Language：English   Publishing type：Research paper (scientific journal)  

The hepatotoxin microcystin-LR is a strong inhibitor of serine/threonine protein phosphatase (PP) 1 and PP2A. The onset of its cytotoxicity depends on its selective uptake via the hepatocyte uptake transporters, organic anion transporting polypeptide (OATP) 1B1 and OATP1B3. Understanding and preventing the cytotoxicity of microcystin-LR is crucial to maintain human health. This chemoprevention study demonstrates that the herbal plant extract of iwajisha (20 µg/mL) reduced microcystin-LR cytotoxicity in OATP1B3-expressing cells by approximately six times. In addition, 20 µM acteoside, which is one of the major compounds in iwajisha, reduced microcystin-LR cytotoxicity by approximately 7.4 times. Acteoside could also reduce the cytotoxicity of other compounds, such as okadaic acid and nodularin, which are both substrates of OATP1B3 and inhibitors of PP1/PP2A. To investigate the mechanism by which the cytotoxicity of microcystin-LR is attenuated by acteosides, microcystin-LR and microcystin-LR-binding proteins in cells were examined after microcystin-LR and acteosides were co-exposed. Thus, acteoside noncompetitively inhibited microcystin-LR uptake by OATP1B3-expressing cells. Furthermore, acteoside inhibited the intracellular interaction of microcystin-LR with its binding protein(s), including the 22 kDa protein. Furthermore, using immunoblot analysis, acteoside induced the phosphorylation of extracellular signal-regulated kinase (ERK), which is one of the survival signaling molecules. These results suggest that acteoside reduces microcystin-LR cytotoxicity through several mechanisms, including the inhibition of microcystin-LR uptake via OATP1B3, and decreased interaction between microcystin-LR and its binding protein(s), and that ERK signaling activation contributes to the attenuation effect of acteoside against microcystin-LR cytotoxicity.

DOI： 10.1055/a-1978-8768

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Ninjinyoeito, Hochuekkito, and Juzentaihoto are the three types of Kampo-hozai used to support the treatment of various diseases by energizing patients through improved mental health. While Kampo-hozais are clinically used to improve mental energy decline, a comparison between their effects on neuropsychiatric symptoms like anxiety and sociability and the strength of their effects has not been conducted. Therefore, this study compared the effects of Ninjinyoeito, Hochuekkito, and Juzentaihoto on psychiatric symptoms using neuropeptide Y knockout (NPY-KO) zebrafish, a suitable animal model for anxiety and low sociability. Neuropeptide Y knockout zebrafish were fed a Ninjinyoeito, Hochuekkito, or Juzentaihoto-supplemented diet for 4 days. Then, sociability was analyzed using a three-Chambers test and anxiety-like behavior was evaluated using the cold stress and novel tank tests. The results showed that Ninjinyoeito treatment improved the low sociability of neuropeptide Y knockout, while Hochuekkito and Juzentaihoto did not. Neuropeptide Y knockout exhibited anxiety-like behaviors, such as freezing and swimming in the wall area under cold stress, but Ninjinyoeito treatment improved these behaviors. However, these anxiety-like behaviors were not improved by Hochuekkito and Juzentaihoto. Ninjinyoeito treatment also improved anxiety-like behaviors of neuropeptide Y knockout in the novel tank test. However, no improvement was shown in the Hochuekkito and Juzentaihoto groups. This trend was also confirmed in the low water stress test using wild-type zebrafish. This study exhibits that among the three types of Kampo-hozai, Ninjinyoeito is the most effective in psychiatric disorders associated with anxiety and low sociability.

DOI： 10.3389/fphar.2023.1168229

PubMed

Language：English  

DOI： 10.3389/fphar.2023.1213252

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Neu1 is a glycosidase that releases sialic acids from the non-reducing ends of glycoconjugates, and its enzymatic properties are conserved among vertebrates. Recently, Neu1-KO zebrafish were generated using genome editing technology, and the KO fish showed abnormal emotional behavior, such as low schooling, low aggressiveness, and excess exploratory behavior, accompanied by the downregulation of anxiety-related genes. To examine the alteration of neuronal and glial cells in Neu1-KO zebrafish, we analyzed the molecular profiles in the zebrafish brain, focusing on the midbrain and telencephalon. Using immunohistochemistry, we found that signals of Maackia amurensis (MAM) lectin that recognizes Sia α2-3 linked glycoconjugates were highly increased in Neu1-KO zebrafish brains, accompanied by an increase in Lamp1a. Neu1-KO zebrafish suppressed the gene expression of AMPA-type glutamate receptors such as gria1a, gria2a, and gria3b, and vesicular glutamate transporter 1. Additionally, Neu1-KO zebrafish induced the hyperactivation of astrocytes accompanied by an increase in Gfap and phosphorylated ERK levels, while the mRNA levels of astrocyte glutamate transporters (eaat1a, eaat1c, and eaat2) were downregulated. The mRNA levels of sypb and ho1b, which are markers of synaptic plasticity, were also suppressed by Neu1 deficiency. Abnormal activity of microglia was also revealed by IHC, and the expressions of iNOS and IL-1β, an inflammatory cytokine, were increased in Neu1-KO zebrafish. Furthermore, drastic neuronal degeneration was detected in Neu1-KO zebrafish using Fluoro-Jade B staining. Collectively, the neuronal and glial abnormalities in Neu1-KO zebrafish may be caused by changes in the excitatory neurotransmitter glutamate and involved in the emotional abnormalities.

DOI： 10.1007/s10719-022-10074-8

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Neu1 is a lysosomal glycosidase that catalyzes the removal of sialic acids from glycoconjugates. Although Neu1 sialidase is highly conserved among vertebrates, the role of fish Neu1 is not fully understood because of its unique aquatic living situation. Compared to land animals, fish have a higher chance of bacterial infection, and to understand the role of fish Neu1, the susceptibility of Neu1 knockout zebrafish (Neu1-KO) was evaluated using Edwardsiella piscicida, a fish pathogen. Neu1-KO larvae showed high susceptibility to E. piscicida, despite the activation of macrophages, and presented increased lysosomal signals induced by the accumulation of Sia α2-3 linked oligosaccharides. The accumulation coincided with the signal of the macrophage marker, suggesting that the dysfunction of lysosomes in macrophages would result in a high susceptibility of Neu1-KO to E. piscicida. Chloroquine, an inhibitor of lysosomal degradation, induced high mortality of wild type zebrafish with E. piscicida infection accompanied by increased lysosomal accumulation, similar to Neu1-KO zebrafish. This study revealed that Neu1 sialidase plays a crucial role in the lysosomal degradation of macrophages with a bacterial infection.

DOI： 10.1016/j.gene.2022.146667

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Sialic acid and its catabolism are involved in bacterial pathogenicity. N-acetylneuraminate lyase (NAL), which catalyzes the reversible aldol cleavage of sialic acid to form N-acetyl-D-mannosamine in the first step of sialic acid degradation, has been recently investigated to elucidate whether NAL enhances bacterial virulence; however, the role of NAL in bacterial pathogenicity remains unclear. In the present study, we demonstrated that the existence of two enzymes in Edwardsiella piscicida, referred to as dihydrodipicolinate synthase (DHDPS) and NAL, induced the cleavage/condensation activity toward sialic acids such as N-acetylneuraminic acid, N-glycolylneuraminic acid and 3-deoxy-D-glycero-D-galacto-non-2-ulopyranosonic acid. NAL enhanced cellular infection in vitro and suppressed the survival rate in zebrafish larvae in bath-infection in vivo, whereas DHDPS did not. Furthermore, NAL strongly activated the expression of E. piscicida phenotypes such as biofilm formation and motility, whereas DHDPS did not. Besides, the gene expression level of nanK, nanE, and glmU were up-regulated in the NAL-overexpressing strain, along with an increase in the total amount of N-acetylglucosamine.

