ABSTRACT:The purpose of the present study was to determine the absolute protein expression levels of multiple drug-metabolizing enzymes and transporters in 17 human liver biopsies, and to compare them with the mRNA expression levels and functional activities to evaluate the suitability of the three measures as parameters of hepatic metabolism. Absolute protein expression levels of 13 cytochrome P450 (P450) enzymes, NADPH-P450 reductase (P450R) and 6 UDPglucuronosyltransferase (UGT) enzymes in microsomal fraction, and 22 transporters in plasma membrane fraction were determined using liquid chromatography/tandem mass spectrometry. CYP2C9, CYP2E1, CYP3A4, CYP2A6, UGT1A6, UGT2B7, UGT2B15, and P450R were abundantly expressed (more than 50 pmol/mg protein) in human liver microsomes. The protein expression levels of CYP3A4, CYP2B6, and CYP2C8 were each highly correlated with the corresponding enzyme activity and mRNA expression levels, whereas for other P450s, the protein expression levels were better correlated with the enzyme activities than the mRNA expression levels were. Among transporters, the protein expression level of organic anion-transporting polypeptide 1B1 was relatively highly correlated with the mRNA expression level. However, other transporters showed almost no correlation. These findings indicate that protein expression levels determined by the present simultaneous quantification method are a useful parameter to assess differences of hepatic function between individuals.

IFN-␥ treatment of cells leads to tyrosine phosphorylation of signal transducer and activator of transcription (STAT) 1 followed by dimerization through a reciprocal Src homology 2-phosphotyrosine interaction near the -COOH end of each monomer, forming a parallel structure that accumulates in the nucleus to drive transcription. Prompt dephosphorylation and return to the cytoplasm completes the activation-inactivation cycle. Nonphosphorylated STATs dimerize, and a previously described interface between N-terminal domain (ND) dimers has been implicated in this dimerization. A new crystal structure of nonphosphorylated STAT1 containing the ND dimer has two possible configurations for the body of STAT1, one of which is antiparallel. In this antiparallel structure, the Src homology 2 domains are at opposite ends of the dimer, with the coiled:coil domain of one monomer interacting reciprocally with the DNA-binding domain of its partner. Here, we find that mutations in either the coiled:coil͞DNA-binding domain interface or the ND dimer interface block dimerization of nonphosphorylated molecules and cause a resistance to dephosphorylation in vivo and resistance to a tyrosine phosphatase in vitro. We conclude that a parallel STAT1 phosphodimer not bound to DNA most likely undergoes a conformational rearrangement (parallel to antiparallel) to present the phosphotyrosine efficiently for dephosphorylation.dephosphorylation ͉ structural rearrangement S ignal transducers and activators of transcription (STATs) are latent cytoplasmic transcription factors that can be activated by a variety of tyrosine kinases in response to many different cytokine, growth factor, and peptide ligands binding to their respective cell surface receptors. Accumulation in the nucleus of tyrosine phosphorylated STAT dimers is followed by DNA binding, activation of target gene transcription, dephosphorylation, and return to the cytoplasm (1). The crystal structure of the phosphodimer core (amino acids 130-712), bound to DNA, showed a reciprocal phosphotyrosine (pY)-Src homology 2 (SH2) interaction at one end of a parallel dimer (2), with the DNA separating the monomers along the long axis of the monomers. Mao et al. (3) have now described a dimeric structure of nonphosphorylated STAT1, including the core (amino acids 130-712), as well as the core plus the N-terminal domain (ND) (amino acids 1-683). (The terminal 29 aa, 684-712, in the core were unstructured and omitted from study.) The body of each of the monomers in the nonphosphorylated structure is identical to the monomers in the previously reported phosphorylated dimer (2). However, the monomers in the nonphosphorylated core structure are arranged differently in space. In contrast to the dimeric, parallel structure of the phosphorylated STAT1, the SH2 domains in the nonphosphorylated core structure project from the opposite ends of an antiparallel dimer with a dimeric interface formed by reciprocal contacts between the coiled:coil (CC) and DNA-binding domains (DBD) of the monomers. In the crystal...

No abstract

AimsPre-clinical data suggest that the racemic phyto-oestrogen 8-prenylnaringenin (8-PN) may have beneficial effects in postmenopausal women and may become an alternative to classical hormone replacement therapy (HRT) treatment regimes. The aim of this study was to investigate the pharmacokinetics, endocrine effects and tolerability of chemically synthesized 8-PN in postmenopausal women. MethodsThe study was performed using a randomized, double-blind, placebo-controlled, doseescalation design with three groups of eight healthy postmenopausal women. In each group six subjects received 8-PN and two subjects placebo. 8-PN was given orally in doses of 50, 250 or 750 mg. Drug concentrations in serum, urine and faeces were measured up to 48 h and follicle-stimulating hormone/luteinizing hormone (LH) concentrations up to 24 h. ResultsAll treatments were well tolerated and associated with a low incidence of (drug unrelated) adverse events. Serum concentrations of free 8-P N showed rapid drug absorption and secondary peaks suggestive of marked enterohepatic recirculation. Independent of the treatment group, approximately 30% of the dose was recovered in excreta as free compound or conjugates over the 48-h observation period. The first C max and AUC 0 − 48 h showed dose linearity with ratios of 1 : 4.5 : 13.6 ( C max ) and 1 : 5.2 : 17.1 (AUC). The750-mg dose decreased LH concentrations by 16.7% (95% confidence interval 0.5, 30.2). ConclusionSingle oral doses of up to 750 mg 8-PN were well tolerated by postmenopausal women. The pharmacokinetic profile of 8-PN was characterized by rapid and probably complete enteral absorption, high metabolic stability, pronounced enterohepatic recirculation and tight dose linearity. The decrease in LH serum concentrations found after the highest dose demonstrates the ability of 8-PN to exert systemic endocrine effects in postmenopausal women.