Study on the Free Radical Scavenging Activity of Sea Cucumber （Paracaudina chinens vat.） Gelatin Hydrolysate

Gelatin from the sea cucumber （Paracaudina chinens var.） was hydrolyzed by bromelain and the hydrolysate was found to have a high free radical scavenging activity. The hydrolysate was fractionated through an ultrafiltration membrane with 5 kDa molecular weight cutoff （MWCO）. The portion （less than 5 kDa） was further separated by Sephadex G-25. The active peak was collected and assayed for free radical scavenging activity. The scavenging rates for superoxide anion radicals （O2·^-） and hydroxyl radicals （·OH） of the fraction with the highest activity were 29.02% and 75.41%, respectively. A rabbit liver mitochondrial free radical damage model was adopted to study the free radical scavenging activity of the fraction. The results showed that the sea cucumber gelatin hydrolysate can prevent the damage of rabbit liver and mitochondria.     

Study on the Free Radical Scavenging Activity of Sea Cucumber (Paracaudina chinens var.) Gelatin Hydrolysate

Gelatin from the sea cucumber(Paracaudina chinens var.) was hydrolyzed by bromelain and the hydrolysate was found to have a high free radical scavenging activity. The hydrolysate was fractionated through an ultrafiltration membrane with 5 kDa molecular weight cutoff(MWCO). The portion(less than 5 kDa) was further separated by Sephadex G-25. The active peak was col-lected and assayed for free radical scavenging activity. The scavenging rates for superoxide anion radicals(O2·-) and hydroxyl radi-cals(·OH) of the fraction with the highest activity were 29.02% and 75.41%,respectively. A rabbit liver mitochondrial free radical damage model was adopted to study the free radical scavenging activity of the fraction. The results showed that the sea cucumber gelatin hydrolysate can prevent the damage of rabbit liver and mitochondria.     

Two-dimensional electrophoresis analysis of proteins extracted from <Emphasis Type="Italic">Alexandrium</Emphasis> sp. LC3

Two-dimensional electrophoresis(2-DE) of protein extracted and purified from Alexandrium sp. LC3 was conducted. In the SDS-PAGE study, the relative molecular weights of the proteins were mainly in the range of 14 kDa?31 kDa and 43 kDa?66 kDa, and more proteins were detected between 14 kDa and 31 kDa. With the improved protein preparation, the two-dimensional electrophoresis patterns indicated that the relative molecular weights of the proteins were between 14 kDa and 100 kDa, and most of them ranged from 14 kDa to 31 kDa. This was consistent with the result of the SDS-PAGE analysis. The isoelectric points were found to lie between 3.0 and 8.0, and most of them were in the range of 3.0?6.0. Better separation effect was acquired with pre-prepared immobilized gradient (IPG) strip (pH 3?5.6), and about 320 protein spots could be visualized on the 2-DE map by staining. Within pH 3?10 and pH 3?5.6 strips, the protein samples of Alexandrium sp. LC3 could be separated well.     

Purification and characterization of iron-cofactored superoxide dismutase from Enteromorpha linza

A superoxide dismutase was purified from Enteromorpha linza using a simple and safe procedure, which comprised phosphate buffer extraction, ammonium sulphate precipitation, ion exchange chromatography on Q-sepharose column, and gel filtration chromatography on Superdex 200 10/300GL. The E. linza superoxide dismutase （E/SOD） was purified 103.6-fold, and a yield of 19.1% and a specific activity of 1 750 U/rag protein were obtained. The SDS-PAGE exhibited E/SOD a single band near 23 kDa and the gel filtration study showed E/SOD＇s molecular weight is near 46 kDa in nondenatured condition, indicating it＇s a homodimeric protein. E/SOD is an iron-cofactored superoxide dismutase （Fe-SOD） because it was inhibited by hydrogen peroxide, insensitive to potassium cyanide. The optimal temperature for its maximal enzyme activity was 35℃, and it still had 29.8% relative activity at 0℃, then E/SOD can be classified as a cold-adapted enzyme. E/SOD was stable when temperature was below 40℃ or the pH was within the range of 5 10. The first 11 N-terminal amino acids orE/SOD were ALELKAPPYEL, comparison of its N-terminal sequence with other Fe-SOD N-terminal sequences at the same position suggests it is possibly a chloroplastic Fe-SOD.     

