An Escherichia coli MutY mutant without the six-helix barrel domain is a dimer in solution and assembles cooperatively into multisubunit complexes with DNA

Escherichia coli MutY is an adenine and weak guanine DNA glycosylase involved in reducing the mutagenic effects of 7,8-dihydro-8-oxoguanine (GO). MutY contains three structural domains: an iron-sulfur module, a six-helix barrel module with the helix-hairpin-helix motif, and a C-terminal domain. Here, we demonstrate that the mutant MutY(Delta26-134), which lacks the six-helix barrel domain, cannot complement the mutator phenotype of a mutY mutant in vivo. However, the mutant can still bind DNA and has weak catalytic activity at high enzyme concentrations. The mutant is a dimer in solution and assembled into two and multiple (up to five) complexes with 20- and 44-bp DNA fragments, respectively, in a concentration-dependent manner. Higher order complexes with DNA substrates containing A/GO mismatches were formed at lower protein concentrations than with the A/G mismatch and homoduplex DNA. Measurement of equilibrium binding using fluorescence anisotropy showed that the mutant protein retains some specificity for A/GO-containing DNA substrates and that the binding event is highly cooperative. This is consistent with the MutY structure determined, which indicates that GO specificity is contributed by both the six-helix barrel and C-terminal domains. The nonspecific binding of MutY(Delta26-134) to DNA suggests a model in which the specific binding of mismatched DNA by MutY involves sequential interactions, in which one MutY molecule scans the DNA and enhances binding of another MutY molecule to the A/GO mismatch.     

Genome sequencing and comparison of two nonhuman primate animal models, the cynomolgus and Chinese rhesus macaques

The nonhuman primates most commonly used in medical research are from the genus Macaca. To better understand the genetic differences between these animal models, we present high-quality draft genome sequences from two macaque species, the cynomolgus/crab-eating macaque and the Chinese rhesus macaque. Comparison with the previously sequenced Indian rhesus macaque reveals that all three macaques maintain abundant genetic heterogeneity, including millions of single-nucleotide substitutions and many insertions, deletions and gross chromosomal rearrangements. By assessing genetic regions with reduced variability, we identify genes in each macaque species that may have experienced positive selection. Genetic divergence patterns suggest that the cynomolgus macaque genome has been shaped by introgression after hybridization with the Chinese rhesus macaque. Macaque genes display a high degree of sequence similarity with human disease gene orthologs and drug targets. However, we identify several putatively dysfunctional genetic differences between the three macaque species, which may explain functional differences between them previously observed in clinical studies.     

Effects of domestic wastewater slow infiltration on the growth of poplar 'Zhonglin 2001' plantation

A slow infiltration experiment with different hydraulic loads (0, 3, 6, 9, 12, and 15 cm per week) of domestic wastewater was conducted in a 'Zhonglin 2001' poplar plantation to study the effects of the wastewater slow infiltration on the growth of the plantation. Comparing with the control (0 cm), the other five treatments increased the soil organic matter, total N, total P, total K, and Na+ contents in the plantation averagely by 1.940 g x kg(-1), 0.115 g x kg(-1), 0.029 g x kg(-1), 1.454 g x kg(-1) and 0.030 g x kg(-1), respectively. At lower hydraulic loads (3-12 cm per week), the poplar biomass growth and the N, P and Na+ contents in different poplar organs averagely increased by 17.583 t x hm(-2) x a(-1), 3.086 g x kg(-1), 0.645 g x kg(-1), and 0.121 g x kg(-1), with the maximum (36.252 t x hm(-2) x a(-1), 13.162 g x kg(-1), 5.137 g x kg(-1), and 0.361 g x kg(-1), respectively) at hydraulic loads 6-12 cm per week. The further increase of the hydraulic load decreased the poplar biomass growth and the N, P and Na+ contents in different poplar organs. The K content in different poplar organs decreased with increasing hydraulic load. Treating with domestic wastewater increased the leaf length, decreased the leaf asymmetry, and delayed leaf-falling. At high hydraulic load (15 cm per week), the higher soil Na+ and water contents would threat the poplar growth. The proper domestic wastewater hydraulic loads for the growth of poplar 'Zhonglin 2001' plantation would be 3-12 cm per week.     

