Major, minor and trace-elemental contents analysis in northeast soybeans by ICP-AES

Major, minor and trace elemental contents in northeast China soybeans were determined by using inductively, coupled plasma atomic emission spectrometry (ICP-AES). Three different sample digestion methods including two wet digestions, HNO3-HClO4 and HNO3-H2SO4 and a dry ash method were compared. Owing to the high oil content in soybeans, long time is needed and access acid should be added, with mixed acid digestion methods, which may result in higher sample blank. Therefore, the dry ask method would be more proper for the pre-treatment of soybean samples. Potassium and phosphorus are major elements in soybeans, so the effect of potassium and phosphorus on the other elements was investigated. Results showed that the potassium and phosphorus did not affect the determination. of other trace elements. There are not significant differences in trace elemental contents for the eleven northeast China soybeans.

Preparation and mass spectrometric study of egg yolk antibody (IgY) against rabies virus

Rabies virus was used as the antigen to immunize laying chickens. Anti-rabies virus immunoglobulin Y(IgY) was isolated from yolks of the eggs laid by these chickens using a two-step salt precipitation and one-step gel filtration protocol. The purified IgY was reduced with dithiothreitol, and heavy chains (HC) and light chains (LC) were obtained. In addition, the purified IgY was digested with pepsin and the fragment with specific antigen binding properties (Fab) was produced. Using matrix-assisted laser desorption/ionization mass spectrometry (MALDI-TOFMS), the average molecular weights of IgY, HC, LC, and Fab were determined as 167 250, 65 105, 18 660, and 45,359 Da, respectively. IgY has two structural differences compared with mammalian IgGs. First, the molecular weight of the heavy chain of IgY is larger than that of its mammalian counterpart, while the molecular weight of the light chain of IgY is smaller. Second, upon pepsin digestion, anti-rabies virus IgY is degraded into Feb, in contrast to mammalian IgG, which has been reported to be degraded into F(ab')(2) under the same conditions. Copyright (C) 2001 John Wiley & Sons, Ltd.

Determination of rare earth elements in biological samples by inductively coupled plasma mass spectrometry

A method for the determiantion of rare earth elements in biological sampels by inductively coupled plasma mass spectrometry was developed. Oxide ion yield of the rare earth elements (RFE) decreased with the increasing of RF power and the sampling depth, or with the decreasing of carrier gas flow rate. The spectral interference arising from (PrO)-Pr-141-O-16 on Gd-157 must be corrected. if the concentration of Ba was high enough, it was necessary to correct the spectral interference arising from (BO)-B-135-O-16 on Eu-151, and it was not necessary to correct spectral interference arising from (NdO)-Nd-143-O-16 on Tb-159 etc. in the biological samples under the selected operation parameters. In the biological sample, the major matrix elements, such as K, Na and Ca, result in the suppression of REEs signals and the suppression degree of the Ca is grezter than that of the K and Na. The mussel sample was digested by thd dry ashing, wet digestion with HNO3 + H2O2 and HNO3 + HClO4, respectively. The analytical results of REEs were consistent with each other. Detection limits for REEs are 0.001 similar to 0.013 mu g/L. Recoveries of standard addition are 91.7% similar to 125%. REEs in biological samples were determined directly without separation and preconcentration procedure.

Analysis of mussel standard reference material by inductively coupled plasma atomic emission spectrometry and inductively coupled plasma mass spectrometry

