Exercise-induced changes in protein metabolism

Mono-specific antibodies against the human cytochrome P450 (P450) enzymes CYP1A1, CYP1A2, CYP1B1, CYP2A6, CYP2B6, CYP2D6, CYP2E1, CYP3A4, CYP3A5 and CYP4A11 and an antibody that binds to CYP2C8, CYP2C9 and CYP2C19 have been produced by immunising rabbits with synthetic peptides representing small regions of each of these P450 enzymes. The specificity of the antibodies was confirmed by immunoblotting using recombinant P450 enzymes and samples of human hepatic microsomal fraction. Each of the antibodies bound only to their respective target P450 enzyme(s). The relative intensity of immunoreactive bands was compared with a variety of P450 activities and correlations were found between CYP1A2 and phenacetin O-deethylase activity, CYP2A6 and coumarin 7-hydroxylase activity, CYP2C9 and tolbutamide 4-hydroxylase activity, CYP2C19 and S-mephenytoin 4-hydroxylase activity, CYP2D6 and debrisoquine 4-hydroxylase activity, CYP2E1 and chlorzoxazone 6-hydroxylase activity, CYP3A4 and midazolam 1'-hydroxylase activity, and CYP4A11 and lauric acid 12-hydroxylase activity. A proportion of the 30 liver samples examined lacked CYP2A6 (7%), CYP2C19 (10%) or CYP2D6 (13%), consistent with the polymorphic expression of these P450 enzymes in human liver. Although CYP3A5 was detected in most individuals (97%), expression was polymorphic with 20% containing substantially higher levels. CYP2B6 was expressed in 20% of the human liver samples, with one sample containing a particularly high level. No immunodetectable CYP1A1 or CYP1B1 was found, consistent with the low level of expression of these P450 enzymes in human liver. The results demonstrate the utility of the antipeptide approach for producing specific antibodies against human P450 enzymes, enabling a comprehensive panel of antibodies against human P450 enzymes to be produced.     

Structure-function relationships of human liver cytochromes P450 3A: aflatoxin B1 metabolism as a probe

Cytochromes P450 3A4 and 3A5, the dominant drug-metabolizing enzymes in the human liver, share >85% primary amino acid sequence identity yet exhibit different regioselectivity toward aflatoxin B1 (AFB1) biotransformation [Gillam et al., (1995) Arch. Biochem. Biophys. 317, 374-384]. P450 3A4 apparently prefers AFB1 3alpha-hydroxylation, which results in detoxification and subsequent elimination of the hepatotoxin, over AFB1 exo-8,9-oxidation. In contrast, P450 3A5 is incapable of appreciable AFB1 3alpha-hydroxylation and converts it predominantly to the exo-8,9-oxide which is genotoxic. To elucidate the structural features that govern the regioselectivity of the human liver 3A enzymes in AFB1 metabolism and bioactivation, a combination of approaches including sequence alignment, homology modeling, and site-directed mutagenesis was employed. Specifically, the switch in AFB1 regioselectivity was examined after individual substitution of the divergent amino acids in each of the six putative substrate recognition sites (SRSs) of P450 3A4 with the corresponding amino acid of P450 3A5. Of the P450 3A4 mutants examined, P107S, F108L, N206S, L210F, V376T, S478D, and L479T mutations resulted in a significant switch of P450 3A4 regioselectivity toward that of P450 3A5. The results confirmed the importance of some of these residues in substrate contact in the active site, with residue N206 (SRS-2) being critical for AFB1 detoxification via 3alpha-hydroxylation. Moreover, the P450 3A4 mutant N206S most closely mimicked P450 3A5, not only in its regioselectivity of AFB1 metabolism but also in its overall functional capacity. Furthermore, the other SRS-2 mutant, L210F, also resembled P450 3A5 in its overall AFB1 metabolism and regioselectivity. These findings reveal that a single P450 3A5 SRS domain (SRS-2) is capable of conferring the P450 3A5 phenotype on P450 3A4. In addition, some of these P450 3A4 mutations that affected AFB1 regioselectivity had little influence on testosterone 6beta-hydroxylation, thereby confirming that each substrate-P450 active site fit is indeed unique.     

