Sensitivity of a bone-equivalent polymer gel dosimeter for measuring the dose to bone during radiation therapy.

Treatment planning systems that use the Monte Carlo algorithm can calculate the dose to the medium (Dm) in non-water-equivalent tissues such as bones. However, Dm cannot be verified using actual measurements; therefore, it is necessary to develop tissue-equivalent dosimeters. In this study, we developed a bone-equivalent polymer gel dosimeter (BPGD) that can measure the dose absorbed by the bone and investigated its sensitivity. The BPGDs were prepared by adding 3.0 mol of calcium hydrogen phosphate dihydrate as a component of bone to an improved dose-sensitive polyacrylamide gelatin and tetrakis hydroxymethyl phosphonium chloride (iPAGAT). One day after preparation, the BPGDs were irradiated with a field size of 15 × 15 cm2 using a 10 MV X-ray beam to evaluate the dose sensitivity, dose-rate dependence, and dose-integration dependence. One day after dose exposure, the BPGDs were scanned using a 0.4 T MRI APERTO Eterna (Hitachi, Tokyo, Japan) to obtain R2 values. The difference between the R2 values of 6 Gy and 0 Gy was up to 5 s-1, and the R2 curve plateaued in the high-dose region. Moreover, the BPGD did not depend on the integration of the dose and dose rates. Therefore, the BPGDs that we developed can determine the radiation dose to bones.

Expression profiles of types 2 and 3 iodothyronine deiodinase genes in relation to vitellogenesis in a tropical damselfish, Chrysiptera cyanea.

Thyroid hormone (TH) is involved in regulating the reproduction of vertebrates. Its physiological action in the target tissues is due to the conversion of TH by iodothyronine deiodinases. In this study, we aimed to clone and characterize type 2 (sdDio2) and type 3 (sdDio3) of the sapphire devil Chrysiptera cyanea, a tropical damselfish that undergoes active reproduction under long-day conditions, and to study the involvement of THs in the ovarian development of this species. When the cDNAs of sdDio2 and sdDio3 were partially cloned, they had deduced amino acid sequences of lengths 271 and 267, respectively, both of which were characterized by one selenocysteine residue. Real-time quantitative PCR (qPCR) revealed that both genes are highly expressed in the whole brain, and sdDio2 and sdDio3 are highly transcribed in the liver and ovary, respectively. In situ hybridization analyses showed positive signals of sdDio2 and sdDio3 transcripts in the hypothalamic area of the brain. Little change in mRNA abundance of sdDio2 and sdDio3 in the brain was observed during the vitellogenic phases. It is assumed that simultaneous activation and inactivation of THs occur in this area because oral administration of triiodothyronine (T3), but not of thyroxine (T4), upregulated mRNA abundance of both genes in the brain. The transcript levels of sdDio2 in the liver and sdDio3 in the ovary increased as vitellogenesis progressed, suggesting that, through the metabolism of THs, sdDio2 and sdDio3 play a role in vitellogenin synthesis in the liver and yolk accumulation/E2 synthesis in the ovary. Taken together, these results suggest that iodothyronine deiodinases act as a driver for vitellogenesis in tropical damselfish by conversion of THs in certain peripheral tissues.

Effects of temperature and melatonin on day-night expression patterns of arginine vasotocin and isotocin mRNA in the diencephalon of a temperate wrasse Halichoeres tenuispinis.

Most wrasses are protogynous species that swim to feed, reproduce during the daytime, and bury themselves under the sandy bottom at night. In temperate and subtropical wrasses, low temperature influences emergence from the sandy bottom in the morning, and induces a hibernation-like state in winter. We cloned and characterized the prohormone complementary DNAs (cDNAs) of arginine vasotocin (AVT) and isotocin (IT) in a temperate wrasse (Halichoeres tenuispinis) and examined the effects of day/night and temperature on their expression in the diencephalon, because these neurohypophysial peptides are related to the sex behavior of wrasses. The full-length cDNAs of pro-AVT and pro-IT were 938 base pairs (154 amino acids) and 759 base pairs (156 amino acids) in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Reverse-transcription polymerase chain reaction (RT-PCR) revealed that pro-AVT mRNA expression was specifically observed in the diencephalon, whereas pro-IT mRNA expression was seen in the whole brain. Quantitative RT-PCR revealed that the mRNA abundance of pro-AVT and pro-IT was higher at midday (zeitgeber time 6; ZT6) than at midnight (ZT18) under 12 h light and 12 h darkness (LD 12:12) conditions, but not under constant light. Intraperitoneal injection of melatonin decreased the mRNA abundance of pro-AVT, but not of pro-IT. When fish were reared under LD 12:12 conditions at 25, 20, and 15 °C, day high and night low mRNA expressions of pro-AVT and pro-IT were maintained. A field survey revealed seasonal variation in the number of swimming fish at observatory sites; many fish emerged from the sandy bottom in summer, but not in winter, suggesting a hibernation-like state under the sandy bottom under low temperature conditions. We conclude that the day-night fluctuation of pro-AVT and pro-IT mRNA abundance in the brain is not affected by temperature and repeated under the sandy bottom in winter.

Hypothalamic expression and moonlight-independent changes of Cry3 and Per4 implicate their roles in lunar clock oscillators of the lunar-responsive Goldlined spinefoot.

