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.

Role of TRAF6 from obscure puffer (Takifugu obscurus) in immune response against Edwardsiella tarda infection.

Tumor necrosis factor receptor-associated factor 6 (TRAF6) is a vital molecule of inflammatory signaling pathways in innate immune response against pathogens. To elucidate its role in defense against Edwardsiella tarda infection in teleost fish, TRAF6 homologue was identified from obscure puffer (Takifugu obscurus) and functionally analyzed in this study. The obscure puffer TRAF6 (ToTRAF6) is a protein of 565 amino acids containing conserved RING domain, zinc finger-TRAF and MATH_TRAF6 domain. ToTRAF6 mRNA distributed in various healthy tissues of obscure puffer and was upregulated in the immune related tissues after E. tarda infection. ToTRAF6 protein was localized in the cytoplasm and aggregate as dots around the nuclei in FHM cells. The overexpression of ToTRAF6 in FHM cells decreased the quantity of E. tarda and induced the significant upregulation of downstream MAPK signaling pathway genes. These data suggest that ToTRAF6 is a key molecule of MAPK signaling pathway in defense against E. tarda infection.

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.

Three novel L-type lectins from obscure puffer Takifugu obscurus promote antimicrobial immune response.

L-type lectins (LTLs) have leguminous lectin domains that bind to high-mannose-type oligosaccharides. LTLs are involved in glycoprotein secretory pathways and associated with many immune responses. In the present research, three LTL homologs from obscure puffer Takifugu obscurus, designated as ToVIP36-1, ToVIP36-2, and ToVIP36-3, were first cloned and identified. The open reading frames of ToVIP36-1, ToVIP36-2, and ToVIP36-3 were 1068, 1002, and 1086 bp in length, respectively, and encode polypeptides with 355, 333, and 361 amino acids, respectively. Key conserved residues and functional domains, including lectin_leg-like domain (LTLD), transmembrane region, and C-terminal trafficking signal KRFY, were identified in all ToVIP36s. Quantitative real-time PCR analysis showed that the three ToVIP36s were widely expressed in six examined tissues and had relatively high expression levels in the liver and intestine. The expression levels of ToVIP36s were remarkably altered in the liver and kidney after induction by Vibrio harveyi and Staphylococcus aureus. Subsequently, the recombinant LTLDs of ToVIP36s (rToVIP36-LTLDs) were prepared by prokaryotic expression. Three rToVIP36-LTLD proteins agglutinated with S. aureus, V. harveyi, Vibrio parahaemolyticus, and Aeromonas hydrophila in a calcium-dependent manner. In the absence of calcium, rToVIP36-LTLD proteins bound to the bacteria by binding to lipopolysaccharides, peptidoglycans, d-mannose, and d-galactose and inhibited the growth of S. aureus and V. harveyi. Our results indicated that ToVIP36s function as pattern-recognition receptors in T. obscurus immunity, providing insights into the role of LTLs in the antibacterial immunity of fishes.

Estrodiol-17ß and aromatase inhibitor treatment induced alternations of genome-wide DNA methylation pattern in Takifugu rubripes gonads.

