Period2 3'-UTR and microRNA-24 regulate circadian rhythms by repressing PERIOD2 protein accumulation.

We previously created two PER2::LUCIFERASE (PER2::LUC) circadian reporter knockin mice that differ only in the Per2 3'-UTR region: Per2::Luc, which retains the endogenous Per2 3'-UTR and Per2::LucSV, where the endogenous Per2 3'-UTR was replaced by an SV40 late poly(A) signal. To delineate the in vivo functions of Per2 3'-UTR, we analyzed circadian rhythms of Per2::LucSV mice. Interestingly, Per2::LucSV mice displayed more than threefold stronger amplitude in bioluminescence rhythms than Per2::Luc mice, and also exhibited lengthened free-running periods (∼24.0 h), greater phase delays following light pulse, and enhanced temperature compensation relative to Per2::Luc Analysis of the Per2 3'-UTR sequence revealed that miR-24, and to a lesser degree miR-30, suppressed PER2 protein translation, and the reversal of this inhibition in Per2::LucSV augmented PER2::LUC protein level and oscillatory amplitude. Interestingly, Bmal1 mRNA and protein oscillatory amplitude as well as CRY1 protein oscillation were increased in Per2::LucSV mice, suggesting rhythmic overexpression of PER2 enhances expression of Per2 and other core clock genes. Together, these studies provide important mechanistic insights into the regulatory roles of Per2 3'-UTR, miR-24, and PER2 in Per2 expression and core clock function.

Evaluation of canine T-cell dependent antibody response to the primary and secondary immunization with keyhole limpet hemocyanin.

T-cell dependent antibody response (TDAR) incorporating both primary and secondary responses to keyhole limpet hemocyanin (KLH) in canine models have not yet been fully understood. To develop a practical dog TDAR model, we characterized primary and secondary antibody responses by intravenous or intramuscular immunization of KLH twice at intervals of 8 days during a 28-day course of study. Primary immunization with KLH by both routes induced a maximum IgM response on 6 to 8 days after the treatment, whereas the IgG response started 6 to 8 days after the treatment with relatively low levels. Remarkable increases in anti-KLH IgG levels (about 10-times compared with the primary response) were produced 5 to 7 days after the secondary KLH immunization by both routes. These results indicate that IgM-predominant and IgG-predominant responses were respectively induced by the primary and secondary immunization. Furthermore, the intravenous route showed higher baseline titers of primary and secondary anti-KLH IgM responses, suggesting that intravenous immunization of KLH might be a more suitable method for immunotoxicity evaluation. No remarkable inter-individual variability was noted in our canine models. Treatment with cyclophosphamide at 2 mg/kg/day for a consecutive 28 days significantly suppressed primary and secondary anti-KLH IgM and IgG responses induced by KLH injection on Days 15 and 23 of CPA treatment. These results demonstrate that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in dogs when exposed to potential immunomodulatory drugs.

Evaluation of primary and secondary responses to a T-cell-dependent antigen, keyhole limpet hemocyanin, in rats.

To develop a rat T-cell-dependent antibody response (TDAR) model evaluating both primary and secondary antibody responses, keyhole limpet hemocyanin (KLH) was used to immunize rats twice during a 14-day course of study, a pattern closely linked to that of a short-term general toxicity study. Female rats of four representative strains (e.g., Sprague-Dawley, Wistar, Fischer, and Lewis) were immunized twice with intravenous administrations of KLH (300 µg/rat) on Days 5 and 9 during a 14-day treatment regimen with cyclophosphamide (CPA) at 1, 3, or 6 mg/kg/day. The primary and secondary immunizations of KLH markedly elevated serum anti-KLH IgM and IgG levels in all strains on Days 9 and 15. Remarkable higher levels of anti-KLH IgG (≈ 1000 µg/ml) were noted in all strains, which were more than 4-times compared with those of anti-KLH IgM levels at Day 9, indicating that predominant IgG reactions were induced by the dual immunizations. A large inter-individual variability in KLH-specific IgM and IgG production was observed in all strains. However, levels of the KLH-specific antibodies were considered sufficient for the evaluation, even in Sprague-Dawley and Wistar rats reported as strains with a wide range of variability since immunosuppression of CPA on responses in both anti-KLH IgM and IgG were observed in all strains to the same extent. In addition, the sensitivity of the KLH-ELISA assay system detecting the immunosuppressive effects of CPA was comparable to other assay systems with PFC assay or ELISA using SRBC. The results here demonstrated that these experimental designs could provide valuable information about the influence on both the primary and secondary humoral immune responses in rats when exposed to potential immunomodulatory drugs. Furthermore, the design of the presented TDAR study would support comprehensive evaluation together with the outcome of the conventional general toxicity study.

