bluuebunny/crossref_metadata_2025 huggingface dataset:
Quantitative evaluation of print density uniformity using MTF fractal dimension
Authors: Nakamoto Jun , Yamashita Hiroshi , Kaneko Hiroshi
Published: January 2003

The unevenness of the density of a printed product which should ideally be even is evaluated by a printer's visual sense, and thus is affected by each printer's individual characteristics. There have been no quantitative standards for such an evaluation.

This paper proposes to use the fractal dimension, which is originally an index representing the complexity of an object, for the quantitative evaluation of print density uniformity. It has been found that the modulation transfer function (MTF) fractal dimension produces results similar to those obtained by visual evaluation. It has also been found that the MTF fractal dimension with the visual spatial‐frequency characteristic (sensitivity of appearance) can emphasize the differences in print density uniformity. These findings will help in establishing the quantitative evaluation of print density uniformity. © 2003 Wiley Periodicals, Inc. Syst Comp Jpn, 34(2): 92–99, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/scj.1193
Speech recognition using recurrent neural prediction model
Authors: Uchiyama Toru , Takahashi Haruhisa
Published: January 2003

The neural prediction model (NPM) proposed by Iso and Watanabe is a successful example of a speech recognition neural network with a high recognition rate. This model uses multilayer perceptrons for pattern prediction (not for pattern recognition), and achieves a recognition rate as high as 99.8% for speaker‐independent isolated words. This paper proposes a recurrent neural prediction model (RNPM), and a recurrent network architecture for this model. The proposed model very significantly reduces the size of the network, with as high a recognition rate as the original model, and with a high efficiency of learning, for speaker‐independent isolated words. © 2003 Wiley Periodicals, Inc. Syst Comp Jpn, 34(2): 100–107, 2003; Published online in Wiley InterScience (www.interscience.wiley.com). DOI 10.1002/scj.1194
A comparative evaluation of generic programming in Java and C++
Authors: Saiedian Hossein , Hill Steve
Published: January 2003

Generic programming has been defined as ‘programming with concepts’ where a concept refers to a family of abstractions. The criteria for generic programming include independence of collections from data types, independence of algorithms that operate on the collection, and the adaptability of the collections. This paper examines and evaluates the support for generic programming in the Java Development Kit (JDK) in comparison to C++'s Standard Template Library (STL). The evaluation will consider both the ‘qualitative’ factors as well as certain ‘quantitative’ factors (i.e. factors that can be measured). The qualitative factors that are considered include: 1. a comparison of the structure and APIs; 2. homogeneity versus heterogeneity; and 3. ease of use (including ease of converting to collection classes, ease of changing collection type, and ease of error handling).

The quantitative factors include: 1. compiled size; 2. runtime memory usage; and 3. performance. The results of our evaluative comparisons based on the above factors and certain other criteria are presented at the end. Based on the results, we conclude that the support provided for generic programming in C++'s STL is superior to that provided by JDK. Copyright © 2003 John Wiley & Sons, Ltd.
DrawCAD: using deductive object‐relational databases in CAD
Authors: Liu Mengchi
Published: January 2003

Computer‐aided design (CAD) involves the use of computers in the various stages of engineering design. CAD has large volumes of data with complex structures that need to be stored and managed effectively and properly. Database systems provide general purpose programs that can be used to access and manipulate large amounts of data stored in the database. They also provide an independence between the program accessing data and the database. It is therefore important to use database systems to store CAD data in the most efficient and effective manner for easy retrieval and better management.

Graphical objects can be created, in CAD, by reusing previously created objects. The data of these objects have references to the other objects they contain. Deductive object‐relational databases not only provide direct support for the effective storage and efficient access to large amounts of data with complex structures on disk, but also perform the inferences and computations to obtain the complete data of graphical objects that reuse other objects. They should be able to play a major role in CAD systems. This is the idea behind the development of the DrawCAD system. DrawCAD is a CAD system built on top of the Relationlog object‐relational deductive database system. It facilitates the creation of graphical objects by reusing previously created objects. The DrawCAD system illustrates how CAD systems can be developed, using database systems to store and manage data and also perform the inferences and computations that are normally performed by the application program. Copyright © 2003 John Wiley & Sons, Ltd.
A music notation construction engine for optical music recognition
Authors: Bainbridge David , Bell Tim
Published: January 2003

Optical music recognition (OMR) systems are used to convert music scanned from paper into a format suitable for playing or editing on a computer. These systems generally have two phases: recognizing the graphical symbols (such as note‐heads and lines) and determining the musical meaning and relationships of the symbols (such as the pitch and rhythm of the notes). In this paper we explore the second phase and give a two‐step approach that admits an economical representation of the parsing rules for the system. The approach is flexible and allows the system to be extended to new notations with little effort—the current system can parse common music notation, Sacred Harp notation and plainsong. It is based on a string grammar and a customizable graph that specifies relationships between musical objects. We observe that this graph can be related to printing as well as recognizing music notation, bringing the opportunity for cross‐fertilization between the two areas of research. Copyright © 2003 John Wiley & Sons, Ltd.
The emergence of career services and their important role in working with employers
Authors: McGrath Gary L.
Published: January 2003

