万安玻璃红鲤鱼PKR的克隆鉴定与系统进化分析

本研究以万安玻璃红鲤鱼为材料,通过同源克隆的方法克隆了万安玻璃红鲤鱼双链RNA激活的蛋白激酶（PKRCcvwPKR）,并首次克隆到CcvwPKR的剪接异构体。CcvwPKR全长为2145 bp,编码714个氨基酸,CcvwPKR剪接异构体全长1431 bp,编码476个氨基酸。CcvwPKR的剪接异构体是CcvwPKR第477个密码子的G突变为T（gaa477taa）,从而使得CcvwPKR的翻译提前终止。氨基酸序列比对和空间结构SMART预测显示,CcvwPKR和CcvwPKR剪接异构体都含有三个双链RNA结合模体（dsRBM）,CcvwPKR剪接异构体缺失PKR的C端的激酶结构域。系统进化分析表明,万安玻璃红鲤鱼PKR在进化上与鲤鱼和鲫鱼密切相关。万安玻璃红鲤鱼和目前已有研究的鲤科鱼类PKR一样,分子结构都含有三个dsRBM,三个dsRBM使得其具有更强的结合dsRNA的作用,推测可能与其较强的抗病能力有关。     

Structure of the kinase domain of human RNA-dependent protein kinase with K296R mutation reveals a face-to-face dimer

RNA-dependent protein kinase (PKR) is crucial for the innate immune response, cell growth, proliferation, signal transduction and apoptosis. The activation process of PKR has been studied for many years and is still under debate. To obtain new insight into the mechanism of PKR activation, we solved the crystal structure of a latent mutant of the PKR kinase domain (PKR-KD) in the apo form at a resolution of 2.9 Å. The overall structure of PKR-KD is similar to previously reported structures. Structural analysis revealed a classical back-to-back dimer and a newly defined face-to-face dimer. Analytical ultracentrifugation experiments, electrostatic surface maps and the model of PKR-KD in complex with the eIF2α substrate all support that the face-to-face dimer is more reflective of PKR in solution. Our results provide new information on PKR dimerization and its activation mechanism.     

流感病毒NS1蛋白在复制中的作用研究

流感病毒NS1基因敲除毒株(delNS1毒株)的获得可以使我们建立更有效的生物学关系,主要表现在流感病毒繁殖周期和致病的过程中,流感病毒NS1蛋白和双链RNA活性蛋白激酶(PKR)之间的生物学关系.实验结果表明,缺少功能性的PKR可以使delNS1毒株在其他非允许宿主中繁殖,这表明流感病毒NS1蛋白的主要功能是对抗或阻止PKR调解抗病毒效应.     

Cyclic AMP response element binding protein (CREB) participates in the heat-inducible expression of humanhsp90β gene

Human heat shock protein 90b gene ( hsp90b ) is a constitutively expressed heat shock gene existing in most of cell types tested that can be further induced by heat shock. Chloramphenical acetyl transferase (CAT) reporter plasmids driven by different regulatory fragments of hsp90b gene were constructed and transfected into Jurkat cells to explore the role of a cAMP response element (CRE) in the upstream of the gene. Results show that, in comparison with the wild type construct, a severe reduction (~2/3) in the increased folds of promoter activity induced by heat shock at 42℃ for 1 h was observed in a construct with CRE-containing fragment (-173/-91bp) deleted. Electrophoretic mobility shift assays (EMSA) showed that phosphorylated CRE-binding protein (CREB) in the nuclear extract of heat shocked Jurkat cells is specifically bound to the fragment. Additionally, both of the phosphorylation on CREB and the activity of protein kinase A (PKA) were found in Jurkat cells to be enhanced with extending time of heat shock treatment. Our results indicate that in addition to the intronic HSE/HSF pathway, phosphorylated CREB also participates in the heat shock induced expression of human hsp90b gene via its interaction with CRE which may be regulated by PKA-sig- naling pathway.     