DOI： 10.1007/s10719-022-10045-z

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Edwardsiella piscicida is a gram-negative bacterium that causes Edwardsiellosis in cultured fish. Edwardsiellosis is accompanied by symptoms such as skin lesions, hemorrhage, and necrosis in fish organs, which leads to significant economic losses in the aquaculture industry. Recently, we found that bacterial sialoglycoconjugates may be involved in the infectivity of E. piscicida. The more infectious strains of E. piscicida contain more sialic acid in the bacterial body, and the mRNA level of putative CMP-Neu5Ac synthase (css) is upregulated compared to that in the non-pathogenic strain. However, this putative css gene is yet to be cloned, and the involvement of CSS in E. piscicida pathogenicity remains unclear. Here, we cloned and transferred the css gene from E. piscicida into the FPC498 strain. CSS promoted infection in cultured cells originating from different fish species, and enhanced the mortality of E. piscicida-infected zebrafish larvae. CSS enhanced cell attachment and motility in E. piscicida, which differs from the decreased bacterial growth observed with the sialic acid-supplemented M9 medium. Both fractions (chloroform-methanol)-soluble and -insoluble fraction) prepared from E. piscicida pellet exhibited the increment of sialo-conjugates induced by CSS. Further, lectin blotting revealed the increment of Sia α2-3- and α2-6-, but not α2-8-, -linked glycoprotein in CSS-overexpressing E. piscicida. Overall, these findings indicate the physiological significance of CSS and the role of sialylation in E. piscicida pathogenicity.

DOI： 10.1016/j.fsi.2022.04.033

PubMed

Language：Japanese   Publisher：一般社団法人 日本応用糖質科学会  

DOI： 10.5458/bag.12.1_43

Language：English   Publishing type：Research paper (scientific journal)  

Sociability is an essential component of the linkage structure in human and other vertebrate communication. Low sociability is defined as a poor social approach, including social withdrawal and apathy, and is implicated in a variety of psychiatric disorders. Ninjinyoeito (NYT), a traditional Japanese herbal medicine, has been used in the medical field. This study aimed to determine the effect of NYT on low sociality in NPY-KO zebrafish. NPY-KO zebrafish were fed a 3% NYT-supplemented diet for 4 days and subjected to behavioral tests. In the mirror test, NPY-KO zebrafish fed a control diet showed avoidance behavior toward their mirror counterparts. In contrast, the treatment of NPY-KO zebrafish with NYT significantly increased their interaction with their counterparts in the mirror. In addition, a 3-chambers test was conducted to confirm the effect of NYT on the low sociality of NPY-KO zebrafish. NPY-KO zebrafish fed the control diet showed less interaction with fish chambers, while NYT treatment increased the interaction. Phosphorylation of ERK, a marker of neuronal activity, was significantly reduced in the whole brain of NYT-fed NPY-KO zebrafish, compared to the control diet. NYT treatment significantly suppressed hypothalamic-pituitary-adrenal-related genes (gr, pomc, and crh) and sympathetic-adrenal-medullary-related genes (th1, th2, and cck) in NPY-KO zebrafish. NYT administration significantly reduced mRNA levels of gad1b compared to the control diet, suggesting the involvement of GABAergic neurons in NYT-induced improvement of low sociability. Furthermore, the expression of CREB was suppressed when NPY-KO zebrafish were fed NYT. Next, we attempted to identify the effective herb responsible for the NYT-induced improvement of low sociability. NPY-KO zebrafish were fed an experimental diet containing the target herb for 4 days, and its effect on sociability was evaluated using the 3-chambers test. Results showed that Cinnamon Bark and Polygala Root treatments significantly increased time spent in the fish tank area compared to the control diet, while the other 10 herbs did not. We confirmed that these two herbs suppressed the activity of HPA-, SAM-, and GABAergic neurons, as well as NYT-treated zebrafish, accompanied by downregulation of CREB signaling. This study suggests the potential use of NYT as a drug for sociability disorders.

DOI： 10.3389/fphar.2022.905711

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

NEU1 sialidase hydrolyzes sialic acids from glycoconjugates in lysosomes. Deficiency of NEU1 causes sialidosis with symptoms including facial dysmorphism, bone dysplasia, and neurodegeneration. However, the effects of NEU1 deficiency on emotional activity have not been explored. Here, we conducted the behavioral analysis using Neu1-knockout zebrafish (Neu1-KO). Neu1-KO zebrafish showed normal swimming similar to wild-type zebrafish (WT), whereas shoaling was decreased and accompanied by greater inter-fish distance than WT zebrafish. The aggression test showed a reduced aggressive behavior in Neu1-KO zebrafish than in WT zebrafish. In the mirror and 3-chambers test, Neu1-KO zebrafish showed more interest toward the opponent in the mirror and multiple unfamiliar zebrafish, respectively, than WT zebrafish. Furthermore, Neu1-KO zebrafish also showed increased interaction with different fish species, whereas WT zebrafish avoided them. In the black-white preference test, Neu1-KO zebrafish showed an abnormal preference for the white region, whereas WT zebrafish preferred the black region. Neu1-KO zebrafish were characterized by a downregulation of the anxiety-related genes of the hypothalamic-pituitary-adrenal axis and upregulation of lamp1a, an activator of lysosomal exocytosis, with their brains accumulating several sphingoglycolipids. This study revealed that Neu1 deficiency caused abnormal emotional behavior in zebrafish, possibly due to neuronal dysfunction induced by lysosomal exocytosis.

DOI： 10.1038/s41598-021-92778-9

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Edwardsiella tarda is a Gram-negative bacterium causing economic damage in aquaculture. The interaction of E. tarda with microdomains is an important step in the invasion, but the target molecules in microdomains remain undefined. Here, we found that intraperitoneal injection of E. tarda altered splenic glycosphingolipid patterns in the model host medaka (Oryzias latipes) accompanied by alteration of glycosphingolipid metabolism-related gene expressions, suggesting that glycosphingolipid levels are involved in E. tarda infection. To ascertain the significance of glycosphingolipids in the infection, fish cell lines, DIT29 cells with a high amount of lactosylceramide (LacCer) and glucosylceramide (GlcCer), and GAKS cells with a low amount of these lipids, were treated with methyl-β-cyclodextrin to disrupt the microdomain. E. tarda infection was suppressed in DIT29 cells, but not in GAKS cells, suggesting the involvement of microdomain LacCer and GlcCer in the infection. DL-threo-1-phenyl-2-palmitoylamino-3-morpholino-1-propanol, an inhibitor of glycosphingolipid-synthesis, attenuated the infection in DIT29 cells, while Neu3-overexpressing GAKS cells, which accumulated LacCer, enhanced the infection. E. tarda possessed binding ability towards LacCer, but not GlcCer, and LacCer preincubation declined the infection towards fish cells, possibly due to the masking of binding sites. The present study suggests that LacCer may be a positive regulator of E. tarda invasion.

DOI： 10.1111/cmi.13365

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Neurogenesis is an important process for the formation of the central nervous system during ontogenesis. Mammalian sialidases are involved in neurogenesis through desialylation of sialo-glycoconjugates. However, the significance of fish sialidases, unlike that of mammals, in neurogenesis has not been investigated. The present study focuses on Nile tilapia (Oreochromis niloticus) because of its unique profiles of sialidases related to enzymatic properties, subcellular localization, and tissue-specific gene expression. First, the fish were cultured under aphotic condition, which is known to cause the delayed development of the retina and brain in various fish. Next, we investigate the effect of aphotic condition on the levels of tilapia sialidases. Our results revealed that the tilapia showed a decrease in the number of ganglion cell in the retina. The expression level of neu4 mRNA is up-regulated in the eyes from tilapia reared in Dark accompanied by the increase of retinal differentiation markers. These results indicated that tilapia Neu4 is involved in retinal development in Nile tilapia. Furthermore, we tried to clarify the function of tilapia Neu4 in the neuronal cells using two neuroblast cell lines (SH-SY5Y and Neuro2a cell lines). Tilapia Neu4 decreased sialic acid level of both nuclear glycoproteins as well as glycolipids. Moreover, tilapia Neu4 accelerated neurite formation in both two neural cell lines and, increased the acetylcholinesterase activity, but it did not affect cell proliferation. Collectively, these results suggest that Neu4 accelerates neurite differentiation during ontogenesis in tilapia.

