteristic of mature tight junctions. The formation zones resemble tight junctions in terms of size, complexity and the patterns of membrane ridges. Although some of the beaded particle specialization may actually be gap junctions, it is unlikely that all can be interpreted in this way. No other membrane structures were detected that could represent developmental stages of tight junctions. Dexamethasone (at 2 × 10-6 M) apparently stimulated formation of tight junctions. Treated cultures had a greater number of formation zones and mature tight junctions, although no differences in qualitative features of the junctions were noted.
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  • 39
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 61-77 
    ISSN: 0091-7419
    Keywords: lectins ; binding sites ; neuroblastoma cells ; receptor redistribution ; cell surface labeling ; cytochalasin B ; concanavalin A ; wheat germ agglutinin ; fluorescent microscopy ; scanning electron microscopy ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Concanavalin A (Con A), wheat germ agglutinin (WGA), and Ricinus communis agglutinin (RCA) bound with either 125I, fluorescent dyes, or fluorescent polymeric microspheres were used to quantitate and visualize the distribution of lectin binding sites on mouse neuroblastoma cells. As viewed by fluorescent light and scanning electron microscopy, over 107 binding sites for Con A, WGA, and RCA appeared to be distributed randomly over the surface of differentiated and undifferentiated cells. An energy-dependent redistribution of labeled sites into a central spot occurred when the cells were labeled with a saturating dose of fluorescent lectin and maintained at 37°C for 60 min. Reversible labeling using appropriate saccharide inhibitors indicated that the labeled sites had undergone endocytosis by the cell. A difference in the mode of redistribution of WGA or RCA and Con A binding sites was observed in double labeling experiments. When less than 10% of the WGA or RCA lectin binding sites were labeled, only these labeled sites appeared to be removed from the cell surface. In contrast, when less than 10% of the Con A sites were labeled, both labeled and unlabeled Con A binding sites were removed from the cell surface. Cytochalasin B uncoupled the coordinate redistribution of labeled and unlabeled Con A sites, suggesting the involvement of microfilaments. Finally, double labeling experiments employing fluorescein-tagged Con A and rhodamine-tagged WGA indicate that most Con A and WGA binding sites reside on different membrane components and redistribute independenty of each other.
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  • 40
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 137-149 
    ISSN: 0091-7419
    Keywords: cholera toxin ; chemical modification ; amino acid composition ; antigenic relationships ; radioimmunoassay ; adenylate cyclase ; ovine luteinizing hormone ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Chemical modification of intact cholera toxin or its B subunit by either partial nitration or reduction and alkylation did not result in significant loss of biological activity as determined by measurement of cyclic AMP in Chinese hamster overy cells. Complete nitration or succinylation in the presence of guanidine hydrochloride resulted in complete loss of biological activity and significantly affected the immunoreactivity of the toxin and B subunit. Compositional analyses of both the isolated α and γ chains of the toxin were typical of globular proteins and did not reveal significant hydrophobicity. Analysis of antigenic relationships by radioimmunoassay indicated a partial crossreactivity between the α chain and the B subunit of cholera toxin. Since previous structural studies of the β chain of cholera toxin indicated chemical similarity with the glycoprotein hormones [Kurosky et al. Science 195:299 (1977)], radioimmunoassay procedures were employed to investigate for possible crossreactivity. No evidence of crossreactivity between cholera toxin subunits and subunits of ovine luteinizing hormone was found.
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  • 41
    ISSN: 0091-7419
    Keywords: antigenic membrane glycoproteins ; immunoprecipitation ; two-dimensional electrophoresis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sialoglycoprotein subunits of human placental brush border membranes were labeled by sequential treatment with periodate and (3H)-sodium borohydride, which trititates sialic acid, and by lactoperoxidase-catalyzed (125I) iodination of tyrosine residues. The labeled subunits were characterized with respect to their affinity for antisera raised against Triton X-100 extracts of placental brush border membranes. The immunochemically reactive components were analyzed by two-dimensional electrophoresis according to a modification of the O'Farrell technique [20] enabling the assignment of estimated Mr̄ and pI. Of the 33 3H-labeled brush border subunits present in Triton X-100-solubilized membrane preparations, 18 subunits reacted with antiplacental brush border antisera insolubilized on CNBr-activated Sepharose or in immunoprecipitates. Fourteen of these tritiated subunits were also labeled with 125I, confirming that these are glycoproteins.The plasma membranes of normal human liver and microsomes from kidney were examined for the placental brush border glycoprotein subunits by reaction with insolubilized antiplacental brush border antisera and two-dimensional electrophoresis of the reacting tritium-labeled subunits. Comparison of the two-dimensional electrophoretic maps of the immunochemically reacting glycoproteins from liver, kidney, and placenta resulted in the identification of seven placental subunits in common with liver and kidney on the basis of antigenic cross-reactivity, Mr̄, and pI. Four placental glycoproteins were not found in the other tissues and are potentially specific to the placenta. Three of the placental subunits were only seen in placenta and kidney. Three of the subunits ran at the dye front and could not be assigned molecular weights. One of the subunits was poorly labeled by tritiation of sialic acid and was not considered.
    Additional Material: 5 Ill.
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  • 42
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    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 349-357 
    ISSN: 0091-7419
    Keywords: ribosomes ; crystallization ; hypothermia ; chick embryos ; reconstruction from electron micrographs ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ribosome microcrystals have been obtained for the first time in homogenates and extracts of chick embryos mainly in the form of P422 stacks that have average linear dimensions some 40% greater than those obtained in vivo.
    Additional Material: 7 Ill.
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  • 43
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 359-364 
    ISSN: 0091-7419
    Keywords: ribosomes ; crystallization ; hypothermia ; chick embryos ; degeneration ; cell suffering ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The relationship between ribosome crystallization and cell degeneration has been studied in chick embryos at various temperatures, and new methods of inducing ribosome microcrystals are described. A model is discussed that reinterprets the role of low temperatures in these phenomena and provides a unitary explanation of the various cases in which the occurrence of ribosome crystallization in chick embryos has been reported.
    Additional Material: 4 Ill.
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  • 44
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 117-122 
    ISSN: 0091-7419
    Keywords: dynein ATPase ; latency ; high-affinity binding site ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The enhancing effect of low concentrations (eg, 8 μM) of bis(4-fluoro-3-nitrophenyl)sulfone (FNS) on 30S dynein ATPase activity is increased when 1 mM dithiothreitol (DTT) is present. The effect of FNS + DTT is optimal at pH 7.5. Activation of the latent ATPase activity of 30S dynein by FNS + DTT is partially prevented by 1-3 μM ATP. Adenylylimidodiphosphate (AMP-PNP) is less effective than ATP, while β,γ-methylene-adenosine triphosphate (AMP-PCP), though a much stronger inhibitor of ATPase activity than AMP-PNP, does not protect against enhancement. These results demonstrate the presence of a high-affinity ATP-binding site on 30S dynein.
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  • 45
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 139-145 
    ISSN: 0091-7419
    Keywords: fd coat protein ; 1H NMR ; 13C NMR ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The conformations of the major coat protein of a filamentous bacteriophage can be described by nuclear magnetic resonance spectroscopy of the protein and the virus. The NMR experiments involve detection of the 13C and 1H nuclei of the coat protein. Both the 13C and 1H nuclear magnetic resonance (NMR) spectra show that regions of the polypeptide chain have substantially more motion than a typical globular protein. The fd coat protein was purified by gel chromatography of the SDS solubilized virus. Natural abundance 13C NMR spectra at 38 MHz resolve all of the nonprotonated aromatic carbons from the three phenylalanines, two tyrosines, and one tryptophan of the coat protein. The α carbons of the coat protein show at least two different classes of relaxation behavior, indicative of substantial variation in the motion of the backbone carbons in contrast to the rigidity of the α carbons of globular proteins. The 1H spectrum at 360 MHz shows all of the aromatic carbons and many of the amide protons. Titration of a 1H spectra gives the pKas for the tyrosines.
