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  • 1
    ISSN: 0730-2312
    Keywords: cell-substratum adhesion ; cell surface ; integral membrane glycoproteins ; conserved structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Broad spectrum antisera have been raised against surface membrane-derived material from baby hamster kidney cells and mouse mammary tumor epithelial cells. These antisera disrupt cell-substratum adhesion in their respective cell types. Using an antibody neutralization (blocking) assay, adhesion-related glycoproteins have been isolated from non-ionic detergent extracts of each cell type. The purified material in each case consisted of a restricted population of glycoproteins of approximately 120,000-160,000 Mr. Purified material from each system blocked the disruption of adhesion induced by the heterologous antiserum on either cell type. The antisera were capable of disrupting cell-substratum adhesion of a large number of cell types and species sources. In addition, antibody blocking activity could be detected from partially purified extracts of several adult hamster cell types and a variety-of cultured cell types. Thus, in addition to having similar substratum-associated glycoproteins (eg, fibronectin) and cytoskeleton-associated proteins (eg, α-actinin and vinculin) cells from different species and tissue sources appear to have a relatively conserved class of integral membrane glycoproteins involved in cell substratum-adhesion.
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  • 2
    ISSN: 0730-2312
    Keywords: hepatoma cells ; cell surface components ; membrane glycoproteins ; lectin receptor ; sialoglycoproteins ; plasma membrane glycoproteins ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A major cell surface sialoglycoprotein with Concanavalin A receptor activity has been isolated from rat Zajdela ascites hepatoma cells.The sialic acid residues of the plasma membrane glycoproteins were specifically labeled by oxidation with NaIO4 followed by reduction with NaB3H4. Surface-labeled glycoproteins were released by short incubations with TPCK-trypsin at 37°C and then separated by gel filtration on Sepharose 6B column. The predominantly labeled fraction, GP II2, was then purified by chromatography on DEAE-cellulose equilibrated with 0.05 M phosphate buffer, pH 7.5, and eluted with increasing molarities of NaCl. It was shown to be homogeneous by protein and carbohydrate staining on SDS-polyacrylamide gels, isoelectric focusing, rechromatography on DEAE-cellulose and immunoelectrophoresis. It has an apparent molecular weight of 110,000 daltons.The location of GP II2on the cell surface was confirmed by the fact that it could be labeled metabolically with, D-(3H) glucosamine and externally through the nonpenetrating periodate-NaB3H4 system.GP II2could not be removed from the cell surface by high salt concentrations, chelator, or chaotropic agents but was released from the membrane by detergents. This suggests that GP II2could be an integral protein.Analysis of the carbohydrate composition of GP II2 revealed galactose, N-acetylglucosamine, N-acetylgalactosamine, and sialic acid as major constituents and mannose as a minor one. This suggests that it contains carbohydrate chains both O- and N-linked to the polypeptide chain, most of them being O-linked.Finally, GP II2has a potent Concanavalin A receptor activity. It inhibits the interaction between Concanavalin A and hepatoma cells and suppresses its effects on hepatoma cell proliferation.
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  • 3
    ISSN: 0730-2312
    Keywords: E coli ; DNA damage ; excision repair ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Bacteria and eukaryotic cells employ a variety of enzymatic pathways to remove damage from DNA or to lessen its impact upon cellular functions. Most of these processes were discovered in Escherichia coli and have been most extensively analyzed in this organism because suitable mutants have been isolated and characterized. Analogous pathways have been inferred to exist in mammalian cells from the presence of enzyme activities similar to those known to be involved in repair in bacteria, from the analysis of events in cells treated with DNA damaging agents, and from the analysis of the few naturally occurring mutant cell types.Excision repair of pyrimidine dimers produced by UV in E coli is initiated by an incision event catalyzed by a complex composed of uvrA, uvrB, and uvrC gene products. Multiple exonuclease and polymerase activities are available for the subsequent excision and resynthesis steps. In addition to the constitutive pathway, which produces short patches of 20-30 nucleotides, an inducible excision repair process exists that produces much longer patches. This long patch pathway is controlled by the recA-lexA regulatory circuit and also requires the recF gene. It is apparently not responsible for UV-induced mutagenesis. However, the ability to perform inducible long patch repair correlates with enhanced bacterial survival and with a major component of the Weigle reactivation of bacteriophage with double-strand DNA genomes.Mammalian cells possess an excision repair pathway similar to the constitutive pathway in E coli. Although not as well