扁舵鲣抗氧化肽分离纯化及结构鉴定

【目的】从扁舵鲣(Auxis thazard)的木瓜蛋白酶酶解产物中分离鉴定具有较高1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除率的抗氧化肽。【方法】用木瓜蛋白酶水解扁舵鲣,以DPPH自由基清除能力为检测指标,通过超滤、凝胶过滤层析和反向高效液相色谱分离抗氧化肽,再经过超高效液相/三重四级杆飞行时间质谱(UPLC/Xevo G2-XS QTOF)进行结构鉴定。【结果】从酶解产物中获得3种抗氧化肽,其氨基酸序列分别为β-丙氨酸-1-甲基-L-组氨酸(241 u)、Gly-Ala-Gly-Gly-Pro(357 u)和Val-Glu(246 u)。【结论】扁舵鲣的木瓜蛋白酶酶解产物含有抗氧化活性的肽类,可为其抗氧化肽的开发提供理论依据。     

Characterization of angiotensin-І converting enzyme inhibiting peptide from <Emphasis Type="Italic">Venerupis philippinarum</Emphasis> with nano-liquid chromatography in combination with orbitrap mass spectrum detection and molecular docking

The complexity and diversity of peptide mixture from protein hydrolysates make their characterization difficult. In this study, a method combining nano LC-MS/MS with molecular docking was applied to identifying and characterizing a peptide with angiotensin-? converting enzyme (ACE-I) inhibiting activity from Venerupis philippinarum hydrolysate. Firstly, ethanol supernatant of V. philippinarum hydrolysate was separated into active fractions with chromatographic methods such as ion-exchange chromatography and high performance liquid chromatography in combination. Then seven peptides from active fraction were identified according to the searching result of the MS/MS spectra against protein databases. Peptides were synthesized and subjected to ACE-I-inhibition assay. The peptide NTLTLIDTGIGMTK showed the highest potency with an IC50 of 5.75 μmol L^?1. The molecular docking analysis showed that the ACE-I inhibiting peptide NTLTLIDTGIGMTK bond with residues Glu123, Glu403, Arg522, Glu376, Gln281 and Asn285 of ACE-I. Therefore, active peptides could be identified with the present method rather than the traditional purification and identification strategies. It may also be feasible to identify other food-derived peptides which target other enzymes and receptors with the method developed in this study.     

菲律宾蛤仔糖胺聚糖的纯化及单糖组成的研究

菲律宾蛤仔中提取的糖胺聚糖(GAGs)粗品,经DEAE-52-离子交换柱层析和Sephadex G-100凝胶柱纯化后,采用Sephadex G-100凝胶柱层析法测定该糖胺聚糖纯品F-1-1的相对分子质量,并以薄板层析(TLC)法对F-1-1的酸水解液进行单糖组成分析。结果表明:两级纯化得到均一的糖胺聚糖,其相对分子质量为4.4×104,单糖组分主要为氨基半乳糖、半乳糖、葡萄糖;对多糖进行两级纯化效果较好,纯品可进行结构分析;TLC法可快速分析水解多糖的单糖组成。     

Extract of Meretrix meretrix Linnaeus induces angiogenesis in vitro and activates endothelial nitric oxide synthase

Meretrix meretrix Linnaeus has long been used as traditional Chinese medicine in oriental medicine.The angiogentic activity of the extract of M.meretrix was investigated in this study,using human umbilical vein endothelial cells(HUVECs).Extract of M.meretrix Linnaeus(AFG-25) was prepared with acetone and ethanol precipitation,and further separated by Sephadex G-25 column.The results show that AFG-25 promoted proliferation,migration,and capillary-like tube formation in HUVECs,and in the presence of eNOS inhibitor NMA,the tube formation induced by AFG-25 is inhibited significantly.Moreover,AFG25 could also promote the activation of endothelial nitric oxide synthase(eNOS) and the resultant elevation of nitric oxide(NO) production.The results suggested that M.meretrix contains active ingredients with angiogentic activity and eNOS/NO signal pathway is in part involved in the proangiogenesis effect induced by AFG-25.     