This chapter reviews how and why career services have changed since the 1970s and indentifies some ways to effectively utilize employers to enhance the student experience and strengthen institutional relationships.
Substrate effect on the formation of hydrogels with heterogeneous network structure
Authors: Peng Mao , Ping Gong Jian , Osada Yoshihito
Published: January 2003

It was found that when an aqueous solution of vinyl monomers is polymerized on a hydrophobic substrate,
obvious heterogeneity occurs in the region of the interface. This substrate effect was observed on
polytetrafluroethylene (Teflon), polypropylene (PP), polyethylene (PE), polystyrene
(PS), and polyvinylchloride (PVC), but not on hydrophilic substrates. Compared with synthesis on
hydrophilic surfaces, the surfaces of hydrogels synthesized on a hydrophobic substrate exhibit a larger degree of
swelling, a lower surface coefficient of friction and elastic modulus, weaker interfacial adhesion, and reduced
interaction with biological cells. This substrate effect has been observed for many types of aqueous monomer
solutions. It was found that the above properties are related to the loosely cross‐linked architecture,
containing some graft‐like polymer chains, that is formed on the gel surface when the gel is prepared on
a hydrophobic substrate. To understand the mechanism of the substrate effect, two novel optical methods, electric
speckle pattern interferometry (ESPI) and real‐time laser sheet refraction (RT‐LSR),
were developed. It was found that oxygen trapped in the composite interface between the monomer solution and rough
hydrophobic substrates played an important role in the substrate effect. © 2003 The Japan Chemical Journal
Forum and Wiley Periodicals, Inc., Chem Rec 3: 40–50; 2003: Published online in Wiley InterScience
(www.interscience.wiley.com) DOI 10.1002/tcr.10048
Technological novelty and open‐endedness: Two characteristics of interactive exhibits that contribute to the holding of visitor attention in a science museum
Authors: Sandifer Cody
Published: January 2003

This study was undertaken to isolate characteristics of interactive exhibits that are particularly effective in attracting and holding the attention of visitors in a science museum. Forty‐seven visitors were tracked through two adjacent exhibitions, comprising a total of 61 interactive exhibits. Four exhibit characteristics were identified and examined: technological novelty, user‐centeredness, sensory stimulation, and open‐endedness. Regression analyses show that two of these characteristics, technological novelty and open‐endedness, help to account for the variance in average visitor holding time; these characteristics have positive correlations with the amount of time spent by visitors at exhibits. Nonsignificant results are explained in terms of mitigating environmental and exhibit‐related factors. In addition, topics for future study are suggested. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 121–137, 2003
Dialogic inquiry in life science conversations of family groups in a museum
Authors: Ash Doris
Published: January 2003

This research illustrates the efficacy of a new approach for collecting and analyzing family conversational data at museums and other informal settings. This article offers a detailed examination of a small data set (three families) that informs a larger body of work that focuses on conversation as methodology. The dialogic content of this work centers on biological themes, specifically adaptation. The biological principle becomes visible when families talk about survival strategies such as breeding or protection from predators. These themes arise from both the family members and the museum exhibit. This study also analyzes the inquiry skills families use as they make sense of science content. I assume that children and adults offer different interest areas or expertise for dialogic negotiation and that family members use inquiry skills in dialogue to explore matters of importance. This analysis offers educators methodological tools for investigating families' scientific sense‐making in informal settings. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 138–162, 2003
Investigating the impact of prior knowledge and interest on aquarium visitor learning
Authors: Falk John H. , Adelman Leslie M.
Published: January 2003

Most free‐choice science learning institutions, in particular science centers, zoos, aquariums, and natural history museums, define themselves as educational institutions. However, to what extent, and for which visitors, do these free‐choice learning settings accomplish their educational mission? Answering this question has proven challenging, in large part because of the inherent variability of visitors to such settings. We hypothesize that the challenges of measuring free‐choice science learning might be diminished if it were possible to pool populations during analysis in ways that reduced this variability. Specifically, we propose grouping learners according to their entering understanding and attitudes, using qualitative categories such as minimal, moderate, and extensive. In this article, we use data collected at the National Aquarium in Baltimore to determine whether grouping makes it possible to discern more readily the nature of changes in aquarium visitors' conservation knowledge and attitudes. Although analysis revealed that there were significant changes in both conservation knowledge and attitudes, entry to exit, for all 100 visitors studied, a more detailed analysis revealed that gains were not evenly distributed across all visitors. The results support the hypothesis that the grouping of learners into minimal, moderate, and extensive conservation knowledge and attitude categories enabled a more fine‐grained and accurate understanding of changes in aquarium visitor's conservation learning. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 163–176, 2003
Theoretical perspectives on learning in an informal setting
Authors: Anderson David , Lucas Keith B. , Ginns Ian S.
Published: January 2003

