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  • 1
    ISSN: 0020-7608
    Keywords: Computational Chemistry and Molecular Modeling ; Atomic, Molecular and Optical Physics
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology
    Notes: A procedure is described which leads to experimentally based models for the transition-state structures of enzyme-catalyzed reactions. Substrates for an enzymic reaction are synthesized with isotopically enriched atoms at every position in which bonding changes are anticipated at the enzyme-enforced transition state. Kinetic isotope effects are measured for each atomic substitution and corrected for diminution of the isotope effects from nonchemical steps of the enzymic mechanism. A truncated geometric model of the transition-state structure is fitted to the kinetic isotope effects using bond-energy bond-order vibrational analysis. Full molecularity is restored to the transition state while maintaining the geometry of the bonds which define the transition state. Electronic wave functions are calculated for the substrate and the transition-state molecules. The molecular electrostatic potential energies are defined for the van der Waal surfaces of substrate and transition state and displayed in numerical and color-coded constructs. The electronic differences between substrate and transition state reveal characteristics of the transition state which permits the extraordinary binding affinity of enzyme-transition state interactions. The information has been used to characterize several enzymatic transition states and to design powerfully inhibitory transition-state analogues. Enzymatic examples are provided for the reactions catalyzed by AMP deaminase, nucleoside hydrolase, purine nucleoside phosphorylase, and for several bacterial toxins. The results demonstrate that the combination of experimental, classical, and quantum chemistry approaches is capable of providing reliable transition-state structures and sufficient information to permit the design of transition-state inhibitors. © 1996 John Wiley & Sons, Inc.
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  • 2
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Computational Chemistry 9 (1988), S. 608-619 
    ISSN: 0192-8651
    Keywords: Computational Chemistry and Molecular Modeling ; Biochemistry
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Chemistry and Pharmacology , Computer Science
    Notes: The study of the qualitative and quantitative product distribution in a chemical reaction, in particular regioselectivity, is of fundamental importance. Recently, it has been shown that the regioselectivity of some Diels-Alder cycloadditions can be explained by analyzing the interrelations between electron density contours and molecular electrostatic potentials. This problem is related to a central topic of modern theoretical chemistry and biochemistry: the analysis of molecular shape. This work deals with a rigorous, algebraic method to analyze these surfaces. The procedure is based on the computation of the shape groups (symmetry-independent homology groups of algebraic topology) of the molecular surface, using either a fully analytical algorithm requiring no visual inspection, or a precise method for processing pictorial information if the latter is available. The method provides a concise description of the molecular contour surface, that can replace the usually intuitive, and somewhat subjective, visual characterization of density and electrostatic potential contours. The method is illustrated for the case of Diels-Alder reactions by considering a number of monosubstituted dienes. Extensions of the analysis to dienophiles, as well as other types of reactions are also discussed.
    Additional Material: 5 Ill.
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  • 3
    ISSN: 1040-452X
    Keywords: Gap junction protein ; Gene expression ; Compaction ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology
    Notes: De novo assembly of gap junctions begins during compaction in the eight-cell stage of mouse development, and intercellular coupling mediated by gap junctions appears to be required for maintenance of the compacted state. We have begun to explore the expression of the family of genes encoding the connexins, the proteins that form the gap junction channels. We recently reported that a protein with antigenic and size similarity with connexin32, the rat liver gap junction protein, is inherited as an oogenetic product by the mouse zygote, but its gene appears not to be transcribed prior to implantation (Barron et al., Dev Genet 10:318-323, 1989). Here we report that another member of this gene family, connexin43, is transcribed by the embryonic genome from shortly after the time of genomic activation. As revealed by Northern blotting, connexin43 mRNA is absent from ovulated oocytes, becomes detectable in the 4-cell stage, and accumulates steadily thereafter to reach a maximum in blastocysts. In contrast, no transcripts of connexin26 could be detected in any preimplantation stage. A protein with antigenic and size similarity with connexin43 from rat heart was found by Western blotting to accumulate from the four-cell stage onward. Immunofluorescence analysis with embryo whole mounts was used to demonstrate that this protein is incorporated into punctate interblastomeric foci during compaction, consistent with its assembly into gap junction plaques. We conclude that connexin43 is one member of the connexin gene family whose zygotic expression is critical for preimplantation morphogenesis.
    Additional Material: 6 Ill.
