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Sesamin is a major lignan in sesame, and its biological effects such as antioxidant effect, anti-carcinogenic effects, and suppression of hypertension have been extensively studied by many researchers. However, its metabolic pathways and metabolic enzymes in human bodies have not been identified. Recently we demonstrated that CYP2C9 was the most important cytochrome P450 isoform in human liver. Next, we focused on metabolism of sesamin mono-catechol by cytochrome P450 or UDP-glucuronosyltransferase (UGT). Further catecholization of sesamin mono-catechol by cytochrome P450 enhances its anti-oxidant activity, whereas glucuronidation by UGT strongly reduces anti-oxidant activity. In human liver microsomes, glucuronidation activity toward sesamin mono-catechol was much higher than the di-catecholization activity. In contrast, both activities were similar in rat liver microsomes. These results suggest a large species-based difference between humans and rats in sesamin metabolism. In vitro studies using 10 individual human liver microsomes suggested that UGT2B7 was responsible for glucuronidation of sesamin mono-catechol in human liver. In addition, we observed a significant methylation activity toward sesamin mono-catechol by catechol O-methyl transferase (COMT) in human liver cytosol. Based on these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in human liver.

The supplementation of royal jelly (RJ) is known to provide a variety of health benefits, including anti-inflammatory and anti-obesity effects. RJ treatment also reportedly protects against bone loss, but no single factor in RJ has yet been identified as an anti-osteoporosis agent. Here we fractionated RJ and identified 10-hydroxy-2-decenoic acid (10H2DA) as a key component involved in inhibiting osteoclastogenesis based on mass spectrometric analysis. We further demonstrated free fatty acid receptor 4 (FFAR4) as directly interacting with 10H2DA; binding of 10H2DA to FFAR4 on osteoclasts inhibited receptor activator of nuclear factor-κB (NF-κB) ligand (RANKL)-induced activation of NF-κB signaling, thereby attenuating the induction of nuclear factor of activated T cells (NFAT) c1, a key transcription factor for osteoclastogenesis. Oral administration of 10H2DA attenuated bone resorption in ovariectomized mice. These results suggest a potential therapeutic approach of targeting osteoclast differentiation by the supplementation of RJ, and specifically 10H2DA, in cases of pathological bone loss such as occur in postmenopausal osteoporosis.

Cytochrome P450 enzymes belonging to the CYP105 family are predominantly found in bacteria belonging to the phylum Actinobacteria and the order Actinomycetales. In this review, we focused on the protein engineering of P450s belonging to the CYP105 family for industrial use. Two Arg substitutions to Ala of CYP105A1 enhanced its vitamin D3 25- and 1α-hydroxylation activities by 400 and 100-fold, respectively. The coupling efficiency between product formation and NADPH oxidation was largely improved by the R84A mutation. The quintuple mutant Q87W/T115A/H132L/R194W/G294D of CYP105AB3 showed a 20-fold higher activity than the wild-type enzyme. Amino acids at positions 87 and 191 were located at the substrate entrance channel, and that at position 294 was located close to the heme group. Semi-rational engineering of CYP105A3 selected the best performing mutant, T85F/T119S/V194N/N363Y, for producing pravastatin. The T119S and N363Y mutations synergistically had remarkable effects on the interaction between CYP105A3 and putidaredoxin. Although wild-type CYP105AS1 hydroxylated compactin to 6-epi-pravastatin, the quintuple mutant I95T/Q127R/A180V/L236I/A265N converted almost all compactin to pravastatin. Five amino acid substitutions by two rounds of mutagenesis almost completely changed the stereo-selectivity of CYP105AS1. These results strongly suggest that the protein engineering of CYP105 enzymes greatly increase their industrial utility. This article is part of a Special Issue entitled: Cytochrome P450 biodiversity and biotechnology, edited by Erika Plettner, Gianfranco Gilardi, Luet Wong, Vlada Urlacher, Jared Goldstone.

Our previous studies revealed that CYP105A1 can convert vitamin D3 (VD3) to its active form, 1α,25-dihydroxyvitamin D3 (1,25D3). Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity; the activity of double variants R73A/R84A and R73A/R84V was more than 100-fold higher than that of the wild type of CYP105A1. In contrast, these variants had a low ability to convert vitamin D2 (VD2) to 1α,25-dihydroxyvitamin D2 (1,25D2), whereas they catalyzed the sequential hydroxylation at positions C25 and C26 to produce 25,26D2. A comparison of the docking models of 25D2 and 25D3 into the substrate-binding pocket of R73A/R84A suggests that the side chain of the Met239 inhibits the binding of 25D2 for 1α-hydroxylation. Therefore, the Met239 residue of R73A/R84A was substituted for Ala. As expected, the triple variant R73A/R84A/M239A showed a 22-fold higher 1α-hydroxylation activity towards 25D2. To the best of our knowledge, this is the first report on the generation of microbial cytochrome P450 that converts VD2 to 1,25D2 via 25D2.

Our previous studies revealed that the double variants of CYP105A1- R73A/R84A and R73V/R84A-show high levels of activity with respect to conversion of vitamin D3 to its biologically active form, 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). In this study, we found that both the double variants were also capable of converting vitamin D2 to its active form, that is, 1α,25-dihydroxyvitamin D2 (1α,25(OH)2D2), via 25(OH)D2, whereas its 1α-hydroxylation activity toward 25(OH)D2 was much lower than that toward 25(OH)D3. Comparison of the wild type and the double variants revealed that the amino acid substitutions remarkably enhanced both 25- and 26-hydroxylation activity toward vitamin D2. After 25-hydroxylation of vitamin D2, further hydroxylation at C26 may occur frequently without the release of 25(OH)D2 from the substrate-binding pocket. Thus, the double variants of CYP105A1 are quite useful to produce 25,26(OH)2D2 that is one of the metabolites of vitamin D2 detected in human serum.