Research into learning in informal settings such as museums has been in a formative state during the past decade, and much of that research has been descriptive and lacking a theory base. In this article, it is proposed that the human constructivist view of learning can guide research and assist the interpretation of research data because it recognizes an individual's prior knowledge and active involvement in knowledge construction during a museum visit. This proposal is supported by reference to the findings of a previously reported interpretive case study, which included concept mapping and semistructured interviews, of the knowledge transformations of three Year 7 students who had participated in a class visit to a science museum and associated postvisit activities. The findings from that study are shown in this report to be consistent with the human constructivist view of learning in that for all three students, learning was found to be at times incremental and at other times to involve substantial restructuring of knowledge. Thus, we regard that the human constructivist view of learning has much merit and utility for researchers investigating the development of knowledge and understanding emergent from experiences in informal settings. The theoretical and practical implications of these findings for teachers and staff of museums and similar institutions are also discussed. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 177–199, 2003
Investigation of guided school tours, student learning, and science reform recommendations at a museum of natural history
Authors: Cox‐Petersen Anne M. , Marsh David D. , Kisiel James , Melber Leah M.
Published: January 2003

A study of docent‐led guided school tours at a museum of natural history was investigated. Researchers engaged in naturalistic inquiry to describe how natural history content was conveyed to students and what students gained from this model of touring. They also investigated how the content and pedagogy within the guided tour complemented recommendations from formal science standards documents and informal learning literature. About 30 visiting school groups in Grades 2–8 were observed. Teachers (n = 30) and select students (n = 85) were interviewed. Researchers found that tours were organized in a didactic way that conflicted with science education reform documents and research related to learning within informal contexts. Students' responses to interview questions indicated high satisfaction with the tours but low levels of science learning. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 200–218, 2003
Home‐school learning of science: The culture of homes, and pupils' difficult border crossing
Authors: Solomon Joan
Published: January 2003

A British project that explored the way parents and their children of elementary school age carried out simple science activities at home is described and illustrated. Previous research in this field has yielded ambiguous results when evaluated in terms of school science knowledge gained. The basis of the analysis carried out here is largely descriptive using some sociological theory to understand activities in the home. It is argued that home is a special place not only rich in supportive emotions, but also imbued with idiosyncratic attitudes toward science education, which often match with attitudes toward other matters. Schools create different and more uniform cultures for the same children. There has been a long history of calls for collaboration between the two constituencies; however, this article demonstrates that a number of these differences exist which cannot fail to affect children's learning in each situation. Extracts from the children's conversations with their parents during the investigations as well as parents' interpretations of what they are doing will be presented. These vignettes illustrate a wide variation in attitude which affects the children as they daily cross boundaries from one culture to another, trying to preserve what is precious in their home culture. At home the children's participation becomes far more relaxed and personal, just as discussion with their parents is more fluent than at school. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 219–233, 2003
Thinking about television science: How students understand the nature of science from different program genres
Authors: Dhingra Koshi
Published: January 2003

Student views on the nature of science are shaped by a variety of out‐of‐school forces and television‐mediated science is a significant force. To attempt to achieve a science for all, we need to recognize and understand the diverse messages about science that students access and think about on a regular basis. In this work I examine how high school students think about science that is mediated by four different program genres on television: documentary, magazine‐format programming, network news, and dramatic or fictional programming. The following categories of findings are discussed: the ethics and validity of science, final form science, science as portrayed by its practitioners, and school science and television science. Student perceptions of the nature of science depicted on the program sample used in this study ranged from seeing science as comprising tentative knowledge claims to seeing science as a fixed body of facts. © 2003 Wiley Periodicals, Inc. J Res Sci Teach 40: 234–256, 2003
Long-Term Memory Survives Nerve Injury and the Subsequent Regeneration Process
Authors: Lukowiak Ken , Haque Zara , Spencer Gaynor , Varshay Nishi , Sangha Susan , Syed Naweed
Published: January 2003

A three-neuron network (a central pattern generator [CPG]) is both sufficient and necessary to generate aerial respiratory behavior in the pond snail, Lymnaea stagnalis. Aerial respiratory behavior is abolished following a specific nerve crush that results in axotomy to one of the three CPG neurons, RPeD1. Functional regeneration of the crushed neurite occurs within 10 days, allowing aerial respiratory behavior to be restored. Functional regeneration does not occur if the connective is cut rather than crushed. In unaxotomized snails, aerial respiratory behavior can be operantly conditioned, and following memory consolidation, long-term memory (LTM) persists for at least 2 weeks. We used the Lymnaea model system to determine (1) If in naive animals axotomy and the subsequent regeneration result in a nervous system that is competent to mediate associative learning and LTM, and (2) if LTM survives RPeD1 axotomy and the subsequent regenerative process. We show here that (1) A regenerated nervous system is competent to mediate associative memory and LTM, and (2) LTM survives axotomy and the subsequent regenerative process.
Visuomotor Adaptation in Normal Aging
Authors: Buch Ethan R. , Young Sereniti , Contreras-Vidal José L.
Published: January 2003

