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.     

Proteomic aspects of infection by lymphocystis disease virus in Japanese flounder (Paralichthys olivaceus)

To reveal the key factor in self-healing from LCDV (lymphocystis disease virus)-infec Japanese flounder (Paralichthys olivaceus), serum proteins from self-healing and sick Japanese floun were separated by two-dimensional electrophoresis to screen differentially expressed proteins. Prot spots demonstrating changes greater than two-fold in the expression level were digested and furt identified in capillary liquid chromatography tandem mass spectrometry (LC-MS/MS). Two immuni relevant proteins were thus identified as transferrin and the complement component C3 of Japan flounder. These findings suggest that the two proteins may play important roles in the self-healing lymphocystis in Japanese flounder. This is an important theoretical foundation to promote self-healing LCDV-infected Japanese flounder by improving their innate immunity.     

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.     

Optimization of the purification methods for recovery of recombinant growth hormone from Paralichthys olivaceus

This study aimed to optimize the purification of recombinant growth hormone from Paralichthys olivaceus. Recombinant flounder growth hormone (r-fGH) was expressed by Escherichia coli in form of inclusion body or as soluble protein under different inducing conditions. The inclusion body was renatured using two recovery methods, i.e., dilution and dialysis. Thereafter, the refolded protein was purified by Glutathione Sepharase 4B affinity chromatography and r-fGH was obtained by cleavage of thrombin. For soluble products, r-fGH was directly purified from the lysates by Glutathione Sepharase 4B affinity chromatography. ELISA-receptor assay demonstrated that despite its low receptor binding activity, the r-fGH purified from refolded inclusion body had a higher yield (2.605 mgL-1) than that from soluble protein (1.964 mgL-1). Of the tested recovery methods, addition of renaturing buffer (pH 8.5) into denatured inclusion body yielded the best recovery rate (17.9%). This work provided an optimized purification method for high recovery of r-fGH, thus contributing to the application of r-fGH to aquaculture.     

C_(17)-fengycin B,produced by deep-sea-derived Bacillus subtilis,possessing a strong antifungal activity against Fusarium solani

Root rot disease caused by Fusarium solani is the most devastating disease of the tomato and legume crops in China.The metabolites of Bacillus species can inhibit many fungal diseases.In this study,the metabolites of deep-sea-derived bacterium Bacillus subtilis 2 H11 can significantly inhibit the growth of F.solani.The metabolite C_(17)-fengycin B,one of the cyclic lipopeptides,was identified by the combination of silica column chromatography,high-performance liquid chromatography(HPLC),high-energy collision induced dissociation mass spectrometry(HCD-MS) and tandem mass spectrometry(HCD-MS/MS).The results of scanning electron microscopy(SEM) and transmission electron microscopy(TEM) showed that C_(17)-fengycin B could destroy the structure of the hyphae and spores of F.solani.The antifungal activities of C_(17)-fengycin B against F.solani were tested at concentrations ranging from 0.05 mg/mL to 0.20 mg/mL.The results indicated that C_(17)-fengycin B inhibited the growth of F.solani with antifungal index of 89.80%at 0.20 mg/mL,and the antifungal activity of C_(17)-fengycin B was further verified by the pot experiment.In addition,the cytotoxicity experiment showed that C_(17)-fengycin B had good biocompatibility and was a potential candidate for the development of biocontrol pesticide in the future.     

Gene cloning and prokaryotic expression of recombinant flagellin A from Vibrio parahaemolyticus

The Gram-negative Vibrio parahaemolyticus is a common pathogen in humans and marine animals. Bacteria flagellins play an important role during infection and induction of the host immune response. Thus, flagellin proteins are an ideal target for vaccines. We amplified the complete flagellin subunit gene (flaA) from V. parahaemolyticus ATCC 17802. We then cloned and expressed the gene into Escherichia coli BL21 (DE3) cells. The gene coded for a protein that was 62.78 kDa. We purified and characterized the protein using Ni-NTA affinity chromatography and Anti-His antibody Western blotting, respectively. Our results provide a basis for further studies into the utility of the FlaA protein as a vaccine candidate against infection by Vibrio parahaemolyticus. In addition, the purified FlaA protein can be used for further functional and structural studies.     

