bluuebunny/crossref_metadata_2025 huggingface dataset:
Tails in generalized Jackson networks with subexponential service-time distributions
Authors: Baccelli François , Foss Serguei , Lelarge Marc
Published: June 2005

We give the exact asymptotics of the tail of the stationary maximal dater in generalized Jackson networks with subexponential service times. This maximal dater, which is an analogue of the workload in an isolated queue, gives the time taken to clear all customers present at some time t when stopping all arrivals that take place later than t. We use the property that a large deviation of the maximal dater is caused by a single large service time at a single station at some time in the distant past of t, in conjunction with fluid limits of generalized Jackson networks, to derive the relevant asymptotics in closed form.
The Rise of Europe: Atlantic Trade, Institutional Change, and Economic Growth
Authors: Acemoglu Daron , Johnson Simon , Robinson James
Published: June 2005

The rise of Western Europe after 1500 is due largely to growth in countries with access to the Atlantic Ocean and with substantial trade with the New World, Africa, and Asia via the Atlantic. This trade and the associated colonialism affected Europe not only directly, but also indirectly by inducing institutional change. Where “initial” political institutions (those established before 1500) placed significant checks on the monarchy, the growth of Atlantic trade strengthened merchant groups by constraining the power of the monarchy, and helped merchants obtain changes in institutions to protect property rights. These changes were central to subsequent economic growth.
Sunspots in the Laboratory
Authors: Duffy John , Fisher Eric O'N
Published: June 2005

We show that extrinsic or nonfundamental uncertainty influences markets in a controlled environment. This work provides the first direct evidence of sunspot equilibria. These equilibria require a common understanding of the semantics of the sunspot variable, and they appear to be sensitive to the flow of information. Sunspots always occur in a closed-book call market, but they happen only occasionally in a double auction, where inframarginal bids and offers are observable.
The Willingness to Pay–Willingness to Accept Gap, the “Endowment Effect,” Subject Misconceptions, and Experimental Procedures for Eliciting Valuations
Authors: Plott Charles R , Zeiler Kathryn
Published: June 2005

We conduct experiments to explore the possibility that subject misconceptions, as opposed to a particular theory of preferences referred to as the “endowment effect,” account for reported gaps between willingness to pay (“WTP”) and willingness to accept (“WTA”). The literature reveals two important facts. First, there is no consensus regarding the nature or robustness of WTP-WTA gaps. Second, while experimenters are careful to control for subject misconceptions, there is no consensus about the fundamental properties of misconceptions or how to avoid them. Instead, by implementing different types of experimental controls, experimenters have revealed notions of how misconceptions arise. Experimenters have applied these controls separately or in different combinations. Such controls include ensuring subject anonymity, using incentive-compatible elicitation mechanisms, and providing subjects with practice and training on the elicitation mechanism before employing it to measure valuations. The pattern of results reported in the literature suggests that the widely differing reports of WTP-WTA gaps could be due to an incomplete science regarding subject misconceptions. We implement a “revealed theory” methodology to compensate for the lack of a theory of misconceptions. Theories implicit in experimental procedures found in the literature are at the heart of our experimental design. Thus, our approach to addressing subject misconceptions reflects an attempt to control simultaneously for all dimensions of concern over possible subject misconceptions found in the literature. To this end, our procedures modify the Becker-DeGroot-Marschak mechanism used in previous studies to elicit values. In addition, our procedures supplement commonly used procedures by providing extensive training on the elicitation mechanism before subjects provide WTP and WTA responses. Experiments were conducted using both lotteries and mugs, goods frequently used in endowment effect experiments. Using the modified procedures, we observe no gap between WTA and WTP. Therefore, our results call into question the interpretation of observed gaps as evidence of loss aversion or prospect theory. Further evidence is required before convincing interpretations of observed gaps can be advanced.
Are Two Heads Better Than One? Team versus Individual Play in Signaling Games
Authors: Cooper David J , Kagel John H
Published: June 2005

We compare individuals with two-person teams in signaling game experiments. Teams consistently play more strategically than individuals and generate positive synergies in more difficult games, beating a demanding “truth-wins” norm. The superior performance of teams is most striking following changes in payoffs that change the equilibrium outcome. Individuals play less strategically following the change in payoffs than inexperienced subjects playing the same game. In contrast, the teams exhibit positive learning transfer, playing more strategically following the change than inexperienced subjects. Dialogues between teammates are used to identify factors promoting strategic play.
Acceptable Input: Using Decision Analysis to Guide Public Policy Deliberations
Authors: Gregory Robin , Fischhoff Baruch , McDaniels Tim
Published: June 2005

Multiparty deliberative processes have become a popular way to increase public participation in public policy choices. Their legitimacy depends on participants' ability, first, to understand the issues facing them and, then, to form and express their own positions on them. These tasks pose significant cognitive and emotional challenges. This paper argues that decision analysis, informed by behavioral decision research, offers procedures and standards for creating responsible deliberative processes. These involve (a) formal analysis of decisions, identifying the kernel of most relevant information, (b) communication procedures, recognizing the strengths and weaknesses of lay understanding, and (c) interactive elicitation methods, helping individuals to articulate the implications of their values for specific settings. A construct validity criterion assesses the extent to which the resulting valuations are properly sensitive to decision features. Feasible extensions of traditional decision analysis create opportunities to formalize the aspirations of participants and ensure that the intellectual content of deliberative processes is worthy of the political hopes vested in them.
Anticoagulation Monitoring Part 2: Unfractionated Heparin and Low-Molecular-Weight Heparin
Authors: Spinler Sarah A , Wittkowsky Ann K , Nutescu Edith A , Smythe Maureen A
Published: June 2005

OBJECTIVE

To review the availability, mechanisms, limitations, and clinical application of point-of-care (POC) devices used in monitoring anticoagulation with unfractionated heparin (UFH) and low-molecular-weight heparins (LMWHs).

