Biochemical properties of a high fidelity DNA ligase from Thermus species AK16D.

NAD+-dependent DNA ligases from thermophilic bacteria Thermus species are highly homologous with amino acid sequence identities ranging from 85 to 98%. Thermus species AK16D ligase, the most divergent of the seven Thermus isolates collected worldwide, was cloned, expressed in Escherichia coli and purified to homogeneity. This Thermus ligase is similar to Thermus thermophilus HB8 ligase with respect to pH, salt, NAD+, divalent cation profiles and steady-state kinetics.However, the former is more discriminative toward T/G mismatches at the 3'-side of the ligation junction, as judged by the ratios of initial ligation rates of matched and mismatched substrates. The two wild-type Thermus ligases and a Tth ligase mutant (K294R) demonstrate 1-2 orders of magnitude higher fidelity than viral T4 DNA ligase. Both Thermus ligases are active with either the metal cofactor Mg2+, Mn2+or Ca2+but not with Co2+, Ni2+, Cu2+or Zn2+. While the nick closure step with Ca2+becomes rate-limiting which results in the accumulation of DNA-adenylate intermediate, Ni2+only supports intermediate formation to a limited extent. Both Thermus ligases exhibit enhanced mismatch ligation when Mn2+is substituted for Mg2+, but the Tsp. AK16D ligase remains more specific toward perfectly matched substrate.     

High production of laccase by a new basidiomycete, <Emphasis Type="Italic">Trametes</Emphasis> sp

A new basidiomycete, Trametes sp. 420, produced laccase at 6,810 U l⁻¹ (268 mg, 25.4 U mg⁻¹ protein for guaiacol) in glucose medium and 7,870 U l⁻¹ (310 mg) in cellobiose medium with induction by 0.5 mM Cu²⁺ and 6 mM o-toluidine. Laccase isozyme E (LacE) was the sole laccase in the fermentation products. It was stable at pH 5–9 and below 70°C over 30 min. The K _m values of LacE for four substrates (guaiacol ABTS, 2,6-dimethoxyphenol and syringaldazine) varied from 5 to 245 μM. The activity of LacE was strongly inhibited by NaN₃ but not by EDTA or dimethylsulfoxide. LacE at 0.5 U l⁻¹ could decolorize industrial dyes. The open reading frame of the lacE gene was 2,130 bp and was interrupted by 10 introns. It displayed a high homology to laccases from other fungi. Pingui Tong and Yuzhi Hong contributed equally to the study     

Loss of viability during freeze–thaw of intact and adherent human embryonic stem cells with conventional slow-cooling protocols is predominantly due to␣apoptosis rather than cellular necrosis

Summary A major challenge in the widespread application of human embryonic stem (hES) cells in clinical therapy and basic scientific research is the development of efficient cryopreservation protocols. Conventional slow-cooling protocols utilizing standard cryoprotectant concentrations i.e. 10% (v/v) DMSO, yield extremely low survival rates of <5% as reported by previous studies. This study characterized cell death within frozen–thawed hES colonies that were cryopreserved under standard conditions. Surprisingly, our results showed that immediately after post-thaw washing, the overwhelming majority of hES cells were viable (≈98%), as assessed by the trypan blue exclusion test. However, when the freshly-thawed hES colonies were incubated within a 37 °C incubator, there was observed to be a gradual reduction in cell viability over time. The kinetics of cell death was drastically slowed-down by keeping the freshly-thawed hES colonies at 4 °C, with >90% of cells remaining viable after 90 min of incubation at 4 °C. This effect was reversible upon re-exposing the cells to physiological temperature. The vast majority of low temperature-exposed hES colonies gradually underwent cell death upon incubation for a further 90 min at 37 °C. Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP nick-end-labeling (TUNEL) assay confirmed apoptosis-induced nuclear DNA fragmentation in frozen–thawed hES cells after incubation at 37 °C for 90 min. Expression of active caspase-3 enzyme, which is another prominent marker of apoptosis, was confirmed by immunocytochemical staining, while transmission electron microscopy showed typical ultrastructural features of apoptosis such as chromatin condensation and margination to the nuclear membrane. Hence, our results demonstrated that apoptosis instead of cellular necrosis, is the major mechanism of the loss of viability of cryopreserved hES cells during freeze–thawing with conventional slow-cooling protocols.     

