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  • Life and Medical Sciences  (24)
  • 1985-1989  (24)
  • 1950-1954
  • 1985  (24)
  • 1
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 28 (1985), S. 23-29 
    ISSN: 0730-2312
    Keywords: fibronectin ; laminin ; liposomes ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: We have incorporated antibodies against fibronectin or laminin into liposomes and studied their interaction with insoluble forms of these antigens. The antibodies, after modification by palmitoylchloride, were incorporated into the lipid bilayer by the cholate dialysis method. The antibodies in the liposomes recognized their specific antigen with little reaction to the alternative attachment protein or to albumin (〈2%). The binding of antibody-containing liposomes to insoluble antigen was inhibited by soluble antibodies to the respective antigens but not by antibodies to other antigens. The affinity constant of the liposome-antibody complex with the antigen was estimated at 1-10 × 10-9 M liposomes. Thus, antibodies in liposomes retain their reactivity and specificity, and the reaction constant is comparable to that observed for immune complexes.
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  • 2
    ISSN: 0148-7280
    Keywords: boar ; binding proteins ; plasma membrane proteins ; sperm ; zona pellucida ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: Boar sperm plasma membrane proteins (PMPs) with affinity for the zona pellucida were partially purified from columns of dextran sulfate using a linear salt gradient and a buffered detergent that retained their ability to block directly the binding of uncapacitated and capacitated sperm to isolated porcine oocytes. PMPs that bound most strongly to dextran sulfate (fraction IV) were also most effective in blocking sperm binding to porcine oocytes. These tightly bound proteins also bound to isolated zonae to a greater extent than other fractions. Monovalent antibodies to fraction IV PMPs completely blocked sperm binding to isolated eggs. Fraction IV PMPs lost the ability to inhibit directly the binding to eggs when treated with chaotropic agents and trypsin; the fraction also displayed a tendency to aggregate in the absence of high salt. This property and the affinity of proteins in this fraction for sulfated polysaccharides indicate that specific hydrophilic interactions may play a significant role in sperm-zona attachments.
    Additional Material: 4 Ill.
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  • 3
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Morphology 184 (1985), S. 41-49 
    ISSN: 0362-2525
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Upon fertilization, the zebrafish egg undergoes marked physiological and structural changes, one of which involves blastodisc formation. Before fertilization, yolk globules are rounded and the endoplasm extends throughout the oocyte. During blastodisc formation, the yolk globules become angular and the endoplasm is restricted to streamers among the yolk globules. The streamers are oriented in an anterior-posterior axis of the egg. During blastodisc formation the cytoskeleton consists of an extensive array of filamentous structures of variable width in both the cortex as well as within elongate endoplasmic streamers. Although the filamentous components in the cortex and endoplasmic streamers probably include both microfilaments and microtubules, frequently they are somewhat wider than the usual dimensions, and possible reasons for this are suggested. From their arrangement in both the cortex and endoplasm, it seems likely that the components of the cytoskeleton (e.g., microfilaments and microtubules) may provide, through contraction, the major force responsible for the streaming of the endoplasm into the forming blastodisc. It is assumed that the surface tension of the vegetal hemisphere exceeds that of the animal hemisphere, thus forcing, through differential contraction, the endoplasm to flow in the direction of the forming blastodisc. No distinct barrier between the yolk and forming blastodisc was observed. The compressed condition of the larger and many-sided yolk globules could prevent their movement into the blastodisc. Scanning electron microscopy is limited in the resolution with which it can depict the cytoskeleton, but nonetheless it provides useful information about structural interrelationships.
    Additional Material: 12 Ill.