DATA SOURCES

Articles were identified through a MEDLINE search (1966–August 2004), device manufacturer Web sites, additional references listed in articles and Web sites, and abstracts from scientific meetings.

STUDY SELECTION AND DATA EXTRACTION

English-language literature from clinical trials was reviewed to evaluate the accuracy, reliability, and clinical application of POC monitoring devices.

DATA SYNTHESIS

The activated partial thromboplastin time (aPTT) and activated clotting time (ACT) are common tests for monitoring anticoagulation with UFH. Multiple devices are available for POC aPTT, ACT, and heparin concentration testing. The aPTT therapeutic range for UFH will vary depending upon the reagent and instrument employed. Although recommended by the American College of Chest Physicians Seventh Conference on Antithrombotic and Thrombolytic Therapy, establishing a heparin concentration–derived therapeutic range for UFH is rarely performed. Additional research evaluating anti-factor Xa monitoring of LMWHs using POC testing is necessary.

CONCLUSIONS

Multiple POC devices are available to monitor anticoagulation with UFH. For each test, there is some variability in results between devices and between reagents used in the same device. Despite these limitations, POC anticoagulation monitoring of UFH using aPTT and, more often, ACT is common in clinical practice, particularly when evaluating anticoagulation associated with interventional cardiology procedures and cardiopulmonary bypass surgery.
Photosensitized one-electron oxidation of DNA
Authors: Kawai Kiyohiko , Majima Tetsuro
Published: June 2005

Photosensitized one-electron oxidation of DNA has attracted much interest because it causes oxidative damage which leads to mutation, and because it is involved in the basic mechanism of photodynamic therapy. In the present article, we describe the mechanistic study of photosensitized DNA damage, especially addressing the kinetics of hole transfer by adenine(A)-hopping and its effect on the DNA damage. The combination of the transient absorption measurement and DNA damage quantification by high-performance liquid chromatography clearly demonstrate that the yield of the DNA damage correlates well with the lifetime of the charge-separated state caused by A-hopping, showing that hole transfer helps DNA damage. These findings led us to propose a new method to accomplish the efficient DNA damage using a combination of two-color, two-laser irradiation.
Photooxygenation in polymer matrices: En route to highly active antimalarial peroxides
Authors: Griesbeck Axel G. , El-Idreesy Tamer T. , Bartoschek Anna
Published: June 2005

Photooxygenation involving the first excited singlet state of molecular oxygen is a versatile method for the generation of a multitude of oxy-functionalized target molecules often with high regio- and stereoselectivities. The efficiency of singlet-oxygen reactions is largely dependent on the nonradiative deactivation paths, mainly induced by the solvent and the substrate intrinsically. The intrinsic (physical) quenching properties as well as the selectivity-determining factors of the (chemical) quenching can be modified by adjusting the microenvironment of the reactive substrate. Tetraarylporphyrins or protoporphyrin IX were embedded in polystyrene (PS) beads and in polymer films or covalently linked into PS during emulsion polymerization. These polymer matrices are suitable for a broad variety of (solvent-free) photooxygenation reactions. One specific example discussed in detail is the ene reaction of singlet oxygen with chiral allylic alcohols yielding unsaturated β-hydroperoxy alcohols in (threo) diastereoselectivities, which depended on the polarity and hydrogen-bonding capacity of the polymer matrix. These products were applied for the synthesis of mono- and spirobicyclic 1,2,4-trioxanes, molecules that showed moderate to high antimalarial properties. Subsequent structure optimization resulted in in vitro activities that surpassed that of the naturally occurring sesquiterpene-peroxide artemisinin.
Infrared Multiple Specular Reflectance of Mono- and Submonomolecular Layers of p-Bromobenzoic Acid on Oxidized Metals
Authors: Sondag A. H. M. , Raas M. C.
Published: June 2005

The advantages of Multiple Specular Reflection (MSR) over single reflection grazing incidence infrared spectroscopy are discussed in this paper. A calculation which incorporates the experimental constraints for a multiple reflection setup is performed. The results concerning the angular dependence are confirmed by experiment. The main advantages are a wide angular acceptance and a release of the grazing angle of incidence geometry. Approximately the same sensitivity can be obtained as is theoretically possible with a single reflection at grazing incidence. However, in practice a somewhat higher sensitivity can be obtained with multiple specular reflectance spectroscopy. The sensitivity is illustrated by the measurement of submonolayers of p-bromobenzoic acid chemi-sorbed on an oxidized aluminium flat surface. Comparison with neutron activation analysis data shows that the reflection-absorption intensities are proportional to the surface coverage. A dependence of spectral features on surface coverage can be observed.
Deconvolution versus Derivative Spectroscopy
Authors: Kalkandjiev T. K. , Petrov V. P. , Nickolov J. B.
Published: June 2005