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  • 46
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 123-138 
    ISSN: 0091-7419
    Keywords: pea stem membranes ; L-cell membranes ; polyisoprenyl oligosaccharides ; glycoprotein synthesis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Membrane preparations from growing regions of pea stems and activelydividing mouse L-cells form lipid-linked saccharides from GDP-mannose and UDP-N-acetylglucosamine. These lipids have properties which are consistent with those of mono-and di-phosphoryl polyisoprenyl derivatives.In experiments using plant membranes, the monophosphoryl derivative labeled with GDP-(14C) mannose contains mannose only, while the diphosphoryl derivative labeled with the same nucleotide sugar is heterogeneous, containing oligosaccharides corresponding to mannosaccharides of 5, 7, and 9-12 residues. Only the diphosphoryl polyisoprenyl derivatives are labeled with UDP-(14C)glucosamine and these contain predominantly chitobiose and N-acetylglucosamine itself. Unlabeled GDP-mannose added after UDP-N-acetyl (14C)glucosamine results in the formation of higher lipid-linked oligosaccharides which are apparently the same as those which are labeled with GDP-(14C)mannose alone. Incubation of the membranes with GDP-(14C)mannose in the presence of Mn2+, unlabeled UDP-glucose or unlabeled UDP-N-acetylglucosamine results in marked changes in the accumulation of both the polyisoprenyl monophosphoryl mannose and polyisoprenyl diphosphoryl oligosaccharides.Animal cell membranes synthesise lipid-linked oligosaccharides when incubated with UDP-N-acetylglucosamine and GDP-mannose. These oligosaccharides are similar in size to those synthesised by the plant membranes but their formation is more efficient. The potential roles of these compounds in glycoprotein biosynthesis in both plant and animal tissues is discussed.
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  • 47
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 175-187 
    ISSN: 0091-7419
    Keywords: growth control ; 3T3 cells ; Schwann cells ; neurites ; plasma membranes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Control of cell growth by cell to cell contact is reviewed with particular emphasis on two systems - contact inhibition of growth observed with Swiss 3T3 cells and the mitogenic stimulation of Schwann cells by dorsal root ganglia neurites. In both cases the biological effect can be reproduced by the addition of surface membranes to the corresponding cells. In the case of contact inhibition of 3T3 cells, biological activity appears to correlate with membrane binding to the cells. An octylglucoside extract of 3T3 plasma membranes retains the biological activity (growth inhibition) of the original membranes.
    Additional Material: 7 Ill.
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  • 48
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 251-258 
    ISSN: 0091-7419
    Keywords: tumorigenicity ; cyclic AMP-dependent protein kinase ; tyrosinase MSH-growth-resistant variant ; mouse melanoma ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A variant of B-16 F1 mouse melanoma was selected for its ability to survive and replicate in the presence of melanocyte-stimulating hormone (MSH). Although the variant (MR-4) was completely resistant to growth inhibition by MSH, cyclic AMP was still able to block cell replication. Tyrosinase activity in MR-4 cells was considerably lower than in B-16 F1 cells. MSH induced a twofold to three-fold increase in tyrosinase activity in both cell types, but the absolute activity in MR-4 remained significantly less than in the parental cells. MR-4 cells were also found to have a markedly depressed cyclic AMP-dependent protein kinase activity relative to B-16 F1 cells. The protein kinase from both cell types was stimulated by cyclic AMP, but the level of MR-4 kinase activity at maximal cyclic AMP concentrations remained considerably lower than B-16 F1 kinase activity under the same conditions. In both cell types adenylate cyclase activity was markedly stimulated by MSH. When equal numbers of viable F1 and MR-4 cells were injected subcutaneously into C57/B1 mice, the MR-4 cells formed tumors earlier and killed the host sooner than the parental F1 cells. We conclude that the biochemical alteration which allows MR-4 cells to replicate in the presence of MSH is a low level of tyrosinase activity, which in turn may be the result of low cyclic AMP-dependent protein kinase activity.
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  • 49
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 237-250 
    ISSN: 0091-7419
    Keywords: coated vesicles ; coat dissembly ; coat reassembly ; coat dissociation ; clathrin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Disruption of the coat of coated vesicles is accompanied by the release of clathrin and other proteins in soluble form. The ability of solubilized coated vesicle proteins to reassemble into empty coats is influenced by Mg2+, Tris ion concentration, pH, and ionic strength. The proteins solubilized by 2 M urea spontaneously reassemble into empty coats following dialysis into isolation buffer (0.1 M MES-1 mM EGTA-1 mM MgCl2-0.02% NaN3, pH 6.8). Such reassembled coats have sedimentation properties similar to untreated coated vesicles. Clathrin is the predominant protein of reassembled coats; most of the other proteins present in native coated vesicles are absent. We have found that Mg2+ is important in the coat assembly reaction. At pH 8 in 0.01 M or 0.1 M Tris, coats dissociate; however, 10 mM MgCl2 prevents dissociation. If the coats are first dissociated at pH 8 and then the MgCl2 raised to 10 mM, reassembly occurs. These results suggest that Mg2+ stabilizes the coat lattice and promotes reassembly. This hypothesis is supported by our observations that increasing Mg2+ (10 μM-10 mM) increases reassembly whereas chelation of Mg2+ by (EGTA) inhibits reassembly. Coats reassembled in low-Tris (0.01 M, pH 8) supernatants containing 10 mM MgCl2 do not sediment, but upon dialysis into isolation buffer (pH 6.8), these coats become sedimentable. Nonsedimentable coats are noted also either when partially purified clathrin (peak I from Sepharose CL4B columns) is dialyzed into low-ionic-strength buffer or when peaks I and II are dialyzed into isolation buffer. Such nonsedimentable coats may represent intermediates in the assembly reaction which have normal morphology but lack some of the physical properties of native coats. We present a model suggesting that tightly intertwined antiparallel clathrin dimers form the edges of the coat lattice.
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  • 50
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 259-267 
    ISSN: 0091-7419
    Keywords: cell surface receptors ; proteolysis of receptors ; positive or negative regulation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Addition of highly purified thrombin t o cultures of several kinds of nondividing fibroblasts brings about cell division. This stimulation occurs in serum-free medium, permitting studies on its mechanism under chemically defined conditions. Previous studies have shown that action of thrombin a t the cell surface is sufficient to cause cell division and that the proteolytic activity of thrombin is required for its mitogenic effect. These results prompted experiments which showed that there is a cell surface receptor for thrombin and that thrombin must hind to its receptor and cleave it to stimulate cell division. Some of the thrombin that hinds to its receptors becomes attached to them by a linkage that appears to be covalent. However, it is presently unknown whether this direct thrombin receptor complex plays a role in the stimulation.These results raise a number of question that should be explored in future studies. They also provide a foundation on which to build hypotheses about tentative molecular mechanisms that might be involved in the stimulation. Knowledge that thrombin must cleave its receptor to bring about cell division suggests two alternative mechanisms for stimulation by proteolysis. In one the receptor is a negative effector which prevents cell division when it is intact, but not after it has been cleaved. Alternatively, a fragment of the receptor could be a positive effector. In this mechanism, proteolysis by thrombin would produce a specific receptor fragment which brings about cell proliferation. If every protease which cleaves the receptor also stimulates cell division, the receptor is probably a negative effector. In contrast, if certain proteases cleave the receptor but do not stimulate the cells, a fragment of the receptor is likely a positive effector. With negative regulation by the receptor, the controlling events would occur before proteolysis of it, and it might be possible to find putative regulatory molecules by identification of nearest neighbors of the receptor. This should be possible by using bifunctional crosslinking reagents. If a fragment of the thrombin receptor turns out to be a positive effector, it should be possible to identify and study fragments by analyzing the metabolic fate of the receptor. Techniques are now available for this kind of analysis and it should also be possible to determine whether receptor fragments remain in the membrane or whether they are translocated to specific sites within the cell. A critical question to be asked is which of these events and interactions involving the thrombin receptor are necessary for stimulation of cell division. It now appears that the best way to answer this question is to examine these events in a large number of cloned cell populations that are responsive or unresponsive to the mitogenic action of thrombin. If a thrombin-mediated event occurs in all responsive clones but is altered or absent in sonie unresponsive clones, it is probably necessary for stimulation of cell division.
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  • 51
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 52
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. i 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 53
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 283-293 
    ISSN: 0091-7419
    Keywords: cytochalasins ; muscle and platelet actin ; microfilaments ; cell motility ; viscosity changes ; electron microscopy ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The cytochalasins (CE, CD, CB and H2CB) inhibit numerous cellular processes which require the interaction of actin with other structural and contractile proteins. In this report we describe the effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin. The cytochalasins decreased the viscosity of F-actin solutions. The effect of H2CB, CB and CD on F-actin viscosity was maximal at concentrations of 20-50μM and did not increase with time. In contrast, CE caused a progressive decrease in the viscosity of F-actin solutions which was dependent upon the concentration of CE and the duration of incubation of the CE-actin mixture. After two hours of incubation of drug-actin mixtures, the relative effectiveness of the cytochalasins in reducing the viscosity of F-actin was CE 〉 CD 〉 CB = H2CB. The effects of CD and CE were paralleled by morphologic changes in negatively stained actin filaments. The effects of the cytochalasins on the viscosity and morphology of muscle and platelet actin were the same whether the drugs were added before or after the polymerization of the protein.These studies show that the interaction of the cytochalasins with actin is highly specific. Because the relative potencies of these drugs for affecting motile processes and the relative affinities of the drugs for binding sites within a variety of cells are CE 〉 CD 〉 CB = H2CB, the effects of cytochalasins on actin described here may contribute to some of the biological effects of the drugs on motile processes.