Our previous studies revealed that CYP105A1 can convert vitamin D

Our previous studies revealed that C2α-substituted-1α,25(OH)(2)D(3) analogs had unique biological activities. For example, 19-nor-2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (MART-10), which has a high affinity for vitamin D receptor (VDR), is more bioavailable and more potent than 1α,25(OH)(2)D(3) in inhibiting cancer cell growth and invasion because of its weaker binding to vitamin D binding protein (DBP), and more resistance to CYP24A1-dependent metabolism. In this study, we examined the metabolism of MART-10 and two other 2α-substituted analogs, 2α-(3-hydroxypropoxy)-1α,25(OH)(2)D(3) (O2C3) and 2α-(3-hydroxypropyl)-1α,25(OH)(2)D(3) (O1C3) by using human liver microsomes and human P450s. We demonstrated that O2C3 was converted to 1α,2α,25(OH)(3)D(3) in human liver microsomes, whereas both O1C3 and MART-10 were hardly metabolized. The metabolism of O2C3 was significantly inhibited by ketoconazole, and the recombinant human CYP3A4 converted O2C3 to 1α,2α,25(OH)(3)D(3), which suggests that CYP3A4 is responsible for the metabolism of O2C3 in human liver. The k(cat)/K(m) values of CYP3A4 for O1C3 and MART-10 are much smaller than that for O2C3. The k(cat)/K(m) values of human CYP24A1 for the three analogs are 1% (MART-10), 3% (O2C3), and 4% (O1C3) of that for 1α,25(OH)(2)D(3), indicating that MART-10 is the most resistant to CYP24A1 hydroxylation. On the other hand, 1α,2α,25(OH)(3)D(3), the metabolite of O2C3 by CYP3A4, was metabolized by CYP24A1 via multiple pathways similar to 1α,25(OH)(2)D(3), which suggests that O2C3 can be metabolized by two sequential hydroxylations, first by CYP3A4 and then by CYP24A1 in human body. These results suggest that modification at C-2α position and C-19 demethylenation markedly change metabolic profiles and biological activities of vitamin D analogs.

Glucuronidation is one of the most common pathways in mammals for detoxification and elimination of hydrophobic xenobiotic compounds, including many drugs. Metabolites, however, can form active or toxic compounds, such as acyl glucuronides, and their safety assessment is often needed. The absence of efficient means for in vitro synthesis of correct glucuronide metabolites frequently limits such toxicological analyses. To overcome this hurdle we have developed a new approach, the essence of which is a coexpression system containing a human, or another mammalian UDP-glucuronosyltransferases (UGTs), as well as UDP-glucose-6-dehydrogenase (UGDH), within the budding yeast, Saccharomyces cerevisiae. The system was first tested using resting yeast cells coexpressing UGDH and human UGT1A6, 7-hydroxycoumarin as the substrate, in a reaction medium containing 8% glucose, serving as a source of UDP-glucuronic acid. Glucuronides were readily formed and recovered from the medium. Subsequently, by selecting suitable mammalian UGT enzyme for the coexpression system we could obtain the desired glucuronides of various compounds, including molecules with multiple conjugation sites and acyl glucuronides of several carboxylic acid containing drugs, namely, mefenamic acid, flufenamic acid, and zomepirac. In conclusion, a new and flexible yeast system with mammalian UGTs has been developed that exhibits a capacity for efficient production of various glucuronides, including acyl glucuronides.

Sesamin and episesamin are two epimeric lignans that are found in refined sesame oil. Commercially available sesamin supplements contain both sesamin and episesamin at an approximate 1:1 ratio. Our previous study clarified the sequential metabolism of sesamin by cytochrome P450 (P450) and UDP-glucuronosyltransferase in human liver. In addition, we revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9, the P450 enzyme responsible for sesamin monocatecholization. In the present study, we compared the metabolism and the MBI of episesamin with those of sesamin. Episesamin was first metabolized to the two epimers of monocatechol, S- and R-monocatechols in human liver microsomes. The P450 enzymes responsible for S- and R-monocatechol formation were CYP2C9 and CYP1A2, respectively. The contribution of CYP2C9 was much larger than that of CYP1A2 in sesamin metabolism, whereas the contribution of CYP2C9 was almost equal to that of CYP1A2 in episesamin metabolism. Docking of episesamin to the active site of CYP1A2 explained the stereoselectivity in CYP1A2-dependent episesamin monocatecholization. Similar to sesamin, the episesamin S- and R-monocatechols were further metabolized to dicatechol, glucuronide, and methylate metabolites in human liver; however, the contribution of each reaction was significantly different between sesamin and episesamin. The liver microsomes from CYP2C19 ultra-rapid metabolizers showed a significant amount of episesamin dicatechol. In this study, we have revealed significantly different metabolism by P450, UDP-glucuronosyltransferase, and catechol-O-methyltransferase for sesamin and episesamin, resulting in different biological effects.

According to the prevailing paradigm, 1α-hydroxylation of 25-hydroxyvitamin D3 (25(OH)D3) and its analogs is a pre-requisite step for their biological effects. We previously reported that 25-hydroxy-19-nor-vitamin D3 (25(OH)-19-nor-D3) had anti-proliferative activity in a cell line, PZ-HPV-7, which was derived from human non-cancerous prostate tissue, and suggested that 25(OH)-19-nor-D3 acted after 1α-hydroxylation by vitamin D 1α-hydroxylase (CYP27B1). However, metabolic studies of 25(OH)-19-nor-D3 using recombinant CYP27B1 revealed that 25(OH)-19-nor-D3 was rarely subjected to 1α-hydroxylation. Therefore, in this report, we attempted to clarify the mechanism of 25(OH)-19-nor-D3 action in intact cells using PZ-HPV-7 prostate cells. After incubating the cells with 25(OH)-19-nor-D3, eight metabolites of 24-hydroxylase (CYP24A1) were detected, whereas no products of CYP27B1 including 1α,25-dihydroxy-19-nor-vitamin D3 (1α,25(OH)2-19-nor-D3) were found. Furthermore, the time-dependent nuclear translocation of vitamin D receptor (VDR) and the subsequent transactivation of cyp24A1 gene in the presence of 25(OH)-19-nor-D3 were almost identical as those induced by 1α,25(OH)2-19-nor-D3. These results strongly suggest that 25(OH)-19-nor-D3 directly binds to VDR as a ligand and transports VDR into the nucleus to induce transcription of cyp24A1 gene. In addition, knock down of cyp27B1 gene did not affect the anti-proliferative activity of 25(OH)-19-nor-D3, whereas knock down of VDR attenuated the inhibitory effect. Thus, our results clearly demonstrate that the anti-proliferative activity of 25(OH)-19-nor-D3 is VDR dependent but 1α-hydroxylation independent, suggesting that 25(OH)D3 analogs such as 25(OH)-19-nor-D3 could be attractive candidates for anticancer therapy.

Bioconversion processes, including specific hydroxylations, promise to be useful for practical applications because chemical syntheses often involve complex procedures. One of the successful applications of P450 reactions is the bioconversion of vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Recently, a cytochrome P450 gene encoding a vitamin D hydroxylase from the CYP107 family was cloned from Pseudonocardia autotrophica and is now applied in the bioconversion process that produces 1α,25-dihydroxyvitamin D₃. In addition, the directed evolution study of CYP107 has significantly enhanced its activity. On the other hand, we found that Streptomyces griseolus CYP105A1 can convert vitamin D₃ to 1α,25-dihydroxyvitamin D₃. Site-directed mutagenesis of CYP105A1 based on its crystal structure dramatically enhanced its activity. To date, multiple vitamin D hydroxylases have been found in bacteria, fungi, and mammals, suggesting that vitamin D is a popular substrate of the enzymes belonging to the P450 superfamily. A combination of these cytochrome P450s would produce a large number of compounds from vitamin D and its analogs. Therefore, we believe that the bioconversion of vitamin D and its analogs is one of the most promising P450 reactions in terms of practical application.

