bluuebunny/crossref_metadata_2025 huggingface dataset:
Dichloroethane Removal from Gas Streams by an Extractive Membrane Bioreactor
Authors: Freitas Santos Luisa M. Dos , Hömmerich Uwe , Livingston Andrew G.
Published: March 2005

A novel extractive membrane bioreactor (EMB) system, in which a silicone rubber membrane separates a contaminated gas stream from an aerated biomedium, extracted and biodegraded 1,2‐dichloroethane (DCE) from a gaseous waste stream. Selective transfer of the organic compound takes place through the silicone membrane to the biomedium, where biodegradation occurs. At a gas flow rate of 770 mL min−1 and an average DCE concentration of 0.65 mg L−1, the EMB, with 2.5 m2 of membrane surface contained in a spirally wound module, degraded 91% of the DCE. A biofilm grew on the membrane surface and caused a 600 mbar pressure drop at steady state conditions. The chloride ions released by DCE mineralization were 99% of the stoichiometric amount, while 60% of the total amount of carbon crossing the membrane as DCE was bioconverted to CO2. The overall mass transfer coefficient across the membrane in the absence of microbial activity was (0.8‐1.3) × 10−6 m s−1 and was controlled by the liquid film resistance. Extensive biofilm growth significantly lowered the DCE flux. Comparison of the EMB to a direct bioscrubbing process showed that the EMB system has superior volumetric efficiency.
Membrane‐Based Cell Affinity Chromatography To Retrieve Viable Cells
Authors: Mandrusov Evgenia , Houng Abraham , Klein Elias , Leonard Edward F.
Published: March 2005

A novel scheme for the separation and live recovery of one cell type from a mixture of cells using a cell affinity chromatography (CAC) system is demonstrated. An antimurine IgG was chemically immobilized to a cellophane support via a carbonyldiimidazole (CDI) link. Murine splenocytes flowed over the support, and B‐cells were allowed to attach at a shear rate of 15 s−1. Once loading was terminated, the support was washed at a shear rate of 315 s−1 to remove nonspecifically bound cells. Elution of the B‐cells was initiated by the transmembrane diffusion of hydrochloric acid (pH 1), supplied to the side of the membrane opposite the cells. At the same time, a shear flow of normal saline was established on the cell side of the membrane, and cells, freed by acid, were retrieved. Results showed that, on average, 250 cells/mm2 attached to antibody immobilized on cellophane surfaces, at a shear rate of 15 s−1, and that attached cells were successfully displaced by acid supplied to the side of the membrane opposite that holding the cells. On average, at least 60% of the B‐cells removed by this elution appeared viable, based on a Trypan Blue dye exclusion assay.
Extraction of Charged Fusion Proteins in Reversed Micelles: Comparison between Different Surfactant Systems
Authors: Forney Craig E. , Glatz Charles E.
Published: March 2005

Behavior of a series of fusion proteins of varying charge in reversed micellar extraction was studied. The proteins consisted of the enzyme glucoamylase from Aspergillus awamori joined to short peptides containing from 0–10 additional aspartate residues. The fusions were partitioned into two different cationic surfactant systems, one based on the surfactant trioctylmethylammonium chloride (TOMAC) and the other on cetyltrimethylammonium bromide (CTAB). These two systems differed chiefly in micelle size as measured by the water to surfactant ratio, w0. Water numbers were determined for the TOMAC system, with values of approximately 10, and as a function of pH and ionic strength for CTAB for each of the mutant enzymes. For the CTAB system, water numbers were as low as 50 with NaCl concentrations of 500 mM and as high as 68 at 300 mM NaCl (95% confidence level of 2.4). The enzyme partitioned most strongly using CTAB, with maximal recoveries approaching 95%. However, in the CTAB system, there were no significant differences in behavior between the mutants because of the relatively large micellar size, even under high salt concentrations. Extraction of the control enzyme from clarified cell broth indicated that broth components did not significantly interfere with partitioning.
Investigation of the Ethanol Tolerance of Clostridium thermosaccharolyticum in Continuous Culture
Authors: Baskaran Sunitha , Ahn Hyung ‐Jun , Lynd Lee R.
Published: March 2005

The ethanol tolerance of Clostridium thermosaccharolyticum HG8 has been studied in continuous culture using a new technique that requires knowledge of the kinetic constants and measurement of substrate concentrations at various concentrations of the inhibitor. Endogenously produced ethanol was supplemented with exogenously supplied ethanol to achieve various inhibitor concentrations. The ethanol tolerance of C. thermosaccharolyticum was significantly greater than expected on the basis of most previous reports, which may be explained in part by acclimation occurring over time periods exceeding those typical of batch systems studied previously. An ethanol concentration of 40 g/L is required for 50% growth inhibition of C. thermosaccharolyticum at 55 °C. Process considerations suggest that the ethanol tolerance of C. thermosaccharolyticum is unlikely to significantly constrain its use for ethanol production from cellulosic biomass. Ester‐linked phospholipid membrane analyses (ELPFA) revealed that growth in the presence of high concentrations of ethanol (33 g/L) resulted in a membrane profile having increased fluidity and molecular diversity. Ethanol‐induced changes included a significant increase in shorter chain unsaturated fatty acids (C15:0) at the expense of longer chain unsaturated fatty acids (C17:0) and a slight increase in the amount of mono‐unsaturated fatty acids.
Metabolic Control Analysis of the Penicillin Biosynthetic Pathway in a High‐Yielding Strain of Penicillium chrysogenum
Authors: Nielsen Jens , Jørgensen Henrik S.
Published: March 2005