Studies on the antitumor activity and isolation of the glycoprotein from the gonad ofChlamys (Azumapecten) farreri

After being degreased with acetone, the female gonad ofChlamys (Azumapecten) farreri was extracted with water, and then subjected to DEAE—cellulose column chromatography and purified by Sephadex G-100 chromatography to obtain a pure female gonad glycoprotein (FGGP). The IR spectra, electrophoresis and chemical analysis of FGGP indicated that it was a kind of acid glycoprotein and that the content of total protein and sugar were 54.8% and 40%, respectively. Pharmacological tests showed that FGGP could inhibit (inhibition rate of 40.87%) the growth of Sarcoma 180 of mice.     

Purification and characterization of lectin from humoral fluids of Charybdis feriatus

1 INTRODUCTION Lectins have been regarded as having a puta- tive role in non-self recognition in vertebrate and invertebrate immunities (Arason, 1996; Matsushita, 1996; Olafsen, 1996; Vasta et al., 1999; Wilson, et al., 1999; Marques and Barracco, 2000). …     

An extracellular oligopeptide permease may be a potential virulence factor of Vibrio harveyi

An oligopeptide permease A(OppA)was purified from the extracellular product of Vibrio harveyi SF-1.The molecular weight of the purified protein was estimated to be 58 kDa on SDS-PAGE.The purified protein showed phospholipase C activity at the optimal values of temperature 50℃ and pH 8.0.The enzymatic activity decreased when the temperature increased to 40℃.The N-terminal sequence of the purified protein was determined as ADVPAGTKLA,which is similar to that of OppA.The OppA pre-cursor gene was cloned from th...     

Coccolith Assemblages and Primary Productivity Variations in the Central Western Pacific Warm Pool Over the Last 380 kyr

Coccolith assemblages in two gravity cores(KX21-2 and KX12-1) from the central Western Pacific Warm Pool(WPWP) have been analyzed with SYRACO. The variations of nutricline and primary productivity(PP) have been reconstructed based on these assemblages. The results show that the coccolith assemblages were dominated by Florisphaera profunda, Gephyrocapsa and Emiliania huxleyi over the last 380 kyr. Variations of nutricline and primary productivity can be divided into three intervals. Interval I(about 380–300 kyr): PP was high and nutricline was shallow; Interval II(about 300–160 kyr): PP decreased dramatically for a short time after the acme of G. caribbeanica in Mid-Brunhes while nutricline became deeper; Interval III(about 160 kyr–present): PP fluctuated at low levels and nutricline was deep. Variations of each coccolith taxon and PP were highly correlated in the two cores, which means that the geological environment is similar in the two cores. Spectrum analysis is performed for all coccolith taxons and PP, and the 19-kyr cycle is the most prominent. It means that the production of coccolithophores in the WPWP is mainly controlled by precession.     