Metabolism of sesamin by cytochrome P450 (P450) was examined using yeast expression system and human liver microsomes. Saccharomyces cerevisiae cells expressing each of human P450 isoforms (CYP1A1, 1A2, 2A6, 2B6, 2C8, 2C9, 2C18, 2C19, 2D6, 2E1, and 3A4) were cultivated with sesamin, and monocatechol metabolite was observed in most of P450s. Kinetic analysis using the microsomal fractions of the recombinant S. cerevisiae cells revealed that CYP2C19 had the largest k(cat)/K(m) value. Based on the kinetic data and average contents of the P450 isoforms in the human liver, the putative contribution of P450s for sesamin metabolism was large in the order of CYP2C9, 1A2, 2C19, and 2D6. A good correlation was observed between sesamin catecholization activity and CYP2C9-specific activity in in vitro studies using 10 individual human liver microsomes, strongly suggesting that CYP2C9 is the most important P450 isoform for sesamin catecholization in human liver. Inhibition studies using each anti-P450 isoform-specific antibody confirmed that CYP2C9 was the most important, and the secondary most important P450 was CYP1A2. We also examined the inhibitory effect of sesamin for P450 isoform-specific activities and found a mechanism-based inhibition of CYP2C9 by sesamin. In contrast, no mechanism-based inhibition by sesamin was observed in CYP1A2-specific activity. Our findings strongly suggest that further studies are needed to reveal the interaction between sesamin and therapeutic drugs mainly metabolized by CYP2C9.

Our previous studies revealed that the double variant of cytochrome P450 (CYP)105A1, R73V/R84A, has a high ability to convert vitamin D(3) to its biologically active form, 1α,25-dihydroxyvitamin D(3) [1α,25(OH)(2)D(3)], suggesting the possibility for R73V/R84A to produce 1α,25(OH)(2)D(3). Because Actinomycetes, including Streptomyces, exhibit properties that have potential advantages in the synthesis of secondary metabolites of industrial and medical importance, we examined the expression of R73V/R84A in Streptomyces lividans TK23 cells under the control of the tipA promoter. As expected, the metabolites 25-hydroxyvitamin D(3) [25(OH)D(3)] and 1α,25(OH)(2)D(3) were detected in the cell culture of the recombinant S. lividans. A large amount of 1α,25(OH)(2)D(3), the second-step metabolite of vitamin D(3), was observed, although a considerable amount of vitamin D(3) still remained in the culture. In addition, novel polar metabolites 1α,25(R),26(OH)(3)D(3) and 1α,25(S),26(OH)(3)D(3), both of which are known to have high antiproliferative activity and low calcemic activity, were observed at a ratio of 5:1. The crystal structure of the double variant with 1α,25(OH)(2)D(3) and a docking model of 1α,25(OH)(2)D(3) in its active site strongly suggest a hydrogen-bond network including the 1α-hydroxyl group, and several water molecules play an important role in the substrate-binding for 26-hydroxylation. In conclusion, we have demonstrated that R73V/R84A can catalyze hydroxylations at C25, C1 and C26 (C27) positions of vitamin D(3) to produce biologically useful compounds.

Vitamin D 3 (VD 3), a prohormone in mammals, plays a crucial role in the maintenance of calcium and phosphorus concentrations in serum. Activation of VD 3 requires 25-hydroxylation in the liver and 1alpha-hydroxylation in the kidney by cytochrome P450 (CYP) enzymes. Bacterial CYP105A1 converts VD 3 into 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) in two independent reactions, despite its low sequence identity with mammalian enzymes (21% identity). The present study determined the crystal structures of a highly active mutant (R84A) of CYP105A1 from Streptomyces griseolus in complex and not in complex with 1alpha,25(OH) 2D 3. The compound 1alpha,25(OH) 2D 3 is positioned 11 A from the iron atom along the I helix within the pocket. A similar binding mode is observed in the structure of the human CYP2R1-VD 3 complex, indicating a common substrate-binding mechanism for 25-hydroxylation. A comparison with the structure of wild-type CYP105A1 suggests that the loss of two hydrogen bonds in the R84A mutant increases the adaptability of the B' and F helices, creating a transient binding site. Further mutational analysis of the active site reveals that 25- and 1alpha-hydroxylations share residues that participate in these reactions. These results provide the structural basis for understanding the mechanism of the two-step hydroxylation that activates VD 3.

Our previous study demonstrated that 25-hydroxy-19-nor-vitamin D(3) [25(OH)-19-nor-D(3)] inhibited the proliferation of immortalized noncancerous PZ-HPV-7 prostate cells similar to 1 alpha,25-dihydroxyvitamin D(3) [1 alpha,25(OH)(2)D(3)], suggesting that 25(OH)-19-nor-D(3) might be converted to 1 alpha,25-dihydroxy-19-nor-vitamin D(3) [1 alpha,25(OH)(2)-19-nor-D(3)] by CYP27B1 before exerting its antiproliferative activity. Using an in vitro cell-free model to study the kinetics of CYP27B1-dependent 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) and 25-hydroxyvitamin D(3) [25(OH)D(3)] and CYP24A1-dependent hydroxylation of 1 alpha,25(OH)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3), we found that k(cat)/K(m) for 1 alpha-hydroxylation of 25(OH)-19-nor-D(3) was less than 0.1% of that for 25(OH)D(3), and the k(cat)/K(m) value for 24-hydroxylation was not significantly different between 1 alpha,25(OH)(2)-19-nor-D(3) and 1 alpha,25(OH)(2)D(3). The data suggest a much slower formation and a similar rate of degradation of 1 alpha,25(OH)(2)-19-nor-D(3) compared with 1 alpha,25(OH)(2)D(3). We then analyzed the metabolites of 25(OH)D(3) and 25(OH)-19-nor-D(3) in PZ-HPV-7 cells by high-performance liquid chromatography. We found that a peak that comigrated with 1 alpha,25(OH)(2)D(3) was detected in cells incubated with 25(OH)D(3), whereas no 1 alpha,25(OH)(2)-19-nor-D(3) was detected in cells incubated with 25(OH)-19-nor-D(3). Thus, the present results do not support our previous hypothesis that 25(OH)-19-nor-D(3) is converted to 1 alpha,25(OH)(2)-19-nor-D(3) by CYP27B1 in prostate cells to inhibit cell proliferation. We hypothesize that 25(OH)-19-nor-D(3) by itself may have a novel mechanism to activate vitamin D receptor or it is metabolized in prostate cells to an unknown metabolite with antiproliferative activity without 1 alpha-hydroxylation. Thus, the results suggest that 25(OH)-19-nor-D(3) has potential as an attractive agent for prostate cancer therapy.