Comparative Genomic Analysis of N2-Fixing and Non-N2-Fixing Paenibacillus spp.: Organization,Evolution and Expression of the Nitrogen Fixation Genes

We provide here a comparative genome analysis of 31 strains within the genus Paenibacillus including 11 new genomic sequences of N₂-fixing strains. The heterogeneity of the 31 genomes (15 N₂-fixing and 16 non-N₂-fixing Paenibacillus strains) was reflected in the large size of the shell genome, which makes up approximately 65.2% of the genes in pan genome. Large numbers of transposable elements might be related to the heterogeneity. We discovered that a minimal and compact nif cluster comprising nine genes nifB, nifH, nifD, nifK, nifE, nifN, nifX, hesA and nifV encoding Mo-nitrogenase is conserved in the 15 N₂-fixing strains. The nif cluster is under control of a σ⁷⁰-depedent promoter and possesses a GlnR/TnrA-binding site in the promoter. Suf system encoding [Fe–S] cluster is highly conserved in N₂-fixing and non-N₂-fixing strains. Furthermore, we demonstrate that the nif cluster enabled Escherichia coli JM109 to fix nitrogen. Phylogeny of the concatenated NifHDK sequences indicates that Paenibacillus and Frankia are sister groups. Phylogeny of the concatenated 275 single-copy core genes suggests that the ancestral Paenibacillus did not fix nitrogen. The N₂-fixing Paenibacillus strains were generated by acquiring the nif cluster via horizontal gene transfer (HGT) from a source related to Frankia. During the history of evolution, the nif cluster was lost, producing some non-N₂-fixing strains, and vnf encoding V-nitrogenase or anf encoding Fe-nitrogenase was acquired, causing further diversification of some strains. In addition, some N₂-fixing strains have additional nif and nif-like genes which may result from gene duplications. The evolution of nitrogen fixation in Paenibacillus involves a mix of gain, loss, HGT and duplication of nif/anf/vnf genes. This study not only reveals the organization and distribution of nitrogen fixation genes in Paenibacillus, but also provides insight into the complex evolutionary history of nitrogen fixation.     

拟南芥根毛衰老死亡过程的PCD检测

根毛是植物体吸收养分的重要器官, 自然条件下根毛的寿命很短, 仅能存活2–3周, 随即脱落死亡。以模式植物拟南芥(Arabidopsis thaliana)根毛为材料, 对根毛死亡的细胞学特征进行了报道。结果发现, 根毛衰老死亡后细胞内的原生质体发生了收缩, 并在胞质中观察到凝集物的出现; 通过原位末端标记(TUNEL)检测, 发现幼根上的根毛细胞核DNA发生了片段化。上述结果表明, 拟南芥根毛的衰老死亡很可能是植物体自主调控的程序性细胞死亡(PCD)。另外, 当根毛衰老死亡后,细胞核大多会迁移到靠近根毛基部的位置, 且正常的长管状根毛发生旋转扭曲。     

电针对帕金森病模型小鼠黑质铁染色细胞和铁蛋白表达的影响

目的探讨电针防治帕金森病的作用机制。方法采用1-甲基-4-苯基1,2,3,6四氢吡啶腹腔注射建立帕金森病小鼠模型,利用组织化学技术以及免疫组织化学技术观察电针对帕金森病小鼠脑黑质铁染色细胞和铁蛋白表达的影响。结果模型组1周、2周、4周小鼠黑质铁染色阳性细胞的数量较正常组明显增多,染色强度也明显增强（P〈0．001;P〈0．01;P〈0．001）,电针在3个存活期内均可明显减少帕金森病小鼠黑质铁阳性细胞的数量和染色强度（P〈0．001;P〈0．05;P〈0．001）;模型组1周、2周黑质部位铁蛋白的表达与正常组相比都有不同程度下降（P〈0．01;P〈0．001）,而电针组2周帕金森病小鼠黑质铁蛋白的表达与模型组相比显著增加（P〈0．01）。结论电针可以通过降低脑内铁的含量,同时增加铁蛋白的表达,从而提高帕金森病模型小鼠脑抗氧化能力,达到保护黑质多巴胺能神经元的目的。     

Novel mutations in ADAMTS13 CUB domains cause abnormal pre-mRNA splicing and defective secretion of ADAMTS13

