小兴安岭大亮子河国家森林公园不同生境下土壤细菌多样性和群落结构

土壤细菌是森林生态系统的一个重要组成成分, 是生态系统中物质循环和能量流动的重要参与者, 细菌群落组成和生物多样性是反映土壤生态功能的重要指标。本文利用高通量测序技术分析了大亮子河国家森林公园内红松(Pinus koraiensis)林、落叶松(Larix gmelinii)林、蒙古栎(Quercus mongolica)林、枫桦(Betula costata)林、针阔混交林、灌木林和草甸等7种不同生境土壤细菌群落组成和多样性的差异性, 探讨该地区土壤细菌群落对不同生境的响应, 为地区森林生态系统的经营管理及生态系统稳定性的维护提供科学理论基础。在门的水平上, 各生境的细菌群落组成基本一致, 其中变形菌门(Proteobacteria)、放线菌门(Actinobacteria)、酸杆菌门(Acidobacteria)和疣微菌门(Verrucomicrobia)在7种生境土壤中相对丰度均大于10.0%, 是细菌中的优势菌门。在属的水平上, 共测得245个菌属, 各样地共有属118个, 占总属数的48.2%, 占总相对丰度的97.8%; 优势菌属分别为Spartobacteria_ genera_incertae_sedis、Gaiella、Gp16、Gp4, 占总相对丰度的47.0%, Spartobacteria_genera_incertae_sedis在7种生境土壤中丰度均最高。7种生境下的土壤细菌多样性和土壤理化因子存在着显著的差异, 红松林的土壤细菌群落多样性和丰富度均高于其他生境。土壤pH是大亮子河森林公园影响土壤细菌多样性的关键因子。     

真盐生植物盐地碱蓬根系边缘细胞在耐盐中的作用初探

以黄河三角洲潮间带盐地碱蓬种子生成的幼苗为材料,研究了NaCl胁迫对盐地碱蓬生长与根系边缘细胞的影响。盐地碱蓬的第一个边缘细胞几乎与根尖同步产生,当根长达到13mm时,边缘细胞数目达到最大值。NaCl胁迫抑制边缘细胞的活性,但低浓度的NaCl处理增加边缘细胞的数目。低浓度NaCl处理时果胶甲基酯酶（PME）的活性比对照有明显增加,超氧化物歧化酶（SOD）活性随着NaCl浓度的增加呈现先上升后下降的趋势,低浓度NaCl可以增加盐地碱蓬根内过氧化氢酶（CAT）的活性,NaCl处理时间和处理浓度都对过氧化物酶（POD）活性的影响不明显。这些结果表明,盐地碱蓬至少部分通过增加调控活性氧（ROS）水平增加PME活性及根系边缘细胞数目来抵抗NaCl胁迫。     

灰绿曲霉β-葡萄糖苷酶的分离及特性

目的：利用灰绿曲霉EU7-22发酵产纤维素酶,从中分离到β-葡萄糖苷酶,分析其理化特性,确定其最佳活性条件。方法：灰绿曲霉EU7-22发酵液离心后,上清液经硫酸铵沉淀、Phenyl 6 Fast Flow（highsub）疏水层析和Sephacryl S-200凝胶层析,获得纯化的β-葡萄糖苷酶。结果：纯酶的比活性为5.1 IU/mg,得率为13.89%。SDS-PAGE凝胶电泳分析表明该酶是单亚基蛋白,其分子量为56.2 kDa。在pH4.0~6.0范围内,β-葡萄糖苷酶具有较高的稳定性,该酶的最适酶促反应pH为5.0。当β-葡萄糖苷酶在温度低于60℃的缓冲液中温育1 h后,酶活损失不大,表现了较好的稳定性;当该酶在温度高于60℃的缓冲液中温育1 h后,酶活迅速丧失。β-葡萄糖苷酶在70℃时具有最大催化活性。结论：灰绿曲霉EU7-22发酵产生的β-葡萄糖苷酶具有较高活性,具有分子量较小、最佳催化温度较高的特点。     