RNA干扰抑制肾小管上皮细胞TSP1表达和TGF-β1活化

血小板反应蛋白1(TSP1)是转化生长因子-β1(TGF-β1)体内重要的活化因子,而后者又是致肾小管间质纤维化的关键因素。观察了针对TSP1的小双链干扰RNA(siRNA-TSP1),抑制由血管紧张素II(AngII)诱导的肾小管上皮细胞TGF-β1过度活化。将根据人TSP1基因序列设计的特异siRNA-TSP1转染人肾小管上皮细胞系(HK-2),利用Western印迹、RT-PCR、流式细胞仪及ELISA等方法,检测了TSP1、TGF-β1及其信号蛋白Smad2与p-Smad2、纤维连接蛋白(FN)和纤溶酶原激活剂抑制物-1(PAI-1)的基因转录水平、蛋白质表达或蛋白质活性。结果显示,siRNA-TSP1能有效转染HK-2细胞,并以剂量依赖方式显著抑制TSP1的基因转录与合成;其对TGF-β1的合成影响较小,但能明显抑制TGF-β1的活化。此外siRNA-TSP1可阻抑TGF-β1依赖的Smad2磷酸化,减少细胞外基质FN以及PAI-1的合成。研究结果提示,由于TSP1是TGF-β1重要的内源性活化因子,故针对TSP1的RNA干扰能在体外有效抑制TSP1表达并相应调抑了TGF-β1的活化。     

Plasma Adiponectin Levels and Metabolic Factors in Nondiabetic Adolescents

Objectives: The relationship of plasma adiponectin levels with various anthropometric and metabolic factors has been surveyed extensively in adults. However, how plasma adiponectin levels are related to various anthropometric indices and cardiovascular risk factors in adolescents is not as vigorously studied. In this study, we investigated this among healthy nondiabetic adolescents. Research Methods and Procedures: Two hundred thirty nondiabetic subjects (125 boys and 105 girls, ～10 to 19 years old) were included. The plasma adiponectin, fasting plasma glucose, insulin, lipids and anthropometric indices including body height, weight, waist circumference, and hip circumference were examined. Body fat mass (FM) and percentage were obtained from DXA scan. The homeostasis model assessment was applied to estimate the degree of insulin resistance. Results: The plasma adiponectin levels were significantly higher in girls (30.79 ± 14.48 μg/mL) than boys (22.87 ± 11.41 μg/mL). The plasma adiponectin levels were negatively related to BMI, FM, FM percentage, waist circumference, waist‐to‐hip ratio, insulin resistance, plasma insulin, triglycerides, and uric acid levels, but positively with high‐density lipoprotein cholesterol (HDL‐C) with the adjustment for age and gender. Using different multivariate linear regression models, only age and HDL‐C were consistently related to the plasma adiponectin levels after adjustment for the other variables. Discussion: The relationship between plasma adiponectin and various anthropometric indices and metabolic factors, especially HDL‐C, previously reported in adults was present in the healthy nondiabetic adolescents. Whether variation of plasma adiponectin levels in healthy nondiabetic adolescents may influence their future coronary artery disease risk warrants further investigation.     

Protective Effect of the Silkworm Protein 30Kc6 on Human Vascular Endothelial Cells Damaged by Oxidized Low Density Lipoprotein (Ox-LDL)

Although the 30K family proteins are important anti-apoptotic molecules in silkworm hemolymph, the underlying mechanism remains to be investigated. This is especially the case in human vascular endothelial cells (HUVECs). In this study, a 30K protein, 30Kc6, was successfully expressed and purified using the Bac-to-Bac baculovirus expression system in silkworm cells. Furthermore, the 30Kc6 expressed in Escherichia coli was used to generate a polyclonal antibody. Western blot analysis revealed that the antibody could react specifically with the purified 30Kc6 expressed in silkworm cells. The In vitro cell apoptosis model of HUVEC that was induced by oxidized low density lipoprotein (Ox-LDL) and in vivo atherosclerosis rabbit model were constructed and were employed to analyze the protective effects of the silkworm protein 30Kc6 on these models. The results demonstrated that the silkworm protein 30Kc6 significantly enhanced the cell viability in HUVEC cells treated with Ox-LDL, decreased the degree of DNA fragmentation and markedly reduced the level of 8-isoprostane. This could be indicative of the silkworm protein 30Kc6 antagonizing the Ox-LDL-induced cell apoptosis by inhibiting the intracellular reactive oxygen species (ROS) generation. Furthermore, Ox-LDL activated the cell mitogen activated protein kinases (MAPK), especially JNK and p38. As demonstrated with Western analysis, 30Kc6 inhibited Ox-LDL-induced cell apoptosis in HUVEC cells by preventing the MAPK signaling pathways. In vivo data have demonstrated that oral feeding of the silkworm protein 30Kc6 dramatically improved the conditions of the atherosclerotic rabbits by decreasing serum levels of total triglyceride (TG), high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C) and total cholesterol (TC). Furthermore, 30Kc6 alleviated the extent of lesions in aorta and liver in the atherosclerotic rabbits. These data are not only helpful in understanding the anti-apoptotic mechanism of the 30K family proteins, but also provide important information on prevention and treatment of human cardiovascular diseases.     