DOI： 10.1016/j.biochi.2021.03.008

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Anxiety induced by excess mental or physical stress is deeply involved in the onset of human psychiatric diseases such as depression, bipolar disorder, and panic disorder. Recently, Kampo medicines have received focus as antidepressant drugs for clinical use because of their synergistic and additive effects. Thus, we evaluated the anxiolytic activity of Ninjinyoeito (NYT) using neuropeptide Y-knockout (NPY-KO) zebrafish that exhibit severe anxiety responses to acute stress. Adult NPY-KO zebrafish were fed either a 3% NYT-supplemented or normal diet (i.e., the control diet) for four days and were then examined via behavioral tests. After short-term cold stress (10 °C, 2 s) was applied, control-fed NPY-KO zebrafish exhibited anxiety behaviors such as freezing, erratic movement, and increased swimming time along the tank wall. On the other hand, NYT-fed NPY-KO zebrafish significantly suppressed these anxiety behaviors, accompanied by a downregulation of tyrosine hydroxylase levels and phosphorylation of extracellular signal-regulated kinases in the brain. To understand the responsible component(s) in NYT, twelve kinds of herbal medicines that composed NYT were tested in behavioral trials with the zebrafish. Among them, nine significantly reduced freezing behavior in NPY-KO zebrafish. In particular, Schisandra fruit induced the most potent effect on abnormal zebrafish behavior, even in the lower amount (0.3% equivalent to NYT), followed by Atractylodes rhizome and Cinnamon bark. Subsequently, four lignans uniquely found in Schisandra fruit (i.e., gomisin A, gomisin N, schizandrin, and schizandrin B) were investigated for their anxiolytic activity in NPY-KO zebrafish. As a result, schizandrin was identified as a responsible compound in the anxiolytic effect of NYT. These results suggest that NYT has a positive effect on mental stress-induced anxiety and may be a promising therapeutic for psychiatric diseases.

DOI： 10.1016/j.npep.2021.102136

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

The effective use of distillery by-products of the shochu sweet potato has long been considered as a rich source of protein, fiber, amino acids, fatty acids, probiotics with a great potential as a functional feed for animals. This study applied fermented by-product of Shochu distillery (FBPSD) and salmon roe emulsion oil for rotifer enrichment to evaluate its effects on the: (i) nutritional composition of rotifers and (ii) feeding responses of Paralichthys olivaceus larvae when fed with enriched rotifers. The salmon roe oil enrichment was set as a control diet and FBPSD was supplemented at different doses (1, 5 and 10 % based on the salmon roe oil amount). The results showed that the rotifers supplemented with FBPSD at 5 and 10 % significantly enhanced protein, carbohydrate, and HUFA content. Similarly, the activity and expression profiles of targeted enzymes including trypsin, chymotrypsin, lipase and amylase effectively responded at early and middle-metamorphosis of the larvae to 5 and 10 % enrichment regiment. These two treatments were also found to be better supported in energy metabolism evidenced by Cytochrome-C-Oxidase (cox) and ATPase-pk (atp) gene expression in the larvae. Moreover, the expression levels of immune-related genes (IL-10, IL-1 beta, TNF-alpha, P2Y(6)) and antioxidant-related genes (SOD, CAT) were significantly improved at 5 and 10 % of FBPSD. Cessation of feeding and delay in growth during the metamorphic process was also molecularly demonstrated. Larvae suffered low survival (24.5-25.3 %) in control and 1 % supplementation of FBPSD, but significantly improved at 5 and 10 % (36.4-38.7%). However, the growth performance of larval fish was not remarkable after 40 days of culture. The results of this study revealed that FBPSD could be considered as a potential supplementary enrichment for live feed in fish larviculture, especially at 5 and 10 % rotifer enrichment dosage.

DOI： 10.1016/j.aquaculture.2021.736352

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Kawakawa tuna (Euthynnus affinis) is a novel target species for aquaculture in Japan and currently there is very limited knowledge on its early ontogenetic development in captivity. The ontogeny of E. affinis larvae from hatching to 20 days after hatching (DAH) was illustrated in the present study through an integrated approach of histology, biochemistry and molecular approaches. Larvae were fed enriched L-type rotifer Brachionus plicatilis species complex from 2 to 10 DAH, followed by enriched Artemia nauplii. Target digestive enzymes; trypsin, pepsin, lipase and amylase were assessed in order to evaluate nutritional condition. The larvae began exogenous feeding from 2 DAH, coinciding with the opening of the oral capacity and anus. The yolk sac was rapidly consumed and completely resorbed at 3 DAH, while the oil globule disappeared by 5 DAH. Enzymatic activity and expression of the target enzymes were detectable at hatching, except pepsin. With the development of the gastric glands, pepsin activity was then detected at 10 DAH, which was followed by rapid increase in pepsin activity at 15 DAH. The larvae showed rapid growth, however, high mortality also occurred at 6-10 DAH which has been highlighted as a critical stage in the larviculture of E. affinis. From 15 to 20 DAH, the histological structures of the digestive tract were completely segmented with the digestive tract resembling that of its juvenile stages. The activity of trypsin, pepsin, and lipase tended to increase sharply, while amylase dramatically dropped, possibly indicating a change in dietary preference. Findings from the present study provides a preliminary insight into the early development of the digestive system and associated digestive enzymes of E. affinis larvae and establishes an initial step towards the rearing and feeding protocol optimization of this novel target species.

DOI： 10.1016/j.aquaculture.2020.735935

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

A good understanding of the morphological and functional changes during early larval fish ontogeny is essential for the enhancement of larviculture techniques. This study applied an integrative approach to describe the ontogenetic development of the Japanese flounder (Paralichthys olivaceus) emphasizing the changes in histology and related functions of its digestive system. Changes in histological structures of the digestive tract, expression and activity of digestive enzymes (trypsin, chymotrypsin, lipase, pepsin, amylase), expression of energy metabolism (ATP synthase (atp), cytochrome-c-oxidase (cox), regulatory factors (neuropeptide Y (npy), cholecystokinin (cck)) and growth (insulin like growth factor 1 (igf1), growth hormone (GH)) were investigated and linked to larval biometrics. The larvae absorbed nutrients from its yolk upon hatching until mouth opening (3 DAH), before beginning its exogenous nutritional phase. The digestive tract differentiations were histologically observed at 5 DAH. The results showed that the targeted enzymes were genetically programmed and activated from hatching, except pepsin which was detected at 25 DAH coinciding with visible development of the gastric glands. The larval stages (3-5 DAH) were marked with high mortality at exogenous feeding transition and also at the onset of metamorphosis (20-30 DAH). Energy metabolism, growth and peptide regulatory factor related genes were transcribed and translated at hatching to assist upon the onset of exogenous feeding. Less feeding and growth delay were noticed between 20 and 30 DAH. The digestive system was fully developed by 35 DAH and larvae transited to settlement life.

