12-360 Sigma-AldrichAcetyl-Histone H3 (Lys9/14) Peptide

The chromatographic behaviour of tamoxifen, toremifene and their major metabolites was investigated by reversed-phase high-performance liquid chromatography on four stationary phases. Two packings were the usual octadecylsilane type and the other two were octylsilane and octadecylsilane of the type specific for basic compounds. The results provide new insight into variations in selectivity with column type for drugs whose basic properties, owing to the presence of an ionizable nitrogen atom, make their chromatography difficult. The results allow an improvement of the separation of metabolites of tamoxifen and toremifene, two triphenylethylene drugs widely used for the treatment of breast cancer. A method is described for the identification and determination of metabolites formed by incubating the parent drugs with human liver microsomal preparations. The assay has been optimized for the identification and quantification of three major metabolites formed by N-oxidative demethylation of the side-chain, 4-hydroxylation of the aromatic ring and a side-chain deamination followed by hydroxylation. These catalytic activities involve cytochrome P450 enzymes.

We have subcloned the Escherichia coli uvrD gene under control of the inducible phage lambda PL promoter and report a procedure for the large-scale purification of helicase II protein. Yields of approximately 60 mg of > 99% pure helicase II protein, free of detectable nuclease activity, are obtained starting from 250 g of induced E. coli cells containing the overexpression plasmid. Overproduction of helicase II protein at these levels is lethal in E. coli. The extinction coefficient of helicase II protein was determined to be epsilon 280 = 1.06 (+/- 0.05) x 10(5) M-1 (monomer) cm-1 [20 mM Tris-HCl (pH 8.3 at 25 degrees C), 0.2 M NaCl, and 20% (v/v) glycerol, 25 degrees C]. We also present a preliminary characterization of the dimerization and DNA binding properties of helicase II and a systematic examination of its solubility properties. The apparent site size of a helicase II monomer on ss-DNA is 10 +/- 2 nucleotides as determined by quenching of the intrinsic tryptophan fluorescence of the protein upon binding poly(dT). In the absence of DNA, helicase II protein can self-assemble to form at least a dimeric species at concentrations > 0.25 microM (monomer) and exists in a monomer-dimer equilibrium under a variety of solution conditions. However, upon binding short oligodeoxynucleotides, the dimeric form of helicase II is stabilized, and dimerization stimulates the ss-DNA-dependent ATPase activity, suggesting that the dimer is functionally important. On the basis of these observations and similarities between helicase II and the E. coli Rep helicase, which appears to function as a dimer [Chao, K., & Lohman, T. (1991) J. Mol. Biol. 221, 1165-1181], we suggest that the active form of helicase II may also be a dimer or larger oligomer.

Morphine and oxycodone were administered to ten patients suffering from severe cancer pain in a double-blind cross-over study. The patients titrated themselves pain-free, first intravenously, using a patient-controlled analgesia device, and then orally. Each titration phase lasted for 48 hours. Blood samples were drawn after 36 hr of each administration phase. The plasma levels of morphine, morphine-6- and morphine-3 glucoronides were determined with high performance liquid chromatography (HPLC), whereas the oxycodone samples were assayed with gas chromatography (GC). Twin samples were analyzed for plasma opioid activity with a radioreceptor assay (RRA) using 3H-dihydromorphine and 3H-naloxone as radioligands. Adequate analgesia was achieved with both morphine and oxycodone. About 30% more oxycodone was needed intravenously, whereas 25% less oxycodone than morphine was consumed orally. There was a good linear correlation between the morphine concentrations measured with HPLC and RRA. The mean morphine-6-glucuronide to morphine concentration ratio was 2.3 after intravenous and 4.6 after oral administration. Results from RRA indicate that oxycodone in vivo is a potent mu-agonist and that at least part of its analgesic action is mediated by active metabolites. In vitro morphine glucuronides enhanced morphine in displacing radioligands from the opioid receptors, thus suggesting their complex interactions in vivo.