A cDNA library was constructed from the liver of Lampetra japonica. 10077 ESTs were obtained by random selecting clones for sequencing. The results demonstrated that 8515 ESTs were assembled into 648 consensus sequenc...A cDNA library was constructed from the liver of Lampetra japonica. 10077 ESTs were obtained by random selecting clones for sequencing. The results demonstrated that 8515 ESTs were assembled into 648 consensus sequences, represented 2210 unique transcripts, 47.06% of which were predicted as full length cDNAs. In addition, 1562 ESTs were singlets. Using the BLAST to align the assembled ESTs, we found that 93.9% (2053) transcripts shared similarity to sequences published in GenBank databases. The functional annotations to assembled ESTs showed that the genes, involved in immu-nology, blood coagulation and metabolism of jawed vertebrates, were highly expressed in the liver of L. japonica. Furthermore, 8 potential novel genes were identified. Further comparing liver transcriptome of L. japonica with Fundulus heteroclitus, Mus musculus, Bos Taurus, and Homo sapiens revealed that the genes of Chitinase and Polysaccharides metabolism were more highly expressed in L. japonica than the others, which implied that they may play an important role in immunity of L. japonica. In addi-tion, using the TargetScan, we marked microRNA target within 3′ UTR of L. japonica liver transcriptome. The data indicated that some microRNA targets were homology with the targets embeded in human cancer genes. The result seems to provide a useful clue to the treatment of human cancer. Therefore, the present work will be an important resource for investigating the functional genomics and pro-teomics of L. japonica as well as evolution of vertebrates.展开更多
Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a bucc...Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 （BGSP-2） from a buccal gland cDNA library of Larnpetrajaponica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of hmnan T lymphocytes stimulated by phytohemagglutinin （PHA） at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.展开更多
A 105-k Da polymer lectin was purified from lamprey（Lampetra japonica） serum by chromatography methods including cation ion-exchange chromatography with a SP-Sepharose TM XL column and size exclusion chromatography ...A 105-k Da polymer lectin was purified from lamprey（Lampetra japonica） serum by chromatography methods including cation ion-exchange chromatography with a SP-Sepharose TM XL column and size exclusion chromatography with a Superdex 200 column. The target fractions were collected according to the direction of hemagglutinating activity. The results revealed that the active fractions could adsorb on SP-Sepharose column and showed a 280 nm UV absorbance peak corresponding to molecular weights of 105 k Da in the following size exclusion chromatography. The target fractions with hemagglutinating activity were further checked by NativePAGE and SDS-PAGE. Two single bands at around 105 k Da and 35 k Da were displayed by two electrophoresis methods respectively, indicating that the protein exists as a trimer in solution. After Native-PAGE and SDS-PAGE,two bands were excised from the gels respectively and further identified by MALDI-TOF/TOF as serum lectin（gi：13094239）. The lectin was able to agglutinate rabbit red blood cells（RRBCs） and sheep red blood cells（SRBCs） in vitro. The lectin isolated from lamprey serum in the current study might be helpful for deeply understanding the innate immune molecules dependent immune defence in jawless vertebrates which have been proved recently that they possess a lymphocyte-based system of anticipatory immunity with variable lymphocyte receptors as mediators.展开更多
The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 ...The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.展开更多
Lamprey is considered to be an excellent model for studying the origin of adaptive immunity.In the present study,three up-regulated and ten down-regulated proteins were identified in the lipopolysaccharide(LPS)induced...Lamprey is considered to be an excellent model for studying the origin of adaptive immunity.In the present study,three up-regulated and ten down-regulated proteins were identified in the lipopolysaccharide(LPS)induced liver proteome of Lampetra morii.Quantitative real-time PCR(qPCR)was used to characterize in the liver the abundance of transcripts encoding the differentially expressed proteins.The abundance of transcripts and the corresponding differentially expressed proteins revealed that expression of cystathionase and