bluuebunny/crossref_metadata_2025 huggingface dataset:
Modeling Fluid Catalytic Cracking in a Novel CREC Riser Simulator: Adsorption Parameters under Reaction Conditions
Authors: Atias Jesus A , Tonetto Gabriela M , de Lasa Hugo Ignacio
Published: October 2005

The complexity of a heavy gas oil feedstock and the multitude of reaction pathways have limited previous attempts to model fluid catalytic cracking (FCC). The demand for more detailed kinetic information motivates the use of pure components to first elucidate the dominant pathways and mechanisms and then determine the associated rate parameters, including adsorption constants and heats of adsorption. The aim of the present work is to evaluate adsorption constants and heats of adsorption, under FCC relevant reaction conditions. The experiments are carried out in a novel CREC Riser Simulator (batch reactor unit) using USY zeolite catalysts with different crystallite sizes (0.4 and 0.9 microns). This study confirms a special feature of the CREC Riser Simulator, as a valuable tool for the study of adsorption phenomena. Adsorption constants and heats of adsorption are evaluated for benzene, toluene, xylene and trimethylbenzene, at initial reaction conditions. Catalytic conversion experiments for 1,2,4-trimethylbenzene help to demonstrate the consistency of the determined adsorption parameters at various temperatures and reaction times. In addition, adsorption constants and heats of adsorption are found to be constant throughout the reaction time and the formation of coke does not hinder the adsorption of 1,2,4-TMB, although it significantly affects the reactivity of this model compound.
Recent Research in Catalytic Inorganic Membrane Reactors
Authors: Dixon Anthony G.
Published: October 2005

The two most important, and often the most expensive, steps in a chemical process are usually the chemical reactor and the separation of the product stream. Both the process economics and the efficient use of natural resources could be improved by the combination of these two operations into a single unit operation, leading to potential savings in energy and reactant consumption and reduced by-product formation. One promising way to accomplish this combination is the use of membrane separation and catalytic reaction together in a multifunctional reactor. Until relatively recently, the use of membranes was restricted to low temperature processes with mild chemical environments, which could be tolerated by polymeric materials. Recent advances in inorganic materials have expanded the range of membrane use, to include high temperature and chemically harsh environments. This has allowed inorganic membranes to be integrated into catalytic reactors. This area was reviewed previously by the present author (Dixon, 1999). The present contribution seeks to review literature and new developments in the succeeding four and a half year period, since the end of 1998. Research directions that were previously considered promising are re-evaluated here, and new ideas since then are presented.
Numerical Aspects of Bubble Column Simulations
Authors: Bove Stefano , Solberg Tron , Hjertager Bjørn H
Published: October 2005

A numerical study of gas-liquid bubble flow in a square cross section bubble column was carried out. The Euler-Euler approach was used to describe the equations of motion. The effects on the solution of the numerical modeling of the convective terms and of the inlet condition were investigated. Upwind first order and higher order, oscillation free, Flux Corrected Transport (FCT) schemes were tested for both the phase fraction equations and for the momentum equations. Different inlet conditions, constrained on the mass flux inlet, were modeled and tested in order to show the sensitivity of the solution on the inlet condition. The solver used to simulate the gas-liquid flow is FLOTRACS-MP-3D, which originally used a Partial Elimination Algorithm (PEA) for solving the momentum equations. It has been upgraded with a coupled solver for the momentum equations. The numerical results are compared with the experimental data of Deen (2001).
Combined Mechanistic and Empirical Modelling
Authors: Duarte Belmiro , Saraiva P. M. , Pantelides C. C.
Published: October 2005

We consider a hybrid modelling approach based on the combination of prior knowledge, in the form of mechanistic models, with tools for the extraction of knowledge from operating data: the first component captures first-principles system behavior features, while the second one accounts for the differences found between mechanistic predictions and real data. The empirical modelling methods tested include backpropagation Artificial Neural Networks (ANN), Multivariate Adaptive Regressive Splines (MARS) and Regression Analysis (RA). In the proposed hybrid structure, mechanistic predictions provide additional inputs for the empirical module. To evaluate the performance of the various combinations of modules and hybrid approaches, we consider two simulated case studies (involving a CSTR with a reversible reaction and a fed-batch penicillin fermentation process). In all of the above, our hybrid structure based on a mechanistic module together with an empirical component has outperformed other approaches, and the mechanistic model/MARS combination resulted in lowest overall prediction errors.
A Laboratory Steam-Cracking Reactor to Characterize Raw Materials
Authors: Pinter Attila , Tungler Antal , Nagy Lajos , Vida László , Kovács Imre , Kerezsi Janos
Published: October 2005

A laboratory steam-cracking reactor was designed, built and tested in order to compare yields under different operating conditions. A typical industrial gasoline was used as feedstock to test the reactor. The design of the reactor was found appropriate to crack hydrocarbon feedstocks and the olefin yields follow the expected trends.
Multi-fluid CFD modelling of fluidised bed reactors
Authors: Hjertager Bjørn H , Solberg Tron , Ibsen Claus H , Hansen Kim G
Published: October 2005

