A key gene of the RNA interference pathway in the black tiger shrimp, Penaeus monodon: Identification and functional characterisation of Dicer-1

RNA interference (RNAi) is an evolutionarily conserved mechanism by which double-stranded RNA (dsRNA) initiates post-transcriptional silencing of homologous genes. Here we report the amplification and characterisation of a full length cDNA from black tiger shrimp (Penaeus monodon) that encodes the bidentate RNAase III Dicer, a key component of the RNAi pathway. The full length of the shrimp Dicer (Pm Dcr1) cDNA is 7629 bp in length, including a 51 untranslated region (UTR) of 130 bp, a 3' UTR of 77 bp, and an open reading frame of 7422 bp encoding a polypeptide of 2473 amino acids with an estimated molecular mass of 277.895 kDa and a predicted isoelectric point of 4.86. Analysis of the deduced amino acid sequence indicated that the mature peptide contains all the seven recognised functional domains and is most similar to the mosquito (Aedes aegypti) Dicer-1 sequence with a similarity of 34.6%. Quantitative RT-PCR analysis showed that Pm Dcr1 mRNA is most highly expressed in haemolymph and lymphoid organ tissues (P 0.05). However, there was no correlation between Pm Dcr1 mRNA levels in lymphoid organ and the viral genetic loads in shrimp naturally infected with gill-associated virus (GAV) and Mourilyan virus (P > 0.05). Treatment with synthetic dsRNA corresponding to Pm Dcr1 sequence resulted in knock-down of Pm Dcr1 mRNA expression in both uninfected shrimp and shrimp infected experimentally with GAV. Knock-down of Pm Dcr1 expression resulted in more rapid mortalities and higher viral loads. These data demonstrated that Dicer is involved in antiviral defence in shrimp. (c) 2007 Elsevier Ltd. All rights reserved.

Characterization of proteolytic bacteria from the Aleutian deep-sea and their proteases

Six deep-sea proteolytic bacteria taken from Aleutian margin sediments were screened; one of them produced a cold-adapted neutral halophilic protease. These bacteria belong to Pseudoalteromonas spp., which were identified by the 16S rDNA sequence. Of the six proteases produced, two were neutral cold-adapted proteases that showed their optimal activity at pH 7-8 and at temperature close to 35 degrees C, and the other four were alkaline proteases that showed their optimal activity at pH 9 and at temperature of 40-45 degrees C. The neutral cold-adapted protease E1 showed its optimal activity at a sodium chloride concentration of 2 M, whereas the activity of the other five proteases decreased at elevated sodium chloride concentrations. Protease E1 was purified to electrophoretic homogeneity and its molecular mass was 34 kDa, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The molecular weight of protease E1 was determined to be 32,411 Da by mass spectrometric analysis. Phenylmethyl sulfonylfluoride (PMSF) did not inhibit the activity of this protease, whereas it was partially inhibited by ethylenediaminetetra-acetic acid sodium salt (EDTA-Na). De novo amino acid sequencing proved protease E1 to be a novel protein.

cDNA cloning, characterization and expression analysis of the antioxidant enzyme gene, catalase, of Chinese shrimp Fenneropenaeus chinensis

Catalase is an important antioxidant protein that protects organisms against various oxidative stresses by eliminating hydrogen peroxide. The full-length catalase cDNA of Chinese shrimp Fenneropenaeus chinensis was cloned from the hepatopancreas using degenerate primers by the method of 3' and 5' rapid amplification of cDNA ends PCR. The cDNA sequence consists of 1892 bp with a 1560 bp open reading frame, encoding 520 amino acids with high identity to invertebrate, vertebrate and even bacterial catalases. The sequence includes the catalytic residues His71, Asn144, and Tyr354. The molecular mass of the predicted protein is 58824.04 Da with an estimated pl of 6.63. Sequence comparison showed that the deduced amino acid sequence of F. chinensis catalase shares 96%, 73%, 71% and 70% identity with that of Pacific white shrimp Litopenaeus vannamei, Abalone Haliotis discus hannai, Zhikong scallop Chlamys farreri and Human Homo sapiens, respectively. Catalase transcripts were detected in hepatopancreas, hemocytes, lymphoid organ, intestine, ovary, muscle and gill. by real-time PCR. The variation of catalase mRNA transcripts in hemocytes and hepatopancreas was also quantified by real-time PCR and the result indicated that the catalase showed up-regulated expression trends in hemocytes at 14 h and in hepatopancreas at 37 h after injection with white spot syndrome virus (WSSV). (c) 2008 Elsevier Ltd. All rights reserved.

