Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ　Ｂｌｕｅ　Ｆ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法。实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦瓜毒素（ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析显示三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光诸明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合。将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕｅｄｅｘ。Ｂｌｕｅｄｅｘ亲和层析是一种经济有效,简单易行,便于在各类实验室中使用的蛋白质亲和层析技术。结果表明：在低盐溶液中ＲＴＡ和ＭＴ均可迅速地与Ｂｌｕｅｄｅｘ结合,而在高盐溶液中（０．６５ｍｏｌ／ＬＮａＣｌ）又极易被洗脱回收。这一技术用于免疫毒素的研究可有效地去除游离抗体,而不影响其杀伤活性。     

天花粉蛋白Glu160在N-糖苷酶活性中的作用

用浸泡法得到了（Ｅ１６０Ａ）天花粉蛋白（ｔｒｉｃｈｏｓａｎｔｈｉｎ, ＴＣＳ）,（Ｅ１６０Ｄ）ＴＣＳ与Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ与ＦＭＰ复合物的晶体．在Ｍａｒ Ｒｅｓｅａｒｃｈ 面探测器系统上分别收集了０．２０ ｎｍ ,０．１９ｎｍ 和０．２０５ ｎｍ 分辨率的Ｘ 射线衍射数据,数据处理用Ｍａｒ Ｓｃａｌｅ 程序系统完成．用同晶差值Ｆｏｕｒｉｅｒ法解析了（Ｅ１６０Ａ）ＴＣＳ－Ａｄｅ,（Ｅ１６０Ｄ）ＴＣＳ－Ａｄｅ 和（Ｅ１６０Ａ）ＴＣＳ－ＦＭＰ的晶体结构,结构修正利用Ｘ－ＰＬＯＲ程序,修正结果,晶体学Ｒ因子分别为０．１６６,０．１７６,０．１７９．键长和键角的ＲＭＳ偏差分别为０．００１０ ｎｍ 和２．５０３°,０．００１３ ｎｍ 和２．６６５°,０．００１２ ｎｍ 和２．６７６°．在这三个结构中均未见到Ｇｌｕ１８９侧链方向的改变．Ａｄｅ 或ＦＭＰ仍结合在Ｎ－糖苷酶活性口袋之中,它夹在Ｔｙｒ７０和Ｔｙｒ１１１两个侧链环之间,与Ｔｙｒ７０环近乎平行．这一结果表明：ＴＣＳ中的Ｇｌｕ１６０分别突变成Ａｌａ 和Ａｓｐ,仍能与ＡＭＰ发生Ｎ－糖苷酶反应,但是活性降低了一些．可见Ｇｌｕ１６０对ＴＣＳ与ＡＭＰ的作用是重要的,但不是必要的．     

蛇毒蛋白Echistatin的C端突变基因的构建,表达及其活性研究

通过ＰＣＲ定点突变的技术,将蛇毒蛋白Ｅｃｈｉｓｔａｔｉｎ基因的Ｃ端进行了突变（Ａｌａ４８→Ａｒｇ４８→,Ｔｈｒ４９→Ｖａｌ４９）,模拟纤维蛋白Ｎ端的四肽（Ｇｌｙ－Ｐｒｏ－Ａｒｇ－Ｖａｌ）,以期增加Ｅｃｓ（Ｅｃｈｉｓｔａｔｉｎ）的活性,突变的基因重组到表达质粒ｐＪＣ２６４上,经ＩＰＴＧ诱导,以ＣｈｅＹ－Ｅｃｓ融合蛋白方式进行了表达,表达量占菌体总蛋白的１５￣２０％,Ｓｅｐｈａｄｅｘ Ｇ－７５初步纯化     

Cibacron Blue亲和层析及应用

以Ｆ３ＧＡ（Ｃｉｂａｃｒｏｎ ＢｌｕｅＦ３ＧＡ）为配基建立了一种可用于免疫毒素（ＩＴ）分离纯化的亲和层析方法,实验中用三种不同来源的核糖体灭活蛋白（ＲＩＰ）,即蓖麻毒素Ａ链（ＲＴＡ）,苦撤毒素（Ｍｏｍｏｒｄｉｎ,ＭＴ）和Ｓａｐｏｒｉｎ,以探讨ＲＩＰ与Ｆ３ＧＡ的相互作用。分析三种ＲＩＰ均能引起Ｆ３ＧＡ吸收光谱明显红移,提示ＲＩＰ均可与Ｆ３ＧＡ发生特异结合,将Ｆ３ＧＡ与Ｓｅｐｈａｄｅｘ交联可获得Ｂｌｕ     

