产朊假丝酵母全细胞转化合成谷胱甘肽的营养条件

为了进一步提高产朊假丝酵母全细胞转化生物合成谷胱甘肽(GSH)的能力,利用响应面分析方法对酵母培养的发酵培养基进行优化。在单因素实验的基础上,通过Plackett-Burman设计筛选出显著影响GSH转化力的2个主要因素:葡萄糖和KH2PO4。采用最陡爬坡试验和响应面设计预测了葡萄糖和KH2PO4的最佳质量浓度分别为58.5和17.2 g/L。验证实验结果表明,在该优化培养基条件下,酵母细胞的GSH转化力为1.54 mg/(g·h),比优化前提高了1倍。该结果为类似的采用全细胞转化法高效合成有用化学品的研究与开发提供了可行的优化思路。     

展示南极假丝酵母脂肪酶B的毕赤酵母全细胞催化合成短链芳香酯

展示酶的酵母细胞作为全细胞催化剂,既具有固定化酶的优点,又有制备简单、成本较低的特点。本研究将细胞表面展示南极假丝酵母脂肪酶B(Candida antarctica lipase B,CALB)的重组毕赤酵母用于非水相中催化合成短链芳香酯,通过滴定和气相色谱的方法测定底物酸的转化率,从底物的碳链长度、醇的结构、酵母冻干粉的添加量、底物浓度及底物的酸醇摩尔比等方面考察了展示CALB的毕赤酵母全细胞催化合成短链芳香酯的特性。研究结果表明:该全细胞催化剂可催化C10以下的酸和醇直接酯化合成多种短链芳香酯,酸的转化率达到90%以上;其中己酸和乙醇为酶的最适底物;酵母冻干粉的添加量20g/L(306.0U/g-drycell)、己酸浓度0.8mol/L、酸醇摩尔比1:1.1是合成己酸乙酯的最佳条件。在此条件下反应1.5h,己酸的转化率达到97.3%。在现有的关于脂肪酶非水相催化合成短链芳香酯的报道中,该全细胞催化剂显示出较好的底物耐受性以及较高的催化反应速率。因此,展示CALB的毕赤酵母全细胞催化剂在合成短链芳香酯方面具有较大的商业化应用潜能。     

南极假丝酵母脂肪酶A的表面展示及酶学性质研究

采用a凝集素作为载体蛋白,首次将南极假丝酵母脂肪酶A展示在酿酒酵母细胞表面,通过MD平板筛选获得表面展示型的CALA酵母工程菌株。免疫荧光检测显示CALA被成功展示在酵母细胞壁表面,重组子经诱导后能在三丁酸甘油酯板上形成透明圈,说明展示的CALA具有活性。重组酵母在液体培养基培养72 h,活性达到最高,为80.4 U/g干细胞。酿酒酵母展示的CALA最适温度及pH值为70°C和pH 8.0。经50°C保温2 h,仍含有60%水解酶活力。展示的CALA在pH 7.0和pH 8.0溶液中比较稳定。经DMSO处理2 h,展示的CALA仍保持70%的活性。以上结果表明酵母展示的CALA可作为一种有潜质商业用途的全细胞催化剂。     

热带假丝酵母转化烷烃过程中P450酶活的研究

a-、ω-长链二元酸(α-、ω-Long Chin Dicarboxylic Acid,DCA)是一种重要的化工原料,是合成工程塑料、香料、耐寒性增塑剂、涂料和液晶等物质的主要原料.目前,人们主要通过热带假丝酵母(Candidatropicalis)代谢烷烃来生产从DCA11到DCA18等不同碳链长的二元酸[1,2].多年来在各种微生物,尤其是假丝酵母的烷烃氧化途径方面有大量的研究[3,4].在假丝酵母转化烷烃生成长链二元酸的代谢过程中[5-7],烷烃被吸引进入细胞后,首先经过细胞色素P450酶(Cy-tochrome P450)氧化生成a-一元醇,再进一步被氧化生成a-一元酸,引过程称为a-氧化.     

高产小麦旗叶细胞结构与产量关系的研究

本文旨在揭示小麦旗叶结构与产量的关系.通过时四个品种小麦旗叶进行离析、显微观察、拍照、数据统计,并计算出叶肉细胞环数的期望,分别作了同产量的相关分析和回归分析.结果表明,气孔密度、气孔面积、表皮细胞面积、叶肉细胞环数的期望同产量呈正相关关系,并建立了同产量的回归方程.     

