太田 明

Tetrahydrobiopterin is an essential cofactor for nitric oxide synthase (NOS). This study was undertaken to examine the effects of intraperitoneally injected lipopolysaccharide on tetrahydrobiopterin biosynthesis in murine white and brown adipose tissues. Tetrahydrobiopterin content, catalytic activity and mRNA expression level of GTP cyclohydrolase I (GCH), rate-controlling enzyme in de novo biosynthesis of tetrahydrobiopterin, in both adipose tissues were up-regulated by 500-gg lipopolysaccharide at 6 h after the injection. On the contrary, treatment of 3T3-L1 adipocytes with lipopolysaccharide alone did not affect GCH mRNA expression level, whereas the combination of lipopolysaccharide, tumor necrosis factor (TNF)-alpha, and interferon gamma induced the increase in expression levels of GCH mRNA and CD14 mRNA. Collectively, our results showed that tetrahydrobiopterin biosynthesis can be augmented by increased GCH activity caused by a synergistic effect of lipopolysaccharide and cytokines in white and brown adipose tissues. These observations support the view that tetrahydrobiopterin biosynthesis in the adipose tissues is a target of inflammatory events triggered by peripheral LPS injection. (C) 2004 Elsevier B.V All rights reserved.

Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified a functional Egr1 motif overlapping with Sp1 site in the rat TH promoter. Its response to Egr1 also required the presence of an AP1/Ebox motif. Here, we further examined the cross-talk between these sites. Insertion of 10- or 20-bp between the Sp1/Egr1 and AP1/Ebox elements, reduced the ability of Egr1 to upregulate luciferase reporter activity controlled by the proximal 272 nucleotides of the rat TH promoter in PC12 cells. Electrophoretic mobility shift assays with nuclear extracts from TPA treated cells were used to identify the composition of the factors which bound the AP1/Ebox motif and whether there is competition with factors which bind the Sp1/Egr1 motif. The complexes formed with labeled AP1/E box oligonucleotide were reduced or supershifted with antisera to Fos family, c-Fos, Fra-2, and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Fra-2 was a major component of the complex after 2-4 h TPA. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression plasmids elicited lower induction of luciferase activity than observed with Egr1 alone. Our results suggest that although it does not compete for binding to the promoter, Egr1 can modulate the regulation of TH transcription by AP1 factors. © 2003 Elsevier Science B.V. All rights reserved.

Several treatments which regulate tyrosine hydroxylase (TH) transcription, such as stress in vivo, or 12-O-tetradecanoylphorbol-13-acetate (TPA) in cell culture, induce both Egr1 and AP1 factors. Previously, we identified a functional Egr1 motif overlapping with Sp1 site in the rat TH promoter. Its response to Egr1 also required the presence of an AP1/Ebox motif. Here, we further examined the cross-talk between these sites. Insertion of 10- or 20-bp between the Sp1/Egr1 and AP1/Ebox elements, reduced the ability of Egr1 to upregulate luciferase reporter activity controlled by the proximal 272 nucleotides of the rat TH promoter in PC12 cells. Electrophoretic mobility shift assays with nuclear extracts from TPA treated cells were used to identify the composition of the factors which bound the AP1/Ebox motif and whether there is competition with factors which bind the Sp1/Egr1 motif. The complexes formed with labeled AP1/E box oligonucleotide were reduced or supershifted with antisera. to Fos family, c-Fos, Fra-2, and Jun D. Excess Sp1/Egr1 oligonucleotide or anti Egr1 antisera did not compete. Fra-2 was a major component of the complex after 2-4 h TPA. Transfection of PC12 cells with Fra-2 induced reporter activity requiring the AP1, but not the Egr1 motif. However, when cotransfected with Fra-2, Egr1 expression plasmids elicited lower induction of luciferase activity than observed with Egr1 alone. Our results suggest that although it does not compete for binding to the promoter, Egr1 can modulate the regulation of TH transcription by AP1 factors. (C) 2003 Elsevier Science B.V. All rights reserved.

