Among the RFamide peptide family, the LPXRFamide peptide (LPXRFa) group regulates the release of various pituitary hormones and, recently, LPXRFa genes were found to be regulated by photoperiod via melatonin. As a first step towards investigating the role of LPXRFa on reproductive function in grass puffer (Takifugu niphobles), which spawns in semilunar cycles, genes encoding LPXRFa and its receptor (LPXRFa-R) were cloned, and seasonal, diurnal and circadian changes in their absolute amounts of mRNAs in the brain and pituitary were examined by quantitative real-time polymerase chain reaction. The grass puffer LPXRFa precursor contains two putative RFamide peptides and one possible RYamide peptide. LPXRFa and LPXRFa-R genes were extensively expressed in the diencephalon and pituitary. The expression levels of both genes were significantly elevated during the spawning periods in both sexes in the brain and pituitary, although they were low in the spawning fish just after releasing eggs and sperm. The treatment of primary pituitary cultures with goldfish LPXRFa increased the amounts of follicle-stimulating hormone β- and luteinising hormone β-subunit mRNAs. In the diencephalon, LPXRFa and LPXRFa-R genes showed synchronised diurnal and circadian variations with one peak at zeitgeber time 3 and circadian time 15, respectively. The correlated expression patterns of LPXRFa and LPXRFa-R genes in the diencephalon and pituitary and the possible stimulatory effects of LPXRFa on gonadotrophin subunit gene expression suggest the functional significance of the LPXRFa and LPXRFa-R system in the regulation of lunar-synchronised spawning of grass puffer.

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Melatonin receptor gene expression as well as melatonin synthesis and secretion activities were examined in the pineal gland of the grass puffer, which exhibits unique lunar/tidal cycle-synchronized mass spawing: spawning occurs before high tide on the day of spring tide during spawing season. Melatonin synthesizing activity was assessed by the abundance of arylalkylamine N-acetyltransferase 2 (AANAT2) mRNA. The amount of aanat2 mRNA was low during light phase and initiated to increase after the light was turned off. The secretion of melatonin from primary pineal organ culture was stimulated after the light was turned off and ceased immediately after the light was turned on. The expression levels of four melatonin receptor subtype genes (mel1a1.4, mel1a1.7, mel1b, and mel1c) showed synchronous variations, and the levels tended to be high during the dark phase under light/dark conditions. These results suggest that the action of melatonin on the pineal gland is highly dependent on light and photoperiod, possibly with stronger action during night time. Under constant darkness, the expression of four melatonin receptor subtype genes showed unique ultradian oscillations with the period of 14.0–15.4 h, suggesting the presence of a circatidal oscillator in the pineal gland. The present results indicate that melatonin may serve local chronobiological functions in the pineal gland. These cyclic expressions of melatonin receptor genes in the pineal gland may be important in the control of the lunar/tidal cycle-synchronized mass spawning in the grass puffer.

PurposeThe aim of this study was to evaluate the 8th edition of the American Joint Committee on Cancer Staging Manual: Head and Neck Section on oropharyngeal squamous cell cancer (OPSCC) and to clarify the relationship between p16 overexpression and the presence of human papillomavirus (HPV) DNA using fresh frozen samples.MethodsSamples from 100 OPSCC patients were analyzed using polymerase chain reaction (PCR) and p16 immunohistochemistry.ResultsFive-year overall survival (OS) was 73.0%, 93.9%, and 62.2% in all, p16-positive (n = 34), and p16-negative (n = 66) cases, respectively. OS tended to be better aligned with stage in the 8th edition than in the 7th edition. The 5-year OS was 96.0% in never or light smokers (< 40 pack-years), and 87.5% in heavy smokers (≥ 40 pack-years) in the p16-positive group, respectively (p = 0.027). HPV infection was found in 100% of p16-positive and 21.2% of p16-negative cases. The p16-positive cases had higher viral load and integrated physical status than the p16-negative cases. Although 1 case with p16 overexpression showed no PCR amplification using consensus primers, PCR amplification was detected using HPV 16 E6-specific primers.ConclusionsThe 8th edition predicts OPSCC prognosis more accurately than the 7th edition and p16-overexpression is an excellent surrogate marker for detecting HPV infection. Although high-risk-type HPV infection was observed in p16-negative cases, it showed no significant effect in survival outcome.

