Pharmacokinetics and tissue distribution of orally administrated imidazole dipeptides in carnosine synthase gene knockout mice.

Imidazole dipeptides (ID) are abundant in skeletal muscle and the brain and have various functions, such as antioxidant, pH-buffering, metal-ion chelation. However, the physiological significance of ID has not been fully elucidated. In this study, we orally administered ID to conventional carnosine synthase gene-deficient mice (Carns-KO mice) to investigate the pharmacokinetics. Carnosine or anserine was administered at a dose of 500 mg (∼2 mmol) per kilogram of mouse body weight, and ID contents in the tissues were measured. No ID were detected in untreated Carns-KO mice. In the ID treatment groups, the ID concentrations in the tissues increased in a time-dependent manner in the gastrocnemius muscle, soleus muscle, and cerebrum after ID administration. Our findings suggest that the Carns-KO mice are a valuable animal model for directly evaluating the effects of dietary ID and for elucidating the physiological functions of oral ID administration.

Molecular characterization of feline melanocortin 4 receptor and melanocortin 2 receptor accessory protein 2.

Melanocortin 4 receptor (MC4R), which is a member of the G protein-coupled receptor (GPCR) family, mediates regulation of energy homeostasis upon the binding of α-melanocyte-stimulating hormone (α-MSH) in the central nervous system (CNS). Melanocortin 2 receptor accessory protein 2 (MRAP2) modulates the function of MC4R. We performed cDNA cloning of cat MC4R and MRAP2 and characterized their amino acid sequences, mRNA expression patterns in cat tissues, protein-protein interactions, and functions. We found high sequence homology (>88%) with other mammalian MC4R and MRAP2 encoding 332 and 206 amino acid residues, respectively. Reverse transcription-polymerase chain reaction analysis revealed that cat MC4R and MRAP2 mRNA were expressed highly in the CNS. In CHO-K1 cells transfected with cat MC4R, stimulation with α-MSH increased intracellular cyclic adenosine monophosphate (cAMP) concentration in a dose-dependent manner. Furthermore, the presence of MRAP2 enhanced the cat MC4R-mediated cAMP production. These results suggested that cat MC4R acts as a neuronal mediator in the CNS and that its function is modulated by MRAP2. In addition, our NanoBiT study showed the dynamics of their interactions in living cells; stimulation with α-MSH slightly affected the interaction between MC4R and MRAP2, and did not affect MC4R homodimerization, suggesting that they interact in the basal state and that structural change of MC4R by activation may affect the interaction between MC4R and MRAP2.

Canine REIC/Dkk-3 interacts with SGTA and restores androgen receptor signalling in androgen-independent prostate cancer cell lines.

BACKGROUND: The pathological condition of canine prostate cancer resembles that of human androgen-independent prostate cancer. Both canine and human androgen receptor (AR) signalling are inhibited by overexpression of the dimerized co-chaperone small glutamine-rich tetratricopeptide repeat-containing protein α (SGTA), which is considered to cause the development of androgen-independency. Reduced expression in immortalised cells (REIC/Dkk-3) interferes with SGTA dimerization and rescues AR signalling. This study aimed to assess the effects of REIC/Dkk-3 and SGTA interactions on AR signalling in the canine androgen-independent prostate cancer cell line CHP-1. RESULTS: Mammalian two-hybrid and Halo-tagged pull-down assays showed that canine REIC/Dkk-3 interacted with SGTA and interfered with SGTA dimerization. Additionally, reporter assays revealed that canine REIC/Dkk-3 restored AR signalling in both human and canine androgen-independent prostate cancer cells. Therefore, we confirmed the interaction between canine SGTA and REIC/Dkk-3, as well as their role in AR signalling. CONCLUSIONS: Our results suggest that this interaction might contribute to the development of a novel strategy for androgen-independent prostate cancer treatment. Moreover, we established the canine androgen-independent prostate cancer model as a suitable animal model for the study of this type of treatment-refractory human cancer.

Thyrotrophin in the pars tuberalis triggers photoperiodic response.

