Long-term memory (LTM) formation requires gene transcription and de novo protein synthesis. The transcription factor CREB is required for hippocampus-dependent LTM formation, and it is activated by several signaling pathways, including protein kinase A (PKA), the mitogen activated protein/extracellular signal-regulated kinases (MAPK or ERKs), and Ca²⁺/calmodulin kinases (CaMKs). However, it is unknown whether all types of hippocampus-dependent LTM use the same signaling to activate transcription, and whether the transcriptional output is the same. Here we present molecular genetic and behavioral studies to demonstrate that two types of hippocampus-dependent LTM formation, spatial and contextual, require different signaling molecules. This can be illustrated by the CaMK kinases, CaMKKα, and CaMKKβ, which have converse roles. CaMKKα is required for contextual and CaMKKβ is required for spatial LTM formation. This leads to the surprising conclusion that there are distinct types of hippocampus-dependent LTM, which differ in their underlying molecular mechanisms.

View full abstract

Sir2 (silent information regulator 2) is an NAD-dependent deacetylase that is broadly conserved from bacteria to humans. It catalyzes a unique deacetylation reaction using NAD, and specific inhibitors and activators of its activity have been discovered. In yeast, Sir2 deacetylates histones and participates in transcription silencing and the suppression of recombination. Sir2 is also implicated in the regulation of aging, because its increased expression extends the lifespan of yeast and nematodes. Mammalian SIRT1 (Sir2α) is a member of the Sir2 family. Recently, SIRT1 was shown to interact with various transcription factors such as p53, forkhead transcription factor (FOXO) family proteins, and MyoD, and to participate in stress tolerance, differentiation, and development.

View full abstract

Forkhead box transcription factor, class O (FOXO) is a mammalian homologue of DAF-16, which is known to regulate the lifespan of Caenorhabditis elegans and includes subfamilies of forkhead transcription factors such as AFX, FKHRL1, and FKHR. FKHR is phosphorylated on three sites (Thr-24, Ser-256, and Ser-319) in a phosphatidylinositol 3-kinase (PI3K)/Akt-dependent manner, thereby inhibiting death signals. We here documented dephosphorylation of FKHR following transient forebrain ischemia with its concomitant translocation into the nucleus in neurons in gerbil and mouse brains. The activation of FKHR preceded delayed neuronal death in the vulnerable hippocampal regions following ischemic brain injury. The FKHR activation was accompanied by an increase in DNA binding activity for FKHR-responsive element on the Fas ligand promoter. We also defined FKHR-induced downstream targets such as Fas ligand and Bim in brain ischemia. Therefore, we propose a new strategy to rescue neurons from delayed neuronal death by promoting the survival signaling. Sodium orthovanadate, a protein tyrosine phosphatase inhibitor, up-regulated Akt activity in the brain and in turn rescue neurons from delayed neuronal death by inhibiting FKHR-dependent or -independent death signals in neurons.

View full abstract

Although it is widely accepted that an activity-dependent gene transcription is induced by the calcium (Ca²⁺) signals in neurons, it is still unclear how the particular mRNA moieties are transiently accumulated in response to synaptic transmission that evokes multiple intracellular signals including Ca²⁺ and cAMP ones. Promoters of the brain-derived neurotrophic factor (BDNF) and the pituitary adenylate cyclase-ativating polypeptide (PACAP) can commonly be activated through the cAMP-responsive element (CRE), to which the CRE-binding protein (CREB) predominantly bound. The activation of BDNF gene promoter I and III (BDNF-PI and -PIII, respectively) was mediated not only by the CREB but also by the upstream stimulatory factor, whereas that of PACAP gene promoter (PACAP-P) was mediated by only one CRE located at around −200. The PACAP-P was synergistically enhanced by Ca²⁺ and cAMP signals through the CRE, whereas the BDNF-PI did not show such a synergistic activation upon the stimulation with both signals. In addition, we found that the half-lives of PACAP and BDNF mRNA were prolonged by the Ca²⁺ influx into neurons but not that of Arc mRNA, indicating an activity-dependent stabilization of particular mRNA species in neurons. Thus, the activity-dependent gene expression is co-ordinately controlled by Ca²⁺ and cAMP signals not only at the transcriptional level but also at the post-transcriptional level for the cumulative mRNA expression in neurons.