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  • 4
    ISSN: 0730-2312
    Keywords: monoclonal antibody ; A431 ; EGF receptor ; chromosomal location ; internalization ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: A monoclonal antibody of the IgG class, EGFR1, has been isolated using cells of the epidermoid carcinoma line A431 as immunogen. The A431 antigen recognized by EGFR1 has an apparent molecular weight of approximately 175,000, is a cell-surface molecule which can be specifically cross-linked to EGF, exhibits an EGF-stimulated protein kinase activity, binds to EGFR1 in a number of human cell lines to a degree which parallels EGF binding, and shows EGF-dependent internalization in A431 cells and human fibroblasts. We therefore conclude that EGFR1 is directed against an antigenic site on the human EGF receptor. EGFR1 is not mitogenic for human fibroblasts and does not inhibit EGF binding under a variety of assay conditions. The characterization of EGFR1 has allowed the unambiguous assignment of the structural gene for the human EGF receptor to chromosome 7. Preliminary results suggest that a convenient method for isolating a range of anti-EGF receptor monoclonal antibodies can be developed, based on a hybridoma supernatant screening assay in which positive supernatants bind selectively to a human-mouse cell hybrid containing human chromosome 7.
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  • 5
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 77 (1971), S. 135-144 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Glutamine synthetase activity of NCTC clone 929 mouse cells (strain L) was studied as a function of the prior nutritional experience of the cells. Small enzyme increases were recorded in response to either glutamine depletion or chronic serum supplementation of the growth medium. Somewhat greater increases resulted from the administration of cortisol or certain other steroids, particularly if the hormone treatment was combined with glutamine withdrawal. High concentrations of glutamate in the medium did not augment the glutamine synthetase content of the cells and even caused an apparent decrease in it. The presence of glutamine in the culture medium resulted in a fairly rapid rate of disappearance of the glutamine synthetase of previously induced cells. The data suggest that glutamine and cortisol act independently on the cells in regulating the level of the enzyme.
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  • 6
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 121 (1984), S. 298-308 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Platelet-derived growth factor (PDGF) is a potent mitogen for cultured cells of mesenchymal origin. Known sources of PDGF or PDGF-like protein are blood platelets, several transformed cell lines, and cultured endothelial cells (EC). We have examined the regulation of production of a PDGF-like protein in cultures of bovine aortic EC using a specific radioreceptor assay for PDGF. EC constitutively secreted PDGF-like protein into serum-containing or serum-free medium. The rate of production of PDGF-like protein was constant for at least 3 weeks and was not due to release of an internal store, since cell lysis by repeated freeze/thaw cycles did not relase significant amounts of the protein. Synthesis of PDGF-like protein was sensitive to changes in the pH of the media and was maximal at pH 8.5. Production of PDGF-like protein was independent of EC growth rate: rapidly dividing cells and confluent, quiescent cells produced equal amounts per cell. However, sparse, quiescent EC produced more PDGF-like protein per cell than did confluent, quiescent cells. Several phorbol esters stimulated production of PDGF-like protein. At a concentration of 10-6 M, a twofold stimulation was observed upon addition of the tumor promoter 12-O-tetradecanoylphorbol 13-acetate (TPA) and nearly a fourfold stimulation upon addition of the nonpromoting analog, methyl TPA. Incubation of EC with endotoxin (10 μ/ml) resulted in a twofold stimulation of PDGF-like protein production. In all experiments with endotoxin and phorbol esters, an increase in the production of PDGF-like protein was accompanied by morphological changes in the EC cultures. The cells appeared elongated and fibroblastic and exhibited low viability. A mathematical model was developed in which PDGF-like protein production was shown to consist of two separate components - production at a constant rate by healthy cells and a large burst of synthesis and secretion by dying cells. These results suggest that injurious agents may be capable of stimulating production of a growth factor by the endothelium.
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  • 7
    Electronic Resource
    Electronic Resource
    New York, NY [u.a.] : Wiley-Blackwell
    Journal of Cellular Physiology 111 (1982), S. 97-103 
    ISSN: 0021-9541
    Keywords: Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: A postlavage in situ subpopulation of pulmonary macrophages (PM), biochemically distinct from the lavaged population, has recently been isolated from rats. After exhaustive bronchopulmonary lavage to extract the free lung cells, the lungs were excised, homogenized, and filtered, and the resultant cell suspension was allowed to form a monolayer on plastic Petri dishes. Electron microscopic morphometry failed to indicate any morphologic differences in the two populations. The postlavage in situ PM were more active metabolically during phagocytosis of zymosan particles or stimulation by phorbol myristate acetate (PMA) than the corresponding lavage population, as evidenced by greater superoxide generation. Macrophages prepared by either method became more avidly phagocytic when incubated with cell-free medium isolated in the preparation of the in situ population. Peroxidase, an enzyme absent from the granules of PM separated by lavage techniques, was found in a granule-rich fraction of the in situ macrophage. Catalase activity was found in similar amounts in both supernatants and granule-rich fractions of both populations. The results support the concept of subpopulations of PM and suggest that these subpopulations are distinguished by their biochemical properties and their functional abilities.