The set of functions that can be deconvoluted is extended. An explicit form of the deconvolution operator is obtained by generalization of the operator of even derivation. The advantages of the deconvolution to derivation, when the former is used for resolution enhancement, are given analytically.
Direct Sample Introduction of Solid Material into a Pulse-Operated MIP
Authors: Mohamed M. M. , Uchida T. , Minami S.
Published: June 2005

To minimize problems caused by sample introduction into a microwave-induced plasma (MIP), we have developed a system consisting of a spark ablation cell and a pulse-operated microwave-induced plasma (pulsed-MIP). An aerosol is generated from solid samples with the use of a spark discharge. The resultant material is swept into a pulsed-MIP. Design and fabrication criteria for the spark source and the spark ablation cell are presented. The combined sources show enhanced precision, stability, signal-to-noise levels, and detection limits relative to direct spark emission. Analytical calibration curves and detection limit data are shown for nickel, manganese, and chromium in steel samples. Time-resolved data are also shown.
An Efficient Method for Recovering the Optimal Unbiased Linear Spectrum-Estimate from Hadamard Transform Spectrometers Having Nonideal Masks
Authors: Johnson T. W. , Park J. B. , Dyer S. A. , Harms B. K. , Dyer R. A.
Published: June 2005

A spectrum-recovery method is presented which efficiently computes an optimal unbiased linear spectrum-estimate for measurements obtained with Hadamard transform (HT) spectrometers having nonideal masks. This method has the following advantages over other spectrum-recovery techniques: it is computationally efficient, it requires no additional measurements, and it computes an optimal spectrum-estimate. In the method presented, after the mask of the HT spectrometer has been characterized, approximately 3 N preliminary arithmetic operations are performed once for a given spectrometer, where N is both the number of spectral resolution-elements desired and the number of measurements required. Each spectrum-estimate to be recovered then requires only an additional O[ N(log2 N + 4)] arithmetic operations. In contrast, conventional methods for obtaining an optimal unbiased linear spectrum-estimate require O( N3) preliminary operations, and O(2 N2) operations during each spectrum-recovery.
Applications of Microspectroscopy in the Near-Infrared Region
Authors: Smith Matthew J. , Carl Richard T.
Published: June 2005

In this report, several applications of near-infrared microspectroscopy are illustrated using unmodified commercial instrumentation. The principal advantage of near-infrared microspectroscopy is the ability to analyze small samples which are totally absorbing in the mid-infrared region. Near-infrared analysis is shown to yield useful structural information about several different types of samples. Examples from the fields of materials science, single crystals, forensics and biological science are illustrated, and some tentative band assignments are made.
A Fluorescence Lifetime Distribution Measurement System Based on Phase-Resolved Detection Using an Image Dissector Tube
Authors: Wang Xue Feng , Uchida Teruo , Minami Shigeo
Published: June 2005

A new phase-resolved two-dimensional fluorescence measurement system using an image dissector tube (IDT) has been developed. The gain control characteristics of the blanking electrode (located between the aperture plate and the first dynode of the IDT) are utilized to allow heterodyne detection with low-voltage control signals. Heterodyning the modulated fluorescence and reference signals at the blanking electrode of the IDT provides high-frequency response up to 1 GHz, in contrast to the 20 MHz bandwidth of the dynode chain due to electron transit time spread. Significant improvement of time resolution is observed. Fluorescence intensity and lifetime data are derived from the dc and ac components of the phase-resolved signals, respectively. Two-dimensional measurements can be performed by electronically scanning images within the IDT. Details of the instrumental system and typical fluorescence lifetime distribution measurements are presented.
Optically Active Vibrations of Lithium and α-Sodium Uranium Hexafluorides (LiUF6 and α-NaUF6)
Authors: Ohwada Ken
Published: June 2005

Normal coordinate analyses of the optically active vibrations of LiUF6 and α-NaUF6 have been made, with the assumption of an idealized structural model. On the basis of the results of this treatment, all of the observed bands so far reported have been successfully assigned to the intramolecular vibrations (UF6−) as well as the optically active lattice vibrations [M+(Li, Na) … UF6−]. The force constants concerning U-F and M-F bonds have been obtained within the framework of the modified valence force field. It has been found that the present analysis, based on a whole crystal, is powerful not only for assigning the optically active lattice vibrations but also for elucidating the vibronic structure of the electronic absorption spectra obtained at low-temperature measurements.
Considerations on the Simultaneous Behavior of the Resonance Fluorescence and Ionization Signals versus Laser Intensity in Flames
Authors: Omenetto N. , Smith B. W. , Jones B. T. , Winefordner J. D.
Published: June 2005

By the use of a rate equation approach and a disregard for recombination between ions and electrons, the variation of fluorescence and ionization signals as a function of laser intensity is given. Plots of ion and photon yields vs. laser intensity for various values of important parameters are given and are discussed with respect to the significance of collisional ionization and photoionization.
On Absorbance Measurements in Spatially Inhomogeneous Fields
Authors: Brewster Jeffrey D. , Anderson James L.
Published: June 2005

The measured absorbance of a spatially inhomogeneous sample may be strongly affected by the relative spatial distribution of the absorbing medium and the optical beam. The measured absorbance is, in general, not the average of local absorbances across the beam, except when the maximum range of local absorbance values across the entire field is small (<0.1 AU). The measured transmittance of an optically inhomogeneous field is equal to the average transmittance only if incident intensity is everywhere uniform. The general problem is outlined and is illustrated by examples of transient spectroelectrochemical experiments monitoring the time course of reactant consumption or product generation as they traverse a thin optical beam parallel to the electrode.
Rotational Temperature in Helium, Argon, and Oxygen Microwave-Induced Plasmas: Comparison with Translational and Solid Surface Temperatures
Authors: Alandari J. , Diamy A. M. , Guillerme J. M. , Legrand J. C. , Ben-Aim R. I.
Published: June 2005

