Anti-Melanogenic Effects of Takifugu flavidus Muscle Hydrolysate in B16F10 Melanoma Cells and Zebrafish.

Abnormal melanogenesis can lead to hyperpigmentation. Tyrosinase (TYR), a key rate-limiting enzyme in melanin production, is an important therapeutic target for these disorders. We investigated the TYR inhibitory activity of hydrolysates extracted from the muscle tissue of Takifugu flavidus (TFMH). We used computer-aided virtual screening to identify a novel peptide that potently inhibited melanin synthesis, simulated its binding mode to TYR, and evaluated functional efficacy in vitro and in vivo. TFMH inhibited the diphenolase activities of mTYR, reducing TYR substrate binding activity and effectively inhibiting melanin synthesis. TFMH indirectly reduced cAMP response element-binding protein phosphorylation in vitro by downregulating melanocortin 1 receptor expression, thereby inhibiting expression of the microphthalmia-associated transcription factor, further decreasing TYR, tyrosinase related protein 1, and dopachrome tautomerase expression and ultimately impeding melanin synthesis. In zebrafish, TFMH significantly reduced black spot formation. TFMH (200 µg/mL) decreased zebrafish TYR activity by 43% and melanin content by 52%. Molecular dynamics simulations over 100 ns revealed that the FGFRSP (T-6) peptide stably binds mushroom TYR via hydrogen bonds and ionic interactions. T-6 (400 µmol/L) reduced melanin content in B16F10 melanoma cells by 71% and TYR activity by 79%. In zebrafish, T-6 (200 µmol/L) inhibited melanin production by 64%. TFMH and T-6 exhibit good potential for the development of natural skin-whitening cosmetic products.

Affinity Purification and Molecular Characterization of Angiotensin-Converting Enzyme (ACE)-Inhibitory Peptides from Takifugu flavidus.

An affinity chromatography filler of CNBr-activated Sepharose 4B-immobilized ACE was used to purify ACE-inhibitory peptides from Takifugu flavidus protein hydrolysate (<1 kDa). Twenty-four peptides with an average local confidence score (ALC) ≥ 80% from bounded components (eluted by 1 M NaCl) were identified by LC-MS/MS. Among them, a novel peptide, TLRFALHGME, with ACE-inhibitory activity (IC50 = 93.5 µmol·L-1) was selected. Molecular docking revealed that TLRFALHGME may interact with the active site of ACE through H-bond, hydrophobic, and electrostatic interactions. The total binding energy (ΔGbinding) of TLRFALHGME was estimated to be -82.7382 kJ·mol-1 by MD simulations, indicating the favorable binding of peptides with ACE. Furthermore, the binding affinity of TLRFALHGME to ACE was determined by surface plasmon resonance (SPR) with a Kd of 80.9 µmol, indicating that there was a direct molecular interaction between them. TLRFALHGME has great potential for the treatment of hypertension.

Salinity-dependent mitigation of naphthalene toxicity in migratory Takifugu obscurus juveniles: Implications for survival, oxidative stress, and osmoregulation.

Naphthalene, an environmental pollutant classified as a polycyclic aromatic hydrocarbon (PAH), can induce toxicity in fish and other aquatic organisms. Through our investigation, we determined how Takifugu obscurus juveniles were affected by naphthalene (0, 2 mg L-1) exposure in terms of oxidative stress biomarkers and Na+/K+-ATPase activity in various tissues (gill, liver, kidney and muscle) under dissimilar salinities (0, 10 psu). Results suggest that naphthalene exposure significantly affects the survival of T. obscurus juveniles and leads to significant changes in the levels of malondialdehyde, superoxide dismutase, catalase, glutathione, and Na+/K+-ATPase activity, which are indicative of oxidative stress and emphasized the risks associated with osmoregulatory function. The higher salinity affected on the noxious effects of naphthalene can be observed, resulting in decreased biomarker levels and increased Na+/K+-ATPase activity. Salinity levels affected the uptake of naphthalene and its impact on different tissues, with high salinity conditions having mitigating effects on oxidative stress and naphthalene uptake in the liver and kidney tissues. Increased Na+/K+-ATPase activity was observed in all tissues treated with 10 psu and 2 mg L-1 naphthalene. Our findings deepen the understanding of T. obscurus juveniles' physiological responses to naphthalene exposure, and highlight the potential mitigating effects of salinity. These insights can inform the development of appropriate conservation and management practices to protect aquatic organisms from susceptibility.

