bluuebunny/crossref_metadata_2025 huggingface dataset:
Compositional and structural characterization of zirconia films with reticular structure obtained by spray pyrolysis
Authors: Martin F. , Ayouchi R. , Ruiz C. , Ramos‐Barrado J. R. , Leinen D.
Published: October 2002

Zirconia (ZrO2) films with reticular structure, to be used as support in catalysis, have been deposited on AISI‐304 stainless steel held at 473 K by spray pyrolysis using aqueous solutions of zirconyl chloride octahydrate (ZrOCl2·8H2O) as precursor. As‐deposited films and films annealed in air to different temperatures after deposition have been studied by x‐ray diffraction (XRD), SEM and XPS. It was found that the crystal structure of the ZrO2 films changes from amorphous to tetragonal and that the films were uniform and well adhered to the stainless‐steel substrate for annealing temperatures up to 773 K. However, films annealed to 873 K crack and peel off from the substrate owing to the growth of an interfacial oxide layer between the ZrO2 film and the stainless‐steel substrate. The fracture, which leads to delamination of the ZrO2 film, occurs through the interfacial oxide layer. XPS depth profiling revealed that this interfacial oxide layer is of varying composition (containing iron, chromium and nickel) with depth. Copyright © 2002 John Wiley & Sons, Ltd.
Surface chemical changes induced by low‐energy ion bombardment in chromium nitride layers
Authors: Bertóti I. , Mohai M. , Mayrhofer P. H. , Mitterer C.
Published: October 2002

In this work three types of CrNx layers—one close to Cr2N, the other to CrN and a third with high nitrogen content CrN1+x—were prepared by reactive magnetron sputtering. The samples were subjected to bombardment by Ar+ and N2+ ions applied in sequence. The relative atomic concentration and the chemical states of the elements in the surface layer were determined by x‐ray photoelectron spectroscopy. Alternate bombardment with Ar+ and N2+ ions (1–2 keV) resulted in significant compositional and chemical state changes. Although prolonged Ar+ bombardment did not decrease the nitrogen content of Cr2N below the nominal (CrN0.5) composition, it decreased the nitrogen content of the closely stoichiometric CrN appreciably, to about CrN0.8. At bombardment with N2+ ions nitrogen could be implanted into the Cr2N films, changing its composition up to about CrN0.8. It was shown that extra nitrogen could be incorporated also into the CrN samples, increasing the nitrogen content above the stoichiometric composition up to about CrN1.2. The CrN1+x‐type sample showed high stability towards Ar+ bombardment. Two significantly different chemical states of nitrogen were determined for the two types of nitrides: the N 1s peak at 397.5 ± 0.1 eV binding energy was characteristic of N in Cr2N and the peak at 396.5 ± 0.1 eV of N in the CrN state. The effect of Ar+ and N2+ bombardment on the surface composition discussed in this paper may provide information for elaboration of deposition conditions for CrNx coatings with predetermined chemical composition and structure. Copyright © 2002 John Wiley & Sons, Ltd.
Characterization of ultrathin Cr layers on PET by RBS and XRF
Authors: Draxler M. , Zoister S. , Kastner F. , Bergsmann M. , Bauer P.
Published: October 2002

We studied the characterization of thin metallic layers vacuum‐deposited by magnetron sputtering onto commercial polyethylene terephthalate (PET) films in an industrial web coater. Because the technology of metal coating for technical films tends more and more towards nanotechnology, quantitative surface and interface analysis are required to optimize the metallization procedures. An important factor is the thickness of the thin coated layers. A powerful tool to characterize chemical composition and layer thickness of such nanofilms quantitatively is low‐energy Rutherford backscattering spectroscopy (LE‐RBS); it is, however, not suitable for production control. A standard industrial tool for these measurements at higher layer thickness is x‐ray fluorescence spectroscopy (XRF). This method is highly sensitive and reproducible but needs calibration standards.

We show that the thickness of chromium layers in the subnanometre range can be determined by means of LE‐RBS without deterioration of the PET film. We further show that a set of layers of different thickness can be measured by LE‐RBS with sufficiently high accuracy to serve as calibration standards for XRF or optical density measurement. From the correlation of the results obtained by RBS and XRF, we learn about the uncertainties induced by the production process (lateral uniformity) and by the analytical techniques. Copyright © 2002 John Wiley & Sons, Ltd.
Monolayers of simple organic molecules on silicon studied by surface tools
Authors: Scandurra A. , Renna L. , Cerofolini G. , Pignataro S.
Published: October 2002

Simple organic molecules such as 1‐octadecene, 1‐tetradecene, 1‐dodecyne and 1,13‐tetradecadiene have been grafted to hydrogenated B‐doped silicon (100) surfaces. Such substrate has been obtained by etching commercial native oxide‐capped flat crystal silicon wafers with procedures involving dilute HF aqueous solutions, but also by hydrogenation with a new method involving treatment of the wafer in an oven with H2 gas. The functionalization of such a surface has been done by using described wet methodologies involving pure liquids or solutions with thermal activation of the hydride. Contact angle, ESCA, atomic force microscopy (AFM) and time‐of‐flight secondary ion mass spectroscopy (ToF‐SIMS) measurements have been carried out to characterize the above systems. Particular emphasis is devoted to the interpretation of the ToF‐SIMS spectra, seldom used until now to characterize organic monolayers. Copyright © 2002 John Wiley & Sons, Ltd.
Combined application of ion beam slope cutting and SEM/EDX for investigation of the surface layer system on tungsten microwires after tribological treatment
Authors: Hauffe W. , Pannicke S. , Däbritz S. , Schade P.
Published: October 2002