Sequence analysis and functional study of thymidylate synthase from zebrafish, Danio rerio

The thymidylate synthase (TS), an important target for many anticancer drugs, has been cloned from different species. But the cDNA property and function of TS in zebrafish are not well documented. In order to use zebrafish as an animal model for screening novel anticancer agents, we isolated TS cDNA from zebrafish and compared its sequence with those from other species. The open reading frame (ORF) of zebrafish TS cDNA sequence was 954 nucleotides, encoding a 318-amino acid protein with a calculated molecular mass of 36.15 kDa. The deduced amino acid sequence of zebrafish TS was similar to those from other organisms, including rat, mouse and humans. The zebrafish TS protein was expressed in Escherichia coli and purified to homogeneity. The purified zebrafish TS showed maximal activity at 28 degrees C with similar K-m value to human TS. Western immunoblot assay confirmed that TS was expressed in all the developmental stages of zebrafish with a high level of expression at the 1-4 cell stages. To study the function of TS in zebrafish embryo development, a short hairpin RNA (shRNA) expression vector, pSilencer 4.1-CMV/TS, was constructed which targeted the protein-coding region of zebrafish TS mRNA. Significant change in the development of tail and epiboly was found in zebrafish embryos microinjected pSilencer4.1-CMV/TS siRNA expression vector.

The cDNA cloning and mRNA expression of heat shock protein 70 gene in the haemocytes of bay scallop (Argopecten irradians, Lamarck 1819) responding to bacteria challenge and naphthalin stress

Heat shock protein 70 (HSP70) is an important member of the heat shock protein superfamily, and it plays a key role in the process of protecting cells, facilitating the folding of nascent peptides and responding to stress. The cDNA of bay scallop Argopecten irradians HSP70 (designated AIHSP70) was cloned by the techniques of homological cloning and rapid amplification of cDNA end (RACE). The full length of AIHSP70 cDNA was 2651 bp in length, having a 5' untranslated region (UTR) of 96 bp, a 3' UTR of 575 bp, and an open reading frame (ORF) of 1980 bp encoding a polypeptide of 659 amino acids with an estimated molecular mass of 71.80 kDa and an estimated isoelectric point of 5.26. BLAST analysis revealed that the AIHSP70 gene shared high identity with other known HSP70 genes. Three classical HSP signature motifs were detected in AIHSP70 by InterPro, analysis. 3-D structural prediction of AIHSP70 showed that its N terminal ATPase activity domain and,C terminal substrate-binding domain shared high similarity with that in human heat shock protein 70. The results indicated that the AIHSP70 was a member of the heat shock protein 70 family. A semi-quantitive RT-PCR method was used to analyse the expression of AIHSP70 gene after the treatment of naphthalin which is one kind of polycyclic aromatic hydrocarbon (PAH) and the challenge of bacteria. mRNA expression of AIHSP70 in scallop was up-regulated significantly after the stimulation of naphthalin and increased with increasing naphthalin concentration. A clearly time-dependent expression pattern of AIHSP70 was observed after the scallops were infected by Vibrio anguillarum, and the mRNA expression reached a maximum level at 8 h and lasted to 16 h, and then dropped progressively. The results indicated that AIHSP70 could play an important role in mediating the environmental stress and immune response in scallop. (c) 2006 Elsevier Ltd. All rights reserved.

