High-level expression, purification, and crystallization of recombinant rat leukotriene C(4) synthase from the yeast Pichia pastoris

Chemical-enzymatic synthesis of human Epidermal Growth Factor (hEGF) cDNA has been performed, following by cloning into expression vector pTWIN1 (New England Biolabs). The resulting recombinant fusion protein expressed in Escherichia coli consisted of the N-terminal chitin-binding domain, mini-intein Ssp dnaB domain and hEGF polypeptide at the C-terminus. In this construct, mini-intein Ssp dnaB played a role of catalytically active subunit capable under certain conditions of autocatalytic cleavage resulting in separation of the target protein. As the hybrid protein had several cysteins in its sequence-one in chitin-binding domain, one in mini-intein and six in hEGF, it was necessary to work out optimal scheme for refolding and purification of the recombinant hEGF. As a result of this work, two schemes of the recombinant hEGF purification have been developed: according to the first scheme, the recombinant protein with reduced cysteins is bound to the chitin column, the hEGF is cleaved off and eluted, and then refolded to form appropriate cystein bridges. In the second scheme, the entire hybrid protein is first refolded to form disulfide bonds and then loaded to affinity resin; the recombinant hEGF is cleaved off and eluted in its native state. In spite of the fact that the first scheme is more common and suitable for a variety of recombinant proteins, in case of recombinant hEGF, the second scheme proved to be more productive and cost-effective.     

Thermodynamics, kinetics, and mechanism in yeast inorganic pyrophosphatase catalysis of inorganic pyrophosphate: inorganic phosphate equilibration

We have developed two methods for quantitatively measuring inorganic pyrophosphate (PPi) in the presence of 10(3)--10(4) molar excesses of inorganic phosphate (Pi) and used them to measure the extent of enzyme-bound pyrophosphate (EPPi) formation in solutions of yeast inorganic pyrophosphatase and Pi. We have also measured the rate of enzyme-catalyzed H2O--phosphate oxygen exchange. We find both processes to have essentially identical dependence on Mg2+ and Pi concentrations, thus providing important confirmation for the recent proposal by Janson et al. (1979) that oxygen exchange proceeds via EPPi formation. Our results are consistent with a model in which three Mg2+ per active site are required for EPPi formation but inconsistent with a model requiring only two Mg2+ per active site and permit the formulation of an overall scheme for inorganic pyrophosphatase catalysis of PPi--Pi equilibration as well as the evaluation of equilibrium and rate constants in this scheme. The major results and conclusions of our work are the following: (a) the equilibrium constant for PPi (enzyme-bound) in equilibrium with 2Pi (enzyme-bound) is 4.8; (b) following PPi hydrolysis, the first released Pi contains an oxygen from solvent water; (c) the steps for PPi hydrolysis on the enzyme and for release of both product Pi's are all partially rate determining in overall enzyme-catalyzed PPi hydrolysis; (d) PPi formation on the enzyme is rate determining for H2O--Pi oxygen exchange; (e) PPi dissociation from the enzyme is very slow and is the rate-determining step in Pi--PPi exchange (Cohn, 1958; Janson et al., 1979). This also accounts for the observation that the calculated dissociation constant for MgPPi complex binding to enzyme is considerably lower than the measured Km for enzyme-catalyzed MgPPi hydrolysis.     

Efficient simulations of tubulin-driven axonal growth

This work concerns efficient and reliable numerical simulations of the dynamic behaviour of a moving-boundary model for tubulin-driven axonal growth. The model is nonlinear and consists of a coupled set of a partial differential equation (PDE) and two ordinary differential equations. The PDE is defined on a computational domain with a moving boundary, which is part of the solution. Numerical simulations based on standard explicit time-stepping methods are too time consuming due to the small time steps required for numerical stability. On the other hand standard implicit schemes are too complex due to the nonlinear equations that needs to be solved in each step. Instead, we propose to use the Peaceman–Rachford splitting scheme combined with temporal and spatial scalings of the model. Simulations based on this scheme have shown to be efficient, accurate, and reliable which makes it possible to evaluate the model, e.g. its dependency on biological and physical model parameters. These evaluations show among other things that the initial axon growth is very fast, that the active transport is the dominant reason over diffusion for the growth velocity, and that the polymerization rate in the growth cone does not affect the final axon length.     