Visuomotor adaptation to a gradual or sudden screen cursor rotation was investigated in healthy young and elderly subjects. Both age groups were equally divided into two subgroups; one subgroup was exposed to 11.25° step increments of visual feedback rotation, every 45 trials (up to a total of 90°), whereas a second subgroup was subjected to 90° rotation from the onset of exposure. Participants performed discrete, horizontal hand movements to virtual targets in four randomized directions. Targets appeared on a computer screen in front of them, and a board prevented vision of the hand at all times. Differential effects of aging on visuomotor adaptation were found, depending on the time course of the visual distortion. In both age groups, early exposure to the sudden visual feedback distortion resulted in typical spiral-like trajectories, which became straighter by late exposure. However, the final adaptation level was reduced in the aged group, although the aftereffects were similar. When subjects were exposed to the gradual distortion, no statistically significant differences in measures of adaptation with advancing age were found. In this case, both age groups appeared to adapt equally. However, after removal of the distortion, elderly subjects showed reduced aftereffects as compared with the young group. These findings suggest differential effects of aging on adaptation to gradual versus sudden visual feedback distortions, and may help to explain the conflicting results obtained in previous visuomotor adaptation studies.
A Role for ERK MAP Kinase in Physiologic Temporal Integration in Hippocampal Area CA1
Authors: Selcher Joel C. , Weeber Edwin J. , Christian Jill , Nekrasova Tanya , Landreth Gary E. , Sweatt J. David
Published: January 2003

Recent studies demonstrate a requirement for the Extracellular signal Regulated Kinase (ERK) mitogen-activated protein kinase (MAPK) cascade in both the induction of long-lasting forms of hippocampal synaptic plasticity and in hippocampus-dependent associative and spatial learning. In the present studies, we investigated mechanisms by which ERK might contribute to synaptic plasticity at Schaffer collateral synapses in hippocampal slices. We found that long-term potentiation (LTP) induced with a pair of 100-Hz tetani does not require ERK activation in mice whereas it does in rats. However, in mice, inhibition of ERK activation blocked LTP induced by two LTP induction paradigms that mimicked the endogenous θ rhythm. In an additional series of studies, we found that mice specifically deficient in the ERK1 isoform of MAPK showed no impairments in tests of hippocampal physiology. To investigate ERK-dependent mechanisms operating during LTP-inducing stimulation paradigms, we monitored spike production in the cell body layer of the hippocampus during the period of θ-like LTP-inducing stimulation. θ-burst stimulation (TBS) produced a significant amount of postsynaptic spiking, and the likelihood of spike production increased progressively over the course of the three trains of TBS independent of any apparent increase in Excitatory Post-Synaptic Potential (EPSP) magnitude. Inhibition of ERK activation dampened this TBS-associated increase in spiking. These data indicate that, for specific patterns of stimulation, ERK may function in the regulation of neuronal excitability in hippocampal area CA1. Overall, our data indicate that the progressive increase in spiking observed during TBS represents a form of physiologic temporal integration that is dependent on ERK MAPK activity.
Audiospatial and Visuospatial Working Memory in 6–13 Year Old School Children
Authors: Vuontela Virve , Steenari Maija-Riikka , Carlson Synnöve , Koivisto Juha , Fjällberg Mika , Aronen Eeva T.
Published: January 2003

The neural processes subserving working memory, and brain structures underlying this system, continue to develop during childhood. We investigated the effects of age and gender on audiospatial and visuospatial working memory in a nonclinical sample of school-aged children using n-back tasks. The results showed that auditory and visual working memory performance improves with age, suggesting functional maturation of underlying cognitive processes and brain areas. The gender differences found in the performance of working memory tasks suggest a larger degree of immaturity in boys than girls at the age period of 6–10 yr. The differences observed between the mastering of auditory and visual working memory tasks may indicate that visual working memory reaches functional maturity earlier than the corresponding auditory system.
Conflicting Processes in the Extinction of Conditioned Taste Aversion: Behavioral and Molecular Aspects of Latency, Apparent Stagnation, and Spontaneous Recovery
Authors: Berman Diego E. , Hazvi Shoshi , Stehberg Jimmy , Bahar Amir , Dudai Yadin
Published: January 2003

The study of experimental extinction and of the spontaneous recovery of the extinguished memory could cast light on neurobiological mechanisms by which internal representations compete to control behavior. In this work, we use a combination of behavioral and molecular methods to dissect subprocesses of experimental extinction of conditioned taste aversion (CTA). Extinction of CTA becomes apparent only 90 min after the extinction trial. This latency is insensitive to muscarinic and β-adrenergic modulation and to protein synthesis inhibition in the insular cortex (IC). Immediately afterwards, however, the extinguishing trace becomes sensitive to β-adrenergic blockade and protein synthesis inhibition. The subsequent kinetics and magnitude of extinction depend on whether a spaced or massed extinction protocol is used. A massed protocol is highly effective in the short run, but results in apparent stagnation of extinction in the long-run, which conceals fast spontaneous recovery of the preextinguished trace. This recovery can be truncated by a β-adrenergic agonist or a cAMP analog in the insular cortex, suggesting that spontaneous overtaking of the behavioral control by the original association is regulated at least in part by β-adrenergic input, probably operating via the cAMP cascade, long after the offset of the conditioned stimulus. Hence, the performance of the subject in experimental extinction is the sum total of multiple, sometimes conflicting, time-dependent processes.
Size and Reversal Learning in the Beagle Dog as a Measure of Executive Function and Inhibitory Control in Aging
Authors: Tapp P. Dwight , Siwak Christina T. , Estrada Jimena , Head Elizabeth , Muggenburg Bruce A. , Cotman Carl W. , Milgram Norton W.
Published: January 2003

