尖吻蝮蛇毒碱性磷脂酶A2的表达及其生化特征

将尖吻蝮蛇毒碱性磷脂酶A₂（A.aBPLA₂）基因克隆至温敏表达载体pBLMVL2,在大肠杆菌RR1中成功诱导表达.表达产物A.aBPLA₂约占细菌蛋白质总量的20％,并以包涵体的形式存在.纯化包涵体后,将产物变性、复性,然后用FPLC Superose^TM12纯化,产物经过SDS-聚丙烯酰胺凝胶电泳检测只有单一条带.对纯化后的表达A.aBPLA₂进行了酶活性、抑制血小板聚集活性和溶血活性的测定.结果显示,表达A.aBPLA₂的酶活性与变性后复性江浙蝮蛇酸性磷脂酶A₂酶活性相近,具有类似变性后复性江浙蝮蛇碱性磷脂酶A₂的溶血活性,没有抑制血小板聚集活性.最后对磷脂酶A₂的结构与这些活性的关系进行了讨论.     

GM3抑制人白血病J6－2细胞肌醇磷脂代谢循环

采用无载体 ³²P和[ ³H]肌醇标记磷脂,观察了促分化剂神经节苷脂GM₃对人单核样白血病J6-2细胞肌醇磷脂代谢的影响.GM₃抑制[ ³²P]Pi和[ ³H]肌醇掺入J6-2细胞磷脂酰肌醇(PI),促进[ ³²P]Pi和[ ³H]肌醇掺入磷脂酰肌醇-4,5-二磷酸(PIP₂),抑制[ ³²P]Pi掺入磷脂酸(PA),抑制[ ³H]肌醇掺入三磷酸肌醇(IP₃).GM₃的上述作用均为浓度依赖性的,随GM₃浓度的提高而增强.上述结果表明,GM₃抑制J6-2细胞的肌醇磷脂代谢循环.     

NaHCO3胁迫对紫根水葫芦形态学指标和光合参数的影响

为研究NaHCO₃胁迫对紫根水葫芦的形态学指标及光合参数的影响,该文以紫根水葫芦为材料,采用不同浓度碱性盐NaHCO₃溶液处理成株紫根水葫芦,测定在NaHCO₃胁迫下紫根水葫芦的植株株高、根长、根冠比、生物量、含水量和光合参数[净光合速率(P_n)、胞间CO₂浓度(C_i)、蒸腾速率(T_r)、气孔导度(G_s)]。结果表明:紫根水葫芦在20 mmol·L^-1 NaHCO₃浓度下的水溶液pH值最为平缓; 在低浓度NaHCO₃溶液中(≤40 mmol·L^-1),相比CK,紫根水葫芦的形态学指标呈现增长或无显著影响情况,而在高浓度NaHCO₃溶液中(≥60 mmol·L^-1),随着NaHCO₃浓度的升高,紫根水葫芦形态学指标显著降低,且与NaHCO₃浓度呈负相关; NaHCO₃胁迫对紫根水葫芦的光合参数影响显著,随着NaHCO₃浓度的增加及试验处理时间的延长,紫根水葫芦的P_n呈持续下降的趋势,C_i、T_r和G_s整体呈上升趋势,其光合作用受到的主要是非气孔限制。综合分析显示,紫根水葫芦具有一定的耐NaHCO₃能力,能正常生存在不超过40 mmol·L^-1 NaHCO₃浓度的水体中,且能改善低NaHCO₃浓度下的水体pH值。     

β-Agkistrodotoxin的聚合与异构体观察

中国江浙产短尾蝮蛇突触前神经毒素(β₁-Agkistrodotoxin,β₁-AgTx)经制备电泳被进一步纯化.经SDS-PAGE鉴定, 观察到天然β₁-AgTx以及经电泳纯化获得的碱溶β₁-AgTx-OH^-成分在电泳图谱上出现的聚合谱带, 然而这些聚合谱带的含量似乎并不随时程的延长而明显地增强.另经双向电泳分析发现, 无论是天然的β₁-AgTx或经电泳纯化获得的碱溶β₁-AgTx-OH^-成分均含有三个具有不同等电点(pI 7.0, 5.8和5.4)及不同含量比例的异构体谱带.天然β₁-AgTx中三个异构体的含量比并不随时程延长而明显改变, 碱溶β₁-AgTx-OH^-成分中的三个异构体的含量比则随着时程的延长而发生显著的变化.     