Anterior pituitary cell population control: basal cell turnover and the effects of adrenalectomy and dexamethasone treatment

There is a need for methodology to predict clinically significant drug-drug interactions so that clinical studies can be directed toward interactions which are likely to be clinically relevant. To this end, we evaluated selective assays for the seven drug-metabolizing cytochrome P450 (P450) isozymes 1A2 (caffeine N3-demethylation), 2A6 (coumarin 7-hydroxylation), 2C9 (tolbutamide hydroxylation), 2C19 (S-mephenytoin 4-hydroxylation), 2D6 (dextromethorphan O-demethylation), 2E1 (chlorzoxazone 6-hydroxylation), and 3A4/5 (dextromethorphan N-demethylation). Using initial rate conditions, we determined the Km and Vmax values of each reaction in human liver microsomes from three individuals. Because organic solvents (usually methanol) are frequently used as solubilization aids for drugs/inhibitors, we also screened several solvents for inhibitory activity. Methanol was the least inhibitory toward P450s 2A6, 2D6, and 3A4, dimethylformamide was the least inhibitory toward P450s 1A2 and 2C9, and acetonitrile was the least inhibitory toward P450s 2C19 and 2E1. Using substrate concentrations close to the determined Km and an appropriate solvent (where necessary), we used the selective inhibitors furafylline (1A2), 8-methoxypsoralen (2A6), sulfaphenazole (2C9), S-mephenytoin (2C19), quinidine (2D6), diethyldithiocarbamate (2E1), and troleandomycin (3A4) to assess the limitations of each probe assay as an indicator of the P450 isoform in question. Our results were consistent with these inhibitors and probes, being selective tools for studying P450 drug metabolism.     

Effect of methanol, ethanol, dimethyl sulfoxide, and acetonitrile on in vitro activities of cDNA-expressed human cytochromes P-450

The effects of methanol, ethanol, dimethyl sulfoxide (DMSO), and acetonitrile were studied in vitro on nine individual, cDNAexpressed cytochrome P-450 activities (phenacetin O-deethylase for CYP1A1 and CYP1A2, coumarin 7-hydroxylase for CYP2A6, testosterone 6beta-hydroxylase for CYP3A4, 7-ethoxy-4-trifluoromethylcoumarin deethylase for CYP2B6, paclitaxel 6alpha-hydroxylase for CYP2C8, diclofenac 4'-hydroxylase for CYP2C9, S-mephenytoin 4-hydroxylase for CYP2C19, and (+/-)-bufuralol 1'-hydroxylase for CYP2D6) in commercially available human lymphoblastoid microsomes. These data show that specific solvents have enzyme-selective effects on P-450 activities. Methanol did not substantially inhibit (相似文献   

In vitro evaluation of the antigenotoxic activity of a strain of L. bulgaricus isolated from commercial yogurt

Sequential oxidations at the arylamine moiety of the procainamide molecule leading to the formation of N-hydroxyprocainamide and its nitroso derivative may be responsible for lupus erythematosus observed in patients treated with the drug. The objective of the present study was to characterize major cytochrome P450 isozyme(s) involved in the N-hydroxylation of procainamide. Firstly, incubations were performed with microsomes from either lymphoblastoid cells or yeast transfected with cDNA encoding for specific human cytochrome P450 isozymes. Experiments performed with these enzyme expression systems indicated that the highest formation rate of N-hydroxyprocainamide was observed in the presence of CYP2D6 enriched microsomes. Additional experiments demonstrated that the formation rate of N-hydroxyprocainamide by CYP2D6 enriched microsomes was decreased from 45 +/- 4% to 93 +/- 1% by quinidine at concentrations ranging from 30 nM to 100 microM (all p < 0.05 vs control) and by approximately 75% by antibodies directed against CYP2D6. Secondly, incubations were performed with microsomes prepared from 15 human liver samples. Using this approach, an excellent correlation was observed between the formation rate of N-hydroxyprocainamide and dextromethorphan O-demethylase activity (CYP2D6; r = 0.9305; p < 0.0001). In contrast, no correlation could be established between N-hydroxyprocainamide formation rate and caffeine N3-demethylase (CYP1A2), coumarin 7-hydroxylase (CYP2A6), S-mephenytoin N-demethylase (CYP2B6), tolbutamide methlhydroxylase (CYP2C9), S-mephenytoin 4'-hydroxylase (CYP2C19), chlorzoxazone 6-hydroxylase (CYP2E1), dextromethorphan N-demethylase (CYP3A4), testosterone 6 beta-hydroxylase (CYP3A4/5) or lauric acid 12-hydroxylase (CYP4A11) activities. Furthermore, formation rate of N-hydroxyprocainamide was decreased in a concentration-dependent manner by quinidine (300 nM to 100 microM) and by antibodies directed against CYP2D6 but not by furafylline 20 microM (CYP1A2), ketoconazole 1 microM (CYP3A4), sulfaphenazole 10 microM (CYP2C9) or antibodies directed against CYP1A1/1A2, CYP2C, CYP2A6, CYP2E1 or CYP3A4/3A5. In conclusion, the results obtained in the present study demonstrate that CYP2D6 is the major human cytochrome P450 isozyme involved in the formation of the reactive metabolite of procainamide, namely N-hydroxyprocainamide.     