Metabolic control analysis is used to identify the rate‐limiting step in the penicillin biosynthetic pathway in Penicillium chrysogenum. The analysis is carried out using a kinetic model for the first two steps in the pathway, i. e., the ACV synthetase (ACVS) and the isopenicillin N synthetase (IPNS). The kinetic model is based on Michaelis—Menten type kinetics, with noncompetitive inhibition of the ACVS by ACV and competitive inhibition of the IPNS by glutathione. From measurements of the enzyme activities and intracellular metabolites during a fed‐batch cultivation, the kinetic model is used to predict the flux through the pathway. The model prediction corresponds well with the measured rate of penicillin biosynthesis. From measurement of the activity of the acyl‐CoA:isopenicillin acyltransferase, which catalyzes the third and last reaction in the pathway, it is concluded that the rate‐limiting step is either at the ACVS or at the IPNS. From the kinetic model, the elasticity coefficients and the flux control coefficients are calculated throughout the fed‐batch cultivations, and it is found that there is a shift in the flux control from the ACVS to. the IPNS during the cultivation.
Tracking of Individual Cell Cohorts in Asynchronous Saccharomyces cerevisiae Populations
Authors: Porro Danilo , Srienc Friedrich
Published: March 2005

A novel flow cytometric procedure has been developed with the aim to obtain the growth properties of individual Saccharomyces cerevisiae cells in asynchronous culture. The method is based on labeling of the cell surface with FITC‐conjugated concanavalin A and detection of the single‐cell fluorescence with flow cytometry after cell exposure to growth conditions. Because the formation of new cell wall material in budded cells is restricted to the bud tip, exposure of the stained cells to growth conditions results in three cell types: (i) stained cells, (ii) partially stained cells, and (iii) unstained cells Analysis of the staining pattern over time permits the determination of the specifice growth rate of the cell population, the length of the budded cell cycle phase, and the growth pattern during the cell cycle of newly formed, partially stained daughter cells. The procedure has been tested with yeast cell populations growing at different rates. The data suggest an exponential increase in the size of individual cells during the cell cycle, as reflected by the forward angle light scattering (FALS) signals. It has been found that the apparent single‐cell specific cell size growth rates, determined by FALS intensity, are significantly lower than the specific growth rates of the overall population. This could indicate that the tracking of a cohort of cells is significantly perturbed by a distribution of staining levels of daughter cells at cell division and that FALS may not be a good indicator of the cell size.
Process Characterization of a Novel Cross‐Regulation System for Cloned Protein Production in Escherichia coli
Authors: Chen Wilfred , Kallio Pauli T. , Bailey James E.
Published: March 2005

A novel cross‐regulation expression system has been shown previously to be very effective for regulated recombinant protein production. Earlier studies established that this system offers better control of basal expression and higher maximal induced expression than more traditional vectors. Using production of cloned chloramphenicol acetyltransferase (CAT) as a model system, several factors determining the performance of this system were examined. Specifically, the effects of varying induction times and inducer (IPTG) concentrations on cell growth and the rate of CAT production were examined. The CAT expression was maximally induced with at least 0.5 mM IPTG added at the midexponential growth phase. Specific CAT content (on a total protein basis) was correlated with the CAT mRNA level. CAT message levels were minimal preinduction and far above background postinduction, consistent with prior simulation results. Cessation of CAT accumulation as the culture entered the stationary phase coincided with a corresponding 10‐fold decrease in the level of CAT mRNA which was likely caused by an increased mRNA degradation rate. Maintenance of significant CAT message levels with a concomitant 2‐fold increase in CAT accumulation was achieved by extending cell erowth in a fed‐batch process.
Fluidized‐Bed Bioreactors
Authors: Gòdia Francesc , Solà Caries
Published: March 2005

Fluidized‐bed reactors present a number of advantages that make them an attractive alternative in processes involving biocatalysts. However, fluidized‐bed bioreactors are also realtively complex, basically for two reasons. First, their use requires the biocatalyst, commonly cells or enzymes, to be immobilized into or onto a solid support. Second, the hydrodynamic characterization is difficult, especially in those systems where three phases (gas—liquid—solid) are involved. The mathematical model of a fluidized‐bed bioreactor needs to take into account those hydrodynamic aspects that will determine the flux model in the reactor. Moreover, the description of other aspects is also required: the mechanisms of transport between the different phases, the kinetic equations for the phenomena taking place in the biocatalytic particles, such as cell growth, product formation, substrate consumption, enzyme deactivation, and the mass balance equations in the reactor. In addition to these aspects, the application of fluidized‐bed bioreactors to different kind of processes is also discussed. The potential of this type of bioreactor is also emphasized from the point of view of the different number of possible modifications in the design both of the bioreactor and the biocatalyst particles, in order to enhance its operation.
Phosphorescence Probes of the Glassy State in Amorphous Sucrose
Authors: Shahs Naina K. , Ludescher Richard D.
Published: March 2005

