Structural characterization of a fucoidan from Ascophyllum nodosum and comparison of its protective effect against cellular oxidative stress with its analogues.

In the present study, a fucoidan fraction (ANP-3) was isolated from Ascophyllum nodosum, and the combined application of desulfation, methylation, HPGPC, HPLC-MSn, FT-IR, GC-MS, NMR, and Congo red test elucidated ANP-3 (124.5 kDa) as a triple-helical sulfated polysaccharide constituted by →2)-α-Fucp3S-(1→, →3)-α-Fucp2S4S-(1→, →3,6)-ß-Galp4S-(1→, →3,6)-ß-Manp4S-(1→, →3,6)-ß-Galp4S-(1→，→6)-ß-Manp-(1→, →3)-ß-Galp-(1→, α-Fucp-(1→, and α-GlcAp-(1→ residues. To better understand the relationship between the fucoidan structure of A. nodosum and protective effects against oxidative stress, two fractions ANP-6 and ANP-7 were used as contrast. ANP-6 (63.2 kDa) exhibited no protective effect against H2O2-induced oxidative stress. However, ANP-3 and ANP-7 with the same molecular weight of 124.5 kDa could protect against oxidative stress by down-regulating reactive oxygen species (ROS) and malondialdehyde (MDA) levels and up-regulating total antioxidant capability (T-AOC), superoxide dismutase (SOD), catalase (CAT), and glutathione peroxidase (GPX) activities. Then metabolites analysis indicated that arginine biosynthesis and phenylalanine, tyrosine, and tryptophan biosynthesis metabolic pathways and metabolic biomarkers such as betaine were involved in the effects of ANP-3 and ANP-7. The better protective effect of ANP-7 compared to that of ANP-3 could be attributed to its relatively higher molecular weight, sulfate substitution and →6)-ß-Galp-(1→ content, and lower uronic acid content.

Oral administration of sea cucumber (Stichopus japonicus) protein exerts wound healing effects via the PI3K/AKT/mTOR signaling pathway.

This study aimed to investigate the effect of the oral administration of sea cucumber protein (SCP) on wound healing. SCP was isolated and purified from the body wall of Stichopus japonicus. A mouse skin incision model was operated on to evaluate the wound repair effect of SCP. The histological changes in the skin at the wound sites of BALB/c mice were observed by staining with haematoxylin and eosin (H&E) and Masson's trichrome. The enzyme-linked immunosorbent assay (ELISA) was used to analyze the expression of inflammatory cytokines in BALB/c mice. The boost cell migration ability was detected by a scratch assay after HaCaT cells were cultured with digested SCP (dSCP). Western blotting and RT-PCR assays were performed to determine the mechanism of SCP promoting wound healing. As a result, the wound healing rate in the SCP high dose group was 1.3-fold, compared to that in the blank group on day 14. Also, increased epidermal thickness and 1.79-fold collagen deposition contrasted with the blank group. Additionally, SCP could up-regulate the levels of pro-inflammatory factors (IL-1ß, IL-6, TNF-α) from day 3 to 7 firstly and decreased from day 7 to 14. IL-8 expression continuously decreased while the level of anti-inflammatory factor (IL-10) increased during the healing stage. Furthermore, the cell closure area reached 67% after being treated with 50 µg mL-1 of dSCP for 48 h. Cell proliferation was associated with the dSCP-activated PI3K/AKT/mTOR pathway. Taken together, SCP can be orally used as an effective agent for wound repair.

Ultraviolet-Ray-Induced Sea Cucumber (Stichopus japonicus) Melting Is Mediated by the Caspase-Dependent Mitochondrial Apoptotic Pathway.

Sea cucumber body-wall melting occurs under certain circumstances. We have shown that apoptosis but not autolysis plays a critical role in the initial stage. However, it is still unclear how apoptosis is triggered in this process. In this study, we examined the levels of reactive oxygen species (ROS), the levels of B-cell lymphoma 2 (Bcl-2) and Bcl-2-associated X (Bax) proteins, the depolarization of mitochondrial transmembrane potentials, and cytochrome c (Cyt c) release during sea cucumber melting induced by ultraviolet (UV) exposure. We also investigated the contribution of caspase in this process by injecting a pan-caspase inhibitor. Our data showed that UV exposure stimulates ROS production, dysfunction of mitochondria, and the release of Cyt c in sea cucumber coelomic fluid cells and body walls. We found a decrease of Bcl-2 and increase of Bax in the mitochondria after UV exposure. We also demonstrated that these changes are associated with elevated caspase-9 and -3 activity. Finally, our data showed that the inhibition of caspases-9 and -3 using an inhibitor suppresses UV-induced sea cucumber melting. These results suggest that apoptosis during sea cucumber melting is mediated by mitochondrial dysfunction and follows the activation of the caspase-signaling pathway. This study presents a novel insight into the mechanism of sea cucumber melting.