The surface layer system on tungsten wires developed after different technological processing steps and depending on the tribological treatment during the production technology has been investigated. The ion beam slope cutting (IBSC) technique in combination with scanning electron microscopy (SEM) and energy‐dispersive x‐ray (EDX) analysis has been applied for full three‐dimensional determination of the phase and grain microstructure of the border zones of tungsten wires. The IBSC technique was changed for wire‐shaped samples and modified by additional ion beam processing steps to characterize the near‐surface regions of microwires. Transversal and longitudinal cuts through the border zones of the microwires have been performed for SEM observation and EDX microanalysis of the cut areas. The inhomogeneous three‐dimensional distribution of tungsten oxide and graphite (as a lubricant) as well as the fine‐grained tungsten structure could be determined with SEM resolution. Copyright © 2002 John Wiley & Sons, Ltd.
Quantitative XPS analysis of aluminium in the presence of copper
Authors: Hazell Leonard B.
Published: October 2002

Quantification of aluminium in the presence of copper is a fundamental problem in XPS analysis due to peak overlap. This article presents and demonstrates the utility of a simple approach to alleviate this difficulty based on measurement of the P : S combination peak area ratio. It is particularly appropriate for inclusion in this Festshrift edition of the journal dedicated to Professor Jim Castle, who has worked extensively on copper–aluminium alloys throughout his long career. Incorporation of the method into commercial data systems and, particularly, an expert system is recommended so that the analyst can be alerted to the likely presence of aluminium and provided with a means to quantify the amount. Copyright © 2002 John Wiley & Sons, Ltd.
Surface evolution of Ni–V transparent oxide films upon Li insertion reactions
Authors: Zanoni R. , Decker F. , Coluzza C. , Artuso F. , Cimino N. , Di Santo G. , Masetti E.
Published: October 2002

A detailed picture is reported of the nature of an Li insertion Ni–V mixed‐oxide electrode and of its evolution upon prolonged intercalation/deintercalation cycles. A combination of bulk and surface‐sensitive techniques is used, including electrochemical analysis (cyclic voltammetry, impedance spectroscopy), atomic force microscopy (AFM) and XPS. The Ni–V oxide film, obtained by r.f. sputtering as a thin film on conductive glass for use as a transparent electrode in electrochromic windows, is an Li insertion material with high charge capacity, very good reversibility and a surface morphology characterized by a low initial roughness. Its surface composition shows an initial Ni enrichment. The nominal Ni–V stoichiometry is recovered already after ten cycles. Extended Li charge–discharge cycles induce progressive changes in the electrode reversibility (capacity fading) and in the reaction kinetics (increase of impedance). The surface evolution consists of the progressive accumulation of an Li carbonate layer, with thickness and roughness increasing with the number of cycles. The constancy of the core level and Auger peaks of Ni and V after 100 cycles excludes major changes in the electronic structure of the host oxide. However, electrochemical and AFM experiments performed after 1000 cycles indicate that the carbonate deposition on the electrode progresses further, therefore the surface layer is unable to yield a permanent electrode passivation and stabilization. Copyright © 2002 John Wiley & Sons, Ltd.
Surface chemistry of cement pastes: a study by x‐ray photoelectron spectroscopy
Authors: Baeta Neves Maria Inês , Oliva Victor , Mrabet Béchir , Connan Carole , Chehimi Mohamed M. , Delamar Michel , Hutton Simon , Roberts Adam , Benzarti Karim
Published: October 2002

Two cement pastes commonly used in concrete formulations were characterized by XPS before and after coating with an epoxy resin and a hardener. The XPS allowed the surface chemistry change induced by the organic coating to be monitored. In particular, the C 1s spectra were peak fitted in order to deduce the contribution of the organic materials to the total carbon content on the surface. Inspection of the N 1s peak at the cement/hardener interface indicates a certain degree of donor–acceptor interaction, where the aminated hardener plays the role of the Lewis base (donor) and the cement acts as the Lewis acid.

A relationship has been established between the dispersive contribution to the surface free energy (γSd) of the cement pastes and the surface chemical composition. A substantial decrease in the surface energy (from 65 to ∼30 mJ m−2) of the materials has been observed for a higher surface content in organic materials or, alternatively, for a more homogeneous organic coating even at a lower mass loading of the resin or hardener. Copyright © 2002 John Wiley & Sons, Ltd.
Degradation during ARXPS measurements of polystyrene treated with an oxygen plasma
Authors: Paynter R. W.
Published: October 2002

Thin polystyrene films, deposited on glass substrates, were exposed to an oxygen / helium plasma for 3s, 7s, 15s or 30s. Following exposure to air for four weeks, the samples were analyzed by ARXPS in a VG ESCALAB 220i X‐ray photoelectron spectrometer and in a VG Theta Probe instrument. Comparison of the results obtained demonstrates the effect of the oxygen loss during analysis in the VG ESCALAB 220i, which flattens the angle‐resolved profiles obtained and leads to the calculation of an oxygen depth profile which extends deeper into the surface and in which the oxygen concentration is lower. A simple procedure for the correction of the data obtained in the VG ESCALAB 220i is only partially successful at reconciling the derived oxygen depth profile with that calculated from the Theta Probe data. Copyright © 2002 John Wiley & Sons, Ltd.
Estimation of the area under the ROC curve
Authors: Faraggi David , Reiser Benjamin
Published: October 2002