We have used phosphorescence from the luminescent chromophores N‐acetyltryp‐tophanamide (NATA) and erythrosin B (Ery B) to probe the molecular dynamics of the glassy state and the glass‐to‐rubber transition in amorphous sucrose. These measurements exploit the known sensitivity of phosphorescence intensity to the rate and extent of molecular collisions with the local environment. Both probes exhibit intense, well‐defined phosphorescence emission spectra in the glassy sugar matrix. Time‐resolved phosphorescence intensity decays for each probe were multiexponential both above and below the glass transition temperature, indicating that the sucrose matrix is heterogeneous on the molecular level; local microviscosities around the NATA probe were estimated in the range from 104 to 5 × 107 Pa·s in the glassy state at 25 °C. Plots of phosphorescence lifetime versus temperature for both probes are linear above and below the glass transition temperature of sucrose (62 °C), but exhibit a break in slope near approximately 50 °C characteristic of a dramatic change in the rate of molecular collision between the probe and the local environment. The similar sensitivity of two chemically different spectroscopic probes (the indole ring of NATA and the xanthene ring of Ery B) to molecular changes near the glass transition in this sugar indicates that phosphorescence may provide a generally applicable, site‐specific probe of the glassy state and changes in it in biological materials.
Comparative Enzyme Biodegradability of Xylan, Cellulose, and Starch Derivatives†
Authors: Glasser Wolfgang G. , Ravindran G. , Jain Rajesh K. , Samaranayake Gamini , Todd Jason
Published: March 2005

The enzyme biodegradability of xylan and of two of its prominent derivatives, (hydroxypropyl)xylan and xylan acetate, were examined comparatively vis‐à‐vis similar cellulose and starch derivatives, using a commercially available xylanase‐rich enzyme preparation. The results indicate that xylan, like cellulose and its derivatives, is subject to enzymatic depolymerization in relation to degree of substitution. Degradation paralleled the presence of unmodified xylose units in the polysaccharide. Degradation proceeded rapidly, with greater than 80% of total degradation being complete after only 1 day. A comparison with starch and cellulose derivatives revealed that hydroxypropyl xylan suffers less biodegradability loss as substitution rises to 0.25 or 0.4, after which loss of degradability parallels that of the corresponding cellulose and starch derivatives. This apparent tolerance to low degrees of substitution is attributed to molecular heterogeneity which suggests that constituents other than xylose become propoxylated initially. Xylan acetate was found to lose enzymatic biodegradability at a rate which parallels that of cellulose acetate, and this is 35% slower (with respect to DS) than corresponding starch acetates. Size exclusion chromatography results suggest that xylan degrades to monosaccharides, that xylan derivatives degrade to monosaccharides and oligosaccharides, and that substitution with lignin prevents xylan from being recognized by xylanolytic enzymes, resulting in loss of degradability.
Mathematical Model for Analysis of Mass Transfer for Immobilized Cells in Lactic Acid Fermentation
Authors: Wang Henian , Seki Minoru , Furusaki Shintaro
Published: March 2005

A new mathematical model is proposed to analyze the mass transfer behavior in lactic acid fermentation using immobilized cells entrapped in calcium alginate. The model is comprised of material balance equations for glucose, free lactic acid, and several ions. The dissociation rate of lactic acid (rdiss) is involved in the proposed equations. To solve the equations numerically, a modified calculating method is proposed. Through model analysis, the possible mass transfer behavior in the gel bead was discussed. The model is validated by comparisons with the experimental results obtained from batch and continuous fermentation. The simulations using the model were made to investigate the mass transfer limitation in the gel beads. The results showed that the cell density gradient was formed in the gel beads and it was caused by the accumulation of the inhibitory product (free lactic acid), not by substrate starvation. Moreover, unusual mass transfer behavior of lactate ion in the immobilization support was pointed out.
Development of a Continuous Lactulose Process: Separation and Purification
Authors: Kozempel Michael F. , Kurantz Michael J. , Craig Jr. James C. , Hicks Kevin B.
Published: March 2005

We developed a continuous pilot plant process to produce lactulose from lactose using boric acid to boost the conversion to about 75%. The process consists of three reactors (two continuous stirred tank reactors followed by a tubular reactor), pH adjustment, a crystallizer—filter—evaporator recycle initial separation section, and a chromatographic purification system to produce lactulose containing less than 1–5 ppm of boric acid. The continuous process operates at 0.53 kg/min (20% solids) through the reactors and about 0.3 kg/min (55% solids) out of the initial separation section. The chromatographic separation is at the prep scale with a flow rate of 2.0 mL/min. The final product is a 55% solids syrup containing no detectable boric acid.
Studies of High Solidity Ratio Hydrofoil Impellers for Aerated Bioreactors. 1. Review
Authors: Mcfarlane Caroline M. , Nienow Alvin W.
Published: March 2005

This first part of a four‐part series of papers presents a review of the literature on high solidity ratio axial flow hydrofoil impellers. Interest in the application of high‐flow, low power number, hydrofoil impellers, such as the Prochem Maxflo T and the Lightnin A315, has developed over recent years and has largely been stimulated by reports of improved fermentation performance relative to the Rushton disc turbine. Initially, the review briefly examines the characteristics of the radial flow Rushton turbine, as traditionally used in fermenters, with particular reference to its weaknesses. It also discusses the use of pitched blade turbines (mixed flow impellers) for air dispersion since these show some characteristics similar to those of the axial flow hydrofoils. The consideration of these two impeller types provides the background to explain the advantages and potential problems associated with high solidity ratio, axial flow, downward pumping hydrofoils. These aspects are discussed in relation to existing literature which is still relatively scarce. Finally, the advantages of retrofitting are introduced. Parts 2–4 report on a recent, detailed, fluid dynamic study of the Prochem Maxflo T and Lightnin A315 hydrofoil impellers which extends our knowledge of these important impeller types.
Conditions Promoting Metal‐Catalyzed Oxidations during Immobilized Cu—Iminodiacetic Acid Metal Affinity Chromatography
Authors: Krishnamurthy Rajesh , Madurawe Rapti D. , Bush Kristen D. , Lumpkin Janice A.
Published: March 2005