A method for the analysis of mussel standard reference material by inductively coupled plasma atomic emission spectrometry( ICP-AES) and inductively coupled plasma mass spectrometry(ICP-MS) was developed. K, Na, Ca, Mg, P, Al, Fe, Zn, Mn and Sr were determined by ICP-AES and As, B, Cd, Co, Cr, Cu, Ga, Ge, Mn, Mo, Ni, Pb, Se, Sr, U and V by ICP-MS, The interference coefficients at the Mn-55, Se-78, Cu-63, Co-59, Ni-58, Ni-60, As-75, Se-77, V-51, Cr-53 and Cr-52 originating from polyatomic ion of the matrix elements (KO)-K-39-O-16, K-39(2), (ArNa)-Ar-40-Na-23, (CaO)-Ca-43-O-16, (CaO)-Ca-42-O-16, (CaO)-Ca-44-O-16, (PO2)-P-31-O-16, (ArCl)-Ar-40-Cl-35, (ArCl)-Ar-40-Cl-37, (ClO)-Cl-35-O-16, (ClO)-Cl-37-O-16 and (ArC)-Ar-40-C-12 were determined under the selected operation parameters. The major matrix elements, such as K, Na and Ca, result in the suppression of analytes signals. The apparent concentration at the significant biological element which was produced by the different digestion methods, (.) HNO3 + H2O2 (3 + 2), HNO3 + HClO4 (3 + 0.5) and HNO3 + H2SO4 (3 + 0.5),was determined. The sample digested by HNO3 + H2O2 did not give rise to interfere on the analyte, and the backgrounds of Se-77, Ga-69, Zn-67, As-75, V-51, Cr-53 and Cr-52 were increased by HNO3 + HClO4 digestion method, that affected the determination of these elements, especially the monoisotope As and V. Sample digested by HNO3 + H2SO4 increased the backgrounds at Cu-65, Zn-64 and Zn-67. Detection limits of ICP-AES are 0.001 similar to 0.75 mg/L and those of ICP-MS are 0.005 similar to 1.01 mu g/L. The relative standard derivations of ICP-AES and ICP-MS are 2.7% similar to 12.8%, 3.4% similar to 24.8%, respectively.

A simple method for DNA extraction from sporophyte in the brown alga Laminaria japonica

A simple method was developed for extracting DNA from brown algae Laminaria japonica, which possess large amounts of acidic polysaccharides. Firstly, the sporophyte were washed by eliminating polysaccaride buffer to remove the polysaccharides and then ground in liquid nitrogen. Secondly, the powders were treated with lysing buffer. Thirdly, KAc was used to eliminate the remaining acidic polysaccharides. The extracted DNA was purified using a chloroform-isoamyl alcohol ( 24: 1 v/v), and precipitated in cold isopropanol. The yield was from 18.7 to 37.5 mu g g(-1) (wet weight) and the purity of total DNA was determined spectrophotometrically as the ratio of A(260)/A(280), which was about 1.7 - 1.9. The extracted DNA was of high quality and suitable for molecular analyses, such as PCR, restriction enzyme digestion. This method is a reproducible, simple, and rapid technique for routine DNA extraction from sporophyte in Laminaria japonica. Furthermore, the low cost of this method makes it attractive for large-scale studies.

An efficient method for DNA isolation from red algae

A simple, inexpensive and efficient method was developed for rapid isolation of total genomic DNA from 15 red algal species. It resulted in 0.1 mug high quality DNA from 1 mg fresh algal material, with an A(260)/A(280) ratio of 1.68 - 1.90. Using this rapidly isolated DNA, the 18S ribosomal RNA genes ( rDNA) and the nuclear ribosomal DNA of the internal transcribed spacer (ITS) regions were amplified. The tested DNA was suitable for restriction endonuclease digestion, genetic marker analysis and polymerase chain reaction (PCR) amplification, and may be valid for other genetic manipulation.

Large-scale recovery of C-phycocyanin from Spirulina platensis using expanded bed adsorption chromatography

C-phycocyanin was purified on a large scale by a combination of expanded bed adsorption, anion-exchange chromatography and hydroxyapatite chromatography from inferior Spirulina platensis that cannot be used for human consumption. First, phycobiliproteins were extracted by a simple, scaleable method and then were recovered by Phenyl-Sepharose chromatography in an expanded bed column. The purity (the A(620)/A(280) ratio) of C-phycocyanin isolated with STREAMLINE (TM) Column was up to 2.87, and the yield was as high as 31 mg/g of dried S. platensis. After the first step, we used conventional anion-exchange chromatography for the purification steps, with a yield of 7.7 mg/g of dried S. platensis at a purity greater than 3.2 and with an A(620)/A(650) index higher than 5.0. The fractions from anion-exchange chromatography with a level of purity that did not conform to the above standard were subjected to hydroxyapatite chromatography, with a C-PC yield of 4.45 mg/g of dried S. platensis with a purity greater than 3.2. The protein from both purification methods showed one absolute absorption peak at 620 nm and a fluorescence maximum at 650 nm, which is consistent with the typical spectrum of C-phycocyanin. SDS-PAGE gave two bands corresponding to 21 and 18 kDa. In-gel digestion and LC-ESI-MS showed that the protein is C-phycocyanin. (c) 2006 Elsevier B.V. All rights reserved.