Estradiol-17ß (E2) and aromatase inhibitor (AI) exposure can change the phenotypic sex of fish gonads. To investigated whether alterations in DNA methylation is involved in this process, the level of genome-wide DNA methylation in Takifugu rubripes gonads was quantitatively analyzed during the E2-induced feminization and AI-induced masculinization processes in this study. The methylation levels of the total cytosine (C) in control-XX(C-XX), control-XY (C-XY), E2-treated-XY (E-XY) and AI-treated-XX (AI-XX) were 9.11%, 9.19%, 8.63% and 9.23%, respectively. In the C-XX vs C-XY comparison, 4,196 differentially methylated regions (DMRs) overlapped with the gene body of 2,497 genes and 608 DMRs overlapped with the promoter of 575 genes. In the E-XY vs C-XY comparison, 6,539 DMRs overlapped with the gene body of 3,416 genes and 856 DMRs overlapped with the promoter of 776 genes. In the AI-XX vs C-XX comparison, 2,843 DMRs overlapped with the gene body of 1,831 genes and 461 DMRs overlapped with the promoter of 421 genes. Gonadal genomic methylation mainly occurred at CG sites and the genes that overlapped with DMRs on CG context were most enriched in the signaling pathways related to gonad differentiation, such as the Wnt, TGF-ß, MAPK, CAM and GnRH pathways. The DNA methylation levels of steroid synthesis genes and estrogen receptor genes promoter or gene body were negative correlated with their expression. After bisulfite sequencing verification, the DNA methylation level of the amhr2 promoter in XY was increased after E2 treatment, which consistent with the data from the genome-wide DNA methylation sequencing. In C-XY group, the expression of amhr2 was significantly higher than that in E-XY (p < 0.05). Additionally, dnmt1, which is responsible for methylation maintenance, expressed at similar level in four groups (p > 0.05). dnmt3, tet2, and setd1b, which were responsible for methylation modification, expressed at significantly higher levels in E-XY compared to the C-XY (p < 0.05). Dnmt3 and tet2 were expressed at significantly higher levels in AI-XX than that in C-XX (p < 0.05). These results indicated that E2 and AI treatment lead to the aberrant genome-wide DNA methylation level and expression level of dnmt3, tet2, and setd1b in T. rubripes gonad.

Copper induced intestinal inflammation response through oxidative stress induced endoplasmic reticulum stress in Takifugu fasciatus.

Copper (Cu) pollution in aquaculture water has seriously threatened the healthy and sustainable development of the aquaculture industry. Recently, many researchers have studied the toxic effects of Cu exposure on fish. However, the relationship between endoplasmic reticulum stress (ERS) and the inflammatory response, as well as its possible mechanisms, remain unclear. Particularly, information related to fish intestines must be expanded. Our study initially investigated the mechanisms underlying intestinal toxicity and inflammation resulting from Cu-induced ERS in vivo and in vitro in Takifugu fasciatus. In vivo study, T. fasciatus were treated with different concentrations (control, 20, and 100 µg/L) of Cu exposure for 28 days, causing intestinal oxidative stress, ERS, inflammatory responses, and histopathological and ultrastructural damage. Transcriptomic data further showed that Cu exposure caused ERS, as well as inflammatory responses, in the intestinal tracts of T. fasciatus. In vitro experiments on the intestinal cells of T. fasciatus showed that Cu exposure treatment (7.5 µg/mL) for 24 h induced ERS and increased mitochondrial numbers and inflammatory responses. In contrast, the addition of 4-phenylbutyric acid (4-PBA) alleviated ERS and inflammatory response in the Cu-exposed group. Furthermore, the reactive oxygen species (ROS) inhibitor, N-Acetyl-l-cysteine (NAC), effectively alleviated Cu-induced ERS. In conclusion, our in vivo and in vitro studies have confirmed that oxidative stress triggers the ERS pathway, which is involved in the intestinal inflammatory response. Our study provides new insights into the relationship among Cu-induced oxidative stress, ERS, and inflammatory responses in fish, as well as for the healthy culture of fish in aqueous environments.

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.

Recovery of Fatty Acid and Volatile Flavor Compound Composition in Farmed Tiger Puffer (Takifugu rubripes) with a Fish Oil-Finishing Strategy.

Booming fish farming results in a relative shortage of fish oil (FO) supply, meaning that alternative oils are increasingly used in fish feeds, which leads to reduction of long-chain polyunsaturated fatty acids (LC-PUFAs) and other relevant changes in fish products. This study investigated the efficacy of an FO-finishing strategy in recovering the muscle quality of farmed tiger puffer. An eight-week feeding trial (growing-out period) was conducted with five experimental diets, in which graded levels (0 (control), 25, 50, 75, and 100%) of added FO were replaced by poultry oil (PO). Following the growing-out period was a four-week FO-finishing period, during which fish in all groups were fed the control diet. Dietary PO significantly decreased the muscle LC-PUFA content, whereas in general, the FO-finishing strategy recovered it to a level comparable with that of the group fed FO continuously. The recovery efficiency of EPA was higher than that of DHA. Dietary PO also led to changes of volatile flavor compounds in the muscle, such as butanol, pentenal, and hexenal, whereas the FO-finishing strategy mitigated the changes. In conclusion, the FO-finishing strategy is promising in recovering the LC-PUFA and volatile-flavor-compound composition in farmed tiger puffer after the feeding of PO-based diets.