Several studies converge on the idea that executive processes age earlier than other cognitive processes. As part of a larger effort to investigate age-related changes in executive processes in the dog, inhibitory control was measured in young, middle-aged, old, and senior dogs using size discrimination learning and reversal procedures. Compared to young and middle-aged dogs, old and senior dogs were impaired on both the initial learning of the size task and the reversal of original reward contingencies. Impaired performance in the two aged groups was characterized as a delay in learning the correct stimulus-reward contingencies and, among the senior dogs in particular, an increase in perseverative responding. These separate patterns of reversal impairments in the old and senior dogs may reflect different rates of aging in subregions of the frontal cortex.
Mitral Cell β1 and 5-HT2A Receptor Colocalization and cAMP Coregulation: A New Model of Norepinephrine-Induced Learning in the Olfactory Bulb
Authors: Yuan Qi , Harley Carolyn W. , McLean John H.
Published: January 2003

In the present study we assess a new model for classical conditioning of odor preference learning in rat pups. In preference learning β1-adrenoceptors activated by the locus coeruleus mediate the unconditioned stimulus, whereas olfactory nerve input mediates the conditioned stimulus, odor. Serotonin (5-HT) depletion prevents odor learning, with 5-HT2A/2C agonists correcting the deficit. Our new model proposes that the interaction of noradrenergic and serotonergic input with odor occurs in the mitral cells of the olfactory bulb through activation of cyclic adenosine monophosphate (cAMP). Here, using selective antibodies and immunofluorescence examined with confocal microscopy, we demonstrate that β1-adrenoceptors and 5-HT2A receptors colocalize primarily on mitral cells. Using a cAMP assay and cAMP immunocytochemistry, we find that β-adrenoceptor activation by isoproterenol, at learning-effective and higher doses, significantly increases bulbar cAMP, as does stroking. As predicted by our model, the cAMP increases are localized to mitral cells. 5-HT depletion of the olfactory bulb does not affect basal levels of cAMP but prevents isoproterenol-induced cAMP elevation. These results support the model. We suggest the mitral-cell cAMP cascade converges with a Ca2+pathway activated by odor to recruit CREB phosphorylation and memory-associated changes in the olfactory bulb. The dose-related increase in cAMP with isoproterenol implies a critical cAMP window because the highest dose of isoproterenol does not produce learning.
Using an Aplysia Two-Hybrid System to Examine the Interactions Between Transcription Factors Involved in Long-Term Facilitation in the Nervous System of Aplysia
Authors: Choi Jung-Hwan , Lee Jin-A , Yim Seok-Won , Lim Chae-Seok , Lee Chi-Hoon , Lee Young-Don , Bartsch Dusan , Kandel Eric R. , Kaang Bong-Kiun
Published: January 2003

Interactions between ApCREB1a, ApCREB2, and ApC/EBP have been studied using conventional methods such as the yeast two-hybrid system. However, it is unclear whether these memory-related transcription factors actually interact in the native environment in neurons. To clarify this question, we have developed an Aplysia two-hybrid system and found, consistent with previous studies that ApCREB2 interacts with ApCREB1a and ApC/EBP, and that ApC/EBP forms homodimers. We have also found that ApCREB1a and ApC/EBP do not interact. Therefore, our study shows that formerly described interactions between the proteins actually occur in the Aplysia neurons and that interactions between these transcription factors are specific.
Foamy Virus Envelope Glycoprotein Is Sufficient for Particle Budding and Release
Authors: Shaw Kit L. , Lindemann Dirk , Mulligan Mark J. , Goepfert Paul A.
Published: January 2003

Foamy viruses (FVs) are classified in the family
Retroviridae
, but recent data have shown that they are not conventional retroviruses. Notably, several characteristics of their particle replication strategies are more similar to those of hepatitis B virus (HBV) than those of typical retroviruses. Compared to conventional retroviruses, which require only Gag proteins for budding and release of virus-like particles (VLPs), both FV and HBV require Env proteins. In the case of HBV, Env (S protein) alone is sufficient to form subviral particles (SVPs). Because FVs also depend on Env for budding, we tested whether FV Env alone could produce SVPs. The Env proteins of FV and murine leukemia virus (MuLV) were both released into cell culture supernatants and migrated into isopycnic gradients; however, unlike MuLV Env, FV Env displayed characteristics of SVPs. FV Env particles were of greater density than those of MuLV (1.11 versus 1.07 g/ml, respectively), which strongly suggested that the released proteins of FV Env were particulate. When we examined FV SVPs by immunoelectron microscopy, we found particles that were consistent in morphology, size, and staining with gold beads, similar to FV VLPs and unlike the particle-like structures of MuLV Env, which were more consistent with vesicles produced from nonspecific membrane “blebbing.” Taken together, our results demonstrated that FV Env alone is sufficient for particle budding. This finding is unique among retroviruses and further demonstrated the similarities between FV and HBV.
Identification of Small Molecule Compounds That Selectively Inhibit Varicella-Zoster Virus Replication
Authors: Visalli Robert J. , Fairhurst Jeanette , Srinivas Shamala , Hu William , Feld Boris , DiGrandi Martin , Curran Kevin , Ross Adma , Bloom Jonathan D. , van Zeijl Marja , Jones Thomas R. , O'Connell John , Cohen Jeffrey I.
Published: January 2003