Progress in modelling and simulation of flow processes in gas/particle systems carried out at the authors research group are presented. Particular emphasis is given to computational fluid dynamics (CFD) models that use the multi dimensional multi fluid techniques. Turbulence modelling strategies for gas/particle flows based on the kinetic theory for granular flows are given. Sub models for the interfacial transfer processes and chemical kinetics modelling are presented. Examples are shown for several gas/particle systems including flow in risers, segregation by size and reacting systems.
Characterization and Selection of Materials for Air Biofiltration in Fluidized Beds
Authors: Leslous Amel , Delebarre Arnaud , Pré Pascaline , Warlus Stéphane , Zhang Nan
Published: October 2005

A methodology is described for the selection of the best material to employ in a fluidized biofilter applied to volatil organic compounds (VOC) treatment. Two different supports were considered: one natural, scrap wood, and one synthetic, polyurethane foam. In a first part, the main objective was to establish the fluidizability of the selected solids. Particles of increasing moisture content were tested in rigs equipped with different air distribution technologies. Maps of flow regimes were drawn by varying air velocity and bed height. In the second part, the sorption capacities of the selected materials were evaluated at different humidity levels for a soluble organic compound (ethanol) and an insoluble one (toluene). Their ability for supporting viable microorganisms was then measured. Fine particles with 45% moisture content fluidized with the tuyere distributor in channeling and slugging regimes, whereas the conical distributor generated a spouted bed regime. Coarse wood particles with 45% moisture commonly used in fixed bed biofilters were considered as less suitable due to limitations borne by their fluidization regimes. Finally polyurethane foam filled with Agar gel could not endure the mechanical stress to which particles are subjected in fluidized beds. It was shown that a 45% moisture content was optimum to ensure transfer for both soluble and hydrophobic compounds. Considering the biological criterion, it appeared that a 45% humidity level was sufficient to ensure biomass growth and biodegradation of pollutants on fine scrap wood particles.
Flow Generated by Radial Flow Impellers: PIV Measurements and CFD Simulations
Authors: Khopkar Avinash , Aubin Joella , Rubio-Atoche C , Xuereb Catherine , Le Sauze Nathalie , Bertrand Joel , Ranade Vivek V
Published: October 2005

Particle image velocimetry (PIV) and computational fluid dynamics (CFD) have been used to investigate the single phase and gas-liquid flow generated by a Scaba SRGT turbine. The key details of the trailing vortices, the turbulent flow around the impeller blades and the accumulation of gas have been studied by using PIV measurements and CFD simulations. Both the experimental and numerical results show that the flow and the trailing vortices are not altered significantly upon gassing. The simulated results are generally in good agreement with the experimental findings. The CFD simulations also show that only small low-pressure regions exist behind the blades of the Scaba turbine compared with the very large low-pressure zones formed by the Rushton turbine. These results enable better understanding of the improved performance of the Scaba turbine for gas-liquid dispersions compared with the Rushton turbine.
Numerical and Experimental Investigations into The Effect of Gap Between Baffle and Wall on Mixing in an Oscillatory Baffled Column
Authors: Ni Xiong-Wei , Fitch Andrew , Jian Hongbing
Published: October 2005

We report our numerical and experimental investigation of the effect on mixing of implementing 0, 2, 4 and 6 mm gaps between the outer edge of baffles and the inner diameter of an Oscillatory Baffled Column (OBC). Large eddy simulation and digital particle image velocimetry techniques have been used to obtain the flow patterns and quantify the flow field and turbulence, such as the velocity ratio, integral length scale and mixing time scale versus the oscillatory Reynolds numbers. It is shown that the global mixing intensity and the turbulence length scale decrease, while the Kolmogorov time scale increases with increasing gap size in the range of experimental conditions tested in this work, which suggests a need for new configuration of OBC with baffle gap for certain applications involving particulates.
Wastewater Minimisation Using a Heuristic Procedure
Authors: Liu Zhi-Yong , Zhang Zisheng Jason , Hu Linna , Wu Zhaoliang
Published: October 2005

A two-stage procedure is proposed for the minimisation of wastewater. In the first stage, heuristic rules are used to find the initial feasible flowsheet; in the second stage, the flow rates of streams in the initial flowsheet are determined by mass balance of the contaminants. The operations considered in this work include re-use, regeneration, mixing and bypassing of water streams. The proposed procedure is simple and the results of the examples studied are comparable with or better than those found in the literature.
Hydrodynamic and Mass Transfer Parameters in Agitated Reactors Part I: Critical Mixing Speed, Induced Gas Flow Rate, and Wavy Surface in SARs and GIRs
Authors: Lemoine Romain , Fillion Benoit , Morsi Badie I
Published: October 2005