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  • 54
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 295-309 
    ISSN: 0091-7419
    Keywords: threshold effects ; liposomes ; aggregation ; ricin ; concanavalin A ; synthetic glycolipids ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Cholesterol analogs containing sugar residues linked by spacer groups to the cholesterol O can be incorporated into egg yolk lecithin small unilamellar liposomes. The synthetic glycolipid analogs distribute evenly on both sides of the bilayer. These liposomes are aggregated by the appropriate lectin. For example, when the sugar residue is a β-galactoside the liposomes are aggregated by ricin and when it is an α-mannoside they are aggregated by Con A. The lectin-mediated aggregation of these liposomes is reversed by the addition of the appropriate sugar. The rates but not the extents of aggregation of these liposomes are highly sensitive to the amount of glycolipid incorporated. Below approximately 5% glycolipid incorporation the rate of the lectin-mediated aggregation of these liposomes is exceedingly slow, whereas above this level rapid aggregation proceeds. At all concentrations studied the synthetic glycolipids are incorporated in a unimodal fashion so that the observed threshold effects cannot be based on possible differences in the manner in which the glycolipids are incorporated at different concentrations. This conclusion is based on (1) studies with galactose oxidase that show that the percentage of galactose oxidation in a liposome prepared from a galactosyl-containing glycolipid is independent of glycolipid concentration, and (2) studies on the aggregation of liposomes containing mixed glycolipids in which the glycolipids are shown to behave independently. The importance of a critical density of membrane-bound receptors in order for aggregation to occur is discussed.
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  • 55
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    Journal of Supramolecular Structure 11 (1979), S. 311-317 
    ISSN: 0091-7419
    Keywords: dynein ; flagella ; ATPase ; sperm motility ; sea urchin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A high-resolution sodium dodecyl sulfate polyacrylamide gel electrophoresis system has been used to show the presence, in both whole sperm and isolated flagellar axonemes, of eight polypeptides migrating in the 300,000-350,000 molecular weight range characteristic of the heavy chains of dynein ATPase. Previously, only five such chains have been discernible. Extraction of isolated axonemes for 10 min at 4°C with a solution containing 0.6 M NaCl, pH 7, releases a mixture of particles that separate, in sucrose density gradient centrifugation, into a major peak, dynein 1 ATPase, sedimenting at 21 S and a minor peak at 12-14S. The polypeptide compositions of these two peaks are different. The dynein 1 peak, which contains most of the protein on the gradient, contains approximately equal quantities of two closely migrating heavy chains, with a small amount of a third, more slowly migrating chain; no other heavy chains appear in this peak. Two groups of smaller polypeptides (three intermediate chains, within the apparent molecular weight range 76,000-122,000 and four newly discovered light chains, within the apparent molecular weight range 14,000-24,000) cosediment with the 21 S peak. The heavy chain composition of the 12-14S peak is more complex, all eight heavy chains occurring in approximately the same ratios as occur in intact axonemes.
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  • 56
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    Journal of Supramolecular Structure 11 (1979), S. 371-390 
    ISSN: 0091-7419
    Keywords: viral transformation ; iron starvation ; membrane proteins ; procollagen ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have analyzed the surface proteins of cultured normal rat kidney (NRK) cells and virus-transfromed NRK cells subjected to iron deprivation. Such a treatment specifically induces two transformation-sensitive plasma membrane-associated glycoproteins with a subunit molecular wegiht of 160,000 (160 K) and 130,000 (130 K) daltons in NRK cells. In these cells the 160 K glycoprotein is readily available to lactoperoxidase-mediated iodination, and the 130 K is apparently inaccessible to iodination. Major differences were revealed when iodinated membrane proteins of normal and virus-transformed cells subjected to iron deprivation were compared. In Kirsten sarcoma virus-transformed NRK cells the 160 K glycoprotein was weakly labeled. In two clones of simian virus 40-transformed NRK cells the 160 K glycoprotein was weakly labeled or not at all. The 130 K glycoprotein was inaccessible to iodination in all the virus-transformed cell lines.The 160 K and 130 K glycoproteins were isolated form plasma membranes of NRK cells using preparative SDS gel electrophoresis. Antibodies generated against these glycoproteins stained the external surfaces of NRK cells and induced antigen redistribution. Evidence presented suggests that 160 K and 130 K are plasma membrane-associated procollagen molecules. A possible interaction of these proteins with transferrin is also described. The data suggest that these proteins may have an important role in the sequence of events leading to transformation.
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  • 57
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 58
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 15-22 
    ISSN: 0091-7419
    Keywords: glycosaminoglycans ; glycocalyx ; liver and kidney plasma membranes ; hyaluronic acid ; chondroitin sulfates ; heparin sulfates ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Glycosaminoglycans were isolated from plasma membranes of hepatic and renal tubule cells of guinea pig. Plasmalemma of renal tubule cells contained more total glycosaminoglycans, hyaluronic acid, chondroitin-4 sulfates and chondroitin-6 sulfates, and less dermatan sulfates and heparin sulfates than liver plasma membranes. These glycocalyx components, owing to their polyanionic properties, may have a role in the transport of water, ions, and macromolecules across the cell membrane.
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  • 59
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    Journal of Supramolecular Structure 12 (1979), S. 23-34 
    ISSN: 0091-7419
    Keywords: thiourea ; cilia ; dyneins ; ATPase ; sulfhydryl groups ; turbidity response ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The effects of thioura and of several substituted thioureas-phenylthiourea, α-naphtylthiourea, metiamide, and burimamide-on dynein ATPase have been studied. The substituted thioureas are over 30 times more potent than thiourea in causing enhancement of 30S dynein ATPase activity and inhibition of 14S dynein ATPase activity. The effects of thiourea and phenylthiourea can be prevented by very low concentrations of β-mercaptoethanol or dithiotheritol. Axonemal ATPase is also enhanced by the thioureas, but the reaction proceeds more slowly than for solubilized 30S dynein. Enhancement of 30S dynein ATPase by metiamide is prevented by low (∼ 1 μM) concentrations of ATP and, less effectively, by AMP-PNP, but not by AMP-PCP even though the latter is a stronger inhibitor of 30S dynein ATPase than is AMP-PNP.The thioureas inhibit the ATP-induced decrease in turbidity (measured as ΔA350) of axonemal suspensions. Inhibition of the turbidity response is also prevented by low concentrations of β-mercaptoethanol, but, in contrast to the irreversible enhancement of ATPase activity, inhibition of the turbidity response is largely reversible. The ability of 30S dynein to rebind onto twice extracted axonemes is not changed by treatment with phenylthiourea or metiamide.These observations indicate that the thioureas react with at least two sets of SH or S-S groups on axonemes. Reaction with the group(s) on the 30S dynein causes an apparently irreversible enhancement of ATPase activity. Reaction with another group(s) causes a reversible inhibition of the turbidity response.
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  • 60
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    Journal of Supramolecular Structure 12 (1979), S. 35-46 
    ISSN: 0091-7419
    Keywords: MuLV ; uninfectious particles ; interferon ; virus assembly ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Interferon treatment of JLSV-6 cells chronically infected with Rauscher MuLV leads to the formation of noninfectious particles (interferon virions) containing the structural proteins of env and gag genes as well as additional viral polypeptides. In the control virions the major glycoprotein detected is gp71, interferon virions contain in addition to gp71 and 85k dalton (gp85) glucosamine-containing, fucose-deficient glycoprotein which is recognized by antiserum to MuLV but not by the gp71 antiserum. The surface iodination of the intact virions indicates that both gp71 and gp85 are the major components of the external virions envelope. However, unlike the control virions in which gp71 associates with p15E (gp90), the gp71-p15E complex was not detected in interferon virions. The analysis of the iodinated proteins of the disrupted interferon virions revealed the presence of 85k and 65k dalton polypeptides preciptable with antiserum against MuLV, which are not present in the control virions. The difference in the polypeptide pattern of virions produced in the presence of interferon does not seem to be a consequence of the slowdown in the synthesis of viral proteins or their processing in the interferon-treated cells. Both the structural proteins of env and gag genes seem to be synthesized and processed at a comparable rate in the interferon-treated and -untreated cells. These results indicate an alteration of virus assembly in the presence of interferon.