Genistein inhibits human TNF-α-induced porcine endothelial cell adhesiveness for human monocytes and natural killer cells

Cellular immune response is a major barrier to xenotransplantation. Human tumor necrosis factor-α (hTNF-α) possesses cross-species activity and directly amplifies the immune rejection via the upregulation of adhesion molecules on porcine endothelium. We investigated the role of protein tyrosine phosphorylation in the induction of expression of E-sclectin and vascular cell adhesion molecule-1 (VCAM-1), and the augmentation of adhesion of human peripheral blood monocytes (PBMo) and natural killer cells (PBNK), after rhTNF-α-stimulation of porcine aortic endothelial cells (PAEC) in vitro, rhTNF-α-increased adhesiveness of PAEC for both PBMo and PBNK was dose-dependently reduced by pretreatment of PAEC with the selective protein tyrosine kinase (PTK) inhibitor genistein. The inhibitory effect occurred at the early time of PAEC activation triggered by rhTNF-α, and was completely reversible. PTK activity assay indicated that genistein also suppressed rhTNF-α stimulated activation of protein tyrosine kinases (PTKs) in PAEC in a dose-dependent manner. Flow cytometric analysis showed that genistein inhibited the upregulation of E-selectin and VCAM-1 by rhTNF-α. These results suggest that PTKs may regulate the expression of E-selectin and VCAM-1 on PAEC and the adherence of PBMo and PBNK induced by rhTNF-α. Moreover, dietary genistein, used as an adhesion antagonist, may contribute to managing the cell-mediated rejection in the clinical application.     

植物ERECTA基因家族特征及其非生物胁迫功能

ERECTA是首次从拟南芥中分离到的一个类受体激酶基因。ERECTA在植物的叶片形态发生、花序形成、气孔发育，以及生物和非生物胁迫应答过程中都发挥重要作用。该文综述了植物ERECTA家族成员的组成、蛋白质序列特征及其在非生物胁迫应答过程中的功能，以期为深入研究ERECTA功能提供新线索。     

Analysis of IGF2 mRNA expression and its methylation status between cattle yaks and their parents

Cattle yaks are an F1 hybrid between cattle and yaks and exhibit significant hybrid vigor. However, cattle yaks are sterile males. To study the epigenetic aspects of this reproductive isolation, IGF2 mRNA expression in cattle yaks and their parents and the patterns of the IGF2 differentially methylated regions (DMR) were examined in blood and testes. The results showed that IGF2 expression in cattle yaks’ testes was lower than in their parents (P < 0.01). The IGF2 DMR was highly methylated (above 90%) in blood and testes of cattle yaks, yaks and cattle, with the highest level in cattle yaks (P > 0.05). Our study showed that IGF2 plays an important role in bovine spermatogenesis and might be involved in cattle-yak male sterility. The methylation level of the IGF2 DMR was irrelevant to the lower expression of IGF2 in cattle yaks; other factors perhaps play roles in its expression.     

PEG-g-poly（aspartamide-co-N,N-dimethylethylenediamino aspartamide）： Synthesis, characterization and its application as a drug delivery system

PEG-g-poly(aspartamide-co-N,N-dimethylethylenediamino aspartamide) (PEG-DMEDA-PASP) was synthesized by two-step ring-opening reactions of polysuccinimide (PSI) with α-methoxy-ω-amino-poly(ethylene glycol) and N,N-dimethylethylenediamine. The polymer structure was confirmed by ¹H NMR and FT-IR. The resultant PEG-DMEDA-PASP with ammonium glycyrrhizinate (AMG) could form polymeric micelles in aqueous solution. The results of transmission electron microscopy (TEM) and dynamic light scattering (DLS) measurements revealed that these polymeric micelles were spherical particles with a narrow diameter distribution and that their average diameter was ca. 70 nm. These polymeric micelles had high-loading capacity (58%) and encapsulation efficiency (70%) for AMG. The results of in vitro release experiments showed that these polymeric micelles possessed sustained-release effects, with a release rate of 25% within 3 h and 90% within 24 h.     

Evolution of the serum resistance-associated SRA gene in African trypanosomes

Serum resistance-associated (SRA) protein, a protein unique for Trypanosoma brucei rhodesiense, is responsible for resistance of this parasite to the lysis by normal human serum (NHS) and is a vital molecular marker to distinguish this species from other African trypanosomes. We cloned and sequenced the SRA basic copy (SRAbc) gene from T. b. rhodesiense and related species and found that this gene is confined to the subgenus Trypanozoon. The average 82% identity among the sequenced SRAbc genes indicates that they may have a common origin and are highly conserved. Since SRAbc coexists in the T. b. rhodesiense genome with SRA, we propose that SRAbc might be the ‘donor VSG’, which after duplication became inserted into the expression site by recombination. Under natural selection, SRAbc could reform into SRA following mosaic formation. Supported by National Natural Science Foundation of China (Grant Nos. 30570245, 30670275), Changjiang Scholars and Innovative Research Team in University (Grant No. DPCKSCU/IRT0447), International Foundation for Science of Sweden (Grant No. B/4318-1), Grant Agency of the Czech Republic (Grant No. Z60220518) and Education Foundation of the Czech Republic (Grant No. 2B06129)     