The critical mixing speed for gas entrainment (NCRE), for gas induction (NCRI), induced gas flow rate (QGI) as well as the wavy gas-liquid interfacial area (aWave) of N2 and air were measured in pure toluene and three mixtures of organic liquids (toluene-benzoic acid-benzaldehyde mixtures) under wide ranges of temperatures, T (300-453K), pressures, P (1-15 bar), mixing speeds, N (13.3-23.3Hz) and liquid heights, H (0.171-0.268m) using a 4-liter, see-through agitated autoclave operating as a surface-aeration reactor (SAR) and gas-inducing reactor (GIR).NCRE and NCRI as well as aWave were estimated by analyzing the videos taken with an on-line high-speed Phantom camera through the reactors Jerguson windows. In the GIR, QGI was determined using a highly sensitive Coriolis mass flow meter. The Central Composite Statistical Design and analysis technique was used to study the effect of operating conditions on these hydrodynamic parameters.NCRE and NCRI appeared to increase with liquid height and decrease with temperature, whereas, the pressure and gas nature did not significantly affect both parameters. The liquid physicochemical properties were found to strongly affect NCRE and NCRI, and QGI. Increasing mixing speed or decreasing liquid height increased QGI. Increasing temperature or decreasing liquid viscosity initially increased and then decreased QGI. Increasing pressure or gas density on the other hand decreased QGI. Increasing mixing speed and temperature or decreasing liquid height significantly enhanced aWave, as compared to the flat liquid surface. At high pressures, however, lower values of aWave were obtained. Empirical and statistical correlations were also developed to accurately predict NCRE, NCRI, QGI and aWave.
Microchannel Reactors: Applications and Use in Process Development
Authors: Gokhale Sagar V. , Tayal Rajiv K. , Jayaraman Valadi K. , Kulkarni Bhaskar D.
Published: October 2005

Recent research results on microchannel reactors are reviewed with particular reference to their applications and use as a cost effective tool during process development tasks. The high surface to volume ratio, efficient heat and mass transfer characteristics, vastly improved fluid mixing etc., allow precision control of reaction with improved conversions, selectivities and yields of desired products. The reaction times are shorter as compared to the conventional reactors with less degradation and side products. Scalability and optimization are also significantly easier. The article reviews variety of applications to catalysis, polymers, advanced materials, biosystems, organic reaction etc., with emphasis on the related engineering science issues.
3-D Modeling of Liquid Injection into Fluidized Beds
Authors: Werther Joachim , Bruhns Stefan
Published: October 2005

A three-dimensional model has been developed to describe the injection of liquid reactants into fluidized bed reactors operating in the bubbling fluidized bed regime. The model considers the processes of liquid transport and evaporation in the vicinity of the point of injection. The underlying idea, which is supported by previous measurements, is that the particles in the dense suspension phase are wetted by the liquid or gas-liquid spray. The wetted particles are subsequently dried while they are following the gross solids circulation within the bed. The model considers the flow structure of the bubbling fluidized bed and the solids mixing with the aid of a hybrid model which combines semi-empirical models for bubble growth by coalescence and for bubble splitting with a CFD approach for the continuous emulsion phase surrounding the bubbles. Submodels for heat and mass transfer are used to describe the temperature and concentration fields in the vicinity of the injection nozzle and the drying process of the wetted particles with the resulting release of the vaporized injection liquid. The model was validated separately against flow structure measurements, solids tracer measurements and experiments with the injection of water and ethanol, respectively, into beds of FCC particles.
A Neural Network Approach for Prediction of the CuO-ZnO-Al2O3 Catalyst Deactivation
Authors: Zahedi Gholamreza , Jahanmiri Abdolhossein , Rahimpor M. R.
Published: October 2005

In this work an Artificial Neural Networks (ANN) approach for estimation of catalyst deactivation during methanol synthesis has been proposed. The approach predicts deactivation of the catalyst at different operating conditions as a function of time. Experimental data of a typical differential reactor were pre-scaled and used for training. Among the various training algorithms, Exact Radial Basis (RBE) method had the best prediction performance and was used for simulation of the reactor. The proposed approach interprets deactivation data, while there are not enough data versus time at different inlet conditions. By using the model, sufficient data were generated which vary with time and agree very well with experimental data. Capability of the model in generating deactivation data at different temperatures, pressures and feed compositions was excellent. The proposed method has great potential as a means to compensate for lack of the phenomenological kinetic modeling techniques.
A Kinetic Model for Toluene Oxidation Comprising Benzylperoxy Benzoate Ester as Reactive Intermediate in the Formation of Benzaldehyde
Authors: Hoorn J.A.A. , Alsters P.L. , Versteeg G.F.
Published: October 2005

During the oxidation of toluene under semibatch conditions, the formation of benzyl alcohol is initially equal to the rate of formation of benzaldehyde. As the overall conversion increases the benzyl alcohol concentration at first decreases much faster than benzaldehyde, but this decrease slows down causing the benzyl alcohol concentration to reduce to zero only very slowly. To account for this phenomenon a new reaction pathway has been proposed where the formation of benzaldehyde out of benzylhydroperoxide is catalysed by benzoic acid. Incorporation of this new reaction in a model improves the description of benzyl alcohol concentration prophiles while maintaining good predictions for benzaldehyde and benzoic acid.
Photocatalytic Inactivation of Bioaerosols by TiO2 Coated Membrane
Authors: Pal Amrita , Min Xu , Yu Liya E , Pehkonen Simo O , Ray Madhumita B.
Published: October 2005