View full abstract

We have previously shown a marked but transient increase in DNA binding of the nuclear transcription factor activator protein-1 after brief exposure to static magnetic fields in cultured rat hippocampal neurons, suggesting that exposure to static magnetism would lead to long-term consolidation as well as amplification of different functional alterations through modulation of de novo protein synthesis at the level of gene transcription in the hippocampus. Hippocampal neurons were cultured under sustained exposure to static magnetic fields at 100 mT, followed by extraction of total RNA for differential display (DD) analysis using random primers. The first and the second DD polymerase chain reaction similarly showed the downregulation of particular genes in response to sustained magnetism. Nucleotide sequence analysis followed by BLASTN homology searching revealed high homology of these 2 DD-PCR products to the 3' non-coding regions of the mouse basic helix-loop-helix transcription factor ALF1 and that of histone H3.3A, respectively. On Northern blot analysis using the 2 cloned differentially expressed fragments labeled with [α-³²P]dCTP by the random primer method, a marked decrease was seen in expression of mRNA for ALF1 and histone H3.3A in hippocampal neurons cultured under sustained exposure to static magnetic fields at 100 mT. It thus appears that static magnetism may modulate cellular integrity and functionality through expression of a variety of responsive genes required for gene transcription and translation, proliferation, differentiation, maturation, survival, and so on in cultured rat hippocampal neurons.

View full abstract

Leptin is an adipose tissue-derived secretory hormone that suppresses appetite by inhibition of neuropepeptide Y (NPY) gene expression in arcuate nucleus (ARC) in the hypothalamus. To investigate the transcriptional regulation of NPY gene by leptin, we carried out a luciferase assay using NPY gene promotor plasmid (NPY-luc) in NPY expressing cells such as N18TG2, NG108-15, and PC12 cells. In these cells, the NPY gene was transactivated by leptin through activation of leptin receptor. Leptin-induced transactivation was mediated through the 221-bp region of the NPY gene promotor, which possesses two putative STAT3 binding sites. To investigate the mechanism of in vivo suppression of NPY gene transcription in ARC by leptin, the effect of SOCS members on the leptin-induced transactivation of NPY gene was studied. In vivo SOCS2 and SOCS3 mRNAs were induced in mouse hypothalamus by leptin. Although leptin (125 ng/ml) induced significant increase in NPY gene transcriptional activity in mock-transfected cells, the leptin-induced NPY gene transcriptional activity was completely abolished in SOCS3-transfected cells. SOCS3 also suppressed the basal NPY gene transcription. These finding suggested that leptin inhibits NPY gene transcription in the hypothalamus in vivo and SOCS3 is a negative regulator of the NPY gene.

View full abstract

We studied the long-term effects of repeated doses of nicotine, causing dependence, 120 days after its withdrawal on feeding behavior and on brain nitric oxide (NO) formation in female mice. Nicotine dependence was induced by subcutaneous (s.c.) nicotine injection (2 mg/kg, four injections daily) for 14 days. Daily food intake was evaluated for the entire observational period (120 days). Moreover, 30, 60, and 120 days after nicotine withdrawal, we evaluated food intake, nitrite/nitrate levels, and nitric oxide synthase (NOS) activity and expression in the hypothalamus after food deprivation (24 h). In animals in which nicotine dependence was induced (NM), daily food intake was similar to that of controls (M). However, following food deprivation, NM mice showed i) a significant increase in food intake, ii) changes in weight gain and in hypothalamic nitrite/nitrate levels, and iii) enhancement of hypothalamic neuronal NOS (nNOS) activity. Results indicate that high doses of nicotine producing dependence induce long-term changes in feeding behavior consequent to food deprivation associated to alterations in the brain nitrergic system.

View full abstract

NELL2 is a neuron-specific thrombospondin-1-like extracellular protein containing six epidermal growth factor-like domains. We previously disrupted the NELL2 gene in mice by gene targeting and showed that long-term potentiation is enhanced in vivo in the dentate gyrus of NELL2-deficient mice. To further elucidate the physiological roles of NELL2, we performed a behavioral characterization of NELL2^−/− and their heterozygous control mice. NELL2-deficient mice exhibited learning impairment in the Morris water maze task. However, we observed no difference in passive avoidance learning between NELL2^−/− and NELL2^+/− mice. These observations suggest that NELL2 plays an important role in hippocampus-dependent spatial learning and that emotional learning does not depend critically on NELL2.

