Masashi Sekimoto

IMMOBILIZATION STRESS REDUCED THE EXPRESSION OF NEUROTROPHINS AND THEIR RECEPTORS IN THE RAT BRAIN

Exposure to stressful events and elevated level of stress hormones are associated with impaired spatial memory and neuronal damage in the hippocampus. These neurons are considered to be maintained by neurotrophins such as nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) and trk family of neurotrophin receptors. Male Wistar rats (6 weeks old) were exposed to immobilization stress for 8 h and their brains were processed for in situ hybridization histochemistry. Exposure to long-lasting immobilization stress reduced mRNA levels for neurotrophins and their high affinity receptors in the brain, especially in the hippocampus. Our results provide, some new information that may be relevant to the pathogenesis of stress-induced disturbances of memory and learning.

5-HT2A receptor subtype in the peripheral branch of sensory fibers is involved in the potentiation of inflammatory pain in rats

&NA; One of the major serotonin (5‐HT) receptor subtypes expressed in the rat dorsal root ganglion (DRG) neurons is the 5‐HT2A receptor. We have previously shown that 5‐HT2A receptors in the peripheral sensory terminals are responsible for 5‐HT‐induced pain and hyperalgesia. In the present study, we characterized neurons expressing 5‐HT2A receptors in the rat DRG neurons by means of in situ hybridization, immunohistochemistry, reverse transcription‐polymerase chain reaction (RT‐PCR) and behavioral tests. In situ hybridization on consecutive sections revealed that 5‐HT2A receptor mRNA is colocalized with calcitonin‐gene related peptide (CGRP) mRNA (100/104; 96.2%) but not with c‐Ret mRNA (1/115; 0.9%). Signals for 5‐HT2A receptor mRNA were found in 9.4±2.2% of normal DRG (L5) neurons, most of which were small to medium in size. Four days of complete Freunds adjuvant‐induced inflammation of the hindpaw doubled the incidence of 5‐HT2A receptor mRNA‐expressing neurons to 19.3±2.8%. The level of 5‐HT2A receptor mRNA in DRGs of normal and various pathological conditions was then determined by RT‐PCR. The level was up‐regulated by peripheral inflammation, but not by axotomy or chronic constriction of the peripheral nerve. Systemic administration of 5‐HT2A receptor antagonist (Sarpogrelate HCI) produced analgesic effects on thermal hyperalgesia caused by peripheral inflammation, but failed to attenuate thermal hyperalgesia in chronic constriction injury model. These findings suggest that 5‐HT2A receptors are mainly expressed in CGRP‐synthesizing small DRG neurons and may be involved in the potentiation of inflammatory pain in the periphery.

Gene Expression of Receptors for IL-6, LIF, and CNTF in Regenerating Skeletal Muscles

The biological actions of interleukin-6 (IL-6), leukemia inhibitory factor (LIF), and ciliary neurotrophic factor (CNTF) are mediated via respective functional receptor complexes consisting of a common signal-transducing component, gp130, and other specific receptor components, IL-6 receptor α (IL-6R), LIF receptor β (LIFR), and CNTF receptor α (CNTFR). IL-6, LIF, and CNTF are implicated in skeletal muscle regeneration. However, the cell populations that express these receptor components in regenerating muscles are unknown. Using in situ hybridization histochemistry, we examined spatiotemporal expression patterns of gp130, IL-6R, LIFR, and CNTFR mRNAs in regenerating muscles after muscle contusion. At the early stages of regeneration (from 3 hr to Day 2 post contusion), significant signals for gp130 and LIFR mRNAs were detected in myonuclei and/or nuclei of muscle precursor cells (mpcs) and in mononuclear cells located in extracellular spaces between myofibers after muscle contusion, but IL-6R mRNA was expressed only in mononuclear cells. At Day 7 post contusion, signals for gp130, LIFR, and IL-6R mRNAs were not detected in newly formed myotubes, whereas the CNTFR mRNA level was upregulated in myotubes. These findings suggest that the upregulation of receptor subunits in distinct cell populations plays an important role in the effective regeneration of both myofibers and motor neurons.

Potentiation of antitumor effect of NKT cell ligand, alpha-galactosylceramide by combination with IL-12 on lung metastasis of malignant melanoma cells.

