Takaharu Mizutani

Characterization of mSelB, a novel mammalian elongation factor for selenoprotein translation

Decoding of UGA selenocysteine codons in eubacteria is mediated by the specialized elongation factor SelB, which conveys the charged tRNASec to the A site of the ribosome, through binding to the SECIS mRNA hairpin. In an attempt to isolate the eukaryotic homolog of SelB, a database search in this work identified a mouse expressed sequence tag containing the complete cDNA encoding a novel protein of 583 amino acids, which we called mSelB. Several lines of evidence enabled us to establish that mSelB is the bona fide mammalian elongation factor for selenoprotein translation: it binds GTP, recognizes the Sec‐tRNASec in vitro and in vivo, and is required for efficient selenoprotein translation in vivo. In contrast to the eubacterial SelB, the recombinant mSelB alone is unable to bind specifically the eukaryotic SECIS RNA hairpin. However, complementation with HeLa cell extracts led to the formation of a SECIS‐dependent complex containing mSelB and at least another factor. Therefore, the role carried out by a single elongation factor in eubacterial selenoprotein translation is devoted to two or more specialized proteins in eukaryotes.

PM frequencies of major CYPs in Asians and Caucasians.

Many administered drugs are first activated by phase I drug-metabolizing enzymes, such as cytochrome P450 (CYP), and then conjugated with ligands such as UDPGA, PAPS, and glutathione by phase II drug-metabolizing enzymes, and finally excreted by transporters. There are some defective activity mutants due to CYP polymorphisms. In these cases, drugs are not metabolized [poor metabolizer (PM)], the high drug levels in blood are maintained, and toxic effects appear in the patients. To clarify the ratio of PMs, in the general population, it is necessary to estimate the drug level to not only prevent toxic reactions, but also to provide more efficient drug therapies, according to their polymorphic information about CYPs. In Caucasians and Asians, PM and allele frequency levels of CYPs (CYP2A6, CYP2C9, CYP2C19, CYP2D6, and CYP3A4) are summarized from previous findings. In Caucasians, high PM ratios (7%) of CYP2D6 deriving from the high frequency of CYP2D6*4 and CYP2D6*5, and 2% CYP2C19 from CYP2C19*2, were found. Meanwhile, in Asians, high PM ratios (19%) of CYP2C19 from high frequencies of CYP2C19*2 and CYP2C19*3, and 2% to 4% CYP2A6 from CYP2A6*4, were found. In both populations, the PM frequencies of the CYP3A4 of major drug-metabolizing CYP and CYP2C9 were low.

Interaction Between Valproic Acid and Carbapenem Antibiotics

The serum concentration of valproic acid (VPA) in epilepsy patients decreased by the administration of carbapenem antibiotics, such as meropenem, panipenem or imipenem, to a sub-therapeutic level. This review summarized several case reports of this interaction between VPA (1–4 g dose) and carbapenem antibiotics to elucidate the possible mechanisms decreasing VPA concentration by carbapenem antibiotics. Studies to explain the decrease were carried out using rats by the following sites: absorption of VPA in the intestine, glucuronidation in the liver, disposition in blood and renal excretion. In the intestinal absorption site, there are two possible mechanisms: inhibition of the intestinal transporter for VPA absorption by carbapenem antibiotics, and the decrease of β-glucuronidase supplied from enteric bacteria, which were killed by antibiotics. This is consistent with a view that the decrease of VPA originated from VPA-Glu, relating to entero-hepatic circulation. The second key site is in the liver, because of no decreased in VPA level by carbapenem antibiotics in hepatectomized rats. There are three possible mechanisms in the liver to explain the decreased phenomenon: first, decrease of the UDPGA level by carbapenem antibiotics. UDPGA is a co-factor for UDP-glucuronosyltransferase (UGT)-mediated glucuronidation of VPA. Second, the direct activation of UGT by carbapenem antibiotics. This activation was observed after pre-incubation of human liver microsomes with carbapenem antibiotics. Third, the inhibition of β-glucuronidase in liver by carbapenem antibiotics and the decreased VPA amount liberated from VPA-Glu. The third site is the distribution of VPA in blood (erythrocytes and plasma). Plasma VPA distributed to erythrocytes by the inhibition of transporters (Mrp4), which efflux VPA from erythrocytes to plasma, by carbapenem antibiotics. The increase of renal excretion of VPA as VPA-Glu depends on the increase of VPA-Glu level by UGT. One or a combination of some factors in these mechanisms might relate to the carbapenem-mediated decrease of the plasma VPA level.