Indoor air pollution by microbial contaminants is increasingly receiving attention as a public health problem. Under a suitable environment, such as in heating, ventilation and air conditioning (HVAC) system, airborne bacteria are able to proliferate and grow causing various allergies and illnesses. This can be particularly serious in tropical regions due to high relative humidity and warm temperatures all round the year. Application of photocatalysis using UV-A and TiO2 to inactivate air-borne bacteria is relatively new and systematic parametric study is required for the engineering design of a process based on this technology. This study investigates the effects of TiO2 mediated inactivation of various bacterial species in batch and continuous systems using different TiO2 loadings and radiation intensities. Gram-negative bacteria, E. coli and two Gram-positive bacteria, Microbacterium sp. and Bacillus subtilis were used for the inactivation studies. In both systems, inactivation rates of Gram-negative E. coli are higher than the Gram-positive Bacillus subtilis and Microbacterium sp. and the inactivation rates increased in presence of TiO2 for all bacteria. Depending on the type of bacteria, TiO2 loading and light intensity, an increase of 1.3-5.8 times in the inactivation rates was obtained from those in the absence of TiO2. The inactivation rates in the batch and continuous systems were reasonably comparable. Inactivation rates in the continuous system are somewhat higher than those in the batch system due to the unaccounted loss of bacteria via adsorption and settling on the reactor walls in the flow system. The study demonstrates an approach that can be used for the designing of large scale systems for the treatment of bioaerosol.
Scale Effects on Fluidized Bed Hydrodynamics
Authors: Mabrouk Rachid , Radmanesh Ramin , Chaouki Jamal , Guy Christophe
Published: October 2005

Industrial history is full of events related to scale-up challenges. Failure at the scale-up stage is no longer surprising. Engineers and scientists have been trying to gather all the key parameters for decades, but, unfortunately, there are still no exact and well-established rules ensuring an accurate transition from one scale to another. Even the minimum lab-scale fluidized bed is still undefined.In this work, the effect of bed diameter on gas-solid behavior is investigated in three fluidized beds, 152mm, 78mm, and 50mm in diameter, in which the conventional scale-up rules are respected. The experiments were carried out using sand and alumina particles.The results were obtained and confirmed using fiber optic techniques and radioactive particle tracking, respectively. The results show that radial solid hold-up behavior on a small bed diameter scale is completely different from that on the intermediate bed diameter scale. The radial profile of solid hold-up on a small and very small bed diameter indicates an increase from a low value near the wall to a high value at the center at different heights from the distributor. By contrast, the opposite profile is observed on the intermediate bed diameter, similar to what is usually reported in the literature.
The Effect of Solvent on the Kinetics and Mass Transfer Resistances for 4,6 DMDBT HDS
Authors: Alvarado-Perea Leo , Galindo-Esquivel Ignacio R. , Perez-Cisneros Eduardo S , de los Reyes Heredia José A. , Viveros Tomás
Published: October 2005

The effect of different solvents for model hydrodesulfurization (HDS) reactions on both the kinetics and the mass transfer resistances on a wide range of hydrogen partial pressures has been investigated. Special attention was directed to study the effect of low hydrogen partial pressures. While some researchers associate solvent effects to competitive adsorption of the solvent molecules on the catalytic surface, there is evidence that other effects may also play an important role in the modification of the kinetics. In this work, HDS experiments were performed in a slurry stirred tank reactor using the model molecule 4,6-dimethyldibenzothiophene (4,6-DMDBT) over a commercial NiMoP/alumina catalyst. The effect of hydrogen pressure on the transformation of 4,6 DMDBT in different solvents was studied. Thermodynamic calculations for hydrogen solubility under the whole set of conditions was considered. From our experiments, modification of the catalyst activity was observed to occur only at low hydrogen pressures (less than 34.7 bar) when n-dodecane was the used solvent. On the other hand, when using decaline there seems to be practically no effect in the catalytic activity of the hydrogen pressure over the entire range of pressures that were studied (18.0-55.5 bar). Additionally, the selectivity to direct desulfurization (DDS) products increased, while simultaneously decreasing the selectivity to the hydrogenation (HYD) pathway products, as the hydrogen partial pressure diminished below 34.7 bar when both solvents were used. It was concluded that hydrogen solubility effects become important and modify the activity at pressures below 34.7 bar.
Alkylation of Bromobenzene with Allyl Acetate on Zeolite H-USY: Catalytic Testing and Reaction Modeling
Authors: Van der Beken Sophie , Dejaegere Eileen , Verelst Harry , Baron Gino V , Denayer Joeri F.M.
Published: October 2005

The alkylation of bromobenzene with allyl acetate on zeolite H-USY (Si/Al 30) was studied in liquid phase conditions, at 150 °C. Reactions were performed at different space velocities (LHSV = 60, 120, 240 and 480 h-1) in tubular plug flow reactors using a high-throughput frontal analysis setup. Besides the desired alkylation products, cis-propenyl bromobenzene (cis-PBB) and trans-propenyl bromobenzene (trans-PBB) and a dialkylated product, light and heavy side products were formed. Polymerization reactions starting from allyl acetate lead to blocking of the zeolite pores, and result in catalyst deactivation. Based on the experimental data, several molecular kinetic models were proposed, and implemented in a reactor model, accounting for axial dispersion, mass transfer into the catalyst crystals, competitive adsorption effects and catalyst deactivation. Model discrimination revealed the underlying reaction mechanisms.
Alternate Co-Catalysts For Ziegler-Natta High Temperature Olefin Polymerization
Authors: Vela-Estrada Jesus
Published: October 2005