DOI： 10.1016/j.aquaculture.2020.735855

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Japan Consortium for Glycobiology and Glycotechnology  

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

2-keto-3-deoxy-D-glycero-D-galacto-nononic acid (KDN) is a minor component of sialic acids detected in vertebrates, such as human cancer cells, rat liver, and fish tissues. Although the enzyme activity of KDN-cleaving sialidase (KDN-sialidase) has been detected in rainbow trout, the gene responsible for its expression has not been identified in vertebrates. We evaluated sialidases in human and various fish for their KDN-cleaving activity using an artificial substrate, methylumbelliferyl-KDN (MU-KDN). Four of the human sialidases tested (NEU1, NEU2, NEU3, and NEU4) did not hydrolyze MU-KDN. Although most fish Neu1s showed negligible KDN-sialidase activity, two Neu1b sialidases from Oreochromis niloticus and Astyanax mexicanus, a paralog of Neu1, exhibited a potent KDN-sialidase activity. Further, O. niloticus and Oryzias latipes Neu3a exhibited a drastically high KDN-sialidase activity, while Danio rerio Neu3.1 showed moderate activities and other Neu3 proteins exhibited little activity. All the Neu4 sialidases tested in fish cleaved KDN and Neu5Ac from MU-KDN and MU-Neu5Ac, respectively, with equivalent potential. To our knowledge, this is the first report to identify KDN-sialidase genes in vertebrates and we believe that KDN-sialidase activity could be conserved among fish Neu4s.

DOI： 10.1007/s10719-020-09948-6

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Mammalian sialidase Neu1 is involved in various physiological functions, including cell adhesion, differentiation, cancer metastasis, and diabetes through lysosomal catabolism and desialylation of glycoproteins at the plasma membrane. Various animal models have been established to further explore the functions of vertebrate Neu1. The present study focused on zebrafish (Danio rerio) belonging to Cypriniformes as an experimental animal model with neu1 gene deficiency. The results revealed that the zebrafish Neu1 desialyzed both α2-3 and α2-6 sialic acid linkages from oligosaccharides and glycoproteins at pH 4.5, and it is highly conserved with other fish species and mammalian Neu1. Furthermore, Neu1-knockout zebrafish (Neu1-KO) was established through CRISPR/Cas9 genome editing. Neu1-KO fish exhibited slight abnormal embryogenesis with the accumulation of pleural effusion; however, no embryonic lethality was observed. Although Neu1-KO fish were able to be maintained as homozygous, they showed smaller body length and weight than the wild-type (WT) fish, and muscle atrophy and curvature of the vertebra were observed in adult Neu1-KO fish (8 months). The expression patterns of myod and myog transcription factors regulating muscle differentiation varied between Neu1-KO and WT fish embryo. Expression of lysosomal-related genes, including ctsa, lamp1a, and tfeb were up-regulated in adult Neu1-KO muscle as compared with WT. Furthermore, the expression pattern of genes involved in bone remodeling (runx2a, runx2b, and mmp9) was decreased in Neu1-KO fish. These phenotypes were quite similar to those of Neu1-KO mice and human sialidosis patients, indicating the effectiveness of the established Neu1-KO zebrafish for the study of vertebrate Neu1 sialidase.

DOI： 10.1042/BCJ20200348

PubMed

Publisher：ELSEVIER  

DOI： 10.1016/j.algal.2020.101905

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER  

Lysosomal desialylation is the initial step in the degradation of sialo-glycopeptides that is essential for regenerating sialo-glycoconjugates. Neu1 sialidase is the enzyme responsible for the removal of sialic acid in the mammalian lysosome. Although Neu1 sialidases are conserved in fish similar to mammals, their physiological functions remain to be fully understood. Nile tilapia (Oreochromis niloticus) is known to possess two putative Neu1 sialidases (Neu1a and Neu1b) in the genome that may have arisen by gene duplication (specifically in cichlidae family members). This suggests that understanding the Neu1 sialidase in fish, particularly cichlids, could provide insights into the (novel) physiological functions of these genes. Moreover, characterization of the tilapia Neu1 sialidase is paramount to ensure clarity of the desialylation reaction performed by the fish sialidases (like the characterized tilapia sialidases Neu3 and Neu4). Therefore, this study focused on the characterization of the tilapia Neu1 sialidases. Neu1b exhibited narrow substrate specificity when compared with Neu1a, whereas the properties of these two Neu1 sialidases, such as cathepsin A-induced activation, optimal pH, and lysosomal localization, were conserved. Neu1a mRNA levels were detected in various tissues of tilapia as compared to the mRNA levels of Neu1b. Although the cloned construct of Neu1b contained an extra exon unlike tilapia Neu1a, the exon did not affect the enzymatic properties of Neu1b. This study suggests that tilapia Neu1a profiles were highly conserved with other vertebrate Neu1 isoforms, while Neu1b probably evolved independently in other members of the cichlidae family. Moreover, the expression of sialidase genes (neu1a, neu1b, neu3a, and neu4) were determined in various stages of tilapia embryogenesis using real-time PCR; sialidase gene expression is reported to be drastically and individually altered during embryogenesis in Japanese medaka (Oryzias latipes). The mRNA levels of neu1a drastically increased between 72 and 84 hpf and mildly decreased from 84 to 144 hpf. In contrast, the transcript levels of neu1b did not change between 84 and 144 hpf and the expression of neu3a gradually increased between 84 and 120 hpf and drastically decreased at 144 hpf. The highest level of the neu4 transcripts was detected at 84 hpf. These expression patterns were different from those in Japanese medaka, possibly due to the different developmental program found in the tilapia embryo accompanied with the unique profiles of the tilapia sialidases.

DOI： 10.1016/j.gene.2020.144538

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

NeuropeptideY (NPY) controls energy homeostasis including orexigenic actions in mammalians and non-mammalians. Recently, NPY has attracted attention as a mediator of emotional behaviour and psychosomatic diseases. However, its functions are not fully understood. We established npy gene-deficient (NPY-KO) zebrafish (Danio rerio) to assess the relationship between NPY and emotional behaviours. The NPY-KO zebrafish exhibited similar growth, but pomc and avp mRNA levels in the brain were higher as compared to wild-type fish. NPY-KO zebrafish exhibited several anxiety-like behaviours, such as a decrease in social interaction in mirror test and decreased locomotion in black-white test. The acute cold stress-treated NPY-KO zebrafish exhibited anxiety-like behaviours such as remaining stationary and swimming along the side of the tank in the mirror test. Moreover, expression levels of anxiety-associated genes (orx and cck) and catecholamine production (gr, mr, th1 and th2) were significantly higher in NPY-KO zebrafish than in wild-type fish. We demonstrated that NPY-KO zebrafish have an anxiety phenotype and a stress-vulnerability like NPY-KO mice, whereby orx and/or catecholamine signalling may be involved in the mechanism actions.

DOI： 10.1038/s41598-020-62699-0

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Fish store triglycerides (TGs) in the liver, muscle, and adipose tissue and TGs constitute an energy source upon metabolic demand. The liver generally plays important roles in lipid metabolism. Recent studies have suggested the possibility of hepatic lipid metabolic regulation by ganglioside in mammals; however, ganglioside-mediated regulation of lipid metabolism is unclear in fish. This study aimed to clarify the role of ganglioside in fish TG metabolism, with particular reference to Neu3a, a ganglioside-specific sialidase expressed in the fish liver. Under fasting conditions, there was a decrease in hepatic TG contents, and neu3a mRNA level was significantly up-regulated in the medaka liver. To determine the role of Neu3a in hepatic lipid metabolism, Neu3a stable transfectants were generated using fish liver Hepa-T1 cells. After treating Neu3a cells with oleic acid, reduction of TG was detected in comparison with the mock cells. Furthermore, lipase activity was greater in Neu3a cells than in mock cells. To examine which ganglioside regulates these events, alterations of ganglioside composition in Neu3a cells were analyzed. Neu3a cells exhibited increased level of lactosylceramide (LacCer), a Neu3 enzymatic product originating from GM3. In addition, exposure of LacCer toward Hepa-T1 cells resulted in an increase of neutral lipase activity. The present results suggest that Neu3a up-regulation in medaka under fasting condition accelerates hepatic TG degradation for energy production via GM3 desialylation.