Molecular cloning and functional analysis of cathepsin B in nutrient metabolism during larval development in Meretrix meretrix

Seed rearing is an important part in large scale clam culture industry. Since the nutritional history affects early development in bivalve, the condition of larval nutrition plays a key role in successful seed rearing. So far, the molecular mechanism of nutrient uptake in bivalve larvae is unclear. As one of the important proteolytic enzymes, cathepsin B of several organisms has been reported to be involved in digestion. We intended to analyze whether cathepsin B is involved in larval nutrient metabolism in the economic bivalve, clam Meretrix meretrix. The full length of M. meretrix cathepsin B (MmeCB) cDNA was cloned, which is 1647 bp with an open reading frame of 1014 bp. The deduced amino acid sequence encoded a preproenzyme of 337 residues with Cys-114, His-282 and Asn-302 composing cathepsin B activity center. The temporal and spatial expressions of MmeCB mRNA were examined from trochophore to post larva stages by whole mount in situ hybridization. In trochophore stage, no detectable signal was found. In the later three stages, MmeCB mRNA was detected in the digestive gland, suggesting a possible role of MmeCB in digestion. Moreover, MmeCB mRNA was also observed in the epidermal cells in D-veligers. Cathepsin B specific inhibitor (CA074 methyl ester) was applied to block the activity of cathepsin B in unfed larvae. The average shell lengths of treated larvae were smaller than that in control groups. The results of mRNA epidermal distribution and inhibitor treatment in D-veligers indicated that MmeCB may be also associated with other pathway of nutrient metabolism in larval epidermis. The overall results in this paper revealed that MmeCB might play a role in larval nutrient metabolism. (C) 2008 Elsevier B.V. All rights reserved.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondria 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitocliondrial 12S rRNA gene, which were about 450 bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR amplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, L. erythopterus from L. argentintaculatus, L. malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for differentiation of L. sattguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The seminested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Published by Elsevier Ltd.

Biosynthesis of fluorescent cyanobacterial allophycocyanin trimer in Escherichia coli

Allophycocyanin (APC), a cyanobacterial photosynthetic phycobiliprotein, functions in energy transfer as a light-harvesting protein. One of the prominent spectroscopic characteristics of APC is a strong red-shift in the absorption and emission maxima when monomers are assembled into a trimer. Previously, holo-APC alpha and beta subunits (holo-ApcA and ApcB) were successfully synthesized in Escherichia coli. In this study, both holo-subunits from Synechocystis sp. PCC 6803 were co-expressed in E. coli, and found to self-assemble into trimers. The recombinant APC trimer was purified by metal affinity and size-exclusion chromatography, and had a native structure identical to native APC, as determined by characteristic spectroscopic measurements, fluorescence quantum yield, tryptic digestion analysis, and molecular weight measurements. Combined with results from a study in which only the monomer was formed, our results indicate that bilin synthesis and the subsequent attachment to apo-subunits are important for the successful assembly of APC trimers. This is the first study to report on the assembly of recombinant ApcA and ApcB into a trimer with native structure. Our study provides a promising method for producing better fluorescent tags, as well as a method to facilitate the genetic analysis of APC trimer assembly and biological function.

Identification and molecular characterization of a peritrophin-like protein from fleshy prawn (Fenneropenaeus chinensis)