Autoantibodies against CYP2D6 and other drug-metabolizing enzymes in autoimmune hepatitis type 2.

Autoimmune hepatitis (AIH) is a disease of unknown etiology, characterized by liver-related autoantibodies. Autoimmune hepatitis is subdivided into two major types: AIH type 1 is characterized by the detection of ANA, SMA, ANCA, anti-ASGP-R, and anti-SLA/LP. Autoimmune hepatitis type 2 is characterized to be mainly related with drug-metabolizing enzymes as autoantigens, such as anti-LKM (liver-kidney microsomal antigen)-1 against CYP2D6, anti-LKM-2 against CYP2C9-tienilic acid, anti-LKM-3 against UGT1A, and anti-LC1 (liver cytosol antigen)-1 and anti-APS (autoimmune polyglandular syndrome type-1) against CYP1A2, CYP2A6, and others. Anti-LKM-1 sera inhibited CYP2D6 activity in vitro but did not inhibit cellular drug metabolism in vivo. CYP2D6 is the major target autoantigen of LKM-1 and expressed on plasma membrane (PM) of hepatocytes, suggesting a pathogenic role for anti-LKM-1 in liver injury as a trigger. Anti-CYP1A2 was observed in dihydralazine-induced hepatitis, and radiolabeled CYP1A2 disappeared from the PM with a half-life of less than 30 min, whereas microsomal CYP1A2 was stably radiolabeled for several hours. Main antigenic epitopes on CYP2D6 are aa 193–212, aa 257–269, and aa 321–351; and D263 is essential. The third epitope is located on the surface of the protein CYP2D6 and displays a hydrophobic patch that is situated between an aromatic residue (W316) and histidine (H326). Some drugs such as anticonvulsants (phenobarbital, phenytoin, and carbamazepine) and halothane are suggested to induce hepatitis with anti-CYP3A and anti-CYP2E1, respectively. Autoantibodies against CYP11A1, CYP17, and/or CYP21 involved in the synthesis of steroid hormones are also detected in patients with adrenal failure, gonadal failure, and/or Addison disease.

Adsorption of cationic biological materials on controlled pore glass

Abstract Controlled pore glass (1 g, 97 m 2 ) adsorbed approximately S μmol of cationic biological materials, such as lysine, histidine, arginine, hexosamines, and cytidine, in a distilled water medium. The amount of other amino acids and neutral carbohydrates adsorbed to controlled pore glass was 0–0.8 μmol/g of controlled pore glass. Glucose and glucosamine were clearly separated on a column of controlled pore glass.

Study of mammalian selenocysteyl-tRNA synthesis with [75Se]HSe.

The mechanisms of the synthesis of mammalian selenocysteyl‐(Scy)‐tRNA were studied using [75Se]H2Se. H2Se was prepared from [75Se]selenite, glutathione, NADPH and glutathione reductase, and was purified by chromatography. It was confirmed that this H2Se was a Se donor in the reaction of the synthesis of Scy‐tRNA. [75Se]Scy, liberated from aminoacyl‐tRNA, was analyzed by TLC on silica gel an subsequent autoradiography. The activity of Scy‐tRNA synthesis was found in the supernatant at 105 000 × g of the murine liver extract, but not in the precipitate. The supernatant was chromatographed on DEAE‐cellulose, and the activity was eluted at a concentration of 0.17 M KCl. This position is at the front shoulder of the peak of seryl‐tRNA synthetase which was eluted at 0.20 M KCl. Major serine tRNAIGA is not a substrate on which to synthesize Scy‐tRNA, but natural opal suppressor serine tRNA is. On a chromatographic pattern of a Scy‐tRNA preparation on Sephacryl S‐200, the radioactivity of 75Se was eluted at the tRNA peak. This showed that Scy bound to tRNA. The active protein fraction from DEAE‐cellulose did not contain tRNA kinase, therefore Scy‐tRNA must be directly synthesized from seryl‐tRNA, not through phosphoseryl‐tRNA. This mechanism is similar to that seen in Escherichia coli [1991, J. Biol. Chem. 266, 6324].

