SRC-3/AIB1 is an important growth coactivator whose activity should be tightly regulated since excess activation results in oncogenesis. Herein, we provide evidence that coordinated phosphorylation-dependent ubiquitination regulates SRC-3 coactivator activation and transcriptional specificity. We discovered a critical "actron/degron" element in SRC-3 that is required for this phosphorylation-dependent ubiquitination event and identified GSK3 and SCF(Fbw7alpha) as the respective responsible kinase and E3 ubiquitin ligase. Interestingly, despite that SCF(Fbw7alpha) enhances ubiquitination and promotes eventual transcription-coupled degradation of SRC-3 in a phosphorylation- and Fbw7alpha dosage-dependent manner, our results also uncovered a nonproteolytic "activation" code for SRC-3 ubiquitination induced by Fbw7alpha. We propose that ubiquitination of SRC-3 is a phospho-mediated biphasic event and that a transition from multi-(mono)ubiquitination (SRC-3 activation) to long-chain polyubiquitination (SRC-3 degradation) is processive during the transcriptional coactivation of select transcription factors and can serve as a "transcriptional time clock" to control both the activation and the functional lifetime of coactivators.

A sensitive and selective method was developed for the determination of (R)-ketoprofen ((R)-kt) and (S)-ketoprofen ((S)-kt) in human plasma using chiral liquid chromatography/tandem mass spectrometry (LC/MS/MS). Plasma samples spiked with stable-isotope-labeled [(13)C(1), (2)H(3)]-(R and S)-ketoprofen, for use as the internal standards, were prepared for analysis using automated solid-phase extraction (SPE) in the 96-well microtiter format. The enantiomers were separated on an (R)-1-naphthylglycine and 3,5-dinitrobenzoic acid (Chirex 3005) 250x2.0 mm i.d. analytical column, equipped with a 30x2.0 mm i.d. guard column using isocratic mobile phase conditions. The (R)- and (S)-kt levels were quantifiable from 0.05 to 2500 ng ml(-1) by constructing two separate curves from calibration standards covering the same range. The first curve ranged from 0.05 to 100 and the second from 100 to 2500 ng ml(-1). A concentration of 0.05 ng ml(-1) of either enantiomer was easily detected using a 1 ml plasma sample volume. The average method accuracy, evaluated at four levels over an extended period, was better than +/-3% over the entire range. The precision for the same set of quality control samples ranged from 4.0 to 7.0 % RSD (n = 24). The method was applied to the evaluation of pharmacokinetic parameters in human plasma obtained from volunteers who received 25 mg of kt by peroral administration of Actron caplets or by topical administration of Oruvail gel.

Chiral separation of nonsteroidal anti-inflammatory drugs (profens), 1,1'-binaphthyl-2,2'-diyl-hydrogen phosphate, N-[1-(1-naphthyl)ethyl]phthalamic acid, and derivatized amino acids by a cationic cyclodextrin, quaternary ammonium beta-cyclodextrin (QA-beta-CD), was investigated by capillary electrophoresis (CE) in both aqueous and nonaqueous media. Several profens and amino acids could only be separated by QA-beta-CD in pure formamide system. No chiral separation of the profens was achieved in the following solvents: N-methylformamide, methanol, dimethyl sulfoxide, and water; however, chiral separations of most of the amino acids were obtained in all of these solvents. The effects of other experimental parameters such as the CD concentration and apparent pH (pH*) were also investigated. The first application of nonaqueous CE chiral separation of ketoprofen in a commercially available sample, Actron, was also examined. In addition, the reversal of electroosmotic flow by QA-beta-CD was observed in water, formamide, N-methylformamide, methanol, and dimethyl sulfoxide media.

Plasma membranes were isolated from the livers of control and Streptococcus pneumoniae-infected rats. This work, therefore, represents the first isolation of plasma membranes from infected actron microscopy and by the use of enzyme markers for microsomes (glucose-6-phosphatase), mitochondria (glutamate and malate dehydrogenases), and lysosomes (acid phosphatase). Plasma membranes from infected cells banded at the same sucrose density as plasma membranes from uninfected cells. Moreover, equivalent amounts of plasma membranes could be isolated from control and infected rat livers. There were, however, significant alterations in the enzyme complement of the plasma membrane after infection. 5'-Nucleotidase activity was significantly decreased, whereas alkaline phosphatase activity was significantly increased. Kinetic analysis demonstrated that only the Vmax and not the Km of these two enzymes was changed, suggesting that the altered affinity of the enzymes for substrate was not the mechanism responsible for the observed alterations. No change in the mitochondrial enzyme markers was observed after infection, but the specific activity of microsomal glucose-6-phosphatase decreased significantly. Possible explanations for the observed alterations are discussed.

Martin Marietta/NASA subcontracted the design, fabrication, testing, and installation of the multiple docking adapter S-190 experiment window assembly to Actron. In this paper we primarily treat the thermal-optical analysis aspect of the design phase. The window is a pane of BK 7 glass 59.18 cm x 44.91 cm x 4.06 cm. Though it must meet many mechanical specifications, the optical requirements, dictated by the high acuity, distortion-free S-190 multiband camera system, are the most severe. Under operating conditions over any 7.6-cm circular area, the maximum rms deviation from the best-fitting plane must be less than 12.0 nm, and from the reference plane through the entire window, 60.0 nm.