Screening for DAX1/EWS-FLI1 functional inhibitors identified dihydroorotate dehydrogenase as a therapeutic target for Ewing's sarcoma.

OBJECTIVE: EWS-FLI1 is the most common oncogenic fusion protein in Ewing's sarcoma family tumors (ESFTs). DAX1, an orphan member of the nuclear receptor superfamily, is up-regulated by EWS-FLI1 and plays a key role in the transformed phenotype of ESFTs. METHODS: To discover a functional inhibitor of DAX1 and EWS-FLI1, we screened small-molecular inhibitors using a DAX1 reporter assay system. RESULTS: K-234 and its derivatives, which were dihydroorotate dehydrogenase (DHODH) inhibitors, showed inhibitory effects in the reporter assay. K-234 inhibited the growth of Ewing's sarcoma with various fusion types, and K-234 derivatives altered the expression of EWS-FLI1-regulated genes. The DAX1 expression had no effect on the growth inhibitory effect of the K-234 derivatives, while DHODH overexpression or uridine treatment attenuated their inhibitory effects, suggesting that inhibition by K-234 derivatives occurs through DHODH inhibition. An in vivo study showed that a K-234 derivative clearly inhibited tumor growth in an Ewing's sarcoma xenograft mouse model. CONCLUSION: Taken together, the present results suggest that DHODH inhibitors can inhibit the function of DAX1/EWS-FLI1 in ESFTs and might be a therapeutic agent with potent anti-tumor activity for Ewing's sarcoma patients.

The Association of Family Functioning With Possible Major Depressive Disorders and Complicated Grief Among Bereaved Family Members of Patients With Cancer: Results From the J-HOPE4 Study, a Nationwide Cross-Sectional Follow-Up Survey in Japan.

CONTEXT: Family functioning is a modifiable factor associated with major depressive disorder (MDD) and complicated grief (CG) among the bereaved families of patients with advanced cancer; however, the evidence regarding this association is limited. OBJECTIVES: We aimed to explore the association of family functioning with possible MDD and CG among the bereaved families of patients with advanced cancer who died in palliative care units. METHODS: This study is a part of the J-HOPE4 study, a nationwide cross-sectional multi-purpose questionnaire survey conducted in 2018. We recruited potential participants from 164 inpatient palliative care units in Japan and assessed family functioning with the Family Relations Index (FRI). Family functioning was classified into three categories (Well-functioning: FRI ≥ 10, Intermediate: FRI = 8〜9, Dysfunctioning ≤ 7). The Patient Health Questionnaire 9 (PHQ-9) and the Brief Grief Questionnaire (BGQ) were used to assess depression (PHQ-9 score ≥ 10) and complicated grief (BGQ score ≥ 8), respectively. Multinomial logistic regression analysis was performed with possible MDD and CG and factors the family functioning. RESULTS: A total of 615 questionnaires were returned, of which 54.0 % (n = 510) age of questionnaires could be used. Although family functioning was associated with possible MDD (21.1% in Dysfunctional; 9.3% in Well-functional, P = 0.016), it was not associated with possible CG (14.8% in Dysfunctional; 9.9% in Well-functional, P = 0.929). Possible MDD and CG were significantly associated with deteriorated family relationships (OR:8.29; P = 0.004 and OR:34.00; P < 0.001, respectively), and consulting with health care providers about their concerns (OR:0.23; P = 0.003 and OR:0.23; P = 0.003, respectively). CONCLUSIONS: Family function was affected by post-bereavement possible MDD and not by CG. Our findings suggest that health care providers can identify risk factors for MDD among bereaved, dysfunctional family members.

The dual pocket binding novel tankyrase inhibitor K-476 enhances the efficacy of immune checkpoint inhibitor by attracting CD8+ T cells to tumors.