CYP27A1 catalyzes vitamin D(3) 25-hydroxylation and further hydroxylation at C-1alpha, C-24 or C-26(27). Molecular modeling of human CYP27A1 and docking with 25-hydroxyvitamin D(3) predicted that Gln 85 might be important for 1alpha-hydroxylation activity of CYP27A1 by forming a hydrogen bond with the 25-OH group of 25-hydroxyvitamin D(3). Expectedly, the mutant Q85H expressed in Escherichia coli showed no detectable 1alpha-hydroxylation activity toward 25-hydroxyvitamin D(3). In addition, Q85H prefers 24-hydroxylation toward 25-hydroxyvitamin D(3) whereas the wild-type prefers 26(27)-hydroxylation. A molecular modeling study also suggests that Gln 85 of CYP27A1 simultaneously interacts with Asn 107 and the hydroxyl group of the substrate. The fact that Q85L did not contain a heme molecule suggests that the hydrogen bond between Gln 85 and Asn 107 is important for protein folding of CYP27A1. Based on these results, it is possible that Gln 85 plays essential roles in both substrate-binding and protein folding.

Our previous studies revealed that sesamin caused a mechanism-based inhibition (MBI) of CYP2C9 in human liver microsomes. Additionally, we observed a similar MBI of CYP2C by sesamin in the rat liver microsomes. Sesamin-induced difference spectra of rat or human liver microsomes in the presence of NADPH showed a peak at 459 nm, suggesting the formation of a metabolic-intermediate (MI) complex of cytochrome P450 and the methylenedioxyphenyl group of sesamin. However, the peak disappeared in both liver microsomes within 30 min after the termination of the metabolism. These results suggest that the MI complex of cytochrome P450 and sesamin is unstable, and the effects of sesamin on human CYP2C9- or rat CYP2C-mediated drug metabolism may be small. To confirm this, in vivo studies using rats were performed. The pharmacokinetics of diclofenac, which is mainly metabolized by CYP2C11 in male rats, were investigated after a 3-days administration of sesamin (0, 10, and 100 mg/kg bw). No significant differences were observed among the three groups in the pharmacokinetic parameters, C max, T max, and AUC. Furthermore, administration of sesamin to rats for 7 days had no significant effects on diclofenac hydroxylation activity in rat liver microsomes. These results demonstrate that no significant interaction occurs between diclofenac and sesamin in rats. Moreover, the results of these in vitro and in vivo studies suggest that no significant interaction may occur between sesamin and diclofenac when sesamin is administered to humans as a supplement, since the standard sesamin dose in humans is much lower than that administered to rats in this study.

The metabolism of eldecalcitol (ED-71), a 2β-hydroxypropoxylated analog of the active form of vitamin D3 was investigated by using in vitro systems. ED-71 was metabolized to 1α,2β,25-trihydroxyvitamin D3 (1α,2β,25(OH)3D3) in human small intestine and liver microsomes. To identify the enzymes involved in this metabolism, we examined NADPH-dependent metabolism by recombinant P450 isoforms belonging to the CYP1, 2, and 3 families, and revealed that CYP3A4 had the activity. However, the CYP3A4 -specific inhibitor, ketoconazole, decreased the activity in human liver microsomes by only 36%, suggesting that other enzymes could be involved in ED-71 metabolism. Because metabolism was dramatically inhibited by cyanide, we assumed that sterol C4-methyl oxidase like gene product (SC4MOL) might contribute to the metabolism of ED-71. It is noted that SC4MOL is physiologically essential for cholesterol synthesis. Recombinant human SC4MOL expressed in COS7, Saccharomyces cerevisiae, or Escherichia coli cells converted ED-71 to 1α,2β,25(OH)3D3. Furthermore, we evaluated the metabolism of ED-71 by recombinant CYP24A1, which plays an important role in the metabolism of the active form of vitamin D3 (1α,25(OH)2D3) and its analogs. The k cat/K m value for 24- or 23-hydroxylation of ED-71 was only 3% of that for 1α,25(OH)2D3, indicating that ED-71 was resistant to CYP24A1-dependent catabolism. Among the three enzymes catalyzing ED-71, SC4MOL appears to be most important in the metabolism of ED-71. To the best of our knowledge, this is the first study showing that SC4MOL can function as a drug-metabolizing enzyme. The yeast and E. coli expression systems for SC4MOL could be useful for structure-function analyses of SC4MOL.

Royal jelly (RJ) has various pharmacological actions, including hypolipidaemic, hypocholesterolaemic and anti-atherosclerotic effects, in experimental animals but the molecular mechanisms involved remain unclear. Here, we investigated changes in the expression of lipid metabolism-associated genes in the liver of RJ-treated mice by means of a DNA microarray technique to obtain clues to the mechanism of the hypocholesterolaemic action of RJ. We compared the hepatic gene expression profiles in three groups of mice fed a diet containing 5% RJ, a diet containing 5% RJ stored at 40°C for 7 days (40-7d RJ) or a control diet which provided the same total energy as the other diets. RJ decreased gene expression of squalene epoxidase (SQLE), which is a key enzyme in cholesterol biosynthesis, and sterol regulatory element-binding protein (SREB)-1, which may be a transcriptional factor of SQLE. It increased gene expression of low-density lipoprotein receptor (LDLR), which is involved in cholesterol incorporation in liver. Thus, the hypocholesterolaemic action of RJ appears to be associated with a decrease of SQLE and an increase of LDLR in mice.

A molecular modeling study of CYP27B1 suggests that Arg458 of mouse CYP27B1 is involved in interaction with adrenodoxin (ADX). Thus, we generated CYP27B1 mutants R458K and R458Q and revealed their enzymatic properties. Substrate-induced difference spectra and K(m) values for 1alpha-hydroxylation of 25(OH)D3 indicate that the replacement of Arg458 with Lys or Gln does not affect substrate binding. However, these mutants showed remarkable decreases of both kcat values and the ratio of product formation to NADPH oxidation (coupling efficiency). A high K(m) value of R458Q for ADX concentration and a decrease of rate constant of the first electron transfer seem reasonable considering that the conversion from Arg to noncharged Gln abolishes salt-bridge formation with the acidic residue of ADX. On the other hand, R458K showed atypical kinetics for ADX concentration with Hill's constant of 2.0 and high catalytic activity at high ADX concentration by increase of coupling efficiency. These results suggest that conformational change of R458K by binding the two ADX molecules is essential for 1alpha-hydroxylation of 25(OH)D3. On the other hand, binding one ADX molecule is sufficient for the conformational change of the wild-type CYP27B1, judging from its Michaelis-Menten-type kinetics for ADX concentration with high coupling efficiency. These results suggest that ADX functions as an effector for the oxygen transfer reaction in addition to being an electron donor for CYP27B1.