Translational temperature ( Tg), rotational temperature of OH ( Tr), and surface temperature ( Ts are determined in helium, argon, and oxygen plasmas. Measurements are carried out at different pressures and different flow rates as a function of microwave power input by using various methods (line broadening, pressure rise, spectroscopy, thermocouple, pyrometry). The three temperatures depend on the nature of the gas (oxygen < helium > argon) and increase with power input and pressure. Results show that Tg is of the same order of magnitude as Ts. But in all experimental conditions, Tr is always higher than Tg. Therefore, Tr is inadequate for estimating Tg. When no direct determination of Tg is available, Ts seems to give a better estimation of this important parameter.
Convenient Fiber-Optic-Based Sample Cell for Shpol'skii and Low-Temperature Phosphorescence Spectrometry
Authors: Madison Richard T. , Carroll Mary K. , Hieftje Gary M.
Published: June 2005

A sample cell for observing the Shpol'skii effect at 77 K is described and analytically assessed. The cell employs fiber-optic light guides to transport excitation and emission radiation. The system is compact, inexpensive, and simple to construct from commercially available laboratory components, and it alleviates several problems inherent in conventional refrigerated-cell designs. Detection limits for anthracene, coronene, and pyrene obtained with the sample cell are 8.8 × 10−8 M, 8.4 × 10−7 M, and 3.5 × 10−7 M, respectively. The linear dynamic range for each compound is 2 to 3 orders of magnitude.
FT-IR Characterization of Metal Acetates in Aqueous Solution
Authors: Tackett James E.
Published: June 2005

A correlation has been developed between antisymmetric and symmetric carboxylate group infrared absorption frequencies, cationic radii, and acetate group types that can be used to differentiate between ionic, unidentate, bidentate, and bridging acetate groups in aqueous metal acetate solutions. In solutions containing the alkali metal, alkaline earth, and Mn(II), Co(II), and Ni(II) ions, the acetate group is primarily ionic. The bound acetate groups in Ce(III), Pb(II), Sm(III), Y(III), and Yb(III) systems are bidentate. A solution of Cr(III) acetate has bridging acetate groups. Cu(II), Zn(II), and Cd(II) appear to form H-bonded unidentate structures in which an acetate oxygen not coordinated to the metal is hydrogen bonded to a coordinated water molecule. Hg(II) acetate has bridging acetate groups and might also have unidentate acetate groups.
Photodiode Array Systems for Inductively Coupled Plasma-Atomic Emission Spectrometry
Authors: Lepla Keith C. , Horlick Gary
Published: June 2005

Details are presented for the construction of photodiode array (PDA) measurement systems from commercial components. The PDA systems described include the Hamamatsu S2304–1024Q, a Reticon 1024S using the RC1000 and RC1001 circuit boards, and a Reticon 1024S using the RC1024S circuit board. Detals are presented for computer-controlled clocking and timing circuits, ADC sub-systems, and Peltier cooling subsystems. The measurement characteristics (sensitivity and detection limits) for all arrays are intercompared with the use of analyte emission signals from an inductively coupled plasma.
Preconcentration of Trace Elements in Aluminum Alloys Using a Chelating Ion-Exchanger and Determination by ICP-AES
Authors: Dumont Jacques , Côté Martin , Hubert Joseph
Published: June 2005

The Hyphan resin, a chelating ion-exchanger, is used to preconcentrate several metals (Cd, Co, Cr, Cu, Fe, Mg, Mn, Ni, Ti, and Zn) in aluminum alloys, before analysis by ICP atomic emission. The recovery of the preconcentration procedure is 100% for most metals. The accuracy of the method is evaluated by analysis of ALCAN and NIST standard reference materials. The results are in good agreement with the certified values. The preconcentration method can be used in a batch or in a flow analyzer mode in conjunction with an ICP/atomic emission spectrometer.
Elimination of Interferences in Spectral Data
Authors: Lacey Richard F.
Published: June 2005

A general-purpose method for eliminating one or more interferences from spectra in a completely objective way is described. The method, called least-squares subtraction, depends only on the linearity of the spectra and the linear independence of the analyte's spectrum from those of the interferences. The spectrum that results after correction is modified from the spectrum of the pure analyte, but it can still be used for quantitation and identification. Because of this modification, the method is best applied to problems where the presence of the analyte is obscured by the interferences, or when fast, automatic removal of one or more time-varying interferences from a series of spectra is required. The method is illustrated by the removal of a strong interference from the ATR measurement of the infrared spectrum of an enzyme-substrate complex and the removal of a changing chromatographic baseline from the output of a liquid chromatograph's diode array detector.
Raman Spectroscopic Investigation of the Denaturation Process of Silk Fibroin
Authors: Zheng Siding , Li Guanxian , Yao Wenhuo , Yu Tongyin
Published: June 2005

