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Hsu H.-Y.,National Yang Ming University | Hsu H.-Y.,Academia Sinica, Taiwan | Lin T.-Y.,National Yang Ming University | Hwang P.-A.,Taiwanese Fisheries Research Institute | And 5 more authors.
Carcinogenesis | Year: 2013

Fucoidan, a polysaccharide extracted from brown seaweeds, reduces tumor cell proliferation. Fucoidan inhibits the growth of breast cancer cells such as 4T1 and MDA-MB-231 and decreases their cell colony formation. Moreover, fucoidan reduces metastatic lung nodules in 4T1 xenograft female Balb/c mice. The molecular network of transforming growth factor β (TGFβ) receptors (TGFRs) plays an important role in the regulation of the epithelial to mesenchymal transition (EMT) in cancer cells. Using 4T1 and MDA-MB-231 cells, we found that fucoidan effectively reverses TGFR-induced EMT morphological changes, upregulates epithelial markers, downregulates mesenchymal markers and decreases the expression of transcriptional repressors Snail, Slug and Twist. Moreover, fucoidan inhibits migration and invasion during the EMT, suggesting the involvement of TGFR-mediated signaling in breast cancer cells. Fucoidan decreases TGFRI and TGFRII proteins and affects downstream signaling molecules, including Smad2/3 phosphorylation and Smad4 expression. In order to elucidate how fucoidan decreases TGFRI and TGFRII proteins in MDA-MB-231 cells, we investigated ubiquitination activity downregulation of TGFRs. It was found that fucoidan enhances proteasome-mediated degradation/ubiquitination of TGFR. This study is the first to identify a novel mechanism for fucoidan antitumor activity, namely regulation of the EMT via modulation of TGFR/Smad-dependent signaling, which leads to an inhibition of breast cancer cell growth in vitro and in vivo. Our current findings indicate that fucoidan is a potential therapeutic agent for breast cancer and acts via an ubiquitin-dependent degradation pathway that affects the TGFR/Smad/Snail, Slug, Twist and EMT axes. © The Author 2012. Published by Oxford University Press. All rights reserved.


Kai Y.-H.,National Taiwan University | Su H.-M.,Taiwanese Fisheries Research Institute | Tai K.-T.,Long Diann Marine Biotechnology Co. | Chi S.-C.,National Taiwan University
Vaccine | Year: 2010

Nervous necrosis virus (NNV) has caused mass mortality in many species of cultured fish at larval stage. Strong evidence of vertical transmission of NNV has been reported in the carrier broodstock of striped jack and sea bass. An effective immunization program was developed and monitored in adult groupers (Epinephelus coioides) with average body weight of 1.35 kg. The highest neutralizing antibody titers were found in the fish intramuscularly injected with adjuvanted NNV vaccine at 109 TCID50 kg-1, and the enhanced 2-fold neutralization activity could sustain up to 17 months post-vaccination (mpv). An immunization program was applied in the broodstocks of grouper (Epinephelus tukula) with body weight of 35-60 kg. The levels of NNV-specific antibodies detected, from 1 to 5 mpi, in the homogenates of the eggs from the vaccinated broodfish were elevated than that from non-vaccinated fish. By nested RT-PCR, NNV was detectable in the eggs from the non-vaccinated fish at Month 5, but not in the eggs from vaccinated fish. It is therefore suggested that vaccination will be a potentially practical measure to reduce the risk of vertical transmission of NNV of grouper broodfish under stress of repeated spawning. © 2009 Elsevier Ltd. All rights reserved.


Chang C.-S.,National Taiwan University | Huang S.-L.,Taiwanese Fisheries Research Institute | Chen S.,National Taiwan University | Chen S.-N.,National Taiwan University
Fish and Shellfish Immunology | Year: 2013

This study attempts to describe the effects of innate immunity responses and field application of mushroom beta-glucan mixture (MBG) in cultured orange-sported grouper, Epinephelus coioides. Chemical analysis for MBG showed that the mixture contains 34.06% of macro-molecular polymers with bio-active linkage such as 3-; 3,4- and 4,6-glucopyranosyl and 6-linked galactopyranosyl residues. Study performed on the innate immunity showed that oral ingestion of MBG at 1.0g and 2.0g per kilogram of feed levels may significantly enhance the lysozyme activity, alternative complement activity, phagocytic activity and respiration burst of the experimental groupers.Observation on the experimental challenge of pathogen showed that uses of MBG at 0.1% and 0.2% levels in feed might significantly enhance the protection of grouper against Vibrio alginolyticus.Field trials performed on short and long-term culture showed that feeding of diet containing 0.1% or 0.2% of MBG may significantly enhance the survival of cultured groupers up to 16% when compared with those obtained from controls. © 2013 Elsevier Ltd.


