bluuebunny/crossref_metadata_2025 huggingface dataset:
Bias correction of two‐state latent Markov process parameter estimates under misclassification
Authors: Rosychuk Rhonda J. , Thompson Mary E.
Published: June 2003

A discretely observed two‐state process may misclassify the state of an unobservable continuous‐time, two‐state Markov process. We examine the behaviour of maximum likelihood transition probability estimates as functions of known misclassification probabilities. Since maximum likelihood estimators are not available in closed form, we provide two alternatives for bias‐adjusted estimation. In the case of large samples, the asymptotic bias is quantified and estimators are constructed iteratively using transition counts and specified misclassification probabilities. For finite samples, we provide an approximation based on partial derivatives. Estimators that are bias‐adjusted to a first approximation are easily constructed and may serve well when misclassification probabilities are known to be small. Simulation studies reveal the effect of misclassification on estimation. Repeated diagnostic testing data illustrate the approaches. Copyright © 2003 John Wiley & Sons, Ltd.
EASYLOCAL++: an object‐oriented framework for the flexible design of local‐search algorithms
Authors: Di Gaspero Luca , Schaerf Andrea
Published: June 2003

Local search is a paradigm for search and optimization problems, which has recently evidenced to be very effective for a large number of combinatorial problems. Despite the increasing interest of the research community in this subject, there is still a lack of a widely‐accepted software tools for local search.

We propose EASYLOCAL, an object‐oriented framework for the design and the analysis of local‐search algorithms. The abstract classes that compose the framework specify and implement the invariant part of the algorithm and are meant to be specialized by concrete classes that supply the problem‐dependent part. The framework provides the full control structures of the algorithms, and the user has only to write the problem‐specific code. Furthermore, the framework comes with some tools that simplify the analysis of the algorithms.

The architecture of EASYLOCAL provides a principled modularization for the solution of combinatorial problems by local search and helps the user by deriving a neat conceptual scheme of the application. It also supports the design of combinations of basic techniques and/or neighborhood structures.

The framework has been tested in some applicative domains and has proved to be flexible enough in the implementation of algorithms for the solution of various scheduling problems. Copyright © 2003 John Wiley & Sons, Ltd.
Trends in the Design, Manufacture and Evaluation of Wind Turbine Blades
Authors: Veers Paul S. , Ashwill Thomas D. , Sutherland Herbert J. , Laird Daniel L. , Lobitz Donald W. , Griffin Dayton A. , Mandell John F. , Musial Walter D. , Jackson Kevin , Zuteck Michael , Miravete Antonio , Tsai Stephen W. , Richmond James L.
Published: June 2003

Wind turbine blades continue to be the target of technological improvements by the use of better designs, materials, manufacturing, analysis and testing. As the size of turbines has grown over the past decade, designers have restrained the associated growth in blade weight to less than would have been possible through simple scaling‐up of past approaches. These past improvements are briefly summarized. Manufacturing trends and design drivers are presented, as are the ways these design drivers have changed. Issues related to blade material choices are described, first for the currently dominant glass fibre technology and then for the potential use of carbon fibres. Some possible directions for future blade design options are presented, namely new planforms, aerofoils and aeroelastic tailoring. The significant improvement in sophistication of stress analysis and full‐scale blade testing are also discussed. Copyright © 2003 John Wiley & Sons, Ltd.
Wind power and the conditions at a liberalized power market
Authors: Morthorst P. E.
Published: June 2003

Wind power is undergoing a rapid development nationally as well as globally and in a number of countries covers an increasing part of the power supply. At the same time an ongoing liberalization of power markets is taking place and to an increasing extent the owners of wind power plants will themselves have to be responsible for trading the power at the spot market and financially handling the balancing. In the western part of Denmark (Jutland/Funen area), wind‐generated power from time to time covers almost 100% of total power consumption. Therefore some examples are chosen from this area to analyse in more detail how well large amounts of wind power in the short‐term are handled at the power spot market. It turns out that there is a tendency that more wind power in the system in the short run leads to relatively lower spot prices, while less wind power implies relatively higher spot prices, although, with the exception of December 2002, in general no strong relationship is found. A stronger relationship is found at the regulating market, where there is a fairly clear tendency that the more wind power produced, the higher is the need for down‐regulation, and, correspondingly, the less wind power produced, the higher is the need for up‐regulation. In general for the Jutland/Funen area the average cost of down‐regulation is calculated as 1·2 c€/kWh regulated for 2002, while the cost of up‐regulation amounts to 0·7 c€/kWh regulated. Copyright © 2003 John Wiley & Sons, Ltd.
Wind Resource Estimation—An Overview
Authors: Landberg Lars , Myllerup Lisbeth , Rathmann Ole , Petersen Erik Lundtang , Jørgensen Bo Hoffmann , Badger Jake , Mortensen Niels Gylling
Published: June 2003

This article gives an overview of the different ways the wind resource at a site can be estimated. Eight separate ways have been identified. Each of these will be described in some detail, and advantages and disadvantages of each of them will be discussed. Copyright © 2003 John Wiley & Sons, Ltd.
Short‐term Prediction—An Overview
Authors: Landberg Lars , Giebel Gregor , Nielsen Henrik Aalborg , Nielsen Torben , Madsen Henrik
Published: June 2003

This article gives an overview of the different methods used today for predicting the power output from wind farms on the 1–2 day time horizon. It describes the general set‐up of such prediction systems and also gives examples of their performance. Copyright © 2003 John Wiley & Sons, Ltd.
Review of Aerodynamics for Wind Turbines
Authors: Snel Herman
Published: June 2003