急性淋巴细胞白血病患儿血清25羟维生素D3、乳酸脱氢酶、白细胞介素-6水平与危险度分层和预后不良的关系研究

摘要 目的：探讨急性淋巴细胞白血病（ALL）患儿血清25羟维生素D3（25-OH-D3）、乳酸脱氢酶（LDH）、白细胞介素-6（IL-6）水平与危险度分层和预后不良的关系。方法：选择2018年6月至2019年8月在我院住院治疗的ALL患儿60例为病例组,另选取同期到我院健康查体的同龄健康儿童60例为对照组。采用酶联免疫吸附试验（ELISA）检测血清25-OH-D3和IL-6水平,采用全自动生化分析仪检测血清LDH水平,对比病例组与对照组血清25-OH-D3、LDH、IL-6水平,对比不同危险度分层ALL患儿血清25-OH-D3、LDH、IL-6水平,分析其与危险度分层的相关性。ALL患儿进行规范化治疗,根据治疗结果分为预后良好组和预后不良组,对比预后良好组和预后不良组临床特征及血清25-OH-D3、LDH、IL-6水平,采用COX比例风险回归模型分析预后不良的危险因素。结果：病例组患儿血清25-OH-D3水平低于对照组,血清LDH、IL-6水平高于对照组（P＜0.05）。低危型、中危型ALL患儿血清25-OH-D3水平高于高危型,且低危型高于中危型（P＜0.05）;低危型、中危型ALL患儿血清LDH、IL-6水平低于高危型,且低危型低于中危型（P＜0.05）。预后不良组高危型患儿及白细胞计数＞100×10⁹/L的患儿所占比例、血清LDH及IL-6水平高于预后良好组（P＜0.05）,血清25-OH-D3水平低于预后良好组（P＜0.05）。血清25-OH-D3水平与ALL患儿危险度分层呈负相关（P＜0.05）,血清LDH、IL-6水平与ALL患儿危险度分层呈正相关（P＜0.05）。危险度分层及血清25-OH-D3、LDH、IL-6是ALL患儿预后不良的危险因素（P＜0.05）。结论：ALL患儿血清25-OH-D3、LDH、IL-6水平异常改变,与危险度分层相关,是预后不良的危险因素。     

A Link between Hinge-Bending Domain Motions and the Temperature Dependence of Catalysis in 3-Isopropylmalate Dehydrogenase

Enzyme function depends on specific conformational motions. We show that the temperature dependence of enzyme kinetic parameters can provide insight into these functionally relevant motions. While investigating the catalytic properties of IPMDH from Escherichia coli, we found that its catalytic efficiency (k_cat/K_M,IPM) for the substrate IPM has an unusual temperature dependence, showing a local minimum at ∼35°C. In search of an explanation, we measured the individual constants k_cat and K_M,IPM as a function of temperature, and found that the van 't Hoff plot of K_M,IPM shows sigmoid-like transition in the 20-40°C temperature range. By means of various measurements including hydrogen-deuterium exchange and fluorescence resonance energy transfer, we showed that the conformational fluctuations, including hinge-bending domain motions increase more steeply with temperatures >30°C. The thermodynamic parameters of ligand binding determined by isothermal titration calorimetry as a function of temperature were found to be strongly correlated to the conformational fluctuations of the enzyme. Because the binding of IPM is associated with a hinge-bending domain closure, the more intense hinge-bending fluctuations at higher temperatures increasingly interfere with IPM binding, thereby abruptly increasing its dissociation constant and leading to the observed unusual temperature dependence of the catalytic efficiency.     

Regulation of Glycerol Phosphate Dehydrogenase and Lactate Dehydrogenase Activity by Forskolin and Dibutyryl Cyclic AMP in the C6 Glial Cells

We have compared the effects of norepinephrine, forskolin, and dibutyryl cyclic AMP (Bt2cAMP) on the regulation of the cytosolic enzyme glycerol phosphate dehydrogenase (GPDH) in the C6 rat glioma cell line. Forskolin and Bt2cAMP elicit a dose-dependent increase in the levels of the enzyme that was, however, unaffected by norepinephrine. The half-maximal effect of forskolin was obtained at 7-8 microM, and the effect was maximal at 30 microM. Dexamethasone at a 50 nM concentration produced a two- to sixfold induction of GPDH after 48 h. The combination of dexamethasone with forskolin or Bt2cAMP leads to an elevation in GPDH levels that is higher than that produced by one of the compounds alone. This potentiation is found when both agents are added together with or after the glucocorticoid. The increase in uninduced and dexamethasone-induced GPDH activity was blocked by cycloheximide and actinomycin D, indicating that de novo protein and RNA synthesis are required. The activity of cytosolic lactate dehydrogenase activity did not change after incubation with dexamethasone, but increased with forskolin or Bt2cAMP.     