Liu Y.-T.,National Kaohsiung Marine University | Chang C.-I.,Taiwanese Fisheries Research Institute | Hseu J.-R.,Taiwanese Fisheries Research Institute | Liu K.-F.,Taiwanese Fisheries Research Institute | Tsai J.-M.,National Kaohsiung Marine University
Molecular Immunology | Year: 2013

Prophenoloxidase (proPO) and cytosolic manganese superoxide dismutase (cytMnSOD) play crucial roles in crustacean innate immunity. In the present study, both of the above genes were cloned from hemocytes of the red claw crayfish Cherax quadricarinatus. A phylogenetic analysis of the amino acid sequences showed that C. quadricarinatus proPO and cytMnSOD were more closely related to the proPO and cytMnSOD of other crayfish than to those of penaeids, crabs, lobsters, or freshwater prawns. A tissue distribution analysis revealed that proPO was primarily expressed in hemocytes, gills, and the heart, while cytMnSOD was detected in all tissues examined. All of the crayfish artificially infected with white spot syndrome virus (WSSV) died within 4 days. According to a non-lethal dose, there was no mortality in crayfish when infected deliberately with Aeromonas hydrophila. Total hemocyte counts (THCs) had significantly decreased in crayfish at 48 and 72. h after infection with WSSV compared to the control group. In contrast, THCs of crayfish after A. hydrophila challenge had recovered by 48 and 72. h from a lower level at 24. h. There were similar responses in enzyme activities toward WSSV and A. hydrophila infection. Phenoloxidase (PO) and superoxide dismutase (SOD) activities per hemocyte significantly increased from 48 to 72. h compared to the control group. After WSSV challenge, expressions of proPO and cytMnSOD transcripts in hemocytes significantly decreased at 12. h, then had respectively recovered and increased at 24. h. At 48-72. h, transcript levels were finally downregulated. No significant differences in the expression profiles of proPO and cytMnSOD were observed between the A. hydrophila-infected and control groups, besides the significant upregulation at 24. h post-infection. These results implicate proPO and cytMnSOD in the immune response, and they presented similar expression patterns, although different defense mechanisms may exist for crayfish induced by WSSV and A. hydrophila. © 2013 Elsevier Ltd.


Grateloupia huangiae S.-M. Lin & H.-Y. Liang sp. nov. was previously recorded as G. okamurae Yamada [currently recognized as Polyopes lancifolius (Harvey) Kawaguchi & H.W. Wang] in Taiwan. Molecular phylogenetic analysis shows that G. huangiae is unrelated to P. lancifolius but is closely related to Grateloupia taiwanensis S.-M. Lin & H.-Y. Liang. Although G. huangiae superficially resembles P. lancifolius in having a leafy thallus and short proliferations borne on the thallus surface, the structure of the auxiliary-cell ampulla is very different. The auxiliary-cell ampulla of G. huangiae is composed of three orders of unbranched filaments before diploidization, each of which is 11-13 cells long. The auxiliary cell is the first cell of the third-order ampullar filament, which is cut off from the first cell of the secondorder ampullar filament. This type of auxiliary-cell ampulla is the same as that found in G. taiwanensis. On the other hand, P. lancifolius has a more complex auxiliary-cell ampulla in which the ampullar filaments may bear up to five to six orders of branches before diploidization. A detailed study of the development of auxiliary-cell ampullae before and after diploidization is needed in other species and genera within the Halymeniaceae to better delineate generic concepts in the family.