Expression and localization of a novel phosducin-like protein from amphioxus Branchiostoma belcheri

A full length amphioxus cDNA, encoding a novel phosducin-like protein (Amphi-PhLP), was identified for the first time from the gut cDNA library of Branchiostoma belcheri. It is comprised of 1 550 bp and an open reading frame (ORF) of 241 amino acids, with a predicted molecular mass of approximately 28 kDa. In situ hybridization histochemistry revealed a tissue-specific expression pattern of Amphi-PhLP with the high levels in the ovary, and at a lower level in the hind gut and testis, hepatic caecum, gill, endostyle, and epipharyngeal groove, while it was absent in the muscle, neural tube and notochord. In the Chinese Hamster Ovary (CHO) cells transfected with the expression plasmid pEGFP-N1/Amphi-PhLP, the fusion protein was targeted in the cytoplasm of CHO cells, suggesting that Amphi-PhLP is a cytosolic protein. This work may provide a framework for further understanding of the physiological function of Amphi-PhLP in B. belcheri.

中国明对虾免疫系统中抗氧化相关基因的克隆与表达分析

对虾病害在世界范围内的广泛传播，给水产养殖和沿海农村经济造成了重大损失。深入开展对虾免疫机制研究并在此基础上寻找对虾疾病防治的有效方法已成为当务之急。研究表明，当对虾等甲壳动物受到外界病原刺激时，其体内的吞噬细胞在吞噬活动中会激活磷酸己糖支路的代谢，引起呼吸爆发，产生多种活性氧分子。另外，受到病原侵染的对虾还会产生其他多种免疫反应，这些免疫反应将消耗大量的能量（ATP），产能的呼吸链会加速运转，由此也会引发大量活性氧的产生。这些活性氧分子可以杀灭入侵的病原微生物，但同时由于活性氧分子反应的非特异性，它们也会对宿主的细胞、组织和器官造成严重伤害，进而导致对虾生理机能的损伤和免疫系统的破坏。所以，消除对虾体内因过度免疫反应产生的过量氧自由基将能够增强其抵御病原侵染的能力，提高免疫力。本论文从中国明对虾体内克隆了线粒体型超氧化物歧化酶（mMnSOD）、胞质型超氧化物歧化酶（cMnSOD）、过氧化氢酶（Catalase）和过氧化物还原酶（Peroxiredoxin）等四种与免疫系统相关的抗氧化酶基因，分析了它们的分子结构特征，组织分布及应答不同病原刺激的表达变化模式，并对其中的mMnSOD基因和Peroxiredoxin基因进行了体外重组表达、分离纯化和酶活性分析。 采用RACE技术从中国明对虾血细胞中克隆了两个超氧化物歧化酶（SOD）基因，通过序列比对分析发现，其中一个为mMnSOD基因，另一个为cMnSOD基因。mMnSOD基因的cDNA全长为1185个碱基，其中开放阅读框为660个碱基，编码220个氨基酸，其中推测的信号肽为20个氨基酸。多序列比对结果显示中国明对虾mMnSOD基因的推导氨基酸序列与罗氏沼虾、蓝蟹的推导氨基酸序列同源性分别为88%和82%。Northern blot结果表明，该基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。半定量RT-PCR结果显示，对虾感染病毒3 h时，该基因在血细胞和肝胰脏中的转录水平显著升高。此外，通过构建原核表达载体，本研究对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活分析。cMnSOD基因的cDNA全长为1284个碱基，其中开放阅读框为861个碱基，编码287个氨基酸。多序列比对结果显示中国明对虾cMnSOD基因的推导氨基酸序列与斑节对虾和凡纳滨对虾的同源性高达98%和94%。组织半定量结果显示，cMnSOD基因在对虾被检测的各个组织中均有表达。 另外，半定量RT-PCR结果表明，对虾感染病毒23h时，该基因在肝胰脏中的转录上升到正常水平的3.5倍；而感染后59 h时，该基因在血细胞中的转录上升到正常水平的2.5倍。 利用根据其他生物过氧化氢酶保守氨基酸序列设计的简并引物，结合RACE技术，从中国明对虾肝胰脏中克隆到了过氧化氢酶基因的部分片段，片段长1725个碱基。多序列比对结果发现目前所得中国明对虾Catalase基因部分片段的推导氨基酸序列与罗氏沼虾和皱纹盘鲍Catalase氨基酸序列的同源性分别达到95%和73%。通过实时荧光定量PCR技术对中国明对虾Catalase基因在各个组织中的分布情况及病毒感染后该基因在血细胞和肝胰脏中的转录变化进行了研究。结果发现，该基因在肝胰脏、鳃、肠和血细胞中表达水平较高，在卵巢、淋巴器官和肌肉中的表达水平相对较弱；感染病毒23 h和37 h时，对虾血细胞和肝胰脏中该基因mRNA的表达量分别出现显著性上升。 依据中国明对虾头胸部cDNA文库提供的部分片段信息，结合SMART-RACE技术，从中国明对虾肝胰脏中克隆到了过氧化物还原酶基因（Peroxiredoxin）， 该基因的cDNA全长为942个碱基，其中开放阅读框为594个碱基，编码198个氨基酸。中国明对虾Peroxiredoxin基因的推断氨基酸序列与伊蚊、文昌鱼和果蝇等Peroxiredoxin基因的推断氨基酸序列同源性分别为77%、76%和73%。其蛋白理论分子量为22041.17 Da，pI为5.17。Northern blot结果表明，Peroxiredoxin基因在对虾的肝胰脏、血细胞、淋巴器官、肠、卵巢、肌肉和鳃等组织中均有表达。实时荧光定量PCR结果显示，弧菌感染后，该基因在对虾血细胞和肝胰脏中的转录水平都有明显变化并且表达模式不同。另外，对该基因进行了体外重组表达，并对纯化的重组蛋白进行了质谱鉴定和酶活性分析。酶活性分析表明，复性后的重组蛋白能在DTT存在的条件下还原H2O2。