THE INDUCTION AND EXTRACTION OF METALLOTHIONEINS IN ARTEMIA

A small amount of heavy metal binding protein,identified by BioRad Protein Assay,has been iso-lated from the adult brine shrimp,Artemia franciscanus.This protein has an apparent molecular weight be-tween 6000 to 9000 dalton.a UV absorption peak at 260 instead of 280 nm like most proteins;andhas high affinity towards binding with radioactive labeled ~(109)Cd.These characteristics are similarto that of metallothioneins reported for many vertebrate and invertebrate,marine and terrestrialanimals.After the brine shrimp is exposed to a small amount of Cd~(2+)for 24 h,a large amount ofmetallothionein can be isolated,showing the inducibility of this detoxifying protein in the adult Artemiain a short period of time.     

Economic and Qualitative Traits of Italian Alps Saffron

Saffron, obtained from the flower stigmas of Crocus sativus L., is one of the most expensive food spices. The introduction of saffron in alpine areas could help to broaden and diversify the activities of mountain multifunctional farms, with a positive impact on economy and land management. According to ISO 3632(2010/2011), saffron can be classified into three categories of quality(I, II, III) depending on the concentration of the three main metabolites responsible for its characteristic colour, flavor and aroma: Crocin, Picrocrocin and Safranal. This study represents the first investigation of the quality of saffron produced in the Italian Alps evaluated with spectrophotometry, HPLC, solid-phase microextraction(SPME), and gas chromatographic analysis combined with mass spectrometry(GC/MS). The experiments used Crocus sativus stigmas produced in 2012-2013 in different areas of the Central Italian Alps were located at an altitude between 720 and 1200 m a.s.l.. Results obtained were compared to commercial saffron. The analyses confirmed that all samples can be classified in the first quality category according to the ISO classification. This high quality is also confirmed by HPLC analysis. Moreover, the SPME-GC/MS analysis identified some differences in the aromatic profile of saffron samples, in particular regarding safranal concentration. A preliminary assessment of the economic viability of high quality saffron production for local markets was also performed. Our study provides valid information regarding the quality and economic sustainability of saffron production in the alpine area confirming this crop as a good candidate for a new source of income for multifunctional farms in mountain areas.     

The induction and extraction of metallothioneins inArtemia

A small amount of heavy metal binding protein, identified by BioRad Protein Assay, has been isolated from the adult brine shrimp,Artemia franciscanus. This protein has an apparent molecular weight between 6000 to 9000 dalton. a UV absorption peak at 260 instead of 280 nm like most proteins; and has high affinity towards binding with radioactive labeled¹⁰⁹Cd. These characteristics are similar to that of metallothioneins reported for many vertebrate and invertebrate, marine and terrestrial animals. After the brine shrimp is exposed to a small amount of Cd²⁺ for 24 h, a large amount of metallothionein can be isolated, showing the inducibility of this detoxifying protein in the adultArtemia in a short period of time.     

Prevalence and risk assessment of antibiotics in riverine estuarine waters of Larut and Sangga Besar River,Perak

Antibiotics released into the environment through anthropogenic activities exert selective pressure,driving bacteria towards increasing antimicrobial resistance.The prevalence of antibiotics and the ecological risks posed in the riverine estuarine of Larut River and Sangga Besar River,which included wastewater effluents from hospital,zoo,and poultry slaughterhouse sources were investigated.Solid phase extraction(SPE) followed by high-performance liquid chromatography tandem mass chromatography(HPLC-MS/MS) were used to extract and quantify the antibiotic residues from 22 antibiotics belonging to six major antibiotic classes(sulfonamide,macrolide,fluoroquinolone,phenicol,trimethoprim,and tetracycline).Sixteen antibiotic residues were detected with concentrations ranging from limit of detection(LOD) to 1 262.3 ng/L.Fluoroquinolones and macrolides were the most frequently detected compounds.Erythromycin,clarithromycin and ofloxacin detected in hospital and zoo effluents posed a high risk to algae while tetracycline had low to medium ecological risks toward all the relevant organisms from aquatic environments(algae,invertebrate Daphnia magna,and fish).     

