Metabolism of testosterone and progesterone by cytochrome P450 2C19 allelic variants.

CYP2C19 is a member of the human microsomal cytochrome P450 (CYP). Significant variation in CYP2C19 levels and activity can be attributed to polymorphisms in this gene. Wildtype CYP2C19 and 13 mutants (CYP2C19.1B, CYP2C19.5A, CYP2C19.5B, CYP2C19.6, CYP2C19.8, CYP2C19.9, CYP2C19.10, CYP2C19.11, CYP2C19.13, CYP2C19.16, CYP2C19.19, CYP2C19.23, CYP2C19.30, and CYP2C19.33) were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. Hydroxylase activity toward testosterone and progesterone was also examined. Ten CYP2C19 variants showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra. CYP2C19.11 and CYP2C19.23 showed higher testosterone 11α, 16α-/17- and progesterone 6ß-,21-,16α-/17α-hydroxylase activities than CYP2C19.1B. CYP2C19.6, CYP2C19.16, CYP2C19.19, and CYP2C19.30 showed lower activity than CYP2C19.1B. CYP2C19.9, CYP2C19.10. CYP2C19.13, and CYP2C19.33 showed different hydroxylation activities than CYP2C19.1B. These results indicated that CYP2C19 variants have very different substrate specificities for testosterone and progesterone.

Localization of SNARE proteins in the brain and corpus allatum of Bombyx mori.

Soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) make up the core machinery that mediates membrane fusion. SNAREs, syntaxin, synaptosome-associated protein (SNAP), and synaptobrevin form a tight SNARE complex that brings the vesicle and plasma membranes together and is essential for membrane fusion. The cDNAs of SNAP-25, VAMP2, and Syntaxin 1A from Bombyx mori were inserted into a plasmid, transformed into Escherichia coli, and purified. We then produced antibodies against the SNAP-25, VAMP2, and Syntaxin 1A of Bombyx mori of rabbits and rats, which were used for immunohistochemistry. Immunohistochemistry results revealed that the expression of VAMP2 was restricted to neurons in the pars intercerebralis (PI), dorsolateral protocerebrum (DL), and central complex (CX) of the brain. SNAP-25 was restricted to neurons in the PI and the CX of the brain. Syntaxin 1A was restricted to neurons in the PI and DL of the brain. VAMP2 co-localized with SNAP-25 in the CX, and with Syntaxin 1A in the PI and DL. VAMP2, SNAP-25, and Syntaxin 1A are present in the CA. Bombyxin-immunohistochemical reactivities (IRs) of brain and CA overlapped with VAMP2-, SNAP-25, and Syntaxin 1A-IRs. VAMP2 and Syntaxin 1A are present in the prothoracicotropic hormone (PTTH)-secretory neurons of the brain.

A defective prostaglandin E synthase could affect egg formation in the silkworm Bombyx mori.

We had previously reported a prostaglandin E synthase (bmPGES) in the silkworm Bombyx mori that catalyzes the isomerization of PGH2 to PGE2. The present study aimed to provide a genome-editing characterization of bmPGES in B. mori. Results showed bmPGES gene disruption to result in a reduced content of PGE2. The change affected the expression of chorion genes and egg formation in silkworms. Collectively, the results indicated that bmPGES could be involved in reproduction of B. mori. Therefore, this study provides insights into the physiological role of bmPGES and PGE2 in silkworms.

Z-505, an Oral Ghrelin Receptor Agonist, Attenuates Anorexia After Total Gastrectomy in Rats.

BACKGROUND: Anorexia is a serious problem in patients with gastric cancer who have undergone gastrectomy. Ghrelin, an orexigenic hormone primarily secreted from the stomach, has been proposed to prevent anorexia. Significant reduction in plasma ghrelin levels after gastrectomy may contribute to lack of appetite and weight loss. In this study, we investigated the effects of Z-505, a ghrelin receptor agonist, on anorexia after total gastrectomy (TG) in a rat model. METHODS AND MATERIALS: Male Sprague-Dawley rats were used to establish a TG model, and then sham-operated (control) and TG rats were randomly assigned to four subgroups receiving administration of Z-505 (100 mg/kg, p.o., once daily) or vehicle for 14 d from day 14 to day 27 after TG. The food intake, body weight, and fat weight were evaluated during the test period. Moreover, the neuronal activity in the hypothalamus was evaluated on day 21 to investigate the mechanism of action of Z-505. RESULTS: In TG rats, Z-505 significantly improved the decrease in cumulative food intake induced by the surgery over 14 d (TG + vehicle; 213.8 ± 15.3 g, n = 12 versus TG + Z-505; 258.2 ± 13.1 g, n = 14, P < 0.05). Z-505 also significantly increased fat weight and had a milder effect on body weight over 14 d. In addition, Z-505 significantly increased the number of c-Fos-positive cells in the hypothalamic arcuate nucleus (TG + vehicle; 17.8 ± 2.0, n = 12 versus TG + Z-505; 72.2 ± 11.8, n = 12, P < 0.001). CONCLUSIONS: Z-505 may be a useful therapeutic treatment for anorexia after TG.

