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  • 1
    ISSN: 1573-4919
    Keywords: 14-3-3 proteins ; kinase C ; electrospray mass spectrometry ; isoforms ; post-translational modifications ; phosphorylation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This report compares the ability of individual members of the 14-3-3 protein family to inhibit particular protein kinase C (PKC) isoforms. We also show that two of these 14-3-3 isoforms (α and δ) specific to mammalian and avian brain arein vivo post-translationally modified forms of β and ζ respectively. The presence of this modification enhances the activity of 14-3-3 as an inhibitor of protein kinase C nearly two fold. A method for analysing isoforms of 14-3-3 on acid-urea gels is also described. This permits the complete separation of all major isoforms and their unequivocal identification by a range of isoform specific antisera. The activity of recombinant 14-3-3 and isoforms renatured by a novel method after separation by reverse phase HPLC are compared. The effects of diacylglycerol and the phorbol ester, PMA (phorbol 12-myristate 13 acetate) on the inhibition suggest that one of the sites of interaction of 14-3-3 may be the cysteine-rich (C1) domain in PKC.
    Type of Medium: Electronic Resource
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  • 2
    ISSN: 1573-4943
    Keywords: 14-3-3 ; signal transduction complexes ; phosphorylation ; kinases ; protein structure
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The 14-3-3 family are homo- and heterodimeric proteins whose biological role has been unclear for some time, although they are now gaining acceptance as a novel type of ‘adaptor’ protein that modulates interactions between components of signal transduction pathways, rather than by direct activation or inhibition. It is becoming apparent that phosphorylation of the binding partner and possibly also the 14-3-3 proteins may regulate these interactions. 14-3-3 isoforms interact with a novel phosphoserine (Sp) motif on many proteins, RSX1,2SpXP. The two isoforms that interact with Raf-1 are phosphorylated in vivo on Ser185 in a consensus sequence motif for proline-directed kinases. The crystal structure of 14-3-3 indicates that this phosphorylation could regulate interaction of 14-3-3 with its target proteins. We have now identified a number of additional phosphorylation sites on distinct mammalian and yeast isoforms.
    Type of Medium: Electronic Resource
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  • 3
    Electronic Resource
    Electronic Resource
    Springer
    Experimental astronomy 6 (1995), S. 163-210 
    ISSN: 1572-9508
    Keywords: CCDs ; data analysis ; instrumentation ; photometry ; software ; array detectors
    Source: Springer Online Journal Archives 1860-2000
    Topics: Physics
    Notes: Abstract We have constructed a computer model for simulation of point-sources imaged on two-dimensional detectors. An attempt has been made to ensure that the model produces “data” that mimic real data taken with 2-D detectors. To be realistic, such simulations must include randomly generated noise of the appropriate type from all sources (e.g. source, background, and detector). The model is generic and accepts input values for parameters such as pixel size, read noise, source magnitude, and sky brightness. Point-source profiles are then generated with noise and detector characteristics added via our model. The synthetic data are output as simple integrations (onedimensional), as radial slices (two-dimensional), and as intensity-contour plots (three-dimensional). Each noise source can be turned on or off so that they can be studied separately as well as in combination to yield a realistic view of an image. This paper presents the basic properties of the model and some examples of how it can be used to simulate the effects of changing image position, image scale, signal strength, noise characteristics, and data reduction procedures. Use of the model has allowed us to confirm and quantify three points: 1) The use of traditionalsize apertures for photometry of faint point-sources adds substantial noise to the measurement which can significantly degrade the quality of the observation; 2) The number of pixels used to estimate the background is important and must be considered when estimating errors; and 3) The “CCD equation” normally used by the astronomical community consistently overestimates the signal-to-noise obtainable by a measurement while a revised equation, discussed here, provides a better estimator.
    Type of Medium: Electronic Resource
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  • 4
    Electronic Resource
    Electronic Resource
    Chicester [u.a.] : Wiley-Blackwell
    Journal of Molecular Recognition 11 (1998), S. 200-203 
    ISSN: 0952-3499
    Keywords: antibody fragment ; scFv ; immobilization ; biosensor ; binding site ; scanning electron microscopy ; CM5 ; Chemistry ; Biochemistry and Biotechnology
    Source: Wiley InterScience Backfile Collection 1832-2000
    Topics: Medicine
    Notes: There are numerous chemical methods published that enable protein coupling to carboxymethyl (CM) dextran. Here we have taken traditional amine coupling using N-hydroxysuccinimide (NHS) and N′-(3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and coupled an antibody fragment (scFv) to CM dextran at a very high density. Using an upgraded BIAlite™ from Biacore AB, more than 7000 RU of scFv was coupled to a CM dextran biosensor chip. In addition, scanning electron microscopy was performed on CM dextran biosensor chips following amine coupling of 30 nm gold anti-IgG particles. This showed that amine coupling was uniform across the biosensor chip surface. Calculations show that 7620 RU of an scFv coupled to such a surface results in a mean distance between binding sites of 8.8 nm. This equates to a packing volume of approximately 20% of the available space occupied by the antibody fragment. Comparisons made with densities of covalently coupled IgG show that a greater number of antibody fragment molecules can be coupled per unit area. This is most likely due to the smaller size of an antibody fragment (scFv), which has a volume of less than 20% of an IgG molecule. The significance of these findings is discussed. Copyright © 1998 John Wiley & Sons, Ltd.
    Additional Material: 4 Ill.
    Type of Medium: Electronic Resource
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