cDNA cloning and characterization of the warm-temperature-acclimation-associated protein Wap65 from carp, Cyprinus carpio

We determined full-length cDNA of carp warm-temperature-acclimation-associated 65-kDa protein (Wap65). It encoded 439 amino acid residues with a signal peptide of 22 residues and showed an amino acid sequence identity of 88% to that of goldfish reported before (J. Biol. Chem. 1995. 270: 17087–17092). The number of potential N-linked glycosylation sites of carp Wap65 was two in contrast to three for goldfish. In addition, molecular mass determined by SDS-PAGE was apparently different from that of goldfish. These results suggest that the amount of oligosaccharide is different between the carp and goldfish protein. As in goldfish, carp Wap65 mRNA showed marked accumulation in hepatopancreas of the 30 °C- acclimated fish, which was 8-fold higher than that of the 10 °C-acclimated fish. Carp Wap65 showed 30% amino acid identity to mammalian hemopexins, which appeared to be considerably low in comparison with those among mammalian hemopexins (72 to 80%), or among carp Wap65 and rainbow trout hemopexin-like protein (70%). However, although mammalian hemopexins contain residues comprising the heme binding pocket, carp Wap65 lacked one of the two histidine residues to serve as heme axial ligands in hemopexins. Our data on carp protein substantiates the previous observation for goldfish and indicates that Wap65 might have some important functions in warm-temperature-acclimation of fish.     

Dopamine functions as a growth hormone-releasing factor in the goldfish,Carassius auratus

In vivo and in vitro approaches have been used to examine the role of dopamine (DA) as a growth hormone (GH)-releasing factor in the goldfish. DA stimulated GH release from perifused pituitary fragments of goldfish in a dose-dependent manner. The GH-releasing effect of DA was seasonal, being the highest in sexually regressed fish, intermediate in recrudescent fish, and the lowest in sexually mature (prespawning) fish. The GH response to DA was blocked by the D1 antagonist (+)SCH23390, confirming the involvement of D1 receptors in DA-stimulated GH release. In studies using static incubation of pituitary cells, somatostatin, a known physiological GH-release inhibitor in the goldfish, abolished the GH response to DA. Intraperitoneal injection of apomorphine, a non-selective DA agonist, also increased the plasma GH levels and enhanced the linear body growth of goldfish. These results strongly suggest that DA, by acting through DA D1 receptors, functions as a GH-releasing factor in the goldfish.     

Partial characterization of the gene encoding myoadenylate deaminase from the teleost fish <Emphasis Type="Italic">Platichthys flesus</Emphasis>

AMP-deaminase (AMPD, EC 3.5.4.6), which catalyzes the irreversible hydrolytic deamination of AMP to IMP and ammonia, is an important energy-related enzyme. The partial genomic sequence of the gene encoding myoadenylate deaminase (AMPD1) from the teleost fish Platichthys flesus was determined. The amino acid sequence of P. flesus AMPD1 shows 82% homology with that of the teleost fish Danio rerio. Comparison of genomic sequences of P. flesus and Rattus norvegicus reveals a high degree of conservation of both sequence and structural organization. A phylogenetic analysis of AMPD sequences shows that bony fish and mammalian AMPD1s arise by duplication of a common primordial gene.     

Occurrence of two distinct molecular species of cathepsin B in carp Cyprinus carpio

ABSTRACT:   We purified cathepsins B₁ and B₂ from the ordinary muscle of carp Cyprinus carpio . The N-terminal amino acid sequences (12 residues) of 29 kDa bands of cathepsins B₁ and B₂ are the same and showed high homology of 75% and 83%, respectively, with the heavy chain of rat and human cathepsins B. Based on conserved sequences of other cathepsins B and the N-terminal amino acid sequences of 29 kDa bands, we cloned carp cathepsin B cDNA. The nucleotide sequence of carp cathepsin B cDNA consists of 1470 bp including a 993 bp open reading frame, encoding a deduced protein of 330 amino acids. The deduced amino acid sequence of carp cathepsin B has similarity of 80% to rainbow trout cathepsin B and of 76–78% to other vertebrate cathepsins B. The sequence of its isoform was also determined during molecular cloning, which has 94.8% similarity with first cloned cathepsin B. They are completely same in N-terminal amino acid sequence of heavy chain, active site and potential N-glycosylation site. This indicates there are at least two kinds of cathepsin B functioning in vivo in carp.     