Science Letters: Transient expression of chicken alpha interferon gene in lettuce

We investigated the possibility of producing chicken alpha interferon （ChlFN-α） in transgenic plants. The cDNA encoding ChlFN-α was introduced into lettuce （Lactuca sativa L.） plants by using an agro-infiltration transient expression system. The ChlFN-a gene was correctly transcribed and translated in the lettuce plants according to RT-PCR and ELISA assays. Recombinant protein exhibited antiviral activity in vitro by inhibition of vesicular stomatitis virus （VSV） replication on chicken embryonic fibroblast （CEF）. The results demonstrate that biologically active avian cytokine with potential pharmaceutical applications could be expressed in transgenic lettuce plants and that it is possible to generate interferon protein in forage plants for preventing infectious diseases of poultry.     

Molecular cloning of a fulllength cDNA for ECBP21 from Angelica dahurica

ECBP21 is an extracellular calmodulin-binding protein which was first detected and purified from extracellular extracts of suspension-cultured cells of Angelica dahurica. The purified protein was electroblotted onto PVDF membrane and the amino acid sequences from 1 to 20 were determined. Using degenerate oligonucleotides of the sequence, a full-length cDNA coding for ECBP21 was isolated by a combination of RT-PCR and 5′-RACE cloning. The cDNA contains 947 nucleotides and codes for a precursor protein of 216 amino acids. The N-terminal 1–25 amino acid sequence is a predicted signal peptide and the other 26–216 amino acid sequence is a mature peptide. The 26–45 amino acid sequence shows identity with the N-terminal amino acid sequence of purified ECBP21 from Angelica dahurica. The fragment of encoding the mature protein was cloned into pET-28b(+) and transformed into E. coli BL21(DE3). A protein with relative molecular mass 21 ku was expressed in E. coli. Using a biotinylated-CaM gel overlay technique, the expression protein was tested for its ability to bind CaM. The results indicated that the expression protein is a Ca²⁺-dependent CaM-binding protein. Thus, these results further defined the cDNA clone for ECBP21. This work laid a foundation for elucidating biological functions of ECBP21 by using molecular biological means.     

菠菜乙醇酸氧化酶基因家族的鉴定及表达分析

在菠菜全基因组中鉴定了菠菜(Spinacia oleracea L.)乙醇酸氧化酶(GLO)家族成员,并对其理化性质、亚细胞定位、基因结构、保守基序、同源关系及基因表达进行了分析,发现菠菜中存在5个SoGLOs蛋白,通过进化树分析,菠菜GLO蛋白与甜菜GLO蛋白亲缘关系较近.通过基因结构分析发现该家族基因由9～11个外显子构成.实时荧光定量聚合酶链式反应(qRT-PCR)结果表明:硝态氮仅能短期诱导SoGLOs的表达,而铵态氮可以持续抑制SoGLOs的表达,从而影响菠菜草酸的含量.在胁迫处理后,SoGLOs的表达均有明显变化,SoGLO1,SoGLO3和SoGLO5对盐胁迫的响应最明显,SoGLOs可能在菠菜的抗盐、耐高温、耐寒、抗旱以及抗氧化过程中起作用.植物激素的喷施普遍使SoGLOs的表达在短时间内增加,这可能引起菠菜体内草酸的快速积累.     

Molecular cloning, identification and characteristics of NYD-SP9: Gene coding protein kinase presumably involved in spermatogenesis

Using cDNA microarray hybridization from a human testicular cDNA library, one gene exhibiting ten-fold difference at expression level between adult and embryo human testes was cloned and named NYD-SP9, which was believed to be involved in spermatogenesis. Southern blot hybridization results showed that NYD-SP9 expressed highly in testis but low in ovary. Protein motif analysis of this cDNA sequence revealed a cluster of phosphorylation sites, indicating its potential involvement in signal pathways during spermatogenesis. Furthermore, one transmembrane helix was predicted in N-terminal region, indicating that putative NYD-SP6 may be served as a transmembrane protein. The proximity of these potential phosphorylation sites to each other indicates that there may be interaction among these sites to regulate spermatogenesis. These findings suggested that protein kinase NYD-SP9 might play a role in male germ cell differentiation.     