Norepinephrine turnover rate in the murine locus coeruleus (LC) is known to be enhanced by the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Approximately 40% of LC neurons are also known to project to the olfactory bulb (OB) and the anterior olfactory nucleus (AON). Therefore, we investigated whether an i.p. injection of 500mug LPS could modulate the catecholamine biosynthesis in these sites in 8-week-old C3H/HeN male mice. Unexpectedly, the content of norepinephrine was not elevated in both sites during 6-h-observation after LPS injection. The contents of dopamine and its metabolites in the AON were highly increased at 4h after LPS injection, whereas those in the OB were not elevated during 6-h-observation. Although the AON has been considered not to belong to the dopaminergic neuron system, our report is the first to show an elevated dopamine content in the AON under a stressful condition such as endotoxemia.

Norepinephrine turnover rate in the murine locus coeruleus (LC) is known to be enhanced by the intraperitoneal (i.p.) injection of lipopolysaccharide (LPS). Approximately 40% of LC neurons are also known to project to the olfactory bulb (OB) and the anterior olfactory nucleus (AON). Therefore, we investigated whether an i.p. injection of 500mug LPS could modulate the catecholamine biosynthesis in these sites in 8-week-old C3H/HeN male mice. Unexpectedly, the content of norepinephrine was not elevated in both sites during 6-h-observation after LPS injection. The contents of dopamine and its metabolites in the AON were highly increased at 4h after LPS injection, whereas those in the OB were not elevated during 6-h-observation. Although the AON has been considered not to belong to the dopaminergic neuron system, our report is the first to show an elevated dopamine content in the AON under a stressful condition such as endotoxemia.

1. Risperidone is an atypical antipsychotic drug that possesses 5-hydroxytryptamine 5-HT2 receptor antagonism combined with milder dopamine D2 receptor antagonism. 2. Excessive bodyweight gain is one of the side-effects of antipsychotics. Risperidone treatment causes a greater increase in the body mass of patients than treatment with conventional antipsychotics, such as haloperidol. Therefore, the present study was undertaken in order to address the aetiology of the risperidone-induced bodyweight change in rats by examining the expression of leptin, an appetite-regulating hormone produced in white adipose tissue (WAT), and uncoupling protein (UCP)-1, a substance promoting energy expenditure in the brown adipose tissues (BAT). 3. Eight-week-old male rats were injected subcutaneously with risperidone (0.005, 0.05 or 0.5 mg/kg) twice daily for 21 days. Both bodyweight and food intake were monitored daily. On day 21, rats were decapitated and their serum leptin and prolactin concentrations were measured. Expression levels of leptin , Ucp1 and beta(3)-adrenoceptor (beta(3)-AR ) genes in WAT and BAT were quantified using real-time polymerase chain reaction amplification. 4. Injection of 0.005 mg/kg risperidone into rats increased food intake and the rate of bodyweight gain, as well as the augmentation of leptin gene expression in WAT. Injection of 0.05 mg/kg risperidone increased food intake and leptin gene expression in WAT, but the rate of bodyweight gain was not affected. Injection of 0.5 mg/kg risperidone caused a reduction in bodyweight gain, as well as enhanced Ucp1 gene expression in BAT and serum prolactin concentrations. The serum leptin concentration and beta(3)-AR gene expression in WAT and BAT were not affected by injection of 0.5 mg/kg risperidone. 5. Although the changes in food intake observed in risperidone-injected rats were rationalized neither by serum leptin nor prolactin concentrations, the reduction in the rate of bodyweight gain following injection of 0.5 mg/kg can be explained, in part, by increased energy expenditure, as revealed by the remarkable increase in the UCP-1 mRNA expression level in BAT. The role of leptin in risperidone-induced alterations in bodyweight gain remain to be clarified.