Effect of winter feeding frequency on growth performance, biochemical blood parameters, oxidative stress, and appetite-related genes in Takifugu rubripes.

Tiger pufferfish (Takifugu rubripes) is one of Asia's most economically valuable aquaculture species. However, winter production of this species in North China is limited by low water temperature and unavailability of high-quality feed, resulting in high mortality and low profitability. Therefore, the aim of this study was to evaluate the effect of feeding frequency (F1: one daily meal; F2: two daily meals; F3: four daily meals; F4: continuous diurnal feeding using a belt feeder) on the growth performance, plasma biochemistry, digestive and antioxidant enzyme activities, and expression of appetite-related genes in T. rubripes (initial weight: 266.80 ± 12.32 g) cultured during winter (18.0 ± 1.0 °C) for 60 days. The results showed that fish in the F3 group had the highest final weight, weight gain rate, specific growth rate, survival rate, and best feed conversion ratio. Additionally, daily feed intake increased significantly with increasing feeding frequency. The protein efficiency and lipid efficiency ratios of fish in the F3 group were significantly higher than those of fish in the other groups. Furthermore, total cholesterol, triglycerides, and glucose levels increased with increasing feeding frequency, peaking in the F2 group and decreasing under higher feeding frequencies. The antioxidant (superoxide dismutase, catalase, glutathione, and glutathione peroxidase) and digestive (trypsin, amylase, and lipase) enzyme activities of fish in the F1 group were significantly higher than those of fish in the F3 and F4 groups. Additionally, there was a decrease in orexin expression with increasing feeding frequency. In contrast, the expression levels of tachykinin, cholecystokinin, and leptin increased with increasing feeding frequency, peaking in the F4 group. Overall, the findings of this study indicated that a feeding frequency of four meals per day was optimal for improved growth performance of pufferfish juveniles cultured during winter.

Kisspeptin Exhibits Stimulatory Effects on Expression of the Genes for Kisspeptin Receptor, GnRH1 and GTH Subunits in a Gonadal Stage-Dependent Manner in the Grass Puffer, a Semilunar-Synchronized Spawner.

Kisspeptin has an important role in the regulation of reproduction by directly stimulating the secretion of gonadotropin-releasing hormone (GnRH) in mammals. In non-mammalian vertebrates, there are multiple kisspeptins (Kiss1 and Kiss2) and kisspeptin receptor types, and the two kisspeptins in teleosts have different effects depending on fish species and reproductive stages, serving reproductive and non-reproductive functions. In the grass puffer, Takifugu alboplumbeus, which has only a single pair of kiss2 and kissr2, both genes display seasonal, diurnal, and circadian oscillations in expression in association with the periodic changes in reproductive functions. To elucidate the role of kisspeptin in this species, homologous kisspeptin peptide (gpKiss2) was administered at different reproductive stages (immature, mature and regressed) and the expression levels of the genes that constitute hypothalamo-pituitary-gonadal axis were examined in male grass puffer. gpKiss2 significantly elevated the expression levels of kissr2 and gnrh1 in the brain and kissr2, fshb and lhb in the pituitary of the immature and mature fish. No noticeable effect was observed for kiss2, gnih, gnihr, gnrh2 and gnrh3 in the brain and gpa in the pituitary. In the regressed fish, gpKiss2 was ineffective in stimulating the expression of the gnrh1 and GTH subunit genes, while it stimulated and downregulated the kissr2 expression in the brain and pituitary, respectively. The present results indicate that Kiss2 has a stimulatory role in the expression of GnRH1/GTH subunit genes by upregulating the kissr2 expression in the brain and pituitary at both immature and mature stages, but this role is mostly ineffective at regressed stage in the grass puffer.

Structures, evolutionary relationships and expression profiles of the tumour necrosis factor superfamily and their receptors in black rockfish (Sebastes schlegelii).