Identification of amino acid substitutions that confer a high affinity for sulfaphenazole binding and a high catalytic efficiency for warfarin metabolism to P450 2C19

Human cytochrome P450s 2C9 and 2C19 metabolize many important drugs including tolbutamide, phenytoin, and (S)-warfarin. Although they differ at only 43 of 490 amino acids, sulfaphenazole (SFZ) is a potent and selective inhibitor of P450 2C9 with an IC50 and a spectrally determined binding constant, KS, of <1 microM. P450 2C19 is not affected by SFZ at concentrations up to 100 microM. A panel of CYP2C9/2C19 chimeric proteins was constructed in order to identify the sequence differences that underlie this difference in SFZ binding. Replacement of amino acids 227-338 in 2C19 with the corresponding region of 2C9 resulted in high-affinity SFZ binding (KS approximately 4 microM) that was not seen when a shorter fragment of 2C9 was substituted (227-282). However, replacement of amino acids 283-338 resulted in extremely low holoenzyme expression levels in Escherichia coli, indicating protein instability. A single mutation, E241K, which homology modeling indicated would restore a favorable charge pair interaction between K241 in helix G and E288 in helix I, led to successful expression of this chimera that exhibited a KS < 10 microM for SFZ. Systematic replacement of the remaining differing amino acids revealed that two amino acid substitutions in 2C19 (N286S, I289N) confer high-affinity SFZ binding (KS < 5 microM). When combined with a third substitution, E241K, the resulting 2C19 triple mutant exhibited a high cataltyic efficiency for warfarin metabolism with the relaxed stereo- and regiospecificity of 2C19 and a lower KM for (S)-warfarin metabolism (<10 microM) typical of 2C9.     

Evaluation of omeprazole and lansoprazole as inhibitors of cytochrome P450 isoforms

The human clearance of omeprazole and lansoprazole is conducted primarily by the hepatic cytochrome P450 (CYP) system. Efficacy data indicate few differences between these two drugs, but they may exhibit discrete drug interaction profiles. To compare the potency and specificity of these drugs as inhibitors of CYP isoforms, we performed in vitro studies with human liver microsomal preparations. Both drugs were potent, competitive inhibitors of CYP2C19, as measured by the conversion of S-mephenytoin to 4-hydroxymephenytoin (k(i) = 3.1 +/- 2.2 microM for omeprazole, K(i) = 3.2 +/- 1.3 microM for lansoprazole). For omeprazole, the highest concentration at which >70% inhibition of CYP2C19 was observed with no significant inhibitory effect on other isoforms was at least 20 times greater than K(i). Both drugs were competitive inhibitors of CYP2C9-catalyzed conversion of tolbutamide to 4-hydroxytolbutamide (K(i) = 40.1 +/- 14.8 microM for omeprazole, K(i) = 52.1 +/- 1.4 microM for lansoprazole) and were noncompetitive inhibitors of CYP3A-catalyzed conversion of dextromethorphan to 3-methoxymorphinan (K(i) = 84.4 +/- 4.0 microM for omeprazole, K(i) = 170.4 +/- 7.1 microM for lansoprazole). Lansoprazole was at least 5 times more potent (K(i) = 44.7 +/- 22.0 microM) than omeprazole (k(i) = 240.7 +/- 102.0 microM) as an inhibitor of CYP2D6-mediated conversion of dextromethorphan to dextrorphan. No inhibition of CYP1A2, assessed by measuring the conversion of phenacetin to acetaminophen, was noted. Our data suggest that whereas the inhibitory profiles of these two drugs are similar, lansoprazole may be the more important in vitro inhibitor of CYP2D6. Since its inhibition is very potent and has a broad "window of selectivity," omeprazole seems to be a useful, selective inhibitor of CYP2C19.     