Molecular mechanisms regulating animal seasonal breeding in response to changing photoperiod are not well understood. Rapid induction of gene expression of thyroid-hormone-activating enzyme (type 2 deiodinase, DIO2) in the mediobasal hypothalamus (MBH) of the Japanese quail (Coturnix japonica) is the earliest event yet recorded in the photoperiodic signal transduction pathway. Here we show cascades of gene expression in the quail MBH associated with the initiation of photoinduced secretion of luteinizing hormone. We identified two waves of gene expression. The first was initiated about 14 h after dawn of the first long day and included increased thyrotrophin (TSH) beta-subunit expression in the pars tuberalis; the second occurred approximately 4 h later and included increased expression of DIO2. Intracerebroventricular (ICV) administration of TSH to short-day quail stimulated gonadal growth and expression of DIO2 which was shown to be mediated through a TSH receptor-cyclic AMP (cAMP) signalling pathway. Increased TSH in the pars tuberalis therefore seems to trigger long-day photoinduced seasonal breeding.

Central injection of neuropeptide B induces luteinizing hormone release in male and female rats.

Neuropeptide B (NPB) has been identified as an endogenous peptide ligand for the orphan receptor NPBWR1. However, the effect of NPB on the central regulatory mechanisms of reproductive functions remains unclear. Our findings indicated the presence of Npb, Npw (which is another ligand for NPBWR1), and Npbwr1 mRNA in the hypothalamus of male and female rats at each stage of the estrous cycle. Npb mRNA expression was found to be significantly higher in diestrus compared to estrus. The expression of Npw mRNA was one order of magnitude lower than that of Npb mRNA, and Npw mRNA expression in diestrus was significantly higher than that in the other stages of the estrous cycle. Furthermore, Npbwr1 mRNA expression was found to be significantly higher in diestrus compared to the other stages of the estrous cycle and intact males. Notably, estrogen did not alter the expression of Npb, Npw, and Npbwr1 mRNAs in the hypothalamus of females. Central injection of NPB increased plasma luteinizing hormone (LH) levels in both intact males and estrogen-primed ovariectomized females but not in ovariectomized females. These results suggest that NPB-NPBWR1 signaling would be a facilitatory regulatory mechanism in the reproductive function of male and female rats. To the best of our knowledge, this study is the first report to describe the central role of NPB-NPBWR1 signaling in LH regulation in mammals.

Immunoglobulin G4-related Disease with Marked Eosinophilia: A Case Report and Literature Review.

We encountered a 78-year-old Japanese man with IgG4-related sialoadenitis complicated with marked eosinophilia. We diagnosed him with IgG4-RD (related disease) with a submandibular gland tumor, serum IgG4 elevation, IgG4-positive plasma cell infiltration, and storiform fibrosis. During follow-up after total incision of the submandibular gland, the peripheral eosinophil count was markedly elevated to 29,480/µL. The differential diagnosis of severe eosinophilia without IgG4-RD was excluded. The patient exhibited a prompt response to corticosteroid therapy. His peripheral blood eosinophil count was the highest ever reported among similar cases. We also review previous cases of IgG4-RD with severe eosinophilia.

Two chicken neuromedin U receptors: characterization of primary structure, biological activity and tissue distribution.

Neuromedin U (NMU) is a bioactive peptide that is involved in a variety of physiological functions. Two of its receptors, NMUR1 and NMUR2, have been identified and characterized in mammals. In this study, we performed cDNA cloning of chicken NMUR1 and NMUR2, and characterized their primary structure, biological activity, and expression patterns in chicken tissues. The chicken NMUR1 and NMUR2 cDNAs encoded 438 and 395 amino acid sequences, respectively. Chicken NMUR1 showed 54.8%-56.5% sequence identity with human, rat, and mouse NMUR1, and NMUR2 shared 67.3%-70.1% sequence identity with mammalian orthologs. Both chicken receptors have typical characteristics of G-protein-coupled receptors with seven transmembrane domains and the D/ERY motif. An increase in intracellular Ca(2+) mobilization was observed in HEK293 cells transfected with chicken NMUR1 or NMUR2 cDNA and treated with chicken or rat NMU. Real-time PCR analysis revealed that NMUR1 mRNA was preferentially expressed in the intestinal tissues such as the duodenum, jejunum, ileum, cecum, and colon/rectum, and brain regions such as the midbrain and optic lobe, and the ovary in adult hens. NMUR2 mRNA was exclusively expressed in the brain regions such as the cerebrum and midbrain. These results indicate that NMUR1 and NMUR2 mRNAs, which encode functional receptor proteins, are expressed in chicken tissues with different distribution patterns.