Members of tumour necrosis factor superfamily (TNFSF) and TNF receptor superfamily (TNFRSF) have crucial roles in many important biological processes such as cell proliferation, cell death, development, survival, immunity, and various diseases. The human TNFSF consists of 19 ligands and 29 receptors. Compared with those in human, fish have most of the TNFSF and receptors that have been found in mammals, while some of the homologues are specific or lost in fish. Especially, no systematic report on the identification of TNFSF ligands and their receptors in S. schlegelii. Therefore, to investigate the characterization and molecular evolution of TNFSF and TNFRSF genes in Sebastes schlegelii, we performed a genome-wide survey and identified 14 TNFSFs and 24 TNFRSFs from S. schlegelii. In S. schlegelii, we found duplication events occurred in TNFSF2, TNFSF6, TNFSF10, TNFSF13, TNFSF14, TNFRSF5, TNFRSF6, TNFRSF6B, TNFRSF10B, TNFRSF16, and TNFRSF19 genes. Among which, the tandem duplications events occurred in TNFSF13 and TNFRSF6, and the whole genome duplications events occurred in the remaining TNFSF and TNFRSF genes. Based on the molecular phylogenetic analysis, 14 TNFSFs were divided into three different clusters and 24 TNFRSFs were classed as three distinct subgroups, respectively. Meanwhile, protein domains and motifs analysis revealed that TNFSF contain homology domain (THD), and TNFRSF have typical cysteine-rich domains (CRDs). Synteny results indicates that the TNFSFs and TNFRSFs neighborhood genes have taken place great changes compared to those in human, fugu and zebrafish. Meanwhile, qRT-PCR results demonstrated that most TNFSFs and TNFSRSFs were significantly differentially expressed in gill, skin and intestine after E. tarda infection with time-dependent manners. In addition, protein-protein interaction network (PPI) analysis indicated that the most related genes connecting to TNFSF and TNFRSFs were TNFSF ligands and receptors. In summary, this study provided a new understanding for characterization and evolution of the TNFSF genes and their receptors in S. schlegelii.

Comparison of physicochemical and umami characterization of aqueous and ethanolic Takifugu obscurus muscle extracts.

Most umami substances were developed in aqueous extracts. In this study, we compared the molecular weight distributions and sensory characteristics of ethanol and aqueous Takifugu obscurus muscle extracts, and assessed their taste-related metabolites and peptide profile (<3 kDa) using nuclear magnetic resonance and nano liquid chromatography-mass spectrometry. The potential antioxidant peptide in ethanolic fraction was screened using Peptide Ranker, BIOPEP and quantum chemical simulations. The results indicated that 60% ethanolic extract fraction (60%-F) had the highest umami intensity and more palatable overall taste among all pufferfish extracts. It can be caused by more umami enhancing components such as Asp, Asn, Ala and 5'-AMP, and considerable umami-potential smaller peptides in 60%-F. 60%-F also showed an antioxidant activity, and several antioxidant peptides was screened. The present study indicated the relationship between extract solution and taste characterization, which provided more possibility for the exploitation of umami substances and screening potential activity peptides.

Gene Expression of Takifugu rubripes Gonads During AI- or MT-induced Masculinization and E2-induced Feminization.

Elucidating the global molecular changes that occur during aromatase inhibitor (AI)- or 17α-methyltestosterone (MT)-induced masculinization and estradiol-17ß (E2)-induced feminization is critical to understanding the roles that endocrine and genetic factors play in regulating the process of sex differentiation in fish. Here, fugu larvae were treated with AI (letrozole), MT, or E2 from 25 to 80 days after hatching (dah), and gonadal transcriptomic analysis at 80 dah was performed. The expression of dmrt1, gsdf, foxl2, and other key genes (star, hsd3b1, cyp11c1, cyp19a1a, etc.) involved in the steroid hormone biosynthesis pathway were found be altered. The expression of dmrt1, gsdf, cyp19a1a, and foxl2 was further verified by quantitative polymerase chain reaction. In the control group, the expression of dmrt1 and gsdf was significantly higher in XY larvae than in XX larvae, while the expression of foxl2 and cyp19a1a was significantly higher in XX larvae than in XY larvae (P < .05). AI treatment suppressed the expression of foxl2 and cyp19a1a, and induced the expression of dmrt1 and gsdf in XX larvae. MT treatment suppressed the expression of foxl2, cyp19a1a, dmrt1, and gsdf in XX larvae. E2 treatment suppressed the expression of dmrt1 and gsdf, but did not restore the expression of foxl2 and cyp19a1a in XY larvae. The shared response following AI, MT, and E2 treatment suggested that these genes are essential for sex differentiation. This finding offers some insight into AI or MT-induced masculinization, and E2-induced femininization in fugu.