This article reviews the state of the art of wind turbine rotor aerodynamics. It addresses present uncertainties in rotor design and load calculations, recent modelling efforts to reduce these uncertainties, and validation activities regarding the modelling and results thereof. Copyright © 2003 John Wiley & Sons, Ltd.
Quantifying shade using a standard environment
Authors: Langman V.A. , Rowe M. , Forthman D. , Langman N. , Black J. , Walker T.
Published: June 2003

The purpose of this study was to quantify the thermal microclimate provided by a shade structure in the African elephant enclosure at Zoo Atlanta. The hypothesis was that the interior of a weather instrument shelter (a Stevenson screen) would provide the maximum environmental shielding and the coolest possible ambient conditions without artificial heating or cooling. The ambient conditions inside the Stevenson screen were compared with the ambient conditions in the shaded and nonshaded sections of the exhibit to quantify the extremes possible under the environmental conditions. The Stevenson screen reduced the radiant heat load by 766 W m−2. The shade structure in the elephant enclosure reduced the radiant heat load by 278 W m−2, which was 37% of the total possible reduction represented by the interior of the Stevenson screen. The longwave radiant heat was 10% greater in the direct sun and 37% greater in the shaded area than the shortwave radiant heat. The shade structure reduced the shortwave radiant heat by 254 W m−2 or 43%, but only reduced the longwave radiant heat by 24Wm−2 or 3%. Shade structures alone may not provide adequate protection from radiant heat for captive species. A cool microclimate in an artificial enclosure should be designed to reduce all sources of radiant heat. Zoo Biol 22:253–260, 2003. © 2003 Wiley‐Liss, Inc.
Effect of solar eclipse on the behavior of a captive group of hamadryas baboons (Papio hamadryas)
Authors: Gil‐Burmann Carlos , Beltrami Marcial
Published: June 2003

To date there have been no systematic studies on the effects of a solar eclipse on the behavior of nonhuman primates. However, some descriptive studies have reported an increase in inactivity levels and congregation of individuals during a solar eclipse. The aim of the present work was to carry out a systematic study of the effect of a solar eclipse on the behavior of baboons at an individual, gender, and one‐male unit (OMU) level. On 3 November 1994, Chile experienced a solar eclipse. A hamadryas baboon colony grouped in two OMUs at the Zoo of Santiago de Chile was observed 2 days before the solar eclipse (control situation), and during the eclipse for the same time period. Two observers simultaneously sampled the data. An all‐occurrence sampling method was used to collect data on threats and attacks made by adult individuals. Simultaneously, instantaneous samplings of 1 min each were used to record behavioral states of the two males and two females. In relation to the control situation, the effect of the solar eclipse on baboon behavior was a reduction in the rates of locomotion and threats, and an increase in the rates of inactivity and grooming, but not significantly so in all the focal individuals. Behavioral changes were more marked in males than in females, but did not differ between the two OMUs. Air temperature and humidity were similar in the two situations, and can therefore be discarded as causal effects on the behavioral changes of the baboons during the eclipse. Zoo Biol 22:299–303, 2003. © 2003 Wiley‐Sons, Inc.
Nutritional status of free‐ranging Mexican howler monkeys (Alouatta palliata mexicana) in Veracruz, Mexico: Serum chemistry; lipoprotein profile; vitamins D, A, and E; carotenoids; and minerals
Authors: Crissey Susan D. , Silva Juan Carlos Serio , Meehan Tom , Slifka Kerri A. , Bowen Phyllis E. , Stacewicz‐Sapuntzakis Maria , Holick Michael F. , Chen Tai C. , Mathieu Jeffrey , Meerdink Gavin
Published: June 2003

The purpose of this work was to measure important nutritional status parameters for a group of free‐ranging Mexican mantled howler monkeys (A. palliata mexicana) and compare those data to published data for primates. The nutritional status of six free‐ranging Mexican mantled howler monkeys was examined using biochemical analysis. Blood samples were analyzed for serum chemistry; lipids; vitamins D, A, and E; carotenoids; and minerals. Serum chemistries were somewhat different from published values, but did not indicate clear abnormalities. Circulating lipids were not different from those in captive primates. Circulating vitamin D metabolites (83±16.3 for 25(OH)D ng/mL; 563±53.8 for 1,25(OH)2D pg/mL) were similar to those in wild‐caught tamarins (Saguinus oedipus), lower than some published data for captive Cebidae and Callitrichidae, and higher than for Old World primates. Serum concentrations of retinol (16.5±1.64 μg/dl) were similar to those in captive spider monkeys (Ateles geoffroyi). Retinyl palmitate and retinyl stearate was present in howler samples and may have reflected recent dietary intake. Circulating α‐tocopherol (997±97.6 μg/dl) was similar to published values for other primates. Carotenoid levels in howlers were within the ranges reported for many primates. A significant finding was the presence of cadmium in samples that should be further studied. The number of individuals sampled was limited, and further investigation into the effects of seasonality is needed. However, this information provides new data for howler monkeys and for free‐ranging primates in general. Zoo Biol 22:239–251, 2003. © 2003 Wiley‐Liss, Inc.
Cool temperatures elicit reproduction in a biologically invasive predator, the brown treesnake (Boiga irregularis)
Authors: Mathies Tom , Miller Lowell A.
Published: June 2003

