The Influence of κ-Carrageenan-R-Phycoerythrin Hydrogel on In Vitro Wound Healing and Biological Function.

Wound healing is widely recognized as a critical issue impacting the healthcare sector in numerous countries. The application of wound dressings multiple times in such instances can result in tissue damage, thereby increasing the complexity of wound healing. With the aim of tackling this necessity, in the present study, we have formulated a hydrogel using natural polysaccharide κ-carrageenan and phycobiliprotein R-phycoerythrin from Pyropia yezoensis. The formulated hydrogel κ-Carrageenan-R-Phycoerythrin (κ-CRG-R-PE) was analyzed for its antioxidant and antimicrobial activity. The wound healing potential of the κ-CRG-R-PE was evaluated in Hs27 cells by the wound scratch assay method. The hydrogel showed dose-dependent antioxidant activity and significant antimicrobial activity at 100 µg/mL concentration. κ-CRG-R-PE hydrogels promoted more rapid and complete wound closure than κ-Carrageenan (κ-CRG) hydrogel at 24 and 48 h. κ-CRG-R-PE hydrogels also filled the wound within 48 h of incubation, indicating that they positively affect fibroblast migration and wound healing.

Antioxidant and chemoprotective peptides from simulated gastrointestinal digested (SGID) protein hydrolysate of Pyropia yezoensis against acetaminophen-induced HepG2 cells.

Excessive production of reactive oxygen and nitrogen species may result in oxidative damage to tissues and organs. Oxidative stress is a pathological mechanism that contributes to the initiation and progression of liver injury. In the present study, antioxidative peptides purified from simulated gastrointestinal-digested (SGID) protein hydrolysate of Pyropia yezoensis, showed significant antioxidant activity and also showed a protective effect against acetaminophen (N-acetyl-p-aminophenol, APAP) -induced injury in HepG2 (human liver cancer cells) cells. The antioxidant activity was increased in a dose-dependent manner. Higher cell viability (73.26 ± 0.9%) and decreasing NO levels (107.6 ± 8.9%) were observed in 15 mM APAP-induced cells when treated with the concentration of (100 µg ml-1) Pyropia peptide. Py. (pep). The sequences of the eight identified peptides present in the active fractions of the protein hydrolysate included hydrophobic and aromatic amino acids, which may have been responsible for their chemoprotective and antioxidant activities. Results indicated that the treatment with the Pyropia-peptides significantly promoted the proliferation of HepG2 cells, protecting them against APAP-mediated injury, and showed a significant antioxidant capacity. This study revealed that the Py. (pep) will be beneficial in treating drug-induced oxidative stress and liver damage conditions. Py. (pep) can also serve as a better alternative for synthetic antioxidant drugs.

Biogenic preparation and characterization of Pyropia yezoensis silver nanoparticles (P.y AgNPs) and their antibacterial activity against Pseudomonas aeruginosa.

Marine algae play key roles in several medical, pharmaceutical, agricultural, and aquacultural applications. Furthermore, biosynthesized nanomaterials are becoming an alternative to conventional antibiotics in cost-effective, biocompatible, and non-toxic treatments for bacterial infections. This study features biogenic synthesis of silver nanoparticles using an aqueous extract of the marine red algae Pyropia yezoensis. The formation of silver nanoparticles was initially confirmed by UV-Vis spectroscopy and FTIR spectra were used to identify functional groups. The average crystalline size of the silver nanoparticles was around 20-22 nm, as determined by XRD analysis. Particle size was confirmed by SEM and TEM analyses, which also showed spherical particles without agglomeration. The antibacterial properties of the nanoparticles were assessed against both Gram-positive and Gram-negative bacterial cultures with significant activity observed against Gram negative P. aeruginosa. Our Pyropia yezoensis silver nanoparticles (P.y AgNPs) reduced the growth of P. aeruginosa at concentrations of 200 and 400 µg/ml. Our results strongly imply that P.y AgNPs may be useful in treating bacterial infections.

Cytotoxicity against human breast carcinoma cells of silver nanoparticles biosynthesized using Capsosiphon fulvescens extract.