Peritrophin, one of the components of the peritrophic matrix, was first isolated from the intestine of insects. It is thought to protect insects from invasion of microorganisms and to stimulate digestion of food. Peritrophin-like proteins have also been found in crustaceans, as a component of the egg layer. In this study, one fragment of the peritrophin-like gene was obtained from fleshy prawn (Chinese shrimp) (Fenneropenaeus chinensis) by panning the T7 phage display library constructed with the shrimp hemocyte cDNA. The total sequence of the peritrophin cDNA was cloned by modified SMART cDNA and LD-PCR methods. The full cDNA is 1048 bp and the deduced protein is composed of 274 amino acids, including 21 amino acid signal peptide, and four peritrophin A domains and the latter three forming three chitin-binding domains. Similarity analysis results showed that the peritrophin-like protein from F chinensis has significant similarities with peritrophin-like and cortical rod proteins from other shrimp. It was inducing expression in hemocytes, heart, stomach, gut, and gills of the infected shrimp, and constitutive expression in the ovaries. No expression signal was detected in the hepatopancreas of either infected or noninfected shrimp. The recombinant peritrophin-like protein has the activity of binding Gram-negative bacteria and strong binding activity to chitin. Therefore, the bacteria and chitin binding activities of the peritrophin-like protein suggest that it may plays a role in immune defense and other physiological resposes. (c) 2005 Elsevier Ltd. All rights reserved.

Cloning, expression and identification of ferritin from Chinese shrimp, Fenneropenaeus chinensis

Ferritin, the iron storage protein, plays a key role in iron metabolism. A cDNA encoding ferritin (FcFer) was cloned from hepatopancreas of Chinese shrimp, Fenneropenaeus chinensis. The predicted protein contains 170 amino acid residues with a predicted molecular weight (MW) about 19, 422.89 Da and theoretical isoelectric point (PI) of 4.73. Amino acid alignment of FcFer revealed 97% homology with Litopenaeus vannamei ferritin. Results of the RT-PCR showed that the expression of FcFer mRNA was up-regulated after shrimp was challenged with either white spot syndrome virus (WSSV) or heavy metal ions (Zn2+ and Cu2+) in the laboratory. A fusion protein containing FcFer was produced and the purified recombinant protein exhibited similar function of iron uptake in vitro. The result of in-gel digestion and identification using LC-ESI-MS showed that two peptide fragments (-DDVALPGFAK- and -LLEDEYLEEQVDS1KK-) of the recombinant protein were identical to the corresponding sequence of L. vannamei ferritin. The recombinant FcFer protein will be proved useful for study on the structure and function of ferritin in F chinensis. (c) 2006 Elsevier B.V. All rights reserved.

Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Assembly of the protoplasm of Codium fragile (Bryopsidales, Chlorophyta) into new protoplasts

The cell organelles of the coenocytic alga Codium fragile (Sur.) Hariot aggregated rapidly and protoplasts were formed when its protoplasm was extruded out in seawater. Continuous observation showed that there were long and gelatinous threads connecting the cell organelles. The threads contracted, and thus the cell organelles aggregated into protoplasmic masses. The enzyme digestion experiments and Coomassie Brilliant Blue and Anthrone stainings showed that the long and gelatinous threads involved in the formation of the protoplasts might include protein and saccharides as structure components. Nile Red staining indicated that the protoplast primary envelope was non-lipid at first, and then lipid materials integrated into its surface gradually. The fluorescent brightener staining indicated that the cell wall did not regenerate in the newly formed protoplasts and they all disintegrated within 72 h after formation. Transmission electron microscopy of the cell wall of wild C. fragile showed electron-dense material embedded in the whole cell wall at regular intervals. The experiments indicated that C. fragile would be a suitable model alga for studying the formation of protoplasts.