Two different temperature regimes for eliciting reproduction in male and females of the Guam form of the brown treesnake were investigated. Males and females maintained at 24°C followed by a 60‐day cool period at 19°C exhibited substantial reproductive activity, and the females that produced clutches did so during a brief period after return to 24°C. In contrast, individuals maintained at 28°C followed by an identical 19°C cooling period exhibited relatively little reproductive activity, and although some females became vitellogenic, none produced eggs. Reproductive activity was virtually absent in all individuals in both groups 7 months after the end of the cool period. Thus, a period of cool temperatures elicits reproductive activity in both sexes and the effect is transitory. Temperatures experienced during the cool period were much lower than the snakes would experience on Guam, and temperatures there are also relatively invariant. Thus, it is possible that only minor fluctuations in temperature are sufficient to elicit reproduction in the Guam population. Because the Guam form does well under, and responds reproductively to, unusually cold temperatures for a lowland tropical reptile, concern that it may have the capacity to invade extralimital temperate areas is warranted. Zoo Biol 22:227–238, 2003. Published 2003 by Wiley‐Liss, Inc.
Activity‐based exhibition of five mammalian species: Evaluation of behavioral changes
Authors: White Brent C. , Houser Lisa A. , Fuller Jodi A. , Taylor Steve , Elliott Jennifer L.L.
Published: June 2003

Activity‐based management of captive animals involves the training and movement of animals among several exhibits and holding areas. We studied the effectiveness of this system in producing variation in behavior, controlling stereotypies, and eliciting natural behaviors. Twelve animals representing five species of mammals (orangutans, siamangs, tapirs, babirusa, and Sumatran and Siberian tigers) were the subjects of focal observations measuring activity levels, stereotypies, natural behaviors, and space utilization. Statistical analysis was used to assess the association between variation in behavior and the movement among the four exhibits. For several animals, the persistence of behavioral changes was studied over a period of 3 years. We also examined the influence of the previous animal in the exhibit on the focal animal. Moving animals among the exhibits affected activity levels and/or space utilization in all animals in the activity‐based management system. In cases for which 3‐year data were available, there was evidence of habituation to the novelty of changing exhibits. Stereotypies, usually in the form of pacing, were affected by exhibits, providing the opportunity to manipulate these behavior patterns by exhibit placement. Natural behaviors in the form of urine‐spraying by the female tapir and the Sumatran tiger were affected by which animal had previously been in the exhibit. The results support the conclusion that exposure to varying exhibits produces variation in the behavior of the animals and elicits natural behaviors that would be unlikely to occur in a traditional single‐species exhibit. Activity‐based management provides unique opportunities for the behavioral enrichment of captive animals. Zoo Biol 22:269–285, 2003. © 2003 Wiley‐Liss, Inc.
Evaluation of the genetic management of the endangered black‐footed ferret (Mustela nigripes)
Authors: Wisely Samantha M. , McDonald David B. , Buskirk Steven W.
Published: June 2003

Empirical support for the genetic management strategies employed by captive breeding and reintroduction programs is scarce. We evaluated the genetic management plan for the highly endangered black‐footed ferret (Mustela nigripes) developed by the American Zoo and Aquarium Associations (AZA) as a part of the species survival plan (SSP). We contrasted data collected from five microsatellite loci to predictions from a pedigree‐based kinship matrix analysis of the captive black‐footed ferret population. We compared genetic diversity among captive populations managed for continued captive breeding or reintroduction, and among wild‐born individuals from two reintroduced populations. Microsatellite data gave an accurate but only moderately precise estimate of heterozygosity. Genetic diversity was similar in captive populations maintained for breeding and release, and it appears that the recovery program will achieve its goal of maintaining 80% of the genetic diversity of the founder population over 25 years. Wild‐born individuals from reintroduced populations maintained genetic diversity and avoided close inbreeding. We detected small but measurable genetic differentiation between the reintroduced populations. The model of random mating predicted only slightly lower levels of heterozygosity retention compared to the SSP strategy. The random mating strategy may be a viable alternative for managing large, stable, captive populations such as that of the black‐footed ferret. Zoo Biol 22:287–298, 2003. © 2003 Wiley‐Liss, Inc.
Considerations on the diet composition and feeding rate of dermersal sharks in 15 European public aquaria
Authors: Janse Max
Published: June 2003

To get an overview of the feeding practices applied to dermersal sharks in captivity, a questionnaire was sent to 15 European public aquaria. The most common dermersal shark species kept in the participating aquaria are Carcharhinus plumbeus (n=9), Carcharias taurus (n=7), and C. melanopterus (n=6). Body weight (BW) was calculated using captive estimated body length (BL) and actual weight data obtained from commercial fisheries. The estimated BW for C. plumbeus in all participating aquaria is higher than the calculated BW. The feeding rate (FR) for adult C. plumbeus varied widely within participating aquaria (2.3–11.5% BWcal/week or 32–112 kcal/kg BWcal/week). The FR for adult C. plumbeus was lower (1.2–7.9% BWest/week or 17–99 kcal/kg BWest/week) using the estimated BW. Adult Carcharias taurus were fed at 1.3–5.0% BWcal/week or 70–170 kcal/kg BWcal/week. Negaprion brevirostris was fed at 5.0% BWest/week or 57 kcal/BWest/week. The relationship between FR and estimated BW is expressed as FR=54.5 e−0.033BW. In this study, the feeding frequency for C. plumbeus varied between 1–7 times a week, with an average of 3.3 times a week. Diet in all aquaria consisted of both lean and fatty fish, and sometimes squid or crustaceans. Supplementation of vitamins is common practice in 80% of the surveyed aquaria, although a wide variety of quantity and quality was observed. Ten aquaria (66%) add iodine, as potassium iodide (0.4–12.5 mg KI/kg BW/week) or calcium‐iodate (322–1,500 mg/kg food/week), to their food. Zoo Biol 22:203–226, 2003. © 2003 Wiley‐Liss, Inc.
A ferredoxin Arg‐Glu pair important for efficient electron transfer between ferredoxin and ferredoxin‐NADP+ reductase
Authors: Teshima Keizo , Fujita Shinobu , Hirose Syuuichi , Nishiyama Daisuke , Kurisu Genji , Kusunoki Masami , Kimata-Ariga Yoko , Hase Toshiharu
Published: June 2003