Among polychlorinated dibenzo-p-dioxins (PCDDs), 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TetraCDD) is the most toxic one. Recently, we reported that rat CYP1A1 mutant, F240A, expressed in yeast showed metabolic activity toward 2,3,7,8-TetraCDD. In this study, we successfully expressed N-terminal truncated P450s (Delta1A1 and DeltaF240A) in Escherichia coli cells. Kinetic analysis using membrane fractions prepared from the recombinant E. coli cells revealed that DeltaF240A has enzymatic properties similar to F240A expressed in yeast. The metabolism of PCDDs by recombinant E. coli cells expressing both DeltaF240A and human NADPH-P450 reductase was also examined. When 2,3,7-TriCDD was added to the E. coli cell culture at a final concentration of 10 microM, approximately 90% of the 2,3,7-TriCDD was converted into multiple metabolites within 8 h. These results indicate the possible application of prokaryotic cells expressing DeltaF240A to the bioremediation of PCDD-contaminated soil.

The worker honeybees of Apis mellifera L. age-dependently change from nurse bees which take care of their brood to forager bees which collect for nectar and convert it into honey during their lifespan of 30-40 days. A 56-kDa protein (p56kP-4) was shown to be present only in the nurse-bee hypopharyngeal gland. Here, we investigated the physiological effects of p56kP-4 on primary-cultured rat hepatocytes in the absence of serum. The p56kP-4 recombinant protein expressed in Escherichia coli significantly stimulated hepatocyte DNA synthesis and protected cells from apoptosis induced by serum deprivation. It also activated mitogen-activated protein kinase, as well as protein kinase B, which is a key regulator of cell survival. These findings suggest that p56kP-4 has a growth factor-like action on primary-cultured rat hepatocytes.

Central fatigue is an indispensable biosignal for maintaining life, but the neuronal and molecular mechanisms involved remain unclear. In this study, we searched for genes differentially expressed in the hippocampus of fatigued mice to elucidate the mechanisms underlying fatigue. Mice were forced to swim in an adjustable-current water pool, and the maximum swimming time (endurance) until fatigue was measured thrice. Fatigued and nonfatigued mice with equal swimming capacity and body weight were compared. We found that the genes of GluR1 and B-cell receptor-associated protein 31 (Bap31), which acts as a transport molecule in the secretory pathway or as a mediator of apoptosis, were upregulated in the hippocampus of fatigued mice, and increases of GluR1 and Bap31 were confirmed by Northern blotting and real-time PCR. No change of gene expression of AMPA receptor subunits other than GluR1 was observed. These results suggest that a compositional change of AMPA receptor (increase of GluR1) and upregulation of the Bap31 gene may be implicated in fatigue in mice.

We previously reported the three-dimensional structure of human CYP27B1 (25-hydroxyvitamin D3 1alpha-hydroxylase) constructed by homology modeling. Using the three-dimensional model we studied the docking of the substrate, 25-hydroxyvitamin D3, into the substrate binding pocket of CYP27B1. In this study, we focused on the amino acid residues whose point mutations cause vitamin D-dependent rickets type 1, especially unconserved residues among mitochondrial CYPs such as Gln65 and Thr409. Recently, we successfully overexpressed mouse CYP27B1 by using a GroEL/ES co-expression system. In a mutation study of mouse CYP27B1 that included spectroscopic analysis, we concluded that in a 1alpha-hydroxylation process, Ser408 of mouse CYP27B1 corresponding to Thr409 of human CYP27B1 forms a hydrogen bond with the 25-hydroxyl group of 25-hydroxyvitamin D3. This is the first report that shows a critical amino acid residue recognizing the 25-hydroxyl group of the vitamin D3.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.

We have previously shown that ethylenediaminetetraacetic acid (EDTA) suppresses the storage-dependent degradation of 57-kDa protein, which is a possible marker for freshness and quality of royal jelly (RJ) through the inhibition of a proteinase in RJ. We suggested that EDTA could be useful as a preservative agent to maintain the quality of RJ. Here, we report the effects of other metal chelators, di- or tricarboxylic acids such as citric acid and malic acid, on proteinase activity in RJ and the specific degradation of 57-kDa protein during storage. Various carboxylic acids inhibited the proteinase activity in RJ, but did not suppress storage-dependent degradation of 57-kDa protein. However, when RJ was stored with various carboxylate buffers (pH 4.0) at 40 degrees C, the degradation of 57-kDa protein during storage was suppressed through the inhibition of proteinase in RJ. Among the buffers, citrate buffer (pH 4.0) effectively inhibited the decrease of 57-kDa protein concentration in RJ during storage. These results suggest that citrate buffer (pH 4.0) could be available as a new preservative agent to maintain the quality of RJ.

The expression of mouse CYP27B1 in Escherichia coli has been dramatically enhanced by coexpression of GroEL/ES. To reveal the enzymatic properties of CYP27B1, we measured its hydroxylation activity toward vitamin D3 and 1alpha-hydroxyvitamin D3 (1alpha(OH)D3) in addition to the physiological substrate 25(OH)D3. Surprisingly, CYP27B1 converted vitamin D3 to 1alpha,25(OH)D3. Both 1alpha-hydroxylation activity toward vitamin D3, and 25-hydroxylation activity toward 1alpha(OH)D3 were observed. The Km and Vmax values for 25-hydroxylation activity toward 1alpha(OH)D3 were estimated to be 1.7 microM and 0.51 mol/min/mol P450, respectively, while those for 1alpha-hydroxylation activity toward 25(OH)D3 were 0.050 microM and 2.73 mol/min/mol P450, respectively. Note that the substrate must be fixed in the opposite direction in the substrate-binding pocket of CYP27B1 between 1alpha-hydroxylation and 25-hydroxylation. Based on these results and the fact that human CYP27A1 and Streptomyces CYP105A1 also convert vitamin D3 to 1alpha,25(OH)D3, 1alpha-hydroxylation, and 25-hydroxylation of vitamin D3 appear to be closely linked together.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M. (2001) Biosci. Biotechnol. Biochem. 65, 277-284.). Here, we investigated the physiological effects of 57-kDa protein on primary cultured rat hepatocytes in the absence of serum. The 57-kDa protein and RJ significantly stimulated hepatocyte DNA synthesis, whereas bovine serum albumin and RJ stored at 40 degrees C for 7 days, which lacks 57-kDa protein, did not. The mitogenic activity of 57-kDa protein was lost after treatment with trypsin. These results indicate that 57-kDa protein acts as a mitogen. The stimulatory effect of 57-kDa protein was dose-dependent and was more potent at lower than at higher cell densities. The 57-kDa protein also prolonged the cell proliferation of primary cultured rat hepatocytes, with an enhancement of albumin production compared to untreated cells. Therefore, 57-kDa protein is likely to promote liver regeneration and may have a cytoprotective action on hepatocytes.

