Structural effect of a recombinant monoclonal antibody on hinge region peptide bond hydrolysis

IgG hinge region peptide bonds are susceptible to degradation by hydrolysis. To study the effect of Fab and Fc on hinge region peptide bond hydrolysis, a recombinant humanized monoclonal IgG1 antibody, its F(ab')2 fragment, and a model peptide with amino acid sequence corresponding to the hinge region were incubated at 40 degrees C in formulation buffer including complete protease inhibitor and EDTA for 0, 2, 4, 6 and 8 weeks. Two major cleavage sites were identified in the hinge region of the intact recombinant humanized monoclonal antibody and its F(ab')2 fragment, but only one major cleavage site of the model peptide was identified. Hinge region peptide bond hydrolysis of the intact antibody and its F(ab')2 fragment degraded at comparable rates, while the model peptide degraded much faster. It was concluded that Fab region of the IgG, but not Fc portion had significant effect on preventing peptide bond cleavage by direct hydrolysis. Hydrolysis of hinge region peptide bonds was accelerated under both acidic and basic conditions.     

Phylogenetic analyses of Cornales based on 26S rRNA and combined 26S rDNA-MATK-RBCL sequence data

Nuclear 26S rDNA sequences were used to corroborate and test previously published matK-rbcL-based hypotheses of phylogenetic relationships in Cornales. Sequences were generated for 53 taxa including Alangium, Camptotheca, Cornus, Curtisia, Davidia, Diplopanax, Mastixia, Nyssa, and four families: Grubbiaceae, Hydrangeaceae, Hydrostachyaceae, and Loasaceae. Fifteen taxa from asterids were used as outgroups. The 26S rDNA sequences were initially analyzed separately and then combined with matK-rbcL sequences, using both parsimony and maximum likelihood methods. Eight strongly supported major clades were identified within Cornales by all analyses: Cornus, Alangium, nyssoids (Nyssa, Davidia, and Camptotheca), mastixioids (Mastixia and Diplopanax), Hydrangeaceae, Loasaceae, Grubbia-Curtisia, and Hydrostachys. However, relationships among the major lineages are not strongly supported in either 26S rDNA or combined 26S rDNA-matK-rbcL topologies, except for the sister relationships between Cornus and Alangium and between nyssoids and mastixioids in the tree from combined data. Discrepancies in relationships among major lineages, especially the placement of the long-branched Hydrostachys, were found between parsimony and maximum likelihood trees in all analyses. Incongruence between the 26S rDNA and matK-rbcL data sets was suggested, where Hydrangeaceae was found to be largely responsible for the incongruence. The long branch of Hydrostachys revealed in previous analyses was reduced significantly with more sampling. Maximum likelihood analysis of combined 26S rDNA-matK-rbcL sequences suggested that Hydrostachys might be sister to the remainder of Cornales, that Cornus-Alangium are sisters, that nyssoids-mastixioids are sisters, and that Hydrangeaceae-Loasaceae are sisters, consistent with previous analyses of matK-rbcL sequence data.     

Mitochondrial genome nucleotide substitution pattern between domesticated silkmoth, Bombyx mori, and its wild ancestors, Chinese Bombyx mandarina and Japanese Bombyx mandarina

Bombyx mori and Bombyx mandarina are morphologically and physiologically similar. In this study, we compared the nucleotide variations in the complete mitochondrial (mt) genomes between the domesticated silkmoth, B. mori, and its wild ancestors, Chinese B. mandarina (ChBm) and Japanese B. mandarina (JaBm). The sequence divergence and transition mutation ratio between B. mori and ChBm are significantly smaller than those observed between B. mori and JaBm. The preference of transition by DNA strands between B. mori and ChBm is consistent with that between B. mori and JaBm, however, the regional variation in nucleotide substitution rate shows a different feature. These results suggest that the ChBm mt genome is not undergoing the same evolutionary process as JaBm, providing evidence for selection on mtDNA. Moreover, investigation of the nucleotide sequence divergence in the A+T-rich region of Bombyx mt genomes also provides evidence for the assumption that the A+T-rich region might not be the fastest evolving region of the mtDNA of insects.     

Pristimerin Induces Autophagy‐Mediated Cell Death in K562 Cells through the ROS/JNK Signaling Pathway

Chronic myeloid leukemia (CML) is a lethal malignancy, and the progress toward long‐term survival has stagnated in recent decades. Pristimerin, a quinone methide triterpenoid isolated from the Celastraceae and Hippocrateaceae families, is well‐known to exert potential anticancer activities. In this study, we investigated the effects and the mechanisms of action on CML. We found that pristimerin inhibited cell proliferation of K562 CML cells by causing G1 phase arrest. Furthermore, we demonstrated that pristimerin triggered autophagy and apoptosis. Intriguingly, pristimerin‐induced cell death was restored by an autophagy inhibitor, suggesting that autophagy is cross‐linked with pristimerin‐induced apoptosis. Further studies revealed that pristimerin could produce excessive reactive oxygen species (ROS), which then induce JNK activation. These findings provide clear evidence that pristimerin might be clinical benefit to patients with CML.     