Targeting cancer cells with small nanoparticles is a novel and promising approach to cancer therapy. Breast cancer is the most common cancer afflicting women worldwide. In the present study, silver nanoparticles (AgNPs) were synthesized using the aqueous extract of the marine alga Capsosiphon (C.) fulvescens, and the cytotoxicity and anti-cancer activities of the nanoparticles against MCF-7 breast cancer cells were analyzed. Nanoparticle formation was confirmed by solution color change and UV-Vis spectroscopy. The size and distribution of the C. fulvescens-biosynthesized silver nanoparticles (CfAgNPs) were then examined using various analytical methods; the particle size was around 20-22 nm and spherical in shape with no agglomeration. Cytotoxicity analysis revealed that the inhibitory concentration (IC50) of CfAgNPs was 50 µg/ml. MCF-7 cell viability decreased with increasing concentrations of CfAgNPs. MCF-7 cells were evaluated for morphological changes before and after treatment with the CfAgNPs; cells treated with C. fulvescens aqueous algal extract (without CfAgNPs) showed irregular confluent aggregates with round polygonal cells, similar to the untreated control. Tamoxifen- (TMX) and CfAgNPs-treated cells show significant morphological changes. An apoptosis study was conducted using 4',6-diamidino-2-phenylindole (DAPI) staining, in which CfAgNP-treated MCF-7 cells generated bright blue fluorescence and shortened, disjointed chromatin was evident; control cells displayed less bright fluorescence. Flow cytometry analysis revealed that the percentage of cells in late apoptosis was high following treatment with TMX (77.2%) and CfAgNP (74.6%). A novel anti-cancer agent, developed by generating silver nanoparticles from C. fulvescens extract, showed strong cytotoxic activity against MCF-7 cells.

Extraction and Purification of R-Phycoerythrin Alpha Subunit from the Marine Red Algae Pyropia Yezoensis and Its Biological Activities.

Phycoerythrin is a major light-harvesting pigment of red algae and cyanobacteria that is widely used as a fluorescent probe or as a colorant in the food and cosmetic industries. In this study, phycoerythrin was extracted from the red algae Pyropia yezoensis and purified by ammonium sulfate precipitation and various chromatography methods. The purified phycoerythrin was analyzed by UV-visible and fluorescence spectroscopy. The isolated pigment had the typical spectrum of R-phycoerythrin, with a trimmer state with absorbance maxima at 497, 536, and 565 nm. It was further purified and identified by LC-MS/MS and Mascot search. It showed a 100% sequence similarity with the R-phycoerythrin alpha subunit of Pyropia yezoensis. The molecular mass was 17.97 kDa. The antioxidant activity of the purified R-phycoerythrin alpha subunit was analyzed. It showed significant antioxidant activity in ABTS and FRAP assays and had significant cytotoxicity against HepG2 cells.

Peptidyl-prolyl isomerase and the biological activities of recombinant protein cyclophilin from Pyropia yezoensis (PyCyp).

Cyclophilins are highly conserved proteins associated with peptidyl-prolyl cis-trans isomerase activity (PPIase). The present study was designed to analyze the biological activity of recombinant cyclophilin from the marine red algae Pyropia yezoensis (PyCyp). The cyclophilin gene from P. yezoensis was cloned into the pPROEX-HTA expression vector. The plasmid was transformed into BL21 Escherichia coli by high efficiency transformation. Recombinant protein was expressed using 0.1 mM IPTG and the fusion protein was purified by affinity column chromatography. The His-tag was removed by TEV protease. The recombinant protein was further purified on a HiPrep Sephacryl S-200 HR column and by reversed-phase high performance liquid chromatography with a Sep-pak plus C18 column. Purified cyclophilin was characterized by a variety of analytical methods and analyzed for its peptidyl-prolyl isomerase activity. Our recombinant PyCyp was shown to catalyze cis-trans isomerization. PyCyp was also evaluated for antimicrobial activity against both Gram-positive and Gram-negative bacteria cultures and showed significant antibacterial activity against tested pathogens. PyCyp was shown to permeabilize bacterial membranes as evidenced by increased fluorescence intensity in SYTOX Green uptake assays with Staphylococcus aureus. The radical scavenging activity of PyCyp increased in a dose-dependent manner, indicating significant antioxidant activity. This study provides information for the development of therapeutic proteins from marine algae.

Hydrophilic Glycoproteins of an Edible Green Alga Capsosiphon fulvescens Prevent Aging-Induced Spatial Memory Impairment by Suppressing GSK-3ß-Mediated ER Stress in Dorsal Hippocampus.