Molecular characterization of structure and tissue distribution of chicken neurotensin receptor.

Neurotensin, a tridecapeptide, is distributed in a wide range of tissues and exhibits multiple functions through its receptors. Hitherto molecular characterization of the neurotensin receptor has been reported in mammalian, amphibian, and fish species but not in avian species. In this study, we cloned the cDNA encoding chicken neurotensin receptor from the duodenum and characterized its primary structure, biological activity and distribution in the gastrointestinal tract. The cDNA encoded a protein consisting of 399 amino acids that had significant overall sequence homology to other vertebrate neurotensin receptor 1 with higher extent in the seven transmembrane domains. Chicken neurotensin increased intracellular Ca(2+) concentrations in human embryonic kidney 293 cells transiently expressing the chicken neurotensin receptor 1. Real-time PCR analysis showed that chicken neurotensin receptor 1 mRNA is expressed throughout the gastrointestinal tract with markedly higher level in the colon/rectum. These results indicate that the chicken neurotensin receptor 1 is involved in gastrointestinal functions through an intracellular signaling pathway accompanied by an increase in Ca(2+) levels.

Photoperiod-Specific Expression of Eyes Absent 3 Splice Variant in the Pars Tuberalis of the Japanese Quail.

The molecular mechanism underlying photoperiodic response in seasonal breeding animals such as the Japanese quail, red jungle fowl, sheep, mouse, and hamster involves thyroid-stimulating hormone beta subunit (TSHß) mRNA expression in the pars tuberalis stimulated by the extension in day length. Furthermore, this mechanism is regulated by eyes absent 3 (Eya3) in mammals. Even in birds, the expression of both TSHß and EYA3 is induced in the pars tuberalis by the extension in day length; however, the relationship between the two genes is unknown. To clarify the function of EYA3 in quail photoperiodism, in the present study, we performed mRNA structure analysis of the Japanese quail EYA3 mRNA using reverse transcription-polymerase chain reaction (RT-PCR) and Southern blot analysis. The results revealed that there are four types of splice variants within regions of exons 7, 8, and 9 of quail EYA3 mRNA. Among the four splice variants of quail EYA3, the splice variant containing exon 7 was expressed in the pars tuberalis on the first long day, when quails were transferred from the short-day condition to the long-day condition. The results indicate that EYA3 splice variant containing exon 7 is involved in the photoperiodic response of the pars tuberalis in the Japanese quail.

Identification of the photoperiodic signaling pathway regulating seasonal reproduction using the functional genomics approach.

Animals measure photoperiod (daylength) and adapt to seasonal changes in the environment by altering their physiology and behavior accordingly. Although this photoperiodic response has long been of interest, the underlying mechanism has only recently begun to be uncovered at the molecular level. Japanese quail provide an excellent model to study the molecular mechanism underlying the vertebrate photoperiodic response. The recent sequencing of the chicken genome allowed a system-level analysis of photoperiodic time measurement in quail, and this approach uncovered the key event in the photoperiodic signaling cascade that regulates seasonal reproduction. Long photoperiod-induced expression of thyrotropin in the pars tuberalis of the pituitary gland was found to trigger local thyroid hormone catabolism in the mediobasal hypothalamus, which increases the activity of the reproductive neuroendocrine system resulting in gonadal development. Since thyrotropin was only known to stimulate the thyroid gland, a traditional hypothesis-driven approach would not have been expected to predict this discovery. Thus, a functional genomics approach, which is a discovery-driven approach, provides new insights in the field of endocrinology.

Cholecystokinin induces crowing in chickens.