Distinct expression of three estrogen receptors in response to bisphenol A and nonylphenol in male Nile tilapias (Oreochromis niloticus)

Environmental estrogens, such as bisphenol A (BisA) and nonylphenol (NP), have been shown to affect the estrogen receptor (ER) expression and induce male reproductive abnormalities. To elucidate molecular mechanisms of action of xenoestrogenic chemicals on the expression of estrogen receptors in the testes of Nile tilapia (Oreochromis niloticus), three full-length cDNAs respectively encoding ntERα, ntERβ1 and ntERβ2 were cloned from testes. The amino acid sequences of ntERα, ntERβ1 and ntERβ2 showed a high degree of similarity to the relevant fish species. Tissue-specific expression study showed that three receptors were highly expressed in pituitary, liver, testis, kidney and intestine tissues. The ntERα, ntERβ1 and ntERβ2 mRNA expressions were significantly higher at the sexual early recrudescing stage than at other recrudesced stages. After being exposed to xenoestrogens from weeks 2 to 4, the ntERα mRNA levels were increased significantly in testes after NP treatment at all sampling times or after 4 weeks of exposure to BPA. The ntERβ1 mRNA levels remained unchanged, while a significant decrease of the ntERβ2 mRNA level was observed in testes after exposure to NP and BPA. The present study demonstrates that the regulation of all three ntER subtypes in testes may act via different molecular mechanisms of exposure to NP and BPA.     

Insulin-like growth factor-I cDNA cloning,gene expression and potential use as a growth rate indicator in Nile tilapia,Oreochromis niloticus

IGF-I is a mitogenic polypeptide that is an important regulator of growth in fish. The potential of IGF-I mRNA abundance as a rapid growth indicator in the Nile tilapia, Oreochromis niloticus, was evaluated. Hepatic IGF-I cDNA was isolated and partially cloned. The partial sequence having 539 bases encodes for the signal peptide, mature protein and a portion of the E domain. The deduced 68 amino acid sequence for mature IGF-I showed 84–90% and 77–79% sequence identity with fish and mammalian counterparts, respectively. The deduced amino acid sequence for domains B and A was most conserved (93–97%) relative to other fishes. A sensitive TaqMan real time qRT-PCR assay for O. niloticus was developed based on the mature IGF-I peptide for measures of hepatic IGF-I mRNA levels. Hepatic IGF-I mRNA levels were found to be significantly correlated with growth rate of fish reared under different feeding regimes and temperature conditions. Higher feed consumption and water temperature produced faster-growing fish and increased hepatic IGF-I mRNA expression. These findings suggest that hepatic-derived IGF-I plays a key role in controlling growth in O. niloticus and indicates that IGF-I mRNA quantification could prove useful for the rapid assessment of growth rate in this species.     

Studies on teleost corpuscles of Stannius: Physiological and biochemical aspects of synthesis and release of hypocalcin in trout,goldfish and eel

Hypercalcemia (induced by CaCl₂-injection or seawater exposure of the fish) reduced the hypocalcin content of corpuscles of Stannius (CS) in trout, goldfish and eel; concomitantly the synthetic activity of CS of hypercalcemic fish, as determinedin vitro, was enhanced. The monomeric forms of prohypocalcin and of hypocalcin of trout and goldfish are 32 and 28 kDA M_r glycoprotein species respectively; those of the eel are 2 kDa bigger,viz. 34 and 30 kDa respectively. Moreover, eel CS producein vitro an enigmatic 70 kDa glycoprotein with affinity for concanavalin-A. It is concluded that plasma calcium levels control storage and synthesis rates of hypocalcin in the CS.     

欧洲鳗鲡MyD88基因的克隆及其免疫功能分析

髓样分化因子(myeloid differentiation factor 88,My D88)是TLR(toll-like receptor)信号通路的关键接头蛋白,在先天性免疫中发挥重要作用。实验首次克隆了欧洲鳗鲡My D88基因c DNA全长序列,命名为Aa My D88,其全长为1 539 bp,开放阅读框为846 bp,编码282个氨基酸。该蛋白三维丝带空间结构图与人类的My D88十分相似,具有My D88家族典型的死亡结构域和TIR(toll-like/IL-1 receptor)结构域,其中TIR结构域中含有3个序列高度保守的box1、box2和box3。同源性分析显示,欧洲鳗鲡My D88氨基酸序列与斑点叉尾相似性最高,为76.3%,与其他鱼类相似性为67.5%~73.2%,与哺乳动物相似性较低,为61.6%~62.6%。欧洲鳗鲡My D88在系统进化树中与其他鱼类My D88聚为一支,哺乳类以及两栖类My D88分别聚为一支。实时荧光定量PCR检测发现,Aa My D88基因在欧洲鳗鲡各组织器官中均有表达,其中在肝脏中的表达量最高,心脏、肠、脾脏以及肾脏中也有较高的表达,而肌肉和鳃中的表达水平较低;欧洲鳗鲡经山羊Ig G肌肉注射后,肾脏Aa My D88基因在第7天表达量有显著性提高,14 d后恢复至正常水平,而脾脏Aa My D88基因表达水平从第7~21天持续显著上调,于第7天达到峰值,其表达量为肾脏的1.7倍;欧洲鳗鲡鳍细胞系经poly I:C处理后,Aa My D88基因表达水平在3 h显著降低,6~48 h均有显著升高,于12 h达到峰值。LPS处理后的欧洲鳗鲡鳍细胞系Aa My D88基因表达水平在3 h显著降低,6和12 h显著升高,于12 h达到峰值,24 h后恢复至正常水平。poly I:C处理组My D88基因表达水平在12~48 h均显著高于LPS处理组。研究表明,My D88在欧洲鳗鲡抵御外源微生物的免疫应答反应中发挥重要作用。     