黑龙江铜木蚜蝇雌性的描述

黑龙江铜木蚜蝇雌性的描述ＤＥＳＣＲＩＰＴＩＯＮＯＦＴＨＥＦＥＭＡＬＥＣＨＡＬＣＯＳＹＲＰＨＵＳ（ＸＹＬＯＴＯＭＩＭＡ）ＡＭＵＲＥＮＳＩＳ（ＳＴＡＣＫＥＬＢＥＲＧ）（ＤＩＰＴＥＲＡ：ＳＹＲＰＨＩＤＡＥ）￥ＨＥＪｉｌｏｎｇ;ＣＨＵＸｉｐｉｎｇ（Ｄｅｐａｒ...     

药物诱导后的人大肠癌细胞的[Ca^2＋]i的变化

采用荧光分光光度计法检测维甲酸（ＲＡ）、１,２５（ＯＨ）２ＶＤ３及佛波酯（ＰＭＡ）诱导ＣＣＬ２２９细胞分化后［Ｃａ２＋］ｉ变化,并观察内质网（ＥＲ）特异的Ｃａ２＋－ＡＴＰａｓｅ抑制剂Ｔｈａｐｓｉｇａｒｇｉｎ（ＴＧ）、ＩＰ３受体抑制剂Ｈｅｐａｒｉｎ对ＲＡ诱导［Ｃａ２＋］ｉ变化的影响,从而探讨ＲＡ诱导［Ｃａ２＋］ｉ变化与ＥＲ的关系。结果显示：ＲＡ和１,２５（ＯＨ）２ＶＤ３在数秒内引起［Ｃａ２＋］ｉ显著升高。在ＥＧＴＡ和Ｖｅｒａｐａｍｉｌ预处理细胞条件下,ＴＧ不能抑制ＲＡ引起Ｃａ２＋从细胞内钙池中外流,ＲＡ作用后ＴＧ仍能升高［Ｃａ２＋］ｉ。另外,Ｈｅｐａｒｉｎ也不能完全抑制ＲＡ升高［Ｃａ２＋］ｉ。提示ＲＡ诱导大肠癌细胞升高［Ｃａ２＋］ｉ可能通过ＥＲ上ＩＰ３敏感性和非敏感性钙池,亦可能细胞内存在除ＥＲ外对ＲＡ敏感的钙池。     

天花粉蛋白Y14F/R22L定点突变及其活性研究

利用多聚酶链式反应（ＰＣＲ）技术,对天然天花粉蛋白（ｎＴＣＳ）基因在Ｔｙｒ１４和Ａｒｇ２２两个保守残基处同时进行定点突变,即Ｔｙｒ１４变成Ｐｈｅ,Ａｒｇ２２变成Ｌｅｕ,然后克隆到ｐＥＴ－８ｃ高效表达载体上,构建成重组质粒ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ．经序列分析,定点突变的结果与预先设计的完全一致,突变后的天花粉蛋白命名为Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ．将ｐＥＴＹ１４Ｆ／Ｒ２２Ｌ转化到Ｅ．ｃｏｌｉＢＬ２１（ＤＥ３,ｐＬｙｓＳ）中,进行表达．经ＣＭ－ＳｅｐｈａｒｏｓｅＣＬ－６Ｂ柱纯化,ＳＤＳ－ＰＡＧＥ鉴定,纯度可达９０％．ＲＩＰ活性测定显示,Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ的活性比ｎＴＣＳ降低了７．５倍,活性变化不显著,因此,ＴＣＳ的Ｔｒｙ１４和Ａｒｇ２２对维持其活性部位构象并不是必需的．但由于Ｙ１４Ｆ／Ｒ２２ＬＴＣＳ在Ｅ．ｃｏｌｉ中的表达量与ｎＴＣＳ相比明显下降,因此,Ｔｙｒ１４和Ａｒｇ２２可能与ＴＣＳ翻译后的折叠有关．     