In order to find a marker for freshness of royal jelly (RJ), the composition change of RJ during storage was investigated. The contents of 10-hydroxy-2-decenoic acid, a bioactive component of RJ, and several vitamins did not change during storage at 40 degrees C for 7 days. However, a specific protein, designated royal jelly protein-1 (RJP-1), was gradually degraded during storage under various conditions (from 4 degrees C to 50 degrees C for up to 7 days). The specific degradation of RJP-1 was proportional to storage temperature and storage period. RJP-1 was purified to homogeneity and characterized as a monomeric glycoprotein with a molecular mass of 57 kDa. These results suggest that 57-kDa protein in RJ can be used as a marker for freshness of RJ, reflecting the conditions under which RJ has been stored.

1α-Hydroxylation of 25-hydroxyvitamin D3 is believed to be essential for its biological effects. In this study, we evaluated the biological activity of 25(OH)D3 itself comparing with the effect of cell-derived 1α,25-dihydroxyvitamin D3 (1α,25(OH)2D3). First, we measured the cell-derived 1α,25(OH)2D3 level in immortalized human prostate cell (PZ-HPV-7) using [(3)H]-25(OH)D3. The effects of the cell-derived 1α,25(OH)2D3 on vitamin D3 24-hydroxylase (CYP24A1) mRNA level and the cell growth inhibition were significantly lower than the effects of 25(OH)D3 itself added to cell culture. 25-Hydroxyvitamin D3 1α-hydroxylase (CYP27B1) gene knockdown had no significant effects on the 25(OH)D3-dependent effects, whereas vitamin D receptor (VDR) gene knockdown resulted in a significant decrease in the 25(OH)D3-dependent effects. These results strongly suggest that 25(OH)D3 can directly bind to VDR and exerts its biological functions. DNA microarray and real-time RT-PCR analyses suggest that semaphorin 3B, cystatin E/M, and cystatin D may be involved in the antiproliferative effect of 25(OH)D3.

CYP2R1 is known to be a physiologically important vitamin D 25-hydroxylase. We have successfully expressed human CYP2R1 in Saccharomyces cerevisiae to reveal its enzymatic properties. In this study, we examined production of 25-hydroxylated vitamin D using whole recombinant yeast cells that expressed CYP2R1. When vitamin D3 or vitamin D2 was added to the cell suspension of CYP2R1-expressing yeast cells in a buffer containing glucose and β-cyclodextrin, the vitamins were converted into their 25-hydroxylated products. Next, we irradiated the cell suspension with UVB and incubated at 37 °C. Surprisingly, the 25-hydroxy vitamin D2 was produced without additional vitamin D2. Endogenous ergosterol was likely converted into vitamin D2 by UV irradiation and thermal isomerization, and then the resulting vitamin D2 was converted to 25-hydroxyvitamin D2 by CYP2R1. This novel method for producing 25-hydroxyvitamin D2 without a substrate could be useful for practical purposes.

The honeybee (Apis mellifera) forms two female castes: the queen and the worker. This dimorphism depends not on genetic differences, but on ingestion of royal jelly, although the mechanism through which royal jelly regulates caste differentiation has long remained unknown. Here I show that a 57-kDa protein in royal jelly, previously designated as royalactin, induces the differentiation of honeybee larvae into queens. Royalactin increased body size and ovary development and shortened developmental time in honeybees. Surprisingly, it also showed similar effects in the fruitfly (Drosophila melanogaster). Mechanistic studies revealed that royalactin activated p70 S6 kinase, which was responsible for the increase of body size, increased the activity of mitogen-activated protein kinase, which was involved in the decreased developmental time, and increased the titre of juvenile hormone, an essential hormone for ovary development. Knockdown of epidermal growth factor receptor (Egfr) expression in the fat body of honeybees and fruitflies resulted in a defect of all phenotypes induced by royalactin, showing that Egfr mediates these actions. These findings indicate that a specific factor in royal jelly, royalactin, drives queen development through an Egfr-mediated signalling pathway.

Hansenula mrakii IFO 0895 induces glutathione peroxidase as an oxidative stress response. Activities of other antioxidant enzymes—superoxide dismutase, catalase and cytochrome c peroxidase—were assayed. The superoxide dismutase activity did not change when the cells were cultured in the presence of lipid hydroperoxide, whereas the catalase and cytochrome c peroxidase activities decreased. The intracellular glutathione content and the activities of enzymes involved in glutathione biosynthesis were not changed by oxidative stress. Glucose-6-phosphate dehydrogenase, which is involved in the glutathione recycling system, was induced by oxidative stress to supply NADPH which is consumed by glutathione reductase coupling with the glutathione peroxidase reaction.

Our previous study revealed that CYP2C9 played a central role in sesamin monocatecholization. In this study, we focused on the metabolism of sesamin monocatechol that was further converted into the dicatechol form by cytochrome P450 (P450) or the glucuronide by UDP-glucuronosyltransferase (UGT). Catecholization of sesamin monocatechol enhances its antioxidant activity, whereas glucuronidation strongly reduces its antioxidant activity. In human liver microsomes, the glucuronidation activity was much higher than the catecholization activity toward sesamin monocatechol. In contrast, in rat liver microsomes, catecholization is predominant over glucuronidation. In addition, rat liver produced two isomers of the glucuronide, whereas human liver produced only one glucuronide. These results suggest a significant species-based difference in the metabolism of sesamin between humans and rats. Kinetic studies using recombinant human UGT isoforms identified UGT2B7 as the most important UGT isoform for glucuronidation of sesamin monocatechol. In addition, a good correlation was observed between the glucuronidation activity and UGT2B7-specific activity in in vitro studies using 10 individual human liver microsomes. These results strongly suggest that UGT2B7 plays an important role in glucuronidation of sesamin monocatechol. Interindividual difference among the 10 human liver microsomes is approximately 2-fold. These results, together with our previous results on the metabolism of sesamin by human P450, suggest a small interindividual difference in sesamin metabolism. We observed the methylation activity toward sesamin monocatechol by catechol O-methyl transferase (COMT) in human liver cytosol. On the basis of these results, we concluded that CYP2C9, UGT2B7, and COMT played essential roles in the metabolism of sesamin in the human liver.