1. Risperidone is an atypical antipsychotic drug that possesses 5-hydroxytryptamine 5-HT2 receptor antagonism combined with milder dopamine D2 receptor antagonism. 2. Excessive bodyweight gain is one of the side-effects of antipsychotics. Risperidone treatment causes a greater increase in the body mass of patients than treatment with conventional antipsychotics, such as haloperidol. Therefore, the present study was undertaken in order to address the aetiology of the risperidone-induced bodyweight change in rats by examining the expression of leptin, an appetite-regulating hormone produced in white adipose tissue (WAT), and uncoupling protein (UCP)-1, a substance promoting energy expenditure in the brown adipose tissues (BAT). 3. Eight-week-old male rats were injected subcutaneously with risperidone (0.005, 0.05 or 0.5 mg/kg) twice daily for 21 days. Both bodyweight and food intake were monitored daily. On day 21, rats were decapitated and their serum leptin and prolactin concentrations were measured. Expression levels of leptin , Ucp1 and beta(3)-adrenoceptor (beta(3)-AR ) genes in WAT and BAT were quantified using real-time polymerase chain reaction amplification. 4. Injection of 0.005 mg/kg risperidone into rats increased food intake and the rate of bodyweight gain, as well as the augmentation of leptin gene expression in WAT. Injection of 0.05 mg/kg risperidone increased food intake and leptin gene expression in WAT, but the rate of bodyweight gain was not affected. Injection of 0.5 mg/kg risperidone caused a reduction in bodyweight gain, as well as enhanced Ucp1 gene expression in BAT and serum prolactin concentrations. The serum leptin concentration and beta(3)-AR gene expression in WAT and BAT were not affected by injection of 0.5 mg/kg risperidone. 5. Although the changes in food intake observed in risperidone-injected rats were rationalized neither by serum leptin nor prolactin concentrations, the reduction in the rate of bodyweight gain following injection of 0.5 mg/kg can be explained, in part, by increased energy expenditure, as revealed by the remarkable increase in the UCP-1 mRNA expression level in BAT. The role of leptin in risperidone-induced alterations in bodyweight gain remain to be clarified.

The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38 (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38 (iii) S40D, Ser40 replaced by Asp40 and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE &gt S40D &gt S40A = RR-GG &gt wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.

The sequence Arg37-Arg38 of tyrosine hydroxylase (TH) is known to play a significant role in the feedback inhibition by the end product DA. To clarify how deeply the sequence Arg37-Arg38 and the phosphorylated Ser40 of human TH type 1 (hTH1) are involved in the regulation of this feedback inhibition in mammalian cells, we generated the following mutants: (i) RR-GG, Arg37-Arg38 replaced by Gly37-Gly38 (ii) RR-EE, Arg37-Arg38 replaced by Glu37-Glu38 (iii) S40D, Ser40 replaced by Asp40 and (iv) S40A, Ser40 replaced by Ala40. In a cell-free system, the level of the DA inhibition of the RR-EE mutant enzyme was to the same or smaller degree than that of the phosphorylation-mimicking S40D. Next, AtT-20 neuroendocrine cells were transfected with wild-type and mutated TH genes because these cells were earlier shown to be capable of fully converting L-3,4-dihydroxyphenylalanine into DA, whereby the catalytic activity of TH would be expected to be inhibited by the end product DA accumulating in the cells. The level of DA accumulation in AtT-20 cells expressing the TH gene was in the order: RR-EE &gt S40D &gt S40A = RR-GG &gt wild-type, which was in accordance with the observations for the cell-free system. These results suggest that the sequence Arg37-Arg38 of hTH1 is a more potent determinant of the efficient production of DA in mammalian cells than is the phosphorylated Ser40-hTH1.

Tetrahydrobiopterin in the murine locus coeruleus was measured as its fully oxidized form, biopterin, using a HPLC coupled to a fluorescence detector, because tetrahydrobiopterin itself cannot be detected by such means. The differential oxidization method distinguished tetrahydrobiopterin-derived biopterin and dihydrobiopterin-derived biopterin. The protocol reported here is a rapid and sensitive method that facilitates the measurement of tissue and/or cellular tetrahydrobiopterin. Using this assay protocol, we were able to detect and quantify variations in the tetrahydrobiopterin content in the murine locus coeruleus. (C) 2001 Elsevier Science B V All rights reserved.