Hereditary thrombotic thrombocytopenic purpura (TTP) is an autosomal recessive thrombosis disorder, caused by loss-of-function mutations in ADAMTS13. Mutations in the CUB domains of ADAMTS13 are rare, and the exact mechanisms through which these mutations result in the development of TTP have not yet been fully elucidated. In this study, we identified two novel mutations in the CUB domains in a TTP family with an acceptor splice-site mutation (c.3569−1, G>A, intron 25) and a point missense mutation (c.3923, G>A, exon 28), resulting in a glycine to aspartic acid substitution (p.G1308D). In vitro splicing analysis revealed that the intronic mutation resulted in abnormal pre-mRNA splicing, and an in vitro expression assay revealed that the missense mutation significantly impaired ADAMTS13 secretion. Although both the patient and her brother displayed significantly reduced ADAMTS13 activity and increased levels of ultra-large VWF (ULVWF) multimers in plasma, only the female developed acute episodes of TTP. Our findings indicate the importance of the CUB domains for the protein stability and extracellular secretion of ADAMTS13.     

Combination effects of chronic cadmium exposure and gamma-irradiation on the genotoxicity and cytotoxicity of peripheral blood lymphocytes and bone marrow cells in rats

This work investigated the effects of chronic cadmium (Cd) exposure combined with γ-ray irradiation on the cytotoxicity and genotoxicity of peripheral blood cells and bone marrow cells in rats. Results showed that when the rats were exposed to low dose (LD) Cd of 0.1mg CdCl?/(kgd) for 8 and 12 weeks, the Cd concentration in blood reached to 135-140 μg/L and no toxic effects on peripheral blood lymphocytes, white blood cells (WBC) and granulocyte-monocyte (GM) progenitor cells were observed except polychromatic erythrocytes (PCE) of bone marrow. Moreover, this chronic LD Cd exposure significantly decreased irradiation-induced micronucleus (MN) formation and hypoxanthine-guanine phosphoribosyl transferase (hprt) mutation in lymphocytes and PCE, while the combination of LD Cd exposure and irradiation induced the additive metallothionein (MT) protein expression in bone marrow cells. When the rats were exposed to a high dose (HD) Cd of 0.5mg CdCl/?(kgd) for 8 and 12 weeks, the blood Cd level approached to 458-613 μg/L and an inflammatory response was induced, meanwhile, MN formation and hprt mutation were markedly increased, and the ratio of PCE/NCE (normochromatic erythrocyte) was significantly decreased. Furthermore, when the rats were exposed to HD Cd plus 2 Gy irradiation, additive toxic effects on MN formation, hprt mutation, PCE damage and GM progenitor cell proliferation were observed, while this combination treatment resulted in an obvious reduction of MT protein compared to HD Cd group. In conclusion, chronic exposure to LD Cd induced the adaptive response to irradiation in the genotoxicity of peripheral blood lymphocytes and PCE of bone marrow by the up-regulation of Cd-induced MT protein, but the combination of HD Cd exposure and irradiation generated the additive effects on the cytotoxicity and genotoxicity in peripheral blood lymphocytes and bone marrow cells.     

Deciphering the proteome of the in vivo diagnostic reagent "purified protein derivative" from Mycobacterium tuberculosis

Purified protein derivative (PPD) has served as a safe and effective diagnostic reagent for 60 years and is the only broadly available material to diagnose latent tuberculosis infections. This reagent is also used as a standard control for a number of in vitro immunological assays. Nevertheless, the molecular composition and specific products that contribute to the extraordinary immunological reactivity of PPD are poorly defined. Here, a proteomic approach was applied to elucidate the gene products in the U.S. Food and Drug Administration (FDA) standard PPD-S2. Many known Mycobacterium tuberculosis T-cell antigens were detected. Of significance, four heat shock proteins (HSPs) (GroES, GroEL2, HspX, and DnaK) dominated the composition of PPD. The chaperone activities and capacity of these proteins to influence immunological responses may explain the exquisite solubility and immunological potency of PPD. Spectral counting analysis of three separate PPD reagents revealed significant quantitative variances. Gross delayed-type hypersensitivity (DTH) responses in M. tuberculosis infected guinea pigs were comparable among these PPD preparations; however, detailed histopathology of the DTH lesions exposed unique differences, which may be explained by the variability observed in the presence and abundance of early secretory system (Esx) proteins. Variability in PPD reagents may explain differences in DTH responses reported among populations.