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  • 4
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 29 (1985), S. 195-208 
    ISSN: 0730-2312
    Keywords: epidermal growth factor receptor ; protein-tyrosine kinase ; self-phosphorylation ; protein kinase C ; oncogene ; growth factor ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The receptor for epidermal growth factor (EGF) is a glycosylated transmembrane phosphoprotein that exhibits EGF-stimulable protein tyrosine kinase activity. On EGF stimulation, the receptor undergoes a self-phosphorylation reaction at tyrosine residues located primarily in the extreme carboxyl-terminal region of the protein. Using enzymatically active EGF receptor purified by immunoaffinity chromatography from A431 human epidermoid carcinoma cells, the self-phosphorylation reaction has been characterized as a rapid, intramolecular process which is maximal at 30-37°C and exhibits a very low Km for ATP (0.2 μM). When phosphorylation of exogenous peptide substrates was measured as a function of receptor self-phosphorylation, tyrosine kinase activity was found to be enhanced two to threefold at 1-2 mol of phosphate per mol of receptor. Analysis of the dependence of the tyrosine kinase activity on ATP concentration yielded hyperbolic kinetics when plotted in double-reciprocal fashion, indicating that ATP can serve as an activator of the enzyme. Higher concentrations of peptide substrates were found to inhibit both the self- and peptide phosphorylation, but this inhibition could be overcome by first self-phosphorylating the enzyme. These results suggest that self-phosphorylation can remove a competitive/inhibitory constraint so that certain exogenous substrates can have greater access to the enzyme active site. In addition to self-phosphorylation, the EGF receptor can be phosphorylated on threonine residues by the calcium- and phospholipid-dependent protein kinase C. The sites on the EGF receptor phosphorylated in vitro by protein kinase C are identical to the sites phosphorylated on the receptor isolated from A431 cells exposed to the tumor promoters 12-O-tetradecanoylphorbol 13-acetate or teleocidin. This phosphorylation of the EGF receptor results in a suppression of its tyrosine kinase and EGF binding activities both in vivo and in vitro. The EGF receptor can thus be variably regulated by phosphorylation: self-phosphorylation can enhance tyrosine kinase activity whereas protein kinase C-catalyzed phosphorylation can depress enzyme activity. Because these two phosphorylations account for only a fraction of the phosphate present in the EGF receptor in vivo, other protein kinases can apparently phosphorylate the receptor and these may exert additional controls on EGF receptor/kinase function.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 27 (1985), S. 1-12 
    ISSN: 0730-2312
    Keywords: helper T cell-B cell interactions ; surface immunoglobulin ; Ia molecules ; membrane receptors ; signal transducers ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: The question of whether surface immunoglobulin and Ia molecules have a signalling function in helper T cell-dependent activation of B cells has been evaluated. Two sources of B cells have been used, one a purified population of haptenbinding B cells, the other a B-cell lymphoma, CH12, with known antigen specificity. Evidence is presented that both immunoglobulin and Ia molecules are receptors actively involved in the initial activation of resting B cells. Nevertheless, the requirements for ligand binding to either receptor can be bypassed under appropriate conditions, and the implications of this result for the function of these molecules is discussed. With respect to B-cell Ia, the authors present data that demonstrate two distinct functions of this molecule, one as a restricting element for T-cell activation, the second as a signalling receptor for B-cell excitation. On the CH12 surface, the I-A molecule fulfills the former function, but T-cell interactions with I-A fail to result in B-cell stimulation, suggesting that B-cell la may limit helper T cell-B cell interactions. We suggest that the binding of antigen surface immunoglobulin and binding of helper T-cell receptors to the appropriate Ia molecule(s) results in the activation of genes that encode for a third class of membrane B-cell receptors, those that bind B-cell stimulating factors.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 122 (1985), S. 159-165 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A method has been developed to quantitate megakaryocytopoiesis in culture by measuring acetylcholinesterase synthesized in vitro. Murine marrow cells, treated with diisopropylfluorosphosphate (DFP) to inactivate initial acetylcholinesterase (AchE) present in megakaryocytes and contaminating blood, were set up in Iscove's medium supplemented with 15% DFP-treated horse serum ± pokeweed mitogen-stimulated spleen cell conditioned medium (PWM-SCM) in 96-well microplates. Following the culture period, Triton X-100, dithiobisnitrobenzoic acid (DTNB), and acetylthiocholine iodide were added to each well. AchE synthesized in culture cleaved acetylthiocholine to thiocholine, which stochiometrically reduced the colorless indicator DTNB to a highly colored product. Thirty minutes following the addition of substrate, the plates were assayed for activity with a vertical recording photometer. When platelets, freshly prepared bone marrow cells, or cultured marrow were assayed by this method, a linear relationship was observed between optical density (OD) and the number of cells assayed. Moreover, a linear relationship between the number of AchE-positive megakaryocytes determined histochemically and AchE activity determined spectrophotometrically was observed. Red cells exhibited no activity. Inhibitor studies demonstrated that the activity measured was true AchE. Separation of marrow by density gradient centrifugation showed that the megakaryocyte enriched fraction contained all the AchE while the megakaryocyte depleted fraction contained none. From the data we conclude that this rapid, semiautomated method quantitates megakaryocytic AchE synthesis in culture, and that this method will be a useful assay system for the detection of factors that influence megakaryocytopoiesis.