Lunar cycle-associated physiology has been found in a wide variety of organisms. Studies suggest the presence of a circalunar clock in some animals, but the location of the lunar clock is unclear. We previously found lunar-associated expression of transcripts for Cryptochrome3 gene (SgCry3) in the brain of a lunar phase-responsive fish, the Goldlined spinefoot (Siganus guttatus). Then we proposed a photoperiodic model for the lunar phase response, in which SgCry3 might function as a phase-specific light response gene and/or an oscillatory factor in unidentified circalunar clock. In this study, we have developed an anti-SgCRY3 antibody to identify SgCRY3-immunoreactive cells in the brain. We found immunoreactions in the subependymal cells located in the mediobasal region of the diencephalon, a crucial site for photoperiodic seasonal responses in birds. For further assessment of the lunar-responding mechanism and the circalunar clock, we investigated mRNA levels of Cry3 as well as those of the other clock(-related) genes, Period (Per2 and Per4), in S. guttatus reared under nocturnal moonlight interruption or natural conditions. Not only SgCry3 but SgPer4 mRNA levels showed lunar phase-dependent variations in the diencephalon without depending on light condition during the night. These results suggest that the expressions of SgCry3 and SgPer4 are not directly regulated by moonlight stimulation but endogenously mediated in the brain, and implicate that circadian clock(-related) genes may be involved in the circalunar clock locating within the mediobasal region of the diencephalon.

Expression of type II iodothyronine deiodinase gene in the brain of a tropical spinefoot, Siganus guttatus.

Type II iodothyronine deiodinase (D2) converts 3,5,3',5'-tetraiodothyronine to 3,5,3'-triiodothyronine and is involved in regulating thyroid hormone-dependent processes in various tissues. D2 mRNA expression in the mediobasal hypothalamus is affected by photoperiod, which influences reproductive processes in temperate birds and mammals. We examined whether D2 mRNA is expressed in the hypothalamus (located in the forebrain within the diencephalon area) and whether its abundance is affected by day length, temperature, or food availability in the tropical spinefoot, Siganus guttatus, which is endemic to tropical monsoon areas. The reverse transcription-polymerase chain reaction (RT-PCR) revealed that D2 mRNA is expressed in various brain regions. The abundance of hypothalamic D2 mRNA was higher at 12.00h than at 06.00h or 24.00h. Rearing fish under constant dark conditions resulted in a decrease in D2 mRNA abundance during the subjective night. A single injection of melatonin lowered D2 mRNA abundance within 3h. Collectively, it appears that hypothalamic D2 mRNA abundance is regulated by the circadian system and/or melatonin. No differences in D2 mRNA abundance were observed, when fish were reared at 20, 25, and 30°C. However, food deprivation stimulated D2 mRNA expression during the daytime. These results suggest that photoperiodic and nutritive conditions affect hypothalamic D2 mRNA expression in S. guttatus.

Isolation and characterization of DMRT1 and its putative regulatory region in the protogynous wrasse, Halichoeres tenuispinis.

A full-length cDNA of doublesex and mab-3-related transcription factor 1 gene (DMRT1) from wrasse testis was isolated by cDNA library screening. Wrasse DMRT1 was 3116 bp in size and contained the DM domain, with a zinc finger DNA-binding motif, and the male-specific motif. Northern blot analysis identified a 3.2-kb transcript approximately equal in size to the DMRT1 nucleotide sequence detected in the testis, but not in the ovary, confirming that this sequence is male-specific in protogynous wrasse. Southern blot analysis suggested that the wrasse genome contains two copies of the DMRT1 gene. The ORF consisted of five exons and four introns with conserved donor-acceptor splice sites at all exon-intron junctions. The 5'-flanking region of the wrasse DMRT1 gene was isolated by DNA walking, and putative regulatory sites were identified by searching data bases. The 5'-flanking region was divided into 9 elements, then 17 DMRT-luciferase chimeric plasmids were constructed. By transient transfection into Cos-1 and TM4 cells, distal element I which contains GATA-binding sites and proximal element B containing the sex-determining region on Y chromosome gene (SRY) binding site were revealed to have an important role in transcriptional regulation of the wrasse DMRT1 when an enhancer sequence was provided.

Expression of the melatonin receptor Mel(1c) in neural tissues of the reef fish Siganus guttatus.

The golden rabbitfish, Siganus guttatus, is a reef fish exhibiting a restricted lunar-related rhythm in behavior and reproduction. Here, to understand the circadian rhythm of this lunar-synchronized spawner, a melatonin receptor subtype-Mel(1c)-was cloned. The full-length Mel(1c) melatonin receptor cDNA comprised 1747 bp with a single open reading frame (1062 bp) that encodes a 353-amino acid protein, which included 7 presumed transmembrane domains. Real-time PCR revealed high Mel(1c) mRNA expression in the retina and brain but not in the peripheral tissues. When the fish were reared under light/dark (LD 12:12) conditions, Mel(1c) mRNA in the retina and brain was expressed with daily variations and increased during nighttime. Similar variations were noted under constant conditions, suggesting that Mel(1c) mRNA expression is regulated by the circadian clock system. Daily variations of Mel(1c) mRNA expression with a peak at zeitgeber time (ZT) 12 were observed in the cultured pineal gland under LD 12:12. Exposure of the cultured pineal gland to light at ZT17 resulted in a decrease in Mel(1c) mRNA expression. When light was obstructed at ZT5, the opposite effect was obtained. These results suggest that light exerts certain effects on Mel(1c) mRNA expression directly or indirectly through melatonin actions.