Distribution patterns of chlorophyll <Emphasis Type="Italic">a</Emphasis> in spring and autumn in association with hydrological features in the southern Yellow Sea and northern East China Sea

Two field studies were conducted to measure pigments in the Southern Yellow Sea (SYS) and the northern East China Sea (NECS) in April (spring) and September (autumn) to evaluate the distribution pattern of phytoplankton stock (Chl a concentration) and the impact of hydrological features such as water mass, mixing and tidal front on these patterns. The results indicated that the Chl a concentration was 2.43±2.64 (Mean ± SD) mg m^?3 in April (range, 0.35 to 17.02 mg m^?3) and 1.75±3.10 mg m^?3 in September (from 0.07 to 36.54 mg m^?3) in 2003. Additionally, four areas with higher Chl a concentrations were observed in the surface water in April, while two were observed in September, and these areas were located within or near the point at which different water masses converged (temperature front area). The distribution pattern of Chl a was generally consistent between onshore and offshore stations at different depths in April and September. Specifically, higher Chl a concentrations were observed along the coastal line in September, which consisted of a mixing area and a tidal front area, although the distributional pattern of Chl a concentrations varied along transects in April. The maximum Chl a concentration at each station was observed in the surface and subsurface layer (0–10 m) for onshore stations and the thermocline layer (10–30 m) for offshore stations in September, while the greatest concentrations were generally observed in surface and subsurface water (0–10 m) in April. The formation of the Chl a distributional pattern in the SYS and NECS and its relationship with possible influencing factors is also discussed. Although physical forces had a close relationship with Chl a distribution, more data are required to clearly and comprehensively elucidate the spatial pattern dynamics of Chl a in the SYS and NECS.     

Spatial variations of <Emphasis Type="Italic">b</Emphasis>-values in the coastal area of Guangdong

We used earthquake catalogs recorded by Guangdong Seismological Network from 2008 to 2014 to resolve the spatial variations of b-values in the coastal area of Guangdong, particularly in three key research areas (Yangjiang, Heyuan, and offshore Nanao Island) with strong seismicity. Our results revealed that b-values exhibited significant spatial variations, and zones with low b-values could indicate the most likely seismogenic area of large earthquakes. We observed three clear low b-value patches in the offshore Nanao Island. We found a distinct high b-value peak at the depth of 11 km and two minimum peaks at about 14 and 7–8 km in the Yangjiang area. The overall b-values generally decrease with depth in the Heyuan area. The spatial variations of b-values reflect tectonic anomalies; that is, the ‘low-high-low’ distribution of b-values in the offshore Nanao Island and the Yangjiang area may indicate the anomaly of the crustal structure with a weak layer. The b-values of reservoir-induced seismicity are obviously lower than that induced by tectonism. This finding indicates that the reservoir area is generally at high stress state under the condition of high pore pressure. We inferred that large earthquakes might be prone to occur at 10–12 km depth in the offshore Nanao Island, at 12–15 km depth in the Yangjiang area, and at the lower part of the seismic activity zone in the Heyuan area. Moreover, the upstream area of the Xinfengjiang reservoir is the most likely area of future large earthquakes in the Heyuan area.     

Biochemical Composition and Nutritional Value of Different Shell Color Strains of Pacific Oyster <Emphasis Type="Italic">Crassostrea gigas</Emphasis>

The shell color of Pacific oyster (Crassostrea gigas) is a desirable trait; but the nutritional studies on C. gigas with different shell colors have not been conducted. Through successive selective breeding, five shell color strains of black (B), purple (P), orange (O), golden (G) and white (W) C. gigas have been developed. The aim of this study was to evaluate the chemical composition and nutritional value of five shell color strains and one commercial population with a common color. The biochemical composition including moisture, total protein, glycogen, ash, total fat, fatty acids (FA), amino acids and minerals was detected. The results indicated that the protein (50.76%–56.57%) was the major component. The content of glycogen showed a significant difference between orange shell and golden shell strains, as well as between commercial population and golden shell strain. In addition, all shell color strains contained a large amount of essential amino acids (12.20–14.15 g (100 g)^?1), of them leucine (2.81–3.29 g (100 g)^?1) and lysine (2.79–3.28 g (100 g)^?1) were predominant. The oysters were rich in polyunsaturated fatty acids (42.26%–45.24% of total fatty acid) with high levels of DHA (18.53%–21.16% of total fatty acid) and EPA (17.23%–18.68% of total fatty acid). Significant differences of mineral contents (Mg, Zn, Fe and Cu) were identified among the six populations. These results indicated that C. gigas with different shell colors presented rich nutritional value with high protein, glycogen, essential amino acids and polyunsaturated fatty acids. The biochemical composition obtained in this study is useful for selective breeding of C. gigas with different shell colors.     

