Se Jae Kim

Mitochondrial thioredoxin-2 from disk abalone (Haliotis discus discus): Molecular characterization, tissue expression and DNA protection activity of its recombinant protein

Thioredoxin-2 is a mitochondria-specific member of the thioredoxin (TRx) super-family that plays an important role as a component of the mitochondrial antioxidant system. The gene coding mitochondrial TRx-2 was isolated from the disk abalone (Haliotis discus discus) cDNA library, denoted as AbTRx-2. It contains 1214-bp full length with 519-bp open reading frame, encoding 173 amino acids. AbTRx-2 showed characteristic TRx active site at (96)WCGPC(100) and mitochondrial targeting peptide at the N-terminal amino acid sequence. The deduced amino acid comparison showed that AbTRx-2 shares 43 and 42% identity with Xenopus laevis and human TRx-2, respectively. Purified recombinant AbTRx-2 fusion protein was shown to catalyze insulin reduction and protect supercoiled plasmid DNA from damages induced by metal-catalyzed generation of reactive oxygen species. Constitutive AbTRx-2 mRNA was detected in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes, in a tissue specific manner. The AbTRx-2 mRNA was up-regulated in gill and digestive tract tissues initially at 3 h post-injection of H(2)O(2) and maintained higher level at 6 h. Our results suggest that abalone TRx-2 may play an important role in regulating oxidative stress in mitochondria by catalyzing protein disulfide reduction, scavenging of ROS, and minimizing the DNA damage.

Vitellogenin synthesis via androgens in primary cultures of tilapia hepatocytes

Involvement of androgens in vitellogenin (VTG) synthesis was investigated using the primary hepatocyte cultures of tilapia, Oreochromis mossambicus. Concentration of VTG in the medium was assessed by enzyme-linked immunosorbent assay. When the hepatocytes of females were treated with testosterone (T), 17 alpha-methyltestosterone (MT) and 5 alpha-dihydrotestosterone (DHT), VTG concentration in the medium slightly increased or maintained. DHT, but not T and MT, increased VTG in the medium of male hepatocyte cultures. However, VTG production in the male hepatocytes, which were previously treated with estradiol-17 beta (E(2)), maintained high level by treatment of T. Similarly, co-treatment of E(2) and the androgens to the male hepatocytes enhanced VTG concentration in the medium. These results suggest that the androgens have some roles in VTG synthesis in the hepatocytes. Tamoxifen, a nonsteroidal antiestrogen, reduced VTG synthesis by the androgens. On the other hand, co-treatment of T and fadrozole, an aromatase inhibitor, failed to inhibit the effect of VTG synthesis by T alone. Analysis with RT-PCR did not demonstrate expression of the brain and the ovarian types of aromatase mRNA in the liver. These results suggest that the possibility of local aromatization of the androgens in the tilapia liver is low and that androgens bind estrogen receptor and, consequently, exert estrogenic action. Treatment of cyproterone acetate, an antiandrogen reagent, increased production of VTG with DHT. Involvement of androgens might not be ignored in regulation of VTG synthesis in the liver.

Comparative study of two thioredoxin peroxidases from disk abalone (Haliotis discus discus): cloning, recombinant protein purification, characterization of antioxidant activities and expression analysis.

Thioredoxin peroxidase (TPx), also named peroxiredoxin (Prx), is an important peroxidase, which can protect organisms against various oxidative stresses. Two TPxs were isolated from a disk abalone (Haliotis discus discus) cDNA library, named as AbTPx1 and AbTPx2, respectively. AbTPx1 and AbTPx2 consist of 1315 and 1045 bp full-length cDNA with 753 and 597 bp open reading frames encoding 251 and 199 amino acids, respectively. The TPx signature motif 1 (FYPLDFTFVCPTEI) and motif 2 (GEVCPA) were conserved in both AbTPx1 and AbTPx2 amino acid sequences. Purified recombinant abalone TPx fusion proteins catalyzed the reduction of H2O2 and butyl hydroperoxide in peroxidase assays. Furthermore, both AbTPx fusion proteins were shown to protect super-coiled DNA from damage by metal-catalyzed oxidation (MCO) in vitro. Escherichia coli cells transformed with AbTPx1 and AbTPx2 coding sequences in pMAL-c2x showed resistance to H2O2 at 0.8 mM concentration by in vivo H2O2 tolerance assay. AbTPx1 and AbTPx2 mRNA were constitutively expressed in gill, mantle, abductor muscle and digestive tract in a tissue specific manner. Additionally, both TPxs mRNA were up-regulated in gill and digestive tract tissues against H2O2 at 3h post injection. The results indicate that AbTPx1 and AbTPx2 gene expressions are induced by oxidative stress and their respective proteins function in the detoxification of different ROS molecules to maintain efficient antioxidant defense in disk abalone.