The Wnt/ß-catenin pathway, which is associated with disease progression, is activated in many cancers. Tankyrase (TNKS) has received attention as a target molecule for Wnt/ß-catenin pathway inhibition. We identified K-476, a novel TNKS inhibitor, a dual pocket binder that binds to both the nicotinamide and ADP-ribose pockets. In a human colon cancer cell line, K-476 specifically and potently inhibited TNKS and led to stabilization of the Axin protein, resulting in Wnt/ß-catenin pathway suppression. Aberrant Wnt/ß-catenin pathway activation was recently reported as a possible mechanism of ineffectiveness in immune checkpoint inhibitor (ICI) treatment. Because the Wnt/ß-catenin pathway activation causes dendritic cell inactivation and suppresses chemokine production, resulting in a paucity of CD8+ T cells in tumor tissue, which is an important effector of ICIs. Thus, TNKS inhibitors may enhance the efficacy of ICIs. To examine whether K-476 enhances the antitumor effect of anti-PD-L1 antibodies, K-476 was administered orally with an anti-PD-L1 antibody to melanoma-bearing C57BL/6J mice. Although K-476 was ineffective as a monotherapy, it significantly enhanced the antitumor effect in combination with anti-PD-L1 antibody. In mice, intra-tumor infiltration of CD8+ T cells was increased by combination treatment. K-476 upregulated the chemokine expression (e.g., Ccl3 and Ccl4), which attracted CD8+ T cells. This was considered to contribute to the increased CD8+ T cells in the tumor microenvironment. Furthermore, while the potential gastrointestinal toxicity of TNKS inhibitors has been reported, it was not observed at effective doses. Thus, K-476 could be an attractive therapeutic option to enhance the efficacy of ICIs.

Multiple Chemosensory Neurons Mediate Avoidance Behavior to Rare Earth Ions in Caenorhabditis elegans.

As rare earth (RE) metals are abundantly present in the soil, in spite of their name, it is conceivable that organisms may encounter and interact with RE ions. In the present study, we demonstrated that the soil nematode Caenorhabditis elegans avoids RE ions, such as yttrium and all examined lanthanide ions, which exhibit toxic effects on nematodes. We also demonstrated that the chemosensory system of this animal mediates avoidance behavior toward RE ions similar to heavy metal (HM) ion avoidance. The C. elegans dyf-11(pe554) mutant is unable to respond to chemosensory cues because it lacks all ciliated endings of the chemosensory neurons required for the detection of environmental chemicals. Cell-specific rescue of the dyf-11 mutant and cell-specific genetic ablation studies revealed that the avoidance behavior toward HM and RE ions was mediated by a partially overlapping but distinct subset of chemosensory neurons (ASH, ADL, ASE, ADF, and ASK). With the help of multiple chemosensory neurons, worms may improve the fidelity of avoidance behavior to evade RE ions. Among the chemosensory neurons in C. elegans, ADF and ASK neurons were involved in RE avoidance, but not in HM avoidance. These results suggested that ADF and ASK neurons in C. elegans have RE-selective mechanisms to mediate the avoidance response.

Species differences in ocular pharmacokinetics and pharmacological activities of regorafenib and pazopanib eye-drops among rats, rabbits and monkeys.

Age-related macular degeneration (AMD) is the leading cause of severe vision impairment in patients over the age of 60 years. Choroidal neovascularization (CNV) is the hallmark of neovascular AMD and vascular endothelial growth factor (VEGF) plays a causal role in the formation of CNV. Although regorafenib and pazopanib, small molecule VEGF receptor (VEGFR) inhibitors, were developed as eye-drops, their efficacies were insufficient in clinical. In this study, we evaluated ocular pharmacokinetics and pharmacological activities of regorafenib and pazopanib after ocular instillation in multiple animal species. In rats, both regorafenib and pazopanib showed high enough concentrations in the posterior eye tissues to inhibit VEGFR. In laser-induced rat CNV model, regorafenib showed clear reduction in CNV area. On the other hand, the concentrations of regorafenib and pazopanib in the posterior eye tissues were much lower after ocular instillation in rabbits and monkeys compared to those in rats. Pazopanib did not show any improvement in monkey model. Regorafenib was nano-crystalized to improve its drug delivery to the posterior eye tissues. The nano-crystalized formulation of regorafenib showed higher concentrations in the posterior segments in rabbits compared to its microcrystal suspension. From these studies, large interspecies differences were found in ocular delivery to the posterior segments after ocular instillation. Such large interspecies difference could be the reason for the insufficient efficacies of regorafenib and pazopanib in clinical studies. Nano-crystallization was suggested to be one of the effective ways to overcome this issue.

The discovery and characterization of K-563, a novel inhibitor of the Keap1/Nrf2 pathway produced by Streptomyces sp.