Many solutions that contain oxygen and/or hydrogen peroxide, transition metals, and reductants undergo metal‐catalyzed oxidation (MCO) reactions. These reactions produce highly reactive radical intermediates which can cause damage to a variety of biomolecules. Some of the types of damage caused by MCO reactions to proteins are activity losses, irreversible amino acid modifications, increased susceptibility to proteolysis, and/or fragmentation. The occurrence of such reactions in immobilized metal affinity chromatography (IMAC) systems has not been reported, nor has it been well studied. We report here enzyme activity studies of lactate dehydrogenase (LDH) during chromatography on an immobilized Cu2+‐‐iminodiacetic acid (IDA) metal affinity column and document the occurrence of MCO reactions under various chromatography conditions. Chromatography in the presence of the reducing agent ascorbate or the oxidant hydrogen peroxide caused LDH inactivation, and the presence of both reagents greatly enhanced the loss of activity. Increasing concentrations of reducing agent or hydrogen peroxide led to increased levels of damage. Chromatography under anaerobic conditions reduced LDH inactivation. Enzyme inactivation on the column was consistent with activity losses observed in solutions containing dissolved Cu2+‐‐IDA. Other reducing agents such as glutathione, β‐mercaptoethanol, and cysteine also caused LDH inactivation during chromatography. During chromatography in the presence of a reducing agent and/or peroxide, Cu+ and hydroxyl radicals were generated on the column and metal ions were removed from the column. Studies with the Cu+‐specific chelator bicinchoninic acid indicated that Cu+ was an essential component for the observed protein inactivation. The loss of enzyme activity in the presence of ascorbate and/or peroxide is most likely due to the occurrence of MCO reactions on the column. During chromatography in the absence of added reagents, the loss of LDH activity and the occurrence of MCO reactions were not detected over the chromatography times used in this study. However, LDH inactivation did occur in solutions containing dissolved Cu2+‐‐IDA. An understanding of the conditions under which MCO reactions occur during IMAC would aid the design of better downstream processing operations utilizing metal affinity methods.
Hollow Fiber Array Affinity Chromatography
Authors: Schisla D. K. , Carr P. W. , Cussler E. L.
Published: March 2005

Preparative separations of creatine phosphokinase and bovine albumin are carried out by hollow fiber liquid chromatography. The hollow fibers used are poly(propylene), 240μm in diameter. They are uniformly coated with cross‐linked poly(vinyl alcohol) and then reacted with the triazine dye Procion Blue MX‐R. Experiments with these fibers show good protein separations, including those of the phosphokinase from the rabbit heart and the rabbit brain. Other experiments show that overloading can be successfully predicted. In terms of resolution at low sample loads, hollow fiber chromatography is currently inferior to analytical columns, though it is potentially superior for preparative separations.
Effective Bioconversion with Continuous Product Recovery Using AOT/Lecithin Mixed Reverse Micellar Systems and Centrifugal Partition Chromatography as a Novel Bioreactor
Authors: Yamada Yasushi , Kuboi Ryoichi , Komasawa Isao
Published: March 2005

The application of reverse micellar micro‐bioreactor systems for enzymatic conversion with continuous recovery of water‐soluble products was demonstrated, based on the production of galactose by micelles containing β‐galactosidase (β‐gal). The β‐gal was firmly encapsulated in AOT (bis(2‐ethylhexyl)sodium sulfosuccinate)/Lecithin mixed micelles. The addition of lecithin to the AOT reverse micelles stabilized the micellar interface, and the leakage of the encapsulated β‐gal to the bulk aqueous phase was therefore negligible, while substrate and product were free to transfer and partition owing to their low molecular weights. The stability of the solubilized β‐gal was also increased in the AOT/Lecithin mixed micelles as compared to the conventional AOT micelles. A considerably higher activity was observed in the present systems than in the bulk buffer solution. Enzymatic conversion and product recovery was carried out in a continuous operation mode by use of centrifugal partition chromatography (CPC), employing the reverse micellar solution with encapsulated β‐gal as the stationary phase and the substrate aqueous solution as the mobile phase. Use of the CPC contactor eliminated any mass transfer limitation on the substrate and the product, and galactose was continuously recovered in the effluent. The recovery of galactose continued for a period of 5 h with more than a 85% conversion at the optimum operating condition. This reverse micellar micro‐bioreactor system was thus found to be very effective for the continuous production and recovery of hydrophilic products.
Sensational Interests, Mating Effort, and Personality: Evidence for Cross-Cultural Validity
Authors: Egan Vincent , Figueredo Aurelio José , Wolf Pedro , McBride Kara , Sefcek Jon , Vasquez Geneva , Charles Kathy
Published: March 2005