The area under the receiver operating characteristic curve is frequently used as a measure for the effectiveness of diagnostic markers. In this paper we discuss and compare estimation procedures for this area. These are based on (i) the Mann–Whitney statistic; (ii) kernel smoothing; (iii) normal assumptions; (iv) empirical transformations to normality. These are compared in terms of bias and root mean square error in a large variety of situations by means of an extensive simulation study. Overall we find that transforming to normality usually is to be preferred except for bimodal cases where kernel methods can be effective. Copyright 2002 John Wiley & Sons, Ltd.
Role of the Data and Safety Monitoring Committee (DSMC)
Authors: Wilhelmsen Lars
Published: October 2002

The Data and Safety Monitoring Committee (DSMC) is a committee independent of both the Steering Committee (SC) and any sponsor. It advises these bodies on continuation or stopping based upon safety and efficacy considerations. The primary objective is to assure safety for the patients in the trial. Blinding of the DSMC regarding type of treatment is usually avoided. The DSMC is composed of three to five members and should include one or two clinicians knowledgeable in the field of the trial, one or two statisticians and in some situations a pharmacologist and an ethicist. It is important that the members have experience from other trials and have high integrity. Rules for the statistical analyses have to be set up in the beginning of the trial. However, the DSMC cannot rely only upon statistical rules. Information from other sources may cause stopping before the scheduled end of the trial. The DSMC has to check that the overall quality of the data is good. Of special importance is that the Endpoint Committee is current with classification of endpoints. Timing of the DSMC meetings is dependent on several factors: (i) the incidence of events; (ii) the power of the study design; (iii) the effectiveness of the intervention. Legal requirements to report serious adverse events to legal authorities may be taken over by the DSMC, which ideally is the only body that has access to unblinded data. Copyright © 2002 John Wiley & Sons, Ltd.
Evaluating the costs and effectiveness of cardiovascular therapies: who cares about economic analyses?
Authors: Califf Robert M.
Published: October 2002

The field of cardiovascular medicine is providing important insights about how the costs and effectiveness of diagnostic and therapeutic technologies are managed. As health‐care budgets have taken up more of the economy and as employers and patients have become concerned about the escalating costs of health care, we have entered an era in which individual practitioners must become concerned with the costs of a service relative to its benefits. Further, increasing numbers of effective therapies in a time when finances are notably constrained, we clearly cannot use all effective therapies in all patients. Increasingly, therefore, we must assess the value derived from spending incremental money on health care products and services. This exercise has been termed cost‐effectiveness analysis. The case of the platelet glycoprotein IIb/IIIa inhibitor, abciximab, is used to illustrate many of the issues surrounding the collection, interpretation, and (mis) application of cost‐effectiveness data. Copyright © 2002 John Wiley & Sons, Ltd.
Bayesian subset analysis: application to studying treatment‐by‐gender interactions
Authors: Simon Richard
Published: October 2002

Evaluating treatment effects within subsets of patients plays a major part of the analysis of many major clinical trials. Clinicians are often impressed by the heterogeneity of patient populations in clinical trials and hence are interested in examining subset effects. Statisticians generally discourage subset analysis or suggest that clinicians ‘do subset analysis but do not believe it’. This advice, however, is a sign of the inadequacy of the analytic methods generally used for subset analysis. Separate analysis of many subsets, and basing conclusions on whether the observed treatment difference achieves significance at the 0.05 level, is likely to yield erroneous conclusions. Making the separate analysis of subsets dependent on demonstration of a statistically significant treatment‐by‐subset interaction is also not an effective analytic strategy because of the limited power of interaction tests. This paper describes a Bayesian approach to subset analysis developed by Simon, Dixon and Freidlin. The method avoids many of the problems of subset analysis because it is not ‘separate’ analysis of subsets. Instead, subset‐specific treatment effects are estimated as an average of observed within‐subset differences and overall differences; the two components are weighted by thea prioriestimate of the likelihood of qualitative treatment by subset interactions. Hence, the Bayesian method proposed permits subset analyses incorporating the assumption that qualitative interactions are unlikely. The methodology is applied to the problem of designing and analysing clinical trials to estimate treatment effects for males and females. Copyright © 2002 John Wiley & Sons, Ltd.
Anti‐stress effect of yoga‐type breathing: modification of salivary cortisol, heart rate and blood pressure following a step‐climbing exercise
Authors: Monnazzi Paola , Leri Oriana , Guizzardi Liliana , Mattioli Domenico , Patacchioli F. R.
Published: October 2002

This paper reports salivary cortisol levels and cardiovascular adjustments monitored after the performance of physical exercise in a subject (healthy man, 58 years old) who had practised and taught yoga techniques for a number of years. The subject performed two different step‐climbing trials which were varied according to the different breathing recovery techniques used. Immediately after the step‐climbing, the subject was asked either to get his breath back spontaneously (as reference) or by 3 min breathing using the ‘Istity Method’, based on the prolongation or amplification of the steps of the breath (breathing in–out) with short regular rest intervals. The measurements of the basal heart rate, blood pressure and cortisol concentration in the saliva were made just before starting, and 5, 15 and 30 min after the end of step‐climbing. Results suggest that the Istity breathing method can induce rapid modification of the cardiovascular function and attenuation of the cortisol level after exposure to physical stress. It might be of interest to pursue further studies of this effect, perhaps not confined to yoga experts, since the ‘Istity Method’ can be implemented by anyone after a short and simple training period. Copyright © 2002 John Wiley & Sons, Ltd.
Structural systems: a marriage of structural engineering and system science
Authors: Skelton Robert E.
Published: October 2002