DOI： 10.1007/s10695-019-00730-6

Web of Science

PubMed

Publisher：ELSEVIER  

The use of live feed to meet larval nutritional requirements has caused a bottleneck in marine fish larviculture whilst the use of micro-diets has had limited success in completely replacing live feeds. In this study, the variations in enzyme activity and its related gene expressions during early ontogenetic development of red sea bream (Pagrus major) were investigated. The experiment included four treatments: (i) using live feed (enriched L-type rotifers of the Brachionus plicatilis sp. complex) until 15 days after hatching (DAH) and gradually switching to Anemia; (ii) using micro-diet only; (iii) co-feeding diet (using both live feed and micro-diet) and, (iv) starvation (no food supplied). This study lasted 40 days and the activity and expression of digestive enzymes (trypsin (try), chymotrypsin (ctrb), pepsin (pep), amylase (amy2 alpha), pancreatic lipase (pl), expression of energy metabolism (ATP synthase (atp), cytochrome-c-oxidase (cox1), cholecystokinin (cck) and growth (insulin like growth factor 1 (igf1)) were linked to larval biometrics. After 40 DAH, no significant difference was observed in growth (igf1), energy metabolism (cox1, atp) and survival rate between live feed and co-feeding diet treatments. However, there were significantly higher differences observed when these were compared to the starvation and micro-diet only treatments. The pancreatic enzymes and cck were regulated by dietary compositions and detectable from hatching and fluctuated throughout larval ontogeny. While pep was initially observed only after 15 DAH. Limits of larval survival and low enzyme activity were observed when the larvae were subjected to starvation for 10 days and were able to adapt to micro-diet only after 15 DAH accompanied with a 5-day co-feeding transitionary period.

DOI： 10.1016/j.aquaculture.2019.734721

Web of Science

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Intracellular bacterial infections affect all vertebrates. Cultured fish are particularly vulnerable because no effective protection measures have been established since such infections emerged approximately 50 years ago. As in other vertebrates, the induction of cell-mediated immunity (CMI) plays an important role in protecting fish against infection. However, details of the mechanism of CMI induction in fish have not been clarified. In the present study, we focused on the production of interleukin 12 (IL-12), an important factor in CMI induction in fish. Using several different approaches, we investigated IL-12 regulation in amberjack (Seriola dumerili), the species most vulnerable to intracellular bacterial disease. The results of promoter assays and transcription factor gene expression analyses showed that the expression of interferon regulatory factor-1 (IRF-1) and activator protein-1 (AP-1) is necessary for IL-12 production. Phagocytosis of living cells (LCs) of Nocardia seriolae bacteria induced IL-12 production in neutrophils, accompanied by IRF-1 and AP-1 gene expression. Bacteria in which the exported repetitive protein (Erp)-like gene was deleted (Delta erp-L) could not establish intracellular parasitism or induce IRF-1 and AP-1 expression or IL-12 production, despite being phagocytosed by neutrophils. These data suggest that IL-12 production is regulated by (i) two transcription factors, IRF-1 and AP-1, (ii) phagocytosis of LCs by neutrophils, and (iii) one or more cell components of LCs. Our results enhance the understanding of the immune response to intracellular bacterial infections in vertebrates and could facilitate the discovery of new agents to prevent intracellular bacterial disease.

DOI： 10.1128/IAI.00459-19

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)   Publisher：PORTLAND PRESS LTD  

Edwardsiella tarda is a gram-negative bacterium causing significant economic losses to aquaculture. E. tarda possesses NanA sialidase which removes sialic acids from alpha 2-3 sialo-glycoprotein of host cells. However, the relationship between NanA sialidase activity and E. tarda invasiveness remains poorly understood. Furthermore, the pathway of sialic acid metabolism in E. tarda remains to be elucidated. We studied sialidase activity in several E. tarda strains and found that the pathogenic strains exhibited higher sialidase activity and greater up-regulation of the NanA mRNA level than non-pathogenic strain. Pathogenic strains also showed higher rates of infection in GAKS cells, and the infection was drastically suppressed by sialidase inhibitor. Additionally, NanA gene overexpression significantly increased infection and treatment of E. tarda with free sialic acid enhanced the rate of infection in GAKS cells. Sialic acid treatment enhanced mRNA levels of two N-acetylneuraminate lyases and one N-acetylneuraminate cytidylyltransferase. E. tarda uses sialic acid as a carbon source for growth via N-acetylneuraminate lyases. The strains with high N-acetylneuraminate cytidylyltransferase level showed greater sialylation of the lipopolysaccharides and glycoproteins. Our study establishes the significance of desialylation by E. tarda sialidase in the regulation of its invasiveness.

DOI： 10.1042/BCJ20190367

Web of Science

PubMed

Publisher：ELSEVIER  

Red sea bream (Pagrus major) is an important aquaculture species in Japan. This study aimed to determine the ontogenetic development of the digestive system, enzyme activity and related gene expressions of red sea bream larvae from hatching to post-metamorphosis (40 days after hatching, DAH). The larvae were fed with the euryhaline rotifer Brachionus plicatilis species complex L-type enriched with Chlorella vulgaris containing DHA from 3 to 15 DAH followed by Anemia nauplii from 16 to 40 DAH. Post culture analysis included, histological analysis of the larval gut, enzyme assays (trypsin, chymotrypsin, lipase, pepsin) and real time PCR with larvae sampled from 1 to 40 DAH. M hatching, the larval digestive tract was undifferentiated and resembled a straight tube. There was low detection of enzyme activity and gene expression of trypsin, chymotrypsin, lipase, and amylase during yolk-sac stage, before mouth opened. At 3 DAH, the mouth opened and the larvae began to exogenous feeding followed by the differentiation of the digestive tract into buccopharyngeal capacity and intestine at 5 DAH. The pancreas, liver, and gallbladder were functional indicating the transition from endogenous to exogenous feeding. The enzyme activity and gene expression of trypsin, chymotrypsin, lipase, and amylase increased and fluctuated throughout the larval development, a pattern that is common among marine fish species. Pepsin activity and gene expression were not detected until 15 DAH and strongly increased from 20 DAH. Metamorphosis was complete by 40 DAH following the regular arranging of microvilli and the abundance of vacuoles in intestine.

DOI： 10.1016/j.aquaculture.2019.734283

Web of Science

Publisher：ELSEVIER  

Euryhaline rotifer belonging to the genera Brachionus are often used as live feed for first feeding marine finfish larviculture. Two common rotifer enrichments, a single usage of docosahexaenoic acid (DHA)-enriched Chlorella vulgaris and a combination of DHA-enriched C. vulgaris and salmon roe emulsion oil were used to assess: (i) nutritional status of rotifers and, (ii) performance when rotifers were fed to Pagrus major and Paralichthys olivaceus larvae from 2 to 15 days after hatching (DAH). Both rotifer enrichment treatments were found to be nutritionally effective with high unsaturated fatty acid (HUFA) content, especially in the combined treatment with significantly higher eicosapentaenoic acid (EPA). Despite deficient levels of EPA, DHA-enriched C. vulgaris was found to be a suitable enrichment for rotifer and also had positive response in survival, growth and trypsin activity in both P. major and P. olivaceus. DHA-enriched C. vulgaris only treatment had higher rotifer population growth (13% higher), egg bearing capacity (7% higher), almost double the soluble protein content and generally higher free amino acid content. Oxidation of lipids in the combined diet due to its high-saturated fatty acid content could be a possible cause to the inferior performance of the combined treatment. Both P. major (20.1 +/- 10.1%) and P. olivaceus (12.3 +/- 3.87%) suffered low survival and lower gut content in the combined treatment especially during periods of significant growth in both species (7-11 DAH). Despite high HUFA content in the combined treatment, this study revealed that consideration of free amino acids and soluble protein content as parameters of nutritional indicators for first feeding P. major and P. olivaceus should not be discounted.