Functional characterization of insect-specific RabX6 of Bombyx mori.

Rab proteins are low-molecular weight (20-25 kDa) monomeric GTPases that are central to the control and regulation of vesicle trafficking. RabX6 is an insect-specific Rab protein that has no close homolog in vertebrates. However, little information about insect-specific Rab proteins is available. In this study, RabX6 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX6 were produced in rabbits and rats for western immunoblotting and immunohistochemistry. Western blotting of testis tissues revealed two bands, at positions corresponding to a molecular weight of approximately 26 kDa. RabX6-like immunohistochemical reactivity (RabX6-ir) was identified at the face of the testis, not in the spermatogonia, and was specifically detected at a pair of tritocerebral cells of the male brain. Furthermore, RNA interference of RabX6 was shown to decrease testicular growth. These findings suggest that RabX6 is involved in the regulation of testicular growth and male-specific neuropeptide secretion in the brain of B. mori.

Metabolism of steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and ten mutants were co-expressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward steroids were examined. CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.48 and CYP2C9.52 had higher testosterone 6ß-hydroxylation than CYP2C9.1. CYP2C9.4 showed higher progesterone 6ß-hydroxylation activity than CYP2C9.1. CYP2C9.28 and CYP2C9.48 showed higher progesterone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.48 showed higher progesterone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.2, CYP2C9.3, CYP2C9.16 and CYP2C9.30 had higher estrone 16α-hydroxylation activity than CYP2C9.1. CYP2C9.3 had higher estrone 11α-hydroxylation activity than CYP2C9.1. CYP2C9.39 and CYP2C9.57 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.39 and CYP2C9.57 was not changed, but CYP2C9.2, CYP2C9.3, CYP2C9.4, CYP2C9.16, CYP2C9.28, CYP2C9.30, CYP2C9.48 and CYP2C9.52 showed different hydroxylation activities toward steroids compared with CYP2C9.1.

Modification of N-Terminal Amino Acids of Fungal Benzoate Hydroxylase (CYP53A15) for the Production of p-Hydroxybenzoate and Optimization of Bioproduction Conditions in Escherichia coli.

The aromatic compound p-hydroxybenzoate (PHBA) is an important material with multiple applications, including as a building block of liquid crystal polymers in chemical industries. The cytochrome P450 (CYP) enzymes are beneficial monooxygenases for the synthesis of chemicals, and CYP53A15 from fungus Cochliobolus lunatus is capable of executing the hydroxylation from benzoate to PHBA. Here, we constructed a system for the bioconversion of benzoate to PHBA in Escherichia coli cells coexpressing CYP53A15 and human NADPH-P450 oxidoreductase (CPR) genes as a redox partner. For suitable coexpression of CYP53A15 and CPR, we originally constructed five plasmids in which we replaced the N-terminal transmembrane region of CYP53A15 with a portion of the N-terminus of various mammalian P450s. PHBA productivity was the greatest when CYP53A15 expression was induced at 20°C in 2×YT medium in host E. coli strain ΔgcvR transformed with an N-terminal transmembrane region of rabbit CYP2C3. By optimizing each reaction condition (reaction temperature, substrate concentration, reaction time, and E. coli cell concentration), we achieved 90% whole-cell conversion of benzoate. Our data demonstrate that the described novel E. coli bioconversion system is a more efficient tool for PHBA production from benzoate than the previously described yeast system.

Molecular and functional characterization of the American cockroach, Periplaneta americana, Rab5: the first exopterygotan low molecular weight ovarian GTPase during oogenesis.