Forkhead transcription factor O1 (FoxO1) in torafugu pufferfish Takifugu rubripes: Molecular cloning, in vitro DNA binding, and target gene screening in fish metagenome.

The fish insulin/insulin-like growth factor (IGF) pathway has weak control over carbohydrate metabolism. To understand the molecular basis for the metabolic diversity, we characterized the forkhead box transcription factor O1A (FoxO1A), a downstream target of the insulin/IGF pathway, in torafugu Takifugu rubripes. The cloned torafugu FoxO1A cDNA contained all conserved features critical for its transcriptional activity and a unique unspliced intron encoding a poly-glutamine stretch. Torafugu FoxO1A showed the IGF-dependent nuclear exclusion and in vitro binding to the well-conserved FoxO1 binding site, DAF-16 binding element (DBE), but failed to bind to the insulin-responsive element by which mammalian FoxO1 mediates insulin effects. The subsequent in silico genomic screening provided a list of 587 potential torafugu FoxO1A target genes containing the DBE. Some carbohydrate metabolic genes regulated by FoxO1 in mammals were not included in the list. We further identified about 250 potential fish FoxO1 target genes by integrating results of the DBE screening against fish metagenome that contained 262 species. Neuronal processes appeared to be the common major function of fish FoxO1, although further annotation of the potential target genes is required. These results provide a part of the molecular basis underlying the weak association between the insulin/IGF pathway and carbohydrate metabolism in fish.

Capacity for freshwater acclimation and differences in the transcription of ion transporter genes underlying different migratory life histories of Takifugu fish.

The genus Takifugu is a group of approximately 20 species of puffer fishes living in a wide range of salinity environments around East Asian countries. This group presents a broad spectrum of evolutionary stages adapted to anadromy as a result of speciation that occurred a short time (2-5 million years) ago on an evolutionary timescale. This group thus can be considered as a model for studying the evolutionary mechanisms of anadromy. We firstly conducted a transfer experiment from seawater to low-salinity waters on five Takifugu species: two anadromous species T. obscurus and T. ocellatus, two euryhaline wanderer marine species T. rubripes and T. niphobles, and a strictly marine species T. snyderi, and confirmed that the capacity for acclimation to hypotonic environments was associated with their life history strategies. Next, transcriptomes of the gill and intestine of these species in hypotonic condition were compared to those under hypertonic condition for each species using RNA-Sequencing so as to determine possible candidate transporters playing an important role on freshwater adaptation. As this analysis suggested that cftr, encoding an important ion transporter for seawater acclimation in the gill, and ncc, encoding a transporter that is suggested to play important osmoregulatory roles in the intestine, are important candidates, their expression was validated by quantitative real-time PCR analysis. Expression of cftr was downregulated in the gills of the four euryhaline species under the hypotonic condition, but no change was detected in the gill of stenohaline T. snyderi, which may be one reason for the poor hypotonic acclimation capacity of T. snyderi. Expression of ncc was clearly upregulated in the intestines of the two anadromous species under the hypotonic condition, but not in other three species. Different ion transporter expression patterns between the five species indicate that the transcriptional regulation of cftr in the gill and ncc in the intestine may be important for the improvement of hypotonic acclimation capacity and evolution of anadromy in the Takifugu species.

Immunohistochemical characterization and change in location of branchial ionocytes after transfer from freshwater to seawater in the euryhaline obscure puffer, Takifugu obscurus.

The obscure puffer Takifugu obscurus is a euryhaline fish species suitable for studying the molecular mechanism of osmoregulation. The distributional changes of branchial ionocytes were detected following the transfer from freshwater (FW) to seawater (SW) based on two main ion transporters, Na+/K+-ATPase (NKA) and Na+/K+/ 2Cl- cotransporter 1 (NKCC1). The mRNA and protein expression levels of NKA and NKCC1 in the gills all increased rapidly in the first four days after transfer to SW. Double immunofluorescence staining showed that NKCC1 and NKA were colocalized in the branchial ionocytes and the immunoreaction of NKCC1 was stronger after transfer. Moreover, following transfer to SW, the number of lamellar ionocytes in the gills is reduced and the number of filament ionocytes is increased significantly. Taken together, these findings indicated that SW transfer of obscure puffer promotes the changes of distribution, function and size of branchial ionocytes.