Tetrahydrobiopterin in the murine locus coeruleus was measured as its fully oxidized form, biopterin, using a HPLC coupled to a fluorescence detector, because tetrahydrobiopterin itself cannot be detected by such means. The differential oxidization method distinguished tetrahydrobiopterin-derived biopterin and dihydrobiopterin-derived biopterin. The protocol reported here is a rapid and sensitive method that facilitates the measurement of tissue and/or cellular tetrahydrobiopterin. Using this assay protocol, we were able to detect and quantify variations in the tetrahydrobiopterin content in the murine locus coeruleus. (C) 2001 Elsevier Science B V All rights reserved.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in dopamine (DA) biosynthesis.(1,2) Exon 3 of the human TH gene encodes the sequence from Ser31 to Glu104 of type 1 enzyme,(3,4) which contains the critical parts for regulation of the catalytic activity. The amino acid residues Gly36-Arg37-Arg38 were identified as a key sequence for DA to exert its inhibitory effect on catalytic activity.(5-7) Therefore, we screened the nucleotide sequences of exon 3 from 201 Japanese patients with schizophrenia to explain the elevation in the synaptic or presynaptic DA concentrations in the schizophrenic brain,(8-11) based on the hypothesis that any mutation changing the amino acid sequence Gly36-Arg37-Arg38 would result in the elevation of DA synthesis, due to a reduced inhibitory effect of DA on the catalytic activity.(5-7) However, no mutated sequences of exon 3 and both exon-intron boundaries were detected in any of the patients examined. Polymorphisms generating Val81 and Met81 were compared of the distributions of genotype and allele between the patients and 175 Japanese healthy controls, which did not suggest an association between the polymorphism and schizophrenia. These results indicate that exon 3 of the human TH gene lacks association with schizophrenia in Japanese patients.

Among the enzymes involved in the system for catecholamine biosynthesis, GTP cyclohydrolase I (GCH) contributes to the system as the first and rate-limiting enzyme for the de novo biosynthesis of tetrahydrobiopterin (BH4), which is the cofactor for tyrosine hydroxylase (TH). Therefore, we investigated whether the endotoxemia caused by an intraperitoneal (i.p.) injection of lipopolysaccharide (LPS) can modulate BH4 production in the norepinephrine nuclei, i.e. the locus ceruleus (LC A6) and central caudal pons (A5), in C3H/HeN mice and whether such a change in BH4, if any, can result in the modification of norepinephrine production in these nuclei. After a 5-μg i.p. injection of LPS, the protein expression of GCH and TH in both nuclei was examined by immunohistochemistry. The staining intensity of GCH-positive cells increased at 6 h, whereas no significant change in the staining intensity of TH-positive cells was detected. Next, we measured the contents of BH4, norepinephrine, and its metabolites 4-hydroxy-3-methoxyphenylglycol (MHPG) and dl-4-hydroxy-3-methoxymandelic acid (VMA) in these nuclei after LPS i.p. injection. The BH4 content increased to a statistically significant level at 2 and 4 h after the injection. The contents of MHPG and VMA also showed a time-course similar to that of BH4. These data can be rationalized to indicate that an increased supply of BH4 in the LC increased TH activity and resulted in an increase in norepinephrine production rate at the site. This is the first report that sheds light on BH4 as a molecule that intervenes during endotoxemia to increase norepinephrine production rate in the LC. © 2001 Elsevier Science B.V.

Tyrosine hydroxylase (TH), which converts L-tyrosine to L-3,4-dihydroxyphenylalanine, is a rate-limiting enzyme in the biosynthesis of catecholamines; its activity is regulated by the feedback inhibition of the catecholamine products including dopamine. To rationalize the significant role of the N-terminal sequence Arg(37)-Arg(38) of human TH type 1 (hTH1) in determining the efficiency of feedback inhibition, we produced mutants of which the positively charged Arg(37)-Arg(38) Site was replaced by electrically neutral Gly and/or negatively charged Glu and analyzed the degree of inhibition of these mutant enzymes by dopamine. The replacement of Arg by Gly reduced the inhibitory effect of dopamine on the catalytic activity measured in the basic pH range and the replacement of Arg by Glu was enough to abolish the inhibitory effect, although these mutations brought no significant changes to the circular dichroism spectrum, The prediction of the secondary structure of N-terminal residues 1-60 by computer software specified the location of the Arg(37)-Arg(38) sequence in the turn intervening between the two alpha-helices (residues 16-29 and residues 41-59), These results suggest that the positive charge of the amino acid residues at positions 37 and 38 is one of the main factors that maintains the characteristic of the turn and is responsible for the enzyme inhibition by dopamine. (C) 2000 Federation of European Biochemical Societies.