The small Rab GTPases are key regulators of membrane vesicle trafficking. Ovaries of Periplaneta americana (Linnaeus) (Blattodea: Blattidae) have small molecular weight GTP/ATP-binding proteins during early and late vitellogenic periods of oogenesis. However, the identification and characterization of the detected proteins have not been yet reported. Herein, we cloned a cDNA encoding Rab5 from the American cockroach, P. americana, ovaries (PamRab5). It comprises 796 bp, encoding a protein of 213 amino acid residues with a predicted molecular weight of 23.5 kDa. PamRab5 exists as a single-copy gene in the P. americana genome, as revealed by Southern blot analysis. An approximate 2.6 kb ovarian mRNA was transcribed especially at high levels in the previtellogenic ovaries, detected by Northern blot analysis. The muscle and head tissues also showed high levels of PamRab5 transcript. PamRab5 protein was localized, via immunofluorescence labeling, to germline-derived cells of the oocytes, very early during oocyte differentiation. Immunoblotting detected a ∼25 kDa signal as a membrane-associated form revealed after application of detergent in the extraction buffer, and 23 kDa as a cytosolic form consistent with the predicted molecular weight from amino acid sequence in different tissues including ovary, muscles and head. The PamRab5 during late vitellogenic periods is required to regulate the endocytotic machinery during oogenesis in this cockroach. This is the first report on Rab5 from a hemimetabolan, and presents an inaugural step in probing the molecular premises of insect oocyte endocytotic trafficking important for oogenesis and embryonic development.

Metabolism of 7-ethoxycoumarin, flavanone and steroids by cytochrome P450 2C9 variants.

CYP2C9 is a human microsomal cytochrome P450c (CYP). Much of the variation in CYP2C9 levels and activity can be attributed to polymorphisms of this gene. Wild-type CYP2C9 and mutants were coexpressed with NADPH-cytochrome P450 reductase in Escherichia coli. The hydroxylase activities toward 7-ethoxycoumarin, flavanone and steroids were examined. Six CYP2C9 variants showed Soret peaks (450 nm) typical of P450 in reduced CO-difference spectra. CYP2C9.38 had the highest 7-ethoxycoumarin de-ethylase activity. All the CYP2C9 variants showed lower flavanone 6-hydroxylation activities than CYP2C9.1 (the wild-type). CYP2C9.38 showed higher activities in testosterone 6ß-hydroxylation, progesterone 6ß-/16α-hydroxylation, estrone 11α-hydroxylation and estradiol 6α-hydroxylation than CYP2C9.1. CYP2C9.40 showed higher testosterone 17-oxidase activity than CYP2C9.1; CYP2C9.8 showed higher estrone 16α-hydroxylase activity and CYP2C9.12 showed higher estrone 11α-hydroxylase activity. CYP2C9.9 and CYP2C9.10 showed similar activities to CYP2C9.1. These results indicate that the substrate specificity of CYP2C9.9 and CYP2C9.10 was not changed, but CYP2C9.8, CYP2C9.12 and CYP2C9.40 showed different substrate specificity toward steroids compared with CYP2C9.1; and especially CYP2C9.38 displayed diverse substrate specificities towards 7-ethoxycoumarin and steroids.

Localization and functional analysis of the insect-specific RabX4 in the brain of Bombyx mori.

Rab proteins are small monomeric GTPases/GTP-binding proteins, which form the largest branch of the Ras superfamily. The different Rab GTPases are localized to the cytosolic face of specific intracellular membranes, where they function as regulators of distinct steps in membrane trafficking. RabX4 is an insect-specific Rab protein that has no close homolog in vertebrates. There is little information about insect-specific Rab proteins. RabX4 was expressed in Escherichia coli and subsequently purified. Antibodies against Bombyx mori RabX4 were produced in rabbits for western immunoblotting and immunohistochemistry. Western blotting of neural tissues revealed a single band, at approximately 26 kD. RabX4-like immunohistochemical reactivity was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum in the brain. Further immunohistochemical analysis revealed that RabX4 colocalized with Rab6 and bombyxin in the corpus allatum, a neuronal organ that secretes neuropeptides synthesized in the brain into the hemolymph. RabX4 expression in the frontal ganglion, part of the insect stomatogastric nervous system that is found in most insect orders, was restricted to two neurons on the outer region and did not colocalize with allatotropin or Rab6. Furthermore, RNA interference of RabX4 decreased bombyxin expression levels in the brain. These findings suggest that RabX4 is involved in the neurosecretion of a secretory organ in Bombyx mori.