Moonlight affects nocturnal Period2 transcript levels in the pineal gland of the reef fish Siganus guttatus

Abstract:u2002 The golden rabbitfish Siganus guttatus is a reef fish with a restricted lunar‐synchronized spawning cycle. It is not known how the fish recognizes cues from the moon and exerts moon‐related activities. In order to evaluate the perception and utilization of moonlight by the fish, the present study aimed to clone and characterize Period2 (Per2), a light‐inducible clock gene in lower vertebrates, and to examine daily variations in rabbitfish Per2 (rfPer2) expression as well as the effect of light and moonlight on its expression in the pineal gland. The partially‐cloned rfPer2 cDNA (2933 bp) was highly homologous (72%) to zebrafish Per2. The rfPer2 levels increased at ZT6 and decreased at ZT18 in the whole brain and several peripheral organs. The rfPer2 expression in the pineal gland exhibited a daily variation with an increase during daytime. Exposing the fish to light during nighttime resulted in a rapid increase of its expression in the pineal gland, while the level was decreased by intercepting light during daytime. Two hours after exposing the fish to moonlight at the full moon period, the rfPer2 expression was upregulated. These results suggest that rfPer2 is a light‐inducible clock gene and that its expression is affected not only by daylight but also by moonlight. Since the rfPer2 expression level during the full moon period was higher than that during the new moon period, the monthly variation in the rfPer2 expression is likely to occur with the change in amplitude between the full and new moon periods.

Transcriptional up-regulation of disk abalone selenium dependent glutathione peroxidase by H2O2 oxidative stress and Vibrio alginolyticus bacterial infection

Selenium dependent glutathione peroxidase (Se-GPx) belongs to the family of selenoprotein, which acts mainly as an antioxidant in the cellular defence system. We have identified Se-GPx full length cDNA from disk abalone (Haliotis discus discus) designated as AbSe-GPx. It has a characteristic codon at (223)TGA(225) that corresponds to selenocysteine (Sec) amino acid as U(75). The full length cDNA consists of 675 bp, an open reading frame encoding 225 amino acids. Sequence characterization revealed that AbSe-GPx contains a characteristic GPx signature motif 2 ((97)LGFPCNQF(104)), an active site motif ((183)WNFEKF(188)) and essential residues for the enzymatic function. Additionally, the eukaryotic selenocysteine insertion sequence (SECIS) is conserved in the 3 UTR. The AbSe-GPx amino acid sequence exhibited the highest level of identity (46%) with insect (Ixodes scapularis) GPx, and shares 41% with bivalve (Unio tumidus) Se-GPx. The RT-PCR analysis revealed that AbSe-GPx mRNA was expressed constitutively in gill, mantle, gonad, abductor muscle, digestive tract, and hemocytes in a tissue specific manner. AbSe-GPx mRNA expression was significantly up-regulated in gill and digestive tract tissues after H(2)O(2) injection and Vibrio alginolyticus infection. However, AbSe-GPx expression was not up-regulated after Aroclor 1,254 injection. These results indicate that AbSe-GPx mRNA is expressed at a basal level in abalone tissues, which can be up-regulated transcriptionally by H(2)O(2) oxidative stress and Vibrio alginolyticus infection. Therefore, AbSe-GPx may be involved in a protective role against H(2)O(2) oxidative stress and immune defence against bacterial infection.

Sequence and expression of androgen receptor and estrogen receptor gene in the sex types of protogynous wrasse, Halichoeres trimaculatus.

Sex steroid hormones play important roles in sex change and behavior of wrasses. Their actions are considered to be mediated through the nuclear hormone receptors. In this study, to elucidate the roles of estrogen receptor (ER) and androgen receptor (AR) in the reproduction of the protogynous wrasse, Halichoeres trimaculatus, AR and ER genes were partially cloned using 5- and 3-RACE and their transcript levels in the gonads and the brain were measured by competitive RT-PCR. The amino acid sequence (563 a.a) deduced from 5 truncated cDNA encoding wrasse AR shows about 81%, 69%, 66%, 64%, and 58% identity with those of red seabream (Chrysophrys major) androgen receptor subtype, AR1, and rainbow trout (Oncorhynchus mykiss) ARalpha, ARbeta, Japanese eel (Anguilla japonica) ARalpha, and ARbeta, respectively. The amino acid sequence (458 a.a) deduced from 5() truncated cDNA encoding wrasse ER shows about 81%, 79%, 73%, 66%, and 63% identity with those of red seabream estrogen receptor subtype, ERalpha, Atlantic croaker (Micropogonias undulatus) ERalpha, tilapia (Oreochromis niloticus) ERalpha, rainbow trout ERalpha, and catfish (Ictalurus punctatus) ERalpha, respectively. Among the various tissues tested, AR and ER mRNAs were highly expressed in the gonads and brains. When the transcript levels of ER were measured in the gonads and the brains of females (F), initial phase male (IP), and terminal phase male (TP), no significant changes in the gene expression were observed. The transcript levels of AR in the gonads did not change among different sex types, while those in the brains of TP were higher than F and IP. These results suggest that higher expression of AR in the brains of TP is strongly correlated with behavioral change.