Animals that communicate using sound are found throughout the animal kingdom. Interestingly, in contrast to human vocal learning, most animals can produce species-specific patterns of vocalization without learning them from their parents. This phenomenon is called innate vocalization. The underlying molecular basis of both vocal learning in humans and innate vocalization in animals remains unknown. The crowing of a rooster is also innately controlled, and the upstream center is thought to be localized in the nucleus intercollicularis (ICo) of the midbrain. Here, we show that the cholecystokinin B receptor (CCKBR) is a regulatory gene involved in inducing crowing in roosters. Crowing is known to be a testosterone (T)-dependent behavior, and it follows that roosters crow but not hens. Similarly, T-administration induces chicks to crow. By using RNA-sequencing to compare gene expression in the ICo between the two comparison groups that either crow or do not crow, we found that CCKBR expression was upregulated in T-containing groups. The expression of CCKBR and its ligand, cholecystokinin (CCK), a neurotransmitter, was observed in the ICo. We also showed that crowing was induced by intracerebroventricular administration of an agonist specific for CCKBR. Our findings therefore suggest that the CCK system induces innate vocalization in roosters.

Thyroid hormones and seasonal reproductive neuroendocrine interactions.

Many animals that breed seasonally measure the day length (photoperiod) and use these measurements as predictive information to prepare themselves for annual breeding. For several decades, thyroid hormones have been known to be involved in this biological process; however, their precise roles remain unknown. Recent molecular analyses have revealed that local thyroid hormone activation in the hypothalamus plays a critical role in the regulation of the neuroendocrine axis involved in seasonal reproduction in both birds and mammals. Furthermore, functional genomics analyses have revealed a novel function of the hormone thyrotropin. This hormone plays a key role in signaling day-length changes to the brain and thus triggers seasonal breeding. This review aims to summarize the currently available knowledge on the interactions between elements of the thyroid hormone axis and the neuroendocrine system involved in seasonal reproduction.

Bimodal clock gene expression in mouse suprachiasmatic nucleus and peripheral tissues under a 7-hour light and 5-hour dark schedule.

Using the mPer1::luc real-time monitoring technique, the authors observed the bimodal patterns of mPer1 bioluminescence on each side of the SCN, in parallel with maintaining synchronization between the left and right sides of the SCN under an artificial light:dark:light:dark (LDLD) 7:5:7:5 condition. In situ hybridization analysis of mPer1 and mBmal1 mRNA distribution in the SCN showed that in 1 photophase (morning photophase; M) of LDLD, the mPer1 level in the ventrolateral-like (VL-like) subdivision of the SCN was higher than that in the dorsomedial-like (DM-like) subdivision, and this regional distribution pattern was reversed in another photophase (evening photophase; E). In contrast, the mBmal1 level was higher in the DM-like subdivision than in the VL-like subdivision in the M phase, and this distribution changed in the E phase. The prokineticin 2 (PK2) mRNA that encodes an SCN output molecule that is thought to transmit the circadian locomotor rhythms was reduced in both the DM-like and VL-like SCN and did not clearly correlate with the activity under the LDLD condition. The expression of mPer1 and mPer2 in the liver was clearly bimodal, whereas the expressions of other clock genes were not synchronized to the LDLD condition. These results may provide important insights into the mechanism underlying the splitting or bimodal rhythms that may in turn facilitate the understanding of the ability to measure the seasonal day length in mammals.

Expression of Prolactin Receptor mRNA in Lactotrophs and Somatotrophs of the Chicken Anterior Pituitary Gland.

Prolactin (PRL) is a hormone mainly secreted by the anterior pituitary gland. In birds, PRL exerts a variety of physiological functions in target tissues expressing the PRL receptor (PRLR). In chicken, the PRLR mRNA is abundant in the anterior pituitary gland, but its regional and cellular localization are unknown. In the present study, we investigated the expression of the PRLR mRNA in cephalic and caudal lobes of the chicken anterior pituitary gland. Real-time polymerase chain reaction (PCR) revealed high levels of PRLR mRNA in both cephalic and caudal lobes. In situ hybridization revealed that the PRLR mRNA was distributed in a wide area of both lobes, and co-localized with the PRL and growth hormone (GH) mRNAs in the cephalic and caudal lobes, respectively. These results suggest that PRL exerts autocrine/paracrine effects through PRLR on PRL-producing lactotrophs and GH-producing somatotrophs in the chicken anterior pituitary gland.