Effects of abalone (Haliotis discus hannai Ino) gonad polysaccharides on cholecystokinin release in STC-1 cells and its signaling mechanism.

Abalone gonad polysaccharide (AGP) -31, -32 and -33 prepared in this study had the molecular weight (MW) of 37.8, 32.2 and 27.5kDa, respectively. They all contained mannose, rhamnose, glucuronic acid, glucose, galactose, xylose, arabinose, and fucose, with very similar monosaccharide profile. All the three polysaccharides could significantly increase the secretion of cholecystokinin (CCK) in STC-1 cells. Among them, AGP-32 showed the strongest effect. However, the low-MW fragments of AGP-32 showed significantly lower activity than AGP-32 itself. It was also found that the inhibitors on calcium-sensing receptor (CaSR), protein kinase A (PKA), Ca(2+)/calmodulin-dependent protein kinase (CaMK) II, p38- mitogen-activated protein kinases (MAPK), and an intracellular calcium chelator all inhibited AGP-induced CCK secretion. To conclude, Ca(2+)/calmodulin (CaM)/CaMK, cyclic adenosine monophosphate (cAMP)/PKA and MAPK pathways are all involved in AGP-induced CCK secretion.

Analysis of Apoptosis in Ultraviolet-Induced Sea Cucumber (Stichopus japonicus) Melting Using Terminal Deoxynucleotidyl-Transferase-Mediated dUTP Nick End-Labeling Assay and Cleaved Caspase-3 Immunohistochemistry.

The sea cucumber body wall melting phenomenon occurs under certain circumstances, and the mechanism of this phenomenon remains unclear. This study investigated the apoptosis in the ultraviolet (UV)-induced sea cucumber melting phenomenon. Fresh sea cucumbers (Stichopus japonicus) were exposed to UV radiation for half an hour at an intensity of 0.056 mW/cm(2) and then held at room temperature for melting development. The samples were histologically processed into formalin-fixed paraffin-embedded tissues. The apoptosis of samples was analyzed with the terminal deoxynucleotidyl-transferase-mediated dUTP nick end-labeling (TUNEL) assay and cleaved caspase-3 immunohistochemistry. The emergence of TUNEL-positive cells speeds up between 0.5 and 2 h after UV irradiation. Cleaved caspase-3 positive cells were obviously detected in sample tissues immediately after the UV irradiation. These results demonstrated that sea cucumber melting induced by UV irradiation was triggered by the activation of caspase-3 followed by DNA fragmentation in sea cucumber tissue, which was attributed to apoptosis but was not a consequence of autolysis activity.

Induction of mitogynogenetic diploids and identification of WW super-female using sex-specific SSR markers in half-smooth tongue sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial induction of mitogynogenesis by hydrostatic pressure using heterologous sperm was developed in half-smooth tongue sole in order to assess homozygosity of gynogens and to identify WW super-female. The optimal initiation time for pressure shock of mitogynogenetic embryos was determined to be 21.5 min after insemination when water temperature is at 22-23°C, while the optimal pressure and treatment duration were determined to be 70 MPa for 4 min. About 1,500 mitogynogenetic diploid larvae were obtained. Ten tongue sole microsatellite markers were used for homozygosity analysis of 24 mitogynogenetic larvae. Among the 24 larvae, the percentage of homozygosity ranged from 73.91% to 87.50% with an average homozygosity of 80.54%. Sex-specific simple sequence repeat (SSR) markers, CseF-SSR1, were isolated and used for identifying WW super-female mitogynogens in the tongue sole. The amplification of genomic DNA using the sex-specific SSR marker produced one DNA band of 206 bp in ZZ males, two DNA bands of 206 and 218 bp in ZW females, and one DNA band of 218 bp in WW super-females. Four WW "super-female" gynogens were observed in 39 mitogynogenetic diploids, indicating a ZW sex determination mechanism in the tongue sole. Thus, a protocol for the induction of artificial mitogynogenesis has been developed for the first time in half-smooth tongue sole, and the WW super-female diploids were identified in the mitogynogens by sex-specific SSR markers. These findings lay the foundation and provide important tool for the elaboration of sex determination mechanism, generation of WW super-females, and development of clone line and breeding of all-female stock in the half-smooth tongue sole.