Annual patterns of ocular melatonin level in the female grass puffer, Takifugu alboplumbeus: possible involvement in seasonal reproductive response.

The aim of this study was to investigate the expression patterns of ocular melatonin in the annual reproductive cycle of the female grass puffer. Spawning season of the female grass puffer is from June to July in Jeju, South Korea. Time-resolved fluoroimmunoassay revealed that levels of ocular melatonin, which show an annual change, peaked in May (spawning season). Additionally, expression of reproductive-related genes also showed annual patterns: GnRH1 peaked in August, GnRH2 peaked in February, GnRH3, Kiss2, and LPXRFa peaked in November. These results suggest that ocular melatonin may be related to the annual reproductive cycle in the grass puffer. To better understand the photic regulation of AANAT1a mRNA in the retina, we observed the nocturnal pattern of ocular melatonin levels daily, which shows a nocturnal pattern in both short photoperiod (SD) and long photoperiod (LD) conditions. In the brain, AANAT2 mRNA also shows a nocturnal pattern in both SD and LD; however, the time of peak expression of AANAT2 mRNA was unchanged in both conditions. Following intraperitoneal injection of melatonin for 2 weeks, expression of GnRH2 and LPXRFa mRNA in the brain significantly increased, while that of Kiss2 mRNA was decreased, suggesting that melatonin has a reproduction-related effect. Furthermore, under SD and LD conditions for 14 weeks, the gonadosomatic index more increased and the maturity of the ovary progressed under LD compared with those under SD, suggesting that the SD photoperiodic signal inactivated ovarian development. These results indicate that the ocular melatonin may have a possible role in the reproductive endocrinology of the grass puffer.

Interactive effects of temperature and salinity on the survival, oxidative stress, and Na+/K+-ATPase activity of newly hatched obscure puffer (Takifugu obscurus) larvae.

Obscure puffer (Takifugu obscurus) is an anadromous fish widely distributed around the coastal and inland rivers in East Asia. T. obscurus often encounters fluctuations in temperature and salinity. This study aimed to investigate the effect of the interactions of temperature and salinity on survival and oxidative stress response of newly hatched T. obscurus larvae. A combination of three temperatures (19, 25, and 31 °C) and three salinities (0, 10, and 20 ppt) was applied for 96 h under laboratory conditions. The newly hatched larvae could not tolerate 31 °C for 96 h. No death was recorded at other temperatures during this experiment. Malondialdehyde concentrations increased significantly after 6 h of exposure to high salinity (10 and 20 ppt) and then decreased until the end of the experiment at each temperature. The highest superoxide dismutase activity was observed under the exposure to 20 ppt for 24 h at 31 °C. Na+/K+-ATPase activity significantly increased as salinity increased, especially at low temperatures. With the prolong of exposure time, the integrated biomarker response (IBR) values showed an increase until 48 h and then declined at 96 h in most treatments. The largest IBR value appeared when larvae were exposed to the highest temperature and salinity for 24 h. Our study indicated that high temperature with high salinity may negatively affect the early development of T. obscurus and their combined effects should be considered in the larvae culture.

Differential roles of ice crystal, endogenous proteolytic activities and oxidation in softening of obscure pufferfish (Takifugu obscurus) fillets during frozen storage.

Obscure pufferfish (Takifugu obscurus) softening during frozen storage remains to be solved. This study was therefore aimed to provide explanations by differentiate the roles of three potential factors in fish softening. The influences of ice crystal, endogenous proteolytic activities, and oxidization were distinguished by treatment of fish fillets with liquid nitrogen, iodoacetic acid, and tea polyphenol with ascorbic acid, respectively. This distinguishing method was verified to be effective by investigation in ice crystal microstructure, endogenous proteolytic activities and lipid and protein oxidation. In comparison of three factors, it showed that the shear force of fish fillets with smaller ice crystals was about 15.5% and 13.7% higher than those with the inhibition of endogenous proteolytic activities and oxidation respectively, indicating the dominant role of ice crystal in frozen fish softening. Besides, quality decline of frozen fish was initially fast and then slowed down during the storage.