Zgc:112210-201 was not correlated with protein expression level.Polyl 4-hydroxylase,serum lectin,Zgc:112210-201,and cofilin 1 mRNA expression was significantly upregulated in liver tissue after treatment with LPS.Transcript abundance of differentially expressed proteins in the gill,kidney,heart,intestine,liver,and supraneural body was determined.Transcripts of Zgc:112210-201 were almost undetectable in the tissues while polyl 4-hydroxylase,protein disulfide isomerase family A(member 4),cystathionase,and serum lectin were low abundance unlike the protein which was easily detected.This study represents the first attempt at understanding the LPS induced liver proteome of L.morii and our findings contribute to the identification of novel immune genes in lamprey.展开更多
p21活化蛋白激酶4(p21-activated protein kinase 4,PAK4)是一类丝氨酸/苏氨酸蛋白激酶,参与调控多种信号通路,在高等脊椎动物免疫应答中起重要作用。在低等脊椎动物日本七鳃鳗(Lampetra japonica)中发现一个PAK4同源基因(Lja-PAK4),其...p21活化蛋白激酶4(p21-activated protein kinase 4,PAK4)是一类丝氨酸/苏氨酸蛋白激酶,参与调控多种信号通路,在高等脊椎动物免疫应答中起重要作用。在低等脊椎动物日本七鳃鳗(Lampetra japonica)中发现一个PAK4同源基因(Lja-PAK4),其开放阅读框长度为2544 bp,编码一个含有848个氨基酸的蛋白质。该分子与高等脊椎动物PAK4序列比对一致性最高,且具有该蛋白家族保守的两个功能结构域。利用通过原核表达、纯化的重组Lja-PAK4蛋白为抗原,成功制备了大鼠抗Lja-PAK4的多克隆抗体。用混合菌刺激七鳃鳗,采用实时荧光定量PCR和免疫印迹方法发现,Lja-PAK4的mRNA和蛋白质在外周血淋巴细胞和髓样小体中均显著性上调表达。采用B细胞丝裂原LPS和T细胞丝裂原PHA分别对七鳃鳗进行刺激后发现,经LPS刺激后Lja-PAK4的mRNA和蛋白质在外周血淋巴细胞和髓样小体中呈现出显著性上调表达,而它们在鳃囊中的表达受到抑制。以上结果说明Lja-PAK4参与了LPS介导的七鳃鳗VLRB+淋巴细胞亚群的免疫应答反应。展开更多
基金Supported by the National High-Tech R&D Program (863 Program) (Grant No. 2007AA09Z428)the National Natural Science Foundation of China (Grant Nos. 60305001 and 60575005)+1 种基金the Key Project of Science and Technology of the Educa-tional Ministry of China (Grant No. 206032)the Science and Technology Pro-ject of Dalian, China (Grant No. 2006E11SF068)
文摘A cDNA library was constructed from the liver of Lampetra japonica. 10077 ESTs were obtained by random selecting clones for sequencing. The results demonstrated that 8515 ESTs were assembled into 648 consensus sequences, represented 2210 unique transcripts, 47.06% of which were predicted as full length cDNAs. In addition, 1562 ESTs were singlets. Using the BLAST to align the assembled ESTs, we found that 93.9% (2053) transcripts shared similarity to sequences published in GenBank databases. The functional annotations to assembled ESTs showed that the genes, involved in immu-nology, blood coagulation and metabolism of jawed vertebrates, were highly expressed in the liver of L. japonica. Furthermore, 8 potential novel genes were identified. Further comparing liver transcriptome of L. japonica with Fundulus heteroclitus, Mus musculus, Bos Taurus, and Homo sapiens revealed that the genes of Chitinase and Polysaccharides metabolism were more highly expressed in L. japonica than the others, which implied that they may play an important role in immunity of L. japonica. In addi-tion, using the TargetScan, we marked microRNA target within 3′ UTR of L. japonica liver transcriptome. The data indicated that some microRNA targets were homology with the targets embeded in human cancer genes. The result seems to provide a useful clue to the treatment of human cancer. Therefore, the present work will be an important resource for investigating the functional genomics and pro-teomics of L. japonica as well as evolution of vertebrates.
基金supported by the National High Technology Research and Development Program of China (No. 2007AA09Z428)the National Natural Science Foundation of China (No.30671083)+1 种基金the National Basic Research Program of China (No.2007CB815802)the Program for Innovative Research Team in University of Liaoning Province (No. 2007T089 and 2008T103)
文摘Lamprey is a representative of the agnathans, the most ancient class of vertebrates. Parasitic lampreys secrete anticoagulant from their buccal glands and prevent blood coagulation of host fishes. We identified a buccal gland secretory protein-2 （BGSP-2） from a buccal gland cDNA library of Larnpetrajaponica. The full-length BGSP-2 gene was cloned and the recombinant BGSP-2 protein was generated. The role of BGSP-2 on lymphocyte proliferation was studied by examining its effects on human T lymphocytes. We found that lamprey BGSP-2 was able to effectively block the proliferation of T cells in vitro by inducing G1/S cell cycle arrest. Furthermore, it inhibited the proliferation of hmnan T lymphocytes stimulated by phytohemagglutinin （PHA） at a minimum concentration of 0.1μg/ml. Our data suggest that lamprey BGSP-2 is able to block the mitosis of human T lymphocytes at the G1/S point, and has the potential of anti-proliferative effect on PHA-activated T lymphocytes.