Quality,bioactivity study,and preclinical acute toxicity,safety pharmacology evaluation of PEGylated recombinant human endostatin (M2ES)

Endostar, a potent endogenous antiangiogenic factor, is wildly used in clinics. However, it was easily degraded by enzymes and rapidly cleared by the kidneys. To overcome these shortcomings, PEGylated recombinant human endostatin was developed. In this study, the purity of M₂ES was evaluated by silver stain and reversed‐phase high‐performance liquid chromatography. Ultraviolet spectrum was used to examine the structural of M₂ES and endostar. The bioactivity and antitumor efficacy of M₂ES were evaluated using an in vitro endothelial cell migration model and athymic nude mouse xenograft model of a heterogeneous lung adenocarcinoma, respectively. A preclinical study was performed to evaluate the acute toxicity and safety pharmacology in rhesus monkeys. The purity of M₂ES was more than 98%; PEG modification has no effect on endostatin structure. Compared with the control group, M₂ES dramatically retards endothelial cell migration and tumor growth. After intravenous (IV) infusions of M₂ES at a dose level of three and 75 mg/kg in rhesus monkeys, there was no observable serious adverse event in both acute toxicity and safety pharmacology study. On the basis of the quality and bioactivity study data of M₂ES and the absence of serious side effect in rhesus monkeys, M₂ES was authorized to initiate a phase I clinical trial.     

小麦族几种近缘植物细胞在长期离体培养中的染色体变异

对小麦及其4种近缘属间禾草进行了长时间 (0.5～5.7年, 个别8.6年) 的愈伤组织培养,在继代过程中染色体数目的变异在染色体倍性低的簇毛麦(Haynaldia villosa, 2n=2x=14)及新麦草(Psathyrostachys juncea, 2n=2x=14) 倾向于数目的增大;染色体倍性高的小麦 (Triticum aestivum, 2n=6x=42) 趋向于数目的减少;而倍性居中的羊草 (Leymus chinensis, 2n=4x=28) 既有增大也有减小;染色体倍性最高的高冰草 (Agropyron elongatum, 2n=6x=70) 最为稳定,但也有减少的趋向。愈伤组织的胚性主要与二倍体及亚二倍体的总水平相关。     

分子伴侣及信号肽对毕赤酵母中分泌蛋白表达水平的影响

目的：探索定位于细胞质、内质网膜及内质网腔中的分子伴侣及其组合对于带有不同信号肽的胞外β-1,3-葡聚糖酶（EXGl）在巴斯德毕赤酵母GS200中表达水平的影响。方法：通过融合PCR技术分别构建带有酵母a交配因子引导肽序列（仅MF）、酵母仅交配因子信号肽序列（ccPre）和重链结合蛋白（Bip）信号肽序列的报告蛋白EXGl的表达质粒pPIC9-EXG1,同时构建分子伴侣基因及其组合的表达质粒pBLArg-IV,然后将2种重组质粒共转化至毕赤酵母宿主菌GS200,转化子经筛选获得共表达菌株,通过测定EXG1酶活来评价分子伴侣与信号肽对其表达水平的影响。结果：细胞质及内质网膜上的分子伴侣Sec61a、Sec61B及胞质中的分子伴侣Ydjl、Ssal、Hsp104及其组合对各种信号肽引导的报告蛋白EXG1的表达水平没有显著影响。然而,内质网腔中的分子伴侣Bip、EroI、PDI与HacI组合能显著提高报告蛋白EXG1的表达水平,其中,以aMF或ctPre作为信号肽引导的报告蛋白EXG1的表达水平分别提高了2．6倍和3．8倍,以Bip信号肽引导的报告蛋白EXGl的表达水平提高了20％～45％,而对于以EXG1自身信号肽引导的报告蛋白EXG1的表达水平没有显著影响。结论：在酵母表达体系中,内质网腔中的分子伴侣是报告蛋白EXG1表达水平的重要影响因素．但分子伴侣对于信号肽的选择性还须进一步证明。     