林下植被和凋落物对寒温带森林生长季土壤CH4通量的影响

为进一步探究林下植被和凋落物管理对我国寒温带森林生长季土壤CH₄通量的影响,采用静态箱-气相色谱法对大兴安岭北部4种林型（白桦林、山杨林、樟子松林和兴安落叶松林）4种处理（自然状态、去除凋落物、去除林下植被以及去除林下植被和凋落物）的土壤CH₄通量排放特征进行观测研究。结果表明：该地区森林生长季土壤均表现为CH₄的汇,4种林型不同处理后土壤CH₄通量表现为单峰变化趋势,吸收峰值出现在7月或8月。自然状态4种林型土壤CH₄平均吸收通量表现为白桦林（-79.23±14.92）μg m^-2 h^-1>山杨林（-64.27±9.60）μg m^-2 h^-1>樟子松林（-62.54±15.48）μg m^-2 h^-1>兴安落叶松林（-48.73±12.26）μg m^-2 h^-1,兴安落叶松土壤CH₄平均吸收通量显著小于其他三种林型（P<0.05）。相比于自然状态,4种林型在去除凋落物后土壤CH₄吸收通量提高了2.12%-12.15%,但变化幅度均没有达到显著水平（P>0.05）。去除林下植被后4种林型CH₄吸收通量提高了0.84%-20.55%,且只有山杨林吸收增加达到显著水平（P<0.05）。同时去除林下植被和凋落物后,对白桦林和樟子松土壤CH₄通量影响不显著（P>0.05）,但对山杨林和兴安落叶松林影响显著（P<0.05）。总之,去除凋落物或林下植被均会提高土壤对CH₄吸收,去除林下植被对土壤CH₄通量的影响要大于去除凋落物的影响,但不同林型不同处理之间还存在差异。     

苄基异喹啉类化合物 D20抑制钙调素激活磷酸二酯酶的研究

苄基异喹啉化合物是一类钙调素拮抗剂.对新合成的双苄基异喹啉化合物 D₂₀对钙调素依赖的磷酸二酯酶的抑制作用进行了研究,IC₅₀=5μmol/L,表明其拮抗作用大于三氟啦嗪,是强的拮抗剂.荧光分析表明,钙调素与化合物 D₂₀的结合常数为2.64(μmol/L)^-1,一个化合物 D₂₀分子与两个钙调素分子结合,并显示了结合方向性及空间位阻影响.     

不同CO2浓度升高和氮肥水平对水稻叶绿素荧光特性的影响

为研究不同CO₂浓度升高和氮肥水平对水稻叶绿素荧光特性的影响,利用由开顶式气室（OTC）组成的CO₂浓度自动调控平台开展田间试验。以粳稻9108为试验材料,CO₂浓度设置CK（对照,环境大气CO₂浓度）、C₁（CO₂浓度比CK增加160 μmol/mol）和C₂（CO₂浓度比CK增加200 μmol/mol）3个水平;氮肥设置低氮（N₁：10 g/m²）、中氮（N₂：20 g/m²）和高氮（N₃：30 g/m²）3个水平。结果表明,在低氮条件下,与CK相比,C₁处理使拔节期的F_o上升4.8%（P=0.031）;C₂处理使拔节期的F_o上升6.3%（P=0.015）,F_v/F_m下降4.8%（P=0.003）,使孕穗期的F_o上升12.7%（P=0.039）,F_v/F_o下降18.2%（P=0.039）。在高氮条件下,与CK相比,C₂处理使灌浆期的F_m、F_v和F_v/F_m分别下降3.6%（P=0.039）、4.9%（P=0.013）和1.3%（P=0.039）。在中氮条件下,与CK相比,C₁和C₂处理的影响不明显。在整个生育期内,CO₂浓度升高与施氮处理交互作用对水稻叶绿素荧光特性的影响未到达显著水平。研究表明,大气CO₂浓度升高使水稻叶片光系统Ⅱ受损,抑制其电子传递能力、电子受体Q_A氧化还原情况、最大光化学效率和潜在活性,通过适量施氮可以有效地缓解其负面效应。     

蛋白质和磷脂酶A2等电点的理论预测

应用计算数学方法计算了胰岛素等9种蛋白质的等电点,结果表明计算值同实验测定值比较吻合,二者间的差异不显著(P＞0.05).同时还预测了49种磷脂酶A₂的等电点.这一预测结果不仅对这类酶的分离纯化有一定参考价值,也有助于了解磷脂酶A₂同功酶的性质.     