Functional characterization of CYP52G3 from Aspergillus oryzae and its application for bioconversion and synthesis of hydroxyl flavanone and steroids.

Aspergillus oryzae is a fungus widely used in traditional Japanese fermentation industries. Cytochrome P450 (CYP) proteins are ubiquitously distributed in nature and display a broad range of enzymatic activities. A novel CYP52 (CYP52G3) gene was found in A. oryzae. In this study, we report the functional characterization of CYP52G3. The recombinant protein was expressed heterologously in Escherichia coli, and its membrane fraction isolated. CYP52G3 showed activities for 7-ethoxycoumarin and α-naphtoflavone. Furthermore, CYP52G3 hydroxylated flavanone at the 4' and 6 position and metabolized some hydroxyl-flavanones and steroids. Bioconversion experiments indicated that CYP52G3 could convert flavanone and testosterone in a synthetic medium. The conversion rates of flavanone and testosterone at 24 H were 50% and 70%, respectively. These results support that CYP52G3 could prove a useful enzyme for the efficient production of new compounds from flavonoids and steroids.

Rab proteins in the brain and corpus allatum of Bombyx mori.

In eukaryotic cells, Rab guanosine triphosphate-ases serve as key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab3, Rab6, and Rab27 control the regulatory secretory pathway of neuropeptides and neurotransmitters. The cDNAs of Rab3, Rab6, and Rab27 from B. mori were inserted into a plasmid, transformed into Escherichia coli, and then subsequently purified. We then produced antibodies against Rab3, Rab6, and Rab27 of Bombyx mori in rabbits and rats for use in western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue revealed a single band at approximately 26 kDa. Immunohistochemistry results revealed that Rab3, Rab6, and Rab27 expression was restricted to neurons in the pars intercerebralis and dorsolateral protocerebrum of the brain. Rab3 and Rab6 co-localized with bombyxin, an insect neuropeptide. However, there was no Rab that co-localized with prothoracicotropic hormone. The corpus allatum secretes neuropeptides synthesized in the brain into the hemolymph. Results showed that Rab3 and Rab6 co-localized with bombyxin in the corpus allatum. These findings suggest that Rab3 and Rab6 are involved in neurosecretion in B. mori. This study is the first to report a possible relationship between Rab and neurosecretion in the insect corpus allatum.

Functional characterization of CYP1A9 and CYP1C1 from Anguillus japonica.

We evaluated the metabolism of several herbicides and progesterone by two P450 proteins (CYP1A9 and CYP1C1) from Japanese eel (Anguilla japonica). Expression vectors harboring CYP1A9 and CYP1C1 sequences were introduced into Escherichia coli. E. coli membrane fractions were incubated with each substrate, and the metabolites were analyzed. CYP1A9 and CYP1C1 deethylated 7-ethoxycoumarin and phenacetin, and demethylated chlorotoluron, diuron, and linuron. CYP1C1 specifically hydroxlyated progesterone at the 6ß and 16α positions. Five amino acids of CYP1A9 related to substrate binding were selected for mutation analyses [CYP1A9(F128A), CYP1A9(F229A), CYP1A9(F263A), CYP1A9(V387A), and CYP1A9(I391A)]. Two variants, CYP1A9(F229A) and CYP1A9(F128A), changed the ratio of 16α hydroxyprogesterone to 6ß hydroxyprogesterone. Among all the variants, CYP1A9(F263A) showed the highest activity towards substrates used. CYP1A9(V387A) and CYP1A9(I391A) showed higher activities than that of CYP1A9 toward progesterone. The substrate specificity of CYP1A9 may be altered by replacing an amino acid related to substrate binding.

Point mutation of cytochrome P450 2A6 (a polymorphic variant CYP2A6.25) confers new substrate specificity towards flavonoids.

