bluuebunny/crossref_metadata_2025 huggingface dataset:
Effects of Sowing Treatment and Landscape Position on Establishment of the Perennial Tussock Grass Themeda triandra (Poaceae) in Degraded Eucalyptus Woodlands in Southeastern Australia
Authors: Cole Ian , Lunt Ian D. , Koen Terry
Published: August 2005

Grassy woodlands dominated by Eucalyptus species such as E. albens Benth., E. melliodora A. Cunn. ex Schauer, and E. microcarpa Maiden are poorly conserved in Australia, owing to widespread agricultural development. Understorey restoration is vitally important to enhance degraded remnants but no reliable techniques are available to restore herbaceous understorey species over large areas. Reestablishment of dominant native grasses such as Kangaroo grass (Themeda triandra Forssk.) is particularly important. This study compared Themeda establishment using a variety of sowing techniques across a range of landscape positions in degraded woodlands in central New South Wales. Four localities were sampled at three landscape positions (upper, mid‐, and lower slopes). Existing vegetative cover was sprayed with herbicide and removed by mowing. A randomized block experiment was established at each site with five replicates of five seedbed treatments: all four factorial combinations of soil disturbance (disturbed, nondisturbed) and weed control (atrazine, nonatrazine), plus topsoil scalping followed by soil disturbance. After 117 days, all plots were subject to a common herbicide (atrazine) treatment to control weeds. Themeda establishment was not significantly affected by landscape position, despite significant differences in the cover of exotic species among landscape positions. The scalped/disturbed treatment resulted in significantly better establishment at 400 days (18% or 3.9 plants/m2) than the control (8% or 1.8 plants/m2). All other treatments did not differ significantly from the control suggesting that seedbed treatments are not critical to successful Themeda establishment. These results suggest that Themeda swards can be reestablished in degraded woodlands relatively simply. The similarity in outcomes across all landscape positions suggests that general recommendations for “best‐bet” establishment methods may prove robust under a wide range of environmental and seasonal conditions.
The Cost of Firearm Deaths in the United States: Reduced Life Expectancies and Increased Insurance Costs
Authors: Lemaire Jean
Published: August 2005

Abstract

The United States remains far behind most other affluent countries in terms of life expectancy. One of the possible causes of this life expectancy gap is the widespread availability of firearms and the resulting high number of U.S. firearm fatalities: 10,801 homicides in 2000. The European Union experienced 1,260 homicides, Japan only 22. Using multiple decrement techniques, I show that firearm violence shortens the life of an average American by 104 days (151 days for white males, 362 days for black males). Among all fatal injuries, only motor vehicle accidents have a stronger effect. I estimate that the elimination of all firearm deaths in the United States would increase the male life expectancy more than the total eradication of all colon and prostate cancers. My results suggest that the insurance premium increases paid by Americans as a result of firearm violence are probably of the same order of magnitude as the total medical costs due to gunshots or the increased cost of administering the criminal justice system due to gun crime.
Defining International Entrepreneurship and Modeling the Speed of Internationalization
Authors: Oviatt Benjamin M. , McDougall Patricia P.
Published: August 2005

This article provides a reformulated definition of international entrepreneurship. Consistent with the new definition, a model is presented of how the speed of entrepreneurial internationalization is influenced by various forces. The model begins with an entrepreneurial opportunity and depicts the enabling forces of technology, the motivating forces of competition, the mediating perceptions of entrepreneurs, and the moderating forces of knowledge and networks that collectively determine the speed of internationalization.
The Emergence of Entrepreneurship Education: Development, Trends, and Challenges
Authors: Kuratko Donald F.
Published: August 2005

Entrepreneurship has emerged over the last two decades as arguably the most potent economic force the world has ever experienced. With that expansion has come a similar increase in the field of entrepreneurship education. The recent growth and development in the curricula and programs devoted to entrepreneurship and new–venture creation have been remarkable. The number of colleges and universities that offer courses related to entrepreneurship has grown from a handful in the 1970s to over 1,600 in 2005. In the midst of this huge expansion remains the challenge of complete academic legitimacy for entrepreneurship. While it can be argued that some legitimacy has been attained in the current state of entrepreneurship education, there are critical challenges that lie ahead. This article focuses on the trends and challenges in entrepreneurship education for the 21st century.
The Effects of Strategic Decision Making on Entrepreneurial Self–Efficacy
Authors: Forbes Daniel P.
Published: August 2005

This study builds on the construct of entrepreneurial self–efficacy (ESE), which measures the degree to which individuals believe they are capable of performing the tasks associated with new–venture management. I examined whether entrepreneurs’ levels of ESE are influenced by the ways in which their ventures make strategic decisions. Results show that entrepreneurs exhibit a stronger belief in their own abilities when their ventures make decisions in ways that involve other employees, that are more comprehensive, and that incorporate more current information. Exploratory analyses also provide preliminary evidence that ESE enhances firm performance.
Temperature‐dependent ovariole and testis maturation in the yellow dung fly
Authors: Blanckenhorn Wolf U. , Henseler Cornelia
Published: August 2005