A series of nonnucleoside,
N
-α-methylbenzyl-
N
′-arylthiourea analogs were identified which demonstrated selective activity against varicella-zoster virus (VZV) but were inactive against other human herpesviruses, including herpes simplex virus. Representative compounds had potent activity against VZV early-passage clinical isolates and an acyclovir-resistant isolate. Resistant viruses generated against one inhibitor were also resistant to other compounds in the series, suggesting that this group of related small molecules was acting on the same virus-specific target. Sequencing of the VZV ORF54 gene from two independently derived resistant viruses revealed mutations in ORF54 compared to the parental VZV strain Ellen sequence. Recombinant VZV in which the wild-type ORF54 sequence was replaced with the ORF54 gene from either of the resistant viruses became resistant to the series of inhibitor compounds. Treatment of VZV-infected cells with the inhibitor impaired morphogenesis of capsids. Inhibitor-treated cells lacked DNA-containing dense-core capsids in the nucleus, and only incomplete virions were present on the cell surface. These data suggest that the VZV-specific thiourea inhibitor series block virus replication by interfering with the function of the ORF54 protein and/or other proteins that interact with the ORF54 protein.
Distinct Modes of Regulation of Transcription of Hepatitis B Virus by the Nuclear Receptors HNF4α and COUP-TF1
Authors: Yu Xianming , Mertz Janet E.
Published: January 2003

To study the effects of the nuclear receptors (NRs) HNF4α and COUP-TF1 on the life cycle of hepatitis B virus (HBV), the human hepatoma cell line Huh7 was transiently cotransfected with plasmids containing the HBV genome and encoding these two NRs. Overexpression of HNF4α and COUP-TF1 led to a 9-fold increase and a 7- to 10-fold decrease, respectively, in viral DNA synthesis. These two NRs also exhibited distinct modes of regulation of viral transcription. Overexpression of HNF4α led to a more-than-10-fold increase in synthesis of the pregenomic RNA but to only a 2- to 3-fold increase in synthesis of the pre-C and S RNAs. Moreover, the NR response element within the pre-C promoter, NRRE
preC,
played the major role in activation of pregenomic RNA synthesis by HNF4α. On the other hand, overexpression of COUP-TF1 led to an over-10-fold repression of synthesis of both pre-C and pregenomic RNAs mediated through either NRRE
preC
or NRRE
enhI
. HNF4α and COUP-TF1 antagonized each other's effects on synthesis of pregenomic RNA and viral DNA when they were co-overexpressed. A naturally occurring HBV variant which allows for binding by HNF4α but not COUP-TF1 in its NRRE
preC
exhibited significantly higher levels of synthesis of pregenomic RNA and viral DNA than wild-type HBV in coexpression experiments. Last, deletion analysis revealed that non-NRRE sequences located within both the C and pre-S1 regions are also essential for maximum activation of the pregenomic promoter by HNF4α but not for repression by COUP-TF1. Thus, HNF4α and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes.
Reduction of Natural Adenovirus Tropism to the Liver by both Ablation of Fiber-Coxsackievirus and Adenovirus Receptor Interaction and Use of Replaceable Short Fiber
Authors: Nakamura Takafumi , Sato Kenzo , Hamada Hirofumi
Published: January 2003

The initial recognition and binding of adenovirus vector to the host cell surface is mediated by interaction between the adenovirus fiber knob protein and its receptor, the coxsackievirus and adenovirus receptor (CAR). This natural tropism of adenovirus vector needs to be ablated in order to achieve targeted gene transfer. To this end, we noted that adenovirus serotype 40 (Ad40) contains two distinct long and short fibers; the short fiber is unable to recognize CAR, while the long fiber binds CAR. We generated adenovirus serotype 5-based mutants with chimeric Ad40-derived fibers, which were composed of either long or short shafts together with CAR binding or nonbinding knobs. The capacity of these adenovirus mutants for in vitro and in vivo gene transfer to liver cells was examined. In the case of primary human hepatocytes displaying a high expression level of CAR and αv integrin, both CAR binding ability and fiber shaft length played important roles in efficient transduction. Most significantly, the high transduction efficiency observed in the liver and spleen following intravenous administration of adenovirus vector was dramatically reduced by both ablation of fiber-CAR interaction and the use of replaceable short fiber. In other tissues displaying a low level of transduction, no significant differences in transduction efficiency were observed among adenovirus vector mutants. Furthermore, incorporation of a 7-lysine-residue motif at the C-terminal end of CAR-nonbinding short fiber efficiently achieved transduction of target cells via the heparan-containing receptor. Our results demonstrated that the natural tropism of adenovirus in vivo is influenced not only by fiber-CAR interaction but also by fiber shaft length. Furthermore, our strategy may be useful for retargeting adenovirus to particular tumors and tissue types with specific receptors.
Phosphatidylinositol 3-Kinase Regulates Human Immunodeficiency Virus Type 1 Replication following Viral Entry in Primary CD4+T Lymphocytes and Macrophages
Authors: François Fleur , Klotman Mary E.
Published: January 2003