Abstract. We assessed whether violent or macabre interests (“sensational interests”) were related to mating effort and a higher-order personality construct reflecting the combined features of higher Extraversion, lower Psychoticism, and lower Neuroticism in 1321 participants from English-speaking (Tucson, Arizona, and Glasgow, Scotland) and Spanish-speaking (Hermosillo, Mexico, and Talca, Chile) communities. Participants from Spanish-speaking communities generally had more sensational interests. Mating effort was positively related to sensational interests, though the relationship was slightly weaker in Spanish-speaking communities. Personality effects were modest and entirely moderated by language; personality was negatively related to sensational interests in English-speaking communities, but positively associated to sensational interests in Spanish-speaking communities. These findings suggest that the relationship between mating effort and sensational interests is universal and reflects general intrasexual competition, whereas the relationship between personality and sensational interests is more culture-specific.
Observation of the reactive component of Langmuir wave phase‐bunched electrons
Authors: Kletzing C. A. , Bounds S. R. , LaBelle J. , Samara M.
Published: March 2005

Observations from a new wave‐particle correlator show clear evidence of phase bunching of the electrons by a monochromatic Langmuir wave electric field which varied slowly in amplitude. The electrons are found to be bunched at 90° with respect to the wave field indicating that they are the reactive component of the perturbed distribution function. This is in agreement with theory that predicts this phase relation for the transit of electrons through a very long wave field.
Equatorward shift of the subarctic boundary in the northwestern Pacific during the last deglaciation
Authors: Yamamoto Masanobu , Suemune Ryutaro , Oba Tadamichi
Published: March 2005

We have generated a high‐resolution record of alkenone sea surface temperature (SST) between 10–24 ka from Core MD01‐2421 off central Japan, in the northwestern Pacific. The cooling by 5°C from 21 ka to 12.8 ka implies the equatorward shift of the subarctic boundary in the northwestern Pacific by ∼2.8° in latitude. This shift was a result of the stronger summer Okhotsk High. The Okhotsk High was likely enhanced by the combined effects of El Niño‐like conditions in the tropical Pacific and the heating of the land surface of northeastern Siberia.
Shortwave aerosol radiative forcing over cloud‐free oceans from Terra: 2. Seasonal and global distributions
Authors: Zhang Jianglong , Christopher Sundar A. , Remer Lorraine A. , Kaufman Yoram J.
Published: March 2005

Using 10 months of collocated Clouds and the Earth's Radiant Energy System (CERES) scanner and Moderate Resolution Imaging Spectroradiometer (MODIS) aerosol and cloud data from Terra, we provide estimates of the shortwave aerosol direct radiative forcing (SWARF) and its uncertainties over the cloud‐free global oceans. Newly developed aerosol angular distribution models (ADMs) (Zhang et al., 2005), specifically for different sea surface conditions and aerosol types, are used for inverting the CERES observed radiances to shortwave fluxes while accounting for the effect of aerosol optical properties on the anisotropy of the top of atmosphere (TOA) shortwave radiation fields. The spatial and seasonal distributions of SWARF are presented, and the MODIS retrieved aerosol optical depth (τ0.55) and the independently derived SWARF show a high degree of correlation and can be estimated using the equation SWARF = 0.05 − 74.6 τ0.55 + 18.2 τ0.552 W m−2 (τ0.55 < 0.8). The instantaneous TOA SWARF from Terra overpass time is −6.4 ± 2.6 W m−2 for cloud‐free oceans. Accounting for sample biases and diurnal averaging, we estimate the SWARF over cloud‐free oceans to be −5.3 ± 1.7 W m−2, consistent with previous studies. Our study is an independent measurement‐based assessment of cloud‐free aerosol radiative forcing that could be used as a validation tool for numerical modeling studies.
Determination of the minimal functional ligand-binding domain of the GABAB(1b) receptor
Authors: DERIU Daniela , GASSMANN Martin , FIRBANK Susan , RISTIG Dorothee , LAMPERT Christina , MOSBACHER Johannes , FROESTL Wolfgang , KAUPMANN Klemens , BETTLER Bernhard , GRÜTTER Markus G.
Published: March 2005

In the mammalian central nervous system, slow inhibitory neurotransmission is largely mediated by metabotropic GABAB receptors (where GABA stands for γ-aminobutyric acid), which belong to the G-protein-coupled receptor gene family. Functional GABAB receptors are assembled from two subunits GABAB(1) (GABAB receptor subtype 1) and GABAB(2). For the GABAB(1) subunit, which binds the neurotransmitter GABA, two variants GABAB(1a) (GABAB receptor subtype 1 variant a) and GABAB(1b) have been identified. They differ at the very N-terminus of their large glycosylated ECD (extracellular domain). To simplify the structural characterization, we designed truncated GABAB(1) receptors to identify the minimal functional domain which still binds a competitive radioligand and leads to a functional, GABA-responding receptor when co-expressed with GABAB(2). We show that it is necessary to include all the portion of the ECD encoded by exon 6 to exon 14. Furthermore, we studied mutant GABAB(1b) receptors, in which single or all potential N-glycosylation sites are removed. The absence of oligosaccharides does not impair receptor function, suggesting that the unglycosylated ECD of GABAB(1) can be used for further functional or structural investigations.
Thyroid hormones directly activate the expression of the human and mouse uncoupling protein-3 genes through a thyroid response element in the proximal promoter region
Authors: SOLANES Gemma , PEDRAZA Neus , CALVO Verónica , VIDAL-PUIG Antonio , LOWELL Bradford B. , VILLARROYA Francesc
Published: March 2005