Identification of GIYWHHY as a novel peptide substrate for human p60c-src protein tyrosine kinase

We have recently determined that -Ile-Tyr- were the two critical residues as a peptide substrate for p60c-src protein tyrosine kinase (Lou, Q. et al., Lett. Peptide Sci., 1995, 2, 289). Here, we report on the design and synthesis of a secondary 'one-bead, one-compound' combinatorial peptide library based on this dipeptide motif (XIYXXXX, where X = all 19 eukaryotic amino acids except for cysteine). This secondary library was screened for its ability to be phosphorylated by p60c-src PTK using [gamma 32P]ATP as a tracer. Five of the strongest [32P]-labeled peptide-beads were identified and microsequenced: GIYWHHY, KIYDDYE, EIYEENG, EIYEEYE, and YIYEEED. A solid-phase phosphorylation assay was used to evaluate the structure-activity relationship of GIYWHHY. It was determined that Ile2, Tyr3, His5, and His6 were crucial for its activity as a substrate.     

Mononuclear cell subpopulations in preterm and full-term neonates: independent effects of gestational age, neonatal infection, maternal pre-eclampsia, maternal betamethason therapy, and mode of delivery

We have previously characterized an allelic variant of cytochrome P450 CYP 2C11 from the Gunn rat that differs at three positions (amino acids 4, 116, and 187) from the predominant allele from Wistar rats and that displays a dramatically reduced testosterone hydroxylation activity. To assess the relative contribution of these mutations to the decrease in the enzymatic activity we constructed single and double mutants and coexpressed them with reductase. Testosterone metabolism was determined with a baculovirus/insect cell expression system. None of the identified positions alone is critical for the activity since the reversion of one of these mutations is unable to restore fully the Wistar-type activity. The activity of CYP 2C11 containing either the Asn116Ser substitution or the Phe187Leu represents congruent with30% of the activity of the CYP 2C11 Wistar-type protein. In contrast, the activity of the Val4Ala mutated protein is only 10% that of the Wistar-type protein, close to that of the Gunn-type protein. This study reevaluates the contribution of amino acid 4 to the catalysis by cytochrome P450 2C11 and points out the role of extra SRS residues.     

Identification of human cytochrome P450 isoforms involved in the 3-hydroxylation of quinine by human live microsomes and nine recombinant human cytochromes P450