A second‐generation genetic linkage map for bighead carp (Aristichthys nobilis) based on microsatellite markers

Bighead carp (Aristichthys nobilis) is an important aquaculture fish worldwide. Genetic linkage maps for the species were previously reported, but map resolution remained to be improved. In this study, a second‐generation genetic linkage map was constructed for bighead carp through a pseudo‐testcross strategy using interspecific hybrids between bighead carp and silver carp. Of the 754 microsatellites genotyped in two interspecific mapping families (with 77 progenies for each family), 659 markers were assigned to 24 linkage groups, which were equal to the chromosome numbers of the haploid genome. The consensus map spanned 1917.3 cM covering 92.8% of the estimated bighead carp genome with an average marker interval of 2.9 cM. The length of linkage groups ranged from 52.2 to 133.5 cM with an average of 79.9 cM. The number of markers per linkage group varied from 11 to 55 with an average of 27.5 per linkage group. Normality tests on interval distances of the map showed a non‐normal marker distribution; however, significant correlation was found between the length of linkage group and the number of markers below the 0.01 significance level (two‐tailed). The length of the female map was 1.12 times that of the male map, and the average recombination ratio of female to male was 1.10:1. Visual inspection showed that distorted markers gathered in some linkage groups and in certain regions of the male and female maps. This well‐defined genetic linkage map will provide a basic framework for further genome mapping of quantitative traits, comparative mapping and marker‐assisted breeding in bighead carp.     

生物体衰老与复制衰老--体内与体外研究

体外连续培养的细胞在有人数的细胞分裂后,更新换代合成ＤＮＡ及分裂的能力,最后导致增殖能力的丧失,但基本代谢过程仍能维持,这种现象称为复制衰老。本文讨论了复制衰老现象存在的普遍性,描述了衰老细胞伯特征,对复制衰老和生物体衰老之间的联系进行了重点分析。现有的研究虽然还不完全,但都提示复制衰老是生物体衰老在细胞水平上的反映,并充分肯定了复制衰老是一个较好的研究机体衰老的模型。     

Taxonomic Precision of Different Hypervariable Regions of 16S rRNA Gene and Annotation Methods for Functional Bacterial Groups in Biological Wastewater Treatment

High throughput sequencing of 16S rRNA gene leads us into a deeper understanding on bacterial diversity for complex environmental samples, but introduces blurring due to the relatively low taxonomic capability of short read. For wastewater treatment plant, only those functional bacterial genera categorized as nutrient remediators, bulk/foaming species, and potential pathogens are significant to biological wastewater treatment and environmental impacts. Precise taxonomic assignment of these bacteria at least at genus level is important for microbial ecological research and routine wastewater treatment monitoring. Therefore, the focus of this study was to evaluate the taxonomic precisions of different ribosomal RNA (rRNA) gene hypervariable regions generated from a mix activated sludge sample. In addition, three commonly used classification methods including RDP Classifier, BLAST-based best-hit annotation, and the lowest common ancestor annotation by MEGAN were evaluated by comparing their consistency. Under an unsupervised way, analysis of consistency among different classification methods suggests there are no hypervariable regions with good taxonomic coverage for all genera. Taxonomic assignment based on certain regions of the 16S rRNA genes, e.g. the V1&V2 regions – provide fairly consistent taxonomic assignment for a relatively wide range of genera. Hence, it is recommended to use these regions for studying functional groups in activated sludge. Moreover, the inconsistency among methods also demonstrated that a specific method might not be suitable for identification of some bacterial genera using certain 16S rRNA gene regions. As a general rule, drawing conclusions based only on one sequencing region and one classification method should be avoided due to the potential false negative results.