In order to elucidate the importance of a ferredoxin (Fd) Arg‐Glu pair involved in dynamic exchange from intra‐ to intermolecular salt bridges upon complex formation with ferredoxin‐NADP+ oxidoreductase (FNR), Equisetum arvense FdI and FdII were investigated as normal and the pair‐lacking Fd, respectively. The FdI mutant lacking this pair was unstable and rapidly lost the [2Fe–2S] cluster. The catalytic constant (k
cat) of the electron transfer for FdI is 5.5 times that for FdII and the introduction of this pair into FdII resulted in the increase of k
cat to a level comparable to that for FdI, demonstrating directly that the Arg‐Glu pair is important for efficient electron transfer between Fd and FNR.
Membrane topology of ABC‐type macrolide antibiotic exporter MacB in Escherichia coli
Authors: Kobayashi Nobuyoshi , Nishino Kunihiko , Hirata Takahiro , Yamaguchi Akihito
Published: June 2003

MacB is an ABC‐type membrane protein that exports only macrolide compounds containing 14‐ and 15‐membered lactones, cooperating with a membrane fusion protein, MacA, and a multifunctional outer membrane channel, TolC. We determined the membrane topology of MacB by means of site‐specific competitive chemical modification of single cysteine mutants. As a result, it was revealed that MacB is composed of four transmembrane (TM) segments with a cytoplasmic N‐terminal nucleotide binding domain of about 270 amino acid residues and a periplasmic large hydrophilic polypeptide between TM segments 1 and 2 of about 200 amino acid residues.
Asp‐863 is a key residue for calcium‐dependent activity of Escherichia coli RTX toxin α‐haemolysin
Authors: Cortajarena Aitziber L , Goñi Félix M , Ostolaza Helena
Published: June 2003

α‐Haemolysin is a protein toxin secreted by pathogenic strains of Escherichia coli and requires sub‐millimolar Ca2+ for optimum lytic activity. As a member of the so‐called RTX toxin family it contains a Gly‐rich, Asp‐rich Ca2+‐binding domain, consisting of a series of nonapeptides repeated in tandem. Asp‐863 is located immediately after the last‐but‐one nonapeptide. A mutant in which Asp‐863 has been substituted by Gly displays a requirement for Ca2+ that is 100‐fold higher than the wild‐type. Membrane lytic activity, as well as a conformational change revealed through an increase in intrinsic fluorescence, and the appearance of Ca2+‐bound protein monomers resolvable by fast protein liquid chromatography, are all three dependent on Ca2+ concentrations in the 2–20 mM range. Most RTX toxins have an Asp or Glu residue located at a position homologous to Asp‐863, thus the key role of this residue for Ca2+ requirements of α‐haemolysin may be a general feature of this family of toxins.
Targeting of the AE2 anion exchanger to the Golgi apparatus is cell type‐dependent and correlates with the expression of Ank195, a Golgi membrane skeletal protein
Authors: Holappa Katja , Kellokumpu Sakari
Published: June 2003

Sodium‐independent anion exchangers (AE1–4) show remarkable variability in their tissue‐specific expression and subcellular localization. Currently, isoform‐specific targeting mechanisms are considered to be responsible for this variable localization. Here, we report that targeting can also be cell type‐specific. We show that the full‐length AE2 protein and its green fluorescent protein‐ or DsRed‐tagged variants localize predominantly either to the Golgi apparatus in COS‐7 cells, or to the plasma membrane in HeLa cells. This alternative targeting did not seem to result from either translational or post‐translational differences, but rather from differential expression of at least one of the Golgi membrane skeletal proteins, ankyrin195 (Ank195), between the two cell types. Comparative studies with several different cell lines revealed that the Golgi localization of the AE2 protein correlated strictly with the expression of Ank195 in the cells. The two Golgi‐associated proteins also co‐localized well and similarly resisted detergent extraction in the cold, whereas the plasma membrane‐localized AE2 in Ank195‐deficient cells was mostly detergent‐soluble. Collectively, our results suggest that Ank195 expression is a key determinant for the variable and cell type‐dependent localization of the AE2 protein in the Golgi apparatus in mammalian cells.
Complex effects of different green tea catechins on human platelets
Authors: Lill Gereon , Voit Simone , Schrör Karsten , Weber Artur-Aron
Published: June 2003

Epigallocatechin gallate (EGCG), a major component of green tea, has been previously shown to inhibit platelet aggregation. The effects of other green tea catechins on platelet function are not known. Pre‐incubation with EGCG concentration‐dependently inhibited thrombin‐induced aggregation and phosphorylation of p38 mitogen‐activated protein kinase and extracellular signal‐regulated kinases‐1/2. In contrast EGCG stimulated tyrosine phosphorylation of platelet proteins, including Syk and SLP‐76 but inhibited phosphorylation of focal adhesion kinase. Other catechins did not inhibit platelet aggregation. Interestingly, when EGCG was added to stirred platelets, a tyrosine kinase‐dependent stimulation of platelet aggregation was observed. The two other catechins containing a galloyl group in the 3′ position (catechin gallate, epicatechin gallate) also stimulated platelet aggregation, while catechins without a galloyl group (catechin, epicatechin) or the catechin with a galloyl group in the 2′ position (epigallocatechin) did not.
Homodimerization through coiled‐coil regions enhances activity of the myotonic dystrophy protein kinase
Authors: Zhang Rongxin , Epstein Henry F.
Published: June 2003