Studies using human liver microsomes and nine recombinant human cytochrome P450 (CYP) isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C9, 2C19, 2D6, 2E1 and 3A4) were performed to identify the CYP isoform(s) involved in the major metabolic pathway (3-hydroxylation) of quinine in humans. Eadie-Hofstee plots for the formation of 3-hydroxyquinine exhibited apparently monophasic behavior for all of the 10 different microsomal samples studies. There was interindividual variability in the kinetic parameters, as follows: 1.8-, 3.2- and 3.5-fold for K(m) Vmax and Vmax/K(m), respectively. The mean +/- S.D. values for K(m), Vmax and Vmax/K(m) were 106.1 +/- 19.3 microM, 1.33 +/- 0.48 nmol/mg protein/min and 12.8 +/- 5.1 microliters/mg protein/min, respectively. With 10 different human liver microsomes, the relationships between the 3-hydroxylation of quinine and the metabolic activities for substrates of the respective CYP isoforms were evaluated. The 3-hydroxylation of quinine showed an excellent correlation (r = 0.986, P < .001) with 6 beta-hydroxylation of testosterone, a marker substrate for CYP3A4. A significant correlation (r = 0.768, P < .01) between the quinine 3-hydroxylase and S-mephenytoin 4'-hydroxylase activities was also observed. However, no significant correlation existed between the 3-hydroxylation of quinine and the oxidative activities for substrates for CYP1A2 (phenacetin), 2C9 (diclofenac), 2D6 (desipramine) and 2E1 (chlorzoxazone). Ketoconazole and troleandomycin (inhibitors of CYP3A4) inhibited the 3-hydroxylation of quinine by human liver microsomes with respective mean IC50 values of 0.026 microM and 28.9 microM. Anti-CYP3A antibodies strongly inhibited quinine 3-hydroxylation, whereas weak inhibition was observed in the presence of S-mephenytoin or anti-CYP2C antibodies. Among the nine recombinant human CYP isoforms, CYP3A4 exhibited the highest catalytic activity with respect to the 3-hydroxylation of quinine, compared with the minor activity of CYP2C19 and little discernible or no effect of other CYP isoforms. Collectively, these data suggest that the 3-hydroxylation of quinine is mediated mainly by CYP3A4 and to a minor extent by CYP2C19. Other CYP isoforms used herein appear to be of negligible importance in this major pathway of quinine in humans.     

Use of omeprazole as a probe drug for CYP2C19 phenotype in Swedish Caucasians: comparison with S-mephenytoin hydroxylation phenotype and CYP2C19 genotype

A single oral dose of omeprazole (20 mg) was given orally to 160 healthy Caucasian Swedish subjects and tested as a probe for CYP2C19. The study was nonrandomized and included seven subjects previously classified as poor metabolizers (PM) of S-mephenytoin. The ratio between the plasma concentrations of omeprazole and hydroxyomeprazole (metabolic ratio; MR) was determined by HPLC in a blood sample drawn 3 h after drug intake. In 17 subjects the test was repeated and the MRs of omeprazole on the two occasions were correlated (rs = 0.85; p < 0.0001). There was a significant correlation between the MR of omeprazole and the S/R mephenytoin ratio among 141 subjects, in whom both ratios were determined (rs = 0.63, p < 0.001). All seven PMs of S-mephenytoin had higher MRs of omeprazole (7.1-23.8) than extensive metabolizers (EM) (0.1-4.9). All 160 subjects and another 15 Caucasian Swedish PMs previously phenotyped with mephenytoin were analysed with respect to the presence of the CYP2C19m1 allele by PCR amplification of the intron 4/exon 5 junction followed by Sma I digestion. EMs heterozygous for the CYP2C19m1 gene had MRs of omeprazole and S/R ratios of mephenytoin that were higher than those of subjects who were homozygous for the wild-type allele (p = 0.0001). Nineteen of the 22 PMs were homozygous for the CYP2C19m1 gene. Three were heterozygous for this allele. Thus, 41 of the 44 alleles (93%) of PMs were defective CYP2C19m1. One of the remaining three PM alleles was subsequently found to contain the CYP2C19m2 mutation, which has earlier been shown to be associated with the PM phenotype in Oriental populations. In conclusion, the phenotype determined by omeprazole correlated with that of mephenytoin, and was in good agreement with the genotype.     

Tachykinins (substance P, neurokinin A and neuropeptide gamma) and neurotensin from the intestine of the Burmese python, Python molurus

Peptides with substance P-like immunoreactivity, neurokinin A-like immunoreactivity and neurotensin-like immunoreactivity were isolated in pure form from an extract of the intestine of the Burmese python (Python molurus). The primary structure of python substance P (Arg-Pro-Arg-Pro-Gln-Gln-Phe-Tyr-Gly-Leu- Met-NH2) shows one amino acid substitution (Phe8-->Tyr) compared with chicken/alligator substance P and an additional substitution (Lys3-->Arg) as compared with mammalian substance P. The neurokinin A-like immunoreactivity was separated into two components. Python neuropeptide gamma (Asp-Ala-Gly-Tyr- Ser-Pro-Leu-Ser-His-Lys-Arg-His-Lys-Thr-Asp-Ser-Phe-Val-Gly-Leu-Met-NH2 shows three substitutions (Gly5-->Ser, Gln6-->Pro and Ile7-->Leu) compared with alligator neuropeptide gamma and an additional substitution (His4-->Tyr) compared with mammalian neuropeptide gamma. Python neurokinin A (His-Lys-Thr-Asp-Ser-Phe-Val-Gly- Leu-Met.NH2) is identical to human/chicken/alligator neurokinin A. Python neurotensin (pGlu-Leu-Val-His-Asn-Lys-Ala-Arg-Pro-Tyr-Ile-Leu) is identical to chicken/alligator neurotensin. The data are indicative of differential evolutionary pressure to conserve the amino acid sequences of reptilian gastrointestinal peptides.     