八肽胆囊收缩素对大鼠大脑皮质细胞钙调素活性的影响

为了探讨胆囊收缩素(CCK)受体在中枢神经系统中的信号传递机制,观察了CCK₈和CCK_A受体拮抗剂L-364,718、CCK_B受体拮抗剂L-365,260对大鼠大脑皮质钙调素（CaM）活性的影响.结果表明：a.CCK₈对CaM活性的影响具有时间依赖性,15 min达到最高点后逐渐下降;b.CCK₈在10^－12～10^－7 mol/L范围内可刺激CaM活性的增加,超过10^－7 mol/L后,逐渐趋于饱和.c.CCK_A受体拮抗剂L-364,718、CCK_B受体拮抗剂L-365,260均可抑制10^－7 mol/L CCK₈引起的CaM活性的增加,但两者IC₅₀相差40倍,L-365,260在较低浓度时即能明显拮抗CCK₈引起的CaM活性变化.研究结果提示CCK₈可能通过CCK_B受体引起CaM活性变化,而CaM可能是CCK_B受体的重要信号传递机制之一.     

枯落物输入变化对云南松林地CO2释放的影响

为研究枯落物输入变化对云南松（Pinus yunnanensis）林地CO₂释放的影响。本研究于2018年3月至2020年2月,应用枯落物添加和去除实验（DIRT）,设置对照（CK）、双倍枯落物（DL）、去除枯落物（NL）、去除有机层和A层（O/A-Less）、去除根系（NR）和无输入（NI）6个处理水平,采用Li-6400便携式光合作用测量仪及TRIME-PICO 64/32土壤温度水分测定仪对不同处理样地每月的CO₂通量（R_s）、土壤温度和土壤水分（15cm）进行了测定。结果表明：（1）不同处理样地CO₂通量均呈现出明显的月变化,7至8月最高,1至4月最低,平均值表现为R_{s （DL）}=8.10 μmol m^-2 s^-1 > R_{s （CK）}=6.27 μmol m^-2 s^-1 > R_{s （NL）}=5.44 μmol m^-2 s^-1 > R_{s （NR）}=4.46 μmol m^-2 s^-1 > R_{s （O/A-Less）}=3.86 μmol m^-2 s^-1 > R_{s （NI）}=2.94 μmol m^-2 s^-1。（2）与CK相比,DL样地CO₂通量升高了29.12%,而去除地上枯落物和地下根系样地CO₂通量显著降低,CO₂通量平均变幅分别为α（NR）=-28.85%,α（NI）=-53.14%,α（O/A-Less）=-38.46%,α（NL）=-13.29%。（3）不同处理土壤水分和土壤温度均存在显著的月变化（P<0.01）,NL和O/A-Less的土壤水分显著低于CK,而其余处理与CK间无显著差异（P>0.05）;不同处理间土壤温度表现为NR和NI均显著高于CK,其余处理与CK间无显著差异（P>0.05）。（4）不同处理样地CO₂通量与土壤温度呈显著指数相关（P<0.01）,与土壤水分在NI和O/A-Less处理中无显著相关（P>0.05）;与CK相比,NI、O/A-Less和NL处理的Q₁₀增加,而NR和DL处理的Q₁₀则降低;不同处理林地CO₂通量与土壤水热因子双因素模型能更好的解释林地CO₂通量的变化。本研究表明枯落物不同处理通过改变土壤碳输入和土壤环境因子从而影响生态系统碳排放,研究结果可为未来气候变化和人为干扰下云南松林的碳循环提供基础数据。     

Optimization of 1-Deoxynojirimycin Extraction from Mulberry Leaves by Using Response Surface Methodology

Mulberry 1-deoxynojirimycin (DNJ, a potent α-glycosidase inhibitor) has therapeutic potency against diabetes mellitus. However, the amount of DNJ in mulberry leaves is low (about 0.1%), and a more effective extraction method is needed. Ultrasound-assisted extraction (UAE) was applied in this study for mulberry DNJ extraction, and five factors, the percentage of ethanol in the extraction solvent (x ₁), ratio of the extraction solvent to mulberry sample (x ₂), ultrasonic power (x ₃), extraction temperature (x ₄) and extraction time (x ₅), were investigated by fractional factorial 2^(5-1) design (FFD) to obtain the optimum extraction efficiency (DNJ yield, Y ₁) and extraction productivity (total yield, Y ₂). The results showed that x ₂, x ₃ and x ₅ had significant impact on Y ₁ and Y ₂, and were further optimized by response surface methodology (RSM). Under the optimized conditions (x ₂, x ₃ and x ₅ of 7 ml/g, 180 W and 260 s, respectively), DNJ-enriched powder (0.8%) was produced with high extraction efficiency (98%) and productivity (20%), enabling this product to be used for nutraceutical purposes.     