Huang Y.-C.,National Taiwan Ocean University | Hsiao P.-C.,National Taiwan Ocean University | Chai H.-J.,Taiwanese Fisheries Research Institute
Ceramics International | Year: 2011

In recent years, environmental and economic conditions have raised concerns about the treatment and use of bio-waste. This study analyzed hydroxyapatite extracted from fish scale (FHAP) by enzymatic hydrolysis. The structures of FHAP were characterized by Fourier transform infrared spectroscopy, particle size analyzer, scanning electron microscopy, X-ray diffraction, and energy dispersive spectrometry. The analytical results indicated that FHAP consisted of nano-sized particles with Ca/P ratio of 1.76. After FHAP particles were sintered at 800°C for 4 h, the particles showed increased porosity and surface roughness. The influence of FHAP particles on the proliferation and osteogenic differentiation of MG63 cells was also investigated. Compared with hydroxyapatite from Sigma (SHAP), FHAP significantly increased MG63 growth, whether hydroxyapatite powders were sintered or not. Under osteogenic-inductive culture condition, FHAP particles promoted osteogenic differentiation and mineralization of MG63 cells, which was confirmed by alkaline phosphate assay and von Kossa staining. This study confirms that FHAP extracted by enzymatic hydrolysis is a promising biomaterial for artificial bone fabrication. © 2011 Elsevier Ltd and Techna Group S.r.l.


Tang C.-H.,National Chung Hsing University | Hwang L.-Y.,Taiwanese Fisheries Research Institute | Lee T.-H.,National Chung Hsing University
Journal of Experimental Biology | Year: 2010

Previous studies have reported the mechanisms of ion absorption and secretion by diverse membrane transport proteins in gills of various teleostean species. To date, however, the chloride channel expressed in the basolateral membrane of mitochondrionrich (MR) cells for Cl- uptake in freshwater (FW) fish is still unknown. In this study, the combination of bioinformatics tools [i.e. National Center for Biotechnology Information (NCBI) database, Tetraodon nigroviridis (spotted green pufferfish) genome database (Genoscope), BLAT and BLASTn] were used to identify the gene of ClC-3 (TnClC-3), a member of the CLC chloride channel family in the T. nigroviridis genome. RT-PCR analysis revealed that the gene encoding for the ClC-3 protein was widely expressed in diverse tissues (i.e. gill, kidney, intestine, liver and brain) of FW- and seawater (SW)-acclimated pufferfish. In wholemount double immunofluorescent staining, branchial ClC-3-like immunoreactive protein was localized to the basolateral membrane of Na+/K+-ATPase (NKA) immunoreactive cells in both the FW- and SW-acclimated pufferfish. In response to salinity, the levels of transcript of branchial TnClC-3 were similar between FW and SW fish. Moreover, the membrane fraction of ClC-3-like protein in gills was 2.7-fold higher in FW compared with SW pufferfish. To identify whether the expression of branchial ClC-3-like protein specifically responded to lower environmental [Cl-], the pufferfish were acclimated to artificial waters either with a normal (control) or lower Cl- concentration (low-Cl). Immunoblotting of membrane fractions of gill ClC-3-like protein showed the expression was about 4.3-fold higher in pufferfish acclimated to the low-Cl environment than in the control group. Furthermore, branchial ClC-3-like protein was rapidly elevated in response to acute changes of environmental salinity or [Cl-]. Taken together, pufferfish ClC-3-like protein was expressed in the basolateral membrane of gill MR cells, and the protein amounts were stimulated by hyposmotic and low-Cl environments. The enhancement of ClC-3-like protein may trigger the step of basolateral Cl- absorption of the epithelium to carry out iono- and osmoregulatory functions of euryhaline pufferfish gills. © 2010. Published by The Company of Biologists Ltd.


Yang S.-D.,National Taiwan Ocean University | Liu F.-G.,Taiwanese Fisheries Research Institute | Liou C.-H.,National Taiwan Ocean University
Aquaculture | Year: 2011