CYP2A6 is a major hepatic member of the cytochrome P450 family in humans. Much variation in CYP2A6 levels and activity can be attributed to genetic polymorphisms of this gene. CYP2A6*25 comprises an amino acid substitution, F118L. To clarify the effect of the leucine substitution at position 118 in CYP2A6.25, this variant, wild type CYP2A6 and three additional variants consisting of artificial mutations at the substrate binding site (position 481) suggested by earlier reports using random mutagenesis studies [CYP2A6.1, CYP2A6.25, CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G)], were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activity of these variants toward 7-ethoxycoumarin, coumarin, flavone, α-naphthoflavone, flavanone and hydroxyflavanone were examined. All the mutants had lower activities for coumarin 7-hydroxylation than the wild type. All the mutants showed higher activities for flavone and α-naphthoflavone compared with CYP2A6.1. CYP2A6.1 had the highest flavanone 2'-hydroxylase activity, whereas CYP2A6.25 had the highest 6- and 4'-hydroxylase activities. CYP2A6.1(F118A), CYP2A6.1(A481G) and CYP2A6.25(A481G) had higher flavanone 3'-hydroxylase activities than CYP2A6.1 and CYP2A6.25. Furthermore, 4'-hydroxyflavanone was metabolized by CYP2A6.25. These results indicate that the CYP2A6.25 mutation confers new substrate specificity towards flavonoids.

Characterization of Rab-interacting lysosomal protein in the brain of Bombyx mori.

Rab guanosine triphosphatases in eukaryotic cells are key regulators of membrane-trafficking events, such as exocytosis and endocytosis. Rab7 regulates traffic from early to late endosomes and from late endosomes to vacuoles/lysosomes. The Rab7-interacting lysosomal protein (RILP) was extracted from the silkworm, Bombyx mori (B. mori), and expressed in Escherichia coli (E. coli), followed by its purification. The glutathione sulfotransferase pull-down assay revealed that Rab7 of B. mori interacted with RILP of B. mori. We then produced antibodies against RILP of B. mori in rabbits for their use in Western immunoblotting and immunohistochemistry. Western immunoblotting of brain tissue for RILP revealed a single band, at approximately 50 kD. RILP-like immunohistochemical reactivity (RILP-ir) was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Furthermore, RILP-ir was colocalized with the eclosion hormone-ir and bombyxin-ir. However, RILP-ir was not colocalized with prothoracicotropic hormone-ir. These results were similar to those of Rab7 from our previous study. These findings suggest that RILP and Rab7 are involved in the neurosecretion in a restricted subtype of neurons in B. mori. Thus, our study is the first to report of a possible relationship between an insect Rab effector and neurosecretion.

A cis-element responsible for cGMP in the promoter of the soybean chalcone synthase gene.

The cyclic nucleotides cGMP and cAMP have been reported to play key roles in the regulation of plant processes and responses. We have previously reported that several genes encoding flavonoid biosynthetic enzymes, including chalcone synthase (CHS) in soybean (Glycine max L.), were induced by cGMP but not cAMP. The soybean genome contains nine CHS gene copies (GmCHS1-9). We investigated the responsiveness of several GmCHS genes to cGMP, cAMP, NO, and white light. Quantitative RT-PCR analysis showed that the transcript levels of GmCHS7 and GmCHS8 were increased by 3.6- and 3.8-fold, respectively, with cGMP whereas the transcript levels of GmCHS2 remained constant. Although cAMP had no effect on the transcript levels of the three genes, NO had an activation effect on all three. White light activated the three genes in a transient manner, with GmCHS2, GmCHS7, and GmCHS8 transcript levels increasing 3-fold after 3 h and decreasing to basal levels after 9 h. The GmCHS8 promoter contains several important cis-elements, including the G-box and H-box forming the Unit-I-like sequence and the MYB binding sequence, a target of the GmMYB176 transcription factor regulating the expression of GmCHS8. A transient gene expression assay revealed the activation of the Unit-I-like sequence, but not of the MYB binding sequence, by cGMP. The combination of G-box and H-box was necessary for cGMP responsiveness. Taken together, these results suggest that the Unit-I-like sequence in the promoters of GmCHS7 and GmCHS8 is a cGMP responsive cis-element in these genes and that NO exerts its effect via cis-elements other than the Unit-I-like sequence.

Metabolism of 7-ethoxycoumarin, safrole, flavanone and hydroxyflavanone by cytochrome P450 2A6 variants.