Identification of Novel Antifreeze Peptides from Takifugu obscurus Skin and Molecular Mechanism in Inhibiting Ice Crystal Growth.

Foodborne hydrolyzed antifreeze peptides have been widely used in the food industry and the biomedical field. However, the components of hydrolyzed peptides are complex and the molecular mechanism remains unclear. This study focused on identification and mechanism analysis of novel antifreeze peptides from Takifugu obscurus skin by traditional methods and computer-assisted techniques. Results showed that three peptides (EGPRAGGAPG, GDAGPSGPAGPTG, and GEAGPAGPAG) possessed cryoprotection via reducing the freezing point and inhibiting ice crystal growth. Molecular docking confirmed that the cryoprotective property was related to peptide structure, especially α-helix, and hydrogen bond sites. Moreover, the antifreeze peptides were double-faces, which controlled ice crystals while affecting the arrangement of surrounding water molecules, thus exhibiting a strong antifreeze activity. This investigation deepens the comprehension of the mechanism of antifreeze peptides at molecular scale, and the novel efficient antifreeze peptides can be developed in antifreeze materials design and applied in food industry.

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.

Pituitary luteinizing hormone synthesis starts in aromatase (cyp19a1b)-positive cells expressing esr1 and esr2b at the onset of puberty in Takifugu rubripes (fugu).

Unlike mammals, teleost fish have high aromatase activity (AA) in the pituitary. However, the cells responsible for oestradiol synthesis and the local physiological roles of this hormone remain unclear. Hence, we investigated the effects of age and development on steroidogenic activity, mRNA expression, and cyp19a1b localization in the pituitary gland of the Japanese pufferfish Takifugu rubripes. Under aquaculture conditions, AA was highest after puberty, and the mRNA expression levels of cyp19a1b and the oestrogen receptors esr1 and 2b and the level of serum testosterone (T) were significantly increased after puberty compared with the other developmental stages in male and female pufferfish. Immunohistochemistry using multiple antibodies and in situ hybridization analysis revealed that Cyp19a1b colocalizes with luteinizing hormone (LH) in pituitary cells. Furthermore, Esr1 was localized in the nuclei of all hormone-producing cells, whereas Esr2b was localized only in the nuclei of Cyp19- and LH-positive cells. The administration of an aromatizable androgen (T) or oestrogen (E2) to reproductively inactive females induced LH synthesis in vivo. We prepared spheroids from pituitary cells to investigate the role of local E2 in LH synthesis in vitro. Immunohistochemical analysis of spheroids showed that T-induced LH synthesis could be blocked by an aromatase inhibitor and/or an ER antagonist but not an AR antagonist. Taken together, these findings suggest that LH synthesis is initiated in cyp19a1b-, esr1-, and esr2b-expressing cells at the onset of puberty under the control of steroidal feedback, and both feedback and local oestrogen may be involved in controlling LH synthesis in these cells.

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.

Characterization and functional analysis of a c-type lysozyme gene from obscure puffer Takifugu obscurus.

Lysozyme (Lyz) is an alkaline enzyme that hydrolyzes mucopolysaccharides in bacteria and is highly conserved vertebrates and invertebrates. In this study, a c-type lysozyme gene (named ToLyzC) from the obscure puffer Takifugu obscurus was cloned and characterized. The full-length cDNA of ToLyzC was 432 bp, encoding 143 amino acids, with a predicted molecular mass of 16.2 kDa and a theoretical pI of 8.86. The depicted protein sequence contained a LYZ1 domain from 16 to 142 amino acids, seven conserved cysteine residues. Phylogenetic analysis indicated that ToLyzC clustered with Lyzs from other teleost fishes. Quantitative real-time PCR analysis revealed that ToLyzC mRNA was mainly expressed in the liver. The transcript level of ToLyzC gene was significantly upregulated after Staphylococcus aureus and Vibrio harveyi challenge. The optimal pH and temperature of recombinant ToLyzC protein (rToLyzC) lytic activity was detected to be 7.5 and 35 °C, respectively. rToLyzC exhibited significant antibacterial and bacterial binding activities against S. aureus, Aeromonas hydrophila, V. harveyi, and Edwardsiella tarda at different time points. In addition, the morphological changes of V. harveyi cells treated with rToLyzC were observed under scanning electron microscope, which further confirmed the antibacterial and bacteriolytic activity of rToLyzC. Taken together, our current study indicated that ToLyzC is involved in the immune response to bacterial infection in obscure puffers.