Genetic analysis of gynogenetic and common populations of Verasper moseri using SSR markers

The genetic structure and variation of the artificial meio- gynogenetic population and common population of barfin flounder (Verasper moseri) were analyzed using eight microsatellite markers. A total of 29 alleles were detected, of which 23 alleles were in the artificial gynogenetic population while 29 alleles were in the control group. The average observed heterozygosity (H _O) of eight loci in the control group (0.526 8) was several times higher than that (0.185 8) in the gynogenetic population. The results indicate that the genetic diversity of the control group was much higher than that of the gynogenetic population of barfin flounder (Verasper moseri). Most loci significantly deviated from Hardy-Weinberg equilibrium (HWE) after Bonferroni correction (p < 0.005 56) in the gynogenetic population, while four loci deviated from HWE in the control group. The coefficient of gene differentiation (G _ST) was 0.131 0, and the genetic distance was 0.171 8 between the two populations, suggesting a significant genetic differentiation between the two populations. Biography: MA Hongyu (1979–), male, Ph. D. candidate, research direction: marine biotechnology and molecular genetics.     

Novel role of PKR in inflammasome activation and HMGB1 release

The inflammasome regulates the release of caspase activation-dependent cytokines, including interleukin (IL)-1β, IL-18 and high-mobility group box 1 (HMGB1). By studying HMGB1 release mechanisms, here we identify a role for double-stranded RNA-dependent protein kinase (PKR, also known as EIF2AK2) in inflammasome activation. Exposure of macrophages to inflammasome agonists induced PKR autophosphorylation. PKR inactivation by genetic deletion or pharmacological inhibition severely impaired inflammasome activation in response to double-stranded RNA, ATP, monosodium urate, adjuvant aluminium, rotenone, live Escherichia coli, anthrax lethal toxin, DNA transfection and Salmonella typhimurium infection. PKR deficiency significantly inhibited the secretion of IL-1β, IL-18 and HMGB1 in E. coli-induced peritonitis. PKR physically interacts with several inflammasome components, including NOD-like receptor (NLR) family pyrin domain-containing 3 (NLRP3), NLRP1, NLR family CARD domain-containing protein 4 (NLRC4), absent in melanoma 2 (AIM2), and broadly regulates inflammasome activation. PKR autophosphorylation in a cell-free system with recombinant NLRP3, apoptosis-associated speck-like protein containing a CARD (ASC, also known as PYCARD) and pro-caspase-1 reconstitutes inflammasome activity. These results show a crucial role for PKR in inflammasome activation, and indicate that it should be possible to pharmacologically target this molecule to treat inflammation.     

斑马鱼PKR的dsRBM1和dsRBM3重组及体外功能分析

为了重组斑马鱼(Daniorerio)双链RNA依赖的蛋白激酶(DrPKR)的双链RNA结合模体1(dsRBM1)和双链RNA结合模体3(dsRBM3)基因,并对重组基因表达的蛋白进行鉴定和功能分析,设计定向删除引物,应用PCR技术扩增DrPKR的ds RBM1和dsRBM3的基因片段,通过重叠PCR技术将dsRBM1和dsRBM3基因重组在一起得到dsRBM13基因,再将dsRBM13构建到原核表达质粒pET32a上并表达和纯化蛋白;通过表达蛋白的二聚化实验和pull down实验对重组蛋白dsRBM13进行体外功能分析。结果:DrPKR的ds RBM1和dsRBM3重组的蛋白dsRBM13可以进行二聚化和多聚化,且能结合Poly I:C(人工合成的dsRNA)。因此,重组DrPKR蛋白模体dsRBM13仍然具有二聚化和dsRNA结合活性,提示串联了三个dsRBM的Dr PKR激活过程中,串联两个dsRBM对DrPKR激活是必需的,并且在DrPKR结合dsRNA的激活过程中,因dsRNA的长短和空间结构的多样性,含有三个dsRBM的DrPKR可能存在更强和更多的结合方式,因此具有更强的抗病毒免疫反应功能。     

Overexpression of rice OsLOL2 gene confers disease resistance in tobacco to Pseudomonas syringae pv. Tabaci

LSD1-related proteins of Arabidopsis with LSDl-like zinc finger domains regulate disease resistance and programmed cell death （PCD）. We cloned a rice OsLOL2 gene, orthologous to LSD1 of Arabidopsis and expressed it in a tobacco plant. Transgenic tobacco lines displayed enhanced disease resistance to a virulent bacterium Pseudomonas syringae pv. tabaci （Pst）. RT-PCR analysis showed that overexpression of OsLOL2 in transgenic tobacco lines resulted in upregulation of two pathogenesis-related （PR） protein genes, PR2 and PR5. Our results suggest that overexpression of OsLOL2 in transgenic tobacco enhances the resistance through the induction of PR proteins and hypersensitive response-like reaction.