Involvement of transforming growth factor alpha in the photoperiodic regulation of reproduction in birds.

The molecular mechanism underlying photoperiodism is not well understood in any organism. Long-day-induced conversion of prohormone T(4) to bioactive T(3) within the mediobasal hypothalamus (MBH) is critical for the photoperiodic regulation of reproduction. However, because thyroidectomy does not completely block the photoperiodic response in some species, the existence of a thyroid hormone-independent regulatory mechanism appears certain. To identify this novel mechanism, differential subtractive hybridization analysis was performed using MBH of quail kept under short-day and long-day conditions. This analysis identified a gene encoding TGFalpha. Expression of TGFalpha mRNA was induced in the median eminence by the stimulus of long days, and this induction was observed at dusk on the first long day. This rapid induction of TGFalpha mRNA was similar to induction of the thyroid hormone-activating enzyme gene [Dio2 (type 2 iodothyronine deiodinase)], which is the earliest event yet determined in the photo-induction process. Expression analysis of epidermal growth factor receptors revealed strong expression of erbB4 and weak expression of erbB1 and erbB2 in the median eminence. Intracerebroventricular infusion of physiological dose of TGFalpha induced LH secretion and testicular growth under short-day conditions. Finally, we demonstrate that T(3) implantation and TGFalpha infusion into the MBH, either of which causes testicular growth, do not affect the expression of TGFalpha and Dio2, respectively. Thus, long-day-induced activation of the TGFalpha signaling pathway appears to mediate a thyroid hormone-independent pathway for the photoperiodic regulation of reproduction.

Circadian clock gene regulation of steroidogenic acute regulatory protein gene expression in preovulatory ovarian follicles.

It is now known that circadian clocks are localized not only in the central pacemaker but also in peripheral organs. An example of a clock-dependent peripheral organ is the ovary of domestic poultry in which ovulation is induced by the positive feedback action of ovarian progesterone on the neuroendocrine system to generate a preovulatory release of LH during a daily 6-10 h "open period" of the ovulatory cycle. It has been assumed previously that the timing of ovulation in poultry is controlled solely by a clock-dependent mechanism within the neuroendocrine system. Here, we question this assumption by demonstrating the expression of the clock genes, Per2 (Period 2) and Per3, Clock, and Bmal1 (brain and muscle Arnt-like protein 1), in preovulatory follicles in laying quail. Diurnal changes in Per2 and Per3 expression were seen in the largest preovulatory follicle (F1) but not in smaller follicles. We next sought to identify clock-driven genes in preovulatory follicles focusing on those involved in the synthesis of progesterone. One such gene was identified, encoding steroidogenic acute regulatory protein (StAR), which showed 24-h changes in expression in the F1 follicle coinciding with those of Per2. Evidence that StAR gene expression is clock driven was obtained by showing that its 5' flanking region contains E-box enhancers that bind to CLOCK/BMAL1 heterodimers to activate gene transcription. We also showed that LH administration increased the promoter activity of chicken StAR. We therefore suggest that the timing of ovulation in poultry involves an LH-responsive F1 follicular clock that is involved in the timing of the preovulatory release of progesterone.

Photoperiodic changes in hypothalamic insulin receptor gene expression are regulated by gonadal testosterone.