Endoplasmic reticulum (ER) stress is involved in various neurodegenerative disorders. We previously found that Capsosiphon fulvescens (C. fulvescens) crude proteins enhance spatial memory by increasing the expression of brain-derived neurotrophic factor (BDNF) in rat dorsal hippocampus. The present study investigated whether the chronic oral administration of hydrophilic C. fulvescens glycoproteins (Cf-hGP) reduces aging-induced cognitive dysfunction by regulating ER stress in the dorsal hippocampus. The oral administration of Cf-hGP (15 mg/kg/day) for four weeks attenuated the aging-induced increase in ER stress response protein glucose-regulated protein 78 (GRP78) in the synaptosome of the dorsal hippocampus; this was attenuated by the function-blocking anti-BDNF antibody (1 µg/µL) and a matrix metallopeptidase 9 inhibitor 1 (5 µM). Aging-induced GRP78 expression was associated with glycogen synthase kinase-3 beta (GSK-3ß) (Tyr216)-mediated c-Jun N-terminal kinase phosphorylation, which was downregulated upon Cf-hGP administration. The Cf-hGP-induced increase in GSK-3ß (Ser9) phosphorylation was downregulated by inhibiting tyrosine receptor kinase B and extracellular signal-regulated kinase (ERK)1/2 with cyclotraxin-B (200 nM) and SL327 (10 µM), respectively. Cf-hGP administration or the inhibition of ER stress with salubrinal (1 mg/kg, i.p.) significantly decreased aging-induced spatial memory impairment. These findings suggest that the activation of the synaptosomal BDNF-ERK1/2 signaling in the dorsal hippocampus by Cf-hGP attenuates age-dependent ER stress-induced cognitive dysfunction.

Spirulina Crude Protein Promotes the Migration and Proliferation in IEC-6 Cells by Activating EGFR/MAPK Signaling Pathway.

Spirulina is a type of filamentous blue-green microalgae known to be rich in nutrients and to have pharmacological effects, but the effect of spirulina on the small intestine epithelium is not well understood. Therefore, this study aims to investigate the proliferative effects of spirulina crude protein (SPCP) on a rat intestinal epithelial cells IEC-6 to elucidate the mechanisms underlying its effect. First, the results of wound-healing and cell viability assays demonstrated that SPCP promoted migration and proliferation in a dose-dependent manner. Subsequently, when the mechanisms of migration and proliferation promotion by SPCP were confirmed, we found that the epidermal growth factor receptor (EGFR) and mitogen-activated protein (MAPK) signaling pathways were activated by phosphorylation. Cell cycle progression from G0/G1 to S phase was also promoted by SPCP through upregulation of the expression levels of cyclins and cyclin-dependent kinases (Cdks), which regulate cell cycle progression to the S phase. Meanwhile, the expression of cyclin-dependent kinase inhibitors (CKIs), such as p21 and p27, decreased with SPCP. In conclusion, our results indicate that activation of EGFR and its downstream signaling pathway by SPCP treatment regulates cell cycle progression. Therefore, these results contribute to the research on the molecular mechanism for SPCP promoting the migration and proliferation of rat intestinal epithelial cells.

Wound Healing Potential of Spirulina Protein on CCD-986sk Cells.

Wound healing is a dynamic and complex process. The proliferation and migration of dermal fibroblasts are crucial for wound healing. Recent studies have indicated that the extracts from Spirulina platensis have a positive potential for wound healing. However, its underlying mechanism is not fully understood. Our previous study showed that spirulina crude protein (SPCP) promoted the viability of human dermal fibroblast cell line (CCD-986sk cells). In this study, we further investigated the wound healing effect and corresponding mechanisms of SPCP on CCD-986sk cells. Bromodeoxyuridine (BrdU) assay showed that SPCP promoted the proliferation of CCD-986sk cells. The wound healing assay showed that SPCP promoted the migration of CCD-986sk cells. Furthermore, cell cycle analysis demonstrated that SPCP promoted CCD-986sk cells to enter S and G2/M phases from G0/G1 phase. Western blot results showed that SPCP significantly upregulated the expression of cyclin D1, cyclin E, cyclin-dependent kinase 2 (Cdk2), cyclin-dependent kinase 4 (Cdk4), and cyclin-dependent kinase 6 (Cdk6), as well as inhibited the expression of CDK inhibitors p21 and p27 in CCD-986sk cells. In the meanwhile, SPCP promoted the phosphorylation and activation of phosphoinositide 3-kinase (PI3K) and protein kinase B (Akt). However, the phosphorylation of Akt was significantly blocked by PI3K inhibitor (LY294002), which in turn reduced the SPCP-induced proliferation and migration of CCD-986sk cells. Therefore, the results presenting in this study suggested that SPCP can promote the proliferation and migration of CCD-986sk cells; the PI3K/Akt signaling pathway play a positive and important role in these processes.