This research seeks to provide analytical tools and concepts to unify the disciplines of materials, structures, control, and signal processing. Deficiencies are noted in the existing theories of systems science that fails to provide a theory for system design, integrating the design of components. The biggest challenge is the lack of good systems modeling theories, as opposed to the excellent component modeling theories that exist. Mathematical algorithms are suggested to optimally locate the sensing and actuating functions within the structure to enhance the performance of the algorithms looking for faults. These same techniques can be designed to other objectives, such as heating and air conditioning, thermal detection of people movements and security issues. The ‘economic design problem’ discussed herein will determine both the required precision and the best location for sensors to enable the health monitoring or control algorithms to function within a predetermined precision. Copyright © 2002 John Wiley & Sons, Ltd.
Relationship between low‐dose amphetamine‐induced arousal and extracellular norepinephrine and dopamine levels within prefrontal cortex
Authors: Berridge Craig W. , Stalnaker Thomas A.
Published: October 2002

Despite the well‐known and potent arousal‐enhancing effects of amphetamine (AMPH)‐like stimulants, the neurobiological substrates of AMPH‐induced arousal have rarely been examined explicitly. Available evidence suggests the possible participation of noradrenergic and/or dopaminergic systems in the arousal‐enhancing actions of AMPH‐like stimulants. The current studies examined the extent to which low‐dose AMPH‐induced increases in waking are related to AMPH‐induced increases in extracellular norepinephrine (NE) and dopamine (DA) levels within the prefrontal cortex (PFC), as measured by in vivo microdialysis. Vehicle injections elicited brief epochs of waking. Vehicle‐induced waking was closely associated with a brief and moderate (50% above baseline) increase in NE levels. DA levels were less sensitive to the arousing actions of vehicle injections, with maximal increases of approximately 25% above baseline observed. 0.15 mg/kg and 0.25 mg/kg AMPH increased time spent awake, which resulted primarily from increases in quiet waking. Although the magnitude of the waking response did not differ substantially between the two doses across time, a trend for a more rapid recovery to baseline waking levels was observed at the higher dose, possibly suggesting the development of a relatively rapid‐onset tolerance to the wake‐promoting actions of AMPH at this dose. At the 0.15 mg/kg dose, AMPH elicited maximum increases of approximately 175% and 125% above baseline levels for NE and DA, respectively. The time course of AMPH‐induced increases in waking closely paralleled the time course of AMPH‐induced increases in both NE and DA efflux. These observations are consistent with the hypothesis that both increased DA and NE efflux contribute to the low‐dose behavioral effects of AMPH‐like stimulants, including the arousal‐enhancing actions of these drugs. Additionally, these observations also suggest a possibly greater sensitivity of NE efflux, relative to DA, to moderately arousing conditions including low‐dose AMPH‐like stimulant administration. Synapse 46:140–149, 2002. © 2002 Wiley‐Liss, Inc.
Role of maternal smoking and maternal reproductive history in the etiology of hypospadias in the offspring
Authors: Källén Karin
Published: October 2002

Background

This study was undertaken to evaluate some possible risk factors for hypospadias with special regard to parity, subfertility and maternal smoking.

Methods

With the use of the Swedish health registries, 3,262 infants with a diagnosis of hypospadias were identified among 1,413,811 infants born in 1983–96 with prospectively collected smoking exposure during pregnancy. Odds ratios (OR) and 95% confidence intervals (CI) were calculated after adjustment for various possible confounders.

Results

A negative association between hypospadias and maternal smoking was found (OR: 0.83 95% CI: 0.76–0.90), but this association was only observed among mothers of Parity 1 or 4+, and the possibility of the results being due to confounding was suspected. Primiparity (vs. Parity 2) was positively associated with hypospadias (OR: 1.29 95% CI: 1.19–1.40), and so was subfertility (defined here as at least 1 year of attempts to conceive) (OR for subfertility: 1.16 95% CI: 1.01–1.33). The OR for smoking and primiparity, respectively, were of the same magnitude irrespective of whether subfertility was present or not, and no evidence was found that subfertility in the parents of infants with hypospadias seriously confounded the analyses.

Conclusions

No explanation was found for the negative association between maternal smoking and hypospadias or the positive association between primiparity and hypospadias. Possible causal mechanisms are discussed. Teratology 66:185–191, 2002. © 2002 Wiley‐Liss, Inc.
Learning‐teaching‐assessment paradigms and the on‐line classroom
Authors: Speck Bruce W.
Published: October 2002

Professors can make informed choices about teaching in on‐line classrooms by basing their instructional endeavors on pedagogical theory and evaluating two learning‐teaching‐assessment paradigms.
What professors need to know about technology to assess on‐line student learning
Authors: Jones Marshall G. , Harmon Stephen W.
Published: October 2002

Universities around the world are offering classes on the Internet. This chapter answers questions about the nature of on‐line courses and how assessment is done in these courses to help faculty understand how on‐line learning can enhance student learning.
The influence of microcystin‐LR and hepatotoxic cyanobacterial extract on the water plantSpirodela oligorrhiza
Authors: Romanowska‐Duda Zdzisława , Tarczyńska Małgorzata
Published: October 2002