Myotonic dystrophy protein kinase (DMPK) is the protein product of the human DM‐1 locus on chromosome 19q13.1 and has been implicated in the cardiac and behavioral dysfunctions of the disorder. DMPK contains four distinct regions: a leucine‐rich repeat (L), a serine‐threonine protein kinase catalytic domain (PK), an α‐helical coiled‐coil region (H), and a putative transmembrane‐spanning tail (T). Multiple protein kinases that participate in cytoskeletal and cell cycle functions share homology with DMPK in the PK and H regions. Here we show that the LPKH and PKH subfragments of DMPK formed dimers of 140 000 molecular weight, whereas the LPK subfragment remained a monomer of 62 000 apparent molecular weight. The H domain thus appeared to be required for dimerization of DMPK subfragments. Caspase 1 cleaved LPKH between the PK and H regions. After cleavage, LPKH dimers became LPK‐like monomers, consistent with the H domain mediating dimerization. The V
max and k
cat/K
m of LPKH with a synthetic peptide kinase substrate were over 10‐fold greater than either LPK or caspase‐cleaved LPKH. The K
m of dimeric LPKH was over three‐fold greater than those of the monomeric proteins. Dimerization appeared to significantly affect the catalytic efficiency and substrate binding of DMPK. These interactions are likely to be functionally significant in other members of the myotonic dystrophy family of protein kinases with extensive coiled‐coil domains.
Affinity maturation of Fab antibody fragments by fluorescent‐activated cell sorting of yeast‐displayed libraries
Authors: van den Beucken Twan , Pieters Henk , Steukers Mieke , van der Vaart Marcel , Ladner Robert Charles , Hoogenboom Hennie R. , Hufton Simon E.
Published: June 2003

We report for the first time the affinity maturation of Fab antibody fragments using fluorescent‐activated cell sorting (FACS) of yeast‐displayed repertoires. A single yeast display vector which enables the inducible expression of an anchored heavy chain and a soluble light chain has been constructed. The assembly and functional display on the yeast cell surface of Fab antibodies specific for different protein targets has been demonstrated by flow cytometry and immunofluorescence microscopy. We have affinity matured a Fab antibody specific for the tetravalent antigen streptavidin using FACS of yeast‐displayed repertoires diversified by error‐prone polymerase chain reaction. A panel of variants with up to 10.7‐fold improvement in affinity was obtained after selection. Two leading clones, R2H10 (3.2 nM) and R3B1 (5.5 nM), had mutations in light chain complementarity determining region 1 LC‐CDR1 (H34R) and LC‐CDR3 (Y96H or Y96F) and gave a 10.7‐fold and 6.3‐fold affinity improvement over the starting antibody, respectively. The ability to efficiently affinity mature Fab antibodies is an important component of the antibody development pipeline and we have shown that yeast display is an efficient method for this purpose.
PTP1B: From the sidelines to the front lines!
Authors: Tonks Nicholas K.
Published: June 2003

Although initially viewed as housekeeping enzymes, research over the last 15 years has revealed that the protein tyrosine phosphatases (PTPs) are critical regulators of tyrosine phosphorylation‐dependent signaling events and may represent novel targets for therapeutic intervention in a variety of human diseases. In this review I will describe some of the key advances in the characterization of the structure, regulation and function of the prototypic PTP, PTP1B, and illustrate how our understanding of the properties of this enzyme has revealed principles that apply to the PTP family as a whole.
Silencing of the gene coding for the ϵ subunit of DNA polymerase III slows down the growth rate of Escherichia coli populations
Authors: Stefan Alessandra , Reggiani Luca , Cianchetta Stefano , Radeghieri Annalisa , Gonzalez Vara y Rodriguez Antonio , Hochkoeppler Alejandro
Published: June 2003

Chromosome replication in Escherichia coli is accomplished by the multimeric enzyme DNA polymerase III; the relevance, in vivo, of the ϵ subunit (encoded by dnaQ) for processivity and fidelity of DNA polymerase III has been evaluated. To this aim, dnaQ has been conditionally silenced by means of in vivo expression of different antisense RNAs. Unexpectedly, the presence of the Shine–Dalgarno sequence is essential for the effectiveness of antisense constructs. Silencing of dnaQ induces a severe decrease in growth rate not paralleled by high mutation frequencies, suggesting that the ϵ subunit primarily affects the processivity of DNA polymerase III.
Enhanced suicide gene therapy by chimeric tumor‐specific promoter based on HSF1 transcriptional regulation
Authors: Wang Jinhui , Yao Mingzhong , Zhang Zilai , Gu Jinfa , Zhang Yanhong , Li Binhua , Sun Lanying , Liu Xinyuan
Published: June 2003