Human immunodeficiency virus (HIV) gp120 induces multiple cellular signaling pathways, including the phosphatidylinositol 3-kinase (PI3-kinase) pathway. The role of the PI3-kinase pathway in HIV-1 replication is not understood. Here we examined whether HIV-1 gp120 upregulates the PI3-kinase pathway and whether PI3-kinase activity plays a role in virus replication in primary human CD4+T cells and macrophages. Soluble and virion-associated HIV-1 gp120 induced calcium mobilization and phosphorylation of the PI3-kinase downstream effectors PKB/Akt and p70 S6 kinase. gp120-induced PI3-kinase activity and calcium mobilization were inhibited by pertussis toxin and blocking antibodies directed against CCR5 and CXCR4, suggesting that the signaling is mediated through the chemokine receptor. The PI3-kinase inhibitor LY294002 inhibited infection of CD4+T cells and macrophages with X4 and R5 HIV-1-pseudotyped viruses at concentrations that did not induce cell toxicity or downregulate HIV-1 coreceptor expression. When gp120-induced signaling was bypassed with the vesicular stomatitis virus G envelope protein, infection was still sensitive to PI3-kinase inhibition, suggesting that basal PI3-kinase activity is required for infection. LY294002 inhibited HIV-1 infection when added after viral entry and did not affect formation of the HIV-1 reverse transcriptase products R/U5 and long terminal repeat/Gag in the presence of the inhibitor. However, when the inhibitor was added after viral integration had occurred, no inhibition of HIV infection was observed. Our studies show that inhibition of the PI3-kinase signaling pathway suppresses virus infection post-viral entry and post-reverse transcription but prior to HIV gene expression. This type of host-virus interaction has implications for anti-HIV therapeutics that target cellular signaling machinery.
Pseudotyped Lentivirus Vectors Derived from Simian Immunodeficiency Virus SIVagm with Envelope Glycoproteins from Paramyxovirus
Authors: Kobayashi Masanori , Iida Akihiro , Ueda Yasuji , Hasegawa Mamoru
Published: January 2003

We describe the development of novel lentivirus vectors based on simian immunodeficiency virus from African green monkey (SIVagm) pseudotyped with Sendai virus (SeV) envelope glycoproteins. SeV fusion (F) and hemagglutinin-neuraminidase (HN) proteins were successfully incorporated into the SIVagm-based vector by truncation of the cytoplasmic tail of the F protein and by addition of the cytoplasmic tail of SIVagm transmembrane envelope protein to the N terminus of the HN protein. As with the vesicular stomatitis virus G glycoprotein-pseudotyped vector, the mutant SeV F- and HN-pseudotyped SIVagm vector was able to transduce various types of animal and human cell lines. Furthermore, the vector was able to transduce an enhanced green fluorescent protein reporter gene into polarized epithelial cells of rat trachea from the apical and basolateral sides. Therefore, SeV F- and HN-pseudotyped SIVagm vectors have considerable potential for effective use in gene therapy for various therapies, including respiratory diseases.
Kaposi's Sarcoma-Like Tumors in a Human Herpesvirus 8 ORF74 Transgenic Mouse
Authors: Guo Hong-Guang , Sadowska Mariola , Reid William , Tschachler Erwin , Hayward Gary , Reitz Marvin
Published: January 2003

The product of human herpesvirus 8 (HHV-8) open reading frame 74 (ORF74) is related structurally and functionally to cellular chemokine receptors. ORF74 activates several cellular signaling pathways in the absence of added ligands, and NIH 3T3 cells expressing ORF74 are tumorigenic in nude mice. We have generated a line of transgenic (Tg) mice with ORF74 driven by the simian virus 40 early promoter. A minority (approximately 30%) of the Tg mice, including the founder, developed tumors resembling Kaposi's sarcoma (KS) lesions, which occurred most typically on the tail or legs. The tumors were highly vascularized, had a spindle cell component, expressed VEGF-C mRNA, and contained a majority of CD31
+
cells. CD31 and VEGF-C are typically expressed in KS. Tumors generally (but not always) occurred at single sites and most were relatively indolent, although several mice developed large visceral tumors. ORF74 was expressed in a minority of cells in the Tg tumors and in a few other tissues of mice with tumors; mice without tumors did not express detectable ORF74 in any tissues tested. Cell lines established from tumors expressed ORF74 in a majority of cells, expressed VEGF-C mRNA, and were tumorigenic in nude mice. The resultant tumors grew rapidly, metastasized, and continued to express ORF74. Cell lines established from these secondary tumors also expressed ORF74 and were tumorigenic. These data strongly suggest that ORF74 plays a role in the pathology of KS and confirm and extend previous findings on the tumorigenic potential of ORF74.
Interaction of the Equine Herpesvirus 1 EICP0 Protein with the Immediate-Early (IE) Protein, TFIIB, and TBP May Mediate the Antagonism between the IE and EICP0 Proteins
Authors: Kim Seong K. , Jang Hyung K. , Albrecht Randy A. , Derbigny Wilbert A. , Zhang Yunfei , O'Callaghan Dennis J.
Published: January 2003