MHC polymorphism and disease resistance to Vibrio anguillarum in 8 families of half-smooth tongue sole (Cynoglossus semilaevis).

BACKGROUND: Genes in the major histocompatibility complex (MHC) have a critical role in both the innate and adaptive immune responses because of their involvement in presenting foreign peptides to T cells. However, the nature has remained largely unknown. RESULTS: We examined the genetic variation in MHC class IIB in half-smooth tongue sole (Cynoglossus semilaevis) after challenge with vibrio anguillarum. Two thousand and four hundred fry from 12 half-smooth tongue sole families were challenged with Vibrio anguillarum. To determine any association between alleles and resistance or susceptibility to V. anguillarum, 160 individuals from four high-resistance (HR, < 40.55% mortality) families and four low-resistance (LR, > 73.27% mortality) families were selected for MHC IIB exon2 gene sequence analysis. The MHC IIB exon2 genes of tongue sole displayed a high level of polymorphism and were discovered at least four loci. Meanwhile, the dN/dS [the ratio of non-synonymous (dN) substitutions to synonymous (dS) substitutions] in the peptide-binding region (PBR) was higher than that in the non-peptide-binding region (non-PBR). Eighty-eight alleles were discovered among 160 individuals, and 13 out of 88 alleles were used to analyze the distribution pattern between the resistant and susceptible families. Certain alleles presented in HR and LR with a different frequency, while other alleles were discovered in only the HR or LR families, not both. Five alleles, Cyse-DBB*6501, Cyse-DBB*4002, Cyse-DBB*6102, Cyse-DBB*5601 and Cyse-DBB*2801, were found to be associated with susceptibility to V. anguillarum with a frequency of 1.25%, 1.25%, 1.25%, 1.25% and 2.5% in the HR families, and 35%, 33.75%, 27.5%, 16.25%, 15% in the LR families (p < 0.01, 0.01, 0.01, 0.01, 0.01), respectively. Four alleles, Cyse-DBB*3301, Cyse-DBB*4701, Cyse-DBB*6801 and Cyse-DBB*5901, were found to be associated with resistance to V. anguillarum, with a frequency of 13.75%, 11.25%, 11.25%, 8.75% in the HR families and 1.25%, 1.25%, 1.25%, 1.25% and 1.25% in the LR families (p < 0.01, 0.05, 0.05 and p = 0.064), respectively. CONCLUSIONS: Elucidation of the role of MHC II B genes in half-smooth tongue sole should prove to be helpful to the in-depth development of marker-assisted selective breeding in half-smooth tongue sole.

Growth differences and differential expression analysis of pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) between the sexes in half-smooth tongue sole Cynoglossus semilaevis.

Pituitary adenylate cyclase activating polypeptide (PACAP) and growth hormone-releasing hormone (GHRH) are regulators of growth hormone secretion. In this article, we examined the difference in growth and mRNA expression of PACAP and GHRH between the sexes in half-smooth tongue sole, an important cultured fish species indicating sexually growth dimorphism in China. Firstly, a significant body weight difference between females and males was first observed at 7 months (P<0.05) and at 18 onths the mean body weight of the females (771.0±44.3 g) was as much as 4.9 times higher than that of males (130.6±6.0 g). As a result, half-smooth tongue sole, Cynoglossus semilaevis, is a good model to investigate the effects of growth-related genes expression on sexual growth dimorphism. Secondly, the cDNAs encoding PRP/PACAP and GHRH were isolated. Two differently processed mRNA transcripts of PRP/PACAP (PRP-encoding and PRP splice variant) were found. PACAP and GHRH mRNA was highly abundant in brain and less abundant in other tissues. However, PACAP mRNA was expressed in most brain regions, and was lower in the cerebellum. GHRH mRNA was predominantly expressed in the hypothalamus and weakly expressed in all areas of the brain examined. Ontogenetic expression analysis indicated that PACAP and GHRH mRNA was detected in the early stages of embryogenesis. Finally, differential expression showed that there was no significant difference of the expression level of PACAP or GHRH between the sexes before 8 months of age. However, between 9 and 12 months of age, the GHRH mRNA expression level in males was significantly higher than in females (P<0.05), which might be associated with GH deficiency in males. In contrast, the male PACAP mRNA expression level was not significantly higher than that in females even at 9 and 12 months of age. The present results provide important clues for understanding the sexual growth dimorphism mechanisms in half-smooth tongue sole.