Two tandem cassettes, one containing the telomerase reverse transcriptase gene (hTERT) promoter upstream of a constitutively activated form of heat shock transcription factor 1 (cHSF1) and followed by the other containing the heat shock protein 70B (hsp70B) promoter (HSE) upstream of the cytosine deaminase (CD) gene, could greatly enhance the efficiency of CD gene therapy while retaining tumor specificity in vitro and in vivo. This hTERT‐cHSF1/HSE promoter could restrict gene expression in tumor cells and was about 1.5–3‐fold more potent than the cytomegalovirus (CMV) promoter. hTERT‐cHSF1/HSE‐CD transfection led to tumor cells more sensitive to 5‐fluorocytosine compared with hTERT‐CD and its toxicity was comparable to that of CMV‐CD. Besides enhancement of promoter activity, cHSF1 overexpression itself could enhance the bystander effect of CD gene therapy that could be reversed by anti‐Fas antibody. This system also led to activation of stress‐related genes such as hsp70 in tumor cells, which in the presence of cell killing by the cytotoxic gene is a highly immunostimulatory event. Furthermore, a more potent anti‐tumor effect of hTERT‐cHSF1/HSE‐CD was observed in nude mice inoculated with Bcap37 cells. No obvious activity of the hTERT‐cHSF1/HSE promoter was observed in normal tissues after intravenous administration. These results indicate that the hTERT‐cHSF1/HSE promoter is highly tumor‐specific and strong with potential application in targeted gene therapy, and therefore may be useful for construction of vectors for systemic therapy.
Phospholipase Cδ1 does not mediate Ca2+ responses in neonatal rat cardiomyocytes
Authors: Woodcock Elizabeth A. , Mitchell Christopher J. , Biden Trevor J.
Published: June 2003

Phospholipase C (PLC) activation in neonatal rat ventricular cardiomyocytes (NRVM) generates inositol(1,4,5)trisphosphate (Ins(1,4,5)P3) in response to elevations in Ca2+ or inositol(1,4)bisphosphate in response to G protein stimulation. Overexpression of PLCδ1 increased total [3H]inositol phosphate (InsP) content and elevated [3H]Ins(1,4,5)P3, but failed to increase [3H]InsP responses to the Ca2+ ionophore A23187. Antisense PLCδ1 expression reduced endogenous PLCδ1 content but did not decrease the A23187 response. In permeabilized NRVM, [3H]InsP responses to elevated Ca2+ were not inhibited by Ins(1,4,5)P3, even at concentrations 1000‐fold greater than required for selective inhibition of PLCδ1. Taken together these data provide evidence that PLCδ1 does not mediate the InsP response to elevated Ca2+ in NRVM.
Mutations at the CXCR4 interaction sites for AMD3100 influence anti‐CXCR4 antibody binding and HIV‐1 entry
Authors: Hatse Sigrid , Princen Katrien , Vermeire Kurt , Gerlach Lars-Ole , Rosenkilde Mette M. , Schwartz Thue W. , Bridger Gary , De Clercq Erik , Schols Dominique
Published: June 2003

The interaction of the CXCR4 antagonist AMD3100 with its target is greatly influenced by specific aspartate residues in the receptor protein, including Asp171 and Asp262. We have now found that aspartate‐to‐asparagine substitutions at these positions differentially affect the binding of four different anti‐CXCR4 monoclonal antibodies as well as the infectivity of diverse human immunodeficiency virus type 1 (HIV‐1) strains and clinical isolates. Mutation of Asp262 strongly decreased the coreceptor efficiency of CXCR4 for wild‐type but not for AMD3100‐resistant HIV‐1 NL4.3. Thus, resistance of HIV‐1 NL4.3 to AMD3100 is associated with a decreased dependence of the viral gp120 on Asp262 of CXCR4, pointing to a different mode of interaction of wild‐type versus AMD3100‐resistant virus with CXCR4.
Adenosine‐rich elements present in the 5′‐untranslated region of PABP mRNA can selectively reduce the abundance and translation of CAT mRNAs in vivo
Authors: Melo Eduardo O. , de Melo Neto Osvaldo P. , Martins de Sá Cezar
Published: June 2003

The poly(A)‐binding protein (PABP) is a highly conserved eukaryotic protein whose synthesis is regulated at the post‐transcriptional level. The binding of PABP to the poly(A)‐rich element found in the 5′‐untranslated region (5′UTR) of PABP mRNA specifically inhibits its own translation. In this report, we show that similar adenosine‐rich elements in the 5′UTR of the chloramphenicol acetyl‐transferase (CAT) gene can significantly reduce the reporter mRNA abundance and translation in human 293 cells. The reduction in mRNA level, but not CAT expression, is dependent on the size of the 5′UTR poly(A) element. Furthermore, one 5′UTR‐tethered PABP molecule is enough to inhibit CAT expression without affecting its mRNA level. We propose that the control of PABP synthesis may involve mRNA decay and the repression of translation.
The Physarum polycephalum php gene encodes a unique cold‐adapted serine‐carboxyl peptidase, physarolisin II
Authors: Nishii Wataru , Kuriyama Hiroki , Takahashi Kenji
Published: June 2003

The php gene from a true slime mold, Physarum polycephalum, is a late‐replicating and transcriptionally active gene. The deduced amino acid sequence of the gene product is homologous to those of the serine‐carboxyl peptidase family, including physarolisin I from the same organism, but lacks the propeptide region. In this study, the protein was expressed in Escherichia coli and shown to possess endopeptidase activity with unique substrate specificity. Thus, we named it physarolisin II. The enzyme was revealed to be a kind of cold‐adapted enzyme since it was maximally active at 16–22°C. The active enzyme was markedly unstable due to rapid autolysis (t
1/2=∼5 min, at 18°C). At higher temperature, the enzyme was less active but more stable, despite the fact that no gross conformational change was observed by circular dichroism spectroscopy.
Three New Species of Stigmidium s. l. (lichenicolous ascomycetes) on Acarospora and Squamarina
Authors: Calatayud Vicent , Triebel Dagmar
Published: June 2003