Cloning and expression of prostaglandin E2 receptor subtype 1 (ep 1 ) in Bostrichthys sinensis

Our previous studies suggested that prostaglandin E₂ (PGE₂) is a putative sex pheromone in Chinese black sleeper Bostrichthys sinensis, a fish species that inhabits intertidal zones and mates and spawns inside a muddy burrow. We found immunoreactivities of PGE₂ receptor subtypes (Ep_1–3) expressed in the olfactory sac, but only Ep₁ presented higher density of immunoreactivity in mature fish than that in immature fish in both sexes. To gain a better understanding of the underlying molecular mechanism for the detection of PGE₂ in the olfactory system, we cloned an ep ₁ cDNA from the adult olfactory sac. The open-reading frame of the ep ₁ consisted of 1,134-bp nucleotides that encoded a 378-amino acid-long protein with a seven-transmembrane domain, typical for the G protein-coupled receptors superfamily. Expression of ep ₁ mRNA was observed in all tissues examined, with higher levels obtained in the olfactory sacs and testes. The expression of ep ₁ mRNA in the olfactory sacs and gonads was significantly higher in both sexes of mature fish than in those of immature ones. Taken together, our results suggested that Ep₁, which is highly expressed in the olfactory sacs and gonads of mature fish, is important for the control of reproduction and may be involved in PGE₂-initiated spawning behavior in B. sinensis.     

石斑鱼一氧化氮合酶cDNA的分子克隆及序列分析

一氧化氮合酶(nitricoxidesynthase,NOS)专一性催化L-精氨酸转化为L-瓜氨酸和一氧化氮(nitricoxide,NO),产物NO是一种重要的生物信使分子,对其功能和代谢的研究越来越受人们的重视[1]。国际上,鱼类NOS的研究还刚起步,已有研究者在鱼类中检测到NOS的存在[2-5]。虹鳟[6]、金鱼[4]、大西洋鲑[7]和沟鲶[8]的诱导型NOS(iNOS)和神经型NOS(nNOS)的部分序列已鉴定。国内对哺乳动物的NOS也进行了研究[9-11],尚未见关于鱼类NOS方面的研究报道。JOURNALOFFISHERIESOFCHINA　　　　　　　　　　　Vol.27,No.4　斜带石斑鱼(Epin…     

Yellowtail neuropeptide Y: molecular cloning,tissue distribution,and response to fasting

Neuropeptide Y (NPY) and cholecystokinin (CCK) play important roles in regulating appetite in vertebrates, including mammals and fish. Understanding the appetite mechanism is important in aquaculture to improve production performance. The yellowtail Seriola quinqueradiata is one of the most cultured fish in Japan, but little is known about its appetite hormones. In the present study, complementary DNA encoding for NPY was cloned in yellowtail and consists of 604 bp, in which deduced amino acid sequences show high identity to those of other teleosts. In tissue distribution, the npy and cck mRNAs were detected in all examined tissues (whole brain, telencephalon, optic tectum, hypothalamus, cerebellum, pituitary, retina, stomach, pyloric caeca, anterior intestine, liver, and kidney). In the fasting experiment, only npy mRNA expression in the hypothalamus responded to fasting, showing a significantly high value compared with that in control fish. The expression of cck mRNA in the examined tissues did not change with fasting. The npy mRNA expression in the hypothalamus might be involved in feeding regulation in yellowtail.     

Hormonal influences on in vitro [35S]-sulfate uptake by gill arches from the goldfish (Carassius auratus L.)