Cyclic dipeptides exhibit potency for scavenging radicals.

Twenty kinds of cyclic dipeptides containing l-leucine were synthesized, and their antioxidant activity against ·OH and O(2)(·-) was investigated. Compounds possessing polar amino acid residues, such as Asp, Cys, Glu, Lys, Pro, Ser, and Trp, exhibited higher antioxidant activity against ·OH than vitamin E. However, only cyclo(l-Cys-l-Leu) scavenged O(2)(·-).

Protective effects of cyclo(L-Leu-L-Tyr) against postischemic myocardial dysfunction in guinea-pig hearts.

The protective effects of cyclic dipeptides in alcoholic beverages were investigated in the perfused guinea-pig hearts subjected to ischemia and reperfusion. Subsequently, in order to determine the importance of cyclic dipeptide structure, the effects of cyclo(L-Leu-L-Tyr) (cLY) were compared with those of the newly synthesized non-cyclic dipeptides, L-Leu-L-Tyr (LY) and L-Tyr-L-Leu (YL). After reperfusion, pressure recovery (%) in the left ventricle reached a peak of over 90% in the presence of cLY (10(-6) M and 10(-5) M) (control: 22.9%). The recovery by LY and YL was significantly lower than that by cLY, and ATP levels simultaneously monitored using (31)P-NMR were already lower during the ischemic end period than those observed with cLY treatment. In perfused mitochondrial preparations, cLY significantly inhibited mitochondrial Ca(2+) ([Ca(2+)](m)) elevation in a similar way to that of the mitochondrial permeability transition pore (MPTP) inhibitor cyclosporin A. In vitro electron paramagnetic resonance (EPR) revealed that the active oxygen radicals quenching activity of cLY was greater than those of non-cyclic dipeptides. cLY inhibited caspase-3-induced apoptosis. The cyclic dipeptide structure inhibits opening of the MPTP by preventing [Ca(2+)](m) overload-induced apoptosis related to mitochondrial active oxygen radical accumulation in ischemia-reperfusion hearts.

Contribution of cinnamic acid analogues in rosmarinic acid to inhibition of snake venom induced hemorrhage.

In our previous paper, we reported that rosmarinic acid (1) of Argusia argentea could neutralize snake venom induced hemorrhagic action. Rosmarinic acid (1) consists of two phenylpropanoids: caffeic acid (2) and 3-(3,4-dihydroxyphenyl)lactic acid (3). In this study, we investigated the structural requirements necessary for inhibition of snake venom activity through the use of compounds, which are structurally related to rosmarinic acid (1). By examining anti-hemorrhagic activity of cinnamic acid analogs against Protobothrops flavoviridis (Habu) venom, it was revealed that the presence of the E-enoic acid moiety (-CH=CH-COOH) was critical. Furthermore, among the compound tested, it was concluded that rosmarinic acid (1) (IC(50) 0.15 µM) was the most potent inhibitor against the venom.

Sex and circadian modulatory effects on rat liver as assessed by transcriptome analyses.

