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  • Cell & Developmental Biology  (7)
  • 1985-1989  (7)
  • 1985  (7)
  • 1
    ISSN: 0148-7280
    Keywords: sperm ; membrane ; plasma membrane ; polypeptides ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: In this study a variety of properties of boar sperm plasma membrane proteins were examined. A qualitative and quantitative analysis of proteins washed from boar sperm revealed that large numbers and a variety of polypeptides (Ps) are easily removed from sperm upon washing. Initially (by the second wash), Ps are released from the plasma membrane (PM) of epididymal sperm and primarily correspond to those in epididymal fluid, but eventually (fifth wash) Ps are released that are not seen in epididymal fluid nor as components of the PM. These Ps appear to originate from the sperm cytosol and signal the damaging effects of extensive washing on sperm. Upon washing, ejaculated sperm release Ps characteristic of both epididymal fluid and accessory sex glands. Epididymal Ps are almost completely released by the fourth wash; accessory gland proteins appear to be more tenaciously bound and continue to be released with further washing. Most basic accessory gland Ps bind strongly enough to resist the series of washes necessary for the preparation of PM vesicles. About one-half of ejaculated sperm lose motility after five washes, but evidence of massive release of internal Ps, such as seen in epididymal sperm, is not noted. In the epididymis and after ejaculation, sperm are coated with numerous Ps which are released upon washing; many are released nonspecifically and rapidly, others are more firmly bound. These analyses extend the surface map of boar spermatozoa to include a description of loosely bound proteins and their origin. These results also indicate that the qualitative and quantitative changes in surface membrane protein composition, occurring after simple washing, are significant and may confound the interpretation of surface composition changes in studies which rely solely on immunological or radiolabelling procedures.In order to determine the nature of the binding of major polypeptides (Ps) to the lipid bilayer of boar sperm plasma membranes (PMs), the solubility of Ps in solutions of different ionic strength and in detergents was examined. Several major polypeptides (identified in previously published surface maps) were extracted by hypotonic and hypertonic salt solutions, suggesting that electrostatic interactions play a major role in their binding to the bilayer. Other major proteins were extracted only by detergents, suggesting that these proteins are embedded deeply into the bilayer. These extraction procedures also provided a new strategy for isolating specific Ps in large quantities. Radiolabelling procedures identified about 80 surface-exposed Ps, some of which are major constituents of the PM and others which are quantitatively minor components. Labelling of PM vesicles reveals about sixfold more Ps than does labelling of whole sperm. Increased labelling appears to be the result of surface accessibility of PM constituents after removal of loosely bound Ps from epididymal fluid and seminal plasma during the washings which accompany the preparation of PM vesicles from whole sperm. These results prescribe caution when interpreting changes in surface organization and membrane structure which are dependent solely on the use of radiolabels.
    Additional Material: 10 Ill.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, N.Y. : Wiley-Blackwell
    Journal of Cellular Biochemistry 29 (1985), S. 373-379 
    ISSN: 0730-2312
    Keywords: encystment induction ; Acanthamoeba castellanii ; pinocytotic inhibition ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Monoclonal antibodies that bind a large molecular weight plasma membrane protein of Acanthamoeba castellanii cause the cells to differentiate. A different monoclonal antibody that binds specifically to the major plasma membrane protein has no effect upon cell division or differentiation. The induction of differentiation by the monoclonal antibodies requires a bivalent attachment, more than a single binding cycle of the antibody to the plasma membrane protein, does not require cell-cell contact, and appears to be mediated by an inhibition of pinocytosis. These results suggest one of two alternatives: either (1) this free living amoeba possesses a cell surface receptor that serves to initiate the differentiation process when stimulated, or (2) the specific plasma membrane antigen for the differentiation-inducing monoclonal antibodies is an essential component of the pinocytotic mechanism. While it seems more likely on the basis of available evidence that we are observing the biological effects of a cell surface receptor, either of the two alternative circumstances open up investigative areas of large significance.
    Additional Material: 3 Ill.