Keap1/Nrf2 pathway regulates the antioxidant stress response, detoxification response, and energy metabolism. Previous reports found that aberrant Keap1/Nrf2 pathway activation due to Kelch-like ECH-associated protein 1 (Keap1) mutations or Nuclear factor E2-related factor 2 (Nrf2) mutations induced resistance of cancer cells to chemotherapy and accelerated cell growth via the supply of nutrients. Therefore, Keap1/Nrf2 pathway activation is associated with a poor prognosis in many cancers. These previous findings suggested that inhibition of Keap1/Nrf2 pathway could be a target for anti-cancer therapies. To discover a small-molecule Keap1/Nrf2 pathway inhibitor, we conducted high-throughput screening in Keap1 mutant human lung cancer A549 cells using a transcriptional reporter assay. Through this screening, we identified the novel Keap1/Nrf2 pathway inhibitor K-563, which was isolated from actinomycete Streptomyces sp. K-563 suppressed the expression of Keap1/Nrf2 pathway downstream target genes or the downstream target protein, which induced suppression of GSH production, and activated reactive oxygen species production in A549 cells. K-563 also inhibited the expression of downstream target genes in other Keap1- or Nrf2-mutated cancer cells. Furthermore, K-563 exerted anti-proliferative activities in these mutated cancer cells. These in vitro analyses showed that K-563 was able to inhibit cell growth in Keap1- or Nrf2-mutated cancer cells by Keap1/Nrf2 pathway inhibition. K-563 also exerted synergistic combinational effects with lung cancer chemotherapeutic agents. An in vivo study in mice xenotransplanted with A549 cells to further explore the therapeutic potential of K-563 revealed that it also inhibited Keap1/Nrf2 pathway in lung cancer tumors. K-563, a novel Keap1/Nrf2 pathway inhibitor, may be a lead compound for development as an anti-cancer agent.

A comparative study for detecting CYP3A induction by CYP3A probe drugs and endogenous markers in cynomolgus monkeys.

CYP3A probe drugs such as midazolam and endogenous markers, and plasma 4ß-hydroxycholesterol (4ß-OHC) and urinary 6ß-hydroxycortisol-to-cortisol ratios (6ß-OHC/C) have been used as markers of CYP3A induction in cynomolgus monkeys, as with humans. However, there is limited information on their sensitivity and ability to detect CYP3A induction, as most studies were evaluated only at a high dose of the inducer, rifampicin (RIF; 20 mg/kg). In the present study, the CYP3A induction by RIF over a range doses of 0.2, 2 and 20 mg/kg (n = 4) was examined using CYP3A probe drugs (midazolam, triazolam and alprazolam) and the plasma and urinary endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C). The sensitivity and relationship for detecting CYP3A induction was compared among the markers. Four days repeated oral administration of rifampicin to cynomolgus monkeys reduced the area under the plasma concentration-time curve of all CYP3A probe drugs in a rifampicin dose-dependent manner. Although the endogenous CYP3A markers (4ß-OHC and 6ß-OHC/C) were also changed for the middle (2 mg/kg) and high (20 mg/kg) doses of rifampicin, the fold-changes were relatively small, and CYP3A induction could not be detected at the lowest dose of rifampicin (0.2 mg/kg). In conclusion, CYP3A probe drugs are more sensitive for detecting CYP3A induction than endogenous CYP3A markers in cynomolgus monkeys, even for a short experimental period.

Influence of glucocorticoids on time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor-mediated systemic anaphylaxis in different mouse strains.

A time-of-day-dependent variation in IgE-mediated passive systemic anaphylaxis was previously reported in ICR mice. In the present study, we investigated time-of-day-dependent variations in IgE-, histamine-, and platelet-activating factor (PAF)-mediated systemic anaphylaxis in C57BL/6, BALB/c, and NC/Nga mice at 9:00 h and 21:00 h, and evaluated the potential influence of glucocorticoids (GCs) on these variations. We found significant time-of-day-dependent variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice, and in histamine- and PAF-mediated systemic anaphylaxis in BALB/c mice. Significant daily variations in IgE-, histamine-, and PAF-mediated systemic anaphylaxis were not observed in NC/Nga mice. Pretreatment with dexamethasone and adrenalectomy abolished the daily variations in IgE-mediated systemic anaphylaxis in C57BL/6 mice and in PAF-mediated systemic anaphylaxis in BALB/c mice, suggesting that GCs from adrenal glands are pivotal in regulating these variations. In contrast, pretreatment with dexamethasone and adrenalectomy did not abolish the daily variation in histamine-mediated systemic anaphylaxis in BALB/c mice, suggesting that GC-independent and adrenal gland-independent mechanisms are important for the variation. The present study demonstrated that time-of-day-dependent variations in systemic anaphylaxis differed among inbred mouse strains and with anaphylaxis-inducing substances. Thus, mouse strains, time of experiment, and anaphylaxis-inducing substances used must be considered to obtain appropriate experimental results.