The eutrophication of the Sulejów Reservoir dam in Poland is related to toxicity from cyanobacterial blooms. The main species responsible for hepatotoxic bloom formation isMicrocystis aeruginosa. The aim of this study was to evaluate the influence of toxic cyanobacterial extract on the growth and morphology of the water plantSpirodela oligorrhiza, compared with commercial‐grade microcystin‐LR (MC‐LR). It was found that after 96 h of incubation the highest concentration of cyanobacterial extract, containing 0.344 mg MC‐LR/L, reduced the number of fronds by about 50% in comparison with the control. The extract effected a reduction in the frond mass and a decrease in chlorophyll (a+b) concentration. A reduction in the number of fronds was also observed after the first 24 h of incubation in the presence of 0.2 and 0.1 μg/L of commercial‐grade MC‐LR. Changes in activity of constitutive acid phosphatase and RNase after 7 days of incubation with commercial‐grade MC‐LR were observed. The results confirm the toxicity of cyanobacterial hepatotoxins toSpirodela oligorrhiza, which can be used as a sensitive bioindicator. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 434–440, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10076
Acute toxicity, mutagenicity, and estrogenicity of biodegradation products of bisphenol‐A
Authors: Ike Michihiko , Chen Min‐Yu , Jin Chang‐Suk , Fujita Masanori
Published: October 2002

Biodegradation of bisphenol‐A (BPA), which is known as an estrogenic chemical, proceeds via complicated metabolic routes and leads to formation of several kinds of biodegradation products. Through the major route BPA can be completely mineralized; however, p‐hydroxyacetophenone (p‐HAP), p‐hydroxybenzaldehyde (p‐HBAL), and p‐hydroxybenzoic acid (p‐HBA) are transiently accumulated at relatively high concentrations. On the other hand, degradation of BPA through the minor route tends to cause the accumulation of 2,3‐bis(4‐hydroxyphenyl)‐1,2‐propanediol and p‐hydroxyphenacyl alcohol as the dead‐end products. To fully assess the impact of BPA discharge into the environment, the considerable BPA degradation products p‐HAP, p‐HBAL, and p‐HBA and the mixture of the dead‐end products were examined for their acute toxicity, mutagenicity, and estrogenicity using the Daphtoxkit (Creasel Ltd.), umu test system, and yeast two‐hybrid system, respectively. BPA was moderately toxic to Daphnia magna (48‐h EC50 was 10 mg/L) and weakly estrogenic, with activity that was 5 orders of magnitude lower than that of 17β‐estradiol in the yeast screen, though no mutagenicity was observed. All the tested BPA biodegradation products showed very low acute toxicity compared with BPA, and none was mutagenic. A slight estrogenic activity was detected only for p‐HAP among the tested degradation products. It was concluded that biodegradation can remarkably reduce the toxic effects of BPA. © 2002 Wiley Periodicals, Inc. Environ Toxicol 17: 457–461, 2002; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/tox.10079
Ad hoc networks: not an ad hoc field anymore
Authors: Ephremides Anthony
Published: October 2002

The genesis and growth of the field of ad hoc wireless networks is put into perspective through a brief review of historical highlights and through a focused discussion of some of the fundamental traits of such networks. Copyright © 2002 John Wiley & Sons, Ltd.
Maintenance of cell integrity in the gas1 mutant of Saccharomyces cerevisiae requires the Chs3p‐targeting and activation pathway and involves an unusual Chs3p localization
Authors: Carotti Cristina , Ferrario Laura , Roncero Cesar , Valdivieso M‐Henar , Duran Angel , Popolo Laura
Published: October 2002

Chitin synthase III is essential for the increase in chitin level and for cell integrity in cells lacking Gas1p, a β(1,3)‐glucanosyltransferase. In order to discover whether the upregulation of chitin synthesis proceeds through the canonical transport and activation pathway of Chs3p or through an alternative one, here we studied the effects of the inactivation of the GAS1 and CHS4‐5‐6‐7 genes. All the double‐null mutants showed a temperature‐sensitive cell lysis phenotype that could be suppressed by the presence of an osmotic stabilizer. In liquid YEPD at 30 °C, chs4Δ gas1Δ, chs5Δ gas1Δ, chs6Δgas1Δ and chs7Δgas1Δ mutants were unable to grow, whereas they grew very slowly in minimal medium and showed low viability. High osmolarity suppressed the defective phenotype and restored growth. In chs4 gas1, chs5 gas1 and chs7 gas1, chitin levels did not increase and were reduced to only 10%, while in chs6 gas1 the value of gas1 was reduced to 20–40%. To investigate at which level the upregulation of chitin synthesis could occur, mRNA levels were monitored. The expression of CHS4‐5‐6‐7 did not change significantly in gas1Δ. In strains expressing HA‐tagged forms, the localization of Chs3p and Chs5p was examined. In the gas1 mutant the fluorescence pattern was affected and the proteins appeared abnormally present in the bud. The results indicate that: (a) the function of the CHS4–7 genes is required for chitin hyperaccumulation in gas1 mutant and for cell integrity; (b) homologous genes do not replace their function; (c) the regulation of CHS4–7 genes does not occur at transcriptional level. Control of the position of chitin synthesis could be important in protecting the bud from lysis. Copyright © 2002 John Wiley & Sons, Ltd.
Molecular cloning and sequence analysis of the Zygosaccharomyces bailii HIS3 gene encoding the imidazole glycerolphosphate dehydratase
Authors: Branduardi Paola
Published: October 2002