Relationship Between Activation State of Pyruvate Dehydrogenase Complex and Rate of Pyruvate Oxidation in Isolated Cerebro-Cortical Mitochondria: Effects of Potassium Ions and Adenine Nucleotides

The relation between the activation (phosphorylation) state of pyruvate dehydrogenase complex (PDHC; EC 1.2.4.1, EC 2.3.1.12, and EC 1.6.4.3) and the rate of pyruvate oxidation has been examined in isolated, metabolically active, and tightly coupled mitochondria from rat cerebral cortex. With pyruvate and malate as the substrates, the activation state of PDHC decreased on addition of ADP, while the rates of oxygen uptake and 14CO2 formation from [1-14C]pyruvate increased. The lack of correlation between the activation state of PDHC and rate of pyruvate oxidation was seen in media containing 5, 30, or 100 mM KCl. Both the activation state of PDHC and pyruvate oxidation increased, however, when KCl was increased from 5 to 100 mM. Although the PDHC is inactivated by an ATP-dependent kinase (EC 2.7.1.99), direct measurement of ATP and ADP failed to show a consistent relationship between the activation state of PDHC and either ATP levels or ATP/ADP ratios. Comparison of the activation state of PDHC in uncoupled or oligomycin-treated mitochondria also failed to correlate PDHC activation state to adenine nucleotides. In brain mitochondria, unlike those from other tissues, the activation state of PDHC does not seem to be related clearly to the rate of pyruvate oxidation, or to the mitochondrial adenylate energy charge.     

细胞培养液乳酸脱氢酶漏出率的比色测定及其应用

采用乳酸脱氢酶比色法测定细胞培养液乳酸脱氢酶的漏出率是一个稳定而实用的指标。本法既可客观衡量细胞的受损程度,也可进行药物干预的正确评价,也是进行药物筛选的一种有效方法。     

Export of functional <Emphasis Type="Italic">Streptomyces coelicolor</Emphasis> alditol oxidase to the periplasm or cell surface of <Emphasis Type="Italic">Escherichia coli</Emphasis> and its application in whole-cell biocatalysis

Streptomyces coelicolor A3(2) alditol oxidase (AldO) is a soluble monomeric flavoprotein in which the flavin cofactor is covalently linked to the polypeptide chain. AldO displays high reactivity towards different polyols such as xylitol and sorbitol. These characteristics make AldO industrially relevant, but full biotechnological exploitation of this enzyme is at present restricted by laborious and costly purification steps. To eliminate the need for enzyme purification, this study describes a whole-cell AldO biocatalyst system. To this end, we have directed AldO to the periplasm or cell surface of Escherichia coli. For periplasmic export, AldO was fused to endogenous E. coli signal sequences known to direct their passenger proteins into the SecB, signal recognition particle (SRP), or Twin-arginine translocation (Tat) pathway. In addition, AldO was fused to an ice nucleation protein (INP)-based anchoring motif for surface display. The results show that Tat-exported AldO and INP-surface-displayed AldO are active. The Tat-based system was successfully employed in converting xylitol by whole cells, whereas the use of the INP-based system was most likely restricted by lipopolysaccharide LPS in wild-type cells. It is anticipated that these whole-cell systems will be a valuable tool for further biological and industrial exploitation of AldO and other cofactor-containing enzymes.     

Adaboost算法研究土壤微量元素含量与白血病的关系

目的：本文使用数据挖掘方法研究土壤中微量元素和白血病的相关性。方法：使用CFS-Adaboost算法研究我国29个省、市、自治区白血病死亡率的统计数据和土壤中微量元素含量的对应关系。结果：从29种微量元素中发现了13种微量元素与白血病相关,其中砷（As）的相关性较为明显。基于该13种微量元素,建立了土壤中微量元素和白血病致死率的数学模型,该模型的预报准确率可达到89.7%。结论：土壤中微量元素的含量与白血病有一定关系,其中砷（As）元素含量与白血病死亡率较为密切,这与近年文献报导的少量氧化砷治疗白血病效果显著相符合。以上研究发现,可以为研究土壤中微量元素和白血病的关系提供参考,对白血病的防治工作具有一定意义。     