Mechanics of left ventricular relaxation, early diastolic lengthening, and suction investigated in a mathematical model

We investigated the determinants of ventricular early diastolic lengthening and mechanics of suction using a mathematical model of the left ventricle (LV). The model was based on a force balance between the force represented by LV pressure (LVP) and active and passive myocardial forces. The predicted lengthening velocity (e') from the model agreed well with measurements from 10 dogs during 5 different interventions (R = 0.69, P < 0.001). The model showed that e' was increased when relaxation rate and systolic shortening increased, when passive stiffness was decreased, and when the rate of fall of LVP during early filling was decreased relative to the rate of fall of active stress. We first defined suction as the work the myocardium performed to pull blood into the ventricle. This occurred when contractile active forces decayed below and became weaker than restoring forces, producing a negative LVP. An alternative definition of suction is filling during falling pressure, commonly believed to be caused by release of restoring forces. However, the model showed that this phenomenon also occurred when there had been no systolic compression below unstressed length and therefore in the absence of restoring forces. In conclusion, relaxation rate, LVP, systolic shortening, and passive stiffness were all independent determinants of e'. The model generated a suction effect seen as lengthening occurring during falling pressure. However, this was not equivalent with the myocardium performing pulling work on the blood, which was performed only when restoring forces were higher than remaining active fiber force, corresponding to a negative transmural pressure.     

Modeling of self-sustained discharge-pumped,Ne-buffered XeCl laser kinetics

The aim of this work is to highlight some quantities characterizing the Ne/Xe/HCl gas mixture plasma at high pressure and under uniform preionization conditions. This mixture is used as excitation medium for XeCl excimer lasers. A comprehensive model of discharge kinetics is presented. The model combines the physical processes in the discharge with the chemistry of formation and destruction of the excimer molecule. It is based on an extensive reaction scheme including the major electronic and ionic processes. The importance of excited atomic and molecular states is demonstrated. A parametric study is presented.     

Optimization of skeletal configuration: studies of scoliosis correction biomechanics

A scheme for optimizing configurations in models of skeletal structures is presented. Use of the scheme is illustrated through determination of biomechanically optimal correction of a right-thoracic scoliosis by passive brace and active muscle forces. The locations and magnitudes of the passive brace forces, and the trunk muscle groups and their corresponding contraction intensity magnitudes that would optimally correct the geometric deformities of the spine were determined. The results suggest that, from a biomechanical viewpoint, both brace and muscle forces are capable of substantial correction of a model thoracic scoliosis. However, comparison of model results with long-term clinical results suggests that even under optimal conditions it is unlikely that scoliosis can be fully corrected by passive brace forces or active muscle contractions.     

A large-scale model of the locust antennal lobe

The antennal lobe (AL) is the primary structure within the locust’s brain that receives information from olfactory receptor neurons (ORNs) within the antennae. Different odors activate distinct subsets of ORNs, implying that neuronal signals at the level of the antennae encode odors combinatorially. Within the AL, however, different odors produce signals with long-lasting dynamic transients carried by overlapping neural ensembles, suggesting a more complex coding scheme. In this work we use a large-scale point neuron model of the locust AL to investigate this shift in stimulus encoding and potential consequences for odor discrimination. Consistent with experiment, our model produces stimulus-sensitive, dynamically evolving populations of active AL neurons. Our model relies critically on the persistence time-scale associated with ORN input to the AL, sparse connectivity among projection neurons, and a synaptic slow inhibitory mechanism. Collectively, these architectural features can generate network odor representations of considerably higher dimension than would be generated by a direct feed-forward representation of stimulus space.     

Central Upwind Scheme for a Compressible Two-Phase Flow Model

In this article, a compressible two-phase reduced five-equation flow model is numerically investigated. The model is non-conservative and the governing equations consist of two equations describing the conservation of mass, one for overall momentum and one for total energy. The fifth equation is the energy equation for one of the two phases and it includes source term on the right-hand side which represents the energy exchange between two fluids in the form of mechanical and thermodynamical work. For the numerical approximation of the model a high resolution central upwind scheme is implemented. This is a non-oscillatory upwind biased finite volume scheme which does not require a Riemann solver at each time step. Few numerical case studies of two-phase flows are presented. For validation and comparison, the same model is also solved by using kinetic flux-vector splitting (KFVS) and staggered central schemes. It was found that central upwind scheme produces comparable results to the KFVS scheme.     

Neural adaptive control for vibration suppression in composite fin-tip of aircraft

In this paper, we present a neural adaptive control scheme for active vibration suppression of a composite aircraft fin tip. The mathematical model of a composite aircraft fin tip is derived using the finite element approach. The finite element model is updated experimentally to reflect the natural frequencies and mode shapes very accurately. Piezo-electric actuators and sensors are placed at optimal locations such that the vibration suppression is a maximum. Model-reference direct adaptive neural network control scheme is proposed to force the vibration level within the minimum acceptable limit. In this scheme, Gaussian neural network with linear filters is used to approximate the inverse dynamics of the system and the parameters of the neural controller are estimated using Lyapunov based update law. In order to reduce the computational burden, which is critical for real-time applications, the number of hidden neurons is also estimated in the proposed scheme. The global asymptotic stability of the overall system is ensured using the principles of Lyapunov approach. Simulation studies are carried-out using sinusoidal force functions of varying frequency. Experimental results show that the proposed neural adaptive control scheme is capable of providing significant vibration suppression in the multiple bending modes of interest. The performance of the proposed scheme is better than the H(infinity) control scheme.     