Zygosaccharomyces bailii is a spoilage yeast belonging to the Zygosaccharomyces genus. In recent years these yeasts, due to their exceptional resistance to several stresses, have become more and more interesting as model organisms to study the molecular basis of the said resistance. A Z. bailii cDNA library has been built and the 672 bp nucleotide sequence coding for the HIS3 gene was cloned by complementation of a Saccharomyces cerevisiae his3 mutant strain. The deduced 223 amino acid sequence shares a high degree of homology with His3p homologues in other non‐conventional yeast species. The GeneBank Accession No. is AY050224. Copyright © 2002 John Wiley & Sons, Ltd.
The kex2 gene from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis
Authors: Venancio Emerson J. , Daher Bruno S. , Andrade Rosângela V. , Soares Célia M. A. , Pereira Ildinete Silva , Felipe Maria Sueli S.
Published: October 2002

Kexin‐like protein is a component of the subtilase family of proteinases involved in the processing of proproteins to their active forms. Kexin‐like proteins are also synthesized as a propeptide and this is involved in (auto)inhibition, correct folding and subcellular sorting of proteins. The kexin‐like protein was described as the product of the kex2 gene for Aspergillus niger, Candida albicans, Saccharomyces cerevisiae, Yarrowia lipolytica and other fungi. Disruption of the kex2 gene in C. albicans and Y. lipolytica affects hyphae production and induces morphological cell defects, strongly suggesting a possible role of kexin‐like proteins in dimorphism of human pathogenic fungi. In this work, we report the nucleotide sequence of the kex2 gene cloned from the dimorphic and human pathogenic fungus Paracoccidioides brasiliensis (Pbkex2). An open reading frame (ORF) of 2622 bp was identified in the complete sequence, interrupted by only one intron of 93 bp. The 5′ non‐coding region contains consensus sequences such as canonical TATA, CAAT boxes and putative motifs for transcriptional factors binding sites, such as HSE‐like regulating genes involved in thermo‐dependent processes; Xbp1, reported as a transcriptional factor that may control genes involved in cell morphology; and StuAp, which may regulate spore differentiation and pseudohyphal growth in fungi. In the 3′ non‐coding region were observed the canonical motifs necessary for correct mRNA processing and polyadenylation. The deduced protein sequence consists of 842 amino acid residues, showing identity to kexin‐like proteinases from A. niger (55%), Emericella nidulans (53%) and C. albicans (48%). Comparative sequence analysis of P. brasiliensis kexin‐like protein reveals the presence of homologous regions related to a signal peptide, a propeptide, a subtilisin‐like catalytic domain, a P domain, a S/T rich region and a transmembrane domain. A putative Golgi retrieval signal (YEFEMI) has also been found in the cytoplasmic tail. The complete nucleotide sequence of Pbkex2 and its flanking regions have been submitted to GenBank database under Accession No. AF486805. Copyright © 2002 John Wiley & Sons, Ltd.
Liver pathology in compound heterozygous patients for hemochromatosis mutations
Authors: Schöniger‐Hekele Maximilian , Müller Christian , Polli Claudia , Wrba Friedrich , Penner Edward , Ferenci Peter
Published: October 2002

Abstract: Background: While hepatic pathology of homozygous carriers of the C282Y mutation of the HFE haemochromatosis gene is well defined, the impact of the C282Y/H63D compound heterozygous carrier state is unknown.

Aims: To evaluate the range of hepatic pathology in C282Y/H63D compound heterozygous patients.

Patients: 25 C282Y/H63D compound heterozygous patients with and without known underlying liver disease underwent liver biopsies for evaluation or abnormal liver tests. Eleven cadaveric liver donors with HFE wild type served as controls.

Methods: Mutations in the HFE gene were detected by polyacrylamide gel electrophoresis (PAGE) separation of digested polymerase chain reaction (PCR)‐amplificates. The extent of light microscopic changes of liver architecture were studied on haematoxylin, eosin (H. E.) stains. In addition, the extent and the distribution of iron deposition was graded on Prussian blue‐stained sections and hepatic iron was quantified by atom absorption spectroscopy. Serum ferritin concentration and the transferrin saturation index were measured using routine laboratory methods.

Results: Patients without underlying liver disease (n = 15): Hepatic inflammation was seen in only 8% but fibrosis was found in 36% of compound heterozygous patients. Eighty six percent of those patients had stainable iron predominantly found in Rappaport's zone 1 and 2, but all had a liver iron‐index < 1.9. Transferrin saturation was found elevated in 36% of compound heterozygous patients. Patients with liver fibrosis showed significantly higher ferritin levels than patients without liver fibrosis (1110 ng/mL versus 307 ng/mL, p < 0.05). Patients with underlying disease (n = 10): In compound heterozygous patients, 77% had hepatic inflammation and 88% fibrosis. Stainable iron (44%) was less frequently found than in patients without underlying liver disease. Hepatic iron‐index in patients with underlying liver disease was always below 1.17; transferrin saturation was elevated in only 22% of the compound heterozygous patients. Histologic hepatic iron‐index was significantly lower in patients with underlying disease (median 0.047) as compared to patients without underlying liver disease (median 0.274, P < 0.05).