The mechanical denaturation process of silk fibroin is examined by Raman spectroscopy. The fresh silk fibroins from the middle gland of mature silkworms are drawn to various ratios on a tensile tester ( R = ldrawn/ linitial, where l is length) and their conformations are measured with Raman spectroscopy. Undrawn silk fibroin is mainly in the random coil structure with some α-helical conformation, the characteristic bands appearing at 1252 and 1660 (random coil) and at 942, 1106, and 1270 cm−1 (α-helix). When the samples are drawn up to R = 4 at an extension rate of 500 mm/min, two peaks at 1233 cm−1 (the amide III band) and 1085 cm−1 appear; it is shown that the β-sheet conformation is then formed. With an increase in drawing ratios, the intensities of these β-sheet bands increase and those of the random coil and α-helical bands decrease gradually. These changes indicate that, under the action of stress, the conformation of fibroin is altered from random coil and α-helix to β-sheet structures. This result is quite similar to the results achieved by the spinning of the silkworm. The effect of the water content in liquid silk on this conformational transition process is revealed and discussed.
Applications of Laser-Induced Thermal Emission Spectroscopy to Various Samples
Authors: Tsuge Akira , Uwamino Yoshinori , Ishizuka Toshio
Published: June 2005

The Laser-Induced Thermal Emission (LITE) spectra of several solid samples were measured and compared with the usual thermal emission spectra generated by the heating element. The following advantages in LITE spectroscopy were made clear: (1) the spectral background is reduced; (2) the time required for the stabilization of the thermal emission signal is short; (3) the compounds which are coated on background materials with complex shapes can be measured.
Quantitative Measurements of Adsorbed Aminosilanes on Muscovite Mica Using Diffuse Reflectance Infrared Fourier Transform Spectroscopy
Authors: Vågberg Lena , De Potocki Paul , Stenius Per
Published: June 2005

A method for quantitative analysis of 3-aminopropyltriethoxysilane (γ-APS) and N-(N-vinylbenzyl-2-aminoethyl)-3-aminopropyltrimethoxy-silane hydrochloride (cationic vinylbenzyl silane, CVBS) on muscovite mica powder has been developed with the use of diffuse reflectance FT-IR spectroscopy. The adsorption isotherms determined by this method are reported. The results are compared with a previous study of the adsorption of aminosilanes by ESCA, and possible models of the adsorbed layer are discussed.
Deconvolution of the Chemical Shift Effects in Proton Nuclear Magnetic Resonance Imaging
Authors: Liu Juwhan , Nieminen Antti O. K. , Koenig Jack L.
Published: June 2005

When the gradient strength is not strong enough to suppress the chemical shift effects, it is possible to apply a numerical deconvolution technique to remove the chemical shift artifacts. The Wiener filter and an apodization function are combined to produce an effective deconvolution method for the NMR images obtained by standard spin-echo NMR imaging technique. In the deconvolution process, an NMR spectrum is utilized which is taken at the same condition as the NMR image but with the phase encoding and the read gradients off.
Detection of Halogenated Compounds by Capillary Gas Chromatography with Helium Plasma Mass Spectrometry Detection
Authors: Mohamad A. H. , Creed J. T. , Davidson T. M. , Caruso J. A.
Published: June 2005

A gas Chromatograph was coupled to a helium microwave-induced plasma mass spectrometer for the detection of halogenated hydrocarbons. The absolute detection limits for the chlorinated compounds investigated ranged from 9.2 to 21 picograms, while the detection limits for brominated and iodinated compounds ranged from 0.92 to 1.05 picograms. The linear dynamic ranges were found to be 2.5–3 orders of magnitude. The results obtained are compared with optical emission from this and other laboratories.
Spatially Resolved ESCA Using Hadamard Masks
Authors: Hoffmann Douglas P. , Proctor Andrew , Hercules David M.
Published: June 2005

Use of Hadamard-based movable masks is demonstrated to be an effective method for obtaining spatially resolved information from x-ray photoelectron spectroscopy (XPS or ESCA). The concept of such imaging in electron spectroscopy is discussed in conjunction with the results obtained from a relatively simple mask system which was attached to a modified AEI ES200 photoelectron spectrometer. Resolution on the order of 1 mm2 was obtained with this configuration. The ultimate spatial resolution, on the order of microns, depends on engineering limitations concerned primarily with mask design and fabrication. The advantage of such a system stems from its inherent multiplexing nature, the requirements for only a single-channel detector, and the associated possibility of retro-fitting currently existing instrumentation.
Localization of α-Dystroglycan on the Podocyte: from Top to Toe
Authors: Vogtländer Nils P.J. , Dijkman Henry , Bakker Marinka A.H. , Campbell Kevin P. , van der Vlag Johan , Berden Jo H.M.
Published: June 2005

α-Dystroglycan (DG) is a negatively charged membrane-associated glycoprotein that links the cytoskeleton to the extracellular matrix. Previously, we described that α-DG covers the whole podocyte cell membrane in the rat. However, our finding was challenged by the description of a strictly basolateral localization in human kidney biopsies, using a different antibody against α-DG. Therefore, we studied the exact localization of glomerular α-DG by using these two antibodies in both species. The studies were performed by using monoclonal antibodies (MoAbs) IIH6 and VIA4.1 in immunofluorescence, confocal microscopy, and immunoelectron microscopy on both rat and human kidney sections, as well as on cultured mouse podocytes. The apical localization of α-DG on podocytes was more dominant than the basolateral localization. The basolateral staining with MoAb VIA4.1 was more pronounced than that of MoAb IIH6. With both MoAbs, the staining in rat kidneys was more prominent, in comparison to human kidneys. We conclude that α-DG is expressed at both the basolateral and apical sides of the podocyte. This localization suggests that α-DG plays a dual role in the maintenance of the unique architecture of podocytes by its binding to the glomerular basement membrane, and in the maintenance of the integrity of the filtration slit, respectively.
Chemiluminescence Quantitative Immunohistochemical Determination of MRP2 in Liver Biopsies
Authors: Guardigli Massimo , Marangi Marianna , Casanova Silvia , Grigioni Walther F. , Roda Enrico , Roda Aldo
Published: June 2005