View full abstract

Evidence for a role of phospholipase D (PLD) in cellular proliferation and differentiation is accumulating. We studied PLD activity and expression in normal and hypertrophic rat and human hearts. In rat heart, abdominal aortic banding (constriction to 50% of original lumen) caused hypertrophy in the left ventricle (as shown by weight index and ANP expression) by about 15% after 30 days without histological evidence of fibrosis or signs of decompensation and in the right ventricle after 100 days. The hypertrophy was accompanied by small increases of basal PLD activity and strong potentiation of stimulated PLD activity caused by 4β-phorbol-12β,13α-dibutyrate (PDB) and by phenylephrine. The mRNA expressions of both PLD1 and PLD2 determined by semiquantitative competitive RT-PCR were markedly enhanced after aortic banding. In the caveolar fraction of the rat heart, PLD2 protein determined by Western blot analysis was upregulated in parallel with the expression of caveolin-3. A similar induction of PLD mRNA and protein expression was observed in hypertrophied human hearts of individuals (39-45-year-old) who had died from non-cardiac causes. In conclusion, PLD1 and PLD2 expressions were strongly enhanced both in rat and human heart hypertrophy, which may be responsible for the coincident potentiation of the PLD activation by α-adrenoceptor and protein kinase C stimulation. These results are compatible with a significant role of PLD activation in cell signaling of ventricular pressure-overload hypertrophy.

View full abstract

The chronic inhibition of nitric oxide (NO) synthesis with N^ω-nitro-L-arginine methyl ester (L-NAME) in pregnant rats induces a pre-eclampsia-like syndrome, including hypertension. We have previously reported the beneficial effects of Toki-shakuyaku-san (TS) in this model. In the present study we demonstrated the anti-hypertensive effect of TS in pre-eclampsia produced by prolonged L-NAME-infusion during the postpartum period. Analysis of blood sex steroids suggested that the level of progesterone differs between the TS-effective (gestational day 19 and postpartum day 7) and TS-ineffective (postpartum day 1) periods. Co-administration of TS and progesterone inhibited L-NAME-induced hypertension on postpartum day 1. Furthermore, the anti-hypertensive effect of TS on postpartum day 6 disappeared in the presence of a co-administered progesterone antagonist mifepristone. These data suggest that a certain level of progesterone may be an indispensable prerequisite for an anti-hypertensive effect of TS. Finally, the effects of TS are apparently unrelated to blood levels of NO, calcitonin gene-related peptide, and endothelin-1, which have been reported to modulate systolic blood pressure in the L-NAME-induced pre-eclampsia model. Thus, the use of TS may provide a new therapeutic strategy for pre-eclampsia, although elucidation of the mechanism of action of TS would be necessary to optimize treatment protocols.

View full abstract

We examined the involvement of nitric oxide (NO) and/or endothelium-derived hyperpolarizing factor (EDHF) in decreasing peripheral vascular resistance in the rat hind limb perfusion model and analyzed the identity of EDHF in this model. The potency of carbachol (CCh) to produce relaxation was quantitatively similar to sodium nitroprusside (SNP). CCh-induced relaxation was abolished after endothelial denudation, but resistant to nitroarginine and indomethacin. The relaxation was inhibited by tetraethylammonium, ouabain, charybdotoxin plus apamin, and under depolarization. SNP-induced relaxation was accompanied by increased cGMP production, which was inhibited by ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxaline-l-one). Although CCh produced a similar extent of relaxation to SNP, the cGMP level was 24 times lower than that with SNP. Low KCl produced a definite relaxation, which was inhibited by ouabain, but independent of NO, prostacyclin, and endothelium. 1-EBIO (1-ethyl-2-benzimidazolinone) as an activator of IK_Ca channel also produced a concentration-dependent relaxation, which was inhibited by charybdotoxin, ouabain, and depolarization, but independent of NO and prostacyclin. Clotrimazole and 17-octadecynoic acid as inhibitors of P₄₅₀ monooxygenase inhibited the CCh-induced relaxation. Meanwhile, catalase at a concentration sufficient to inhibit H₂O₂-induced relaxation did not exert definite inhibition of the CCh-induced relaxation. These results suggest that CCh produces an endothelium-dependent, EDHF-dependent, and NO-cGMP-independent relaxation and that K⁺ and metabolite(s) of P₄₅₀ monooxygenase possibly play an important role for this relaxation.