DOI： 10.1016/j.aquaculture.2019.05.039

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

This study investigated whether irradiation of a specific light wavelength could affect the sex differentiation of fish. We first found that the photoreceptor genes responsible for receiving red, green, and ultraviolet light were expressed in the eyes of medaka during the sex differentiation period. Second, we revealed that testes developed in 15.9% of genotypic females reared under green light irradiation. These female-to-male sex-reversed fish (i.e. neo-males) showed male-specific secondary sexual characteristics and produced motile sperm. Finally, progeny tests using the sperm of neo-males (XX) and eggs of normal females (XX) revealed that all F1 offspring were female, indicating for the first time in animals that irradiation with light of a specific wavelength can trigger sex reversal.

DOI： 10.1038/s41598-019-38908-w

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publisher：Japanese Society for Aquaculture Science  

<p>Information on effects of different enrichment on different morphotypes within the <i>Brachionus plicatilis</i> sp. complex is limited. This study investigated the effects of different enrichment diets on population growth, fatty acid content and protein content of L and SS types <i>B. plicatilis</i> sp. complex. Three commercial rotifer enrichment formulations were tested: docosahexaenoic acid (DHA) enriched <i>Chlorella vulgaris</i>, frozen <i>Nannochloropsis oculata</i>, salmon roe emulsion oil, against a control diet of normal <i>C. vulgaris</i>. DHA-enriched C. vulgaris and Frozen <i>N. oculata</i> had significantly higher population growth, fatty acid composition and soluble protein in both morphotypes. However, population growth under salmon roe emulsion treatment was affected negatively despite having high essential fatty acid content in both morphotypes. Variations in fatty acid assimilation in non-polar and polar lipids between morphotypes were observed highlighting enrichment effects and possible dissimilar biochemical roles of these compounds in tissue metabolism in each morphotype. Significantly higher levels of soluble protein were detected in DHA-enriched <i>C. vulgaris</i> and control treatment. SS-type had significantly higher soluble protein content except under the frozen <i>N. oculata</i> treatment. Culture method, enrichment type and dosage are important considerations as different rotifer morphotypes respond differently to different enrichment.</p>

DOI： 10.11233/aquaculturesci.67.139

CiNii Books

Other Link： https://agriknowledge.affrc.go.jp/RN/2010928154

Language：Japanese   Publisher：FCCA(Forum: Carbohydrates Coming of Age)  

<p>Sialidase catalyzes the removal of sialic acids from glycoconjugates. While the physiological functions of mammalian sialidases have been elucidated, sialidases in other vertebrates, including those in fish, are not well understood. Recently, increased genomic resources in fish have become available, which will facilitate research on fish sialidase.</p><p>Here, three fish orders (Cypriniformes, Beloniformes and Perciformes) have been selected to compare the properties of their sialidases. A Neu1 knockdown in medaka by a morpholino oligo resulted in the accumulation of Sia α2-3 linked sialoglycoproteins, leading to malfunctions in heart beating, embryo growth, hatching, and swimming, indicating that lysosomal Neu1 is a crucial factor for embryogenesis.</p><p>Fish Neu3 and Neu4 function varies between fish species. Medaka and tilapia Neu3a localize at the plasma membrane, as is seen in humans. Zebrafish Neu3.1 is an ER sialidase, with an optimal pH (pH 2.6). Neu4 shows high diversity in subcellular localization: medaka and zebrafish Neu4 are detected at the lysosome and ER, respectively. Surprisingly, tilapia Neu4 is detected at nucleus, which makes it the first nuclear sialidase to be identified. Moreover, importin has been identified as a regulator of tilapia Neu4 functions. These results demonstrate the diversity of sialidase functions among fish species and the requirement of further fish research.</p>

DOI： 10.4052/tigg.1518.1J

Web of Science

CiNii Books

Publisher：SPRINGER JAPAN KK  

Two common live feeds, the Brachionus plicatilis species complex SS-type and L-type were used to assess whether there were any differences in protein hydrolysis and digestive trypsin activity in first feeding Japanese flounder. There were no significant differences in hydrolysis activity at 2, 3 and 7 days after hatching (DAH). At 5 DAH, hydrolysis activity was significantly higher in larvae fed SS-type (p < 0.05) at 50 kDa in 1.5- and 3-h incubation whereas L-type treatment had not completely hydrolyzed the proteins after 3 h at the same molecular weight. Larvae fed SS-type had significantly higher (p < 0.05) trypsin activity at 3, 5, 6, 7 DAH. Contribution of live prey to trypsin fraction in larvae showed significantly higher (p < 0.05) fraction for SS-type at 5 DAH (2.18 +/- 0.44%) and 6 DAH (2.04 +/- 0.29%) and the effect of exogenous trypsin from live prey was relatively low when compared to the total trypsin activity in larvae. This study discusses the differences in ability to digest proteins in Japanese flounder when fed different rotifer morphotypes and highlights the adaptability of this species to alternative rotifer morphotypes during its early developmental stages.

DOI： 10.1007/s12562-018-1241-2

Web of Science

Publisher：SPRINGER JAPAN KK  

Interleukin-10 (IL-10) is an anti-inflammatory cytokine and negatively regulates cell-mediated immunity (CMI) induction by inhibiting cytokine production in type 1 T helper cells. IL-10 genes have been isolated from several fish, and inflammatory cytokine inhibition by IL-10 has been well examined. However, a CMI regulator of IL-10 in fish has not yet been identified. In this study, we cloned the IL-10 gene in amberjack Seriola dumerili and analyzed its function using its recombinant protein (rIL-10). In an in vitro culture experiment, gene expression of inflammatory cytokines was suppressed in leukocytes incubated with rIL-10 compared with cells that only received Nocardia seriolae stimulation. This result suggests amberjack IL-10 has conserved function as an inflammatory cytokine inhibitor. Bactericidal activity of amberjack cells against intracellular pathogen stimulation was decreased in a rIL-10 dose-dependent manner. Furthermore, a significant reduction in the T-bet/GATA-3 ratio was observed in N. seriolae living cell (LC) + rIL-10-injected fish. Taken together, these results suggest amberjack rIL-10 suppresses CMI induction both in vitro and in vivo. In addition, the number of IgM(+) cells among spleen leukocytes in N. seriolae + rIL-10-injected fish was higher than in only N. seriolae LC, suggesting that Th2-dominant immunity was induced by adding rIL-10.

DOI： 10.1007/s12562-018-1223-4

Web of Science

Publisher：ELSEVIER SCI LTD  

Mycobacteriosis and nocardiosis in cultured fish caused by infections with acid-fast bacteria, are responsible for large economic losses globally. In this study, we suggest a novel adjuvant using glycolipids that activates host immune systems. The immune response to glycolipids stimulation was investigated using ginbuna crucian carp. Ginbuna vaccinated with FKC (formalin-killed cells) + glycolipids isolated from Mycobacterium sp., upregulated inflammatory- and Th1-related cytokines, and a DTH (delayed-type hypersensitivity) response was confirmed only in ginbuna vaccinated with FKC + glycolipids. These observations suggest that glycolipids activated host innate and cell-mediated immunity. Subsequently, we evaluated the adjuvant effect of glycolipids against amberjack nocardiosis. In a challenge test, a higher survival rate was observed in amberjack vaccinated with FKC + glycolipids emulsified with conventional oil adjuvant than in fish vaccinated with FKC + oil adjuvant without glycolipids. Therefore, glycolipids potentially could be used as a practical, economical and safe adjuvant for aquaculture fish. (C) 2018 Published by Elsevier Ltd.