Wild-type and N-terminal 35-, 38-, and 44-amino acid-deleted mutants of human tyrosine hydroxylase type 1 (hTH1) fused to maltose-binding protein via the target sequence for a restriction protease were expressed in Escherichia coli and purified. The fused protein was treated with the restriction protease factor Xa or enterokinase to isolate hTH1 from the fused form. The treatment of fused wild-type and 35-amino acid-deleted mutant with factor Xa and enterokinase caused non-specific cleavages in the vicinity of the phosphorylation sites, Ser(19) and Ser(40), due to the flexible conformation of the N-terminus of hTH1.

To determine if the antagonism of serotonin 5HT(2A) receptors and dopamine D-2 receptors by risperidone alters the mRNAs encoding the transporters and enzymes that are involved in re-uptake of serotonin and dopamine and in the degradation and refilling of secretory vesicles, we employed semi-quantitative HT-PCR on rats given risperidone. The rats were given twice daily a subcutaneous injection of either 0.3% tartaric acid (vehicle) or risperidone (0.5 mg/kg) for 21 days and sacrificed one hour after the last injection. Risperidone enhanced the level of monoamine oxidase type B (MAO-B) mRNA in the brainstem (P<0.05) and that of vesicular monoamine transporter-2 (VMAT2) mRNA in the hypothalamus (P<0.05) when these levels were compared with the control ones, whereas there were no significant group differences in other mRNAs examined. The antagonism of 5HT(2A) and D-2 receptors may thus affect the monoamine levels by regulating mRNAs encoding MAO-B and VMAT2.

We investigated for the first time the effect of lipopolysaccharide and the signal transduction pathway on the biosynthesis of tetrahydrobiopterin [(6R-L-erythro-1',2'-dihydroxypropyl)-2-amino-4-hydroxy-5,6,7, 8-tetrahydropteridine], the cofactor for the enzymatic hydroxylation of the aromatic amino acids, in the murine neuroblastoma cell line N1E-115, which synthesizes tetrahydrobiopterin constitutively. Activation of N1E-115 cells with 1 mu g/ml lipopolysaccharide resulted in statistically significant increases in both intracellular tetrahydrobiopterin contents and the activity (V-max) of GTP cyclohydrolase I, a rate-limiting enzyme in tetrahydrobiopterin de novo biosynthesis. Following simultaneous addition of the inhibitors of protein tyrosine kinases and GTP-binding proteins into serum-free culture media with lipopolysaccharide, we analyzed the transduction pathway of lipopolysaccharide signal toward the tetrahydrobiopterin biosynthetic system in N1E-115 cells. Our data indicate the following conclusions: (a) Protein tyrosine kinase systems are involved in mediating lipopolysaccharide signal to tetrahydrobiopterin production, and (b) there may be a cross-talk between GTP-binding protein and the protein tyrosine kinase system in mediating lipopolysaccharide signal. These observations suggest that a neuronal cell such as N1E-115, which barely expresses CD14 on its cell surface, responds to lipopolysaccharide like macrophages and monocytes in the absence of soluble CD14.

We examined the release of tetrahydrobiopterin ((6R)-L-erythro-dihydroxypropyl-2-amino-4-hydroxy-5,6,7,8-tetrahydropteridine; BH4) and nitric oxide induced by lipopolysaccharide from mouse neuroblastoma N1E-115 cells by measuring BH4 and nitric oxide derivatives, nitrites and nitrates, harbored in the conditioned media. The stimulation of the cells by 1 mu g/ml of lipopolysaccharide for 24 h induced 2-fold increase in the release of BH4 from the cells, but did not induce the nitric oxide release from the cells. Although such increase in BH4 release from the cells was blocked by the inhibitors of nuclear factor-kappa B or protein tyrosine kinases, the release of nitric oxide was not affected by such inhibitors. Our results may suggest that the inductions of BH4 and nitric oxide in this neuroblastoma cell line are processed in different ways and that this cell line is also different from the immune cells in the central nervous system such as microglia in this respect.