A simple, yet highly accurate, QSAR model captures the complement inhibitory activity of compstatin

Compstatin is a 13-residue cyclic peptide inhibitor of complement activation that was originally identified through phage-mediated presentation of a peptide library to C3b. Recent efforts to improve its activity have led to a rich dataset of complement analogs, with the most active analog being approximately 260 times more active than the parent compstatin. In the present work, a highly transparent quantitative structure-activity relationship model (Radj2=0.89) with four parameters is presented that captures important physico-chemical and geometrical properties of the analog molecules with regard to activity. The number of aromatic bonds and hydrophobicity of the fourth residue of compstatin correlated strongly with activity. Also important were the hydrophobic patch size near the disulfide bond and the solvent-accessible surface area occupied by nitrogen atoms of basic amino acid residues.     

Prediction of substrate consumption rate, average biofilm density and active thickness for a thin spherical biofilm at pseudo-steady state

In this work, a new approach is proposed to evaluate substrate consumption rate, average biofilm density and active thickness of a spherical bioparticle in a completely mixed fluidized bed system. The substrate consumption rate and average biofilm density are predicted for a given biofilm surface substrate concentration and operational biofilm thickness. A diffusion and reaction model is developed with an effective diffusion coefficient that depends on the average biofilm density. This approach, a first in the literature, predicts the optimum average density of a biofilm to yield the maximum substrate consumption rate within the biofilm. A reasonable correlation was observed between the model prediction and experimental results for substrate consumption rate and average biofilm density for thin and fully active biofilms.     

Invasion of brain tissue by primary glioma: evidence for the involvement of urokinase-type plasminogen activator as an activator of type IV collagenase.

The immunocharacterization of a metalloproteinase isolated from rat glioma cell conditioned medium is described and confirms that the enzyme is identical to type IV collagenase. Free, active plasminogen activator (PA) and PA-PAI complexes were identified as being secreted by the same cells. Using affinity-purified metalloproteinase we demonstrate that the enzyme can be partially activated by u-PA but not by plasmin in vitro. On the basis of these findings and previous published work we propose a scheme for the proteolytic degradation of normal brain tissue during tumour invasion.     

A Kinetic Theory of First Order Cyclical Processes—Phytochrome Controlled Red Light Induced Cereal Leaf Unfolding Compared with Theory

The phytochrome controlled unfolding of cereal leaves was studied as a function of irradiation time and light intensity (narrowband red light) over a wide energy range (5 decades). With different intensities, a family of similarly shaped response curves appear with distinct time-dependent maxima and minima. A theoretical kinetic model based upon a cyclical phytochrome photoconversion scheme has been calculated by us. The theoretical calculations and the experimental findings are in excellent agreement. The same model explains the early photoresponses (first maxima) as an effect of one active phytochrome form, P₂, and the delayed photoresponses as an effect of a second active form P_n. The active transitory form, P₂ (although it may not be the primary product), is formed upon light absorption from P₁. The P₂ decays by a first order dark reaction through several inactive intermediates to P_n (active). The effect of the intermediates is mainly to delay the production of the second active product. It is possible to identify the two active products, P₂ and P_n, as P_fr and P*_fr, respectively. The presented cyclical phytochrome reaction scheme is a special case of a general first order kinetic cycle which includes all possible feed back loops. The latter scheme also has been calculated and programmed since it has a more general application.     

Quantitative quality control in microarray experiments and the application in data filtering,normalization and false positive rate prediction

Data preprocessing including proper normalization and adequate quality control before complex data mining is crucial for studies using the cDNA microarray technology. We have developed a simple procedure that integrates data filtering and normalization with quantitative quality control of microarray experiments. Previously we have shown that data variability in a microarray experiment can be very well captured by a quality score q(com) that is defined for every spot, and the ratio distribution depends on q(com). Utilizing this knowledge, our data-filtering scheme allows the investigator to decide on the filtering stringency according to desired data variability, and our normalization procedure corrects the q(com)-dependent dye biases in terms of both the location and the spread of the ratio distribution. In addition, we propose a statistical model for false positive rate determination based on the design and the quality of a microarray experiment. The model predicts that a lower limit of 0.5 for the replicate concordance rate is needed in order to be certain of true positives. Our work demonstrates the importance and advantages of having a quantitative quality control scheme for microarrays.     

