Screening and characterization of astaxanthin-hyperproducing mutants of Haematococcus pluvialis

Haematococcus pluvialis was mutated by UV or ethyl methanesulphonate. Mutants resistant to nicotine, diphenylamine, fluridone or norflurazon were then selected. Several nicotine-resistant mutants showed increased (1.9% to 2.5% vs. 1.2% w/w) astaxanthin production. Mutants maintained high astaxanthin production over 4 months of repeated culture.     

禽致病性大肠杆菌毒力基因多重PCR方法的建立和应用

【目的】建立禽致病性大肠杆菌(Avian pathogenic Escherichia coli,APEC)黏附相关基因、侵袭及毒素相关基因、抗血清存活相关基因及铁转运相关基因的多重PCR方法,实现禽致病性大肠杆菌毒力基因的简便、快速检测。【方法】根据GenBank公布的基因序列,设计合成18对特异性引物,通过条件优化,建立四组多重PCR体系,并通过模板倍比稀释检测各组多重PCR的灵敏性。利用多重PCR检测100株APEC毒力基因的分布,验证多重PCR方法的可行性。【结果】根据PCR扩增片段大小判定,上述四组多重PCR体系均能同时扩增出该组中的各个毒力基因,且灵敏度分别为:103CFU、103CFU、105CFU、105CFU细菌和1ng、1ng、10ng、10ng DNA。100株APEC的毒力因子检测结果显示,多重PCR和单基因PCR结果一致。【结论】建立的四组多重PCR方法能够简便、快速地检测禽致病性大肠杆菌的毒力基因,可用于毒力基因的鉴定以及流行病学调查。     

Enhanced neurite outgrowth of rat neural cortical cells on surface-modified films of poly(lactic-<Emphasis Type="Italic">co</Emphasis>-glycolic acid)

Neural cortical cells, isolated from prenatal rat cerebra, were grown on surface-modified poly(lactic-co-glycolic acid, 65:35) (PLGA) films coated with poly-D-lysine (PDL) with either laminin (LN), fibronectin (FN) or collagen (CN). Immunocytochemistry showed that the isolated cells were highly immunopositive for both neurofilament and MAP-2 with well-organized neurites and somatodendritic localization. The presence of PDL with LN or FN on the PLGA films was essential for increased neural cell growth. Also, PLGA films coated with either PDL/LN or PDL/FN mixtures had higher neurite outgrowth and regular differentiation.Revisions requested 30 September 2004; Revisions received 10 November 2004     

Improving the Secretory Expression of an α-Galactosidase from Aspergillus niger in Pichia pastoris

α-Galactosidases are broadly used in feed, food, chemical, pulp, and pharmaceutical industries. However, there lacks a satisfactory microbial cell factory that is able to produce α-galactosidases efficiently and cost-effectively to date, which prevents these important enzymes from greater application. In this study, the secretory expression of an Aspergillus niger α-galactosidase (AGA) in Pichia pastoris was systematically investigated. Through codon optimization, signal peptide replacement, comparative selection of host strain, and saturation mutagenesis of the P1’ residue of Kex2 protease cleavage site for efficient signal peptide removal, a mutant P. pastoris KM71H (Mut^s) strain of AGA-I with the specific P1’ site substitution (Glu to Ile) demonstrated remarkable extracellular α-galactosidase activity of 1299 U/ml upon a 72 h methanol induction in 2.0 L fermenter. The engineered yeast strain AGA-I demonstrated approximately 12-fold higher extracellular activity compared to the initial P. pastoris strain. To the best of our knowledge, this represents the highest yield and productivity of a secreted α-galactosidase in P. pastoris, thus holding great potential for industrial application.     

The effect of different platelet-rich plasma concentrations on proliferation and differentiation of human periodontal ligament cells in vitro

OBJECTIVES: The use of platelets and platelet products has become increasingly popular clinically as a means of accelerating endosseous wound healing. It is likely that growth factors released by activated platelets at the site of injury play a role in periodontal regeneration by regulating cellular activity. The purpose of this study was to evaluate the biological effects of platelet-rich plasma (PRP) on human periodontal ligament cells (hPDLCs) in vitro. MATERIALS AND METHODS: Primary cultures of hPDLCs were obtained from healthy premolars. PRP was isolated by two-step centrifugation. Two main growth factors present in the thrombin-activated PRP (platelet-derived growth factor [PDGF-AB] and transforming growth factor-beta1 [TGF-beta1]) were evaluated using ELISA assay. Activated PRP or the combination of recombined human TGF-beta1 (rhTGF-beta1) and PDGF-AB (rhPDGF-AB) were added to hPDLCs in different concentrations to assess cell proliferation and osteogenic differentiation. RESULTS: PRP contained high levels of TGF-beta1 and PDGF-AB. Cell attachment, proliferation and ALP activity were enhanced by addition of PRP or rhTGF-beta1 and rhPDGF-AB combination to the cell cultures, while the stimulatory potency of PRP was much greater than the latter. These stimulatory effects presented in a dose-dependant manner, it seemed that PRP with 50~100 ng/ml TGF-beta1 was an ideal concentration. CONCLUSIONS: PRP can enhance hPDLC adhesion, proliferation and induce the differentiation of hPDLC into mineralized tissue formation cell; thereby contribute to the main processes of periodontal tissue regeneration. For economical and biological reasons, PRP has more clinical beneficial than analogous growth factors.     

Mass transfer in blood oxygenators using blood analogue fluids

Mass transfer correlations for hollow fiber blood oxygenators have been determined experimentally using Newtonian and non-Newtonian blood analogue fluids. The Newtonian fluids consisted of deionized water and glycerol/water mixtures. The non-Newtonian fluids were prepared by adding small amounts of xanthan gum to the Newtonian blood analogue fluids. The rheological behavior of the non-Newtonian blood analogue fluids was modeled using the power law. The diffusion of oxygen into and out of the Newtonian and non-Newtonian blood analogue fluids has been studied. The liquid stream flowed outside and across bundles of woven hollow fibers, while the gas stream flowed inside the fibers.     

高羊茅胚性悬浮细胞系的快速建立及其单细胞培养

本研究采用高羊茅(Millennium和Hundog Ⅴ)成熟种子为材料,以MS为基本培养基,通过添加2.5 mg/L CuSO4·5H2O,或提高NH4NO3浓度至2.5 g/L等措施均能明显提高愈伤组织的质量,经过3～5个月的筛选获得疏松干燥、颗粒状、生长旺盛、适合悬浮培养的Ⅱ型胚性愈伤组织.悬浮培养初期需用MSⅠ液体培养基进行一个月的启动培养,之后转用MSⅡ继代保持,约2个月左右即建立起来自高羊茅2个品种的3个悬浮细胞系.生长特性测定结果表明,悬浮培养的初始接种量以1.0～3.0 ml/40 ml为宜,生长周期内其pH值不断波动变化,最适范围在5.0～5.7之间.5～8 d为悬浮系的对数生长期,此时细胞分裂旺盛,增殖较快,是分离单细胞的最佳时期.单细胞培养方式以悬浮培养效果最好.     

树干径流过程的动态响应模型

本文用实验测试法测量了干流过程。结果表明,干流过程曲线与R-L-C电路瞬态响应过程(包括输入、输出曲线)极为相似。据此,可用R-L-C电路瞬态响应过程曲线拟合干流过程,求出解析解。在计算机上调试参数,讨论其水文学意义,定性分析参数与影响干流因子的关系,取得了满意的结果。