Typically a Ziegler-Natta catalyst formulation includes the use of alkyl aluminum compounds and this investigation involves the substitution of triethyl aluminum (TEAL) with n-BuLi. It was found that this lithium compound, used as first co-activator, produced polyethylene at rates high enough for solution polymerization technology. Catalyst activity and molecular weight data were collected from a lab-scale continuous polymerization reactor at 150, 170 and 190oC. A balance between n-BuLi and a second alkyl aluminum compound (AA-2), used as second co-activator in the catalyst formulation, enabled the catalyst to achieve a performance (catalyst activity and polymer molecular weight) similar to that obtained with TEAL. It is proposed that the optimum balance between these two compounds is related to the difference in the reducing power of n-BuLi and AA-2, and one of the chain transfer reactions is to AA-2.
Hydrolysis of Lactose in a Fluidized Bed of Zeolite Pellets Supporting Adsorbed ß - Galactosidase
Authors: Poletto Massimo , Parascandola Palma , Saracino Ivan , Cifarelli Giuseppe
Published: October 2005

The purpose of this work is to verify the possibility of using a simple physical adsorption technique to improve enzyme efficiency in performing lactose hydrolysis. Immobilization experiments of ß -galactosidase on zeolite pellets showed that this process is fairly slow and can be accomplished with contact times shorter than few hours. Comparison between homogeneous and heterogeneous hydrolysis experiments in mixed batch reactors indicates that the enzyme distribution within the zeolite pellets is concentrated in a thin shell under the pellet surface. Continuous conversion experiments were carried out on a purposely-built fluidization column. Results were fitted with a mathematical model for fluidized bed conversion without the use of any further adjustable parameter than those used in the heterogeneous batch conversion. Furthermore, no change of the parameter values was necessary. Scale-up predictions given by the model showed that fluidized columns few meters high of pellets supporting b-galactosidase are sufficient for an industrially suitable process of lactose hydrolysis. which can be performed on columns of few meters and, therefore, these are applicable to relatively small process scales as those found in traditional Italian cheese making factories.
On the Performance of Metal Supported Catalysts Prepared by Microemulsion: Sol Gel and Precipitation of the Support Precursor
Authors: Pérez Gustavo , Suaste J. Luis , Barrales César , De los Reyes J. Antonio , Pérez-Sea Juan , Reyes Marcelino , Viveros Tomás
Published: October 2005

Ni/SiO2, NiMo/Al2O3 and CoMo/Al2O3 were prepared by microemulsion method. Precipitation and sol-gel of the support precursor allowed the synthesis of catalysts with differences in textural, structural and catalytic properties. The main difference between the precipitation and sol-gel microemulsion routes is the role of surfactant (Cetyltrimethyl Ammonium Bromide) in each synthesis method. The aggregation mechanism of support is modified by the synthesis route. There are differences in the catalytic performance of Ni/SiO2 microemulsion catalysts by the precipitation or sol-gel of support precursor. The hydrogenation of benzene, a low structure-sensitive reaction, showed that the precipitation 9%Ni/SiO2 microemulsion catalyst is more active than the sol-gel 9%Ni/SiO2 microemulsion catalyst. On other hand, these catalysts showed opposite result in the hydrogenation of C=C double bond of crotonaldehyde molecule. These results are indicative of the effects of nanostructured support and nanostructured active-phase in the hydrogenation activity. NiMo/Al2O3 and CoMo/Al2O3 catalysts prepared by sol-gel-microemulsion method were used in the hydrodesulphurization (HDS) of dibenzothiophene and 4,6-dimethyldibenzothiophene. The HDS activity was improved as a function of metal content and it motivates the study of NiMo and CoMo microemulsion catalysts with higher contents of metals to enhance the HDS of 4,6-dimethyldibenzothiophene.
A Homogeneous Assay to Assess GABA Transporter Activity
Authors: Kopec Karla K. , McKenna Beth Ann , Pauletti Daniel
Published: October 2005

This unit describes a convenient functional uptake assay for GABA transport into cell lines transiently transfected with GABA transporter‐1 (GAT‐1) and other GAT isoforms. This facile, homogeneous assay allows for the determination of Km, Vmax, and Ki values. The assay utilizes commercially available microtiter plates that contain scintillant embedded in the bottom of the wells. Whereas a signal is generated as the cell accumulates the labeled neurotransmitter, label in the medium is undetected. While GABA uptake is observed in several cell lines transfected with GAT‐1, Km values for GABA uptake may vary with the cell line. This indicates that the choice of cell line is an important consideration when conducting uptake assays.
Adenylyl and Guanylyl Cyclase Assays
Authors: Kelly John J. , Stevens Troy , Thompson W. Joseph , Seifert Roland
Published: October 2005

This unit presents two basic protocols to determine adenylyl cyclase and guanylyl cyclase activity in tissue and cell homogenates, permeabilized cells, or subcellular fractions. Each method is divided into two parts: the enzyme reaction that causes the formation of the labeled cyclic nucleotide, and the separation of cyclic nucleotide products from unreacted nucleotide triphosphates and metabolites using Dowex 50 resin and aluminum oxide chromatographies. In the case of guanylyl cyclase, alternative separation protocols are also provided. Additionally, protocols are provided that describe preparation of both the columns used in the assays and the tissue or cells to be assayed.
Assay of Receptor‐Stimulated Phosphoinositide Turnover
Authors: Kendall David A. , Alexander Stephen P.H.
Published: October 2005