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Neuropeptide tyrosine (NPY) is a potent orexigenic neuropeptide implicated in feeding regulation in rodents. However, the involvement of NPY in feeding behavior has not well been studied in fish. Therefore, we investigated the role of NPY in food intake using a tiger puffer (Takifugu rubripes) model. We observed the distribution of NPY-like immunoreactivity in the brain. Neuronal cell bodies containing NPY were located in the telencephalon, hypothalamus, mesencephalon, and medulla oblongata, and their nerve fibers were also found throughout the brain. We cloned two cDNAs, encoding NPYa and NPYb orthologs, respectively, from the brain, and also confirmed two genes encoding these NPYs in the Takifugu genome database. We examined the distribution of these transcripts in the brain using real-time PCR. Levels of NPYa mRNA in the telencephalon, mesencephalon and hypothalamus were much higher than in the medulla oblongata and cerebellum, whereas levels of NPYb mRNA in the medulla oblongata were higher than in other regions. We also examined prandial effects on the expression level of these transcripts in the telencephalon and hypothalamus. NPYa mRNA levels in the hypothalamus, but not in the telencephalon, obtained from fish fasted for one week were higher than those in fish that had been fed normally. The level was decreased at 2 h after feeding. Levels of NPYb mRNA were not affected by prandial conditions. These results suggest that NPY is present throughout the brain, and that NPYa, but not NPYb, in the hypothalamus is involved in the feeding regulation in the tiger puffer.

BackgroundWe aimed to clarify the possible role of human papillomavirus (HPV) infection in the malignant transformation of sinonasal inverted papilloma (IP).MethodsSubjects comprised 32 patients with chronic rhinosinusitis (CRS), 17 with IP, 5 with IP and squamous cell carcinoma (IP + SCC), and 16 with primary sinonasal SCC. HPV presence, viral loads, and physical status were investigated using polymerase chain reaction. Retinoblastoma (pRb), p53, and p16INK4a gene products were investigated by immunohistochemistry.ResultsHPV DNA was detected in 6.3 % of cases with CRS, 29.4 % with IP, 40 % with IP + SCC, and 25 % with SCC. IP cases had significantly higher HPV presence than CRS cases (p = 0.04). High-risk HPV-16 was the most frequently encountered subtype (10/13, 76.9 %). HPV-16 viral loads varied from 2.5 to 7953 E6 copies/50 ng genomic DNA. Patients in the SCC and IP + SCC groups had significantly higher viral loads than those in the IP and CRS groups (p < 0.01). All SCC and IP + SCC patients with HPV-16 demonstrated mixed-type integration, whereas 4 of 5 HPV-16 patients in the IP and CRS groups showed episomal type infection (p = 0.04). Positivity to pRb was found in 78.1 % of CRS, 35.3 % of IP, and 68.8 % of SCC cases. The presence of HPV DNA negatively correlated with pRb expression in SCC (p = 0.029) and IP (P = 0.049) groups. Although 62.5 % of SCC cases exhibited p53 positivity, only 5.9 % of IP, and no CRS cases were positive. Regardless of HPV status, p16INK4a positivity was frequently detected in IP cases (82.4 %), less in SCC (12.5 %) cases, and was not detected in the CRS group. Neither the IP nor SCC cohorts showed any correlation between HPV presence and the expression of either p53 or p16INK4a.ConclusionsHPV infection was more frequent in the IP, IP + SCC, and SCC groups than the CRS group. Higher viral loads and integration observed in the IP + SCC and SCC groups, and an inverse correlation between HPV presence and positive pRb indicated that persistent infection and integration play a part in tumorigenesis and malignant transformation in certain IP cases. However, p16INK4a is not a reliable surrogate marker for HPV infection in IP.