Evaluation of the slow-release polylactic acid/polyhydroxyalkanoates active film containing oregano essential oil on the quality and flavor of chilled pufferfish (Takifugu obscurus) fillets.

Active packaging is an innovative and effective way to extend the shelf life of food, but few studies have focused on the effect of its active ingredients on food flavor. This study aimed to develop slow-release polylactic acid/polyhydroxyalkanoates (PLA/PHA) active packaging containing oregano essential oil (OEO) and investigate the effect of active composite packaging on the flavor and quality of pufferfish fillets. The plasticizing effect of OEO increased the elongation at break (EAB) of the films from 23.36% to 65.80%. The adsorption of montmorillonite (MMT) reduces the loss of OEO during processing. The amount of active substance (carvacrol) released from PLA/PHA/OEO/MMT film to pufferfish was 9.70 mg/kg. The pufferfish fillets packed in PLA/PHA/OEO/MMT film showed the slightest difference on the 8th day from the beginning of storage. The slow-release composite films could extend the shelf life of pufferfish fillets by 2-3 days at 4 °C ± 1 °C.

Molecular cloning, expressional analyses and functional identification of TRAIL in tilapia, Oreochromis niloticus.

The tumour necrosis factor superfamily (TNFSF) plays critical roles in tumour apoptosis, tissue morphogenesis and lineage determination. TNFSF10 (TRAIL or Apol-2) belongs to the tumour necrosis factor (TNF) cytokine family and induces rapid apoptosis in a wide variety of tumour cell lines upon binding to death-inducing signalling receptors. In this study, we identified TNFSF10 from Nile tilapia (Oreochromis niloticus) and found it was most closely related to Japanese pufferfish (Takifugu rubripes) TNFSF10. Amino acid identity between tilapia TNFSF10 and mandarin fish (Siniperca chuatsi) TRAIL was 69.2%. The highest expression of TNFSF10 mRNA was observed in the liver. In vitro studies showed that the mRNA expression of TNFSF10 was significantly stimulated by LPS in head kidney leucocytes, but remarkably inhibited by Poly I:C in spleen leucocytes. In vivo studies showed Streptococcus agalactiae infection significantly induced the mRNA expression of TNFSF10 in both the head kidney and spleen. The soluble recombinant protein Trx-TNFSF10 could induce cytotoxicity and apoptosis in HeLa cells with cycloheximide as a promoter. Taken together, these results in this study indicate that TNFSF10 may play important roles in the immune system of Nile tilapia.

A new calnexin modulates antibacterial immune response in obscure puffer Takifugu obscurus.

Calnexin (Cnx) is a membrane-bound lectin chaperone of the endoplasmic reticulum. In this study, a novel Cnx homologue from the obscure puffer Takifugu obscurus was characterized, tentatively named ToCnx. The cDNA of ToCnx was 1803 bp, and it contained an open reading frame encoding a polypeptide of 600 amino acid residues with a calculated molecular weight of 67.5 kDa. Multiple alignment of the deduced amino acid sequences of ToCnx and other related fish Cnxs revealed that ToCnx had typical characteristics of fish Cnxs. Sequence comparison and phylogenetic tree analysis showed that ToCnx had the closest relationship with Cnxs from Takifugu flavidus and Takifugu rubripes. ToCnx transcripts were detected in all the tissues examined, and they were mainly expressed in the liver, kidney, and intestine. Upon Vibrio harveyi, Edwardsiella tarda, and Aeromonas hydrophila infection, ToCnx transcripts were all significantly upregulated in the kidneys. The recombinant calreticulin domain of ToCnx (rToCnx) was prepared by prokaryotic expression. In the absence of calcium, rToCnx was able to bind three Gram-negative bacteria (V. harveyi, E. tarda, and A. hydrophila) and two bacterial saccharides, such as lipopolysaccharide and peptidoglycan. In the presence of calcium, rToCnx could agglutinate all the detected microorganisms. In addition, rToCnx possessed the effect of inhibiting the growth of three microbe strains. These observations suggested that ToCnx is an important participant in host immune defense against bacteria.