The equine herpesvirus 1 (EHV-1) immediate-early (IE) and EICP0 proteins are potent
trans
-activators of EHV-1 promoters; however, in transient-transfection assays, the IE protein inhibits the
trans-
activation function of the EICP0 protein. Assays with IE mutant proteins revealed that its DNA-binding domain, TFIIB-binding domain, and nuclear localization signal may be important for the antagonism between the IE and EICP0 proteins. In vitro interaction assays with the purified IE and EICP0 proteins indicated that these proteins interact directly. At late times postinfection, the IE and EICP0 proteins colocalized in the nuclei of infected equine cells. Transient-transfection assays showed that the EICP0 protein
trans
-activated EHV-1 promoters harboring only a minimal promoter region (TATA box and cap site), suggesting that the EICP0 protein
trans
-activates EHV-1 promoters by interactions with general transcription factor(s). In vitro interaction assays revealed that the EICP0 protein interacted directly with the basal transcription factors TFIIB and TBP and that the EICP0 protein (amino acids [aa] 143 to 278) mediated the interaction with aa 125 to 174 of TFIIB. Our unpublished data showed that the IE protein interacts with the same domain (aa 125 to 174) of TFIIB and with TBP. Taken together, these results suggested that interaction of the EICP0 protein with the IE protein, TFIIB, and TBP may mediate the antagonism between the IE and EICP0 proteins.
High Mutant Frequency in Populations of a DNA Virus Allows Evasion from Antibody Therapy in an Immunodeficient Host
Authors: López-Bueno Alberto , Mateu Mauricio G. , Almendral José M.
Published: January 2003

The degree of genetic heterogeneity of DNA virus populations in nature and its consequences for disease control are virtually unknown. The parvovirus minute virus of mice (MVMi) was used here to investigate (i) the frequency of antibody-escape mutants in populations of a DNA virus and (ii) the ability of a DNA virus to evade in the long-term a passive monoclonal antibody (MAb) therapy in an immunodeficient natural host. Independent clonal populations of MVMi harbored a high proportion of mutants resistant to neutralizing MAb (mutant frequency = [2.8 ± 0.5] × 10
−5
) that rapidly evolved under antibody pressure in culture to become mixtures dominated by genotypically diverse escape mutants. Immunodeficient mice naturally infected with clonal populations of MVMi and subsequently treated by intravenous injections of MAb were initially protected from the characteristic viral induced lethal leukopenia. However, some treated animals developed a delayed severe leukopenic syndrome associated with the emergence of genetically heterogeneous populations of MAb-resistant mutants in the MVMi main target organs. The 11 plaque-purified viruses analyzed from an antibody-resistant population obtained from one animal corresponded to four different mutant genotypes, although their consensus sequence remained wild type. All cloned escape mutants harbored single radical amino acid changes within a stretch of seven residues in a surface-exposed loop at the threefold axes of the capsid. This antigenic site, which can tolerate radical changes preserving MVMi pathogenic potential, may thereby allow the virus to evade the immune control. These findings indicate a high genetic heterogeneity and rapid adaptation of populations of a mammal DNA virus in vivo and provide a genetic basis for the failure of passive immunotherapy in the natural host.
Novel Gene Product of
Thogoto Virus
Segment 6 Codes for an Interferon Antagonist
Authors: Hagmaier Kathrin , Jennings Stephanie , Buse Johanna , Weber Friedemann , Kochs Georg
Published: January 2003

Thogoto virus
(THOV) is a tick-transmitted orthomyxovirus with a genome of six negative-stranded RNA segments. The sixth segment encodes two different transcripts: a spliced transcript that is translated into the matrix protein (M) and an unspliced transcript. Here, we report that the unspliced transcript encodes an elongated form of M named ML. A THOV isolate deficient in ML expression was an efficient interferon inducer, whereas ML-expressing wild-type strains were poor interferon inducers. These results were confirmed with recombinant THOVs rescued from cDNAs. Expression of ML efficiently suppressed activation of the beta interferon promoter by double-stranded RNA. These results indicate that ML is an accessory protein that functions as a potent interferon antagonist by blocking transcriptional activation of alpha/beta interferons.