Residues in the membrane-spanning and extracellular loop regions of the parathyroid hormone (PTH)-2 receptor determine signaling selectivity for PTH and PTH-related peptide

The parathyroid hormone (PTH)-2 receptor displays strong ligand selectivity in that it responds fully to PTH but not at all to PTH-related peptide (PTHrP). In contrast, the PTH-1 receptor (PTH/PTHrP receptor) responds fully to both ligands. Previously it was shown that two divergent residues in PTH and PTHrP account for PTH-2 receptor selectivity; position 23 (Trp in PTH and Phe in PTHrP) determines binding selectivity and position 5 (Ile in PTH and His in PTHrP) determines signaling selectivity. To identify sites in the PTH-2 receptor involved in discriminating between His5 and Ile5, we constructed PTH-2 receptor/PTH-1 receptor chimeras, expressed them in COS-7 cells, and tested for cAMP responsiveness to [Trp23] PTHrP-(1-36), and to the nondiscriminating peptide [Ile5, Trp23]PTHrP-(1-36) (the Phe23 --> Trp modification enabled high affinity binding of each ligand to the PTH-2 receptor). The chimeras revealed that the membrane-spanning/loop region of the receptor determined His5/Ile5 signaling selectivity. Subsequent analysis of smaller cassette substitutions and then individual point mutations led to the identification of two single residues that function as major determinants of residue 5 signaling selectivity. These residues, Ile244 at the extracellular end of transmembrane helix 3, and Tyr318 at the COOH-terminal portion of extracellular loop 2, are replaced by Leu and Ile in the PTH-1 receptor, respectively. The results thus indicate a functional interaction between two residues in the core region of the PTH-2 receptor and residue 5 of the ligand.     

Engineering of cytochrome P450 2B1 specificity. Conversion of an androgen 16 beta-hydroxylase to a 15 alpha-hydroxylase

Six site-directed mutants of cytochrome P450 2B1 were constructed, and function was evaluated in COS cell microsomes by monitoring testosterone and androstenedione hydroxylation and inactivation by chloramphenicol. Mutants Ile-114-->Val and Ile-114-->Ala exhibited marked decreases in androgen 16 beta-OH:16 alpha-OH ratios and increases in 15 alpha-OH:16-OH ratios. Since substitution of Gly-478 with Ala or Ser reduces 16 beta-hydroxylation in favor of 15 alpha-hydroxylation, four double mutants containing Val or Ala at position 114 and Ala or Ser at position 478 were examined. For any given residue at position 114 (Ile, Val, or Ala), the 15 alpha-OH:16-OH ratio increased as residue 478 was changed from Gly to Ala to Ser, and for any residue at position 478, this ratio increased as residue 114 was changed from Ile to Val to Ala. As a consequence, the Ile-114-->Ala, Gly-478-->Ser mutant displayed an approximately 1000-fold higher androgen 15 alpha-OH:16-OH ratio compared with the parental enzyme and functionally resembles mouse P450 2A4 much more closely than P450 2B1. All three mutants with Val at position 114 retained susceptibility to inactivation by chloramphenicol, whereas inactivation was suppressed by Ala at this position. The results suggest the feasibility of an empirical approach to P450 engineering involving the appropriate combination of residues at a few critical sites to confer new regio- and stereoselectivity with retention of overall monooxygenase activity.     