Stigmidium cartilagineae Calatayud &Triebel (on the apothecial discs of Squamarina cartilaginea), a lichenicolous fungus belonging to Stigmidium s. str., is described as new. Two other species of Stigmidium s. 1. with a net of branched and anastomosing, rudimentary interascal filaments are also described as new to science: Stigmidium rouxianum Calatayud & Triebel (on Acarospora cervina) and S. squamarinicola Calatayud & Triebel (on the thallus of Squamarina spp.). They are related to Stigmidium psorae and treated here as ‘Stigmidium’ psorae group. To facilitate discussion of the distinguishing features of this group, a table comparing the terms referring to hamathecial hyphae in pyrenocarpous ascomycetes is presented.
Opegrapha cladoniicola, a new lichenicolous fungus from Hawaii
Authors: Ertz Damien , Diederich Paul
Published: June 2003

A new species Opegrapha cladoniicola, lichenicolous on the podetia of Cladonia ochrochlora, is described from Hawaii. It is the first known Opegrapha species found growing on Cladonia.
Buellia tesserata and dimelaena radiata, two closely related species
Authors: Rico Victor J. , Calatayud Vicent , Giralt Mireia
Published: June 2003

The morphology, anatomy, ecology and distribution of Buellia tesserata and Dimelaena radiata are discussed. New data on their chemistry are given. The present study reveals a very close relationship between both taxa and consequently their different generic position is questioned. Buellia fimbriata is reduced to synonymy with Buellia tesserata.
Notes on the genus Polycoccum (Ascomycota, Dacampiaceae) in Spain, with a key to the species
Authors: Atienza Violeta , Calatayud Vicent , Hawksworth David L.
Published: June 2003

Comments on and a key to the 13 Polycoccum species known in Spain are presented, including synopses of their world distributions. Amongst these is P. rubellianae sp. nov., a lichenicolous fungus growing on thalli of Caloplaca rubelliana in eastern Spain (Valencia). It has relatively small ascomata, the lower part pale brown, and also small ascospores which are coarsely verrucose and have a thick gelatinous sheath when young. The new species is associated with a Phoma-like anamorph. The identity and systematic position of P. opulentum requires further study as the name has been applied to different species, and the occurrence of P. marmoratum in Spain is in need of confirmation. The Spanish record of P. arnoldii on Lecania cyrtella probably refers to another yet undescribed species.
Niche partitioning and photosynthetic response of alectorioid lichens from subalpine spruce–fir forest in north-central British Columbia, Canada: the role of canopy microclimate gradients
Authors: Coxson Darwyn S. , Coyle Michelle
Published: June 2003

The distribution of alectorioid lichens in subalpine spruce-fir forests of north-central British Columbia is strongly influenced by vertical position within the canopy; Bryoria dominates upper canopy exposures, while Alectoria dominates lower canopy positions. The hypothesis that this height-related niche partitioning reflects differential growth responses to gradients in canopy microclimate is examined. Field measurements of canopy microclimate, taken over a 2-year period, were used in conjunction with laboratory-based measurements of net photosynthesis (NP) and dark respiration to model net assimilation (NA) response of Alectoria sarmentosa and Bryoria spp. (mixed collections of Bryoria fremontii and B. pseudofuscescens) at two different heights (15 and 4 m) within the canopy. Microclimate measurements indicate that lichen thalli are regularly hydrated from snowmelt events during the winter period (October-April), totalling 26 and 29% of the time, respectively, for Alectoria and Bryoria, though most winter hydration exposure (c. 75%) occurred in the dark. In the summer (May-September), rainfall was the major hydration source, with Alectoria and Bryoria each hydrated c. 16% of the time (45% of this in the dark). The NP temperature optimum (Topt) in light saturated thalli of Alectoria was 18.1 and 22.9°C, for winter and summer measurements, respectively. In Bryoria the corresponding seasonal rise in Topt was smaller, from 15.9 to 16.3°C. Both species showed an increase in maximum rates of NP during the summer period, from 1.52 to 1.92 mg CO2 g−1h−1 for Alectoria, and from 1.79 to 2.33 mg CO2g−1 h−1 for Bryoria. Although lichen hydration events peaked in early winter (October and November), NA modeling predicts that maximum growth should occur during the summer period. In Alectoria, higher rates of NA were predicted for thalli in lower canopy positions, especially during the summer months. In Bryoria, no clear trends of NA uptake with canopy position were observed. Thus, while NP response to gradients of canopy microclimate may provide a basis for niche partitioning in Alectoria, other factors (perhaps exclusionary) may be more important for Bryoria. One such factor is documented, namely the greater sensitivity of Bryoria to extended hydration exposure and we speculate that greater rates of fragmentation in upper canopy exposures may limit upper canopy biomass accumulation in Alectoria. Niche partitioning in these alectorioid lichens may therefore reflect both positive (growth responses) and negative (physical and physiological limitations) responses to gradients in canopy microclimate.
Chemotactically‐induced redistribution of CD43 as related to polarity and locomotion of human polymorphonuclear leucocytes
Authors: Zadeh A. Dehghani , Seveau S. , Halbwachs‐Mecarelli L. , Keller H.U.
Published: June 2003

Leukocyte motility involves pseudopods extension at the leading edge and uropod contraction at the cell rear. Previous studies have shown that the glycoprotein CD43 redistributes to the uropod, when the cells develop polarity and locomotion. The present study addresses the question whether the accumulation of specific membrane molecules, such as CD43 at the contracted uropod precedes or follows development of polarity and locomotion. PMNs were labeled with fluorescent anti‐CD43 antibodies and guided to polarize in the direction of a chemoattractant‐containing micropipette or, once polarized, they were forced to reverse polarity and movement direction by placing the micropipette behind the uropod. This chemotactically‐induced reversal of polarity was used as an efficient tool to analyse the sequence of events. CD43, but not another abundant surface glycoprotein CD45, was concentrated at the uropod. This documents that CD43 redistribution is a selective phenomenon. During reversal of polarity and of locomotion direction, the geometric center of the cell clearly changed direction earlier than the center of anti‐CD43 fluorescence intensity. Thus, CD43 redistribution to the new uropod follows rather than precedes reversal of polarity, suggesting that CD43 redistribution is a consequence rather than a prerequisite for polarity and locomotion. PMNs making a U‐turn maintained the pre‐existing polarity and CD43 remained concentrated at the uropod, even when the front was moving in the opposite direction. Our data show that anterior pseudopod formation, rather than capping of CD43 at the uropod or the position of the uropod determines the direction of locomotion.
MAPK is involved in metaphase I arrest in oyster and mussel oocytes
Authors: Portillo‐López Amelia , Gould Meredith C. , Stephano José Luis
Published: June 2003