The transcription of the human UCP3 (uncoupling protein-3) gene in skeletal muscle is tightly regulated by metabolic signals related to fatty acid availability. However, changes in thyroid status also modulate UCP3 gene expression, albeit by unknown mechanisms. We created transgenic mice bearing the entire human UCP3 gene to investigate the effect of thyroid hormones on human UCP3 gene expression. Treatment of human UCP3 transgenic mice with thyroid hormones induced the expression of the human gene in skeletal muscle. In addition, transient transfection experiments demonstrate that thyroid hormones activate the transcription of the human UCP3 gene promoter when MyoD and the TR (thyroid hormone receptor) were co-transfected. The action of thyroid hormones on UCP3 gene transcription is mediated by the binding of the TR to a proximal region in the UCP3 gene promoter that contains a direct repeat structure. An intact DNA sequence of this site is required for thyroid hormone responsiveness and TR binding. Chromatin immunoprecipitation assays revealed that the TR binds this element in vivo. The murine Ucp3 gene promoter was also dependent on MyoD and responsive to thyroid hormone in transient transfection assays. However, it was much less sensitive to thyroid hormone than the human UCP3 promoter. In summary, UCP3 gene transcription is activated by thyroid hormone treatment in vivo, and this activation is mediated by a TRE (thyroid hormone response element) in the proximal promoter region. Such regulation suggests a link between UCP3 gene expression and the effects of thyroid hormone on mitochondrial function in skeletal muscle.
CLOCK is involved in the circadian transactivation of peroxisome-proliferator-activated receptor α (PPARα) in mice
Authors: OISHI Katsutaka , SHIRAI Hidenori , ISHIDA Norio
Published: March 2005

PPARα (peroxisome-proliferator-activated receptor α) is a member of the nuclear receptor superfamily of ligand-activated transcription factors that regulate the expression of genes associated with lipid metabolism. In the present study, we show that circadian expression of mouse PPARα mRNA requires the basic helix–loop–helix PAS (Per-Arnt-Sim) protein CLOCK, a core component of the negative-feedback loop that drives circadian oscillators in mammals. The circadian expression of PPARα mRNA was abolished in the liver of homozygous Clock mutant mice. Using wild-type and Clock-deficient fibroblasts derived from homozygous Clock mutant mice, we showed that the circadian expression of PPARα mRNA is regulated by the peripheral oscillators in a CLOCK-dependent manner. Transient transfection and EMSAs (electrophoretic mobility-shift assays) revealed that the CLOCK–BMAL1 (brain and muscle Arnt-like protein 1) heterodimer transactivates the PPARα gene via an E-box-rich region located in the second intron. This region contained two perfect E-boxes and four E-box-like motifs within 90 bases. ChIP (chromatin immunoprecipitation) also showed that CLOCK associates with this E-box-rich region in vivo. Circadian expression of PPARα mRNA was intact in the liver of insulin-dependent diabetic and of adrenalectomized mice, suggesting that endogenous insulin and glucocorticoids are not essential for the rhythmic expression of the PPARα gene. These results suggested that CLOCK plays an important role in lipid homoeostasis by regulating the transcription of a key protein, PPARα.
Inhibition sites in F1-ATPase from bovine heart mitochondria
Authors: GLEDHILL Jonathan R. , WALKER John E.
Published: March 2005

High-resolution crystallographic studies of a number of inhibited forms of bovine F1-ATPase have identified four independent types of inhibitory site: the catalytic site, the aurovertin B-binding site, the efrapeptin-binding site and the site to which the natural inhibitor protein IF1 binds. Hitherto, the binding sites for other inhibitors, such as polyphenolic phytochemicals, non-peptidyl lipophilic cations and amphiphilic peptides, have remained undefined. By employing multiple inhibition analysis, we have identified the binding sites for these compounds. Several of them bind to the known inhibitory sites. The amphiphilic peptides melittin and synthetic analogues of the mitochondrial import pre-sequence of yeast cytochrome oxidase subunit IV appear to mimic the natural inhibitor protein, and the polyphenolic phytochemical inhibitors resveratrol and piceatannol compete for the aurovertin B-binding site (or sites). The non-peptidyl lipophilic cation rhodamine 6G acts at a separate unidentified site, indicating that there are at least five inhibitory sites in the F1-ATPase. Each of the above inhibitors has significantly different activity against the bacterial Bacillus PS3 α3β3γ subcomplex compared with that observed with bovine F1-ATPase. IF1 does not inhibit the bacterial enzyme, even in the absence of the ε-subunit. An understanding of these inhibitors may enable rational development of therapeutic agents to act as novel antibiotics against bacterial ATP synthases or for the treatment of several disorders linked to the regulation of the ATP synthase, including ischaemia–reperfusion injury and some cancers.
Kinetics of bidirectional H+ and substrate transport by the proton-dependent amino acid symporter PAT1
Authors: FOLTZ Martin , MERTL Manuela , DIETZ Veronika , BOLL Michael , KOTTRA Gabor , DANIEL Hannelore
Published: March 2005