Molecular cloning of capillary permeability-increasing enzyme-2 from Agkistrodon caliginosus (Korean viper)

The gene of capillary permeability-increasing enzyme-2 (CPI enzyme-2) was cloned from the cDNA library of Agkistrodon caliginosus and its nucleotide sequence was determined. Its sequence indicates that CPI enzyme-2 is synthesized as a pre-zymogen of 258 amino acid residues, including a putative secretory signal peptide of 18 amino acids and a proposed zymogen peptide of 6 amino acids. The amino terminal sequence deduced from the cDNA sequence was exactly consistent with that of CPI enzyme-2 except for the substitution of an amino acid (Gly27-->Ser). The open reading frame is very similar to those of plasminogen activator and thrombin-like proteases cloned from other snakes. The clone encoding CPI enzyme-2 belongs to the serine protease family. The active site of the enzyme is highly conserved at His41, Asp86 and Ser180. Its possible glycosylation sites, Asn-X-Thr/Ser, are located at amino acid residues 20-22, 55-57, 79-81 and 97-99.     

Structures that delineate orphanin FQ and dynorphin A pharmacological selectivities

A genetic polymorphism in the metabolism of the anticonvulsant drug S-mephenytoin has been attributed to defective CYP2C19 alleles. This genetic polymorphism displays large interracial differences with the poor metabolizer (PM) phenotype representing 2-5% of Caucasian and 13-23% of Oriental populations. In the present study, we identified two new mutations in CYP2C19 in a single Swiss Caucasian PM outlier (JOB 1) whose apparent genotype (CYP2C19*1/CYP2C19*2) did not agree with his PM phenotype. These mutations consisted of a single base pair mutation (G395A) in exon 3 resulting in an Arg132-->Gln coding change and a (G276C) mutation in exon 2 resulting in a coding change Glu92-->Asp. However, the G276C mutation and the G395A mutation resided on separate alleles. Genotyping tests of a family study of JOB1 showed that the exon 2 change occurred on the CYP2C19*2 allele, which also contained the known splice mutation in exon 5 (this variant is termed CYP2C19*2B to distinguish it from the original splice variant now termed CYP2C19*2A). The exon 3 mutation resided on a separate allele (termed CYP2C19*6). In all other respects this allele was identical to one of two wild-type alleles, CYP2C19*1B. The incidence of CYP2C19*6 in a European Caucasian population phenotyped for mephenytoin metabolism was 0/344 (99% confidence limits of 0 to 0.9%). Seven of 46 Caucasian CYP2C19*2 alleles were CYP2C19*2B(15%) and 85% were CYP2C19*2A. The Arg132Gln mutation was produced by site-directed mutatgenesis and the recombinant protein expressed in a bacterial cDNA expression system. Recombinant CYP2C19 6 had negligible catalytic activity toward S-mephenytoin compared with CYP2C19 1B, which is consistent with the conclusion that CYP2C19*6 represents a PM allele. Thus, the new CYP2C19*6 allele contributes to the PM phenotype in Caucasians.     

Purification, characterization, and spasmogenic activity of neurotensin from the toad Bufo marinus

Neurotensin (NT) was isolated from an extract of the intestine of the cane toad, Bufo marinus and its primary structure established as: pGlu-Ala-Ile-Val-Ser-Lys-Ala-Arg-Arg-Pro-Tyr-Ile-Leu. This amino acid sequence shows five substitutions (Leu2 --> Ala, Tyr3 --> Ile, Glu4 --> Val, Asn5 --> Ser, and Pro7 --> Ala) compared with bovine NT. Synthetic Bufo NT (pD2 = 8.05 +/- 0.28) was equipotent and equally effective as bovine NT (pD2 = 8.24 +/- 0.38) in producing spasmogenic contraction of isolated segments of toad small intestine. However, the maximum response produced by Bufo NT was only 35 +/- 2% of that produced by substance P. The potencies, but not the maximum responses, to Bufo and bovine NT were significantly (p < 0.05) attenuated by pre-treatment with atropine but neither parameter was significantly diminished by tetrodotoxin and indomethacin. The data suggest that the action of NT involves interaction with receptors on toad intestinal smooth muscle that recognize the C-terminal region of NT (residues 8-13) that has been fully conserved during evolution of tetrapods. Contractile activity is mediated, at least in part, by release of acetylcholine.     