View full abstract

We have demonstrated that the relaxing mechanism of papaverine in phasic muscles such as ileum, urinary bladder, and uterus is different from tonic muscles such as aorta. In this study, we examined the inhibitory mechanism of papaverine on carbachol (CCh)-induced contraction in the bovine trachea. Papaverine inhibited muscle contraction and increase in [Ca²⁺]_i level induced by CCh. Papaverine increased cAMP content but not cGMP content. Papaverine did not affect CCh-induced oxidized flavoproteins fluorescence or reduced pyridine nucleotides fluorescence. Papaverine (30 μM) remarkably inhibited muscle tension, but slightly decreased creatine phosphate and ATP contents. Iberiotoxin restored the inhibitions of muscle contraction and [Ca²⁺]_i level induced by papaverine or dibutyryl-cAMP. These results suggested that the relaxing mechanism of papaverine in the bovine trachea is mainly due to increases of cAMP content by inhibiting phosphodiesterase and the mechanism is partially involved in the activation of BK channel by cAMP.

View full abstract

Osteopontin has been reported to have an important role in cardiac fibrosis. However, little is known about the effects of angiotensin-converting enzyme inhibitor (ACEI) and angiotensin type 1 receptor blockers (ARB) on osteopontin expression in infarcted myocardium. The purpose of this study was to elucidate the effects of an ACEI (perindpril) and an ARB (candesartan cilexitil) on cardiac function as assessed by Doppler echocardiography and cardiac osteopontin expression associated with cardiac remodeling in myocardial infarcted rats. ACEI or ARB was administered after myocardial infarction (MI). At 4 weeks after MI, cardiac function, and mRNAs in non-infarcted myocardium were analyzed. ACEI and ARB equally prevented left ventricular dilatation, reduction of ejection fraction, and the increase in E/A wave velocity ratio and the rate of E wave deceleration by MI. ACEI and ARB significantly suppressed increased mRNA expression of atrial natriuretic peptide, brain natriuretic peptide, osteopontin, and collagen I and III in the non-infarcted ventricle at 4 weeks. Immunohistochemically stained osteopontin was increased in interstitial fibrosis of non-infarcted myocardium. Both ACEI and ARB significantly prevented cardiac fibrosis and osteopontin expression. In conclusion, angiotensin blockade inhibits osteopontin expression in non-infarcted myocardium and prevents cardiac remodeling after MI.

View full abstract

It is well-known that the anticonvulsant drug, phenytoin (PHT), induces gingival overgrowth as a side effect. The mechanism of PHT-induced gingival overgrowth, however, is not well understood. One reason for this is the lack of an adequate animal model for the PHT-induced gingival overgrowth. The purpose of this study was to establish a rat model of the drug-induced gingival overgrowth. Fourteen-day-old rats were randomly divided into 3 groups (5 rats/group). The control rats received only the vehicle. The rats in the experimental group were injected with 50 mg/kg per day (group L) and 100 mg/kg per day (group H) of PHT. They received a subcutaneous injection of vehicle or PHT twice a day for 42 days. A charge-coupled device (CCD) laser displacement sensor was used for measurement of the severity of gingival overgrowth of the mandibles. There was no significant difference in the growth of rats between the PHT-injected and the control groups. The CCD laser displacement sensor can measure minute changes in the gingival overgrowth in rats, and a significant extension of the buccal gingiva was observed in groups L and H. Using the CCD sensor, it is possible to quantify the change in the gingiva under precise control of the PHT dose.