Fish sleeping under sandy bottom: Interplay of melatonin and clock genes

Wrasse species exhibit a definite daily rhythm in locomotor activity and bury themselves in the sand at the bottom of the ocean at night. It remains unclear how their behavior in locomotor activity is endogenously regulated. The aim of the present study was to clarify the involvement of melatonin and clock genes (Per1, Per2, Bmal1, and Cry1) in daily and circadian rhythms of the threespot wrasse, Halichoeres trimaculatus, which is a common species in coral reefs. Daily and circadian rhythms in locomotor activity were monitored under conditions of light-dark cycle (LD=12:12), constant light (LL), and darkness (DD). Daily rhythms in locomotor activity were observed under LD and persisted under LL and DD. Melatonin from a cultured pineal gland showed daily variations with an increase during the nighttime and a decrease during daytime, which persisted under DD. Melatonin treatment induced decreases in locomotor activity and respiratory rate, suggesting that melatonin has a sleep-inducing effect. Per1 and Per2 mRNA abundance in the brain under LD showed daily rhythms with an increase around lights on. Robust oscillation of Per1 and Per2 mRNA expression persisted under DD and LL, respectively. Expression of Bmal1 and Cry1 mRNA also showed daily and circadian patterns. These results suggest that clock genes are related to circadian rhythms in locomotor activity and that melatonin plays a role in inducing a sleep-like state after fish bury themselves in the sand. We conclude that the sleep-wake rhythm of the wrasse is regulated by a coordination of melatonin and clock genes.

Influence of moonlight on mRNA expression patterns of melatonin receptor subtypes in the pineal organ of a tropical fish.

The goldlined spinefoot, Siganus guttatus, is a lunar-synchronized spawner, which repeatedly releases gametes around the first quarter moon during the reproductive season. A previous study reported that manipulating moonlight brightness at night disrupted synchronized spawning, suggesting involvement of this natural light source in lunar synchronization. The present study examined whether the mRNA expression pattern of melatonin receptor subtypes MT1 and Mel1c in the pineal organ of the goldlined spinefoot is related to moonlight. Real-time quantitative polymerase chain reaction analysis revealed that the abundance of MT1 and Mel1c mRNA at midnight increased during the new moon phase and decreased during the full moon phase. Exposing fish to moonlight intensity during the full moon period resulted in a decrease in Mel1c mRNA abundance within 1h. Fluctuations in the melatonin receptor genes according to changes in the moon phase agreed with those of melatonin levels in the blood. These results indicate that periodic changes in cues from the moon influence melatonin receptor mRNA expression levels. The melatonin-melatonin receptor system may play a role in predicting the moon phase through changes in night brightness.

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.

Diurnal expression patterns of neurohypophysial hormone genes in the brain of the threespot wrasse Halichoeres trimaculatus

The aim of this study was to determine the involvement of neurohypophysial hormones in the diurnal patterns of the threespot wrasse Halichoeres trimaculatus, which is common in coral reefs and exhibits daily behavioral periodicity. Prohormone cDNAs of the neurohypophysial peptides, arginine vasotocin (AVT) and isotocin (IT), were cloned by 3- and 5-rapid amplification of cDNA ends (RACE). The distribution and expression patterns of pro-AVT and -IT mRNAs in the brain were determined using reverse-transcription polymerase chain reaction (RT-PCR) and real-time quantitative PCR, respectively. The respective full-length cDNAs of pro-AVT and -IT were 945 and 755 bp in length, respectively. The deduced amino acid sequences for pro-AVT and pro-IT were 154 and 156 residues in length, respectively. Both pro-peptides contained a signal sequence followed by the respective hormones and neurophysin connected by a Gly-Lys-Arg bridge. Pro-AVT mRNA was detected only in the hypothalamus area, while pro-IT mRNA in the whole part of the brain. The relative abundance of pro-AVT and -IT mRNA varied according to time of day; it was significantly greater at 12:00 h than at 24:00 h. Following intraperitoneal administration of melatonin, pro-AVT mRNA abundance in the brain decreased, while pro-IT mRNA abundance remained unchanged. These results demonstrate that daily fluctuations of pro-AVT and pro-IT levels in the brain of threespot wrasse are differentially regulated.