(E160A)和(E160D)天花粉蛋白两种突变体晶体结构研究

培养了（Ｅ１６０Ａ）ＴＣＳ和（Ｅ１６０Ｄ）ＴＣＳ的单晶。在ＭＡＲＲｅｓｅａｒｃｈ面探测器系统上分别收集了０．１９３ｎｍ和０．２０ｎｍ分辨率的Ｘ射线衍射数据。数据处理用ＭＡＲＳＣＡＬＥ程序系统完成。用同晶差值Ｆｏｕｒｉｅｒ法解析了突变体的晶体结构,结构修正利用Ｘ－ＰＬＯＲ程序。修正结果,晶体学Ｒ因子分别为０．１７５,０．１７９,键长和键角的ＲＭＳ偏差分别为０．００１１ｎｍ和２．４５７°,０．００１３ｎｍ和２．６７５°。在这两个突变体的结构中均未见到Ｇｌｕ１８９侧链方向的改变。通过对（Ｅ１６０Ａ）ＴＣＳ和（Ｅ１６０Ｄ）ＴＣＳ的结构比较,说明（Ｅ１６０Ｄ）ＴＣＳ活性低于（Ｅ１６０Ａ）ＴＣＳ的原因：这可能是由于在（Ｅ１６０Ｄ）ＴＣＳ中Ｔｙｒ１１１和Ｔｙｒ７０的侧链都具有较大的运动性,使它们与腺嘌呤碱基的芳香堆垛作用减弱,从而导致活性的降低     

大肠杆菌rpoH基因的克隆,表达及其产物的纯化

本文用ＰＣＲ方法获得大肠杆菌热休克蛋白转录因子σ３２的编码基因ｒｐｏＨ,并克隆在含有ｔａｃ启动子的表达载体ｐＵＨＥ中,经ＩＰＴＧ诱导,在大肠杆菌中表达了Ｃ端融合有６个寡聚组氨酸的σ３２。表达产物经金属螯合层析一步纯化,达到ＳＤＳ－ＰＡＧＥ银染一条带纯度,氨基酸组成分析及Ｎ端序列分析结果与文献报道一致。３５Ｓ细胞内参入实验表明：即使在较低的温度下,表达产物σ３２（Ｈｉｓ）６也能导致热休克蛋白如ＧｒｏＥｌ、ＤｎａＫ、Ｈｔｐ的大量合成．     

大肠杆菌精氨酰－tRNA合成酶的Lys306为酶活力所必需

将大肠杆菌精氨酰ｔＲＮＡ合成酶（ＡｒｇＲＳ）上Ｌｙｓ３０６用基因点突变的方法分别变为Ａｌａ和Ａｒｇ的密码子;得到变种基因ａｒｇｓ３０６ＫＡ和ａｒｇｓ３０６ＫＲ。变种基因重组在ｐＵＣ１８上,转化到大肠杆菌ＴＧ１中,转化子中ＡｒｇＲＳ及其变种ＡｒｇＲＳ３０６ＫＡ和ＡｒｇＲＳ３０６ＫＲ所表达的蛋白量至少为ＴＧ１表达ＡｒｇＲＳ蛋白量的１００倍。细胞粗抽提液中ＡｒｇＲＳ的比活ＴＧ１、转化子ｐＵＣ１８－ａｒｇｓ、ｐＵＣ１８－ａｒｇｓ３０６ＫＡ和ｐＵＣ１８－ａｒｇｓ３０６ＫＲ分别为１．６５、２１０、１．８和３８单位／毫克。结果表明ＡｒｓＲＳ的Ｌｙｓ３０６为Ａｌａ取代使活力完全丧失;若被Ａｒｇ取代,则活力丧失８０％以上。Ｌｙｓ３０６为ＡｒｇＲＳ活力所必需。     

Zinc removal from model solution and wastewater by Arthrospira (Spirulina) Platensis biomass

The biosorption of zinc from model solution as well as wastewater by Arthrospira (Spirulina) platensis biomass was studied. Adsorption capacity of the biosorbent was investigated as a function of contact time between adsorbent and zinc, the initial metals and sorbent concentration, pH value, and temperature. The ability of Arthrospira biomass for zinc biosorption exhibited a maximum at the pH range 4–8. Equilibrium data fitted well with the Langmuir model as well as the Freundlich model with maximum adsorption capacity of 7.1 mg/g. The pseudo second-order model was found to correlate well with the experimental data. Different thermodynamic parameters, ΔG°, ΔH° and ΔS° were evaluated and it has been found that the sorption was feasible, spontaneous, and endothermic in nature. The process of zinc removal from industrial effluent was studied at different time of sorbat–sorbent interaction and different sorbent dosage. Maximum zinc removal (83%) was obtained at sorbent concentration 60 g/L during 1-h experiment. The results indicate that Arthrospira platensis biomass could be effectively used for zinc removal from industrial effluents.     