The responsive expression of heat shock protein 22 gene in zhikong scallop Chlamys farreri against a bacterial challenge

HSP22 is a member of a small HSP subfamily contributing to the growth, transformation and apoptosis of the cell as well as acting as a molecular chaperone. In the present study, CfHSP22 cDNA was cloned from Chlamys farreri by the rapid amplification of cDNA ends technique. The full-length cDNA of CfHSP22 was of 1279 bp, consisting of a 5'-terminal untranslated region (5'UTR) of 122 bp, a 3'UTR of 581 bp with a canonical polyadenylation signal sequence AATAAA and a poly( A) tail, and an open reading frame of 576 bp encoding a polypeptide with a molecular mass of 22.21 kDa and a predicted isoelectric point of 9.69. There was an alpha-crystallin domain, a hallmark of the sHSP subfamily, in the C-terminus, and the deduced amino acid sequence of CfHSP22 showed high similarity to previously identified HSP22s. CfHSP22 was constitutively expressed in the haemocyte, muscle, kidney, gonad, gill, heart and hepatopancreas, and the expression level in the hepatopancreas was higher than that in the other tissues. CfHSP22 transcription was up-regulated and reached a maximal level at 12 h after the bacterial challenge, and then declined progressively to the original level at 48 h. These results suggested that CfHSP22 perhaps play a critical role in response to the bacterial challenge in haemocytes of scallop C. farreri.

Study on cytocompatibility of carboxymethyl-chitosan membranes to skin fibroblasts

Chitosan and carboxymethl-chitosan (CM-chitosan) membranes with different molecular mass were prepared by a casting method. The cytocompatibility of two kinds of polysaccharide membranes to skin fibroblasts that cultured in vitro were studied. The methods were to culture the cells in soaking fluid of membranes and to culture the cells on the membranes directly. The results showed that the soaking fluid had no toxicity to fibroblasts and the biological security of lower molecular mass membranes were better than higher molecular mass membranes, and CM-chitosan membranes were better than chitosan membranes. In addition, the growth of fibroblasts on chitosan membranes was inhibited and the cells would fall off from chitosan membranes after a period of culture. However, the cells adhered and expanded well on CM-chitosan membranes. All these demonstrated that cytocompatibility of CM-chitosan membranes to skin fibroblasts was better than chitosan membranes.