Structure analysis of water-soluble polysaccharide CPPS3 isolated from Codonopsis pilosula.

Codonopsis pilosula is a perennial species of flowering plants propagated in Northeast Asia. A water-soluble polysaccharide, CPPS(3), was extracted from the root of Codonopsis pilosula by boiled water extraction and ethanol precipitation. The molecular weight was estimated to be 7.4 x 10(4) Da determined by using Gel permeation chromatography. Monosaccharide composition and the structure of the polysaccharide were determined by gas spectroscopy, Fourier transform IR (FT-IR) spectroscopy, NMR spectroscopy and mass spectroscopy and some chemical method analysis was made. The components were galactose, arabinose and rhamnose in the molar ratio of 1.13:1.12:1. The main chain of CPPS(3) is illustrated to be (1-->3)-linked-beta-GalpNAc, (1-->3)-linked-alpha-Rhap and (1-->2,3)-beta-Galp.

Construction of a genetic linkage map and mapping of a female-specific DNA marker in half-smooth tongue sole (Cynoglossus semilaevis).

The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, "tongue sole") is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.

Artificial gynogenesis and sex determination in half-smooth tongue sole (Cynoglossus semilaevis).

Half-smooth tongue sole (Cynoglossus semilaevis) is an important cultured marine fish as well as a promising model fish for the study of sex determination mechanisms. In the present study, a protocol for artificial gynogenesis of half-smooth tongue sole was developed in order to identify the sex determination mechanism and to generate all-female stock. The optimal UV-irradiation dose for genetically inactivating sea perch spermatozoa was determined to be > or =30 mJ/cm(2). The optimal initiation time for cold shock of gynogenetic embryos was determined to be 5 min after fertilization, while the optimal temperature and treatment duration were determined to be 20-25 min at 5 degrees C. Chromosomes from common diploids, gynogenetic haploids, and diploids were analyzed. WW chromosomes were discovered in some of the gynogenetic diploids. The microsatellite marker was applied to analyze gynogenetic diploid fry. Among the 30 gynogenetic diploid fry, 11 fry contained only one allele, while 19 contained two alleles, which had the same genotype as their mother. The female-specific DNA marker was observed in four individuals out of ten gynogenetic diploid fry. Ploidy analysis of 20 putative gynogenetic fry showed them all to be diploid. Thus, a protocol for the induction of artificial gynogenesis has been developed for the first time in half smooth tongue sole, and the sex determination mechanism in the tongue sole was determined to be female heterogametic with the ZW chromosome.

A new polysaccharide from leaf of Ginkgo biloba L.

A water-soluble polysaccharide, GPB, was obtained from leaf of Ginkgo biloba L by hot water extraction followed by precipitation with ethanol and fractionation with gel chromatography. The results of HPLC with TSK-GEL column and gel filtration chromatography with Sepharose CL-6B analysis indicated GPB was uniform in polarity and its molecular weight (MW) was about 10 kDa. The structure of GPB was analysed using chemical methods, IR spectroscopy, and NMR spectroscopy. GPB has a high branched structure with polygalactose as core part of backbone. The repeating unit of polygalactose consists of 1,6-linked Galactose (Gal) and 1,3,6-linked Gal.

[The study of raman spectra for ethanol under the pressures of 0.1-900 MPa at 24 degrees C].

Experimental measurement and study of Raman spectra for pure ethanol and 50% ethanol aqueous solution has been conducted at ambient temperature and the pressures of 0.1-900 MPa by using Cubic Zirconia anvil cell. The result shows that the frequency of C-H stretch vibration increases with increasing pressure and each of their relation between the frequency and pressure can be expressed as following: for pure ethanol: v1 = 2881.890 + 0.00127 P + 6.213 x 10(-6) P2; V2 = 2928.707 + 0.004 38 P + 4.772 10(-6) p2; v3 = 2973.457 + 0.0089 P + 3.245 x 10(-6) p2; for 50% ethanol aqueous solution: v1 = 2885.616 + 0.010 8 P-2.699 x 10(-6) P2; v2 = 2932.734 + 0.0137 P - 3.346 x 0(-6) p2; v3 = 2978.115 + 0.0165 P - 4.914 x 10(-6) P2. Furthermore, the authors have observed that at the pressure lower than 550 MPa, the strength of hydrogen bonding becomes stronger with increasing pressure.