Effect of tributyltin chloride (TBT-Cl) exposure on expression of HSP90ß1 in the river pufferfish (Takifugu obscurus): Evidences for its immunologic function involving in exploring process.

HSP90ß1 (known as glyco-protein 96, GP96) is a vital endoplasmic reticulum (ER) depended chaperonin among the HSPs (heat shock proteins) family. Furthermore, it always processes and presents antigen of the tumor and keeps balance for the intracellular environment. In the present study, we explored the effect of tributyltin chloride (TBT-Cl) exposure on HSP90ß1 expression in river pufferfish, Takifugu obscurus. The full length of To-HSP90ß1 was gained with 2775 bp in length, with an ORF (open reading frame) encoding an 803 aa polypeptide. A phylogenetic tree was constructed and showed the close relationship to other fish species. The HSP90ß1 mRNA transcript was expressed in all tissues investigated with higher level in the gill and liver. After the acute and chronic exposure of TBT-Cl, the To-HSP90ß1 mRNA transcript significantly was up-regulated in gills. Moreover, the histology study indicated the different injury degree of TBT-Cl in liver and gill. Immunohistochemistry (IHC) staining results implied the cytoplasm reorganization after TBT-Cl stress and the function of immunoregulation for To-HSP90ß1 to TBT-Cl exposure. All the results indicated that HSP90ß1 may be involved in the resistance to the invasion of TBT-Cl for keeping autoimmune homeostasis.

Molecular Cloning and Characterization of Carbonic Anhydrase XII from Pufferfish (Takifugu rubripes).

In this study, an 1888-bp carbonic anhydrase XII (CA XII) sequence was cloned from the brain of the pufferfish, Takifugu rubripes. The cloned sequence contained a coding region of 1470-bp, which was predicted to translate into a protein of 490 amino acid residues. The predicted protein showed between 68-56% identity with the large yellow croaker (Larimichthys crocea), tilapia (Oreochromis niloticus), and Asian arowana (Scleropages formosus) CA XII proteins. It also exhibited 36% and 53% identity with human CA II and CA XII, respectively. The cloned sequence contained a 22 amino acid NH2-terminal signal sequence and three Asn-Xaa-Ser/Thr sequons, among which one was potentially glycosylated. Four cysteine residues were also identified (Cys-21, Cys-201, Cys-355, and Cys-358), two of which (Cys-21 and Cys-201) could potentially form a disulfide bond. A 22-amino acid COOH-terminal cytoplasmic tail containing a potential site for phosphorylation by protein kinase A was also found. The cloned sequence might be a transmembrane protein, as predicted from in silico and phylogenetic analyses. The active site analysis of the predicted protein showed that its active site residues were highly conserved with tilapia CA XII protein. Homology modeling of the pufferfish CA XII was done using the crystal structure of the extracellular domain of human carbonic anhydrase XII at 1.55 Å resolution as a template. Semi-quantitative reverse transcription (RT)-PCR, quantitative PCR (q-PCR), and in situ hybridization confirmed that pufferfish CA XII is highly expressed in the brain.

Effect of dietary astaxanthin on the growth performance, non-specific immunity, and antioxidant capacity of pufferfish (Takifugu obscurus) under high temperature stress.

The present study was conducted to investigate the effects of astaxanthin on growth performance, biochemical parameters, ROS production, and immune-related gene expressions of the pufferfish (Takifugu obscurus) under high temperature stress. The experimental basal diets supplemented with astaxanthin at the rates of 0 (control), 20, 40, 80, 160, and 320 mg kg-1 were fed to fish for 8 weeks. The results showed that the fish fed diet with 80, 160, and 320 mg kg-1 astaxanthin significantly improved weight gain and specific growth rate. Furthermore, fish fed the moderate dietary astaxanthin increased plasma alkaline phosphatase activities, and decrease plasma aspartate aminotransferase and alanine aminotransferase activities. After the feeding trial, the fish were exposed to high temperature stress for 48 h. The results shown that astaxanthin could suppress ROS production induced by high temperature stress. Meanwhile, compared with the control group, the astaxanthin groups increased SOD, CAT, and HSP70 mRNA levels under high temperature stress. These results showed that the basal diet supplemented with 80-320 mg kg-1 astaxanthin could enhance growth, nonspecific immune responses, and antioxidant defense system and improve resistance against high temperature stress in pufferfish.