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  • 8
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 25 (1993), S. 59-72 
    ISSN: 0886-1544
    Keywords: microtubule dynamics ; photobleaching ; neurite elongation ; microtubule stability ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: In order to study microtubule turnover in elongating neurites, chick embryo sensory neurons were microinjected with x-rhodamine tubulin, and after 6-12 hours, short segments along chosen neurites were photobleached at multiple sites. Previous studies [Lim et al., 1989; 1990] indicated that recovery of fluorescence (FRAP) in neurites occurs by the dynamic turnover of stationary microtubules. In all cases, distal bleached zones recovered fluorescence faster than bleached zones more proximally located along the same neurites. Bleached zones at growth cones completely recovered in 30-40 minutes, while bleached zones located more proximally usually recovered in 50-120 minutes. In the most proximal regions of long neurites, recovery of fluorescence was often incomplete, indicating that a significant fraction of the microtubules in these regions were very stable. These studies indicate that there are differences in microtubule stability along the lenght of growing neurites. These differences may arise from the combined effects of (1) modifications that stabilize and lengthen microtubules in maturing neurites and (2) the dynamic instability of the distally oriented microtubule plus ends. © 1993 Wiley-Liss, Inc.
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  • 9
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 3 (1983), S. 657-669 
    ISSN: 0886-1544
    Keywords: Hela spectrin ; membrane ; cytoskeleton ; filamin ; actin ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: From 30-40 g of Hela-S3 cells grown in suspension, 0.25-0.50 mg of spectrin has been purified by conventional biochemical procedures starting from a low ionic strength extraction at alkaline pH of crude Hela membranes. Hela spectrin consists in its native form of a tetramer α2β2 of two high molecular weight polypeptides (240,000 and 230,000 daltons). Three different populations of Hela membranes depleted of both spectrin and actin have been prepared on discontinuous sucrose gradients. Surprisingly, spectrin will reassociate with only the heavier membrane fraction. This reassociation is specific for Hela spectrin, since three other purified Hela proteins as well as human erythrocyte spectrin do not reassociate under the same conditions. This binding is not due to the presence of traces of actin still present in the membrane fraction since two Hela actin-binding proteins (filamin I and II) do not show any significant binding to this fraction. The nature of the membrane-binding site for Hela spectrin is discussed.
    Additional Material: 7 Ill.
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  • 10
    Electronic Resource
    Electronic Resource
    New York, NY : Wiley-Blackwell
    Cell Motility and the Cytoskeleton 7 (1987), S. 272-281 
    ISSN: 0886-1544
    Keywords: intranuclear mitosis ; spindle formation ; cell cycle ; Life and Medical Sciences ; Cell & Developmental Biology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Biology , Medicine
    Notes: Tubulin synthesis in the naturally synchronous plasmodium of Physarum polycephalum is a markedly periodic event restricted to the late G2 period of the cell cycle. Mitosis in the plasmodium is intranuclear, and there are no cytoplasmic microtubules at any stage of the cell cycle. We have combined a biochemical investigation of the synthesis of the plasmodial tubulin isotypes and their participation in the mitotic spindle with a microscopic study (immunofluorescence) of the development of spindle microtubules throughout the cell cycle.We have shown that all four tubulin isotypes identified in the plasmodium (α1, α2, β1 and β2) are present in the mitotic spindle. The stoichiometry of isotype usage in the mitotic spindle generally reflects the overall abundance of isotypes in the plasmodium as a whole: β2 〉 α1 〉 α2 〉 β1. We have also shown that tubulins synthesized in the G2 period of one cell cycle can be incorporated into the spindles of the immediately ensuing mitosis and have sufficient biological longevity to allow participation in the mitotic divisions of future cell cycles. Thus, the phenomenon of periodic tubulin synthesis does not reflect a restricted use of tubulin to the cell cycle in which it was synthesized. The major polymerization of tubulin in the nucleus occurred less than 30 min before metaphase. A novel tubulin-containing structure was, however, present in the nucleus approximately 60 min before metaphase. Polymerized tubulin is rapidly removed from the nucleus following nucleokinesis.
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