understood, the incision event is at least as complex, and repair resynthesis produces patches of about the same size as the constitutive short patches. In mammalian cells, no patches comparable in size to those produced by the inducible pathway of E coli are observed.Repair in mammalian cells may be more complicated than in bacteria because of the structure of chromatin, which can affect both the distribution of DNA damage and its accessibility to repair enzymes. A coordinated alteration and reassembly of chromatin at sites of repair may be required. We have observed that the sensitivity of digestion by staphylococcal nuclease (SN) of newly synthesized repair patches resulting from excision of furocoumarin adducts changes with time in the same way as that of patches resulting from excision of pyrimidine dimers. Since furocoumarin adducts are formed only in the SN-sensitive linker DNA between nucleosome cores, this suggests that after repair resynthesis is completed, the nucleosome cores in the region of the repair event do not return exactly to their original positions.We have also studied excision repair of UV and chemical damage in the highly repeated 172 base pair α DNA sequence in African green monkey cells. In UV irradiated cells, the rate and extent of repair resynthesis in this sequence is similar to that in bulk DNA. However, in cells containing furocoumarin adducts, repair resynthesis in α DNA is only about 30% of that in bulk DNA. Since the frequency of adducts does not seem to be reduced in α DNA, it appears that certain adducts in this unique DNA may be less accessible to repair.Endonuclease V of bacteriophage T4 incises DNA at pyrimidine dimers by cleaving first the glycosylic bond between deoxyribose and the 5′ pyrimidine of the dimer and then the phosphodiester bond between the two pyrimidines. We have cloned the gene (denV) that codes for this enzyme and have demonstrated its expression in uvrA recA and uvrB recA cells of E coli. Because T4 endonuclease V can alleviate the excision repair deficiency of xeroderma pigmentosum when added to permeabilized cells or to isolated nuclei after UV irradiation, the cloned denV gene may ultimately be of value for analyzing DNA repair pathways in cultured human cells.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 18 (1982), S. 393-393 
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 5
    ISSN: 0730-2312
    Keywords: polyclonal B cell activation ; suppression ; T cells ; regulation ; lipopolysaccharide ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Resident T lymphocytes have been found to exert helper and suppressor regulatory influences with regard to polyclonal activation of murine splenic B lymphocytes elicited by lipopolysaccharide. In the normal adult spleen, only T cell helper influences are exercised over polyclonal B cell activation. This activity is a property of Lyt 1+2- T cells and does not appear to be subject to MHC restriction. Suppressive influence evidently is either latent or it exists at such a low level that its effects are difficult to detect. No regulatory activity can be recovered from the supernatants of T cells, cultured either with or without LPS. However, suppressor T cell function may be evoked by activating splenic T cells with Concanavalin A or by sonicating unstimulated splenic T cells in order to liberate a suppressive potential which is not expressed by these unstimulated cells when intact. The soluble fraction of resident splenic T cell sonicates exerts both helper and suppressor regulatory influences. The soluble helper activity is derived from Lyt 1+2- T cells, whereas suppressor activity is generated from Lyt 1-2+ T cells. The suppressive activity of T cell sonicates is not restricted by the MHC gene complex. Helper and suppressor activities contained in splenic T cell sonicates were separated by gel chromatography; the suppressive activity was found to elute with a molecular weight between 68,000 and 84,000 daltons, and the helper activity eluted with a molecular weight between 15,000 and 23,000 daltons. The data indicate that helper and suppressor activities are distinct molecular entities derived from distinct splenic T lymphocyte subpopulations. The possibility that these molecules are precursors to or components of antigen-specific or nonspecific helper and suppressor factors described in the literature is discussed.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 18 (1982), S. 423-431 
    ISSN: 0730-2312
    Keywords: endocytosis ; macrophage-like cell line ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A macrophage-like cell line (P388D1) has been used to demonstrate that glucocorticoids inhibit the fluid-phase endocytosis of fluorescein-labeled dextran (FITC-dextran). Initial experiments demonstrated that the interaction of FITC-dextran with cells had all the features of fluid-phase uptake, ie, the amount taken up was proportional to the concentration in the medium, the uptake proceeded continuously with time and was blocked at 4°C. Dexamethasone (10-7M) had no effect on endocytosis until 11 hours after addition of the steroid, when it inhibited the uptake of FITC-dextran by 35%. The amount of inhibition increased with longer exposure times to the hormone up to 50% after 22 hours. Although this effect on endocytosis was Observed prior to any effect on growth of the cells, endocytosis as well as cell proliferation were inhibited in a dose-dependent fashion. A preliminary survey of selected steroids has established that the inhibition of endocytosis was restricted to steroids of the glucocorticoid class. The key experiments were also performed using horseradish peroxidase instead of FITC-dextran with, essentially, identical results.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 18 (1982), S. 447-459 