Purification and characterisation of a natural lectin from the serum of the shrimp Litopenaeus vannamei

A natural lectin from the serum of the shrimp Litopenaeus vannamei was purified to homogeneity by a single-step affinity chromatography using fetuin-coupled agarose. The purified serum lectin (named LVL) showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC, chicken RBC and its haemagglutinating (HA) activity was specifically dependent on Ca2+ and reversibly sensitive to EDTA. LVL inactive form had a molecular mass estimate of 172 kDa and was composed of two non-identical subunits (32 and 38 kDa) cross-linked by interchain disulphide bonds. Significant LVL activity was observed between pH 7 and 11. In HA-inhibition assays performed with several carbohydrates and glycoproteins, LVL showed a distinct and unique specificity for GalNAc/GluNAc/NeuAc which had an acetyl group, while glycoproteins fetuin and bovine submaxillary mucin (BSM) had sialic acid. Moreover, this agglutinin appeared to recognise the terminal N- and O-acetyl groups in the oligosaccharide chain of glycoconjugates. The HA activity of L. vannamei lectin was also susceptible to inhibition by lipopolysaccharides from diverse Gram-negative bacteria, which might indicate a significant in vivo role of this humoral agglutinin in the host immune response against bacterial infections. (C) 2006 Elsevier Ltd. All rights reserved.

Purification and characterization of a natural lectin from the plasma of the shrimp Fenneropenaeus chinensis

A natural lectin from the plasma of the shrimp Fenneropenaeus chinensis was purified by singlestep affinity chromatography using fetuin-coupled agarose. The purified plasma lectin showed a strong affinity for human A/B/O erythrocytes (RBC), mouse RBC and chicken RBC. The hemagglutinating (HA) activity of the lectin was dependent on Ca2+ and reversibly sensitive to EDTA. This lectin was named FC-L and its inactive form had a molecular mass estimate of 168 kDa. Fifteen N-terminal amino acid sequences of this protein were determined. We performed HA-inhibition assays with several carbohydrates and glycoproteins. FC-L showed a distinct and unique specificity to N-acetylated sugars, particularly sialic acid and sialoproteins. The FC-L also has binding activity to some Gram-negative bacteria which caused disease in shrimp and fish. The activity of FC-L was inhibited at temperatures greater than 75 degrees C and at a pH less than 7 or greater than 11. These results suggest that FC-L may play a role as pattern recognition proteins in the reorganization and clearance of invaders in shrimp F. chinensis. Crown Copyright (c) 2008 Published by Elsevier Ltd. All rights reserved.

Characterization of polyethylene glycol-modified proteins by semi-aqueous capillary electrophoresis

A new program to characterize polyethylene glycol-modified (PEGylated) proteins is outlined using capillary zone electrophoresis (CZE). PEGylated ribonuclease A and lysozyme were selected as examples. Five separation procedures were compared to select out the mixed buffer of acetonitrile-water (1:1, v/v) at pH 2.5 as the best to characterize the PEGylated proteins without sample pretreatment. Polyethylene oxide (PEO) with a high molecular mass of 8X10(6) was applied to rinse the capillary to form a dynamic coating which would decrease the undesirable proteins adsorbed to the inner wall of the silica. The electroosmotic flow (EOF) mobility of the five procedures was determined, respectively. It is found that acetonitrile is mainly responsible for the good resolution of PEGylated proteins with the help of PEO coating in the semi-aqueous system. The low EOF mobility and current in the semi-aqueous system might also have some responsibility for the high resolution. The semi-aqueous procedure described in this paper also demonstrates higher resolution of natural proteins than aqueous ones. (C) 2001 Elsevier Science B.V. All rights reserved.