Molecular cloning and characterization of secretory carbonic anhydrase VI in pufferfish (Takifugu rubripes).

Carbonic anhydrase VI (CA VI) has been characterized as a secretory isozyme in mammals. Our present study confirmed the occurrence of CA VI in pufferfish (Takifugu rubripes). In this study, genomic sequence information for the CA VI of pufferfish was used for molecular cloning. We cloned a 1821 bp cDNA sequence, which consisted of a complete coding sequence of 1623bp and a deduced amino acid sequence of 540 amino acids from the open reading frame. A BLAST search indicated that this protein exhibits 53%, 79%, and 67% identity with human, tilapia, and gar CA VI, respectively. It also shows 63%-77% identity with other fish CA VI-like sequences (zebrafish, Asian arowana, salmon, and large yellow croaker). Moreover, alignment of two or more sequences revealed that the protein sequence of pufferfish CA VI has 34%-37% identity with mammalian and fish CA II sequences. An NH2-terminal signal peptide of 18 amino acids in length was predicted in the pufferfish CA VI sequence. Three potential N-linked glycosylation sites and two cysteine residues (Cys-28 and Cys-209) that are likely to form one disulfide bond were present in pufferfish CA VI. In silico and phylogenetic analyses revealed that pufferfish CA VI is an extracellular secretory protein. Active site analysis indicated that this protein is a low-activity CA isozymes due to a characteristic Val/Ile substitution at position 207. Homology modeling of puffer CA VI was performed using the crystal structure of human carbonic anhydrase XIV as a template structure, based on high similarity. Reverse transcription-polymerase chain reaction (PCR), quantitative PCR and in situ hybridization results revealed that, the pufferfish CA VI is highly expressed in liver tissue.

Digital gene expression analysis of Takifugu rubripes brain after acute hypoxia exposure using next-generation sequencing.

The adverse effects of hypoxia are confined to biochemical, physiological, developmental and behavioral processes, especially injury of the brain. In this study, a subset of genes in the brain of Takifugu rubripes were analyzed using digital gene expression (DGE) profiles and next-generation sequencing after acute hypoxia. Among 32 differentially expressed genes, 29 were up-regulated and 3 were down-regulated following hypoxia exposure. Using Gene Ontology analysis, it was found that transcription and translation, metabolism, and the stress response were affected by exposure to hypoxia. KEGG analysis revealed that the neuroactive ligand-receptor interaction pathway was significantly enriched in hypoxia-exposed T. rubripes. To further confirm the differential expression of genes, quantitative real-time PCR was performed to test six candidate genes, with the following five genes exhibiting the same expression patterns as the sequencing results: Proto-oncogene c-fos, Kruppel-like factor 2, immediate early response 2, proopiomelanocortin A and rhodopsin. This work is the first to identify and annotate genes in T. rubripes affected by hypoxia stress. This investigation provides data for understanding the molecular mechanism of fish adaptation to hypoxia and provides a reference for rationally setting dissolved oxygen levels in aquaculture.

Molecular and immune response characterizations of a novel Bax inhibitor-1 gene in pufferfish, Takifugu obscurus.

Apoptosis plays a crucial role in many biological processes, including development, cellular homeostasis, and immune responses. Bax inhibitor-1 (BI-1) is an anti-apoptotic protein that protects cells from endoplasmic reticulum stress-induced apoptosis. In this study, a BI-1 gene from the pufferfish Takifugu obscurus (Pf-BI-1) was identified and characterized. The full length of Pf-BI-1 cDNA was 1387 bp, including a 5'-UTR of 82 bp, a 3'-UTR of 591 bp containing a poly-(A) tail, and an open reading frame (ORF) of 714 bp that encodes a polypeptide of 237 amino acids. Pf-BI-1 was ubiquitously expressed in various tissues, with the highest expression levels in the blood, brain, and gill. The expression of Pf-BI-1 was up-regulated in a time-dependent manner after heat shock stress, ammonia stress, and bacterial challenge. Intracellular localization revealed that Pf-BI-1 was primarily localized in the cell cytoplasm. Furthermore, over-expression of Pf-BI-1 could active NF-кB reporter genes in HeLa cells. These results indicated that Pf-BI-1 may be involved in the apoptosis and immunity process against ambient stressors in pufferfish.