The combined therapeutic effect of natural killer T (NKT) cell ligand α-galactosylceramide (α-GalCer) and IL-12 against highly metastatic B16-BL6-HM melanoma cells was investigated. In comparison with a single administration of α-GalCer or IL-12, the combined treatment of tumor-bearing mice with α-GalCer plus IL-12 caused a super-induction of serum IFN-γ levels, though α-GalCer-induced IL-4 production was rather inhibited. In parallel with the augmented IFN-γ production, the natural killing activity against YAC-1 cells and syngeneic B16- BL6-HM melanoma was greatly augmented by the combined therapy. The major effector cells responsible for natural killing activity induced by α-GalCer plus IL- 12 were enriched in both NK1.1+TCRαβ+ NKT cells and NK1.1+TCRαβ− NK cells. The preventing effect of α-GalCer or IL-12 alone against lung metastasis of B16-BL6-HM was also enhanced by the combination therapy. The antitumor activity of α-GalCer was totally abolished in NKT-deficient mice. However, IL- 12-induced antitumor activity was not eliminated in NKT-deficient mice though it was inhibited by anti-asialo GM1 Ab treatment. These findings suggested that α-GalCer synergistically act with IL-12 to activate both NKT cells and NK cells, which may play a critical role in the strong prevention of distant tumor metastasis at early stages of tumor-bearing. These data will provide a novel tool for the prevention of tumor metastasis using NKT-specific ligands α-GalCer and IL-12.

Androgen-mediated down-regulation of CYP1A subfamily genes in the pig liver

In Meishan and Landrace pigs, sex differences in the constitutive expression of hepatic cytochrome P4501A (CYP1A) subfamily enzymes were examined in terms of their mRNA, protein, and enzyme activity. All the results from the real-time RT-PCR, western blotting, and enzyme assays for CYP1A subfamily enzymes indicated that, in 5-month-old Meishan pigs, the expression levels of CYP1A1 and CYP1A2 in males were significantly low as compared with those in females. In contrast, there were no such significant sex differences in Landrace pigs. Castration of male Meishan pigs led to a female-type expression of the CYP1A subfamily enzymes, whereas no such effect was observed in male Landrace pigs after castration. In both breeds of pigs, the administration of testosterone propionate to the females and castrated males led to marked decreases in the expression levels of mRNAs and proteins in the CYP1A subfamily enzymes, and also in their enzyme activities. Furthermore, the correlation analyses between the serum testosterone level and the gene expression levels of CYP1A subfamily enzymes in different sex-matured (1-5-month-old) male pigs revealed that the clear decrease in expression levels of hepatic CYP1A subfamily enzymes occurred at concentrations of more than 33 ng/ml of serum testosterone. Incidentally, the mean concentrations of serum testosterone in 5-month-old Landrace and Meishan pigs were around 18 and 50 ng/ml respectively. In conclusion, we demonstrated for the first time that the serum testosterone level is one of the physiological factors which regulate constitutive expression of hepatic CYP1A1 and CYP1A2 in pigs.

Functional expression of the TrkC gene, encoding a high affinity receptor for NT-3, in antigen-specific T helper type 2 (Th2) cells

Neurotrophins, including nerve growth factor, brain-derived neurotrophic factor and neurotrophin-3 (NT-3) are essential factors for the development of the nervous system. In this report, we demonstrate gene expression of neurotrophins and their receptors in T helper 1 (Th1) and T helper 2 (Th2) cells induced from naïve CD4+ CD45RB+ T cells of ovalbumin-specific DO11.10 T cell receptor transgenic mice. Interestingly, the TrkC gene, which encodes a high affinity receptor for NT-3, was expressed in Th2 cells, but not in Th1 and naïve CD4+ T cells. Expression of the TrkC gene was markedly augmented by addition of anti-IFN-gamma monoclonal antibody (mAb) into the culture, whereas it was blocked by anti-IL-4 mAb. Moreover, NT-3 synergistically enhanced anti-CD3 mAb-induced IL-4 production by Th2 cells, but did not affect IFN-gamma production by Th1 cells. These data suggest that NT-3, through its receptor TrkC, plays a critical role in regulating the Th1/Th2 balance.