Trans‐3,5,4´‐trimethoxystilbene reduced gefitinib resistance in NSCLCs via suppressing MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345 and miR‐498

Despite initial dramatic efficacy of epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (EGFR‐TKIs) in EGFR‐mutant lung cancer patients, subsequent emergence of acquired resistance is almost inevitable. Resveratrol and its derivatives have been found to exert some effects on EGFR‐TKI resistance in non‐small cell lung cancer (NSCLC), but the underlying mechanisms remain unclear. We screened several NSCLC cell lines with gefitinib resistance by MTT assay and analysed the miR‐345/miR‐498 expression levels. NSCLC cells were pre‐treated with a resveratrol derivative, trans‐3,5,4‐trimethoxystilbene (TMS) and subsequently challenged with gefitinib treatment. The changes in apoptosis and miR‐345/miR‐498 expression were analysed by flow cytometry and q‐PCR respectively. The functions of miR‐345/miR‐498 were verified by CCK‐8 assay, cell cycle analysis, dual‐luciferase reporter gene assay and immunoblotting analysis. Our results showed that the expression of miR‐345 and miR‐498 significantly decreased in gefitinib resistant NSCLC cells. TMS pre‐treatment significantly upregulated the expression of miR‐345 and miR‐498 increasing the sensitivity of NSCLC cells to gefitinib and inducing apoptosis. MiR‐345 and miR‐498 were verified to inhibit proliferation by cell cycle arrest and regulate the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways by directly targeting MAPK1 and PIK3R1 respectively. The combination of TMS and gefitinib promoted apoptosis also by miR‐345 and miR‐498 targeting the MAPK/c‐Fos and AKT/Bcl‐2 signalling pathways. Our study demonstrated that TMS reduced gefitinib resistance in NSCLCs via suppression of the MAPK/Akt/Bcl‐2 pathway by upregulation of miR‐345/498. These findings would lay the theoretical basis for the future study of TMS for the treatment of EGFR‐TKI resistance in NSCLCs.     

假单胞菌酶法转化DL-ATC合成L-半胱氨酸

采用微生物酶转化法制备L-半胱氨酸具有周期短、成本低、区域和立体选择性强、反应条件容易控制、环境友好等特点,与传统的毛发水解以及化学合成工艺相比显示出明显的优越性。本文从假单胞菌产酶条件和酶学性质、DL-ATC生物转化途径、固定化细胞转化工艺、基因工程菌的研究、以及L-半胱氨酸脱巯基酶的研究等5个方面介绍了国内外关于生物转化DL-2-氨基-Δ2-噻唑啉-4-羧酸(DL-ATC)合成L-半胱氨酸的研究进展。     

Microbial transformation of ginsenoside Rb1 by Rhizopus stolonifer and Curvularia lunata

Of 49 microbial strains screened for their capabilities to transform ginsenoside Rb₁, Rhizopus stolonifer and Curvularia lunata produced four key metabolites: 3-O-[-d-glucopyranosyl-(1,2)--d-glucopyranosyl]- 20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ene (1), 3-O-[-d-glucopyranosyl-(1,2)--d- glucopyranosyl]-20-O-[-d-glucopyranosyl]-3,12, 20(S)-trihydroxydammar-24-ol (2), 3-O-[-d-gluco- pyranosyl-(1,2)--d-glucopyranosyl]-3, 12, 20(S)-trihydroxydammar-24-ene (3), and 3-O--d-glucopyranosyl-3, 12, 20(S)-trihydroxydammar-24-ene (4), identified by TOF-MS, ¹H- and ¹³C-NMR spectral data. Metabolites 1, 3 and 4 were from the incubation with R. stolonifer, and 1 and 2 from the incubation with C. lunata. Compound 2 was identified as a new compound.