A computer-based advisory system for cereal aphids-field-testing the model

A previously developed computer-based interactive model which can aid farmers in the control of summer aphids on winter wheat was tested in field trials carried out from 1985 to 1988. These trials showed that the model performed very well when compared with other popular types of advice available. Economic comparison of the model with current and previous ADAS advice showed that the model performed as well or better. It also had the advantage that it used the economics of the situation, as well as the size of the pest population, in calculating advice.     

microRNA‐143‐3p attenuated development of hepatic fibrosis in autoimmune hepatitis through regulation of TAK1 phosphorylation

Autoimmune hepatitis (AIH) is a chronic liver disease due to autoimmune system attacks hepatocytes and causes inflammation and fibrosis. Intracellular signalling and miRNA may play an important role in regulation of liver injury. This study aimed to investigate the potential roles of microRNA 143 in a murine AIH model and a hepatocyte injury model. Murine AIH model was induced by hepatic antigen S100, and hepatocyte injury model was induced by LPS. Mice and AML12 cells were separated into six groups with or without the treatment of miRNA‐143. Inflammation and fibrosis as well as gene expression were examined by different cellular and molecular techniques. The model was successfully established with the elevation of ALT and AST as well as inflammatory and fibrotic markers. Infection or transfection of mir‐143 in mice or hepatocytes significantly attenuated the development of alleviation of hepatocyte injury. Moreover, the study demonstrated phosphorylation of TAK1‐mediated miRNA‐143 regulation of hepatic inflammation and fibrosis as well as hepatocyte injury. Our studies demonstrated a significant role of miRNA‐143 in attenuation of liver injury in AIH mice and hepatocytes. miRNA‐143 regulates inflammation and fibrosis through its regulation of TAK1 phosphorylation, which warrants TAK1 as a target for the development of new therapeutic strategy of autoimmune hepatitis.     

A subject-specific pelvic bone model and its application to cemented acetabular replacements

A subject-specific three-dimensional finite element (FE) pelvic bone model has been developed and applied to the study of bone–cement interfacial response in cemented acetabular replacements. The pelvic bone model was developed from CT scan images of a cadaveric pelvis and validated against the experiment data obtained from the same specimen at a simulated single-legged stance. The model was then implanted with a cemented acetabular cup at selected positions to simulate some typical implant conditions due to the misplacement of the cup as well as a standard cup condition. For comparison purposes, a simplified FE model with homogeneous trabecular bone material properties was also generated and similar implant conditions were examined.The results from the homogeneous model are found to underestimate significantly both the peak von Mises stress and the area of the highly stressed region in the cement near the bone–cement interface, compared with those from the subject-specific model. Non-uniform cement thickness and non-standard cup orientation seem to elevate the highly stressed region as well as the peak stress near the bone–cement interface.     

A new graphical method for determining parameters in Michaelis-Menten-type kinetics for enzymatic lactose hydrolysis

A new graphical method was developed to determine the kinetic parameters in the Michaelis-Menten-type equation. This method was then applied to studying the kinetics of lactose hydrolysis by Aspergillus niger beta-galactosidase. In this study, the reaction temperature ranged between 8 and 60 degrees C, and the initial lactose concentration ranged between 2.5 and 20%. A kinetic model similar to the conventional Michaelis-Menten equation with competitive product inhibition by galactose was tested using this graphical method as well as a nonlinear computer regression method. The experimental data and the model fit together fairly well at 50 degrees C. However, a relative large disparity was found for reactions at 30 degrees C. A three-parameter integrated model derived from the reversible reaction mechanism simulates the experimental data very well at all temperatures studied. However, this reversible reaction model does not follow the Arrhenius temperature dependence. Nevertheless, reaction rate constants for the proposed model involving the enzyme-galactose complex (in addition to the Michaelis complex) as an intermediate in lactose hydrolysis follow the Arrhenius temperature dependence fairly well, suggesting that this model can be best used for describing the enzymatic lactose hydrolysis. The lack of fit between the model predictions and data may be largely attributed to the effects of galactose mutarotation and oligosaccharide formation during lactose hydrolysis.     