Vibrational spectroscopic studies of the structure of di-amino acid peptides. Part II: cyclo(L-Asp-L-Asp) in the solid state and in aqueous solution

Solid-state protonated and N,O-deuterated Fourier transform infrared (IR) and Raman scattering spectra together with the protonated and deuterated Raman spectra in aqueous solution of the cyclic di-amino acid peptide cyclo(L-Asp-L-Asp) are reported. Vibrational band assignments have been made on the basis of comparisons with previously cited literature values for diketopiperazine (DKP) derivatives and normal coordinate analyses for both the protonated and deuterated species based upon DFT calculations at the B3-LYP/cc-pVDZ level of the isolated molecule in the gas phase. The calculated minimum energy structure for cyclo(L-Asp-L-Asp), assuming C-2 symmetry, predicts a boat conformation for the DKP ring with both the two L-aspartyl side chains being folded slightly above the ring. The C=O stretching vibrations have been assigned for the side-chain carboxylic acid group (e.g. at 1693 and 1670 cm(-1) in the Raman spectrum) and the cis amide I bands (e.g. at 1660 cm(-1) in the Raman spectrum). The presence of two bands for the carboxylic acid C=O stretching modes in the solid-state Raman spectrum can be accounted for by factor group splitting of the two nonequivalent molecules in a crystallographic unit cell. The cis amide II band is observed at 1489 cm(-1) in the solid-state Raman spectrum, which is in agreement with results for cyclic di-amino acid peptide molecules examined previously in the solid state, where the DKP ring adopts a boat conformation. Additionally, it also appears that as the molecular mass of the substituent on the C-alpha atom is increased, the amide II band wavenumber decreases to below 1500 cm(-1); this may be a consequence of increased strain on the DKP ring. The cis amide II Raman band is characterized by its relatively small deuterium shift (29 cm(-1)), which indicates that this band has a smaller N-H bending contribution than the trans amide II vibrational band observed for linear peptides.

Characterization of the chlorosome antenna of the filamentous anoxygenic phototrophic bacterium Chloronema sp strain UdG9001

The absorption and fluorescence properties of chlorosomes of the filamentous anoxygenic phototrophic bacterium Chloronema sp. strain UdG9001 were analyzed. The chlorosome antenna of Chloronema consists of bacteriochlorophyll (BChl) d and BChl c together with γ-carotene as the main carotenoid. HPLC analysis combined with APCI LC-MS/MS showed that the chlorosomal BChls comprise a highly diverse array of homologues that differ in both the degree of alkylation of the macrocycle at C-8 and/or C-12 and the alcohol moiety esterified to the propionic acid group at C-17. BChl c and BChl d from Chloronema were mainly esterified with geranylgeraniol (33% of the total), heptadecanol (24%), octadecenol (19%), octadecanol (14%), and hexadecenol (9%). Despite this pigment heterogeneity, fluorescence emission of the chlorosomes showed a single peak centered at 765 nm upon excitation at wavelengths ranging from 710 to 740 nm. This single emission, assigned to BChl c, indicates an energy transfer from BChl d to BChl c within the same chlorosome. Likewise, incubation of chlorosomes under reducing conditions caused a weak increase in fluorescence emission, which indicates a small redox-dependent fluorescence. Finally, protein analysis of Chloronema chlorosomes using SDS-PAGE and MALDI-TOF-MS revealed the presence of a chlorosomal polypeptide with a molecular mass of 5.7 kDa, resembling the CsmA protein found in Chloroflexus aurantiacus and Chlorobium tepidum chlorosomes. Several minor polypeptides were also detected but not identified. These results indicate that, compared with other members of filamentous anoxygenic phototrophic bacteria and green sulfur bacteria, Chloronema possesses an antenna system with novel features that may be of interest for further investigations.

Extraction and radioimmunoassay quantitation of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP) from human dental pulp tissue

The measurement of neuropeptides in complex biological tissue samples requires efficient and appropriate extraction methods so that immunoreactivity is retained for subsequent radioimmunoassay detection. Since neuropeptides differ in their molecular mass, charge and hydrophobicity, no single method will suffice for the optimal extraction of various neuropeptides. In this study, dental pulp tissue was obtained from 30 human non-carious teeth. Of the three different neuropeptide extraction methods employed, boiling in acetic acid in the presence of protease inhibitors yielded the highest levels of neuropeptide Y (NPY) and vasoactive intestinal polypeptide (VIP). High pressure liquid chromatography (HPLC) analysis of dental pulp tissue verified the authenticity of the neuropeptides extracted. © 2003 Elsevier Science Ltd. All rights reserved.