A bioassay for insulin-like growth factor (IGF), based on the in vitro incorporation of [³⁵S]-sulfate into gill arch tissue was used to study the hormonal regulation of proteoglycan synthesis in the goldfish (Carassius auratus). [³⁵S]-sulfate incorporation into gill arch tissue was found to be time-dependent with maximal uptake occurring by 48h, suggesting that proteoglycan synthesis in this tissue was maintained for at least 48h in vitro. The addition of human recombinant IGF-I (IGF-I) to the incubation medium was found to significantly stimulate [³⁵S]-sulfate uptake into the gill arches, whereas bovine growth hormone (GH) was without effect. Porcine insulin was also stimulatory, but results indicate that the effects of porcine insulin and IGF-I may be mediated by a single receptor system. Finally, arches from hypophysectomized fish were significantly less responsive to IGF-I than were arches from sham-operated fish. Furthermore, administration of ovine GH in vivo appeared to increase subsequent responsiveness in vitro. Together, these results provide evidence that the growth-promoting actions of GH in the goldfish may be mediated, at least in part, by a peptide related in structure to mammalian IGF-I.     

A novel cytochrome P450 1D1 gene in Nile tilapia fish (Oreochromis niloticus): partial cDNA cloning and expression following benzo-a-pyrene exposure

To understand the detoxification and bioactivation mechanisms for organic contaminants, it is essential to identify the cytochrome P450 (CYP) complement. Therefore, this study aimed to clone a partial cDNA sequence of the novel CYP1D1 gene from the fish Oreochromis niloticus and examine whether intraperitoneal injection of benzo-a-pyrene (BaP), a potent AHR agonist, is capable of inducing CYP1D1 mRNA expression in different tilapia fish tissues. The cloned nucleotide sequence consisted of 713 bp representing a portion of the tilapia CYP1D1 cDNA ORF, encoding 237 amino acids. Amino acid sequence comparison of O. niloticus CYP1D1 with the sequences of CYP1D1 from other species showed that this gene shared the highest identity of 81% with Fundulus heteroclitus CYP1D1. Furthermore, analysis of the percent identities shared by the deduced amino acid sequence of O. niloticus CYP1D1 with the sequences of CYP1 from other species revealed that the highest identities were shared with fish CYP1As. Real-time PCR results revealed that the highest expression level of CYP1D1 mRNA was found in muscles, followed by gills, liver, and intestine, while there was no detectable expression recorded in bile acid. These results indicate that tilapia CYP1D1 plays an important role in the metabolism of xenobiotics, expanding our knowledge regarding the diversity of CYP1 genes in this important model fish species.

     

Activin and its receptors in the goldfish

Activin (_AA, _AB and _BB) is a dimeric protein that belongs to the transforming growth factor- (TGF-) superfamily of growth factors and is involved in the regulation of many physiological and developmental processes. Recently, we have demonstrated that porcine activin stimulated goldfish gonadotropin-II (GTH-II) and growth hormone (GH) secretion from dispersed pituitary cells in static culture and pituitary fragments in perifusion. The action of activin in the goldfish is unique in that it has an acute stimulatory effect on the secretion of GTH-II and GH, whereas in mammals activin usually exhibits long-term stimulatory actions on FSH secretion. The action mechanism of activin is different from that of gonadotropin-releasing hormone (GnRH). Using domain-specific antibodies against mammalian activin subunits, we subsequently demonstrated the existence of immunoreactive activin subunits (_A and _B) in the goldfish ovary, testis, pituitary and brain, suggesting endocrine, paracrine and autocrine roles for activin in the regulation of goldfish reproduction. Both activin _A and _B subunits have been cloned from goldfish genome by polymerase chain reaction (PCR). Using the PCR fragments as probes, we have cloned a full length cDNA coding for activin _B subunit from the goldfish ovary. Both activin _A and _B subunits show high homology to those of other vertebrates with the _B subunit much more conserved (93 and 98% identity with human and zebrafish _B subunit, respectively). The identity of the cloned _B subunit was further confirmed by expression in the Chinese hamster ovary (CHO) cells and detection of the specific activity of activin in the culture medium. The messenger RNA of activin _B subunit is expressed in a variety of goldfish tissues including ovary, testis, brain, pituitary, kidney and liver, suggesting a wide range of physiological roles for activin in the goldfish. We have also cloned a full length cDNA coding for the activin Type IIB receptor from the goldfish ovary, suggesting that activin may have paracrine or autocrine actions on the ovarian functions. The identity of the cloned receptor was confirmed by specific binding of¹²⁵ I-activin on COS-1 cells transfected with the cloned Type IIB receptor.     

Glycoprotein hormone receptor family in medaka (Oryzias latipes)

Four types of GPHR cDNAs have been cloned from ovary and testis of medaka (Oryzias latipes) and their gene constructions have been determined. Two of them are closely related to known fish receptors for FSH and LH, respectively. Changes in their mRNA levels were examined during the course of oogenesis. FSH receptor mRNA could not be detected from 20 h before ovulation, whereas LH mRNA remained 5 h before ovulation.