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  • 61
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    Journal of Supramolecular Structure 12 (1979), S. 115-125 
    ISSN: 0091-7419
    Keywords: glycocalyx ; cell surface ; tumor cells ; Ehrlich ascites tumor ; surface labeling ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ehrlich ascites tumor cells spontaneously release cell surface material (glycocalyx) into isotonic saline medium. Exposure of these cells to tritium-labeled 4,4′-diisothiocyano-1,2′-dihenylethane-2,2′-disulfonic acid (3H2DIDS) at 4°C leads to preferential labeling of the cell surface coat. We have combined studies of the kinetics of 3H2DIDS-label release, the effects of enzymatic treatment, and cell electrophoretic mobility to characterize the 3H2DIDS-labeled components of the cell surface. Approximately 73% of the cell-associated radioactivity is spontaneously released from the cells after 5 h at 23°C. The kinetics of release is consistent with the first-order loss of two fractions; a slow (τ½ = 360 min) component representing 33% of the radioactivity, and a fast (τ½ = 20 min) component representing 26%. The remaining 14% of the labile binding may reflect mechanically induced surface release. Trypsin (1 μ/ml) also removes approximately 73% of the labeled material within 30 min and converts the kinectics of release to that of a single component (τ½ = 5.5 min). The specific activity (SA) of material released by trypsin immediately after labeling is 83% of the SA of the material spontaneously los in 1 h. However, trypsinization following a 2-h period of spontaneous release yields material of reduced (43%) SA. Neither 3H2DIDS labeling nor the initial spontaneous loss of labeled material alters cell electrophoretic mobility. However, extended spontaneous release is accompanied by a significant decrease in surface charge density. Trypsinization immediately following labeling or after spontaneous release (2 h) reduces mobility by 32%. We have tentatively identified the slowly released compartment as contributing to cell surface negativity.
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  • 62
    ISSN: 0091-7419
    Keywords: fibronectin ; CSP-60 ; extracellular matrix ; thrombogenic properties ; low-density lipoprotein ; receptor redistribution ; asymmetry of cell surfaces ; cell morphology ; spatial configuration ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vascular endothelial cells cultured in the presence of fibroblast growth factor (FGF) devide actively when seeded at low or clonal cell densities and upon reachin confluence adopt a morphologic appearance and differentiated properties similar to those of the vascular endothelium in vovi. In this review, we present some of our recent observations regarding the characteristics (both structural and functional) of these endothelial cells and the role of FGF in controlling their proliferation and normal differentation. At confluence the endothelial cells from a monolayer of closely apposed and nondividing cell that have a nonthrombogenic apical surface and can no longer internalize bound ligands such as low-density lipoprotein (LDL). The adoption of these properties is correlated and possibly causally related to changes in the cell surface such as the appearance of a 60,000 molecular weight protein (CSP-60); the disappearance of fibronectin from the apical cell surface and its concomitant accumulation in the basal lamina; and a restriction of the lateral mobility of various cell surface receptor sites. In contrast, endothelial cells that are maintained in the absence of FGF undergo within three passages alterations that are incompatible with their in vivo morphologic apperarance and physiologic beharior. They grow at confluence on top of each other and hence can no longer adopt both the structural (CSP-60, cell surface polarity) and functional (barrier function, nonthrombogenicity) attributes of differentiated endothelial cell. Since these characteristics can be reacquired in response to readdition of FGF, in addition to being a mitogen FGF may also be involved in controlling the differentitation and phenotypic expression of the vascular endothelium.
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  • 63
    ISSN: 0091-7419
    Keywords: tumor promoters ; phorbol esters ; plasminogen activator ; epidermal growth factor ; carcinogenesis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The carcinogenic process is usually multifactor in its causation and multistep in its evolution. It is likely that entirely different molecular mechanisms underlie the many steps in this process. In contrast t o initiating carcinogens, the action of the tumor-promoting phorbol esters does not appear t o involve covalent binding t o cellular DNA and they are not mutagenic. Recent studies in cell culture have revealed two interesting biologic effects of the phorbol esters and related macrocyclic plant diterpenes. The first is that at nanomolar concentrations they induce several changes that resemble those seen in cells transformed by chemical carcinogens or tumor viruses. These include altered morphology and increased saturation density, altered cell surface fucose-glycopeptides, decrease in the LETS protein, increased transport of deoxyglucose, and increased levels of plasminogen activator and ornithine decarboxylase. In transformed cells exposed to phorbol esters the expression of these features is further accentuated. Phorbol esters do not induce normal cells to grow in agar but they do enhance the growth in agar of certain transformed cells. The second effect of the phorbol esters is inhibition of terminal differentiation. This effect extends to a variety of programs of differentiation and is reversible when the agent is removed. With certain cell culture systems induction of differentiation, rather than inhibition, is observed. Both the transformation mimetic and the differentiation effects are exerted by plant diterpenes that have tumor-promoting activity but not by congeners that lack such activity. The primary target of phorbol esters appears to be the cell membrane. Early membrane-related effects include enhanced uptake of 2-deoxyglucose and other nutrients, altered cell adhesion, induction of arachidonic acid release and prostaglandin synthesis, inhibition of the binding of epidermal growth factor t o cell surface receptors, altered lipid metabolism, and modifications in the activities of other cell surface receptors. A model of “two stage” carcinogenesis encompassing the known molecular and cellular effects of initiating carcinogens and tumor promoters is presented. According to this model, initiating carcinogens induce stable alterations in the cellular genome but these are not manifested until tumor promoters modulate programs of gene expression and induce the clonal outgrowth of the initiated cell.
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  • 64
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    Journal of Supramolecular Structure 12 (1979), S. 425-433 
    ISSN: 0091-7419
    Keywords: rat liver ribosomal proteins ; amino-terminus ; yeast ribosomes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The sequence of the amino-terminal region of eleven rat liver ribosomal proteins-S4, S6, S8, L7a, L18, L27, L30, L37a, and L39 - was determined. The analysis confirmed the homogeneity of the proteins and suggests that they are unique, since no extensive common sequences were found. The N-terminal regions of the rat liver proteins were compared with amino acid sequences in Saccharomyces cerevisiae and in Escherichia coli ribosomal proteins. It seems likely that the proteins L37 from rat liver and Y55 from yeast ribosomes are homologous. It is possible that rat liver L7a or L37a or both are related to S cerevisiae Y44, although the similar sequences are at the amino-terminus of the rat liver proteins and in an internal region of Y44. A number of similarities in the sequences of rat liver and E coli ribosomal proteins have been found; however, it is not yet possible to say whether they connote a common ancestry.
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  • 65
    Electronic Resource
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    Journal of Supramolecular Structure 12 (1979), S. 435-455 
    ISSN: 0091-7419
    Keywords: N-acetyl neuraminic acid ; neuraminidase (Vibrio cholerae) ; sialic acid ; wheat germ agglutinin receptor ; membrane glycoprotein ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The surface of the HeLa cell is composed of a heterogeneous population of sialogly coproteins which undergo lectin-mediated endocytosis (Kramer and Canellakis, Biochim Biophys Acta 551:328, 1979). One such sialoglyco-protein, gamma protein, is the major periodate-Schiff-reactive and [3H]-glucosamine-labeled component of the plasma membrane; it has an apparent molecular weight of 165,000. Gamma protein is also the major [125I]-wheat germ agglutinin-binding component in sodium dodecyl sulfate gels. Neuraminidase digestion of HeLa cells abolishes binding of [125I]-wheat germ agglutinin to gamma protein, and pretreatment of cells with wheat germ agglutinin protects gamma protein from desialation by neuraminidase. suggesting that wheat germ agglutinin binds to the sialic acid residues of gamma protein at the cell surface. Gamma protein can be extracted with various detergents but not with high-salt, chelating, or chaotropic agents. Intact inside-out plasma membrane vesicles have been prepared from HeLa cells that had phagocytosed latex particles. Treatment of these isolated vesicles with trypsin reduces the molecular weight of gamma protein. These results suggest that gamma protein is an integral membrane protein that spans the plasma membrane. Gamma protein can be purified to homogeneity by sequential lithium diiodosalicylate-phenol extraction, wheat germ agglutinin-agarose affinity chromatography, and preparative sodium dodecyl sulfate-polyacrylamide gel electrophoresis.
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  • 66
    Electronic Resource
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    Journal of Supramolecular Structure 10 (1979), S. 31-37 
    ISSN: 0091-7419
    Keywords: adenylate cyclase ; liver ; solubilized ; MnATP ; MgATP ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A general feature of membrane-bound adenylate cyclase systems is the “lability” of the basal enzyme to dispersion by detergents. A stable form of the detergentsolubilized enzyme is obtained only if the membrane-bound enzyme is first pretreated with fluoride or Gpp(NH)p. However, we have found with the basal hepatic enzyme that the lability is evident primarily when MgATP is used as substrate; substitution of MnATP for MgATP reveals that substantial basal activity survives detergent treatment. This effect is independent of the detergent; it is seen with either Lubrol PX or with deoxycholate. In addition to the altered substrate requirement, the membrane-bound and solubilized forms of the basal enzyme exhibit other differences. In contrast to the membrane-bound form, the solubilized enzyme shows (1) weak stimulation by Gpp(NH)p; (2) little inhibition by adenosine, (3) strong inhibition by Pi or PPi, and (4) and apparent loss of the Me2+-reactive regulatory site. Such dissimilarities between membranebound and solubilized cyclase are not seen if the membranes are pretreated with Gpp(NH)p prior to exposure to detergents. The characteristics of the solubilized basal hepatic enzyme are similar to those of the naturally occurring soluble adenylate cyclase found in mature rat testes. It would appear that separation of adenylate cyclase from components that confer regulation by divalent cation and guanine nucleotides produces a form of the enzyme that will turnover only MnATP; this may represent the free catalytic moiety. Such preparations could be useful in reconstructing some of the regulatory functions of adenylate cyclase seen in its membrane-bound form.