Modulation of retroviral restriction and proteasome inhibitor-resistant turnover by changes in the TRIM5alpha B-box 2 domain

An intact B-box 2 domain is essential for the antiretroviral activity of TRIM5alpha. We modeled the structure of the B-box 2 domain of TRIM5alpha based on the existing three-dimensional structure of the B-box 2 domain of human TRIM29. Using this model, we altered the residues predicted to be exposed on the surface of this globular structure. Most of the alanine substitutions in these residues exerted little effect on the antiretroviral activity of human TRIM5alphahu or rhesus monkey TRIM5alpharh. However, alteration of arginine 119 of TRIM5alphahu or the corresponding arginine 121 of TRIM5alpharh diminished the abilities of the proteins to restrict retroviral infection without affecting trimerization or recognition of the viral capsid. The abilities of these functionally defective TRIM5alpha proteins to accelerate the uncoating of the targeted retroviral capsid were abolished. Removal of the positively charged side chain from B-box 2 arginines 119/120/121 resulted in diminished proteasome-independent turnover of TRIM5alpha and the related restriction factor TRIMCyp. However, testing of an array of mutants revealed that the rapid turnover and retroviral restriction functions of this B-box 2 region are separable.     

中国产桫椤类配子体在不同基质胁迫下的形态畸变多样性研究

为了明确土壤污染对中国产桫椤科植物的生殖影响,以探讨中国桫椤类乃至其他濒危蕨类濒危的内在原因。该研究以中国产9种桫椤植物为材料,采用各植物原产地地表土壤培养孢子至配子体成熟,全程比较观察配子体畸变类型,统计畸变率;并分别取3个属的代表种笔筒树、大叶黑桫椤和中华桫椤的孢子,以MS培养基为对照,分别用5mg·kg^-1Pb^2+和1mg·kg^-1Cd^2+单因素胁迫培养配子体,比较观察各组的配子体形态畸变类型,统计畸变率。结果表明:(1)中国产桫椤类9种植物配子体培养过程中共出现17种形态畸变类型,其中4种为分化畸变,7种为假根畸变,4种为细胞畸变,2种为性器畸变。(2)不同培养基——对照组(MS培养基)、原产地土壤、Cd^2+胁迫、Pb^2+胁迫对中国产桫椤科9种植物配子体的平均畸变比例分别是2.28%、12.61%、31.58%和33.58%。(3)9种桫椤的配子体畸变类型及其畸变率均与培养基的胁迫程度呈正相关关系,其配子体发育都受到了产地土壤污染的严重伤害。(4)3种桫椤属植物配子体对土壤环境胁迫的耐受力强弱为黑桫椤属>白桫椤属>桫椤属。(5)桫椤科配子体的形态畸变多样性与胁迫条件之间具有稳定的对应关系。     

蓝尾石龙子的生长、两性异形及雌性繁殖

报道蓝尾石龙子（Eumeces elegans)的生长、两性异形和雌性繁殖,性成熟个体体色的两性差异显著,成年雄性体长、头长和头宽显著大于成年雌性,幼体体长生长率无显著的两性差异。成年雄体体长生长率显著大于成年雌体,因此,个体大小的两性异形是性成熟后发生的,体长小于50mm的幼体,头长和头宽无性差异;当体长大于50mm。雄性头长和头宽随体长的生长率显著大于雌性。并导致头部大小的两性异形,并随个体发育变得越来越显著,蓝尾石龙子产卵雌体的最小体长为69.3mm,大于此体长的雌体均年产单窝卵。窝卵数、窝卵重和平均卵重均与雌体体长呈正相关,平均值分别为6.4、2.783和0.554g。窝卵数与雌体产后状态无关,蓝尾石龙子雌体主要通过增加窝卵数和卵大小来增加繁殖输出。     

Proper processing of avian sarcoma/leukosis virus capsid proteins is required for infectivity

The formation of the mature carboxyl terminus of CA in avian sarcoma/leukemia virus is the result of a sequence of cleavage events at three PR sites that lie between CA and NC in the Gag polyprotein. The initial cleavage forms the amino terminus of the NC protein and releases an immature CA, named CA1, with a spacer peptide at its carboxyl terminus. Cleavage of either 9 or 12 amino acids from the carboxyl terminus creates two mature CA species, named CA2 and CA3, that can be detected in avian sarcoma/leukemia virus (R. B. Pepinsky, I. A. Papayannopoulos, E. P. Chow, N. K. Krishna, R. C. Craven, and V. M. Vogt, J. Virol. 69:6430-6438, 1995). To study the importance of each of the three CA proteins, we introduced amino acid substitutions into each CA cleavage junction and studied their effects on CA processing as well as virus assembly and infectivity. Preventing cleavage at any of the three sites produced noninfectious virus. In contrast, a mutant in which cleavage at site 1 was enhanced so that particles contained CA2 and CA3 but little detectable CA1 was infectious. These results support the idea that infectivity of the virus is closely linked to proper processing of the carboxyl terminus to form two mature CA proteins.     

GFP基因在新疆小拟南芥和拟南芥中的表达分析

以质粒pMCB30为模板,扩增GFP基因,连接到载体pCMBIA2300-35S-OCS上,构建过量表达载体p35S:GFP,将其转入农杆菌GV3101.通过农杆菌介导法将p35S:GFP载体分别转入新疆特色植物小拟南芥和拟南芥中.T0代经含有卡那霉素的1/2MS培养基筛选,获得了T1代转基因小拟南芥2株,T1代转基因拟南芥9株.通过激光共聚焦显微镜观察,在转基因小拟南芥和拟南芥的根尖细胞中均可检测到GFP绿色荧光蛋白;对转基因植株进行PCR扩增,均可检测到GFP基因,表明GFP基因已成功转入小拟南芥和拟南芥中.该研究建立了小拟南芥的遗传转化体系,为进一步利用GFP基因和进一步研究小拟南芥的功能基因奠定基础.