Stochastic and deterministic simulations of heterogeneous cell population dynamics

A Monte Carlo algorithm, which can accurately simulate the dynamics of entire heterogeneous cell populations, was developed. The algorithm takes into account the random nature of cell division as well as unequal partitioning of cellular material at cell division. Moreover, it is general in the sense that it can accommodate a variety of single-cell, deterministic reaction kinetics as well as various stochastic division and partitioning mechanisms. The validity of the algorithm was assessed through comparison of its results with those of the corresponding deterministic cell population balance model in cases where stochastic behavior is expected to be quantitatively negligible. Both algorithms were applied to study: (a) linear intracellular kinetics and (b) the expression dynamics of a genetic network with positive feedback architecture, such as the lac operon. The effects of stochastic division as well as those of different division and partitioning mechanisms were assessed in these systems, while the comparison of the stochastic model with a continuum model elucidated the significance of cell population heterogeneity even in cases where only the prediction of average properties is of primary interest.     

A simple kinetic model for myeloma cell culture with consideration of lysine limitation

A simple kinetic model is developed to describe the dynamic behavior of myeloma cell growth and cell metabolism. Glucose, glutamine as well as lysine are considered as growth limiting substrates. The cell growth was restricted as soon as the extracellular lysine is exhausted and then intracellular lysine becomes a growth limiting substrate. In addition, a metabolic regulator model together with the Monod model is used to deal with the growth lag phase after inoculation or feeding. By using these models, concentrations of substrates and metabolites, as well as densities of viable and dead cells are quantitatively described. One batch cultivation and two fed-batch cultivations with pulse feeding of nutrients are used to validate the model.     

Modeling and Design of H-Infinity Controller for Piezoelectric Actuator LIPCA

<正> We proposed a dynamic model identification and design of an H-Infinity (i.e.H_∞) controller using a LightweightPiezo-Composite Actuator (LIPCA).A second-order dynamic model was obtained by using input and output data, and applyingan identification algorithm.The identified model coincides well with the real LIPCA.To reduce the resonating mode that istypical of piezoelectric actuators, a notch filter was used.A feedback controller using the H_∞ control scheme was designed basedon the identified dynamic model; thus, the LIPCA can be easily used as an actuator for biomemetic applications such as artificialmuscles or macro/micro positioning in bioengineering.The control algorithm was implemented using a microprocessor, analogfilters, and power amplifying drivers.Our simulation and experimental results demonstrate that the proposed control algorithmworks well in real environment, providing robust performance and stability with uncertain disturbances.     

A bioenergetic model of the mitochondrial population undergoing permeability transition

Mitochondrial permeability transition (MPT) is a highly regulated complex phenomenon that is a type of ischemia/reperfusion injury that can lead to cell death and ultimately organ dysfunction. A novel population transition and detailed permeability transition pore regulation model were integrated with an existing bioenergetics model to describe MPT induction under a variety of conditions. The framework of the MPT induction model includes the potential states of the mitochondria (aggregated, orthodox and post-transition), their transitions from one state to another as well as their interaction with the extra-mitochondrial environment. The model encodes the three basic necessary conditions for MPT: a high calcium load, alkaline matrix pH and circumstances which favor de-energization. The MPT induction model was able to reproduce the expected bioenergetic trends observed in a population of mitochondria subjected to conditions that favor MPT. The model was corroborated and used to predict that MPT in an acidic environment is mitigated by an increase in activity of the mitochondrial potassium/hydrogen exchanger. The model was also used to present the beneficial impact of reducing the duration mitochondria spend in the orthodox state on preserving the extra-mitochondrial ATP levels. The model serves as a tool for investigators to use to understand the MPT induction phenomenon, explore alternative hypotheses for PTP regulation, as well as identify endogenous pharmacological targets and evaluate potential therapeutics for MPT mitigation.     

Evaluation of mammalian fed-batch cultivations by two different models

Simulations of fed-batch hybridoma cell cultures were performed with two different models, a new model suggested by Nielsen and an older model suggested by Batt and Kompala. The response of the new Nielsen model was less sensitive to changes in feed concentrations and frequencies than the Batt and Kompala model. The new simulations with the Nielsen model as well as those previously with the Batt and Kompala model indicate, that possible benefits of fed-batch cultivations must be sought in other features than in intrinsic metabolic kinetics.