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  • 67
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 68
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    Journal of Supramolecular Structure 10 (1979), S. 111-124 
    ISSN: 0091-7419
    Keywords: growth factors ; gangliosides ; neurogenesis ; cell developmental program ; neuronal cell lines ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: For many permanent cell lines the transition from a growing (P) to a resting (R) state is reversibly controlled by growth factors present in serum. This P-to-R transition was studied in a neuronal cell line (B 104) with respect to the action of serum, dibutyryl cyclic AMP (DBcAMP), gangliosides, and a glioma cell-produced growth factor GGF. In this cell system gangliosides seem to act as differentiation and survival factors. The kinetics of uptake of radioactively labeled gangliosides and survival experiments both support the idea of the stable incorporation of exogenously added gangliosides into the cells. Based on the experimental evidence a new model of cell development is proposed. Thus in addition to the R or G0 state, which in this cell system is rather unstable and probably regulated by cyclic nucleotides, we postulate a differentiated D state, which is controlled by gangliosides and which is characterized by its stability (survival time). This D compartment seems to be closer to the in vivo differentiated neuron than does the R or P state. The possible mechanisms for the action of gangliosides are discussed.
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  • 69
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    Journal of Supramolecular Structure 10 (1979), S. 151-163 
    ISSN: 0091-7419
    Keywords: NAD ; ADP-ribose ; poly ADP-ribose ; ADP-ribosyl protein ; cholera toxin ; adenylate cyclase ; pigeon erythrocyte ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Upon incubation of lysed pigeon erythrocytes with NAD, adenosine diphosphateribose (ADP-ribose) is incorporated into nuclear poly ADP-ribose and into an unidentified acid-insoluble product of the cytosol. The properties of these incorporations have been examined and a method developed for reducing their amount whilst retaining the sensitivity of the lysate to cholera toxin. This method has allowed the detection and description of a set of cholera toxin-specific ADP-ribose transfers to membrane-bound and soluble proteins under conditions that lead to adenylate cyclase activation.
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  • 70
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    Journal of Supramolecular Structure 10 (1979), S. 175-184 
    ISSN: 0091-7419
    Keywords: vasopressin ; nocodazole ; urea transport ; rotenone ; dinitrophenol ; methylene blue ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Vasopressin increases the permeability of receptor cells to water and, in tissues such as toad bladder, to solutes such as urea. While cyclic AMP appears to play a major role in mediating the effects of vasopressin, there is evidence that activation of the water permeability system and the urea permeability system involves separate pathways. In the present study, we have shown that inhibitors of oxidative metabolism (rotenone, dinitrophenol, and methylene blue) selectively inhibit either vasopressin-stimulated water flow or vasopressin-stimulated urea transport. There was no inhibition, however, when exogenous cyclic AMP was substituted for vasopressin, and little to no inhibition when the potent analogue 8-bromoadenosine 3′,5′-cyclic monophosphate (8-Br-cAMP) was employed. Rotenone had no effect on adenylate cyclase activity or cyclic AMP levels within the cell; dinitrophenol decreased adenylate cyclase activity minimally.Additional studies with vinblastine and nocodazole, inhibitors of microtubule assembly, demonstrated an inhibition of vasopressin and cyclic AMP-stimulated water flow but showed no effect on urea transport.We would conclude that water and urea transport, as examples of hormone-stimulated processes, have different links to cell metabolism, and that in addition to cyclic AMP, a non-nucleotide pathway may be involved in the action of vasopressin.
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  • 71
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    Journal of Supramolecular Structure 10 (1979), S. 191-197 
    ISSN: 0091-7419
    Keywords: transmembrane signaling ; cholera toxin ; membranes ; photoreactive probes ; Newcastle disease virus ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Using sodium dodecyl sulfate--polyacrylamide gel electrophoresis and autoradiography, we have shown that 125I-labeled cholera toxin binds to Newcastle disease virus. Pretreatment of Newcastle disease virus with “cold” cholera toxin (at 37°C for 30 minutes) inhibits the binding of 125I-labeled toxin in a subsequent incubation (at 37°C for 30 minutes). These results suggest that cholera toxin binds to Newcastle disease virus in a specific manner. The precise receptor for toxin is unknown in Newcastle disease virus but it is presumed to be the ganglioside GM1. We have previously shown that the photoreactive probe 12-(4-azido-2-nitrophenoxy)stearoylgucosamine[1-14C] labels the membrane proteins of Newcastle disease virus. Since the reactive group of the probe, ie, N3, resides within the membrane bilayer, studies were initiated to determine which, if any, of the subunits of cholera toxin cross the membrane of Newcastle disease virus and become radioactively labeled upon photoactivation of the probe at 360 nm. After a 15-minute incubation of cholera toxin with Newcastle disease virus containing the photoreactive probe, irradiation effected the 14C-labeling of the active A1 subunit of cholera toxin. Irradiation of cholera toxin in solution with an equivalent amount of probe but without virus resulted in no labeling of toxin subunits.
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  • 72
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    Journal of Supramolecular Structure 10 (1979), S. 227-239 
    ISSN: 0091-7419
    Keywords: spectrin ; actin ; red cell membranes ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The human erythrocyte structural protein spectrin and its subunits I, II were isolated in the presence of Na-dodecyl-sulfate by gel filtration and preparative gel electrophoresis. After removal of the detergent, spectrin alpha-helical content is comparable to spectrin isolated without detergent. Subunits I and II formed single bands in isoelectric focusing (pI = 5.6) and in Ornstein-Davis disc gel electrophoresis systems, indicating the individual subunits are homogenous in nature. The molecular weights of the subunits I and II, determined by Ferguson plot, are 237,500 and 238,600, respectively, which is in good agreement with values obtained by the standard SDS gel relative mobility method. Limited tryptic digestion of spectrin and two-dimensional peptide maps of the individual subunits cleaved by S-cyanylation reaction showed dissimilar patterns, suggesting differences in primary structure between the two subunits.
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  • 73
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    Journal of Supramolecular Structure 10 (1979), S. 241-251 
    ISSN: 0091-7419
    Keywords: boundary lipid ; 31PNMR ; spin labels ; glycophorin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Both the MN-glycoprotein from human erythrocytes and the hydrophobic fragment from the protein isolated with trypsin treatment, T(is), have been recombined with egg phosphatidylcholine in bilayers at various phospholipid/protein ratios. In order to investigate the effect of the protein on the phospholipid headgroups, 31P nuclear magnetic resonance spectra were obtained with the MN-glycoprotein recombined with egg phosphtidylcholine, which revealed two classes of phospholipid enviroments, one immobilized and one not immobilized. Electron spin resonance (ESR) of fatty acid methyl ester spin labels provided supporting evidence.Computer analysis of the ESR spectra indicate that 4-5 moles of phospholipid are immobilized per mole of protein over a wide range of lipid-to-protein ratios. The immobilization of the phospholipids appears mediated by both the polar headgroups and the hydrocarbon tails of the phospholipid.
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  • 74
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    Journal of Supramolecular Structure 10 (1979), S. 253-263 
    ISSN: 0091-7419
    Keywords: plasma lipoproteins ; erythrocyte morphology ; erythrocyte phosphatase ; spectrin phosphorylation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Addition of human plasma low-density lipoproteins (LDL) to intact human erythrocytes induces the erythrocytes to undergo morphologic transition from biconcave disks to echinocytes and spherocytes. The transformation is time-dependent. Two hours are required before echinocytes are detected by scanning electron microscopy. After two hours, LDL also decrease the phosphate content of spectrin by 40% relative to the control, suggesting that these lipoproteins modulate cell shape by influencing phosphorylationdephosphorylation of a membrane-associated cytoskeletal protein. LDL do not induce depletion of intracellular adenosine triphosphate (ATP), nor do they inhibit cyclic adenosine monophosphate-independent protein kinases which phosphorylate spectrin. LDL stimulate membrane-bound phosphatases by a factor of two, thereby reducing the amount of phosphate covalently bound to membrane proteins. The observed effects are specific for LDL. High-density lipoproteins (HDL) do not stimulate dephosphorylation of spectrin or alter erythrocyte morphology. However, HDL protect the erythrocytes against LDL-induced alterations. These data suggest that the circulating lipoproteins have a role in maintaining erythrocyte morphology by regulating the extent of phosphorylation of spectrin.