Evaluation of protein expression in tissues and cells by electrophoretic and blotting techniques or by the quantification of the mRNA coding for the target protein is a common procedure in biochemistry research and clinical diagnoses. In this article, an alternative approach, based on an immunohistochemical procedure with chemiluminescent imaging detection, is described. The assay exploited the peculiar characteristics of the chemiluminescent detection of enzyme labels (high sensitivity and specificity, low background, easy quantification of the signal) for performing the direct, simple, and rapid quantitative evaluation of protein expression in tissues. When applied to the study of the levels of MRP2, a member of the human multidrug resistance-associated protein family, in samples obtained from formalin-fixed, paraffin-embedded liver biopsies, it allowed the reliable evaluation of the protein content of the tissue. Moreover, the analysis of clinical samples from patients with primary biliary cirrhosis under therapy with ursodeoxycholic acid gave results in line with those, previously reported in the literature, obtained with conventional protein expression analysis techniques.
Genome-Wide Characterization of Tetrahymena thermophila Chromosome Breakage Sites. I. Cloning and Identification of Functional SitesSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY653004, AY653005, AY653006, AY653007, AY653008, AY653009, AY653010, AY653011, AY653012, AY653013, AY653014, AY653015, AY653016, AY653017, AY653018, AY653019, AY653020, AY653021, AY653022, AY653023, AY653024, AY653025, AY653026, AY653027, AY653028, AY653029.
Authors: Hamilton Eileen , Bruns Peter , Lin Cindy , Merriam Virginia , Orias Eduardo , Vong Linh , Cassidy-Hanley Donna
Published: June 2005

The chromosomes of the macronuclear (expressed) genome of Tetrahymena thermophila are generated by developmental fragmentation of the five micronuclear (germline) chromosomes. This fragmentation is site specific and directed by a conserved 15-bp chromosome breakage sequence (Cbs element). This article reports the construction of a library enriched for chromosome breakage junctions and the development of a successful scheme for the genome-wide isolation and characterization of functional Cbs junctions. Twenty-three new Cbs junctions were characterized and each was assigned to a specific micronuclear chromosome or chromosome arm. Two distinct previously unreported variant chromosome breakage sequences were found, each in two or more functional Cbs elements. Analysis of natural Cbs junctions confirmed that microheterogeneity in the macronuclear telomere addition site is associated with chromosome fragmentation. The physical and genetic characterization of these functional chromosome breakage junctions is reported in the accompanying article in this issue. The whole-genome shotgun sequencing and auto-assembly phase of the Tetrahymena Genome Initiative has recently been completed at The Institute for Genome Research (TIGR). By providing unique sequence from the natural ends of macronuclear chromosomes, Cbs junctions characterized in the work reported here will serve as useful sequence tags for relating macro- and micronuclear genetic, physical, and sequence maps.
The Dominant Inhibitory Chalcone Synthase Allele C2-Idf (Inhibitor diffuse) From Zea mays (L.) Acts via an Endogenous RNA Silencing MechanismSequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession nos. AY728478 [c2 gene chalcone synthase (wild type) C2-W22], AY728476 (Zea mays L. C2-Idf allele; gene copies C2-Idf-I and C2-Idf-II), and AY728477 (Zea mays L. C2-Idf allele; gene copy C2-Idf-III).
Authors: Della Vedova Chris B , Lorbiecke René , Kirsch Helene , Schulte Michael B , Scheets Kay , Borchert Lutz M , Scheffler Brian E , Wienand Udo , Cone Karen C , Birchler James A
Published: June 2005

The flavonoid pigment pathway in plants has been used as a model system for studying gene regulatory mechanisms. C2-Idf is a stable dominant mutation of the chalcone synthase gene, c2, which encodes the first dedicated enzyme in this biosynthetic pathway of maize. Homozygous C2-Idf plants show no pigmentation. This allele also inhibits expression of functional C2 alleles in heterozygotes, producing a less pigmented condition instead of the normal deeply pigmented phenotype. To explore the nature of this effect, the C2-Idf allele was cloned. The gene structure of the C2-Idf haplotype differs substantially from that of the normal c2 gene in that three copies are present. Two of these are located in close proximity to each other in a head-to-head orientation and the third is closely linked. Previous experiments showed that the lower level of pigmentation in heterozygotes is correlated with reduced enzyme activity and low steady-state mRNA levels. We found that c2 transcription occurs in nuclei of C2-Idf/C2 heterozygotes, but mRNA does not accumulate, suggesting that the inhibition is mediated by RNA silencing. Infection of C2-Idf/C2 heterozygotes with viruses that carry suppressors of RNA silencing relieved the phenotypic inhibition, restoring pigment production and mRNA levels. Finally, we detected small interfering RNAs (siRNAs) in plants carrying C2-Idf, but not in plants homozygous for the wild-type C2 allele. Together, our results indicate that the inhibitory effect of C2-Idf occurs through RNA silencing.
Decreased expression of retinoid nuclear receptor (RARα and RARγ) mRNA determined by real-time quantitative RT-PCR in peripheral blood mononuclear cells of hypothyroid patients
Authors: Féart C , Vallortigara J , Higueret D , Gatta B , Tabarin A , Enderlin V , Higueret P , Pallet V
Published: June 2005

