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  • 1
    ISSN: 1432-0827
    Schlagwort(e): Amelogenin ; Expression ; Enamel ; Recombinant DNA ; Tooth
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin , Physik
    Notizen: Abstract A mouse cDNA encoding a 180 amino acid amelogenin was subcloned into the pET expression plasmid (Novagen, Madison, WI) for production in Escherichia coli. A simple growth and purification protocol yields 20–50 mg of 95–99% pure recombinant amelogenin from a 4.5-liter culture. This is the first heterologous expression of an enamel protein. The expressed protein was characterized by partial Edman sequencing, amino acid composition analysis, SDS-PAGE, Western blotting, laser desorption mass spectrometry, and hydroxyapatite binding. The recombinant amelogenin is 179 amino acids in length, has a molecular weight of 20,162 daltons, and hydroxyapatite binding properties similar to the porcine 173 residue amelogenin. Solubility analyses showed that the bacterially expressed protein is only sparingly soluble in the pH range of 6.4–8.0 or in solutions 20% saturated with ammonium sulfate. The purified protein was used to generate rabbit polyclonal anti-amelogenin antibodies which show specific reaction to amelogenins in both Western blot analyses of enamel extracts and in immunostaining of developing mouse molars.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 2
    Digitale Medien
    Digitale Medien
    Springer
    Calcified tissue international 55 (1994), S. 302-310 
    ISSN: 1432-0827
    Schlagwort(e): Amelogenin ; Alternative splicing ; Enamel ; Tooth
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Biologie , Medizin , Physik
    Notizen: Abstract A heterogeneous mixture of amelogenins can be extracted from developing tooth enamel matrix. In an attempt to discover the extent to which alternative splicing of the amelogenin primary RNA transcript can generate unique isoforms, we have conducted a thorough search for cDNAs amplified by reverse transcription-polymerase chain reaction (RT-PCR). Over 2400 colonies were screened by colony hybridization. Seven different alternatively spliced amelogenin mRNAs were isolated. The predicted translation products of the messages are 194, 180, 156, 141, 74, 59, and 44 amino acids in length. RT-PCR amplification products not predicted by these seven amelogenin cDNAs were characterized. The intron separating exons 5 and 6 was cloned and sequenced. Using rapid amplification of cDNA ends (RACE) techniques, the 5′ ends of the amelogenin mRNAs were cloned and characterized. The finding that the same exon 1 is common to all of the cloned mRNAs indicates that mouse amelogenin is transcribed from a single promoter. The mouse amelogenin transcription and translation initiation sites, the 5′ untranslated leader, and the segment encoding the signal peptide were determined. The distinctly nonamelogenin-like exon 4, first observed in human amelogenin cDNAs, has also been found in mice. Antibodies were raised to synthetic exon 4-encoded polypeptides and used to immunostain Western transfers and histologic tooth sections.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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  • 3
    Digitale Medien
    Digitale Medien
    Springer
    Journal of applied electrochemistry 30 (2000), S. 499-506 
    ISSN: 1572-8838
    Schlagwort(e): citrate electrolyte ; fractional factorial design ; hydrogen evolution ; Ni/Mo-deposits
    Quelle: Springer Online Journal Archives 1860-2000
    Thema: Chemie und Pharmazie , Elektrotechnik, Elektronik, Nachrichtentechnik
    Notizen: Abstract Hydrogen evolution on various Ni–Mo deposits was systematically compared using fractional factorial design (FFD) and response surface methodology (RSM). The electroplating variables such as pH, Ni/Mo atomic ratio and citrate concentration were found to be the key factors affecting the hydrogen evolution activity from the FFD study. The effects of Ni/Mo atomic ratio and citrate concentration in the plating bath on the apparent current density, the exchange current density, and the specific activity (based on i/q *) of hydrogen gas evolution, and on the Mo/(Ni + Mo) ratio of the deposits were examined using regression models. These models, represented as response surface contour plots, showed the maximum hydrogen evolving activity occurring on the Ni–Mo deposit electroplated from the bath with a pH of 8, a Ni/Mo ratio of 3.3 and a citrate concentration of 40 g l−1, respectively.
    Materialart: Digitale Medien
    Bibliothek Standort Signatur Band/Heft/Jahr Verfügbarkeit
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