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  • 75
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    Journal of Supramolecular Structure 11 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 76
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    Journal of Supramolecular Structure 10 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 77
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    Journal of Supramolecular Structure 10 (1979), S. 265-275 
    ISSN: 0091-7419
    Keywords: membrane hydration ; membrane-bound water ; ANS fluorescence ; infrared spectra ; water-membrane interactions ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bound water is a major component of biological membranes and is required for the structural stability of the lipid bilayer. It has also been postulated that it is involved in water transport, membrane fusion, and mobility of membrane proteins and lipids. We have measured the fluorescence emission of membrane-bound 1-anilino-8-naphthalenesulfonate (ANS) and the infrared spectra of membranes, both as a function of hydration. ANS fluorescence is sensitive to polarity and fluidity of the membrane-aqueous interface, while infrared absorption is sensitive to the hydrogen bonding and vibrational motion of water and membrane proteins and lipids. The fluorescence results provide evidence of increasing rigidity and/or decreasing polarity of the membrane-aqueous interface with removal of water. The membrane infrared spectra show prominent hydration-dependent changes in a number of bands with possible assignments to cholesterol (vinyl CH bend, OH stretch), protein (amide A, II, V), and bound water (OH stretch). Further characterization of the bound water should allow its incorporation into current models of membrane structure and give insight into the role of membrane hydration in cell surface function.
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  • 78
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    Journal of Supramolecular Structure 11 (1979), S. 51-60 
    ISSN: 0091-7419
    Keywords: cyclic AMP ; transport of nucleosides ; nucleobases ; hexoses ; Chinese hamster ovary cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: In a previous study we have demonstrated that neither extracellular nor intracellular cyclic adenosine monophosphate (AMP) levels directly affect the uptake of nucleosides, nucleobases, or hexoses by various types of cultured mammalian cells. Uptake of these nutrients into cells, however, involves two processes operating in tandem: facilitated transport across the membrane and intracellular phosphorylation; and uptake rates generally reflect the rates of substrate phosphorylation rather than of transport. In the present study we have examined the question of whether substrate transport per se is regulated by intracellular cyclic AMP. Initially various cell lines, grown both in suspension and monolayer culture, were screened for their cyclic AMP response to prostaglandin E1, isoproterenol, and inhibitors of cyclic AMP phosphodiesterase. Prostaglandin E1 treatment of Chinese hamster ovary cells was selected as the systems giving the largest and most consistent (50-fold to 100-fold) elevation of cyclic AMP. Rapid kinetic techniques were used to measure the transport of 3-O-methylglucose, thymidine, adenosine, hypoxanthine, and adenine in wild-type cells and in mutant sublines incapable of phosphorylating these substrates. In no case was an increse in intracellular cyclic AMP accompanied by a singinficant change in the rate of transport of these substrates, although prostaglandin E1 slightly inhibited the transport of various substrates.
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  • 79
    ISSN: 0091-7419
    Keywords: cell adhesion ; adhesion proteins ; fibronectin ; chondronectin ; collagen substrates ; gangliosides ; cell surface ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fibronectin mediates the adhesion of fibroblasts to collagen substrates, binding first to the collagen and then to the cells. We report here that the interaction of the cells with the fibronectin-collagen complex is blocked by specific gangliosides, GD1 a and GT1, and that the sugar moieties of these gangliosides contain the inhibitory activity. The gangliosides act by binding to fibronectin, suggesting that they may be the cell surface receptor for fibronectin. Evidence is presented that other adhesion proteins or mechanisms of attachment exist for chondrocytes, epidermal cells, and transformed tumorigenic cells, since adhesion of these cells is not stimulated by fibronectin. Chondrocytes adhere via a serum factor that is more temperature-sensitive and less basic than fibronectin. Unlike that of fibroblasts chondrocyte adhesion is stimulated by low levels of gangliosides. Epidermal cells adhere preferentially to type IV (basement membrane) collagen but at a much slower rate than fibroblasts or chondrocytes. This suggests that these epidermal cells synthesize their own specific adhesion factor. Metastatic cells cultured from the T241 fibrosarcoma adhere rapidly to type IV collagen in the absence of fibronectin and do not synthesize significant amounts of collagen or fibronectin. Their growth, in contrast to that of normal fibroblasts, is unaffected by a specific inhibitor of collagen synthesis. These data indicate the importance of specific collagens and adhesion proteins in the adhesion of certain cells and suggest that a reduction in the synthesis of collagen and of fibronectin is related to some of the abnormalities observed in transformed cells.
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  • 80
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    Journal of Supramolecular Structure 11 (1979), S. 95-104 
    ISSN: 0091-7419
    Keywords: fibronectin structure and properties ; cytoskeleton ; cell surface proteins ; fibronectin distribution ; fibronectin interactions ; transformation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Fibronectin is a large glycoprotein at the cell surface of many different cell types; a related protein is present in plasma. Fibronectin is a dimer of 230,000-dalton subunits and also occurs in larger aggregates; it forms fibrillar networks at the cell surface, between cells and substrata and between adjacent cells, and it is not a typical membrane protein. Cell surface fibronectin is reduced in amount or absent on transformed cells and in many cases its loss correlates with acquisition of tumorigenicity and, in particular, metastatic ability. Exceptions to the correlations with transformation and tumorigenicity exist. Loss of fibronectin and the resulting reduced adhesion appear to be involved in pleiotrpoic alterations in cell behavior and may be responsible for several aspects of the transformed phenotype in vitro. Fibronectin interacts with other macromolecules (collagen/gelatin, fibrin/fibrinogen, proteoglycans) and is apparently connected to microfilaments inside the cell.
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  • 81
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    Journal of Supramolecular Structure 11 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 82
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    Journal of Supramolecular Structure 11 (1979), S. 105-115 
    ISSN: 0091-7419
    Keywords: tumor metastases ; Fc receptor ; shedding ; tumor variants ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The expression of receptors for the Fc portion of IgG immunoglobin molecules was studied on tumor cell lines with high and low metastatic capacity. Two tumor cell lines from DBA/2 mice that had high metastatic activity, ESb and MDAY-D2, contained a high percentage of Fc receptor positive cells, as detected in a rosette assay with IgG antibody-coated erythrocytes (EA). In contrast, the low metastatic parental line Eb, from which ESb was derived, contained only a low percentage of EA-rosette-forming cells. ESb ascites tumor cells adapted to tissue culture in the presence of 2-mercaptoethanol (2ME) had a high expression of Fc receptors, whereas a cell line adapted to tissue culture in the absence of 2ME had a low expression of Fc receptors.“Soluble” Fc receptors were detectable by their ability to bind to EA and to cause blocking of rosette formation. They were found to be present in fluids from tumor-bearing animals, such as serum and cell-free ascites. Even animals with an ascites tumor of the low-metastatic line Eb contained “soluble” Fc receptors.The results are discussed with regard to their possible significance for tumor metastasis.
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  • 83
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    Journal of Supramolecular Structure 11 (1979), S. 189-195 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The objective of this study was the preliminary characterization of the factors from mitotic HeLa cells that can induce meiotic maturation in Xenopus laevis oocytes. We found that this factor is a heat-labile, Ca2+-sensitive, nondialyzable protein with a sedimentation value of 4-5S. Furthermore, no new protein synthesis was found to be required for this mitotic factor to induce maturation in the amphibian oocytes. These data suggest that the factors involved in the breakdown of nuclear membrane and the condensation of chromosomes that are associated with three different phenomena, mitosis, meiosis, and premature chromosome condensation, are very similar in different animal species.
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  • 84
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 207-216 
    ISSN: 0091-7419
    Keywords: renal epithelium ; primary cultures ; prostaglandins ; mammalian cell growth ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Madin Darby canine kidney cells can grow in synthetic medium supplemented with 5 factors - insulin, transferrin, prostaglandin E1, hydrocortisone and triiodothyronine - as a serum substitute. These 5 factors permit growth for one month in the absence of serum, and a growth rate equivalent to that observed in serum-supplemented medium. Dibutyryl cAMP substitutes for prostaglandin E1 in the medium, suggesting that increased growth of Maden Darby canine kidney cells results from increased intracellular cAMP. Potential applications of the serum-free medium are discussed. The medium permits the selective growth of primary epithelial cell cultures in the absence of fibroblast over-growth, and a defined analysis of the mechanisms by which hormones regulate hemicyst formation.