    ISSN: 0730-2312
    Keywords: acetylcholinesterase ; ligatin ; membrane-bound lectin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Ligatin, a lectin that recognizes phosphorylated sugars, has been demonstrated in mammalian tissues to bind specific hydrolases to cell surfaces. Ligatin exists as a filament that can be released from membranes still complexed with its bound hydrolases by treatment of membrane preparations with CaCl2 and/or pH 8.0. The ligatin-hydrolase complexes subsequently can be dissociated with ethyleneglycol-bis(β-amino-ethyl ether) N, N′-tetraacetic acid, resulting in a concurrent depolymerization of the ligatin filament. From membrane preparations of cerebrum, this procedure solubilized ligatin and a membrane-bound acetylcholinesterase (EC 3.1.1.7). Binding of the cosolubilized acetylcholinesterase to ligatin could be demonstrated in vitro by affinity chromatography using the immobilized lectin. Ligatin-hydrolase complexes have been shown to be dissociated by specific phosphorylated sugars (mannose 6-phosphate and glucose 1-phosphate). These sugars were also effective in eluting bound brain acetylcholinesterase from ligatin affinity columns. Analysis of labeled glycitols produced by tritiated borohydride reduction confirmed the presence of phosphorylated sugars on the ligatin-cosolubilized material from brain.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 18 (1982), S. 479-492 
    ISSN: 0730-2312
    Keywords: spectrin domains ; protease-resistant ; erythrocyte ; membrane ; cytoskeleton ; structural repeat ; domain structure ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Mild treatment of human erythrocyte spectrin with trypsin produces discrete intermediate-sized peptides. The effects of buffer composition, enzyme-substrate ratio, temperature, and other experimental parameters on the resulting peptide pattern have been examined. Spectrin is capable of regaining its proteolytic resistance after NaDodSO4-induced denaturation, permitting the use of isolated subunits to study spectrin structure and function. Tryptic digestion of isolated subunits also has greatly facilitated the identification of the subunit origin of the intermediate-sized peptides. Isolated subunits could also be recombined to form functional units similar but not identical to the native dimeric form of the molecule. Spectrin apparently is composed of numerous large protease-resistant regions or domains connected by small protease sensitive segments. The structural integrity and accessibility of these sites is minimally affected by oligomeric state or proteolytic digestion conditions. The similarities of sizes, isoelectric points, and amino acid compositions of many intermediate-size peptides from areas of both subunits suggest that at least part of spectrin's structure may have evolved via replication of a single gene. A possible structural repeat of approximately 50,000 daltons is hypothesized.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    ISSN: 0730-2312
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
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  • 10
    ISSN: 0730-2312
    Keywords: membrane sidedness ; regulatory subunits ; ejaculated sperm ; photoaffinity ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The photoaffinity probe (32P)8-N3 cAMP was used to label the cAMP binding proteins in washed ejaculated human sperm. Three saturable binding proteins were photolabeled in both intact and disrupted cells with apparent molecular weights of 55,000, 49,000 and 40,000 daltons corresponding to the regulatory subunits of type II and type I cAMP-dependent protein kinase (cAMP-PK) and to an endogenous proteolytic product of the regulatory subunits, respectively. Photoincorporation in the three proteins could be totally blocked by preincubating the cells with cAMP. Cell-free seminal plasma was found to be free of detectable (12P) 8-N3 cAMP-binding proteins. The 8-N3 cAMP was also effective in stimulating endogenous cAMP-PK activity in intact and disrupted sperm. A substantial amount of (32P) 8-N3 cAMP binding to types I and II regulatory subunits and cAMP-PK activity was detected on washed intact cells, intact cells. Intact cell bound 1.80 pmol of (32P) 8-N3 cAMP/mg protein and had cAMP-PK activity of 824 units/108 cells. Disrupted cells bound 3.95 pmol (32P) 8-N3 cAMP mg protein and had a cAMP-PK activity of 2,206 units/108 cells. The data presented support the concept of two classes of cAMP receptors being differentially available to externally added (32P) 8-N3 cAMP and proteases. Cellular membrane integrity and membrane sidedness are discussed as possible explanations for the observation reported.
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