View full abstract

SKI306X was previously found to have cartilage protective effects in the experimental osteoarthritis (OA) model. To investigate the chondro-protective benefits of SKI306X for its capacity in altering changes in cartilage metabolism and molecular mechanisms of cartilage protective action, SKI306X is studied in rabbit cartilage explants culture. To investigate the protective effect of SKI306X on cartilage catabolism, we assessed collagen degradation in rabbit cartilage explants treated with interleukin-1α up to 3 weeks. To examine the reaction mechanism, matrix metalloproteinase (MMPs) were investigated by fluorimetric and Western blotting analysis. In addition, its effects on the activation process of proenzyme MMP-3 were determined by gelatin zymography. SKI306X significantly inhibited collagen degradation and inhibited the activities of several MMPs. Total MMPs activities in cultured medium were substantially increased in the third week at the time of collagen degradation with the absence of SKI306. However, the introduction of SKI306X decreased MMPs activities in cultured medium. Furthermore, Western blotting analysis proved that these inhibitory effects of this drug were the result of inhibiting MMPs expression. SKI306X also inhibited the activation of proenzyme MMP-3 to the active form of MMP-3. These results indicate that SKI306X inhibits matrix degradation by down regulating MMPs expression and secretion, inhibition of MMPs activity, and inhibiting activation of MMP-3 during the collagen breakdown process.

View full abstract

Soybean lecithin transphosphatidylated phosphatidylserine (SB-tPS) is already known to improve the learning ability of aged or drug-induced amnesic rodents. In this study, its effect on normal adult rodents was evaluated using several learning tasks. Firstly, three behavioral tests (open-field, Y-maze, and active avoidance test) were consecutively carried out after the daily oral administration of SB-tPS (50 mg/kg per day, for 34 days). Repeated oral administration of SB-tPS did not affect either exploratory behavior in the open-field test or spontaneous alternation behavior in the Y-maze test, while mice pretreated with SB-tPS showed significant enhancement of conditioned avoidance response. Secondly, the brightness discrimination test was used to evaluate the effect of SB-tPS on learning ability. The daily oral administration of SB-tPS (50 mg/kg per day, for 27 days) to normal rats significantly increased the correct response ratio in the brightness discrimination test. Finally, to elucidate the necessity of SB-tPS pretreatment, another active avoidance test was carried out, and no enhancement of conditioned avoidance response was observed in non-pretreated mice. These results suggest that repeated administration of SB-tPS could enhance the learning ability of normal adult rodents as those of aged ones.

View full abstract

A simple in vivo closed-chest atrial fibrillation (Af) model of rats was developed. Af was reproducibly induced by transesophageal atrial burst pacing for 30 s in each of the pentobarbital-anesthetized rats, whereas the cardiohemodynamic condition as well as the inducibility and duration of Af episode was stable over time. Moreover, the anti-Af effect of the class Ic drug pilsicainide was confirmed in this model, which was essentially the same as those reported previously in other Af animal models and clinical practice. Thus, this new model may become an alternative to current techniques.

View full abstract

Histamine H₄ receptor is considered as a novel therapeutic target for allergic diseases. To enhance the knowledge about species difference, which is essential for drug discovery research, monkey H₄ receptor was identified. Monkey H₄ receptor was characterized to have comparable similarity with its human counterpart. Discovery of monkey H₄ receptor will contribute to a better interpretation of effective drug discovery.

View full abstract

Hot flushes are the most common complaint of menopausal women. In the present study, a new animal model of hot flushes was established. Tail skin temperature was measured with a thermo tracer after mice were subjected to a forced exercise task using a motor driven treadmill. In ovariectomized mice, forced exercise for 10 min was most effective in increasing tail skin temperature over that of sham-operated mice. This elevation was blocked by estradiol replacement (1 mg/kg per week for 3 weeks), suggesting that our model simulates menopausal hot flushes.

View full abstract

We studied the effects of Mao (Ephedrae herba) on lipopolysaccharide (LPS)-induced expression of inducible cyclooxygenase (COX-2) in C6 rat glioma cells. Western blot analysis showed that Mao inhibited LPS-induced expression of COX-2 protein, but not that of constitutive cyclooxygenase. Reporter gene assays showed that Mao reduced LPS-inducible NF-κB-dependent transcription that plays a crucial role in induction of COX-2 gene expression. This crude drug decreased LPS-induced IκBα degradation in a concentration-dependent manner without affecting LPS-induced IκB phosphorylation. These results suggest that Mao prevents LPS-induced NF-κB-dependent transcription by inhibiting IκB degradation and suppresses an increase in COX-2 protein expression in C6 cells.

View full abstract