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  • 85
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 321-326 
    ISSN: 0091-7419
    Keywords: bacteriophage T7 ; procapsid core ; electron microscopy ; mutant lysates ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A cylindrical core previously demonstrated in a bacteriophage T7 procapsid (capsid I) has been further examined by electron microscopy. Fibrous extensions of the core have been observed; these fibers appear to connect the core to the capsid I envelope. After infection of a nonpermissive host with bacteriophage T7 amber mutant in any gene coding for a core protein, the resulting lysates contained more noncapsid assemblies of capsid envelope protien than did wild-type lysates; these assemblies had a mass two to at least 500 times greater than the mass of capsid I. This suggests that the internal core and fibers assist the assembly of subunits in the envelope of capsid I.
    Additional Material: 2 Ill.
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  • 86
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 349-359 
    ISSN: 0091-7419
    Keywords: colostrum ; milk ; serum ; growth factors ; mitogens ; DNA synthesis ; proliferation ; 3T3 cells ; serum-free growth ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bovine milk contains growth promoting factors that stimulate DNA synthesis and cell division in confluent monolayers of quiescent Balb/c 3T3 cells. The growth factor activity was highest in colostrum obtained within 24 hours after birth of a calf. Samples of milk obtained 32 hours and 60 hours after birth were 20% and 1% as active respectively as was a sample obtained 8 hours after birth in stimulating DNA synthesis. No activity was detectable 3 days after birth or thereafter. A similar temporal dependence was found in sheep's milk. Bovine colostrum obtained on the day of a calf's birth can be substituted for serum and will support the growth of sparse Balb/c 3T3 cells to confluence. In Dulbecco's modified Eagle's medium (DMEM) supplemented with 2.5% (vol/vol) bovine colostrum, the number of Balb/c 3T3 cells in a dish increased 35-fold, from 2.0 × 104 cells to 7 × 105 cells. The generation time was approximately 38 hours. Proliferation of cells was characterized by formation of clusters of confluent Balb/c 3T3 cells which were smaller in size and more tightly packed than were Balb/c 3T3 cells grown to confluence in serum. No proliferation was detected in DMEM supplemented with milk obtained 10 days after birth of a calf or in DMEM supplemented with bovine serum albumen.
    Additional Material: 7 Ill.
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  • 87
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979), S. 361-370 
    ISSN: 0091-7419
    Keywords: endothelial cells ; platelet-derived growth factor ; thrombin ; wound healing ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Repair of a vascular wound is mediated by migration and subsequent replication of the endothelial cells that form the inner lining of blood vessels. We have measured the growth response of human umbilical vein endothelial cells (HuE) to two polypeptides that are transiently produced in high concentrations at the site of a wound; the platelet-derived growth factor (PDGF) and the protease thrombin. When 104 HuE cells are seeded as a dense island (2-mm diameter) in the center of a 16-mm tissue culture well in medium containing 20% human serum derived from platelet-poor plasma (PDS), no increase in cell number or colony size is observed. With the addition of 0.5 ng/ml partially purified PDGF, colony size increases and the number of cells after 8 days is 4.8 × 104. When human thrombin (1 μg/ml) is added along with the PDGF, the cell number rises to 9.2 × 104. Thrombin alone stimulates no increase in cell number. Although partially purified PDGF stimulates endothelial cells maintained in PDS as well as those maintained in whole blood serum (WBS), pure PDGF is active only when assayed in medium that contains WBS and is supplemented with thrombin. These results suggest the existence of a second class of platelet-derived factors that enable HuE cells to respond to the mitogenic activity of the purified platelet mitogen and thrombin.
    Additional Material: 5 Ill.
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  • 88
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 11 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Type of Medium: Electronic Resource
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  • 89
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 1-14 
    ISSN: 0091-7419
    Keywords: myosin ; Dictyostelium ; RNA ; motility ; nonmuscle cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Myosin purified from Dictyostelium amoebae has approximately 10% by weight of RNA associated with it, unless specific steps (DEAE cellulose chromatography or R Nase digestion) are taken to remove it. This RNA has significant effects on the structural states formed by the myosin at low ionic strength in the presence of Mg2+.Rapid precipitation of Rna-free myosin by dilution generates bipolar thick filaments (540 nm long, 33 nm thick), often with a bare zone and a 15-nm transverse repeat. Rapid precipitation of myosin with copurified RNA yields linear aggregates of bipolar filaments, showing some lateral association.Slow precipitation of RNA-free myosin by dialysis yields very long filaments or ribbons (〉5 μm, 30-60 nm wide) in which the myosin may be packed diagonally across the filament, similar to the “side-polar” aggregates formed by other nonmuscle myosins and by smooth muscle myosin (Craig R, Megerman J: J Cell Biol 75:990, 1977; Hinssen H, D'Haese J, Small JV, Sobieszek A: J Ultrastruct Res 64:282, 1978). Slow precipitation of myosin with copurified RNA generates linear filaments with repeat intervals of 290 and 650 nm.Other polyanions were tested for their effects on myosin aggregation. Total RNA and ribosomal RNA from Dictyostelium, when added to RNA-free myosin, also induced the extensive linear aggregation seen with the copurified RNA/myosin complex, although higher concentrations of RNA were required to obtain quantitatively the same effect. DNA and heparin were also effective inducers of linear aggregation, whereas homopolymers of nucleotides and of acidic or basic amino acids were poorly effective.
    Additional Material: 7 Ill.
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  • 90
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 63-72 
    ISSN: 0091-7419
    Keywords: tumor promoter ; DNA synthesis ; transformed cells ; serum stimulation ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Induction of DNA synthesis by the tumor promoter tetradecanoyl phorbol acetate (TPA) was studied in a line of cultured rat fibroblasts (Rat-1) and their ffRous sarcoma virus-transformed derivative (Rat-1(RSV)). Following serum deprivation for 54 h to achieve quiescene, semiconservative DNA replication was measured by incubation of cells in BrdUrd and FdUrd after serum stimulation in the presence or absence of TPA. Optimal concentrations of TPA (0.1-0.5 μg/ ml) in serum-free medium induced a small increase (10-15%) in the amount of DNA made over a 30-h period in both Rat-1 and Rat-1 (RSV) cells. When Rat-1 cells were stimulated by a 4-h serum pulse, 30% of the DNA was replicated by 30 h. If the serum pulse was follwed by TPA addition, 702% DNA replication wass observed. If the serum pulse was preceded by TPA addition, the onset of DNA synthesis waas delayed by several hous, but stimulation of DNA synthesis occurred. In contrast, the Rat-1 (RSV) cells did not show an increase in DNA synthesis induced by TPA in similar protocols, but the serum-induced onset on DNA synthesis was delayed by several hours in the presence of TPA. Therefore, TPA acts as a co-inducer of DNA synthesis in the Rat-1 but not in the Rat-(RSV) cells. The parent alcohol, phorbol, was inactive in Rat-1 cells, but delayed the onset of DNA synthesis in the Rat(RSV) cells. We conclude that the co-inducing and delaying activities of TPA on DNA synthesis appear to be distinct and to act at different points in the G1 phase of the cell cycle.
    Additional Material: 5 Ill.
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  • 91
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 47-61 
    ISSN: 0091-7419
    Keywords: plasma membrane ; fluidity ; skeletal muscle ; myogenesis laser ; fluorescence photobleaching recovery ; fluorescence depolarization ; carbocyanine ; perylene ; fluorescence anisotropy ; microviscosity ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Changes in membrane fluidity during myogenesis have been studied by fluorescence microscopy of individual cells growing in monolayer cultures of embryonic chick skeletal muscle cells. Membrane fluidity was determined by the techniques of fluorescence photobleaching recovery (FDR), with the use of a lipidsoluble carbocyanine dye, and by fluorescence depolarization (FD), with perylene used as the lipid probe. The fluidity of myoblast plasma membranes, as determined from FPR measurements in membrane areas above nuclei, increased during the period of myoblast fusion and then returned to its initial level. The membrane fluidity of fibroblasts, also found in these primary cultures, remained constant. The fluidity in specific regions along the length of the myoblast membrane was studied by FD, and it was observed that the extended arms of the myoblast have the highest fluidity on the cell and that the tips at the ends of the arms had the lowest fluidity. However, since the perylene probe used in the FD experiments appeared to label cytoplasmic components, changes in fluidity measured with this probe reflect changes in membrane fluidity as well as in cytoplasmic fluidity. The relative change in each of these compartments cannot yet be ascertained. Tips have specialized surface structures, filopodia and lamellipodia, which may be accompanied by a more immobile membrane as well as a more rigid cytoplasm. Rounded cells, which may also have a more convoluted surface structure, show a lower apparent membrane fluidity than extended cells.