Characterization of platelet-activating factor-induced cutaneous edema and erythema in dogs.

OBJECTIVE To characterize platelet-activating factor (PAF)-induced edema and erythema in the skin of dogs and compare those reactions with histamine-induced cutaneous reactions. ANIMALS 6 healthy Beagles. PROCEDURES Experiments were performed at ≥ 2-week intervals. Each dog received ID injections (5 µg/site) of PAF C16, PAF C18, lyso-PAF, and histamine. Edema (mean diameter) and erythema scores (none, mild, moderate, or severe) were assessed 30 minutes after the injections. Dogs received ID injections of PAF and histamine each with various concentrations of WEB 2086 (PAF receptor antagonist) or underwent ID testing with PAF and histamine before and 3 hours after oral administration of cetirizine hydrochloride or prednisolone (at 2 doses each). RESULTS ID injections of PAF C16 and PAF C18, but not lyso-PAF, induced comparable levels of edema and erythema. The PAF-induced edema and erythema peaked at 30 minutes and lasted for 6 hours after the injection; histamine-induced edema and erythema peaked at 30 minutes and lasted for 3 hours after the injection. Edema sizes and erythema scores were significantly smaller and lower, respectively, for PAF than for histamine. The WEB 2086 inhibited PAF-induced but not histamine-induced edema and erythema. Cetirizine slightly, but significantly, repressed PAF-induced edema and erythema as well as histamine-induced cutaneous reactions. Prednisolone suppressed both PAF-induced and histamine-induced edema and erythema. CONCLUSIONS AND CLINICAL RELEVANCE In canine skin, the duration of PAF-induced inflammation was longer than that of histamine-induced inflammation. The PAF- and histamine-induced cutaneous reactions were effectively suppressed by oral administration of prednisolone. The importance of PAF in dogs with anaphylaxis and allergic disorders warrants further investigation.

Influence of glucocorticoids on a time-of-day-dependent variation in intradermal reactivity to histamine in dogs.

The objectives of this study were to determine daily variation in intradermal reactivity to histamine in dogs and to evaluate a potential influence of glucocorticoids on reactivity. Wheal sizes formed after intradermal injections of histamine were measured every 6 h during a single 24 h period in six healthy dogs. To determine whether glucocorticoids were implicated in daily variation, intradermal reactivity to histamine was evaluated at 9:00 h and at 21:00 h during a single day in dogs that received oral prednisolone (a synthetic glucocorticoid) or oral trilostane (an inhibitor of endogenous glucocorticoid synthesis). Finally, the time required for the histamine reaction to diminish after an intravenous injection of hydrocortisone was also assessed. A significant time-of-day-dependent variation in intradermal reactivity to histamine was detected in dogs, with a larger wheal size observed at 9:00 h than at 21:00 h. Administration of prednisolone or trilostane disrupted this variation. Intradermal reactivity to histamine was significantly reduced 6 h after an intravenous injection of hydrocortisone. These results suggest that glucocorticoid secretion from the adrenal glands could be involved in the regulation of daily variation in histamine-mediated reactions in dogs.

Navigational choice between reversal and curve during acidic pH avoidance behavior in Caenorhabditis elegans.

BACKGROUND: Under experimental conditions, virtually all behaviors of Caenorhabditis elegans are achieved by combinations of simple locomotion, including forward, reversal movement, turning by deep body bending, and gradual shallow turning. To study how worms regulate these locomotion in response to sensory information, acidic pH avoidance behavior was analyzed by using worm tracking system. RESULTS: In the acidic pH avoidance, we characterized two types of behavioral maneuvers that have similar behavioral sequences in chemotaxis and thermotaxis. A stereotypic reversal-turn-forward sequence of reversal avoidance caused an abrupt random reorientation, and a shallow gradual turn in curve avoidance caused non-random reorientation in a less acidic direction to avoid the acidic pH. Our results suggest that these two maneuvers were each triggered by a distinct threshold pH. A simulation study using the two-distinct-threshold model reproduced the avoidance behavior of the real worm, supporting the presence of the threshold. Threshold pH for both reversal and curve avoidance was altered in mutants with reduced or enhanced glutamatergic signaling from acid-sensing neurons. CONCLUSIONS: C. elegans employ two behavioral maneuvers, reversal (klinokinesis) and curve (klinotaxis) to avoid acidic pH. Unlike the chemotaxis in C. elegans, reversal and curve avoidances were triggered by absolute pH rather than temporal derivative of stimulus concentration in this behavior. The pH threshold is different between reversal and curve avoidance. Mutant studies suggested that the difference results from a differential amount of glutamate released from ASH and ASK chemosensory neurons.