Extraction of Structural Extracellular Polymeric Substances from Aerobic Granular Sludge

To evaluate and develop methodologies for the extraction of gel-forming extracellular polymeric substances (EPS), EPS from aerobic granular sludge (AGS) was extracted using six different methods (centrifugation, sonication, ethylenediaminetetraacetic acid (EDTA), formamide with sodium hydroxide (NaOH), formaldehyde with NaOH and sodium carbonate (Na₂CO₃) with heat and constant mixing). AGS was collected from a pilot wastewater treatment reactor. The ionic gel-forming property of the extracted EPS of the six different extraction methods was tested with calcium ions (Ca²⁺). From the six extraction methods used, only the Na₂CO₃ extraction could solubilize the hydrogel matrix of AGS. The alginate-like extracellular polymers (ALE) recovered with this method formed ionic gel beads with Ca²⁺. The Ca²⁺-ALE beads were stable in EDTA, formamide with NaOH and formaldehyde with NaOH, indicating that ALE are one part of the structural polymers in EPS. It is recommended to use an extraction method that combines physical and chemical treatment to solubilize AGS and extract structural EPS.     

Optimization of the purification of mitochondrial F1-adenosine triphosphatase

A simple technique of purification of the soluble pig heart mitochondrial F₁-ATPase is described. It consists of removal of extrinsic proteins from mitochondrial membranes before extraction with chloroform and ammonium sulfate fractionation. A high degree of purity, an excellent stability and a good yield are attained after gel filtration through an Ultrogel ACA 34 column equilibrated in the presence of 50% glycerol. The tested properties of the F₁-ATPase prepared by this method are similar to those of the same enzyme extracted by sonication. The enzyme is virtually devoid of tightly bound nucleotides. In addition, some characteristics of the behaviour of the β subunit are shown.     

Application of Supercritical Fluid Extraction in Biotechnology

ABSTRACT

In the present paper recent investigations on the applications of supercritical fluid extraction (SCE) from post fermentation biomass or in situ extraction of inhibitory fermentation products as a promising method for increasing the yield of extraction have been reviewed. Although supercritical CO₂ (SC-CO₂) is unfriendly, or even toxic, for some living cells and precludes direct fermentation in dense CO₂, it does not rule out other useful applications for in situ extraction of inhibitory fermentation products and fractional extraction of biomass constituents. This technique is a highly desirable method for fractional extraction of biomass constituents, and intracellular metabolites due to the potential of system modification by physical parameters and addition of co-solvents to selectively extract compounds of different polarity, volatility and hydrophilicity without any contamination.     

CTAB-PEG DNA Extraction from Fungi with High Contents of Polysaccharides

Polysaccharides influence concentration and purity of extracted DNA. Here we present rapid and efficient protocol for DNA extraction from samples rich in polysaccharides. The technique has been developed using cultures of Schizophyllum commune and involves a modification of known Cetyltrimethyl Ammonium Bromide (CTAB) protocol. To remove polysaccharides, Polyethylene Glycol (PEG) 8000 was added during DNA precipitation. Genomic DNA obtained with the CTAB-PEG method had high integrity, with average fragment size >30 kb, the concentration higher than 100 ng/μL, and the yield more than 30 μg/g. Presented technique is suitable for DNA extraction from fungi, bacteria, archaea or even mollusks with high polysaccharide content.     

Accelerated solvent extraction of monacolin K from red yeast rice and purification by high-speed counter-current chromatography

Monacolin K from red yeast rice was extracted by accelerated solvent extraction (ASE). The effects of various extraction parameters including extraction temperature, static extraction time and cycle index on yield were investigated using a DIONEX ASE 300 system to select the optimal conditions by an orthogonal test design L₉ (3)³. The optimum extraction conditions were determined as follows: extraction temperature 120 °C, static extraction time 7 min, and cycle index 3. Under the optimal conditions, the yield of ASE extract and monacolin K was 5.35% and 9.26 mg/g of dry red yeast rice, respectively. A separation and purification method of monacolin K was then established using high-speed counter-current chromatography (HSCCC) with a two-phase solvent system composed of n-hexane–ethyl acetate–methanol–water (8:2:5:5, v/v/v/v). From 300 mg of crude extract, 51.2 mg of monacolin K was obtained with the purity of 98.7%. The chemical structure of isolated compound was identified by UV, ESI-MS and ¹H NMR.     