In vivo assessment of the cellular impact of thyroid hormones on target tissues might be of help for physiological studies and to evaluate the consequences of various diseases of the thyroid gland in humans. Given the tenuous relationship between retinoid and tri-iodothyronine (T3) status and that retinoids have also intracellular roles, the aim of this study was to determine the effect of hypothyroidism on the expression of T3 nuclear receptors (TR) and retinoic acid nuclear receptors (RAR, RXR) in human peripheral blood mononuclear cells (PBMC). Using real time RT-PCR, we quantified the relative amount of mRNA of the thyroid (TRα and TRβ) and retinoid (RARα, RARγ, and RXRα) nuclear receptors in PBMC of euthyroid (n=22) compared with hypothyroid (n=22) subjects. Classical plasma parameters (free T3 (FT3), free thyroxine (T4) (FT4), thyroid-stimulating hormone (TSH), retinol (ROH), retinol-binding protein (RBP) and transthyretin (TTR)) were also measured. In hypothyroid subjects, the concentration of TSH was elevated, and dramatically low T3 and T4 concentrations were associated with a decrease in the expression of TRβ. Expression of RARα and RARγ significantly decreased in hypothyroid versus control subjects, while an increased concentration of ROH was emphasised by hypothyroidism. These results first indicated that primary hypothyroidism induces hypoactivation of the retinoid nuclear pathway in PBMC, which was not predicted by the plasma ROH level. Further investigations will be necessary to evaluate these parameters in very small changes in thyroid hormone production such as mild (subclinical) hypothyroidism.
Differential effects of sex steroid hormones on the expression of multiple first exons including a novel first exon of prolactin receptor gene in the rat liver
Authors: Tanaka M , Suzuki M , Kawana T , Segawa M , Yoshikawa M , Mori M , Kobayashi M , Nakai N , Saito T R
Published: June 2005

In addition to the known four alternative first exons E11, E12, E13 and E14 of the rat prolactin receptor (PRL-R) gene, a novel first exon, E15, was identified by cDNA cloning of the 5′-end region of PRL-R mRNA in the rat liver. Genomic fragments containing E15 and its 5′- or 3′-flanking regions were also cloned from rat kidney genomic DNA. A sequence search for E15 revealed that E15 is located 49 kb upstream of exon 2 of the PRL-R gene in rat chromosome 2q16. RT-PCR analysis revealed that E15 was preferentially expressed in the liver, brain and kidney. Expression profiles of E12-, E13- and E15-PRL-R mRNAs in the liver of male and female rats at 5 days of age and those at 8 weeks of age were examined by RT-PCR. The levels of E12-PRL-R mRNA in the female rat increased remarkably in rats at 8 weeks of age compared with those at 5 days of age, and the levels of E15-PRL-R mRNA in the male rat decreased markedly at 8 weeks of age compared with those at 5 days of age. In the female rat, the levels of E12-PRL-R mRNA at 8 weeks of age decreased with ovariectomy performed at 4 weeks of age and recovered with the administration of β-oestradiol. On the contrary, the levels of E15-PRL-R mRNA increased with ovariectomy and decreased with the oestrogen treatment. In the male rat liver, the levels of E12-PRL-R mRNA at 8 weeks of age increased strikingly with castration performed at 4 weeks of age and became undetectable with the administration of testosterone. The levels of E15-PRL-R mRNA increased slightly with castration and were restored by testosterone treatment. Removal of gonadal tissues and sex steroid hormone treatment had no effect on the expression levels of E13-PRL-R mRNA in both female and male rat livers. These results indicated that the expression of the PRL-R gene in the liver is regulated by the differential effects of sex steroid hormones on the transcription of the multiple first exons including the novel one.
Activation of the insulin-like growth factor binding protein-5 promoter by parathyroid hormone in osteosarcoma cells requires activation of an activated protein-2 element
Authors: Erclik Mary S , Mitchell Jane
Published: June 2005

We have previously shown that parathyroid hormone (PTH) stimulates the expression of insulin-like growth factor binding protein-5 (IGFBP-5) transcript levels in the osteosarcoma cell-line, UMR106–01 cells. In the present study we examined the molecular basis for the PTH induction of IGFBP-5 mRNA in these cells. PTH had no effect on the half-life of the IGFBP-5 transcript but did stimulate the transactivation of the proximal 889 base pairs of the rat IGFBP 5′ flanking region in a luciferase fusion construct, suggesting that PTH stimulates transcript levels through transcriptional mechanisms. Progressive 5′ deletions to −59 base pairs of the proximal promoter region had no effect on PTH induction of transactivation, indicating that an element existed within the first −59 base pairs upstream of the transcription start site that was responsive to PTH. Within the −59 base pairs there are CCAAT/enhancer binding protein (C/EBP), E-box, nuclear factor-1 (NF-1) and activator protein-2 (AP-2) elements. Mutation of the C/EBP, E-box or NF-1 elements had no effect on the ability of PTH to induce the transactivation of the IGFBP-5 promoter. Mutation of the AP-2 element resulted in a 40% reduction of PTH-stimulated luciferase activity. When three tandem repeats of the AP-2 consensus sequence were fused to a luciferase reporter, PTH stimulated a 25% increase in reporter activity. Electrophoretic mobility shift assays using UMR106–01 cell nuclear extracts showed that PTH caused a prominent shifted band in a probe spanning the region containing all four elements. The shifted band was almost completely absent when the probe contained a mutated AP-2 element. These results suggest that the AP-2 element functions in the PTH induction of IGFBP-5 gene expression.
Quantitative real-time RT-PCR – a perspective
Authors: Bustin S A , Benes V , Nolan T , Pfaffl M W
Published: June 2005