Involvement of thyrotropin in photoperiodic signal transduction in mice.

Local thyroid hormone catabolism within the mediobasal hypothalamus (MBH) by thyroid hormone-activating (DIO2) and -inactivating (DIO3) enzymes regulates seasonal reproduction in birds and mammals. Recent functional genomics analysis in birds has shown that long days induce thyroid-stimulating hormone production in the pars tuberalis (PT) of the pituitary gland, which triggers DIO2 expression in the ependymal cells (EC) of the MBH. In mammals, nocturnal melatonin secretion provides an endocrine signal of the photoperiod to the PT that contains melatonin receptors in high density, but the interface between the melatonin signal perceived in the PT and the thyroid hormone levels in the MBH remains unclear. Here we provide evidence in mice that TSH participates in this photoperiodic signal transduction. Although most mouse strains are considered to be nonseasonal, a robust photoperiodic response comprising induced expression of TSHB (TSH beta subunit), CGA (TSH alpha subunit), and DIO2, and reduced expression of DIO3, was observed in melatonin-proficient CBA/N mice. These responses could not be elicited in melatonin-deficient C57BL/6J, but treatment of C57BL/6J mice with exogenous melatonin elicited similar effects on the expression of the above-mentioned genes as observed in CBA/N after transfer to short-day conditions. The EC was found to express TSH receptor (TSHR), and ICV injection of TSH induced DIO2 expression. Finally, we show that melatonin administration did not affect the expression of TSHB, DIO2, and DIO3 in TSHR-null mice. Taken together, our findings suggest that melatonin-dependent regulation of thyroid hormone levels in the MBH appears to involve TSH in mammals.

A surface plasmon resonance sensor based on 3,5-diaminopyrazine with a high selectivity for thymine in AP site-containing DNA duplex.

We here report on a surface plasmon resonance (SPR) sensor carrying small organic ligands for the detection of single-nucleotide polymorphisms (SNPs). Two kinds of ligands are prepared, both of which have a hydrogen-bond forming site suitable for nucleobase recognition, and have an active amino group for the immobilization to the sensor chip. While the sensor immobilized flavin does not show any useful responses, the sensor based on 3,5-diaminopyrazine shows a highly selective response to thymine over other nucleobases opposite an abasic site in DNA duplexes (5'-GTT GGA GCT GXG GGC GTA GGC-3'/3'-CAA CCT CGA CNC CCG CAT CCG-5', X = AP site, N = target; G, C, A, T). In PBS buffer (pH 6.4, 0.25 M NaCl, at 5 degrees C), the sensor can detect 10 nM of the sample solution, and the SPR signal for thymine is linear in the concentration range from 10 nM to 100 nM. These sensing functions of the present sensor are discussed for the development of SNPs detection chemistry based on DNA-binding small molecules.

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.

New route to enantiopure MalphaNP acid, a powerful resolution and chiral 1H NMR anisotropy reagent.

MalphaNP acid (+/-)-1, 2-methoxy-2-(1-naphthyl)propionic acid, was enantioresolved by the use of phenylalaninol (S)-(-)-4; a diastereomeric mixture of amides formed from acid (+/-)-1 and amine (S)-(-)-4 was easily separated by fractional recrystallization and/or HPLC on silica gel, yielding amides (R;S)-(-)-5a and (S;S)-(+)-5b. Their absolute configurations were determined by X-ray crystallography by reference to the S configuration of the phenylalaninol moiety. Amide (R;S)-(-)-5a was converted to oxazoline (R;S)-(+)-8a, from which enantiopure MalphaNP acid (R)-(-)-1 was recovered. In a similar way, enantiopure MalphaNP acid (S)-(+)-1 was obtained from amide (S;S)-(+)-5b. These reactions provide a new route for the large-scale preparation of enantiopure MalphaNP acid, a powerful chiral reagent for the enantioresolution of alcohols and simultaneous determination of their absolute configurations by (1)H NMR anisotropy.