CYP 2A6 is a human enzyme that metabolizes many xenobiotics including coumarin, indole, nicotine and carcinogenic nitrosamines. The gene for CYP2A6 is polymorphic. There are few data available to clarify the relationship between P450 genetic variants and the metabolism of materials in food. The CYP 2A6 wild-type protein and 13 mutants (CYP2A6.1, CYP2A6.2, CYP2A6.5, CYP2A6.6, CYP2A6.7, CYP2A6.8, CYP2A6.11, CYP2A6.15, CYP2A6.16, CYP2A6.17, CYP2A6.18, CYP2A6.21, CYP2A6.23 and CYP2A6.25) were co-expressed with NADPH-cytochrome P450 reductase in E. coli. The hydroxylase activities toward 7-ethoxycoumarin, coumarin, safrole, flavanone and hydroxyflavanone were examined. Ten types of CYP2A6 variants except for CYP2A6.2, CYP2A6.5 and CYP2A6.6 showed Soret peaks (450 nm) typical of P450 in the reduced CO-difference spectra and had 7-ethoxycoumarin O-deethylase activities. CYP2A6.15 and CYP2A6.18 showed higher activities for safrole 1'-hydroxylation than CYP2A6.1. CYP2A6.25 and CYP2A6.7 had lower safrole 1'-hydroxylase activities. CYP2A6.7 had lower flavanone 6- and 2'-hydroxylase activities, whereas CYP2A6.25 had higher 6-hydroxylase activity and lower 2'-hydroxylase activity. Hydroxyflavanone was metabolized by CYP2A6.25, but was not metabolized by wild-type CYP2A6.1. These results indicate that CYP2A6.25 possessed new substrate specificity toward flavonoids.

Relationship between the expression of Rab family GTPases and neuropeptide hormones in the brain of Bombyx mori.

Rab proteins are small GTPases that play essential roles in vesicle transport. In this study, we examined the expression of Rab proteins and neuropeptide hormones in the brain of the silkworm, Bombyx mori. We produced antibodies against B. mori Rab1 and Rab14 in rabbits. Immunoblotting of samples of brain tissue from B. mori revealed a single band for each antibody. Rab1 and Rab14 immunohistochemical labeling in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Rab1, Rab7 and Rab14 co-localized with bombyxin. Rab1 and Rab7 co-localized with eclosion hormone. Rab1 co-localized with prothoracicotropic hormone. These results suggest that Rab1, Rab7 and Rab14 may be involved in neuropeptide transport in the brain of B. mori. This is the first report on the specificity of Rab proteins for the secretion of different neuropeptides in insects.

Cytochrome P450 (CYP) in fish.

Cytochrome P450 (CYP) enzymes are members of the hemoprotein superfamily, and are involved in the mono-oxygenation reactions of a wide range of endogenous and exogenous compounds in mammals and plants. Characterization of CYP genes in fish has been carried out intensively over the last 20 years. In Japanese pufferfish (Takifugu rubripes), 54 genes encoding P450s have been identified. Across all species of fish, 137 genes encoding P450s have been identified. These genes are classified into 18 CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP5, CYP7, CYP8, CYP11, CYP17, CYP19, CYP20, CYP21, CYP24, CYP26, CYP27, CYP39, CYP46 and CYP51.We pinpointed eight CYP families: namely, CYP1, CYP2, CYP3, CYP4, CYP11, CYP17, CYP19 and CYP26 in this review because these CYP families are studied in detail. Studies of fish P450s have provided insights into the regulation of P450 genes by environmental stresses including water pollution. In this review, we present an overview of the CYP families in fish.

Small GTPases of the Rab family in the brain of Bombyx mori.

Small GTPases of the Rab family are key regulators of membrane trafficking. We produced antibodies against the Rab7 protein of Bombyx mori (BRab7) in rabbits, and against the Rab11 protein of B. mori (BRab11) in mice. The antibodies recognized BRab7 and BRab11 proteins, but did not recognize other Rab proteins. Immunoblotting of samples from brain tissue of B. mori revealed a single band for each antibody. Rab11 was expressed in most tissues, whereas Rab7 was expressed in the brain, ovary, and testis. Immunohistochemical reactivity of Rab7 and Rab11 in the brain of B. mori was restricted to neurons of the pars intercerebralis and dorsolateral protocerebrum. Double-labeling experiments demonstrated that immunohistochemical reactivity of Rab7 co-localized with that of Rab11 and partially with that of Rab8. Immunohistochemical reactivity of Rab11 and Rab8 co-localized with that of PERIOD, one of the proteins associated with circadian rhythm. These findings suggest that Rab7, Rab8, and Rab11 are involved in protein transport in the neurons of the brain of B. mori and might play a role in the control of circadian rhythm.