Conclusions: The underlying liver disease determines the extent of hepatic pathology seen in livers of compound heterozygous patients. However, considerable histologic fibrosis can also be found in compound heterozygous patients without underlying liver disease.
Cloning of thioredoxin h reductase and characterization of the thioredoxin reductase–thioredoxin h system from wheat
Authors: SERRATO Antonio J. , PÉREZ-RUIZ Juan M. , CEJUDO Francisco J.
Published: October 2002

Thioredoxins h are ubiquitous proteins reduced by NADPH— thioredoxin reductase (NTR). They are able to reduce disulphides in target proteins. In monocots, thioredoxins h accumulate at high level in seeds and show a predominant localization in the nucleus of seed cells. These results suggest that the NTR—thioredoxin h system probably plays an important role in seed physiology. To date, the study of this system in monocots is limited by the lack of information about NTR. In the present study, we describe the cloning of a full-length cDNA encoding NTR from wheat (Triticum aestivum). The polypeptide deduced from this cDNA shows close similarity to NTRs from Arabidopsis, contains FAD- and NADPH-binding domains and a disulphide probably interacting with the disulphide at the active site of thioredoxin h. Wheat NTR was expressed in Escherichia coli as a His-tagged protein. The absorption spectrum of the purified recombinant protein is typical of flavoenzymes. Furthermore, it showed NADPH-dependent thioredoxin h reduction activity, thus confirming that the cDNA clone reported in the present study encodes wheat NTR. Using the His-tagged NTR and TRXhA (wheat thioredoxin h), we successfully reconstituted the wheat NTR—thioredoxin h system in vitro, as shown by the insulin reduction assay. A polyclonal antibody was raised against wheat NTR after immunization of rabbits with the purified His-tagged protein. This antibody efficiently detected a single polypeptide of the corresponding molecular mass in seed extracts and it allowed the analysis of the pattern of accumulation of NTR in different wheat organs and developmental stages. NTR shows a wide distribution in wheat, but, surprisingly, its accumulation in seeds is low, in contrast with the level of thioredoxins h.
Organization of the multifunctional enzyme type 1: interaction between N- and C-terminal domains is required for the hydratase-1/isomerase activity
Authors: KIEMA Tiila-Riikka , TASKINEN Jukka P. , PIRILÄ Päivi L. , KOIVURANTA Kari T. , WIERENGA Rik K. , HILTUNEN J. Kalervo
Published: October 2002

Rat peroxisomal multifunctional enzyme type 1 (perMFE-1) is a monomeric protein of β-oxidation. We have defined five functional domains (A, B, C, D and E) in the perMFE-1 based on comparison of the amino acid sequence with homologous proteins from databases and structural data of the hydratase-1/isomerases (H1/I) and (3S)-hydroxyacyl-CoA dehydrogenases (HAD). Domain A (residues 1—190) comprises the H1/I fold and catalyses both 2-enoyl-CoA hydratase-1 and Δ3—Δ2-enoyl-CoA isomerase reactions. Domain B (residues 191—280) links domain A to the (3S)-dehydrogenase region, which includes both domain C (residues 281—474) and domain D (residues 480—583). Domains C and D carry features of the dinucleotide-binding and the dimerization domains of monofunctional HADs respectively. Domain E (residues 584—722) has sequence similarity to domain D of the perMFE-1, which suggests that it has evolved via partial gene duplication. Experiments with engineered perMFE-1 variants demonstrate that the H1/I competence of domain A requires stabilizing interactions with domains D and E. The variant His-perMFE (residues 288—479)Δ, in which the domain C is deleted, is stable and has hydratase-1 activity. It is proposed that the extreme C-terminal domain E in perMFE-1 serves the following three functions: (i) participation in the folding of the N-terminus into a functionally competent H1/I fold, (ii) stabilization of the dehydrogenation domains by interaction with the domain D and (iii) the targeting of the perMFE-1 to peroxisomes via its C-terminal tripeptide.
Translation initiation at non-AUG codons mediated by weakened association of eukaryotic initiation factor (eIF) 2 subunits
Authors: HASHIMOTO Nilce N. , CARNEVALLI Larissa S. , CASTILHO Beatriz A.
Published: October 2002

The heterotrimeric eukaryotic initiation factor (eIF) 2 binds the initiator methionyl-tRNA in a GTP-dependent mode and delivers it to the 40S ribosomal subunit. In the present study, we have identified amino acid residues in eIF2β required for binding to eIF2γ in yeast. Alteration of six residues in the central region of eIF2β abolished this interaction, as determined by GST-pull down and two-hybrid assays, and leads to cell lethality. Substitution of 131Tyr and 132Ser by alanine residues (131YS), although abolishing the binding to eIF2γ in these assays, resulted in a functional but defective protein in vivo, imparting a temperature-sensitive growth phenotype to cells. A dramatically weakened association of this mutant protein with eIF2γ in vivo was shown by co-immunoprecipitation. The 131YS mutation in eIF2β allows translation to initiate at non-AUG codons, as defined by the ability of cells carrying an initiator codon mutation in the HIS4 mRNA to grow in the absence of histidine. The combination of this mutation with the 264Ser→Tyr alteration, a previously isolated suppressor of initiator codon mutations which has been shown to increase the spontaneous GTP hydrolysis in the ternary complex, caused a recessive lethality, suggesting additive defects. Thus the impaired interaction of these two subunits represents a novel type of defect in eIF2 function, providing in vivo evidence that the strength of interaction between eIF2β and eIF2γ defines the correct usage of the AUG codon for translation initiation.
Comparative positions of kinetoplasts in Trypanosoma musculi and Trypanosoma lewisi during development in vitro
Authors: Ashraf M. , Nesbitt R. A. , Humphrey P. A. , Siewe M. , Lee C. M.
Published: October 2002