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.

Hypothalamic expression of thyroid hormone-activating and -inactivating enzyme genes in relation to photorefractoriness in birds and mammals.

Photorefractoriness is the insensitivity of gonadal development to the stimulatory effects of long photoperiods in birds and to the inhibitory effects of short photoperiods in small mammals. Its molecular mechanism remains unknown. Recently, it has been shown that reciprocal expression of thyroid hormone-activating enzyme [type 2 deiodinase (Dio2)] and -inactivating enzyme [type 3 deiodinase (Dio3)] genes in the mediobasal hypothalamus is critical for photoperiodically induced gonadal growth. Since thyroid hormones are required not only for photoinduction, but also for the induction of photorefractoriness, we examined the expression of these genes in relation to photorefractoriness in birds and mammals. Transfer of birds to long photoperiods induced strong expression of Dio2. This was maintained in tree sparrow when they later became photorefractory, but decreased somewhat in quail. In hamsters, transfer to long photoperiods also induced strong expression of Dio2. High values were not maintained under long photoperiods, and, indeed, expression decreased at the same rate as in animals transferred to short photoperiods. There was no renewed expression of Dio2 associated with testicular growth as animals became refractory to short photoperiods. Expression of Dio3 was high under short photoperiods and low under long photoperiods in all the animals examined, except for the short photoperiod-refractory hamsters. Our present study revealed complex regulation of deiodinase genes in the photoinduction and photorefractory processes in birds and mammals. These gene changes may be involved in the regulation of photorefractoriness, as well as photoinduction.

Differential response of type 2 deiodinase gene expression to photoperiod between photoperiodic Fischer 344 and nonphotoperiodic Wistar rats.

The molecular basis of seasonal or nonseasonal breeding remains unknown. Although laboratory rats are generally regarded as photoperiod-insensitive species, the testicular weight of the Fischer 344 (F344) strain responds to photoperiod. Recently, it was clarified that photoperiodic regulation of type 2 iodothyronine deiodinase (Dio2) in the mediobasal hypothalamus (MBH) is critical in photoperiodic gonadal regulation. Strain-dependent differences in photoperiod sensitivity may now provide the opportunity to address the regulatory mechanism of seasonality by studying Dio2 expression. Therefore, in the present study, we examined the effect of photoperiod on Dio2 expression in photoperiod-sensitive F344 and photoperiod-insensitive Wistar rats. A statistically significant difference was observed between short and long days in terms of testicular weight and Dio2 expression in the F344 strain, while no difference was observed in the Wistar strain. These results suggest that differential responses of the Dio2 gene to photoperiod may determine the strain-dependent differences in photoperiod sensitivity in laboratory rats.

Molecular mechanism of photoperiodic time measurement in the brain of Japanese quail.

In most organisms living in temperate zones, reproduction is under photoperiodic control. Although photoperiodic time measurement has been studied in organisms ranging from plants to vertebrates, the underlying molecular mechanism is not well understood. The Japanese quail (Coturnix japonica) represents an excellent model to study this problem because of the rapid and dramatic photoperiodic response of its hypothalamic-pituitary-gonadal axis. Recent investigations of Japanese quail show that long-day-induced type 2 deiodinase (Dio2) expression in the mediobasal hypothalamus (MBH) plays an important role in the photoperiodic gonadal regulation by catalyzing the conversion of the prohormone thyroxine (T(4)) to bioactive 3,5,3'-triiodothyronine (T3). The T3 content in the MBH is approximately 10-fold higher under long than short days and conditions, and the intracerebroventricular infusion of T3 under short days and conditions mimics the photoperiodic gonadal response. While Dio2 generates active T3 from T4 by outer ring deiodination, type 3 deiodinase (Dio3) catalyzes the conversion of both T3 and T4 into inactive forms by inner ring deiodination. In contrast to Dio2 expression, Dio3 expression in the MBH is suppressed under the long-day condition. Photoperiodic changes in the expression of both genes during the photoinduction process occur before the changes in the level of luteinizing hormone (LH) secretion, suggesting that the reciprocal changes in Dio2 and Dio3 expression act as gene switches of the photoperiodic molecular cascade to trigger induction of LH secretion.

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.