In order to adapt to seasonal changes, animals exhibit robust changes in their reproductive status, body weight, and molt. However, the molecular mechanisms regulating such seasonal changes in physiology and behavior are not fully understood. Here, we report the photoperiodic regulation of the insulin receptor (IR) gene in the infundibular nucleus (anatomically homologous to the mammalian arcuate nucleus) of the Japanese quail. When the birds were transferred from short-day to long-day conditions, a significant increase in the level of IR mRNA was observed on the 10th long day, whereas that in testicular length was observed on the 5th long day. Castration abolished IR mRNA expression induced by long-day conditions, whereas the testosterone administration mimicked induction of IR mRNA expression induced by long-day conditions. These results suggested that the photoperiodic regulation of the IR mRNA in the infundibular nucleus is mediated by testosterone from the testes. It has been known that the central administration of insulin increases luteinizing hormone (LH) secretion, and neuron-specific disruption of IR gene causes impaired gonadal function due to the dysregulation of LH and increased food intake and body weight. Together with these results, the photoperiodic regulation of the IR mRNA in the hypothalamus may enhance the effect of long days in the seasonal response of reproduction and body weight changes.

Possible involvement of organic anion transporting polypeptide 1c1 in the photoperiodic response of gonads in birds.

The photoperiodic response of the gonads requires T3, which is generated photoperiodically from T4 by type 2 iodothyronine deiodinase in the hypothalamus. Although thyroid hormones were long thought to traverse the plasma membrane by passive diffusion due to their lipophilic nature, it is now known that several organic anion transporting polypeptides (Oatp) transport thyroid hormones into target cells. In this study, we have used database searches to isolate DNA sequences encoding members of the chicken Oatp family and constructed a molecular phylogenetic tree. Comprehensive expression analyses using in situ hybridization revealed strong expression of cOatp1c1 and weak expression of cOatp1b1 in the ventro-lateral walls of basal tuberal hypothalamus, whereas expression of four genes (cOatp1a1, cOatp1b1, cOatp1c1, and cOatp3a2) was observed in the choroid plexus. Expression levels of all these genes in both regions were not different between short-day and long-day conditions. Functional expression of cOatp1c1 in Chinese hamster ovary cells revealed that cOatp1c1 is a highly specific transporter for T4 with an apparent Km of 6.8 nm and a Vmax of 1.50 pmol per milligram of protein per minute. These results suggest that cOatp1c1 could be involved in the thyroxine transport necessary for the avian photoperiodic response of the gonads.

Long-day suppressed expression of type 2 deiodinase gene in the mediobasal hypothalamus of the Saanen goat, a short-day breeder: implication for seasonal window of thyroid hormone action on reproductive neuroendocrine axis.

In most animals that live in temperate regions, reproduction is under photoperiodic control. In long-day breeders such as Japanese quail and Djungarian hamsters, type 2 deiodinase (Dio2) plays an important role in the mediobasal hypothalamus, catalyzing the conversion of prohormone T4 to bioactive T3 to regulate the photoperiodic response of the gonads. However, the molecular basis for seasonal reproduction in short-day breeders remains unclear. Because thyroid hormones are also known to be involved in short-day breeders, we examined the effect of an artificial long-day stimulus on Dio2 expression in the male Saanen goat (Capra hircus), a short-day breeder. Dio2 expression was observed in the caudal continuation of the arcuate nucleus, known as the target site for both melatonin and T4 action. In addition, expression of Dio2 and T3 content in the mediobasal hypothalamus was suppressed by artificial long-day conditions, which is the opposite of the results of long-day breeders. Thyroid hormone action on the development of neuroendocrine anestrus is known to be limited to a specific seasonal window. This long-day suppression of Dio2 may provide a mechanism that accounts for the lack of responsiveness to thyroxine during the mid to late anestrus.

Peripheral clock gene expression in CS mice with bimodal locomotor rhythms.

CS mice show unique properties of circadian rhythms: unstable free-running periods and distinct bimodal rhythms (similar to rhythm splitting, but hereafter referred to as bimodal rhythms) under constant darkness. In the present study, we compared clock-related gene expression (mPer1, mBmal1 and Dbp) in the SCN and peripheral tissues (liver, adrenal gland and heart) between CS and C57BL/6J mice. In spite of normal robust oscillation in the SCN of both mice, behavioral rhythms and peripheral rhythms of clock-related genes were significantly different between these mice. However, when daytime restricted feeding was given, no essential differences between the two strains were observed. These results indicate that unusual circadian behaviors and peripheral gene expression in CS mice do not depend on the SCN but rather mechanisms outside of the SCN.