Adaboost算法研究土壤微量元素含量与白血病的关系(英文)

目的:本文使用数据挖掘方法研究土壤中微量元素和白血病的相关性。方法:使用CFS-Adaboost算法研究我国29个省、市、自治区白血病死亡率的统计数据和土壤中微量元素含量的对应关系。结果:从29种微量元素中发现了13种微量元素与白血病相关,其中砷(As)的相关性较为明显。基于该13种微量元素,建立了土壤中微量元素和白血病致死率的数学模型,该模型的预报准确率可达到89.7%。结论:土壤中微量元素的含量与白血病有一定关系,其中砷(As)元素含量与白血病死亡率较为密切,这与近年文献报导的少量氧化砷治疗白血病效果显著相符合。以上研究发现,可以为研究土壤中微量元素和白血病的关系提供参考,对白血病的防治工作具有一定意义。     

循环肿瘤细胞水平与卵巢癌患者临床病理特征及生存率的关系研究

目的:研究循环肿瘤细胞(CTC)水平与卵巢癌患者临床病理特征及生存率的关系。方法:选择从2012年6月到2014年12月在我院就诊的卵巢癌患者94例记为观察组。根据所有患者CTC水平将其分成高CTC组(CTC水平≥7个/5mL)57例及低CTC组(CTC水平7个/5 m L)37例,另选同期在我院体检的卵巢良性囊肿者90例记为对照组,分析CTC阳性与患者病理特征的关系、CTC水平与患者生存率和复发转移的关系以及CTC水平与患者病理特征、生存率、复发转移率的相关性。结果:对照组90例患者均未检测出CTC阳性。观察组94例患者中检测出68例CTC阳性,总阳性率为72.34%;观察组患者临床分期为II期和III期的CTC阳性率均明显高于I期,有淋巴结转移的CTC阳性率明显高于无淋巴结转移(P0.05)。高CTC组的1年、2年、3年生存率与低CTC组相比,差异均无统计学意义(P0.05)。高CTC组的1年、2年、3年复发转移率均高于低CTC组(P0.05)。Spearman相关性分析结果显示,CTC水平与患者的临床分期、淋巴结转移以及复发转移率均呈正相关(P0.05),而与年龄、病理分型、病理分级及生存率均无明显相关性(P0.05)。结论:卵巢癌患者临床分期为Ⅱ期和Ⅲ期和有淋巴结转移的CTC阳性率较高,且CTC水平与复发转移率关系密切,临床通过检测CTC水平,可能有助于评估患者的预后。     

E—cad、EGFR、MMP9与肺癌转移的关系

目的：探讨原发性非小细胞肺癌（NSCLC）组织中E-钙粘蛋白（E—cadherin,E—cad）、表皮生长因子受体（Epidermal Growth Factor Receptor,EGFR）和基质金属蛋白酶9（matrix metalloproteinase-9,MMP9）的表达与术后复发及转移的关系。方法：选择临床IB期非小细胞肺癌．总者的手术切除标本,其中36例于36个月之内发生复发或转移,30例术后无病生存期超过36个月。采用免疫组织化学方法检测组织中E-cad、EGFR、MMP9的表达情况。结果：36例早转移IB期非小细胞肺癌术后患者的手术标本中,E-cad的异常表达率、EGFR及MMP9的阳性表达率,均明显高于30例非早转移IB期非小细胞肺癌术后患者（P〈0．05）,且在早转移IB期NSCLC中,三者的表达与病理分级、肿瘤大小有关（P〈0．05）,与病理类型,肿瘤是否浸润胸膜无关（P〉0．05）。结论：对早期肺癌术后病人,检测E-cad、EGFR、MMP9的表达,对判断其恶性程度,提示肿瘤的复发及转移倾向大小,决定术后治疗方案有重要意义。     

The Relationship between Egg Size and the Rate of Early Development in Arctic charr, Salvelinus alpinus

Previous studies have found no consistent relationship between egg size and the rate of development in fish. This is surprising since there are several hypothesis as to why such a relationship should exist. By using eggs from Arctic charr, Salvelinus alpinus, we examined several developmental features repeatedly on live individuals. In all measured parameters except date of hatching, embryos from smaller eggs were found to develop faster. Time of hatching has often been used as a principal measurement for rate of development in fish, but our results suggest that this should be discontinued, since it is poorly correlated with the rates of development of other early life history features.