Protective Effect of Pyropia yezoensis Peptide on Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Dexamethasone (DEX), a synthetic glucocorticoid, causes skeletal muscle atrophy. This study examined the protective effects of Pyropia yezoensis peptide (PYP15) against DEX-induced myotube atrophy and its association with insulin-like growth factor-I (IGF-I) and the Akt/mammalian target of rapamycin (mTOR)-forkhead box O (FoxO) signaling pathway. To elucidate the molecular mechanisms underlying the effects of PYP15 on DEX-induced myotube atrophy, C2C12 myotubes were treated for 24 h with 100 µM DEX in the presence or absence of 500 ng/mL PYP15. Cell viability assays revealed no PYP15 toxicity in C2C12 myotubes. PYP15 activated the insulin-like growth factor-I receptor (IGF-IR) and Akt-mTORC1 signaling pathway in DEX-induced myotube atrophy. In addition, PYP15 markedly downregulated the nuclear translocation of transcription factors FoxO1 and FoxO3a, and inhibited 20S proteasome activity. Furthermore, PYP15 inhibited the autophagy-lysosomal pathway in DEX-stimulated myotube atrophy. Our findings suggest that PYP15 treatment protected against myotube atrophy by regulating IGF-I and the Akt-mTORC1-FoxO signaling pathway in skeletal muscle. Therefore, PYP15 treatment appears to exert protective effects against skeletal muscle atrophy.

Effect of Cyclophilin from Pyropia Yezoensis on the Proliferation of Intestinal Epithelial Cells by Epidermal Growth Factor Receptor/Ras Signaling Pathway.

Cyclophilin (Cyp) is peptidyl-prolyl isomerase (PPIase), and it has many biological functions, including immune response regulation, antioxidants, etc. Cyp from red algae is known for its antioxidant and antifungal activity. However, the other biological effects of Cyp from Pyropia yezoensis are unclear. In this study, we synthesized Cyp from P. yezoensis (pyCyp) and examined its biological activity on IEC-6 cells. First, the MTS assay showed that pyCyp increased cell proliferation in a dose-dependent manner. pyCyp activated the EGFR signaling pathway that regulates cell growth, proliferation, and survival. It induced intracellular signaling pathways, including the Ras signaling pathway. In addition, we observed cell cycle-related proteins. pyCyp increased the expression of cyclin A, cyclin E, and Cdk2, and decreased the expression of p27 and p21 proteins. These results indicate that pyCyp stimulates cell proliferation via the EGFR signaling pathway and promotes cell cycle progression in intestinal epithelial cells. Therefore, we suggest pyCyp as a potential material to promote the proliferation of intestinal epithelial cells.

Probing Multi-Target Action of Phlorotannins as New Monoamine Oxidase Inhibitors and Dopaminergic Receptor Modulators with the Potential for Treatment of Neuronal Disorders.

Modulation of multiple protein targets with a single compound is essential for the effective treatment of central nervous system disorders. In our previous G protein-coupled receptor (GPCR) cell-based study, a selective human monoamine oxidase (hMAO)-A inhibitor, eckol, stimulated activity of dopamine D3 and D4 receptors. This result led to our interest in marine phlorotannin-mediated modulation of hMAO enzymes and related GPCRs in neuronal disorders. Here, we evaluate the multi-target effects of phloroglucinol, phlorofucofuroeckol-A (PFF-A), and dieckol by screening their modulatory activity against hMAO-A and -B and various neuronal GPCRs. Among the tested phlorotannins, PFF-A showed the strongest inhibitory activity against both hMAO isoforms, with higher selectivity toward hMAO-B than hMAO-A. Enzyme kinetics and docking data revealed that PFF-A noncompetitively acts on hMAOs into the alternative binding pocket of enzymes with allosteric functions. In a functional assay for GPCR screening, dieckol and PFF-A exhibited a multi-target combination of D3R/D4R agonism and D1/5HT1A/NK1 antagonism. In particular, they effectively stimulated D3R and D4R, compared to other GPCRs. Docking analysis confirmed that dieckol and PFF-A successfully docked into the conserved active sites of D3R and D4R and interacted with aspartyl and serine residues in the orthosteric binding pockets of the respective receptors. Based on our experimental and computational data, we established the structure-activity relationship between tested phlorotannins and target proteins, including hMAOs and GPCRs. Our current findings suggest that hMAO inhibitors dieckol and PFF-A, major phlorotannins of edible brown algae with multi-action on GPCRs, are potential agents for treatment of psychological disorders and Parkinson's disease.