The stimulation of phosphoinositide turnover is one of the key means by which receptors evoke responses in target cells and tissues. This is true for both G protein‐coupled receptors and receptors that couple via tyrosine kinase activity. The protocols in this unit allow for pharmacological analysis of receptors coupled to phosphoinositide turnover. In general, the [3H]myo‐inositol prelabeling methodology (described for both tissue slices and cultured cells) is the more widely applicable, since it requires fewer experimental steps and typically gives rise to a better signal‐to‐noise ratio. Individual inositol phosphates can also be determined as described by chromatographic separation on ion‐exchange columns. In some circumstances (for example, when rapid responses to receptor stimulation are to be investigated or when the absolute levels of the active inositol phosphate are to be examined), it is preferable to use the mass assay described here for inositol (1,4,5)‐trisphosphate from either tissue slices and cultured cells. This unit also provides support protocols for the preparation of [3H]myo‐inositol, chromatography columns, tissue slices, and the IP3‐binding protein.
Overview of Pharmacokinetics
Authors: Aimone Lisa D.
Published: October 2005

In addition to having potency against the molecular target, a compound must be able to reach its intended site of action in vivo in sufficient quantity and for the appropriate duration to exert a biological effect. The fate of a compound after in vivo administration depends upon the absorption, distribution, metabolism, and excretion (ADME) of the compound, and the concentration of the compound in the blood, plasma, or other tissues represents the sum of all of these processes. Described in this unit are protocols for administering a compound by a variety of routes and for collecting the appropriate samples to determine the pharmacokinetic profile. The basic terms used in pharmacokinetic studies are defined, and representative examples are given to illustrate important variables to consider.
Large‐Scale Preparation of Conjugated Oligonucleoside Phosphorothioates by the High‐Efficiency Liquid‐Phase (HELP) Method
Authors: Bonora Gian Maria
Published: October 2005

A new process for the preparation of large amounts of thioated oligonucleotides in a quasi‐classical solution condition is described. This method takes advantage of the use of polyethylene glycol as a soluble, inert support during synthesis. The easy purification of intermediates from a moderate excess of reagents allows very high coupling yields and, consequently, efficient production of the long oligonucleotide sequences required for pharmacological applications. The reaction of properly protected and activated oligonucleotides with high‐molecular‐weight polyethylene glycols allows a convenient procedure for the postsynthetic conjugation of those biopolymers in solution. The oligonucleotides are modified at the 5′ terminus using a liquid‐phase procedure with a linker carrying a terminal primary amino group to enhance its nucleophilic reactivity. The use of N, N′‐disuccinimidyl carbonate for the activation of the terminal OH groups of the PEG was preferred.
Synthesis of Covalent Oligonucleotide‐Streptavidin Conjugates and Their Application in DNA‐Directed Immobilization (DDI) of Proteins
Authors: Wacker Ron , Niemeyer Christof M.
Published: October 2005

Semisynthetic DNA‐streptavidin conjugates are synthesized by covalent coupling of thiol‐modified DNA oligonucleotides and streptavidin (STV). The resulting conjugates have binding capacities for four equivalents of biotin and a complementary nucleic acid sequence. The conjugates are purified to homogeneity by ultrafiltration and chromatography, and are characterized by spectrophotometry and gel electrophoresis. Subsequently, the conjugates are applied as molecular connectors in the DNA‐directed immobilization (DDI) of biotinylated antibodies using DNA microarrays as immobilization matrices. The results are protein microarrays that can be used for the multiplexed detection of various antigens.
Synthesis of Acyclic Analogs of Adenosine
Authors: Holý Antonin
Published: October 2005

This unit describes preparation of two groups of chiral acyclic nucleosides that are N‐alkyl derivatives of adenine with hydroxyl group(s) on the alkyl chain. As an example of the neutral group, the synthesis is presented for (S)‐(2,3,dihydroxypropyl)adenine (DHPA), a compound with multiple biological effects in proliferating systems. Its synthesis consists of alkylation of adenine with a suitable chiral synthon based on 1,2‐O‐isopropylideneglycerol. Another group of structurally related biologically active compounds are the 9‐alkyladenines bearing both hydroxyl and carboxyl functions. Large‐scale cyanohydrin reaction of the adenine‐containing acetaldehyde intermediate yields 3‐(adenin‐9‐yl)‐2‐hydroxypropanoic acid (AHPA). Lastly, one of the alternative syntheses of its homolog, the nucleoside antibiotic eritadenine (with a d‐erythro‐2,3‐dihydroxybutanoic acid side‐chain) is described starting from d‐ribose. Procedures for preparation of all intermediates as well as the purification of the resulting crude material are described in detail. All these adenine derivatives inhibit SAM‐dependent methylation reactions via inhibition of SAH hydrolase.
Synthesis of Acyclic Nucleoside Phosphonates
Authors: Holý Antonin
Published: October 2005