Natural Killer T Cell Ligand α-Galactosylceramide Inhibited Lymph Node Metastasis of Highly Metastatic melanoma Cells

The role of natural killer T (NKT) cells in the prevention of multiple tumor metastasis was examined. The i.v. inoculation of a highly metastatic subline of B16‐BL6 (B16‐BL6‐HM) melanoma cells resulted in the formation of metastatic nodules in lymph nodes in addition to lung, intrapleural cavity, and ovary. However, treatment of the mice with the NKT cell ligand α‐galactosylceramide (α‐GalCer) three times from 1 day after B16‐BL6‐HM melanoma inoculation caused a significant inhibition of multiple metastasis. Lymph node metastasis of B16‐BL6‐HM was almost completely blocked by α‐GalCer treatment. This antimetastatic effect of α‐GalCer was abolished in NKT celldeficient mice. These results suggest that α‐GalCer‐activated NKT cells played a critical role in the prevention of lymph node metastasis of melanoma cells.

A capillary electrochromatography-electron spray ionization-mass spectrometry method for simultaneous analysis of charged and neutral constituents of a hepatocarcinoma cell metabolome.

Although metabolome research is a rapidly expanding field in the postgenomic era, no single method exists for complete analysis of all the constituents of a metabolome. In this study, we developed a metabolome analysis method using a combination of capillary electrochromatography and electrospray ionization-mass spectrometry. The capillary electrochromatography column was prepared by surface modification of silica compounds (tetraethoxysilane and octyltriethoxysilane) in a fused-silica capillary column. The method was used to separate more than 100 charged and neutral compounds simultaneously. When 1 mM formic acid was used as the eluent, the cationic compounds were eluted rapidly, and then neutral and anionic compounds were eluted (in that order). The developed system was used to analyze the metabolome of a human hepatocellular carcinoma cell line (HepG2). Thirty-three peaks were detected, and eighteen compounds were identified, including marker compounds of hepatocellular cell activity, such as creatinine and homocysteine. Thus, the system was useful not only for metabolome analysis but also for diagnostic measurements of cell function.

IL-1 regulates the Cyp7a1 gene and serum total cholesterol level at steady state in mice.

We examined the role of hepatic interleukin (IL)-1alpha/beta in serum total cholesterol homeostasis using male and female IL-1-knockout (KO) mice and wild-type (WT) mice. Serum total cholesterol level was higher in males than in females in WT and KO mice. The difference between sexes was closely correlated with the difference in gene expression level of cholesterol 7alpha-hydroxylase (Cyp7a1), a rate-limiting enzyme for bile acid synthesis. No significant sex difference in gene expression level of 3-hydroxy-3-methylglutaryl-CoA reductase, a rate-limiting enzyme for cholesterol synthesis, was observed in WT mice. Interestingly, the gene expression level of hepatic Cyp7a1 was lower in KO mice than in sex-matched WT mice, while the serum total cholesterol level was the opposite. The present findings demonstrate that IL-1alpha and IL-1beta are positive regulators for the Cyp7a1 gene in steady-state mice and that Cyp7a1 is one of the factors that mediate the difference in serum total cholesterol level between sexes.

A hepatocarcinogenic tryptophan–pyrolyzate component, Trp‐P‐1, decreases serum total testosterone level and induces hepatic Cyp1a2 in male mice

Male (BALB/c × DBA/2) F1 mice were given 3‐amino‐1,4‐dimethyl‐5H‐pyrido [4,3‐b] indole acetate (Trp‐P‐1; 20 mg/kg body weight) by gavage at 24‐h intervals for 1 or 2 weeks, and the effects of Trp‐P‐1 on the levels of serum total testosterone and hepatic cytochrome P4501a2 (Cyp1a2) were examined. A significant decrease in serum total testosterone level was observed after treatment with Trp‐P‐1 for 2 weeks, but not for 1 week. Likewise, gene expression levels of testicular androgenic enzymes, including cholesterol side chain cleavage cytochrome P450, 3β‐hydroxysteroid dehydrogenase and steroid 17α‐hydroxylase/C17‐20 lyase, decreased only in the mice treated with Trp‐P‐1 for 2 weeks. In contrast, levels of the mRNA and apoprotein of hepatic Cyp1a2 and its enzyme activity for O‐demethylation of methoxyresorufin significantly increased in the mice treated with Trp‐P‐1 for 2 weeks, but only a small increase was observed in mice treated for 1 week. In the present study, we demonstrate for the first time that treatment of male mice with Trp‐P‐1 results in a decrease in serum total testosterone level through suppression of the gene expression of testicular enzymes responsible for androgen biosynthesis, and this then leads to induction of hepatic Cyp1a2. (Cancer Sci 2006; 97: 32–37)