PAT1 is a recently identified member of the PAT family of proton/amino acid co-transporters with predominant expression in the plasma membrane of enterocytes and in lysosomal membranes of neurons. Previous studies in Xenopus oocytes expressing PAT1 established proton/substrate co-transport associated with positive inward currents for a variety of small neutral amino acids. Here we provide a detailed analysis of the transport mode of the murine PAT1 in oocytes using the two-electrode voltage-clamp technique to measure steady-state and pre-steady-state currents. The GPC (giant patch clamp) technique and efflux studies were employed to characterize the reversed transport mode. Kinetic parameters [Km (Michaelis constant) and Imax (maximum current)] for transport of various substrates revealed a dependence on membrane potential: hyperpolarization increases the substrate affinity and maximal transport velocity. Proton affinity for interaction with PAT1 is almost 100 nM, corresponding to a pH of 7.0 and is independent of substrate. Kinetic analysis revealed that binding of proton most likely occurs before substrate binding and that the proton and substrate are translocated in a simultaneous step. No evidence for a substrate-uncoupled proton shunt was observed. As shown by efflux studies and current measurements by the GPC technique, PAT1 allows bidirectional amino acid transport. Surprisingly, PAT1 exhibits no pre-steady-state currents in the absence of substrate, even at low temperatures, and therefore PAT1 takes an exceptional position among the ion-coupled co-transporters.
Bulk GaN and AlGaN∕GaN heterostructure drift velocity measurements and comparison to theoretical models
Authors: Barker J. M. , Ferry D. K. , Koleske D. D. , Shul R. J.
Published: March 2005

The room-temperature velocity-field characteristics for n-type gallium nitride and AlGaN∕GaN heterostructures, grown epitaxially on sapphire, were determined experimentally. A pulsed voltage input and four-point measurements were used on special geometry samples to determine the electron drift velocity as a function of applied electric field in the basal plane. These measurements show apparent saturation velocities near 2.5×107cm∕s at 180kV∕cm for the n-type gallium nitride and 3.1×107cm∕s at 140kV∕cm for the AlGaN∕GaN heterostructures. A comparison of these studies shows that the experimental velocities are close to previously published simulations based upon Monte Carlo techniques.
Breaking of balanced and unbalanced equatorial waves
Authors: Bouchut F. , Le Sommer J. , Zeitlin V.
Published: March 2005

A clear-cut signature of a wave-breaking event is irreversible modification of the mean flow. In this paper, we provide examples of different breaking mechanisms and show that breaking scenario of equatorial waves in the β-plane shallow water model is determined by the degree of balance between the zonal component of the Coriolis force and the pressure gradient. Our analysis is based on a specially designed numerical method which guarantees two essential conditions to simulate nonlinear equatorial waves: (i) the scheme converges toward weak solutions including shocks and (ii) preserves the steadiness of balanced stationary solutions. This allows for accurate diagnostics of Lagrangian invariants of motion such as passive tracer density or potential vorticity. For unbalanced waves, the lack of balance leads to shock formation in finite time. In shock fronts, the variation of the dissipation rate induces a nonadvective potential vorticity flux and violates the local potential vorticity conservation valid for smooth solutions. This dissipative breaking mechanism is generic for unbalanced waves and is associated with enhanced mixing. For long, balanced (Rossby) waves, breaking consists in appearance of recirculation regions. It results in the formation of propagating patterns, the equatorial modons, which trap fluid particles. Such breaking occurs during the propagation of Rossby wave packets with positive geopotential anomaly and is strengthened by decreasing fluid depth. The modons are robust and collide quasielastically with Kelvin waves.
Ultrafast far-infrared dynamics probed by terahertz pulses: A frequency-domain approach. II. Applications
Authors: Němec H. , Kadlec F. , Kadlec C. , Kužel P. , Jungwirth P.
Published: March 2005

We present data obtained by time-resolved terahertz spectroscopy in selected semiconducting and molecular systems exhibiting subpicosecond far-infrared dynamics. We use a frequency-domain method which eliminates the influence of instrumental functions and artifacts due to frequency mixing and yields a two-dimensional transient conductivity of the photoexcited sample. This technique enables improving the attainable experimental time resolution and allows a simple qualitative interpretation of the results without a priori modeling. The quantitative interpretation is based on the time-dependent Drude and damped harmonic oscillator models.
Field-effect transistors based on a polycyclic aromatic hydrocarbon core as a two-dimensional conductor
Authors: Mori Tomohiko , Takeuchi Hisato , Fujikawa Hisayoshi
Published: March 2005

Hexabenzocoronene (HBC) derivatives which are designed to self-assemble into a two-dimensional conductor have been synthesized. The derivatives were deposited by vacuum sublimation as an active layer in organic field-effect transistors. The dihexyl and tetrahexyl derivatives increased the field-effect mobilities and on∕off ratios by a factor of 10 or more as compared to the unsubstituted hexabenzocoronene and the hexahexyl derivative. This good performance could be explained by the self-assembly in a two-dimensional conductor of the HBC derivatives, i.e., the dihexyl and tetrahexyl derivatives, in contrast to the self-assembly in the one-dimensional conductor of the hexahexyl derivative and low self-assembling property of the unsubstituted HBC.
Model analysis for a current-step-like structure in a high Tc Josephson device coupled to a microwave signal
Authors: Mizugaki Yoshinao , Chen Jian
Published: March 2005