A 10-week rearing trial was conducted to assess the lysine requirement of juvenile silver perch, and to evaluate the effects of dietary lysine levels on the morphological measurements, body composition, hematological traits and muscle carnitine status of the fish. Seven isonitrogenous and isolipidic diets were formulated to contain multiple intact proteins and pre-coated crystalline amino acids. l-lysine HCl was supplemented to the experimental diets at the expense of a dispensable amino acid mixture to achieve lysine levels from 0.90 and 2.70% of dry diet. Each diet was randomly assigned to triplicate groups of 10 fish and was fed to apparent satiation five times daily. At the end of the trial, percent weight gain increased with increasing dietary lysine from 0.90% to 2.10%, then the value decreased slightly. A similar trend was observed in the feed efficiency, protein efficiency ratio, productive protein value and daily protein deposition. The condition factor, hepatosomatic index, intraperitoneal fat and viscerosomatic index were markedly affected by the dietary lysine level. Crude protein contents of the whole body, muscle and liver were significantly depressed in the fish fed lysine-deficient diets, while the whole body lipid content was higher in these fish. The hematocrit value, hemoglobin concentration and free lysine levels in plasma were lower in the silver perch given lower lysine diets. Although muscle carnitine status was not clearly affected by the dietary lysine level, the values of free carnitine and acid soluble carnitine tended to be lower in the fish fed lower lysine diets. Based on the quadratic broken-line model, the dietary lysine requirement for maximum growth of the silver perch juveniles was satisfied at 2.32% lysine in the dry diet, corresponding to 5.96% of the dietary protein. Furthermore, 2.06% of chemically available lysine in the diet (equivalent to 5.30% dietary protein) was required for the optimal growth of the fish. © 2010 Elsevier B.V.


Hseu J.-R.,Taiwanese Fisheries Research Institute | Huang W.-B.,National Dong Hwa University
Aquaculture Research | Year: 2014

Cannibalism is frequently observed in larviculture of orange-spotted grouper Epinephelus coioides. Previously, based on measurements of morphometric characters, a linear equation of total length (TL) of prey to cannibals was proposed: TLprey = 0.80 TLcannibal - 1.50. To verify the reliability of the equation, experiments were performed with pairs of fish with different TLs. Cannibalism occurred only when the cannibal-prey size ratios were equal to or larger than that predicted by the equation. To predict the probability of cannibalism among the grouper of known TLs, a logistic regression model was proposed. The logistic regression model is:P(occurence|TLprey,TLcannibal)=11+e-(0.197TLcannibal-0.402TLprey+3.811) The logistic regression model showed the following: when either TLprey or TLcannibal is constant, the probability of cannibalism increases with increase in the cannibal-prey size ratios; if given a constant cannibal-prey size ratio, probability of cannibalism is lower in early stages than in later stages. The prediction contrasts with that calculated from the linear equation where the minimum cannibal-prey size ratios decrease with size of the cannibal. However, the prediction matches observed rearing experiences: cannibals prefer smaller prey to larger ones and the cannibalism rate declines as fry age. © 2012 John Wiley & Sons Ltd.


Wu F.-C.,National Sun Yat - sen University | Wu F.-C.,Taiwanese Fisheries Research Institute | Chen H.-Y.,National Sun Yat - sen University
Aquaculture | Year: 2012

A 12-wk feeding trial was conducted to determine the dietary effects of α-linolenic (LN; 18:3n-3) and linoleic acids (LA; 18:2n-6) on growth, tissue fatty acids profile and non-specific cellular immune response in kidney of juvenile grouper Epinephelus malabaricus (initial weight: 11.3±0.6g). Eight experimental diets and a reference diet each containing 11.6% lipids were compared. Four experimental diets contained either 1 or 2% diet of pure LN or LA (N 1, N 2, L 1, and L2). The other 4 diets contained oil mixtures of linseed oil, safflower oil, LA, and oleic acid and had LN:LA ratios of 3.3, 1.4, 0.7 and 0.4 (NL 3, NL 1.4, NL 0.7, and NL 0.4). The reference diet contained a mixture of cod liver oil, linseed oil and safflower oil at a ratio of 2:1:1. Weight gain of the grouper was significantly increased with increasing dietary LN:LA ratios. The group that fed on the NL 3 diet showed the highest growth. Weight gains of this group equaled those of the reference group. Dietary inclusion of LN increased the presence of n-3 polyunsaturated fatty acids (PUFAs) in the grouper, except 22:6n-3 and 22:5n-3 in neutral lipid fraction of the liver. 18:2n-6 and 18:3n-6, but not 20:4n-6, were the predominant n-6 PUFAs in the body of the grouper, and their quantities were closely related to their quantitative dietary inclusion. Non-specific cellular immune response was significantly increased with increasing dietary LN:LA ratios. The grouper that fed on the NL 3 diet showed the significantly highest head-kidney leucocyte phagocytic and respiratory burst activities among all treatments. Our results suggest that LN and LA at a dietary level of 2% and a ratio of 3:1 are beneficial to weight gain and non-specific cellular immune responses of the juvenile grouper. © 2011 Elsevier B.V.

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