The real-time reverse transcription polymerase chain reaction (RT-PCR) uses fluorescent reporter molecules to monitor the production of amplification products during each cycle of the PCR reaction. This combines the nucleic acid amplification and detection steps into one homogeneous assay and obviates the need for gel electrophoresis to detect amplification products. Use of appropriate chemistries and data analysis eliminates the need for Southern blotting or DNA sequencing for amplicon identification. Its simplicity, specificity and sensitivity, together with its potential for high throughput and the ongoing introduction of new chemistries, more reliable instrumentation and improved protocols, has made real-time RT-PCR the benchmark technology for the detection and/or comparison of RNA levels.
Interaction of insulin-like growth factor (IGF)-I and -II with IGF binding protein-2: mapping the binding surfaces by nuclear magnetic resonance
Authors: Carrick F E , Hinds M G , McNeil K A , Wallace J C , Forbes B E , Norton R S
Published: June 2005

The interaction of IGF binding protein-2 (IGFBP-2) with IGF-I and -II has been investigated in solution using nuclear magnetic resonance (NMR) spectroscopy. Chemical shift perturbations in 15N- and 2H/15N-labelled IGF-I or -II upon binding to unlabelled thioredoxin-tagged bovine IGFBP-2 (Trx1–279IGFBP-2) have been monitored to identify residues involved directly in the binding interaction as well as any affected by conformational changes associated with the interaction. A key step in obtaining high-quality spectra of the complexes was the use of transverse relaxation optimised spectroscopy (TROSY) methods with partially deuterated ligands. Indeed, because the effects of conformational averaging and aggregation are eliminated in IGF-I and -II bound to IGFBP-2, the spectra of the complexes are actually superior to those of the free ligands. Comparison of our results with the crystal structure of the complex between IGF-I and an N-terminal fragment of IGFBP-5 allowed identification of those residues perturbed by the C-domain of IGFBP-2. Other perturbations, such as those of Gly19 and Asp20 of IGF-I (and the corresponding residues in IGF-II) – which are located in a reverse turn linking N-domain and C-domain interactive surfaces – are due to local conformational changes in the IGF-I and -II. Our results confirm that the C-domain of IGFBP-2 plays a key role in binding regions of IGF-I and -II that are also involved in binding to the type-1 IGF receptor and thereby blocking ligand binding to this receptor.
Expression of recombinant membrane-bound type I iodothyronine deiodinase in yeast
Authors: Kuiper George G J M , Klootwijk Willem , Visser Theo J
Published: June 2005

The bioactivity of thyroid hormone is determined to a large extent by the monodeiodination of the prohormone thyroxine (T4) by the hepatic selenoenzyme type I iodothyronine deiodinase (D1), i.e. by outer ring deiodination (ORD) to the active hormone triiodothyronine (T3) or by inner ring deiodination (IRD) to the inactive metabolite reverse T3 (rT3). Since D1 is a membrane-bound protein with an N-terminal membrane-spanning domain, the enzyme is very difficult to purify in an active state. This study was undertaken in order to develop a heterologous (over)-expression system that would eventually allow the production of large amounts of purified active D1 protein. We have expressed a mutant rat D1 protein, in which the selenocysteine residue in the core catalytic center was replaced by cysteine (D1 Cys) in yeast cells (Saccharomyces cerevisiae). After yeast cell fractionation, kinetic analysis was performed with dithiothreitol as reducing cofactor. ORD activity was associated with membrane fractions, while no activity could be detected in the cytosolic fraction. The D1 Cys protein displayed a tenfold increase in Km (2 μM) for rT3 as compared with native D1 protein in rat liver microsomes. The D1 protein content is about 65 pmol/mg microsomal protein, as compared with about 3 pmol/mg in rat liver microsomal fraction. SDS-PAGE analysis of N-bromoacetyl-[125I]T3 affinity-labeled D1 protein showed several labeled protein isoforms with apparent molecular masses between 27 and 32 kDa. Immunoblot analysis with a specific D1 antiserum confirmed the observed D1 protein heterogeneity. Site-directed mutagenesis of several potential N-linked glycosylation sites, phosphorylation sites and a unique myristoylation site established that D1 heterogeneity is not caused by N-linked glycosylation, but probably by a combination of O-linked glycosylation and phosphorylation. Deletion of the endoplasmic reticulum (ER)-signal sequence and the membrane-spanning domain (amino acid residue 2–35), did not result in the production of a soluble D1 enzyme. Although this mutated D1 protein is inactive, the fact that it is still membrane bound indicates the existence of additional membrane attachment site(s) or membrane-spanning domains. Overall, our studies indicate that yeast cells provide a useful system for the expression of relatively high levels of D1 protein which could be used for further structure–function analysis.