The present study was designed to fully uncover sex and circadian modulatory effects on rat liver. Hepatic transcriptome analyses were performed at 4 hr intervals of a day-night cycle using young adult male and female rats. Sexually dimorphic genes, which were identified by a cross-sex comparison of time series data, included representative sex-predominant genes such as male- or female-predominant cytochrome P450 subfamilies (Cyp2c11, Cyp2c12, Cyp2c13, and Cyp3a2), sulfotransferases, and glutathione S-transferase Yc2. The identified sexually dimorphic genes were over-represented in the metabolism of retinols, xenobiotics, linoleic acids, or androgen and estrogen, or bile acid biosynthesis. Furthermore, transcription factor targets modeling suggested that transcription factors SP1, hepatocyte nuclear factor 4-alpha (HNF4-alpha), and signal transducer and activator of transcription 5b (STAT5b) serve as core nodes in the regulatory networks. On the other hand, Fourier transform analyses extracted universal circadian-regulated genes in both sexes. The circadian-regulated genes included clock or clock-controlled genes such as aryl hydrocarbon receptor nuclear translocator-like (Arntl), period homolog 2 (Per2), and D site albumin promoter binding protein (Dbp). The extracted cyclic genes were over-represented in major tissue activities, e.g. the urea cycle and the metabolism of amino acids, fatty acids, or glucose, indicating that the major liver functions are under circadian control. The transcription factor targets modeling suggested that transcription factors SP1, HNF4-alpha, and c-Myc proto-oncogene protein (c-MYC) serve as major hubs in the circadian-regulatory gene networks. Interestingly, transcription factors SP1 and HNF4-alpha are likely to orchestrate not only sexually dimorphic, but also circadian-regulated genes even though each criterion was rather mutually exclusive. This suggests the cross-talk between those regulations. Sexual dimorphism is likely to interact with circadian rhythmicity via overlapping gene regulatory networks on rat liver.

Circadian modulation of hepatic transcriptome in transgenic rats expressing human growth hormone.

The secretory profile of growth hormone (GH) is sexually dimorphic in rats. In male transgenic (TG) rats expressing human GH (hGH) that we generated, the circulating levels of both hGH and endogenous GH are flattened with no male-type pulsatility. To elucidate the regulatory role of episodic GH profile on the liver, the hepatic transcriptome of male TG rats at the middle of the light and dark phases was characterized by genome-wide analyses as compared with that of male wild-type (WT) rats. Transcripts commonly up- or down-regulated regardless of the lighting conditions in TG rats were mainly enriched in the metabolism of xenobiotics. In TG rats, the gene expression profile was functionally feminized, verifying that the sexually dimorphic profile of GH rather than genetic sexuality is a stronger sex-determining factor on the hepatic transcriptome. The common transcripts which fluctuated during the day in both TG and WT rats were enriched in circadian rhythm signaling, and physiological rhythmicity was considered to be finely interconnected with liver metabolism via sexually dimorphic GH secretion. In contrast, some genes were differentially regulated in TG rats at only one of two time points measured, and others were fluctuated daily in only one genotype. In particular, some genes involved in the GH signaling pathway were included, suggesting the signal transduction is circadian-modulated depending upon the GH profile. Our transcriptome analyses clarified the regulatory role of episodic GH profile on the liver and strengthen the functional link between sexually dimorphic GH secretion, liver metabolism, and its circadian regulation.

Collaborative work on evaluation of ovarian toxicity. 8) Two- or four-week repeated-dose studies and fertility study of Anastrozole in female rats.

The main focus of this study was to determine the optimal administration period in terms of toxic effects on ovarian morphological changes. To assess the morphological and functional changes induced by anastrozole in ovaries, the compound was administered to female rats at dose levels or 0, 0.01, 0.1 and 50 mg/kg for 2 or 4 weeks in the repeated dose toxicity study and at levels of 0, 0.01, 0.1 and 5 mg/kg from 2 weeks prior to mating to Day 7 or pregnancy in the female fertility study. In the repeated dose toxicity study, large abnormal atretic follicles, follicular cysts, a decrease in corpus luteum and depletion of developing corpus luteum were observed in the 1 and/or 50 mg/kg groups of both the 2-week and 4-week studies in a histopathological examination of the ovaries. In the female fertility study, the pregnancy rate was decreased in the 5 mg/kg group. Irregular estrous cycles, such as an extended cycle or no cycle, were observed in the 0.1 and 5 mg/kg groups. At necropsy, decreased numbers of implantations, corpora lutea and live fetuses were noted in the 1 and/or 5 mg/kg groups. Based on these findings, histopathological changes in the ovary are important endpoints for the evaluation of drugs inducing ovarian damage. We conclude that a 2-week administration period is sufficient to detect ovarian toxicity of anastrozole in a repeated dose toxicity study.