    Additional Material: 7 Ill.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 124 (1985), S. 439-445 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Extracts of term human placenta were tested for enhancement of proliferative growth of primary cultures of human keratinocytes. Saline extracts or supernatants from homogenates were dialyzed extensively, lyophilized, and tested in subcultures of keratinocytes in MCDB 153 medium with 0.1 mM Ca++ containing only defined supplements (insulin, hydrocortisone, transferrin, ethanolamine, phosphoethanolamine). Cells plated in the absence of EGF at moderately high densities (1000-3000 cells per cm2) formed colonies and grew in the presence of placental extract at 25-500 μg/ml. Extracts of cord serum or maternal serum were inactive, suggesting that the activity is derived from placental tissue. The activity is not EGF, since the activity in the placental extract, unlike EGF, did not promote growth at low cell density, was synergistic with EGF under some conditions, and did not produce changes in colonial morphology which occurred in the presence of EGF. Unlike keratinocyte growth-promoting activity in bovine hypothalamic extract, the activity is nondialyzable and is destroyed at 100°C. Placental extract could not replace any of the defined components of the medium and is therefore distinct from them. The presence of activity in the placenta with distinctive properties suggests that this is a previously undescribed material with growth-promoting properties for epithelium.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 123 (1985), S. 126-131 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The development of hormonally defined serum-free media (HDM) for the culture of mammalian cells has allowed the study of specific cellular functions in a totally defined environment. Recently, several reports have indicated that there are differences in the basic cellular physiology of cells cultured in HDM when compared to cells cultured by the more traditional method of using serum-supplemented culture media (SM). We report here that there are significant changes in the protein synthetic pattern in MDCK cells grown in HDM. There were no changes exhibited during the first passage in HDM, but following 10 passages in HDM there was an increased isotope ratio of (1) plasma membrane proteins with molecular weights of 12,000, 36,000 and 68,000 and (2) endoplasmic reticulum proteins with molecular weights of 12,000 and 37,000. Additionally, the incorporation of methionine and uridine were significantly increased in cells cultured in HDM for 10 passages. At present, we believe that these changes in the protein synthetic patterns are due at least partially to increased protein synthesis as indicated by monosome/polysome ratios. Therefore, though the use of HDM offers a completely defined system for studying cellular function, results obtained using HDM must be interpreted with caution when comparing them to previous studies that have used SM.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 122 (1985), S. 30-38 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Angiotensin I-converting enzyme (ACE) activity was measured in lysates of cloned and uncloned cultures of bovine fetal aortic endothelial cells. The expression of ACE activity in these cells was complex, and influenced by subcultivation, cell density, serum, cumulative population doublings, and clonal heterogeneity. The ACE specific activity at any point in the in vitro lifespan was determined, at least in part, by interaction of these culture variables. After subcultivation to subconfluent densities, cellular ACE specific activity decreased markedly and did not reach detectable levels until cells attained confluent densities. The use of different suppliers' lots of serum in the growth medium resulted in different cellular ACE specific activities. The ACE specific activity decreased as cultures were serially subcultivated, but remained detectable throughout the lifespan, suggesting a linkage between the proliferative history of an endothelial cell and its remaining capacity to express ACE. Increased ACE activity was observed when cells at the end of their lifespan were cultured at high densities. Cloned strains behaved similarly to the uncloned parent culture, except that they exhibited a wide range of ACE specific activities.
    Additional Material: 8 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 122 (1985), S. 64-72 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: The extracellular ionized calcium and magnesium requirements for lectin-induced lymphocyte DNA synthesis were measured in a serum-free system. The use of this system permitted measurements of the ionized calcium and magnesium concentrations with ion-selective electrodes. Maximal DNA synthesis was observed at 270 μ ionized calcium and at 100 μ ionized magnesium in phytohemagglutinin-treated lymphocytes. Lymphocyte DNA synthesis was much more sensitive to reduction of external ionized calcium than to reduction of ionized magnesium. In calcium-free medium (ionized calcium 25 μM), DNA synthesis was reduced by 90%, but in magnesium-free medium (ionized magnesium concentration 7 μM) DNA synthesis was reduced by only 30%. Fifty percent of DNA synthesis stimulated by phytohemagglutinin (PHA) and concanavalin A (Con A) was observed at external ionized calcium concentrations of 97 and 43 μM, respectively. When lymphocytes were stimulated with PHA and the external calcium was chelated with EGTA, 50% inhibition of DNA synthesis was observed at 98 μM ionized calcium. This value agreed well with the free calcium required for PHA activation of DNA synthesis (97 μM). Cytoplasmic calcium, measured with the fluorescent probe Quin 2, increased following lectin exposure if the extracellular ionized calcium concentration was greater than 80 μM. No increase in cytoplasmic calcium could be detected in lectin-treated lymphocytes below 80 μM extracellular ionized calcium, although substantial DNA synthesis was sustained.
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