Elastic energy savings and active energy cost in a simple model of running

The energetic economy of running benefits from tendon and other tissues that store and return elastic energy, thus saving muscles from costly mechanical work. The classic “Spring-mass” computational model successfully explains the forces, displacements and mechanical power of running, as the outcome of dynamical interactions between the body center of mass and a purely elastic spring for the leg. However, the Spring-mass model does not include active muscles and cannot explain the metabolic energy cost of running, whether on level ground or on a slope. Here we add explicit actuation and dissipation to the Spring-mass model, and show how they explain substantial active (and thus costly) work during human running, and much of the associated energetic cost. Dissipation is modeled as modest energy losses (5% of total mechanical energy for running at 3 m s^-1) from hysteresis and foot-ground collisions, that must be restored by active work each step. Even with substantial elastic energy return (59% of positive work, comparable to empirical observations), the active work could account for most of the metabolic cost of human running (about 68%, assuming human-like muscle efficiency). We also introduce a previously unappreciated energetic cost for rapid production of force, that helps explain the relatively smooth ground reaction forces of running, and why muscles might also actively perform negative work. With both work and rapid force costs, the model reproduces the energetics of human running at a range of speeds on level ground and on slopes. Although elastic return is key to energy savings, there are still losses that require restorative muscle work, which can cost substantial energy during running.     

Systematic interpolation method predicts protein chromatographic elution from batch isotherm data without a detailed mechanistic isotherm model

Predicting protein elution for overloaded ion exchange columns requires models capable of describing protein binding over broad ranges of protein and salt concentrations. Although approximate mechanistic models are available, they do not always have the accuracy needed for precise predictions. The aim of this work is to develop a method to predict protein chromatographic behavior from batch isotherm data without relying on a mechanistic model. The method uses a systematic empirical interpolation (EI) scheme coupled with a lumped kinetic model with rate parameters determined from HETP measurements for non‐binding conditions, to numerically predict the column behavior. For two experimental systems considered in this work, predictions based on the EI scheme are in excellent agreement with experimental elution profiles under highly overloaded conditions without using any adjustable parameters. A qualitative study of the sensitivity of predicting protein elution profiles to the precision, granularity, and extent of the batch adsorption data shows that the EI scheme is relatively insensitive to the properties of the dataset used, requiring only that the experimental ranges of protein and salt concentrations overlap those under which the protein actually elutes from the column and possess a ±10% measurement precision.     

Associations among Active Transportation,Physical Activity,and Weight Status in Young Adults

Objective: To describe prevalence of active, public, and car transit by overweight status and amount of leisure‐time physical activity in a nationally representative cohort of ethnically diverse young adults. Research Methods and Procedures: Questionnaire data on patterns of transportation were collected from U.S. adolescents enrolled in Wave III (2001) of the National Longitudinal Study of Adolescent Health (N = 10, 771). Measured height and weight data were used to calculate BMI and classify adults by overweight status (BMI ≥ 25). Self‐reported physical activity data were used to classify adults into those who achieved ≥ 5 bouts of weekly moderate‐vigorous physical activity and those who did not. Results were stratified by overweight and physical activity status. Results: The vast majority of young adults used car transit (work, 90.4%; school, 74.7%). A small proportion of young adults used active means of transportation to work (8.1%) and school (26.7%), and fewer used public transportation to work or school (<10%). The proportion of individuals using active transportation was higher among the nonoverweight traveling to work (9.2%) and school (29.7%) and among the more active traveling to work (15.2%) and school (37.0%) relative to the overweight and less active young adults. Discussion: The vast majority of young adults of all racial/ethnic backgrounds, particularly blacks and Hispanics, did not use active transportation to school and/or work. Active transportation was more common among nonoverweight and more active young adults, of high socioeconomic status, particularly full‐time students. Population‐level efforts (and environmental supports) to increase non‐leisure physical activity, particularly active transportation, are sorely needed as a means of supporting and promoting overall physical activity.     

A computer model of human thermoregulation for a wide range of environmental conditions: the passive system.

A dynamic model predicting human thermal responses in cold, cool, neutral, warm, and hot environments is presented in a two-part study. This, the first paper, is concerned with aspects of the passive system: 1) modeling the human body, 2) modeling heat-transport mechanisms within the body and at its periphery, and 3) the numerical procedure. A paper in preparation will describe the active system and compare the model predictions with experimental data and the predictions by other models. Here, emphasis is given to a detailed modeling of the heat exchange with the environment: local variations of surface convection, directional radiation exchange, evaporation and moisture collection at the skin, and the nonuniformity of clothing ensembles. Other thermal effects are also modeled: the impact of activity level on work efficacy and the change of the effective radiant body area with posture. A stable and accurate hybrid numerical scheme was used to solve the set of differential equations. Predictions of the passive system model are compared with available analytic solutions for cylinders and spheres and show good agreement and stable numerical behavior even for large time steps.