鮸（Miichthys miiuy）早期生长存活过程和消化生理机制的研究

鮸Miichthys miiuy是中国南方重要的经济养殖种类，早期发育阶段的高死亡率和高畸形率严重制约着鮸养殖的工业化和商品化进程。为了更好的了解鮸早期发育规律，提高其人工养殖技术和管理策略，本论文在实验条件下对鮸早期生长和存活过程、消化生理发生机制及其在胁迫条件下的生态对策进行了研究。 鮸早期发育阶段分为仔鱼期和稚鱼期。个体发育的形态学变化和组织器官分化主要集中在仔鱼期，胚胎期所具有的特征在仔鱼期被具体的功能系统所代替，如呼吸、摄食、运动和消化系统。鮸个体的头、尾部快于躯干部的生长，表明其早期发育过程中摄食和运动器官发育的优先性。鱼类早期是优先发育对其生存起首要作用的器官，然后是对其生存作用次之的器官。 在24℃，鮸仔鱼在3日龄开口摄食，4日龄卵黄囊吸收完毕，6日龄仔鱼如不能建立外源性营养即进入饥饿死亡不可逆点(PNR)，而延迟投饵3天（6日龄）以上的仔鱼在8日龄全部死亡。在7日龄时，正常投喂仔鱼的SGR明显高于延迟投饵仔鱼。延迟3天和饥饿的仔鱼在6日龄后出现明显的负增长。在36日龄时，正常投喂仔鱼与延迟投饵1天仔鱼之间的生长差异消失，与延迟投饵2天的仔鱼之间的生长差异显著。饥饿显著影响开口仔鱼的生长存活，但对后期存活仔鱼的生长存活及体成分的影响不显著。仔鱼在12L条件下存活率（9-16%）要小于其他的各组（12-39%），仔鱼的存活率随着密度的增加逐渐减小。SGR与存活率具有相同的变化趋势，在18L条件下仔鱼的生长（3-10%）要大于12L（2-7%）和24L（2.8-8.7%）条件下仔鱼的生长。 鮸消化系统发育分为三个阶段：从孵化到初次摄食之前，消化道为一细长管道；从外源营养开始到胃腺出现，在此阶段，液泡、杯状细胞开始出现在消化道中，在6日龄胃出现，消化道明显分为口咽腔、食管、胃、前肠和后肠；从20日龄胃腺出现开始，胃在25日龄分为贲门胃、胃体和幽门胃三个部分，幽门盲囊出现，消化系统趋于完善。饥饿严重影响鮸消化器官及消化腺的正常发育过程。饥饿使其组织学的结构和功能明显衰退，肝组织变得疏松，细胞缩小，细胞核解体，稚鱼肝组织内没有脂质积累，细胞质中液泡减少；胰脏组织变得致密，腺泡萎缩，分泌物减少；胃腺细胞收缩，结构不完整，肠微绒毛退化，肠粘膜褶减少，肠上皮细胞遭到破坏。 鮸主要消化酶分为三个发育时期：从初孵到外源营养开始的迅速增长期；继而是从3日龄到25日龄波动期；25日龄后处于相对稳定期。各种消化酶总活性随着个体的发育逐渐增加，30日龄后总活性显著增加。延迟1天投饵的仔鱼和正常摄食的仔鱼其消化酶的活性没有显著差异（P>0.05），但是延迟2天投饵的仔鱼和正常摄食及延迟1天投饵的仔鱼消化酶的活性存在显著差异(P<0.05)，无论是延迟1天还是延迟2天仔鱼消化酶与正常摄食的仔鱼具有相同的发育方式。延迟3天投饵的仔鱼和饥饿仔鱼发育方式类似，从3日龄开始，消化酶活性显著下降。 饥饿对鮸仔稚鱼胰蛋白酶、淀粉酶和脂肪酶的活性有显著的影响。在初次摄食期，摄食仔鱼和饥饿2天的仔鱼各种消化酶活性均存在显著差异（P<0.05）；但是仔鱼后期，经过3天饥饿的仔鱼其消化酶的活性和摄食仔鱼之间存在显著差异（P<0.05），饥饿4天的仔鱼恢复摄食5天，即可恢复到和摄食仔鱼相同水平。饥饿6天的鮸稚鱼其消化酶与正常摄食的稚鱼之间存在显著差异（P<0.05），在恢复摄食5天后其消化酶活性可以恢复到和正常摄食仔鱼相同水平。 光照和饲养密度显著影响了鮸消化酶的活性。胰蛋白酶、淀粉酶和脂肪酶在18L低密度饲养条件下其活性明显高于（P<0.05）其他各组；12L高密度饲养条件下各种消化酶活性明显低于（P<0.05）其他各组；在0L条件下，各种消化酶活性在开口摄食之前明显低于其他各组，开口摄食之后，各种消化酶活性迅速下降。延迟投饵、光照周期和饲养密度虽然对鮸消化酶的活性产生了重要影响，但是对消化酶的发育方式没有显著影响，因此，鮸消化酶的具体发育方式是由内部遗传机制来控制，但受外界环境因素（食物组成、光照和饲养密度）的调节。