Acyclic nucleoside phosphonates (ANPs) are important biologically active nucleotide analogs. They contain an isopolar phosphonomethyl function linked to the hydroxyl group of an acyclic side‐chain via an undegradable ether group. Though their most important activity is antiviral, some exhibit cytostatic or antiprotozoic effects. The three most important groups of ANP are presented here as synthetic procedures for a large laboratory scale. Synthesis follows three principles: (1) introduction of a protected phosphonomethyl group to the hydroxyl on an appropriate alkyl side‐chain of an acyclic nucleoside, (2) alkylation of the heterocyclic base by a synthon with all characteristic features of the future phoshonate‐bearing side‐chain, or (3) transformation of a reactive group at the heterocyclic base. The last step in all these cases is removal of the phosphonate esters. Preparation methods are described in detail for PMEA, PMEG, PMEDAP and its N6‐cyclopropyl derivative, (R)‐PMPA, and (S)‐HPMPA, as well as all intermediates and synthons.
In Vivo Marking of Single Cells in Chick Embryos Using Photoactivation of GFP
Authors: Stark D. A. , Kulesa P. M.
Published: October 2005

Selective marking of a single cell within a living embryo is often difficult due to the inaccuracy and invasiveness of standard techniques. This unit describes a minimally invasive optical protocol that uses 405‐nm laser light to photoactivate a variant of green fluorescent protein (PAGFP). This method takes advantage of the accessibility of the chick embryo to inject PAGFP into a region of interest and uses electroporation to deliver the construct into cells. This unit describes in detail how single and small groups of cells (n<10) that express PAGFP can be made visually distinguishable from the host population using the photoactivation process. Included is a means to maximize the fluorescence increase due to photoactivated GFP signal and to reduce photobleaching. Briefly outlined are previously developed chick culture and time‐lapse imaging techniques to allow for the subsequent monitoring of photoactivated cell migratory behaviors. The technique has the potential to be a less‐invasive, accurate tool for in vivo studies that involve following cell lineage and cell migration.
The Detection of MAPK Signaling
Authors: Shaul Yoav , Seger Rony
Published: October 2005

Mitogen‐activated protein kinase (MAPK) cascades are central pathways that participate in the intracellular transmission of extracellular signals. Each of the MAPK signaling cascades seems to consist of three to five tiers of protein kinases that sequentially activate each other by phosphorylation. Since the majority of MAPK cascade components are kinases, the methods used to detect their activation involve determining phosphorylation state and protein kinase activities. The Basic Protocol describes the use of immunoblotting with specific anti‐phospho antibody to detect activation of MAPK components. Alternative methods described are immunoprecipitation of desired protein kinases followed by phosphorylation of specific substrates and the use of an in‐gel kinase assay. These methods have proven useful in the study of the MAPK signaling cascades.
Stem Cells: An Overview
Authors: Denham Mark , Conley Brock , Olsson Fredrik , Cole Timothy J. , Mollard Richard
Published: October 2005

Stem cells are specialized cells that possess a capacity to undergo self‐renewal while at the same time having the ability to give rise to at least one or more differentiated or mature cell type. They therefore represent a fundamental cornerstone during the life of all vertebrates, playing central roles in the production of new and replacement cells for tissues during development and homeostasis, including repair following disease or injury. This unit is a review of stem cells, their roles in development, and their potentials as therapeutic agents.
Mouse Embryonic Stem Cell Derivation, and Mouse and Human Embryonic Stem Cell Culture and Differentiation as Embryoid Bodies
Authors: Conley Brock J. , Denham Mark , Gulluyan Lerna , Olsson Fredrik , Cole Timothy J. , Mollard Richard
Published: October 2005

Embryonic stem (ES) cells are pluripotent cells derived from developing mouse blastocysts in vitro that maintain long‐term self renewal and the capacity to give rise to all cell types in the adult body (including some extraembryonic cell types) when subjected to the appropriate conditions. It is envisaged that the development of methods enabling controlled differentiation of mouse ES cell counterparts from human blastocysts would enable the provision of an unlimited supply of tissue for cell and tissue transplantation therapies for the repair and replacement of diseased, injured, and senescent tissue. Furthermore, derivation of mouse ES cells has allowed for the generation of thousands of gene‐targeted mouse mutants. Culture of mouse ES cells as embryoid bodies (EBs) has provided a convenient system for studying early mouse developmental processes, including several aspects of extraembryonic lineage and axis formation associated with the pre‐ and peri‐gastrulating mouse embryo. Relatively little is known regarding the corresponding development of the early human embryo due to limitations associated with the acquisition of relevant tissue material for study. The transfer of methods such as EB formation to human systems should, by association, facilitate a more advanced understanding of similar processes associated with early human development. This unit describes protocols for isolating mouse embryonic stem cells and methods for propagating, freezing, and producing EBs from both mouse and human embryonic stem cells.
Using the KEGG Database Resource
Authors: Aoki Kiyoko F. , Kanehisa Minoru
Published: October 2005

KEGG (Kyoto Encyclopedia of Genes and Genomes) is a bioinformatics resource for understanding the functions and utilities of cells and organisms from both high‐level and genomic perspectives. It is a self‐sufficient, integrated resource consisting of genomic, chemical, and network information, with cross‐references to numerous outside databases, containing a complete set of building blocks (genes and molecules) and wiring diagrams (interaction networks) to represent cellular functions. This unit describes protocols for using KEGG, KEGG PATHWAY, KEGG GENES, KEGG SSDB, KEGG EXPRESSION, and KEGG LIGAND. Protocols are also described for how to color maps, compare chemical compounds and glycan chains, analyze Ortholog Clusters, and visualize and analyze microarray data, among other procedures. All of these many protocols enable the user to take advantage of the full breadth of the functionality provided by KEGG.
Representing Structural Information with RasMol
Authors: Goodsell David S.
Published: October 2005