Effects of atmospheric CO2 enrichment, water status and applied nitrogen on water- and nitrogen-use efficiencies of wheat

Atmospheric CO₂ levels are expected to exceed 700 mol mol^–1 by the end of the 21st century. The influence of increased CO₂ concentration on crop plants is of major concern. This study investigated water- and nitrogen-use efficiency (WUE and NUE, respectively, were defined by the amount of biomass accumulated per unit water or N uptake) of spring wheat (Triticum aestivumL.) grown under two atmospheric CO₂ concentrations (350 and 700 mol mol^–1), two soil moisture treatments (well-watered and drought) and five nitrogen amendment treatments. Results showed that enriched CO₂ concentration increased canopy WUE, and more N supply led to higher WUE under the increased CO₂. Canopy WUE was significantly lower in well-watered treatments than in drought treatment, but increased with the increased N supply. Elevated CO₂ reduced the apparent recovery fraction of applied N by the plant root system (N_r, defined as the ratio of the increased N uptake to N applied), but increased the NUE and agronomic N efficiency (NAE, defined as the ratio of the increased biomass to N applied). Water limitation and high N application reduced the N_r, NUE and NAE, indicating a poor N efficiency. In addition, there was a close relationship between the root mass ratio and NUE. Canopy WUE was negatively related to the root mass ratio and NUE. Our results indicated that CO₂ enrichment enhanced WUE more at high N application, but increased NUE more when N application was less.     

Effects of proanthocyanidins on porcine pancreatic lipase: Conformation,activity, kinetics and thermodynamics

The interactions between proanthocyanidins (PC) and porcine pancreatic lipase (PL) were investigated from variant aspects of lipase conformation, activity, kinetics, and thermodynamics. Results show that 34% inhibitory rate of PC on PL is achieved after about 30-min incubation, and the inhibitory rate increases with the increase of PC concentration and then plateaus at the PC/PL ratio of 200. Circular dichroism and fluorescence spectroscopic analyses demonstrate that PC decreases the α-helix content while increases the β-sheet content of PL, but does not change the microenvironment of Trp, and PC quenches the fluorescence of PL both dynamically and statically through the formation of PL–PC complex. PC induces PL aggregation and then stabilizes the lipase aggregates. Kinetic studies reveal that PC does not change the K_m value while decreases the V_max value, implying that PC non-competitively inhibits the PL activity.     

Experimental and Model Studies on Continuous Separation of 2‐Phenylpropionic Acid Enantiomers by Enantioselective Liquid–Liquid Extraction in Centrifugal Contactor Separators

Multistage enantioselective liquid–liquid extraction (ELLE) of 2‐phenylpropionic acid (2‐PPA) enantiomers using hydroxypropyl‐β‐cyclodextrin (HP‐β‐CD) as extractant was studied experimentally in a counter‐current cascade of centrifugal contactor separators (CCSs). Performance of the process was evaluated by purity (enantiomeric excess, ee) and yield (Y). A multistage equilibrium model was established on the basis of single‐stage model for chiral extraction of 2‐PPA enantiomers and the law of mass conservation. A series of experiments on the extract phase/washing phase ratio (W/O ratio), extractant concentration, the pH value of aqueous phase, and the number of stages was conducted to verify the multistage equilibrium model. It was found that model predictions were in good agreement with the experimental results. The model was applied to predict and optimize the symmetrical separation of 2‐PPA enantiomers. The optimal conditions for symmetric separation involves a W/O ratio of 0.6, pH of 2.5, and HP‐β‐CD concentration of 0.1 mol L^?1 at a temperature of 278 K, where ee_eq (equal enantiomeric excess) can reach up to 37% and Y_eq (equal yield) to 69%. By simulation and optimization, the minimum number of stages was evaluated at 98 and 106 for ee_eq > 95% and ee_eq > 97%. Chirality 28:235–244, 2016. © 2016 Wiley Periodicals, Inc. Research highlights are as follows:

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Effects of ethanol and 3,4,-dihydron-2,2-dimethyl-2h-1-benzopyran-6-butyric acid on the solubility of sickle hemoglobin.

The effect of 3,4-dihydro-2,2-dimethyl-2H-1 benzopyran-6-butyric acid (DBA) on the solubility of deoxy-Hb S was evaluated by measuring saturation concentration, c_sat. Plots of c_sat versus DBA concentration in the presence or absence of ethanol gave two parallel lines, indicative of the additive fashion in which ethanol and DBA increase the solubility of deoxy-Hb S. At a $\text{DBA}\text{Hb}$ molar ratio of 10:1, c_sat was increased 16%. Ethanol alone increased c_sat comparably, but at a much higher molar excess (200:1). DBA had no effect on the oxygenation parameters of Hb S. Complementary solubility studies using the salting-out method showed that DBA had no effect on deoxy-Hb S, but decreased the solubility of deoxy-Hb A, oxy-Hb A and oxy-Hb S. Hence, no correlation exists between the effect of DBA on the solubility of deoxy-Hb S measured as c_sat and that measured by the salting-out technique.