Abstract. The development of Trypanosoma musculi and Trypanosoma lewisi were studied in vitro in the presence of adherent splenic cells. Both parasites developed only when attached by their flagellar tips to adherent splenic cells. During the proliferation of T. musculi, the kinetoplast migrated towards the nucleus, and once in the vicinity of the nucleus, the nuclear division was triggered. The kinetoplast of T. lewisi did not migrate towards the nucleus, but remained at its original location. The nucleus and kinetoplast divided at the same time in both parasites, and parasites started dividing from their flagellar ends and T. musculi and T. lewisi daughter cells were formed within 48 h. The unavailability of the adherent splenic cells in vitro led the parasites to transform into round/oval nonviable forms.
Evidence for the genetic control of immunoglobulin E reactivity to the allergens of Alternaria alternata
Authors: Karihaloo C. , Tovey E. R. , Mitakakis T. Z. , Duffy D. L. , Britton W. J.
Published: October 2002

Background The fungus Alternaria alternata contains potent allergens, and sensitization to these allergens is associated with a high risk of respiratory disease. The influence of genetic regulation on sensitization to Alternaria is unknown.

Objective To determine the influence of genetic factors on IgE responses to specific allergens of Alternaria.

Methods The concordance of skin prick test (SPT), radioallergosorbent test (RAST) and IgE‐binding profiles of sera were examined from a large cohort of monozygotic and dizygotic twins.

Results Casewise concordance for a positive SPT response was monozygous (MZ) 66%: dizygous (DZ) 40% (P = 0.002). Logistic regression confirmed that casewise concordance was significantly stronger between MZ than DZ pairs. Immunoblotting against an Alternaria extract revealed 19 allergenic bands. The differences in concordance between the different bands were not significant for either the MZ (P = 0.97) or DZ (P = 0.84) groups. The pooled MZ : DZ difference in concordance was just significant (P = 0.049), suggesting an overall genetic effect on the response to Alternaria. This was reinforced by the comparison of the MZ and DZ correlations for total number of bands recognized (MZ r = 0.65; DZ r = 0.37, P = 0.015). Overall, there was a moderate correlation between the individual SPT weal size and RAST score (r2 = 0.41) and a substantial correlation between the number of immunoblotted bands and RAST scores (r2 = 0.79).

Conclusion There is a strong genetic influence on IgE response to the mixture of Alternaria allergens and a lesser effect on IgE response to individual allergens.
Comparison of bronchial responsiveness to histamine in asthma, allergic rhinitis and allergic sensitization at the age of 7 years
Authors: Nickel R. , Lau S. , Niggemann B. , Sommerfeld C. , Wahn U. ,   the German Multicenter Allergy Study Group
Published: October 2002

Background Bronchial responsiveness (BR) to histamine or methacholin is a common finding in adult non‐asthmatic patients with allergic rhinitis.

Objective We tested whether BR is also present in children with a comparatively short history of allergic rhinitis in a paediatric cohort.

Methods We performed pulmonary function tests and histamine challenges in a total of 654 children (age 7 years, participants of the German Multicenter Allergy Study) and compared PC20 FEV1 values in children with asthma, allergic rhinitis, asymptomatic allergic sensitization and non‐atopic controls.

Results Most pronounced BR to histamine was observed in allergic asthmatics (n = 28), irrespective of the presence or absence of allergic rhinitis. Furthermore, PC20FEV1 values in non‐asthmatic children with allergic rhinitis (n = 24) were not significantly different from those seen in asymptomatic atopic (n = 54) or non‐atopic controls (n = 92).

Conclusions In contrast to adult study populations, 7‐year‐old non‐asthmatic children with allergic rhinitis do not show a higher degree of BR than asymptomatic atopic or non‐atopic controls. Therefore, secondary preventive measures in non‐asthmatic children with allergic rhinitis (such as regular local anti‐inflammatory therapy or specific immunotherapy) should be studied and applied more intensely to prevent bronchial hyper‐responsiveness (BHR) and asthma in this high‐risk group.
Use of a flow‐cell system to investigate virucidal dimethylmethylene blue phototreatment in two RBC additive solutions
Authors: Wagner Stephen , Skripchenko Andrey , Thompson‐Montgomery Dedeene
Published: October 2002

BACKGROUND: Limited photoinactivation kinetics, use of low‐volume 30 percent Hct RBCs, and hemolysis have restricted the practicality of the use of dimethylmethylene blue (DMMB) and light for RBC decontamination. A flow‐cell system was developed to rapidly treat larger volumes of oxygenated 45 percent Hct RBCs with high‐intensity red light.

MATERIALS AND METHODS: CPD‐whole blood was WBC reduced, RBCs were diluted in additive solutions (either Adsol or Erythrosol), and suspensions were subsequently oxygenated by gas overlay. Intracellular or extracellular VSV and DMMB were sequentially added. VSV‐infected RBC suspensions (45% Hct) were passed through 1‐mm‐thick flow cells and illuminated. Samples were titered for VSV, stored for up to 42 days, and assayed for Hb, supernatant potassium, ATP, and MCV.

RESULTS: The use of oxygenated RBCs resulted in rapid and reproducible photoinactivaton of ≥6.6 log extracellular and approximately 4.0 log intracellular VSV independent of additive solution. Phototreated Adsol RBCs exhibited more than 10 times greater hemolysis and 30 percent greater MCV during storage than identically treated Erythrosol RBCs. Phototreatment caused RBC potassium leakage from RBCs in both additive solutions. ATP levels were better preserved in Erythrosol than Adsol RBCs.

CONCLUSION: A rapid, reproducible, and robust method for photoinactivating model virus in RBC suspensions was developed. Despite improved hemolysis and ATP levels in Erythrosol‐phototreated RBCs, storage properties were not maintained for 42 days.