Purification and properties of suppressor seryl-tRNA:ATP phosphotransferase from bovine liver

Seryl‐tRNASer CmCA:ATP phosphotransferase was purified 1200‐fold from bovine liver by ultracentrifugation at 150 000 × g, chromatography on DEAE—cellulose, fractional precipitation with ammonium sulfate, chromatography on hydroxyapatite, gel filtration on Sephacryl S‐300 and affinity chromatography on Blue Sepharose. Molecular mass was estimated as 135–145 kDa. The K m values for ATP and ser‐tRNASer CmCA were 2 mM and 21 nM, respectively. This enzyme did not react with ser‐tRNASer IGA, tyr‐tRNA or thr‐tRNA.

Induction of Human UGT1A1 by Bilirubin Through AhR Dependent Pathway

UDP-glucuronosyltransferase1A1 (UGT1A1) plays a key role to conjugate bilirubin and preventing jaundice, but there is no report showing the induction of human UGT1A1 (UGT1A1) by bilirubin. In this report, we show findings of the induction of the reporter gene (-3475/+14) of UGT1A1 in HepG2 cells by bilirubin at 50 microM, 100 microM, with human aryl hydrocarbon receptor (hAhR). We confirmed that induction of the reporter gene by bilirubin is dependent on the position of the xenobiotic responsive element (XRE) (-3328/-3319) of UGT1A1, because the XRE deletion UGT1A1 gene did not respond to stimulation by a complex of bilirubin and hAhR. alpha-Naphthoflavone (alpha-NF) of a typical AhR antagonist at 50 microM inhibited induction by bilirubin, suggesting that bilirubin stimulates through binding with hAhR. Meanwhile, bilirubin itself did not stimulate the induction of AhR, because we detected no-elevation of the mRNA level of AhR by RT-PCR. These results indicate that the induction of UGT1A1 by bilirubin-AhR did not depend on the elevation of AhR but on ligand binding. From this result, we considered that high bilirubin in neonates must induce the elevation of UGT1A1 after birth to prevent jaundice, and bilirubin in adults also regulates the level of UGT1A1. This is the first report showing direct induction of UGT1A1 by a bilirubin through AhR pathway.

Comparison of elution patterns of proteins chromatographed on controlled-pore glass and carboxymethylcellulose

As controlled-pore glass (CPG) has anionic silanol groups, we compared the elution patterns of proteins chromatographed on CPG with those on carboxymethyl (CM)-cellulose using standard proteins and two protein mixtures, rabbit serum and bovine parotid extracts. The results showed that the order of proteins eluted from CPG was similar to that from CM-cellulose, although some differences were found. The adsorption of proteins on CPG was slightly stronger than that on CM-cellulose and the separation of proteins on CPG was slightly better. Some conditions, such as amounts of proteins loaded on the column, pH, temperature and kind of buffer solution for elution, were investigated. The pH of buffer solutions was important in the adsorption chromatography of proteins on CPG and the useful pH range was 7.0-8.5.

Study of in vitro glucuronidation of hydroxyquinolines with bovine liver microsomes

Glucuronidation of drugs by UDP‐glucuronosyltransferase (UGT) is a major phase II conjugation reaction. Defects in UGT are associated with Crigler–Najjar syndrome and Gilberts syndrome with severe hyperbilirubinaemias and jaundice. We analysed the reactivities of some hydroxyquinoline derivatives, which are naturally produced from quinoline by cytochrome P450. The analyses were carried out using a microassay system for UGT activity in bovine liver microsomes in the range 0.5–100 pmol/assay with the highly sensitive radio‐image analyser Fuji BAS2500 (Fujifilm, Tokyo, Japan). 3‐Hydroxylquinoline is a good substrate for glucuronidation, and the relative Kcat values were 3.1‐fold higher than the values for p‐nitrophenol. 5,6‐Dihydroquinoline‐5,6‐trans‐diol gave a similar Km value to that of 3‐hydroxyquinoline, but the Vmax value was approximately 1/15 of that of p‐nitrophenol and showed weak reactivity. Quinoline N‐oxide gave a low Vmax value and showed marginal activity. The Kcat values of 6‐hydroxyquinoline and 5‐hydroxyquinoline were 2.1‐ and 1.2‐fold higher than that of p‐nitrophenol, respectively. Fluoroquinoline (FQ) derivatives, such as 3FQ, 7,8diFQ and 6,7,8triFQ, did not show any substrate activities. These results suggest that there are therapeutic problems in administration of some quinoline drugs to patients with jaundice.