基金The National Program on Key Basic Research Project(973 Program)of China under contract No.2013CB835304the National Marine Public Projects under contract No.201305016+1 种基金the National Natural Science Foundation of China under contract Nos31772884 and 31601865the Key Projects of Scientific Research Platform of Liaoning Provincial Education Department under contract No.L201683651
文摘A 105-k Da polymer lectin was purified from lamprey（Lampetra japonica） serum by chromatography methods including cation ion-exchange chromatography with a SP-Sepharose TM XL column and size exclusion chromatography with a Superdex 200 column. The target fractions were collected according to the direction of hemagglutinating activity. The results revealed that the active fractions could adsorb on SP-Sepharose column and showed a 280 nm UV absorbance peak corresponding to molecular weights of 105 k Da in the following size exclusion chromatography. The target fractions with hemagglutinating activity were further checked by NativePAGE and SDS-PAGE. Two single bands at around 105 k Da and 35 k Da were displayed by two electrophoresis methods respectively, indicating that the protein exists as a trimer in solution. After Native-PAGE and SDS-PAGE,two bands were excised from the gels respectively and further identified by MALDI-TOF/TOF as serum lectin（gi：13094239）. The lectin was able to agglutinate rabbit red blood cells（RRBCs） and sheep red blood cells（SRBCs） in vitro. The lectin isolated from lamprey serum in the current study might be helpful for deeply understanding the innate immune molecules dependent immune defence in jawless vertebrates which have been proved recently that they possess a lymphocyte-based system of anticipatory immunity with variable lymphocyte receptors as mediators.
基金The National Basic Research Program of China(973 Program)under contract No.2013CB835304the National Marine Public Projects under contract No.201305016+2 种基金the National Natural Science Foundation of China(General Program)under contract No.31601865the Dalian Science and Technology Program under contract No.2013E11SF056the Education Department of the General Scientific Research Project under contract No.L201683651
文摘The Guanine nucleotide exchange factor Vav2（Vav2） is a member of the Vav family that serves as an important regulators for the Rho family of Ras-related GTPases. In the current study, an ortholog（Lj-Vav2） of Vav2 was identified in the lamprey（Lampetra japonica）. To elucidate the phylogenetic relationship of Vav2, the metazoan genome databases were analyzed to mine the ortholog of Vav. It was found that Vav2 genes were only existed in vertebrates and Lj-Vav2 was the original one found in agnathans. The evolutionary dynamics of conserved motifs of Vav2 were explored using combined amino acid sequence as markers, and it is revealed that the Calponin homology（CH） domain, Dbl-homologous（DH） domain, Pleckstrin homology（PH） domain, Cysteine-rich（C1）domains, Src homology 3（SH3） domains and Src homology 2（SH2） domain were conserved throughout the Vav2 gene family in vertebrates during gene evolution. Relative quantitative real-time PCR analysis showed that the LjVav2 was distributed in the heart, kidney, supraneural myeloid body, liver, gill and lymphocyte-like cells. The LjVav2 was found to be expressed in these tissues, and the level of which was upregulated in lymphocyte-like cells after the animal was stimulated with LPS. These results indicated that the Lj-Vav2 might be involved in the immune response of lymphocyte-like cells in lamprey. Meanwhile, our findings provided a foundation for further investigation of the function of Lj-Vav2 in the primary vertebrate.
基金Natural Science Foundation of China(Grant No.31500106)the Youth Foundation of Liaoning Normal University(Grant No.LS2014L009)the Ph.D Start-up Fundation of Liaoning Province,China(Grant No.201501107).
文摘Lamprey is considered to be an excellent model for studying the origin of adaptive immunity.In the present study,three up-regulated and ten down-regulated proteins were identified in the lipopolysaccharide(LPS)induced liver proteome of Lampetra morii.Quantitative real-time PCR(qPCR)was used to characterize in the liver the abundance of transcripts encoding the differentially expressed proteins.The abundance of transcripts and the corresponding differentially expressed proteins revealed that expression of cystathionase and Zgc:112210-201 was not correlated with protein expression level.Polyl 4-hydroxylase,serum lectin,Zgc:112210-201,and cofilin 1 mRNA expression was significantly upregulated in liver tissue after treatment with LPS.Transcript abundance of differentially expressed proteins in the gill,kidney,heart,intestine,liver,and supraneural body was determined.Transcripts of Zgc:112210-201 were almost undetectable in the tissues while polyl 4-hydroxylase,protein disulfide isomerase family A(member 4),cystathionase,and serum lectin were low abundance unlike the protein which was easily detected.This study represents the first attempt at understanding the LPS induced liver proteome of L.morii and our findings contribute to the identification of novel immune genes in lamprey.