DOI： 10.1016/j.dci.2018.04.012

Web of Science

Scopus

PubMed

Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Different from Neu1 and Neu3 sialidases, Neu4 enzymatic properties such as substrate specificity and subcellular localization are not well-conserved among vertebrates. In fish only zebrafish and medaka neu4 genes have been cloned and their polypeptides have been characterized so far. Thus, characterization of Neu4 from other fish species is necessary to evaluate Neu4 physiological functions. Here, Nile tilapia was chosen for the characterization of Neu4 polypeptide considering that it is one of the major cultured fish all over the world and that its genomic sequences are now available. Coding DNA sequence of tilapia Neu4 was identified as 1,497 bp and its recombinant protein showed broad substrate specificity and optimal sialidase enzyme activity pH at 4.0. Neu4 activity was sustained even in neutral and alkali pH. Interestingly, immunofluorescence analysis revealed that major subcellular localization of tilapia Neu4 was nuclear, quite distinct from zebrafish (ER) and medaka Neu4 (lysosome). Bioinformatic analysis showed the existence of putative nuclear localization signal (NLS) in tilapia Neu4. In general, it is known that importin families bind to several proteins via NLS and transfer them into nucleus. Therefore, to determine the involvement of putative NLS in Neu4 nuclear localization, Neu4 mutant deleting NLS was constructed and expressed in cultured cells. As a result, NLS deletion significantly diminished the nuclear localization. Furthermore, treatment of importazole, interrupter of binding importin beta and RanGTP, significantly suppressed Neu4 nuclear localization. In summary, tilapia Neu4 is a unique sialidase localized at nucleus and its transport system into nucleus is regulated by importin. (C) 2018 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

DOI： 10.1016/j.biochi.2018.04.003

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Language：English   Publishing type：Research paper (scientific journal)  

Publisher：ELSEVIER ACADEMIC PRESS INC  

Sialidases are glycosidases responsible for the removal of alpha-glycosidically linked sialic acid residues from carbohydrate portions of glycoproteins and glycolipids, this process being the initial step in the degradation of such glycoconjugates. Sialic acids are considered to play important roles in various biological processes largely in two ways, one related to their hydrophilic and acidic properties exerting physicochemical effects on the glycoconjugates to which they are attached, and the other as recognition sites or in an opposing fashion as masking sites. The removal of sialic acids catalyzed by a sialidase, therefore greatly influences many biological processes through changing the conformation of glycoproteins and through recognition and masking of biological sites of functional molecules. Sialidases are found widely distributed in metazoan animals, from echinoderms to mammals, and are also present in viruses and other microorganisms, including fungi, protozoa, and bacteria even mostly lacking sialic acids. In mammals, there are four forms of sialidase (Neu1, Neu2, Neu3, and Neu4), differing in their major subcellular localization and enzymatic properties. They have been implicated in regulation of various cellular activities, such as cell differentiation, cell growth, and cell adhesion and motility, depending on their particular properties. In contrast, in microorganisms the enzymes appear to play roles limited to nutrition and pathogenesis. In this chapter, the focus is on mammalian sialidases preferentially hydrolyzing gangliosides, mostly Neu3 and Neu4, with an attempt to provide a brief overview of their physiological and pathological roles.

DOI： 10.1016/bs.pmbts.2017.12.005

Web of Science

Scopus

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Nocardiosis causes serious economic damage in the fish farming of Japanese yellowtail fish. Nocardia seriolae identified as pathogenic bacterium is an intracellular-pathogen. In general, induction of cell mediated immunity (CMI) is effective in infection defense against intracellular-pathogen. However, the conventional formalin-killed N. seriolae (FKC) vaccine induces humoral immunity. Interleukin-12 (IL-12) is Th1 type heterodimeric cytokine and induces cell differentiation in mammals. Our previous study showed that recombinant amberjack IL-12 has a role in CMI induction in vitro and could be a possible CMI inducing adjuvant. However, its adjuvant effect of fish IL-12 was not studied. In the present study, six types of amberjack recombinant IL-12 (rIL-12) were mixed and injected into amberjack with FKC. Firstly, we analyzed Th1- and Th2-related gene expression and monitored Th1/Th2 status followed by investigation of antibody titer. As a result, Th1-type immunity was induced in FKC + rIL-12 vaccinated fish. Secondly, we checked Th1/Th2 status of vaccinated fish after 10 days of N. seriolae infection using the expression of related genes. High T-bet/GATA-3 ratio was observed in FKC + rIL-12 vaccinated fish, suggesting that Thl cells possesing antigen memory were induced against N. seriolae infection. Finally, the survival rate in challenge test showed that 88% of FKC + rIL-12 vaccinated fish was survived at 34 days after N. seriolae injection whereas PBS (control) and FKC only were exterminated. These result suggest that i) rIL-12 is viable CMI inducible adjuvant and ii) production of Thl cells having antigen memory resulting from activation of IL-12 signaling pathway is important for defense against N. seriolae infection. (C) 2017 Published by Elsevier Ltd.

DOI： 10.1016/j.fsi.2017.06.025

Web of Science

PubMed

Language：English   Publishing type：Research paper (scientific journal)  

Publisher：ELSEVIER SCIENCE INC  

Microcystin-LR is a hepatotoxin produced by several cyanobacteria. Its toxicity is mainly due to a inhibition of protein phosphatase, PP1 and PP2A. Previously, we used a cell line stably expressing uptake transporter for microcystin-LR, OATPIB3 (HEK293-OATP1B3 cells). In this study, to determine whether overexpression of carboxylesterase (CES), which degrades ester-group and amide-group, attenuates the cytotoxicity of microcystin-LR, we generated the HEK293-OATPIB3/CES2 double-transfected cells. HEK293-OATP1B3/CES2 cells showed high hydrolysis activity of p-nitrophenyl acetate (PNPA), which is an authentic substrate for esterase. CES activity in HEK293-OATP1B3/CES2 cells was approximately 3-fold higher than that in the HEK293-OATP1B3 cells. HEK293-0ATP1B3/CES2 cells (IC50: 25.4 +/- 7.7 nM) showed approximately 2.1-fold resistance to microcystin-LR than HEK293-OATP1B3 cells (IC50: 12.0 +/- 1.5 nM). Moreover, the CES inhibition assay and microcystin-agarose pull down assay showed the possibility of the interaction between CES2 and microcystin-LR. Our results indicated that the overexpression of CES2 attenuates the cytotoxicity of microcystin-LR via interaction with microcystin-LR. (C) 2017 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.cbpc.2017.01.008

Web of Science

PubMed

Publisher：ELSEVIER FRANCE-EDITIONS SCIENTIFIQUES MEDICALES ELSEVIER  

Sialidase catalyzes the removal of sialic acids from glycoconjugates. Recently, medaka sialidase Neu1 has been cloned and its enzymatic properties were investigated. Although enzymatic properties of this sialidase, such as optimal pH and substrate specificity, exhibits high similarity with human NEU1, Neul physiological functions in fish are still unclear. Here, to understand Neu1 significance in medaka embryogenesis, sialidase translation knockdown was carried out with one-cell. stage fertilized egg using morpholino oligo injection.Neul exhibited desialylation of alpha 2-3 sialic acid linkage in vitro and lysosomal localization in medaka caudal fin primary cells. Chloroquine treatment, inhibitor of lysosomal enzymes, caused an accumulation of alpha 2-3 sialo-glycoproteins in the primary cells. During the embryogenesis neu1 mRNA level was elevated until 3.5 day post fertilization (dpf) while an initial decrease of alpha 2-3 sialo-glycoprotein was observed around the same developmental stage. Neu1 knockdown by morpholino oligo induced some abnormal phenotypes such as delay of yolk sac absorption and small embryos. Sialidase-knockdown embryos also showed increase of heart rate in 5.5 and 6.5 dpf. Furthermore, about 37% decrease of hatching rate was observed in Neu1-MO treated embryos compared with control MO. Embryos showing severe phenotypes stopped embryogenesis at the late stage of development. Alteration of embryonic sialo-glycoproteins induced by morpholino injection was examined by lectin blotting to clarify the mechanism of abnormal development. As a result, degradation of several alpha 2-3 sialo-glycoproteins was suppressed in Neu1-MO embryo, possibly induced by the interruption of lysosomal desialylation toward yolk glycoprotein. Our results suggest that medaka Neu1 could be crucial for embryonic development through the degradation of yolk sac nutrition. (C) 2017 Elsevier B.V. and Societe Francaise de Biochimie et Biologie Moleculaire (SFBBM). All rights reserved.