Purification and characterization of an alkaline protease from <Emphasis Type="Italic">Micrococcus</Emphasis> sp. isolated from the South China Sea

Protease is wildly used in various fields, such as food, medicine, washing, leather, cosmetics and other industrial fields. In this study, an alkaline protease secreted by Micrococcus NH54PC02 isolated from the South China Sea was purified and characterized. The growth curve and enzyme activity curve indicated that the cell reached a maximum concentration at the 30th hour and the enzyme activity reached the maximum value at the 36th hour. The protease was purified with 3 steps involving ammonium sulfate precipitation, ion-exchange chromatography and hydrophobic chromatography with 8.22-fold increase in specific activity and 23.68% increase in the recovery. The molecular mass of the protease was estimated to be 25 kDa by SDS-PAGE analysis. The optimum temperature and pH for the protease activity were 50°C and pH 10.0, respectively. The protease showed a strong stability in a wide range of pH values ranging from 6.0–11.0, and maintained 90% enzyme activity in strong alkaline environment with pH 11.0. Inhibitor trials indicated that the protease might be serine protease. But it also possessed the characteristic of metalloprotease as it could be strongly inhibited by EDTA and strongly stimulated by Mn²⁺. Evaluation of matrix-assisted laser desorption ionization/time-of-flight MS (MALDI-TOF-TOF/MS) showed that the protease might belong to the peptidase S8 family.     

Effects of <Emphasis Type="Italic">Trichoderma harzianum</Emphasis> YC459 and soil types on seed germination and seedling growth in rock slope restoration

We carried out experiments with various concentrations of Trichoderma harzianum YC459 in different soil types (forest soil, mixed soil, merchantable soil, and leaf mold soil) to evaluate its effect on seed germination and seedling establishment of four species (Festuca arundinacea Schreb., Dianthus barbatus var. asiaticus Nakai, Lespedeza cyrtobotrya Miq., and Parthenocissus tricuspidata Planch) for rock slope restoration. We also investigated the use of drilled slanted holes on the rock slopes for seedling establishment. The results showed that T. harzianum concentration had significant effects on seed germination, seedling growth, and seedling survival for all the species with different soil types. Seed germination and survival rates peaked at 5% T. harzianum concentration with leaf mold soil and decreased as T. harzianum concentration increased from 5% to 10%. Seedling survival rates of all four species were generally lowest at 0% T. harzianum concentration in all soil types. The height of F. arundinacea and L. cyrtobotrya peaked at 5% T. harzianum concentration whereas that of D. barbatus and P. tricuspidata peaked at 10% T. harzianum concentration. We concluded that 5% T. harzianum concentration with leaf mold soil is appropriate for seed germination and seedling survival rates of most species, thus enhancing seedling establishment. Practical application of the findings of this study will contribute in the vegetation restoration of steep rocks in mountain environments     

Regenerative Role of Soil Seed Banks of Different Successional Stages in A Saline-alkaline Grassland in Northeast China