Purification and Refolding of a Novel β-Agarase from Inclusion Body of E. coli

β-agarase AgaB appears to represent a new family of glycoside hydrolase; it is structurally and functionally different from other known agarases. In the present study, AgaB was expressed with a temperature-inducible expression system in E. coli BL21 (DE3) as a fusion protein bearing a C-terminal hexahistidine tag. The protein existed mainly in the form of inclusion body.After being washed and solubilized, AgaB in inclusion body was denatured and purified to electrophoretic purity by immobilized metal affinity chromatography. The purified AgaB was then refolded using a simple pulse dilution method, and the refolded AgaB showed a high specific hydrolysis activity of about 1600 units/mg protein. Forty milligrams of refolded pure protein were obtained from 1L of culture.     

UP-REGULATION OF HEPATIC RECEPTOR FOR GROWTH HORMONE IN THE FLOUNDER （PARALICHTHYS OLIVACEUS） AFTER ORAL ADMINISTRATION WITH EXOGENOUS GH

The iodination efficiency of salmon GH (sGH) was 38.82%,using a modification of the chloramine-T method. The specific activity of the ^125I-sGH was about 40 μCi/μg protein. The results of binding assay showed a single class of high affinity and low-capacity binding site in flounder liver. Long-term administration with exogenous GH can induce the up-regulation of hepatic GH receptor in total binding capacity though there was no significant difference of association constant among any groups. Con-sidering that there was no significant difference in capacity of free binding sites of livers from control and experimental fish, this result also indicated that the liver from experimental fish, compared to that from control fish, had more occupied binding sites.     

淋巴囊肿病毒中国分离株基因组序列和结构分析

LCDV-cn是分离自中国养殖牙鲆的淋巴囊肿病病原,给中国牙鲆养殖业带来了严重危害。通过PCR、克隆和测序,获得了LCDV-cn部分基因的序列,证明LCDV-cn与LCDV-C的基因组序列是一致的。比较基因组学分析发现,LCDV-cn基因组中有137个ORF是其独有的、与LCDV-1及其它虹彩病毒无同源区域。对LCDV-cn基因组进行生物信息学分析发现,在LCDV-cn独有的ORF中,有的编码与宿主同源的蛋白,有的编码与DNA复制相关的蛋白,某些ORF编码的蛋白含有跨膜区等。LCDV-cn的独有基因或许对其毒力和宿主范围有关。     

Isolation and pharmacological activities of bromophenols from Rhodomela confervoides

Four bromophenols were isolated from the extract of marine red alga, Rhodomela confervoides by column chromatography and HPLC methods. By means of spectroscopic methods including IR, MS, 1D, and 2D-NMR techniques, their structures were elucidated as (1) 3-bromo-4,5-dihydroxy benzoic acid methyl ester; (2) his (2,3-dibromo-4,5-dihydroxybenzyl) ether; (3) 3,4-dibromo-5-(methoxymethyl)-1,2-benzenediol and (4)3-bromo-4,5-dihydroxy-benzaldehyde. Compound 1 was first isolated from the algae in nature, and 1, 4 were found to have selective cytotoxic activities against KB, Bel 7402 and A549 cells, 2 showed powerful antibacterial activities against Staphylococcus aurens and Pseudomonas aeruginosa.     

Structural analysis and anti-complement activity of polysaccharides extracted from Grateloupia livida (Harv.) Yamada

Polysaccharides were extracted from Grateloupia livida(Harv.) Yamada using hot water(extracted product denoted WGW) and then degraded in dilute sulfuric acid(degraded product denoted WGWD). The degraded mixture was then separated into four fractions through anion exchange chromatography on 2-diethylaminoethanol(DEAE)-Bio-Gel Agarose FF gel. Electrospray ionization collision-induced dissociation tandem mass spectrometry(ESI-CID-MS/MS) was performed to elucidate the structural features of all fractions. In combination with nuclear magnetic resonance spectroscopy(NMR)and infrared spectroscopy(IR) data, the major polysaccharide structures were concluded to be μ-carrageenan and κ-carrageenan. μ-Carrageenan usually has a backbone of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-6, while κ-carrageenan consists of alternating 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-3,6-anhydrogalactopyranose residues. Trace v-carrageenan, composed of 1,3-linked β-D-galactopyranose residues sulfated at C-4 and 1,4-linked a-D-galactopyranose residues sulfated at C-2 and C-6, was also detected. Furthermore, the polysaccharide had a backbone comprising 1,3-linked β-D-galactopyranose and1,4-linked α-L-galactopyranose sulfated at C-6, which is the agarose precursor. The hydroxy groups in the galactopyranose were partially substituted by methyl and pyruvic acid acetal(PA) groups. The anticomplementary activities of WGW and its derivatives against classical pathways were measured. The native polysaccharides in WGW had higher activities, while the derivatives had much weaker activities. This indicated that the molecular weight and sulfate content were important factors affecting the anti-complement activity.     