Antioxidant constituents in distillation residue of Awamori spirits.

Constituents in a distillation residue of Awamori (millet spirits) and their antioxidant activity are investigated in this study. The supernatant of the distillation residue obtained by centrifugation was partitioned with n-hexane, chloroform, ethyl acetate, and n-butanol against water to afford the corresponding solubles. Among them, n-hexane and chloroform solubles showed higher antioxidant potency than l-ascorbic acid by the bleomycin-Fe method. In chloroform solubles, seven cyclic dipeptides were identified along with ethyl 2-pyrrolidione-5-carboxylate, tyrosol, and ethyl p-hydoroxyphenyllactate. Antioxidant activity of ethyl p-hydoroxyphenyllactate was 4.2 times that of l-ascorbic acid, whereas cyclic dipeptides showed activity 0.89-1.29 times as strong as that of l-ascorbic acid. On the other hand, scavenging effect of cyclic dipeptides against O(2)(-.) and OH(.) by using electron spin resonance was also investigated. In the results, cyclo(l-Ile-l-Pro) showed significantly strong inhibitory effect against OH(.) (95.4% at 2.5 x 10-3 M) and cyclo(l-Phe-l-Pro), cyclo(l-Pro-l-Val), and cyclo(l-Leu-l-Pro) inhibited OH(.) 64.9, 54.1, and 51.0%, respectively, whereas alpha-tocopherol showed 37.7% inhibition, though only a few cyclic dipeptides weakly inhibited O(2)(-.).

Effects of restricted feeding on daily fluctuations of hepatic functions including p450 monooxygenase activities in rats.

Hepatic P450 monooxygenase activities, assessed by measurement of 7-alkoxycoumarin O-dealkylase (ACD) activities, show obvious daily fluctuations in male rats with high values during the dark period and low values during the light period. We have already confirmed that the ACD activities are controlled by the suprachiasmatic nucleus (SCN), which is well known as the oscillator of circadian rhythm. Recently, it is reported that circadian oscillators exist not only in the SCN but also in peripheral organs. To date, it is unclear which circadian oscillators predominantly drive the daily fluctuations of hepatic ACD activities. To address this question, we examined the effects of restricted feeding, which uncouples the circadian oscillators in the liver from the central pacemaker in the SCN, on the daily fluctuations in hepatic ACD activities in male rats. Here we show that restricted feeding inverts the oscillation phase of the daily fluctuations in hepatic ACD activities. Regarding the hepatic P450 content, there were no fluctuations between the light and dark periods under ad libitum and restricted feeding conditions. Therefore, it is considered that the daily fluctuations in hepatic ACD activities are predominantly driven by the circadian factors in peripheral organs rather than by the oscillator in the SCN directly.

Antioxidant constituents of radish sprout (Kaiware-daikon), Raphanus sativus L.

Methanol extracts of 11 kinds of commonly available vegetables were examined for hydroxyl radical scavenging potency using the bleomycin-Fe method. In this method, the iron ion and bleomycin in water form hydroxyl radicals, and the scavenging activity is monitored by the modified thiobarbituric acid method. All extracts showed scavenging capacity, even though the activity of some of them was lower than that of l-ascorbic acid. Those vegetables were classified into three groups according to their activity, groups showing strong activity, moderate activity, and weak activity, as compared to the activity of l-ascorbic acid at the same concentration. Among them, the methanol extract of radish sprout (Japanese name "kaiware-daikon") exhibited the highest potency (1.8 times as l-ascorbic acid). Then, we investigated the constituents of the methanol extract of radish sprout and the contribution to the overall activity of each compound by examining their activity. As the result, several kinds of sinapinic acid esters and flavonoids were isolated with high radical scavenging potency, which must contribute substantially to the activity.