This unit describes the use of RasMol, a popular interactive molecular graphics program, for the display of biomolecular structures. The unit begins with a general guide to obtaining molecular coordinates from the Protein Data Bank and displaying them with RasMol, and then describes several molecular representations that are useful for different applications, along with methods for ensuring that one is viewing the appropriate biological unit and some ideas for customizing one's molecular graphics session.
Generating a Genome Assembly with PCAP
Authors: Huang Xiaoqiu , Yang Shiaw‐Pyng
Published: October 2005

This unit describes how to use the Parallel Contig Assembly Program (PCAP) to assemble the data produced by a whole‐genome shotgun sequencing project. We present a basic protocol for using PCAP on a multiprocessor computer in a 300‐Mb genome assembly project. A support protocol to prepare input files for PCAP is also described. Another basic protocol for using PCAP on a distributed cluster of computers in a 3‐Gb genome assembly project is presented, in addition to suggestions for understanding results from PCAP.
Laboratory Maintenance of Brucella abortus
Authors: Sun Yao‐Hui , den Hartigh Andreas B. , Tsolis Renée M. , Ficht Thomas A.
Published: October 2005

This unit provides protocols for growth of Brucella abortus on solid or in liquid media and for long‐term storage of laboratory stocks. Two issues affecting the culture and storage of isolates of this slow‐growing bacterium are emphasized: contamination of cultures and outgrowth of attenuated variants lacking a complete lipopolysaccharide. Laboratories planning to work with B. abortus should be aware that Biosafety Level 3 facilities are required. Furthermore, this organism is classified in the U.S. as a Select Agent, which restricts its use to laboratories registered with the U.S. government's Select Agent programs.
Laboratory Maintenance of Treponema denticola
Authors: Fenno J. Christopher
Published: October 2005

This unit describes the methods, media, and equipment necessary for routine laboratory culture and handling of the anaerobic oral spirochete Treponema denticola. Topics discussed include nutrient requirements, recommended media formulations, and expected growth kinetics, as well as methods and equipment necessary to maintain anaerobic conditions. An additional protocol on isolation of T. denticola from clinical samples is included.
Laboratory Maintenance of Pathogenic Leptospira
Authors: Zuerner Richard L.
Published: October 2005

Analysis of Leptospira requires use of specialized media for growth, maintenance, and storage of viable bacteria that can be used in experimental protocols in a predictable manner. However, pathogenic Leptospira are fastidious bacteria with unusual nutritional requirements. These problems make primary isolation, routine propagation, and storage of Leptospira difficult. Defined and complex media are available for routine growth of pathogenic Leptospira, and each medium has unique characteristics that favor the growth or isolation of some strains, while not supporting the growth of others. This unit will describe the preparation of Leptospira media and methods for isolation and storage of these bacteria.
Herpes Simplex Virus: Propagation, Quantification, and Storage
Authors: Blaho John A. , Morton Elise R. , Yedowitz Jamie C.
Published: October 2005

Herpes simplex virus (HSV) is the prototype of a family of large, enveloped, double‐stranded DNA viruses, the Herpesviridae, which cause significant morbidity and mortality in humans. Productive replication of HSV in cells in culture results in definitive changes in cellular physiology and metabolism, ultimately leading to lysis. These definitive aspects of viral‐host interactions enable diagnosis of HSV infections. In this unit, a series of methods are described for the propagation, quantification, and storage of HSV. Infectious center assays are used to measure the titers of HSV stocks. In addition, immunological methods are described for documenting the accumulation of viral polypeptides in infected whole cell extracts, as well as in situ using indirect immunofluorescence. These techniques should be beneficial to basic research virologists utilizing standard laboratory HSV strains, as well as clinical microbiologists interested in characterizing HSV isolated from patients.
Using Satellite Tobacco Mosaic Virus Vectors for Gene Silencing
Authors: Gosselé Véronique , Metzlaff Michael
Published: October 2005

This unit describes the use of satellite tobacco mosaic virus (STMV) vectors in combination with native TMV particles for inducing transient gene silencing in tobacco plants. Target gene fragment selection and insertion, virus delivery procedures, and phenotype screening of silenced plants are described in detail. All critical parameters for tobacco plant cultivation, virus infection, and RNA silencing efficiency are discussed.
In Vivo Translation Studies of Plant Viral RNAs Using Reporter Genes
Authors: Matsuda Daiki , Dreher Theo W.
Published: October 2005

This unit describes the analysis of the translation‐regulating properties of viral RNAs in cell‐based assays using a luciferase reporter system. Electroporation and polyethylene glycol–mediated introduction of luciferase reporter RNA into cowpea protoplasts are also presented. The Commentary section discusses employing the luciferase reporter system to study translational control by the 5′‐ and 3′‐untranslated regions of a viral mRNA. Inoculum RNA quality and half‐life after delivery to the protoplasts should be monitored closely for careful investigation of translational regulation elements.