Fucosterol from an Edible Brown Alga Ecklonia stolonifera Prevents Soluble Amyloid Beta-Induced Cognitive Dysfunction in Aging Rats.

Fucosterol from edible brown seaweeds has various biological activities, including anti-inflammatory, anti-adipogenic, antiphotoaging, anti-acetylcholinesterase, and anti-beta-secretase 1 activities. However, little is known about its effects on soluble amyloid beta peptide (sAß)-induced endoplasmic reticulum (ER) stress and cognitive impairment. Fucosterol was isolated from the edible brown seaweed Ecklonia stolonifera, and its neuroprotective effects were analyzed in primary hippocampal neurons and in aging rats. Fucosterol attenuated sAß1-42-induced decrease in the viability of hippocampal neurons and downregulated sAß1-42-induced increase in glucose-regulated protein 78 (GRP78) expression in hippocampal neurons via activation of tyrosine receptor kinase B-mediated ERK1/2 signaling. Fucosterol co-infusion attenuated sAß1-42-induced cognitive impairment in aging rats via downregulation of GRP78 expression and upregulation of mature brain-derived neurotrophic factor expression in the dentate gyrus. Fucosterol might be beneficial for the management of cognitive dysfunction via suppression of aging-induced ER stress.

Phycoerythrin Peptide from Pyropia yezoensis Alleviates Endoplasmic Reticulum Stress Caused by Perfluorooctane Sulfonate-Induced Calcium Dysregulation.

Perfluorooctane sulfonate (PFOS), a stable fluorosurfactant, causes endoplasmic reticulum (ER) stress in the brain. This study was designed to investigate whether a phycoerythrin-derived peptide of Pyropia yezoensis (PYP) reduces PFOS-induced ER stress associated with calcium dysregulation. The protective effects of PYP were determined by cell viability, immunoblotting for ER stress response protein glucose-regulated protein 78 (GRP78) and calcium-dependent protein kinases in rat frontal cortical neurons. PFOS-induced decrease in cell viability was attenuated by PYP pretreatment (1 µg/mL) for 24 h, which was downregulated by inhibiting tropomyosin-receptor kinase B (TrkB). PYP pretreatment downregulated the increase in intracellular calcium levels and phosphorylation of calcium/calmodulin-dependent protein kinase II and c-Jun N-terminal kinase which are associated with a PFOS-induced increase in GRP78. The PFOS-induced increase in GRP78 was downregulated via activation of TrkB receptor-linked extracellular signal-regulated kinases 1/2 (ERK1/2) by PYP pretreatment. Moreover, PYP microinjections (1 µg/kg, 0.54 nmol) attenuated the GRP78 expression in rat prefrontal cortex caused by PFOS (10 mg/kg) exposure for 2 weeks. These findings demonstrate that PYP enhances frontal cortical neuron viability via activation of TrkB receptor-ERK1/2 signaling and attenuation of ER stress in rat prefrontal cortex against PFOS exposure, suggesting that PYP might prevent neuronal dysfunctions caused by PFOS-induced ER stress.

Pyropia yezoensis Protein Supplementation Prevents Dexamethasone-Induced Muscle Atrophy in C57BL/6 Mice.