CYP105A1 from Streptomyces griseolus has the capability of converting vitamin D 3 (VD 3) to its active form, 1alpha,25-dihydroxyvitamin D 3 (1alpha,25(OH) 2D 3) by a two-step hydroxylation reaction. Our previous structural study has suggested that Arg73 and Arg84 are key residues for the activities of CYP105A1. In this study, we prepared a series of single and double mutants by site-directed mutagenesis focusing on these two residues of CYP105A1 to obtain the hyperactive vitamin D 3 hydroxylase. R84F mutation altered the substrate specificity that gives preference to the 1alpha-hydroxylation of 25-hydroxyvitamin D 3 over the 25-hydroxylation of 1alpha-hydroxyvitamin D 3, opposite to the wild type and other mutants. The double mutant R73V/R84A exhibited 435- and 110-fold higher k cat/ K m values for the 25-hydroxylation of 1alpha-hydroxyvitamin D 3 and 1alpha-hydroxylation of 25-hydroxyvitamin D 3, respectively, compared with the wild-type enzyme. These values notably exceed those of CYP27A1, which is the physiologically essential VD 3 hydroxylase. Thus, we successfully generated useful enzymes of altered substrate preference and hyperactivity. Structural and kinetic analyses of single and double mutants suggest that the amino acid residues at positions 73 and 84 affect the location and conformation of the bound compound in the reaction site and those in the transient binding site, respectively.

Daily behaviors are strongly dominated by internally generated circadian rhythms, but the underlying mechanisms remain unclear. In mammals, photoentrainment of behaviors to light-dark cycles involves signaling from both intrinsically photosensitive retinal ganglion cells and classic photoreceptor pathways to the suprachiasmatic nucleus (SCN). How classic photoreceptor pathways work with the photosensitive ganglion cells, however, is not fully understood. Although cholecystokinin (CCK) peptide has been shown to be present in a variety of vertebrate retinas, its function at a systems level is also unknown. In the present study we examined a possible role of CCK-A receptors in photoentrainment using CCK-A receptor knockout mice. The lacZ reporter gene within a gene-knockout cassette revealed precise localization of CCK-A receptors in the circadian clock system. We demonstrated that CCK-A receptors were located predominately on glycinergic amacrine cells but were rarely found on SCN neurons. Moreover, Ca(2+) imaging analysis demonstrated that the CCK-A agonist, CCK-8 sulfate (CCK-8s), mobilized intracellular Ca(2+) in amacrine cells but not glutamate-receptive SCN neurons. Furthermore, light pulse-induced mPer1/mPer2 gene expression in SCN, behavioral phase shifts, and the pupillary reflex were significantly reduced in CCK-A receptor knockout mice. These data indicate a novel function of CCK-A receptors in the nonimage-forming photoreception presumably via amacrine cell-mediated signal transduction pathways.

Traumatic stress has been believed to result in a variety of unusual alterations of the integrity and the functionality in the hippocampus. In this study, we searched for genes responsive to traumatic stress in the mouse hippocampus to elucidate the underlying mechanisms. Adult male mice were subjected to water-immersion restraint stress (WIRS) for 3 h as an extremely stressful experience, followed by dissection of the hippocampus and subsequent extraction of RNA for differential display polymerase chain reaction (PCR) analysis. The actin-based molecular motor protein myosin VI (Myo6) was identified as a gene markedly upregulated by traumatic stress in the mouse hippocampus 24 h after WIRS. Real-time PCR and Western blotting analyses clearly revealed a significant increase in the expression of both mRNA and corresponding protein for Myo6 in the hippocampus within 24 h after WIRS, while WIRS failed to significantly affect the expression of Myo6 protein in the cerebral cortex, cerebellum and olfactory bulb. Immunohistochemistry analysis revealed that Myo6 protein was ubiquitously expressed throughout the mouse brain, with an extremely high level in the olfactory bulb. These results suggest that Myo6 may be selectively and rapidly upregulated to play a hitherto unidentified role in the maintenance of the integrity and functionality in the hippocampus after traumatic stress.

Our previous studies revealed the species-based difference of CYP24A1-dependent vitamin D metabolism. Although human CYP24A1 catalyzes both C-23 and C-24 oxidation pathways, rat CYP24A1 shows almost no C-23 oxidation pathway. We tried to identify amino acid residues that cause the species-based difference by site-directed mutagenesis. In the putative substrate-binding regions, amino acid residue of rat CYP24A1 was converted to the corresponding residue of human CYP24A1. Among eight mutants examined, T416M and I500T showed C-23 oxidation pathway. In addition, the mutant I500F showed quite a different metabolism of 1alpha,25-dihydroxyvitamin D3 [1alpha,25(OH)2D3] from both human and rat CYP24A1. These results strongly suggest that the amino acid residues at positions 416 and 500 play a crucial role in substrate binding and greatly affect substrate orientation. A three-dimensional model of CYP24A1 indicated that the A-ring and triene part of 1alpha,25(OH)2D3 could be located close to amino acid residues at positions 416 and 500, respectively. Our findings provide useful information for the development of new vitamin D analogs for clinical use.

The human genome project revealed a new member of the P450 family 2, CYP2W1, which has orthologous form in other vertebrate species, suggesting CYP2W1's significant physiological function. Recently, it was reported that CYP2W1 can metabolize arachidonic acid. In this study, we isolated human CYP2W1 cDNA, and successfully expressed truncated CYP2W1 lacking N-terminal 20 amino acids in Escherichia coli cells. In the bicistronic expression system for human CYP2W1 and NADPH-P450 reductase, the formation of blue pigment, indigo, was observed in bacterial cultures. Based on this result, we revealed that CYP2W1 catalyzes the oxidation of indole. In addition, CYP2W1 showed monooxygenase activity towards 3-methylindole and chlorzoxazone. However, no activity was observed towards fatty acids including arachidonic acid. Further analysis using an E. coli expression system will reveal substrate specificity of CYP2W1 and why this P450 isoform is universally conserved in vertebrates.

Recently, we demonstrated that some A-ring-modified vitamin D3 analogs had unique biological activity. Of these analogs, 2alpha-propoxy-1alpha,25(OH)2D3 (C3O1) and 2alpha-(3-hydroxypropoxy)-1alpha,25(OH)2D3 (O2C3) were examined for metabolism by CYP27A1 and CYP24A1. Surprisingly, CYP27A1 catalyzed the conversion from C3O1 to O2C3, which has 3 times more affinity for vitamin D receptor than C3O1. Thus, the conversion from C3O1 to O2C3 by CYP27A1 is considered to be a metabolic activation process. Five metabolites were detected in the metabolism of C3O1 and O2C3 by human CYP24A1 including both C-23 and C-24 oxidation pathways. On the other hand, three metabolites of the C-24 oxidation pathway were detected in their metabolism by rat CYP24A1, indicating a species-based difference in the CYP24A1-dependent metabolism of C3O1 and O2C3 between humans and rats. Kinetic analysis revealed that the Km and kcat values of human CYP24A1 for O2C3 are, respectively, approximately 16 times more and 3 times less than those for 1alpha,25(OH)2D3. Thus, the catalytic efficiency, kcat/Km, of human CYP24A1 for O2C3 is only 2% of 1alpha,25(OH)2D3. These results and a high calcium effect of C3O1 and O2C3 in animal experiments using rats suggest that C3O1 and O2C3 are promising for clinical treatment of osteoporosis.