The effects of selective serotonin reuptake inhibitors and their metabolites on S-mephenytoin 4'-hydroxylase activity in human liver microsomes

The inhibitory effects of four selective serotonin reuptake inhibitors (SSRIs), fluoxetine, sertraline, paroxetine and citalopram, and three metabolites (norfluoxetine, demethylcitalopram and didemethylcitalopram), on S-mephenytoin 4'-hydroxylation activities in human liver microsomes were studied. The 4'-hydroxylation of S-mephenytoin, a representative substrate toward CYP2C19, was competitively inhibited by all the SSRIs and their metabolites studied. The mean Ki values of fluoxetine, norfluoxetine, sertraline, paroxetine, citalopram, demethylcitalopram and didemethylcitalopram were 5.2, 1.1, 2.0, 7.5, 87.3, 55.8 and 7.7 microM, respectively. The findings suggest that some SSRIs and their metabolites with a low Ki value (e.g., fluoxetine, norfluoxetine) may reduce the clearance of drugs metabolized by this isoform of P450, thereby resulting in a possible drug-drug interaction, when administered simultaneously. In addition, SSRIs and their metabolites examined herein may be substrates toward CYP2C19.     

Potent and selective aromatase inhibitor: in vitro and in vivo studies with s-triazine derivative SEF19

We found a potent aromatase inhibitor through the screening of agents for estrogen-dependent breast cancer. SEF19 (2-(imidazol-1-yl)-4,6-dimorphorino-1,3,5-triazine) decreased 50% of human placental aromatase activity in vitro at the concentration of 5.3 nM. In order to clarify the selectivity of SEF19 for enzyme inhibition, we determined the effect of SEF19 on the activities of four steroidogenic cytochrome P450 enzymes in porcine adrenal gland, P450SCC(side-chain cleavage of cholesterol), P450(11 beta) (11 beta-hydroxylase), P450(17 alpha)(17 alpha-hydroxylase/C17,20 lyase) and P450C21 (21-hydroxylase). SEF19 failed to inhibit the activities of porcine adrenal P450SCC, P450(17 alpha) and P450C21 up to the concentration of 100 microM and showed some inhibition on P450(11 beta) activity at 100 microM, while SEF19 completely nullified the aromatase activity at 1 microM. We also determined the potency of SEF19 for the suppression of aromatase activity in vivo. SEF19 suppressed dose-dependently the uterine hypertrophy of immature rats caused by administration of androstenedione (30 mg/kg, s.c.). The ED50 of SEF19 for the suppression of uterine hypertrophy was 0.8 mumol/kg. These results suggest that SEF19 may serve as a potent and selective agent for the treatment of estrogen-dependent breast cancer.     

Identification of a novel proline-rich peptide-binding domain in prolyl 4-hydroxylase

Prolyl 4-hydroxylase (EC 1.14.11.2) catalyzes the hydroxylation of -X-Pro-Gly- sequences and plays a central role in the synthesis of all collagens. The [alpha(I)]2beta2 type I enzyme is effectively inhibited by poly(L-proline), whereas the [alpha(II)]2beta2 type II enzyme is not. We report here that the poly(L-proline) and (Pro-Pro-Gly)10 peptide substrate-binding domain of prolyl 4-hydroxylase is distinct from the catalytic domain and consists of approximately 100 amino acids. Peptides of 10-19 kDa beginning around residue 140 in the 517 residue alpha(I) subunit remained bound to poly(L-proline) agarose after limited proteolysis of the human type I enzyme tetramer. A recombinant polypeptide corresponding to the alpha(I) subunit residues 138-244 and expressed in Escherichia coli was soluble, became effectively bound to poly(L-proline) agarose and could be eluted with (Pro-Pro-Gly)10. This polypeptide is distinct from the SH3 and WW domains, and from profilin, and thus represents a new type of proline-rich peptide-binding module. Studies with enzyme tetramers containing mutated alpha subunits demonstrated that the presence of a glutamate and a glutamine in the alpha(II) subunit in the positions corresponding to Ile182 and Tyr233 in the alpha(I) subunit explains most of the lack of poly(L-proline) binding of the type II prolyl 4-hydroxylase. Keywords: collagen/dioxygenases/peptide-binding domain/ proline-rich/prolyl hydroxylase