We investigated the protective effects of Pyropia yezoensis crude protein (PYCP) against dexamethasone (DEX)-induced myotube atrophy and its underlying mechanisms. DEX (3 mg/kg body weight, intraperitoneal injection) and PYCP (150 and 300 mg/kg body weight, oral) were administrated to mice for 18 days, and the effects of PYCP on DEX-induced muscle atrophy were evaluated. Body weight, calf thickness, and gastrocnemius and tibialis anterior muscle weight were significantly decreased by DEX administration (p < 0.05), while PYCP supplementation effectively prevented the DEX-induced decrease in body weight, calf thickness, and muscle weight. PYCP supplementation also attenuated the DEX-induced increase in serum glucose, creatine kinase, and lactate dehydrogenase levels. Additionally, PYCP supplementation reversed DEX-induced muscle atrophy via the regulation of the insulin-like growth factor-I/protein kinase B/rapamycin-sensitive mTOR complex I/forkhead box O signaling pathway. The mechanistic investigation revealed that PYCP inhibited the ubiquitin-proteasome and autophagy-lysosome pathways in DEX-administrated C57BL/6 mice. These findings demonstrated that PYCP increased protein synthesis and decreased protein breakdown to prevent muscle atrophy. Therefore, PYCP supplementation appears to be useful for preventing muscle atrophy.

Pyropia yezoensis Protein Prevents Dexamethasone-Induced Myotube Atrophy in C2C12 Myotubes.

Glucocorticoids (GCs), which are endocrine hormones released under stress conditions, can cause skeletal muscle atrophy. This study investigated whether Pyropia yezoensis crude protein (PYCP) inhibits synthetic GCs dexamethasone (DEX)-induced myotube atrophy associated with proteolytic systems. Mouse skeletal muscle C2C12 myotubes were treated with DEX in the presence or absence of PYCP. DEX exposure (100 µM) for 24 h significantly decreased myotube diameter and myogenin expression, which were all increased by treatment with 20 and 40 µg/mL PYCP. Additionally, PYCP significantly reduced the nuclear expression of the forkhead box transcription factors, FoxO1 and FoxO3a, and ubiquitin-proteasome pathway activation. Further mechanistic research revealed that PYCP inhibited the autophagy-lysosome pathway in DEX-induced C2C12 myotubes. These findings indicate that PYCP prevents DEX-induced myotube atrophy through the regulation of FoxO transcription factors, followed by the inhibition of the ubiquitin-proteasome and autophagy-lysosome pathways. Therefore, we suggest that inhibiting these two proteolytic processes with FoxO transcription factors is a promising strategy for preventing DEX-related myotube atrophy.

Nrf2 and NF-κB Signaling Pathways Contribute to Porphyra-334-Mediated Inhibition of UVA-Induced Inflammation in Skin Fibroblasts.

In this study, we examined the protective effects of porphyra-334 against UVA-irradiated cellular damage and elucidated the underlying mechanisms. Porphyra-334 prevented UVA-induced cell death and exhibited scavenging activities against intracellular oxidative stress induced by UVA irradiation in skin fibroblasts. We found that porphyra-334 significantly reduced the secretion and expression of IL-6 and TNF-α, reduced nuclear expression of Nuclear factor-κB (NF-κB), and sustained NF-E2-related factor 2 (Nrf2) activation. Further mechanism research revealed that porphyra-334 promoted the Nrf2 signaling pathway in UVA-irradiated skin fibroblasts. Our results show that the antioxidant effect of porphyra-334 is due to the direct scavenging of oxidative stress and its inhibitory effects on NF-κB-dependent inflammatory genes, such as IL-6 and TNF-κ. Therefore, we hypothesize that boosting the Nrf2- NF-κB-dependent response to counteract environmental stress is a promising strategy for the prevention of UVA-related damage.

TOXICITY OF CRYOPROTECTIVE AGENTS AND SIGNALING OF INSULIN-LIKE GROWTH FACTOR IN HEN CLAM (MACTRA CHINENSIS) EMBRYOS.