DOI： 10.1016/j.biochi.2017.01.008

Web of Science

PubMed

Publisher：ELSEVIER SCIENCE BV  

Deep-sea living dwarf gulper shark (Centrophorus atromarginatus) is industrially important resource with lots of squalene in the liver, used as the resource for cosmetic ingredients, while consumption of shark muscle is limited because of ammonia smell caused by bacterial urease. Here, effect of shark muscle lipid on matrix metalloproteinase-7 (MMP-7) activity was estimated to develop the new application of shark muscle. Treatment of shark lipid toward HT-29 cells attenuated MMP-7 activity in conditioned medium accompanied with its decreased extracellular secretion. Ganglioside types in shark lipid were investigated by thin layer chromatography. Among shark gangliosides, GDIa and GM1 attenuated MMP-7 activity. GDla negatively regulated MMP-7 polypeptide and its gene expression in HT-29 cells, whereas MAPKs signaling pathways were not altered. GDIa suppressed c-jun expression in HT-29 cells, which could negatively regulate mmp-7 transcription. This study indicated the possibility of dwarf gulper shark muscle as a material of functional food. (C) 2017 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.jff.2017.01.013

Web of Science

Language：English   Publishing type：Research paper (scientific journal)   Publisher：ACADEMIC PRESS LTD- ELSEVIER SCIENCE LTD  

Edwardsiella tarda (E. tarda) is a gram-negative bacterium, which causes Edwardsiellosis in aquaculture. Previous studies indicate that E. tarda NanA sialidase plays crucial roles in infection through the desialylation of glycoproteins in fish cells. On the other hand, 2-deoxy-2,3-dehydro-N-acetylneuraminic acid, classic sialidase inhibitor, negatively regulates E. tarda infection of goldfish scale GAKS cells. Here, to development the suppression model of E. tarda infection for aquaculture application, the possibility of NanA inhibitory activities in citrus phytochemicals was evaluated as citrus extracts have widely been used as a supplement in fish diets for the improvement of meat quality. Some flavanones such as naringenin, hesperetin, hesperidin and naringin showed sialidase inhibitory activity toward recombinant NanA in vitro. Among them, naringenin showed the most potent inhibitory activity and its inhibitory pattern was non-competitive. Naringenin significantly suppressed E. tarda infection in GAKS cells at 200 and 400 mu M without bactericidal effect on E. tarda. On the other hand, naringin, glycosylation form of naringenin, showed slight suppression of E. tarda infection toward GAKS cells, suggesting the glycosides on flavanone could be important for NanA inhibition. Fluorescence microscopy analysis verified that number of invading E. tarda in GAKS cells was declined by naringenin treatment. The present study exhibited the possibility of naringenin as an effective ingredient in fish diet for the inhibition of E. tarda infection. (C) 2016 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.fsi.2016.12.018

Web of Science

PubMed

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)  

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J-GLOBAL

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：English   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Language：Japanese   Publishing type：Article, review, commentary, editorial, etc. (scientific journal)  

Event date： 2016.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2016.9

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Event date： 2016.9

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Event date： 2016.9

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Event date： 2016.9

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Event date： 2016.3

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Event date： 2016.3

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Event date： 2016.1

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Event date： 2015.11

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Event date： 2015.11

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.11

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.11

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.11

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.9

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.8

Language：Japanese   Presentation type：Symposium, workshop panel (nominated)  

Event date： 2015.7

Language：English   Presentation type：Oral presentation (general)  

Event date： 2015.7

Language：Japanese   Presentation type：Poster presentation  

Event date： 2015.7

Language：English   Presentation type：Poster presentation  

Event date： 2015.7

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.7

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.7

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.7

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.7

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2015.1

Language：Japanese   Presentation type：Oral presentation (general)  

Event date： 2014.11

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2014.11

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：福岡  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：福岡  

国内学会

Event date： 2014.9

Language：Japanese  

Venue：福岡  

国内学会

Event date： 2014.8

Language：Japanese  

Venue：愛知  

国内学会

Event date： 2013.8

Language：Japanese  

Venue：大阪  

国内学会

Event date： 2013.8

Language：Japanese  

Venue：大阪  

国内学会

Event date： 2013.3

Language：Japanese  

Venue：鹿児島  

研究会

Event date： 2012.9

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2012.9

Language：Japanese  

Venue：山口  

国内学会

Event date： 2012.9

Language：Japanese  

Venue：山口  

国内学会

Event date： 2012.9

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2012.1

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2012.1

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2011.9

Language：Japanese  

Venue：京都  

国内学会

Event date： 2011.9

Language：Japanese  

Venue：京都  

国内学会

Event date： 2011.9

Language：Japanese  

Venue：京都  

国内学会

Event date： 2011.7

Language：Japanese  

Venue：新潟  

国内学会

Event date： 2011.3

Language：Japanese  

Venue：鹿児島  

研究会

Event date： 2010.12

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2010.12

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2010.10

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2010.8

Language：English  

Venue：ドイツ  

国際学会

Event date： 2010.7

Language：Japanese  

Venue：岡崎  

研究会

Event date： 2010.7

Language：English  

Venue：神奈川  

国際学会

Event date： 2009.11

Language：Japanese  

Venue：新潟  

研究会

Event date： 2009.10

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2009.10

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2009.10

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2009.9

Language：Japanese  

Venue：岐阜  

国内学会

Event date： 2009.5

Language：Japanese  

Venue：宮城  

国内学会

Event date： 2008.12

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2008.12

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2008.10

Language：Japanese  

Venue：愛知  

国内学会

Event date： 2008.8

Language：Japanese  

Venue：茨城  

国内学会

Event date： 2007.12

Language：Japanese  

Venue：宮城  

研究会

Event date： 2007.12

Language：Japanese  

Venue：神奈川  

国内学会

Event date： 2007.12

Language：Japanese  

Venue：神奈川  

国内学会

Event date： 2007.7

Language：English  

Venue：オーストラリア  

国際学会

Event date： 2007.5

Language：Japanese  

Venue：宮城  

国内学会

Event date： 2006.9

Language：English  

Venue：徳島  

国際学会

Event date： 2006.9

Language：Japanese  

Venue：愛知  

研究会

Event date： 2006.8

Language：English  

Venue：静岡  

国際学会

Event date： 2006.8

Language：Japanese  

Venue：宮城  

国内学会

Event date： 2006.6

Language：English  

Venue：京都  

国際学会

Event date： 2006.5

Language：Japanese  

Venue：青森  

国内学会

Event date： 2005.11

Language：Japanese  

Venue：宮城  

国内学会

Event date： 2005.10

Language：Japanese  

Venue：兵庫  

国内学会

Event date： 2004.10

Language：Japanese  

Venue：神奈川  

国内学会

Event date： 2004.10

Language：Japanese  

Venue：東京  

研究会

Event date： 2004.4

Language：Japanese  

Venue：鹿児島  

国内学会

Event date： 2003.4

Language：Japanese  

Venue：東京  

国内学会

Event date： 2000.9

Language：Japanese  

Venue：福井  

国内学会

Event date： 1998.8

Language：Japanese  

Venue：千葉  

国内学会

Event date： 1998.8

Language：Japanese  

Venue：千葉  

国内学会