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  • 92
    Electronic Resource
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979) 
    ISSN: 0091-7419
    Keywords: Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 93
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 127-137 
    ISSN: 0091-7419
    Keywords: polyoma virus ; middle ; and small tumor antigens ; ts-a ; hr-t ; abortive transformation ; transformed cells ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Stable neoplastic transformation of cells by polyoma virus requires the participation of two viral genes, designated ts-a and hr-t. The effects of mutations in these two genes on the patterns of T-antigen synthesis during productive infection have been previously described: ts- a mutants are affected in the “large” (100K) nuclear T antigen, and hr-t mutants are affected in the “middle” (36K, 56K, 63K) and “small” (22K) T agtigens. The latter are associated predominantly with the plasma membrane (56K) and cytosol fractions, rrespectively.Here we examine the expression of the various forms of polyoma T antigen in nonproductive infection (abortive transformation) as well as in stably transformed cell lines of different species. The results on abortive transformation are essentially the same as those described above for productive infection. In stably transformed cells, the middle and small T antigens are seen to various extents. The large T antigen, however, is often absent or present below the level of detection. Clones lacking the large T antigen are found most often among mouse transformants, but are also seen among rat transformants. Retention of the 100K species in transformed cells therefore appears to be, at least in part, an inverse function of the level of permissivity of the host toward productive viral infection. These findings indicate that the induction of the transformed phenotype in both abortively and stably transformed cells generally does not require the large T antigen, but rather the products of the hr-t gene.
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  • 94
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 355-367 
    ISSN: 0091-7419
    Keywords: Alkaline phosphatase ; chromosome-mediated gene transfer ; human breast tumor cells ; hydrocortisone ; lipochromes ; membrane bound enzymes ; nucleoside uptake ; thymidine kinase ; thymidine transport ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: BOT-2 cells (human breast tumor origin) have an impaired ability to utilize exogenous thymidine. Previous studies revealed this deficiency to be the permeation event rather than phosphorylation, since the cells have active thymidine kinase. Chromosome-mediated gene transfer was used to transfer genetic information in the form of metaphase chromosomes, from HeLa-65 cells to the BOT-2 cells, correcting the permease deficiency. Poly-L-ornithine or lipochromes were used for facilitation of chromosome uptake. After selection on HAT medium, transferant clones were isolated at a frequency of 4 X 10-5 and 1 X 10-5, respectively. Transferants MGP-1 and MGL-1 are stable after 18 months and have been characterized on the bases of purine and pyrimidine nucleoside uptake, relative thymidine kinase activities, alkaline phosphatase activities, and hydrocortisone-induced alkaline phosphatase activity. MGP-1 demonstrates positive thymidine uptake and incorporates radiolabeled thymidine into DNA. MGL-1 remains thymidine transport-deficient and survives on HAT by increasing endogenous dihydrofolate reductase activity. Alkaline phosphatase activity in MGL-1 is similar to HeLa-65, 2% of that in BOT-2, and in addition, is inducible 25-30-fold by 3 μM hydrocortisone. We have separated, genetically, a thymidine permease function from phosphorylation in cells of human origin and have transferred genetic information for the regulation of alkaline phosphatase.
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  • 95
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 335-354 
    ISSN: 0091-7419
    Keywords: knobs or blebs ; transformed cells ; reverse transformation ; time-lapse cinematography ; scanning EM ; transmission EM ; microtubules and microfilaments or microfibrillar system ; colcemid ; cytochalasin B ; dibutyryl cyclic AMP ; indirect immunofluorescence ; antiactin ; antitubulin ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Transformed cells often display knobs (or blebs) distributed over their surface throughout most of interphase. Scanning electron microscopy (SEM) and time-lapse cinematography on CHO-K1 cells reveal roughly spherical knobs of 0.5-4 μm in diameter distributed densely around the cell periphery but sparsely over the central, nuclear hillock and oscillating in and out of the membrane with a period of 15-60 sec. Cyclic AMP derivatives cause the phenomenon of reverse transformation, in which the cell is converted to a fibroblastic morphology with disappearance of the knobs. A model was proposed attributing knob formation to the disorganization of the jointly operating microtubular and microfilamentous structure of the normal fibroblast. Evidence for this model includes the following: (1) Either colcemid or cytochalasin B (CB) prevents the knob disappearance normally produced by cAMP, and can elicit similar knobs from smooth-surfaced cells; (2) knob removal by cAMP is specific, with little effect on microvilli and lamellipodia; (3) immunofluorescence with antiactin sera reveals condensed, amorphous masses directly beneath the membrane of CB-treated cells instead of smooth, parallel fibrous patterns of reversetransformed cells or normal fibroblasts; (4) transmission electron microscopy (TEM) of sections show dense, elongated microfilament bundles and microtubules parallel to the long axis of the reverse-transformed CHO cell, but sparse, random microtubules throughout the transformed cell and an apparent disordered network of 6-nm microfilaments beneath the knobs; (5) cell membranes at the end of telophase, when the spindle disappears and cleavage is complete, display typical knob activity as expected by this picture.
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  • 96
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 467-470 
    ISSN: 0091-7419
    Keywords: epidermal growth factor ; fibroblast growth factor ; cell division ; mitogenesis ; growth control ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The enhanced ability of murine serum to support growth of 3T3 cells, when compared with fetal calf serum, is also evident on variants of 3T3 cells lacking the ability to bind epidermal growth factor (EGF). Variant 3T3 cell lines unable to bind EGF also retain a mitogenic response to fibroblast growth factor.
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  • 97
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 12 (1979), S. 457-466 
    ISSN: 0091-7419
    Keywords: human placental basement membrane ; extracellular matrix ; human chorionic villar basement membrane ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The cell-free extracellular matrix of human placental chorionic villi has been prepared by a procedure employing extraction of the terminal villar fragments with the detergents Triton X-100 and sodium deoxycholate. The isolated human placental extracellular matrix retains an intact, but collapsed, histoarchitecture, as observed by scanning and transmission electron microscopy. It remains intact, in large part because of the presence of continuous sheets of villar basement membranes and associated interstitial collagen fibers and scattered patches of fibrin. The staining characteristics and chemical composition of the isolated human placental extracellular matrix are similar to those reported for basement membranes in several tissues and indicate the presence of collagen-like and glycoprotein components in this preparation. Gel electrophoresis of urea-SDS-mercaptoethanol extracts of the matrix showed that it consists of several polypeptide components of various molecular weights, some of which are associated into high molecular weight complexes by disulfide bonds.
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  • 98
    ISSN: 0091-7419
    Keywords: nuclear matrix ; estrogen ; RNA ; uridine ; Xenopus laevis ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: At various times following estorgen administration, the nuclear matrix was isolated from the liver of male Xenopus laevis by sucrose gradient centrifugation of nuclei treated with a high-salt buffer and DNase I in the presence of a proteolytic inhibitor (PMSC - phenylmethyl sulfonyl chloride). Electron micrographs of the nuclear matrix demonstrate a sponge-like network attached to a well-defined inner envelope with a ribosome-free outer envelope. Chemical analyses show that the HSB-DNase-treated nuclei consist of 16% DNA, 2% RNA, and 82% protein, a composition that is consistent with that of nuclear matrices isolated from other species. The specific activity of the matrix-associated RNA following estrogen treatment appears to be maximally enhanced after 5 h and decreases until approximately 12 h, when the activity begins to increase again.
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  • 99
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 365-375 
    ISSN: 0091-7419
    Keywords: ribosomes ; crystallization ; hypothermia ; chick embryos ; degeneration ; nuclei ; nucleolar extrusions ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ribosome crystallization within nuclei has been studied in chick embryos with procedures which increase its frequency by various orders of magnitude as compared to previous findings. The extrusion of ribosome microcrystals from nuclei is reported for the first time, and a model for the transfer of ribosomes from nucleus to cytoplasm is proposed.
    Additional Material: 11 Ill.
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  • 100
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    New York, N.Y. : Wiley-Blackwell
    Journal of Supramolecular Structure 10 (1979), S. 443-455 
    ISSN: 0091-7419
    Keywords: ribosomes, 30S subunit structure, immunochemistry ; Life Sciences ; Molecular Cell Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Antibodies to Escherichia coli ribosomal protein S4 react with S4 in subribosomal particles, eg, the complex of 16S RNA with S4, S7, S8, S15, S16, S17, and S19 and the RI* reconstitution intermediate, but they do not react with intact 30S subunits. Antibodies were isolated by three different methods from antisera obtained during the immunization of eight rabbits. Some of these antibody preparations, which contained contaminant antibodies directed against other ribosomal proteins, reacted with subunits, but this reaction was not affected by removal of the anti-S4 antibody population. Other antibody preparations did not react with subunits. It is concluded that the antigenic determinants of S4 are accessible in some protein deficient subribosomal particles but not in intact 30S subunits.
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