BACKGROUND: Signaling of Insulin-like growth factor-I (IGF-I) is involved in development, growth, reproduction and aging of organisms. OBJECTIVE: The work investigated the toxicity of glycerol, dimethyl sulfoxide (DMSO), and ethylene glycol (EG) to hen clam (Mactra chinensis) embryos, as well as the possible role of the insulin-like growth factor-I (IGF-I) during the development and growth of embryos after freeze. MATERIALS AND METHODS: Effects of glycerol, DMSO and EG at different concentrations were tested. The relationship between larval viability and signaling of IGF-I receptor after cryoprotective treatment and/or freezing was examined using immuno-blot analysis. RESULTS: Glycerol had the highest toxicity, followed by DMSO or EG. No embryo survived freeze and thaw without CPAs. After freeze, the activation of the IGF-I signaling pathway, including the IGF-I receptor (IGF-IR) ß-subunit, could be detected in freeze-thawed embryos. The level of IGF-IR expression was very weak in freeze-thawed embryos. CONCLUSION: The survival and developmental rate of embryos was closely related to CPA concentration. IGF-IR was activated and regulated the downstream IGF-I signaling in embryos. The reduced activation of IGF-IR could be related to the death of hen clam embryos.

Dietary Hizikia fusiformis glycoprotein-induced IGF-I and IGFBP-3 associated to somatic growth, polyunsaturated fatty acid metabolism, and immunity in juvenile olive flounder Paralichthys olivaceus.

This study was aimed to examine the effect of dietary glycoprotein extracted from the sea mustard Hizikia fusiformis (Phaeophyceae: Sargassaceae) as a dietary supplement on growth performance in association with somatotropin level, proximate compositions, and immunity in juvenile olive flounder Paralichthys olivaceus. Water-ethanol extracted glycoprotein from H. fusiformis was supplemented to three fishmeal-based diets at the concentration of 0, 5, and 10gkg(-1) diet (designated as H0, H5, and H10, respectively). After a 12week-long feeding trial, growth performance and biochemical responses were analyzed including proximate composition, and whole body amino acids and fatty acids. We also measured plasma insulin like growth factor (IGF), IGF-binding protein (IGFBP) and interleukin (IL). The fish fed H5 showed the greatest weight gain among the dietary treatments. In parallel with the growth, the fish fed the diets containing H. fusiformis glycoprotein showed an increased plasma IGF-I activity and increased expression of 43-kDa IGFBP-3 compared to that in the control, whereas an opposite trend was observed for 34-kDa IGFBP-1. Although no differences were found in the level of whole body linoleic acid (C18:2n-6) and linolenic acid (C18:3n-3) among treatments, increases in arachidonic acid (ARA, C20:4n-6), eicosapentaenoic acid (EPA, C20:5n-3) and docosahexaenoic acid (DHA, C22:6n-3) were observed in fish fed H5 compared to control. IL-2 and -6 levels increased significantly in fish fed H10 compared to those in the control indicating increased immunity. These results suggest that supplementation of H. fusiformis glycoprotein in fish diet may be beneficial for fish growth and immunity in juvenile olive flounder.

Influence of the toxicity of cryoprotective agents on the involvement of insulin-like growth factor-I receptor in surf clam (Spisula sachalinensis) larvae.

BACKGROUND: The signaling of insulin-like growth factor-I (IGF-I) is involved in the development, growth, reproduction and aging of vertebrates. However, few studies have investigated the involvement of IGF-I during states of extreme shock, such as those induced by potently toxic cryoprotective agents (CPAs) or low temperature conditions, in bivalves. OBJECTIVE: We investigated the toxicity of CPAs and the potential relationship between larval viability and the IGF-I receptor (IGF-IR) after treatment with CPAs or freezing in surf clam (Spisula sachalinensis) larvae. MATERIALS AND METHODS: The umbo larvae and different concentrations of CPAs (dimethyl sulfoxide, DMSO; ethylene glycol, EG) were used to investigate the toxicity of CPAs and the vitrification of surf clam larvae. The relationship between larval viability and the IGF-I receptor (IGF-IR) after treatment with CPAs or freezing was investigated using immunoblot analysis. RESULTS: An increase in concentration greater than 4M DMSO was fatal in larvae; however, 5M EG combined with a mixture of CPAs had no harmful effects. Moreover, live larvae immersed in a 5M EG solution remained intact and maintained their normal shape and organs. However, even though the larvae survived the CPA toxicity test, none of the vitrified larvae survived. After immersion into CPAs and vitrification, 97-kDa IGF-IR ß-subunits could be detected in all larvae; but tyrosine phosphorylation of the intracellular ß-subunits was detected only in the control and live groups. CONCLUSION: IGF-IR was activated in the umbo larvae but not in dead surf clam larvae treated with CPA and frozen. Activation of IGF-IR has relevance to the umbo larval stage in live surf clams treated with CPAs.