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  • 3
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Fetuin, a major protein of fetal calf serum, partially purified by the method of Pedersen, stimulated growth of aortic smooth muscle cells. More highly purified fetuin preparations stimulated growth less than Pedersen fetuin, as previously described for other cell types, suggesting that this activity is due to a contaminant. Recently bovine alpha2-macroglobulin or “Embryonin” has been proposed as the mitogenic component of crude fetuin preparations. We found that active fetuin preparations did contain alpha2-macroglobulin that stimulated smooth muscle cell growth. However, alpha2-macroglobulin purified directly from platelet-poor bovine plasma or fetuin purified from Pedersen fetuin by gel filtration lacked appreciable mitogenic effect on smooth muscle cells. Since alpha2-macroglobulin can bind platelet-derived growth factor (PDGF), and since highly acidic fetuin might bind the very basic PDGF molecule non-specifically, we measured the PDGF content of various fetuin preparations and found a good correlation between the PDGF content and mitogenic activity. Gel filtration experiments demonstrated that in Pedersen fetuin PDGF occurred both free, and in association with alpha2-macroglobulin. We conclude that the principal mitogenic component for smooth muscle cells in crude fetuin preparations is PDGF, since purified bovine alpha2-macroglobulin or fetuin do not appreciably affect growth of these cells. These results help to resolve a long-standing controversy regarding the nutrition of cultured cells. In addition, we suggest that before alpha2-macroglobulin or “Embryonin” is accepted as a bona fide growth factor for a given cell type, the role of contamination with PDGF should be assessed.
    Additional Material: 7 Ill.
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  • 4
    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.
    Additional Material: 7 Ill.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 125 (1985), S. 301-305 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Myosin light chain (P light chain) is phosphorylated by Ca2+. calmodulindependent myosin light chain kinase. Based on studies with rat skeletal muscles, it has been shown that P light chain phosphorylation correlated to the extent of potentiation of isometric twitch tension. It is not clear whether this correlation exists in rabbit skeletal muscle, which has been the primary source of contractile proteins for biochemical studies. Therefore, phosphorylation of myosin P light chain in rabbit slow-twitch soleus and fast-twitch plantaris muscles in situ was examined. Electrical stimulation (5 Hz, 20 seconds) of plantaris muscle produced an increase in the phosphate content of P light chain from 0.17 to 0.45 mol phosphate/mol P light chain. This increase in phosphate content was accompanied by a 58% increase in maximal isometric twitch tension. Tetanic stimulation (100 Hz, 15 seconds) of rabbit soleus muscle resulted in only a small increase in P light chain phosphate content from 0.02 to 0.10 mol phosphate/mol P light chain, and posttetanic twitch tension did not increase significantly. The correlation between potentiated isometric twitch tension and P light chain phosphorylation in rabbit fast-twitch muscle is similar to that observed in rat skeletal muscle. These results were consistent with the hypothesis that phosphorylation of rabbit skeletal muscle myosin, which results in an increase in actin-activated ATPase activity, may be related to isometric twitch potentiation.
    Additional Material: 1 Ill.
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  • 6
    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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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Journal of Electron Microscopy Technique 2 (1985), S. 489-495 
    ISSN: 0741-0581
    Keywords: Embedment media ; Coagulant and noncoagulant fixatives ; Intracellular surfaces ; Scanning electron microscopy ; Thick sections ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Natural Sciences in General
    Notes: Conventional plant histological specimens fixed in formalin-acetic acid-alcohol, chromic acid-acetic acid-formaldehyde, or glutaraldehyde-osmium and embedded in either paraffin or plastic are examined as possible rapid methods for providing an alternative image of cellular structure by using scanning electron microscopy. Using the mitotic figures of actively growing onion root tips as a study specimen, the organization of the nucleus and spindle apparatus is reasonably well preserved as compared with isolated mitotic spindles and studies of mitosis in endosperm tissue. Relief of internal structure in this technique is obtained through the coagulant nature of the fixative. Used judiciously, this technique can reveal aspects of the three-dimensional nature of internal tissue structure that may otherwise be difficult to discern.
    Additional Material: 14 Ill.
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