Oocytes of Crassostrea gigas and Mytilus galloprovincialis are arrested in metaphase I when they are spawned and ready to be fertilized. To investigate the role of MAP kinase in maintaining metaphase I arrest, oocytes were exposed to the MEK inhibitor U0126, and the effects on chromosome behavior and MAPK activity were examined by bisbenzimide staining and in immunoblots with anti‐phospho MAPK antibodies. Following treatment with 50 μM U0126, active MAPK was undetectable and oocytes resumed meiosis, forming enlarged polar bodies and undergoing chromosome decondensation. Prophase stage oyster oocytes maturing spontaneously in seawater completed germinal vesicle breakdown in the presence of U0126, but failed to arrest in metaphase I, and also formed polar bodies and underwent chromosome decondensation. Treatment of oyster oocytes with the protein synthesis inhibitor, emetine (500 μM), also caused them to resume meiosis, although substantial MAPK activity remained. Levels of phospho‐MEK also decreased during emetine treatment. 35 S‐methionine incorporation in emetine treated oocytes was reduced to only 5% of control values. These data show that, while active MAPK is necessary to maintain metaphase I arrest, other proteins are also required.
Molecular cloning and characterization of an adaptor protein Shc isoform from Xenopus laevis oocytes
Authors: Chesnel Franck , Heligon Christophe , Richard‐Parpaillon Laurent , Boujard Daniel
Published: June 2003

In order to gain further insight into IGF‐1 receptor signaling in Xenopus laevis oocytes and embryos, we have undertaken the characterization of the adapter protein Shc and studied its implication in oocyte maturation induced after IGF‐1 receptor activation, especially since expression of this molecule has been indirectly evidenced in Xenopus oocytes, eggs and embryos. We report herein the cloning from Xenopus postvitellogenic oocytes of a complementary DNA encoding a protein of 470 amino acids which shows the higher identity with the mammalian adaptor protein p52ShcA. Western blot analysis using homologous antibodies evidenced a 60‐kDa protein, p60XlShc, that is predominantly expressed in oocytes and in early embryos. We also demonstrate that, like p60XlShc, Grb2 and the guanine nucleotide exchange factor Sos are expressed in oocytes throughout vitellogenesis and in early embryos and that overexpression of a dominant‐negative form of Grb2 specifically inhibits insulin‐induced resumption of meiosis. We finally show that Grb2 binds to p60Shc in oocytes specifically upon insulin treatment. Altogether, these results suggest that Shc and Grb2‐Sos are implicated in ras‐dependent Xenopus oocyte maturation induced by insulin/IGF‐1; they also indicate that inability of insulin/IGF‐1 to activate the Ras‐MAPK cascade in vitellogenic oocytes does not result from an insufficient expression level of Shc, Grb2 and Sos.
Expression of Tra2β isoforms is developmentally regulated in a tissue‐ and temporal‐specific pattern
Authors: Chen Xianhua , Guo Lanping , Lin Wanmin , Xu Ping
Published: June 2003

The contribution of Tra2‐regulated alternative splicing to mammalian development is unknown. To address the question, we initiated studies by systemically characterizing the gene expression profile of Tra2β isoforms in human fetal tissue at two different stages (11 and 16 weeks). Tra2β has at least five isoforms of transcripts (Tra2β1∼5). Tra2β1 encodes full‐length Tra2β proteins, whilst Tra2β3, 4 and 5 encode the truncated proteins. The results show that Tra2β1 transcript levels are developmentally regulated in a tissue‐ and temporal‐specific pattern, although expression is ubiquitous. Compared to Tra2β5, the expression of Tra2β3 and 4 transcripts is significantly restricted and more vigorously regulated in a tissue‐ and temporal‐specific pattern. Western blot analysis shows that Tra2β1 proteins are ubiquitously expressed, but at a relatively higher level in neural tissues than non‐neural tissues. However, there is no direct correlation between the transcript and protein levels for Tra2β1, suggesting that multiple mechanisms are involved in the regulation of Tra2β during development. This study indicates that Tra2β‐regulated splicing may contribute to the regulation of mammalian development in a tissue‐ and temporal‐specific pattern.
Possible origin of a membrane in the subsurface of the Earth
Authors: Trevors J.T.
Published: June 2003

A location for the origin of life on Earth could have been an oil/water interface in the warm, subsurface environment of the Earth. The physico‐chemical conditions of the subsurface would include elevated, but eventually cooling temperatures, anaerobic conditions, and protection from intense surface radiation. This type of subsurface oil/water environment may have been ideal for the assembly of the first simple membrane(s), where no enzyme catalysis was needed. Once a stable, simple, continuous closed membrane was formed, one central component of the first cell(s) would have been present; a semi‐permeable open system that allowed the passage of both matter and energy in and out of the cell. Such an open system could also acquire novel functions, whereas a closed system would be unable to evolve.