Soil seed banks can act as a potential seed source for natural revegetation and restoration. However, in a saline-alkaline grassland, it remains unclear how the stages of vegetation succession affect the characteristics of soil seed banks and the potential of soil seed banks of different successional stages for vegetation restoration. In this study, seasonal changes of the soil seed bank, and seed production and dispersal dynamics along degradation successional gradients were investigated in a saline-alkaline grassland in Northeast China, where the dominant grass during the 1960 s, Leymus chinensis was replaced with the secondary successional order of Puccinellia chinampoensis, Chloris virgata, and Suaeda salsa, together with bare patches. It was found that the soil seed bank composition varied according to the changing vegetation and had the highest species richness(7–16) in the climax successional stage, but had a low S?rensen similarity(0.22–0.37) with the aboveground vegetation. There was a high seed density of the soil seed bank(21 062–62 166/m2 in August and December) and also high S?rensen similarity index values(0.47–0.60) in the secondary successional stages of P. chinampoensis, C. virgata, and S. salsa. In bare patches, there were many seeds in the soil seed bank and some seedlings also appeared in the aboveground vegetation, indicating the existence of a persistent soil seed bank. Seed density and species richness differed substantially among the different successional stages, which was related to the reproductive characteristics of the standing plants in vegetation communities. Due to the lack of propagules of perennial species, especially the climax species of L. chinensis, in the soil, the successful restoration of the degraded saline-alkaline grassland was not possible. The study proved that in a degraded saline-alkaline grassland dominated by biennial or annual species, the soil seed bank was important for the revegetation of the current dominant plants, but not for the restoration of the original target species. Therefore, it is necessary to induce seeds or other propagules of the target perennial species.     

Ecological Network Analysis Quantifying the Sustainability of Regional Economies: A Case Study of Guangdong Province in China

To meet the challenge of sustainable development, sustainability must be made. Ecological network analysis(ENA) was introduced in this paper as an approach to quantitatively measure the growth, development, and sustainability of an economic system. The Guangdong economic networks from 1987 to 2010 were analyzed by applying the ENA approach. Firstly, a currency flow network among economic sectors was constructed to represent the Guangdong economic system by adapting the input-output(I-O) table data. Then, the network indicators from the ENA framework involving the total system throughput(TST), average mutual information(AMI), ascendency(A), redundancy(R) and development capacity(C) were calculated. Lastly, the network indicators were analyzed to acquire the overall features of Guangdong's economic operations during 1987–2010. The results are as follows: the trends of the network indicators show that the size of the Guangdong economic network grows exponentially at a high rate during 1987–2010, whereas its efficiency does not present a clear trend over its whole period. The growth is the main characteristic of the Guangdong economy during 1987–2010, with no clear evidence regarding its development. The quantitative results of the network also confirmed that the growth contributed to a great majority of the Guangdong economy during 1987–2010, whereas the development's contribution was tiny during the same period. The average value of the sustainability indicator(α) of the Guangdong economic network was 0.222 during 1987–2010, which is less than the theoretically optimal value of 0.37 for a sustainable human-influenced system. The results suggest that the Guangdong economic system needs a further autocatalysis to improve its efficiency to support the system maintaining a sustainable evolvement.     

Shrimp arginine kinase being a binding protein of WSSV envelope protein VP31

Viral entry into the host is the earliest stage of infection in the viral life cycle in which attachment proteins play a key role. VP31 (WSV340/WSSV396), an envelope protein of white spot syndrome virus (WSSV), contains an Arg-Gly-Asp (RGD) peptide domain known as a cellular attachment site. At present, the process of VP31 interacting with shrimp host cells has not been explored. Therefore, the VP31 gene was cloned into pET30a (+), expressed in Escherichia coli strain BL21 and purified with immobilized metal ion affinity chromatography. Four gill cellular proteins of shrimp (Fenneropenaeus chinensis) were pulled down by an affinity column coupled with recombinant VP31 (rVP31), and the amino acid sequences were identified with MALDI-TOF/TOF mass spectrometry. Hemocyanin, beta-actin, arginine kinase (AK), and an unknown protein were suggested as the putative VP31 receptor proteins. SDS-PAGE showed that AK is the predominant binding protein of VP31. An i n vitro binding activity experiment indicated that recombinant AK’s (rAK) binding activity with rVP31 is comparable to that with the same amount of WSSV. These results suggested that AK, as a member of the phosphagen kinase family, plays a role in WSSV infection. This is the first evidence showing that AK is a binding protein of VP31. Further studies on this topic will elucidate WSSV infection mechanism in the future.