镉和铜对海洋浮游植物的毒性效应及其机制研究进展

【目的】综述重金属元素镉Cd和铜Cu对海洋浮游植物的毒性效应研究,总结重金属对海洋浮游植物的毒性作用机制,为开展重金属对海洋浮游植物毒害机理的深入研究提供借鉴。【方法】以毒理实验中常用的重金属镉和铜为代表,总结了重金属对海洋浮游植物毒理研究的常用测试指标,归纳了重金属对海洋浮游植物光合作用、抗氧化系统等方面的毒性作用机制。【结果】重金属对海洋浮游植物毒性作用机制概括为:1)重金属替换与其结构相似的作为酶辅助因子的金属元素,使浮游植物体内某些酶失活;2)重金属直接或间接诱导活性氧的产生,使浮游植物受到氧化胁迫;3)重金属与生物大分子中的某些基团亲和性高而结合,阻断相关的生理生化过程。【结论】重金属的毒性效应因浮游植物种类不同而有所差异,今后相关研究中需更加关注重金属在多种海洋浮游植物共存环境下的毒性效应及机制,将有助于全面系统地理解重金属对海洋浮游植物的致毒机理。     

鱼干曲霉菌分离株鉴定及产毒特性

【目的】探明湛江市售鱼干中曲霉菌(Aspergillus spp.)分离株产AFB₁的特性。【方法】采用马铃薯-葡萄糖琼脂(PDA)对鱼干中的曲霉菌进行分离纯化,根据菌落形态、显微形态观察和ITS序列分析对分离株进行鉴定,采用液相色谱-串联质谱(LC-MS/MS)法分析菌株的AFB₁合成量。【结果】鱼干中曲霉菌的分离率高达47.66%,经形态学鉴定发现以黄曲霉(Aspergillus flavus)A03、烟曲霉(Aspergillus fumigatus)H32、杂色曲霉(Aspergillus versicolor)M23、欧式曲霉(Aspergillus europaeus)M11、棘孢曲霉(Aspergillus aculeatus)B21等为主,其中红笛鲷干(Lutjanus sanguinaus)中黄曲霉菌的含量高达1200 CFU/g。黄曲霉A03在红笛鲷干培养基中常温(27~30℃)培养21 d,产AFB₁能力达到3.8 ng/mL。【结论】鱼干中存在产AFB₁的黄曲霉菌,警示鱼干中存在AFB₁污染的风险。     

光温敏核不育水稻幼穗育性转换期蛋白质组的初步分析

为找寻光温敏核不育水稻幼穗与育性调控有关的蛋白质,采用固相pH梯度-SDS聚丙烯酰胺双向凝胶电泳对光温敏核不育水稻(培矮64S)育性转换期幼穗总蛋白进行了分离,获取凝胶图像并进行重复性分析,通过微量测序-Edman降解方法和原位酶解及MALDI-TOF质谱的方法鉴定了60个蛋白质点。结果表明:本方法可得取分辨率和重复性较好的凝胶图谱。蛋白质点在2D胶上的重复性为:沿等电聚焦方向偏差为1.49±0.22 mm,沿SDS-PAGE方向偏差为1.24±0.18 mm。37个蛋白质点得到了准确的鉴定结果,其中16个是差异蛋白质点。在不育变化为可育的过程中,明显表达上调的蛋白质点包括几丁质酶,丙酮酸脱氢酶等;明显下调的蛋白质包括超氧化物歧化酶,硝酸还原酶脱辅基酶蛋白等;金属硫蛋白RicMT只在可育中存在。