The activation of vitamin D requires 25-hydroxylation in the liver and 1α-hydroxylation in the kidney. However, it remains unclear which enzyme is relevant to vitamin D 25-hydroxylation. Recently, human CYP2R1 has been reported to be a potential candidate for a hepatic vitamin D 25-hydroxylase. Thus, vitamin D metabolism by CYP2R1 was compared with human mitochondrial CYP27A1, which used to be considered a physiologically important vitamin D 3 25-hydroxylase. A clear difference was observed between CYP2R1 and CYP27A1 in the metabolism of vitamin D 2 . CYP2R1 hydroxylated vitamin D 2 at the C-25 position while CYP27A1 hydroxylated it at positions C-24 and C-27. The K m and k cat values for the CYP2R1-dependent 25-hydroxylation activity toward vitamin D 3 were 0.45 μM and 0.97 min −1 , respectively. The k cat / K m value of CYP2R1 was 26-fold higher than that of CYP27A1. These results strongly suggest that CYP2R1 plays a physiologically important role in the vitamin D 25-hydroxylation in humans.

We investigated the antifatigue effect of royal jelly (RJ), which had been stored at -20 degrees C from immediately after collection, in male Std ddY mice. The mice were accustomed to swimming in an adjustable-current swimming pool, then subjected to forced swimming five times during 2 wk, and the total swimming period until exhaustion was measured. They were separated into three groups with equal swimming capacity, which were administered RJ, RJ stored at 40 degrees C for 7 d (40-7d RJ), or the control solution including casein, cornstarch, and soybean oil before swimming. All mice were forced to swim for 15 min once; then the maximum swimming time to fatigue was measured after a rest period. The swimming endurance of the RJ group significantly increased compared with those of the other groups. The mice in the RJ group showed significantly decreased accumulation of serum lactate and serum ammonia and decreased depletion of muscle glycogen after swimming compared with the other groups, whereas there was no significant difference between the 40-7d RJ group and the control group in these parameters after swimming. A quantitative analysis of constituents in RJ showed that 5 7-kDa protein, which we previously identified as a possible freshness marker of RJ, was specifically degraded in RJ stored at 40 degrees C for 7 d, whereas the contents of various vitamins, 10-hydroxy-2-decenoic acid, and other fatty acids in RJ were unchanged. These findings suggest that RJ can ameliorate the physical fatigue after exercise, and this antifatigue effect of RJ in mice seems to be associated with the freshness of RJ, possibly with the content of 5 7-kDa protein.

We have previously shown that 57-kDa protein in royal jelly (RJ) was specifically degraded in proportion to both storage temperature and storage period, and we suggested that it could be useful as a marker of freshness of RJ (Kamakura, M., Fukuda, T., Fukushima, M. and Yonekura, M., Biosci. Biotechnol. Biochem., 65, 277–284 (2001).). Here, we investigated the effects of various proteinase inhibitors on proteinase activity in RJ and on the specific degradation of 57-kDa protein during storage. Ethylenediaminetetraacetic acid (EDTA), but not other inhibitors, inhibited the proteinase activity in RJ, and dose-dependently suppressed storage-dependent degradation of 57-kDa protein. These results suggest that EDTA inhibits a specific proteinase activity in RJ, thereby suppressing the degradation of 57-kDa protein during storage at high temperature.

The yeast Hansenula mrakii IFO 0895 induces glutathione peroxidae (GPx) when the cells are exposed to the oxidative stress such as lipid hydroperoxide, superoxide- and hydroxy radical-generating conditions. To clarify the localization of GPx in H. mrakii cell, distribution of the enzyme was investigated. After centrifugation of the yeast protoplast homogenates at 2500 × g for 10 min, 67% of total GPx activity was recovered from the supernatant (Sup. 1) and 33% was from the pellet (Pellet 1). When the Sup. 1 was fractionated by sucrose density gradient ultracentrifugation, GPx activity was essentially recovered from the mitochondria fraction. Submitochondrial localization of the enzyme showed that 95% and 2.5% of the enzyme was recovered from the inner and outer membrane, respectively. No GPx activity was detected neither in intermembrane space nor in matrix of mitochondria. On the other hand, at least 12% of total GPx activity was recovered from the purified plasma membrane which was obtained from the Pellet 1 by successive sucrose density gradient centrifugation. Thus, the GPx of H. mrakii is present in the inner and outer membrane of mitochondria as well as the plasma membrane.

A 57-kDa protein in royal jelly (RJ) was previously shown to stimulate hepatocyte DNA synthesis and prolongs the proliferation of hepatocytes as well as increasing albumin production [Kamakura, M., Suenobu, N., and Fukushima, M. (2001) Biochem. Biophys. Res. Commun. 282, 865-874]. In this study, I investigated the signal transduction mechanisms involved in the induction of hepatocyte DNA synthesis and the promotion of cell survival by this 57-kDa protein in primary cultures of adult rat hepatocytes. Hepatocyte DNA synthesis induced by the 57-kDa protein was not influenced by several alpha- and beta-adrenoceptor antagonists, but was dose-dependently abolished by an inhibitor of a tyrosine-specific protein kinase, genistein. A phospholipase C inhibitor (U-73122) and a protein kinase C (PKC) inhibitor (sphingosine) inhibited 57-kDa protein-stimulated he-patocyte DNA synthesis, whereas a protein kinase A inhibitor (H-89) did not. The 57-kDa protein also activated PKC in rat hepatocytes. Various inhibitors of intracellular signal transduction elements (PD98059, p21 ras farnesyltransferase inhibitor, wortmannin and rapamycin) also blocked hepatocyte DNA synthesis induced by the 57-kDa protein. Furthermore, the 57-kDa protein activated mitogen-activated protein (MAP) kinase in rat hepatocytes. The activation of MAP kinase by the 57-kDa protein was inhibited by PD98059 and sphingosine. The 57-kDa protein also activated protein kinase B, which is a key regulator of cell survival. These results suggest that, like growth factors, the 57-kDa protein activates several important intracellular signaling factors involved in the stimulation of hepatocyte DNA synthesis and the protection of cells from apoptosis.