A rational tool for the umami evaluation of peptides based on multi-techniques.

Umami peptides have become of key interest in the development of flavoring agents. However, the lack of known umami peptides further prevents the understanding of the umami mechanism. The famous pufferfish (Takifugu flavidus) is a great resource for novel umami peptides, and we further analyze the umami characteristics of peptides based on multi-evaluation. In this study, five novel umami peptides, DF9, TK18, AK11, IK10, and GT12 were found; DF9 having the highest umami intensity, followed by AK11. Moreover, biosensor results showed DF9 with the lowest Ka value of 6.85 × 10-13 mol/L, followed by AK11. These data are mostly in agreement with sensory evaluation and fully reveal the umami mechanism of peptides. Quantum chemical and molecular docking demonstrated active site D in peptides bound with T1R1 receptor. Our results open up new strategies to estimate the taste characteristics of umami peptides and provide rational tools for screening umami peptides in food.

Taste and stability characteristics of two key umami peptides from pufferfish (Takifugu obscurus).

Takifugu obscurus (T. obscurus) is known for its umami taste. Two taste-active peptides, Pro-Val-Ala-Arg-Met-Cys-Arg (PR-7) and Tyr-Gly-Gly-Thr-Pro-Pro-Phe-Val (YV-8), were proved as key compounds that contributed to the typical taste of T. obscurus. However, whether these peptides have the potential as umami supplements is unknown. The purpose of this study was to investigate the taste characteristics of PR-7 and YV-8, as well as stability at different pH values by sensory evaluation, instrumental analysis and quantum chemical calculation. The results indicated that PR-7 and YV-8 presented umami taste at near neutral pH (6.5-8.0) and had umami-enhancing effects. PR-7 also exhibited significant kokumi activity. Additionally, two peptides showed remarkable stability after different pH treatments, especially YV-8; this may be related to its stable structural property. All the results suggest that both peptides have great potential to be applied in complex foods to provide desirable taste, and act as a feasible alternative to monosodium l-glutamate.

A Novel Angiotensin-I-Converting Enzyme (ACE) Inhibitory Peptide from Takifugu flavidus.

Alcalase, neutral protease, and pepsin were used to hydrolyze the skin of Takifugu flavidus. The T. flavidus hydrolysates (TFHs) with the maximum degree of hydrolysis (DH) and angiotensin-I-converting enzyme (ACE)-inhibitory activity were selected and then ultra-filtered to obtain fractions with components of different molecular weights (MWs) (<1, 1-3, 3-10, 10-50, and >50 kDa). The components with MWs < 1 kDa showed the strongest ACE-inhibitory activity with a half-maximal inhibitory concentration (IC50) of 0.58 mg/mL. Purification and identification using semi-preparative liquid chromatography, Sephadex G-15 gel chromatography, RP-HPLC, and LC-MS/MS yielded one new potential ACE-inhibitory peptide, PPLLFAAL (non-competitive suppression mode; IC50 of 28 µmmol·L-1). Molecular docking and molecular dynamics simulations indicated that the peptides should bind well to ACE and interact with amino acid residues and the zinc ion at the ACE active site. Furthermore, a short-term assay of antihypertensive activity in spontaneously hypertensive rats (SHRs) revealed that PPLLFAAL could significantly decrease the systolic blood pressure (SBP) and diastolic blood pressure (DBP) of SHRs after intravenous administration. These results suggested that PPLLFAAL may have potential applications in functional foods or pharmaceuticals as an antihypertensive agent.