The N-terminal 52-, 70-, and 157-amino acids-deleted mutants and wild-type tyrosine hydroxylases were expressed in Escherichia coli and utilized to investigate the roles of the N-terminus in the catecholamine inhibition on enzyme activity. Their lysate's supernatants were used as enzyme samples. Three catecholamines, namely dopamine, norepinephrine, and epinephrine, affected both wild-type and mutant enzymes after preincubation in the mode of mixed inhibition, and the most marked alteration among the kinetic parameters produced by the deletion was the increase in the inhibition constants. The deletions also abolished the catecholamine-induced shift of the pH profile of the enzyme activity toward a more acidic pH optimum. All three mutants responded to catecholamines almost in the same way. These results suggest that the three catecholamine end products exert their inhibition on tyrosine hydroxylase to the same extent and that the N-terminal 52 amino acid residues contain the key sequence in mediating the inhibitory action.

Agonist-induced regulation of adrenergic receptors (ARs) has an important role in controlling physiological functions in response to changes in catecholamine stimulation. We previously generated transgenic mice expressing phenylethanolamine N-methyltransferase (PNMT) under the control of a human dopamine beta-hydroxylase gene promoter to switch catecholamine specificity from the norepinephrine phenotype to the epinephrine phenotype. In the present study, we first examined changes in catecholamine metabolism in peripheral tissues innervated by sympathetic neurons of the transgenic mice, in the transgenic target tissues, a high-level expression of PNMT led to a dramatic increase in the epinephrine levels, whereas the norepinephrine levels were decreased to 48.6-87.9% of the nontransgenic control levels. Analysis of plasma catecholamines in adrenalectomized mice showed large amounts of epinephrine derived from sympathetic neurons in the transgenic mice. Subsequently, we performed radioligand binding assays with (-)-[I-125]iodocyanopindolol to determine changes in binding sites of beta-AR subtypes. In transgenic mice, the number of beta 2-AR binding sites was 56.4-74.9% of their nontransgenic values in the lung, spleen, submaxillary gland, and kidney, whereas the beta 1-AR binding sites were regulated in a different fashion among these tissues. Moreover, northern blot analysis of total RNA from the lung tissues showed that down-regulation of beta 2 binding sites was accompanied by a significant decrease in steady-state levels of the receptor mRNA. These results strongly suggest that alteration of catecholamine specificity in the transgenic sympathetic neurons leads to regulated expression of the beta-AR subtypes in their target tissues.

A series of N-terminal deletion mutants of human tyrosine hydroxylase type 1 has been expressed in Escherichia coli to characterize the N-terminal regulatory domain. The mutants lacking the first 74 to 117 amino acids led to the precipitation into aggregates probably due to improper folding. All of the deletion mutants are active in the lysate supernatant and/or the pellet. The Michaelis constants for pterins are similar among all the mutants examined and the wild-type. (C) 1995 Academic Press, Inc.

We carried out the cloning of a mouse cDNA encoding a sepiapterin reductase which is involved in the final step of tetrahydrobiopterin biosynthesis as a first step toward gene-targeting technique in mice. The sequence contained 1245 nucleotides consisting of an open reading frame of 783 nucleotides encoding a protein of 261 amino acid residues whose molecular weight was 27851, a 5'-untranslated region of 21 nucleotides and a 3'-untranslated region of 441 nucleotides containing poly(A) tail. The amino acid sequence of mouse sepiapterin reductase revealed the identity of 88% with rat and 74% with human sequence.

We investigated the effect of subcutaneous injection of nicotine on in vitro tyrosine hydroxylase (TH) activity in adrenal gland and brain of the transgenic mice carrying an 11-kb fragment containing the entire human TH gene. Injection of 5 mg nicotine/kg (as free base) for 3 days caused a statistically significant increase in in vitro TH activity in the adrenal gland, whereas brain TH activity was not affected at all. The adrenal gland of non-transgenic C57BL/6J mice treated in the same way as for transgenic mice tended to enhance TH activity, although not to a significant level. This observation might indicate the possibility that the machinery used by nicotine in regulating the properties or expression of TH in the adrenal gland should be similar between transgenic and non-transgenic mice.