High Tc Josephson junctions have IcRn products as large as a few mV at liquid helium temperature, and hence, the frequency normalized by IcRn∕Φ0 becomes on the order of 10−3 for a gigahertz (GHz) microwave signal. (Here, Ic, Rn, and Φ0 are the critical current, the normal resistance, and a flux quantum, respectively.) It is well known that a microwave-current-driven Josephson junction does not exhibit clear Shapiro steps under such low frequency conditions. Instead of Shapiro steps, however, we often observe a current-step-like structure in the current–voltage curve of YBa2Cu3O7−δ grain boundary Josephson junctions coupled to a GHz signal. In this article, we present a model analysis for such current-step-like structures. Two types of device models are employed: a simple resistively and capacitively shunted junction model and a rf-field-driven two-junction superconducting quantum interference device model. Numerical results of the latter model agree with the experimental results, which indicates that the envelope of small Shapiro steps, including the effects of microwave-induced motion of flux quanta, appears as a current-step-like structure in a high Tc Josephson device coupled to a GHz microwave.
Use of the degenerated Reynolds equation in solving the microchannel flow problem
Authors: Shen Ching
Published: March 2005

In this paper, the author proposes that the generalized Reynolds equation employed in the gas film lubrication problems, where the flow rates of the Poiseuille flow are calculated from the Boltzmann equation, can be degenerated for solving the microchannel flow problem in the transitional regime. Using this approach, the calculated results of pressure distribution in long microchannels show excellent agreement with the experimental data and the result of the information preservation (IP) method. The results in short microchannels show excellent agreement with the direct simulation Monte Carlo method and the IP method. The lattice Boltzmann method solution of the microchannel flow is examined by comparison with the degenerated Reynolds equation calculations and the disagreement in the pressure distribution confirms that the lattice Boltzmann method is unsuitable for the solution of the microelectromechanical systems (MEMS) flows in transitional regime. For microchannel flows, the degenerated Reynolds equation can serve as a criterion having the merits of kinetic theory for testing various methods intending to solve rarefied gas flow problems in MEMS devices in the transitional flow regime.
Excitation and direct imaging of surface plasmon polariton modes in a two-dimensional grating
Authors: Tetz Kevin A. , Rokitski Rostislav , Nezhad Maziar , Fainman Yeshaiahu
Published: March 2005

We describe the simultaneous excitation and direct far-field imaging of the scattering from surface plasmon polariton modes in a two-dimensional metallic hole array grating. Conditions for the coupling and imaging are discussed, where the coupling is shown to be consistent with both measured and calculated dispersion relations. Excitation is accomplished at several different wavelengths (from 1.31 to 1.57μm), incidence angles, and grating periods, enabling the observation of a number of distinct modes with various in-plane wave vectors.
Evidence of gap state formed by the charge transfer in Alq3∕NaCl∕Al interface studied by ultraviolet and x-ray photoelectron spectroscopy
Authors: Yi Yeonjin , Kang Seong Jun , Cho Kwanghee , Koo Jong Mo , Han Kyul , Park Kyongjin , Noh Myungkeun , Whang Chung-Nam , Jeong Kwangho , Hahn Eun Joo
Published: March 2005

Electronic structures of Alq3∕NaCl∕Al and Alq3∕Al were studied by UV and x-ray photoelectron spectroscopy (XPS). The initial energy level of the highest occupied molecular orbital (HOMO) of Alq3∕Al was shifted when the ultrathin NaCl layer was inserted between them, although the vacuum level was not changed. The measured interface dipole was 1.1 eV, identical for both Alq3∕NaCl∕Al and Alq3∕Al. Our experiment shows that the dipole is formed in very short range (less than 0.1 nm) from the interface. The onset of the HOMO level of Alq3 was shifted 0.2 eV toward high binding energy for the additional NaCl layer, which lowered the barrier height and improved injection characteristics of the device. Moreover, a gap state was observed at 1.1 eV below the Fermi level when the NaCl was inserted between Alq3 and Al. The XPS core-level spectra revealed that the interaction on Alq3 and NaCl was very weak, which generated an unusual gap state without breaking or forming chemical bonds. We suggest that the weak interaction would originate from the charge transfer from Alq3 to NaCl.
Genetically stable and fully effective smallpox vaccine strain constructed from highly attenuated vaccinia LC16m8
Authors: Kidokoro Minoru , Tashiro Masato , Shida Hisatoshi
Published: March 2005

A highly attenuated LC16m8 (m8) smallpox vaccine has been licensed in Japan because of its extremely low neurovirulence profile, which is comparable to that of replication incompetent strains of vaccinia virus. From 1973 to 1975, m8 was administrated to >100,000 infants where it induced levels of immunity similar to that of the originating Lister strain, without any serious side effects. Recently, we observed that m8 reverts spontaneously to large plaque forming clones that possess virulence equivalent to that of LC16mO, a parental virus strain of m8. Here, we report that the
B5R
gene is responsible for the reversion, and that we could construct a more genetically stable virus by deleting
B5R
from m8. The protective immunogenicity of the vaccine candidate proved to be equivalent to that of the U.S.-licensed product Dryvax, and much superior to modified vaccinia Ankara in a mouse model. Furthermore, the vaccine strain never elicited any symptoms in severe combined immunodeficiency disease mice, even at a dose 1,000-fold greater than that used in the immune protection experiments, which is in contrast to the lethal pathogenicity induced by Dryvax inoculation of severe combined immunodeficiency disease mice. Our results suggest that this vaccine strain is a good candidate as a suitable smallpox vaccine and a vector virus, and that B5R is not essential for protective immunity against smallpox.