Temperature is one of the abiotic environmental factors most strongly affecting animal behaviour, physiology, and life history. In insects, lower temperatures generally slow down most physiological processes, reducing growth rate and prolonging the juvenile period. Here, we investigate temperature‐dependent ovariole and testis maturation in the anautogenous yellow dung fly, Scathophaga stercoraria L. (Diptera: Scathophagidae), and relate it to corresponding temperature effects on pre‐adult development time and the adult pre‐reproductive period. Flies were reared in the laboratory at three constant temperatures (18, 22, and 26 °C), and the size of the developing ovarioles and testes (reflecting sperm production) was measured over time (i.e., age). Ovariole size increased asymptotically over the first 12 days of adult life, while the testes continued to fill after day 10. In accordance with the temperature‐size rule, warmer temperatures resulted in smaller ovarioles (eggs) and smaller testes, independent of body size. Warmer temperatures also greatly reduced pre‐adult development time by more than half, from 12 to 25 °C, the larger males always taking 1–3 days longer than the females. Corresponding temperature effects on the adult pre‐reproductive period were small (<1 day between 15 and 25 °C), with males taking 5–6 days and females 10–13 days to first reproduction. Time lost by males during the pre‐adult stage, when ovaries and testes are produced, can thus be more than compensated‐for by time gained during the pre‐reproductive period, when eggs and sperm are produced, so males can nevertheless start reproducing sooner than females.
Why Callosobruchus rhodesianus causes limited damage during storage of cowpea seeds in a tropical humid zone in Togo
Authors: Amevoin Komina , Glitho Isabelle Adolé , Monge Jean Paul , Huignard Jacques
Published: August 2005

The sympatric bruchids Callosobruchus rhodesianus (Pic) and Callosobruchus maculatus (F.) (Coleoptera: Bruchidae) colonize cultures of cowpea, Vigna unguiculata (Walp.) (Papilionaceae), in Togo at the end of the rainy season. Seeds containing larvae of the two bruchid species were introduced into the storage systems. Callosobruchus rhodesianus adults emerged from the seeds at the beginning of storage, but this species disappeared quickly and C. maculatus became dominant. Analysis of the reproduction of females collected at the beginning of storage made it possible to distinguish between four groups of females differing in their reproductive activity. Three groups were sexually active but they differed in their fecundity and the duration of the latency period before the first oviposition phase. The fourth group of females was in reproductive diapause. In interspecific competition, the presence of C. maculatus larvae reduced the survival chances of C. rhodesianus and therefore is disadvantageous for this species. The main factors explaining the rapid decrease of the C. rhodesianus populations under storage conditions were the variability of female reproductive activity with the emergence of insects in reproductive diapause and the significant larval mortality in interspecific competition with C. maculatus.
Testing the efficiency of three 15N‐labeled nitrogen compounds for indirect labeling of grasshoppers via plants in the field
Authors: Unsicker Sybille B. , Renker Carsten , Kahmen Ansgar , Spindler Sonja , Buchmann Nina , Weisser Wolfgang W.
Published: August 2005

In field studies of plant–insect herbivore interactions it is often difficult to establish which herbivore has fed on a particular plant. We investigated the suitability of three different 15N‐labeled nitrogen compounds (ammonium, nitrate, and glycine) for indirect marking of three grasshopper species [Omocestus viridulus (L.), Chorthippus parallelus (Zett.), and Chorthippus biguttulus (L.) (Orthoptera: Acrididae)] through labeling their food plants in the field. In two short‐term experiments grassland plots of 1 m2 were separately labeled with either one of the different nitrogen compounds. Grasshoppers were caged on three food‐plant species [Dactylis glomerata L., Holcus lanatus L. (Poaceae), and Trifolium repens L. (Fabaceae)] present in these plots for 72 h. Significantly enriched δ15N values in grasshoppers were found in all plant/grasshopper combinations. Enrichment in grasshoppers was positively correlated with the enrichment of plants and labeling with nitrate resulted in highest 15N enrichment. In a long‐term experiment, individuals of C. biguttulus were placed in a cage covering an area of 1 m2 for 37 days, with sampling of grasshoppers at regular intervals. δ15N values of the grasshopper and a common food plant, D. glomerata, increased steadily over time, up to 40‐fold by the end of the experiment. Our results demonstrate that 15N‐labeling of plants is an appropriate tool for the investigation of insect–plant interactions under natural conditions.
Spatial price efficiency in Mozambique's post‐reform maize markets
Authors: Tostão Emílio , Brorsen B. Wade
Published: August 2005

Maize (Zea mays) shortages and maize price instability continue to occur in Mozambique in spite of policy reforms and great capacity for increased production in northern Mozambique. This study measured the efficiency of spatial maize price arbitrage in Mozambique's post‐reform period. Spatial price inefficiency does not seem to explain why food shortages continue to occur in Mozambique, suggesting nonprice market constraints such as poor infrastructure and poor market institutions as more likely explanations. Baulch's spatial efficiency test indicated that it is not profitable to ship maize from northern surplus maize regions to southern Mozambique. Records of interregional maize shipments show that indeed grain is not being shipped from the north to the south. Market liberalization may have improved spatial price efficiency, but high transfer costs still limit trade and potential benefits from freeing markets. Under these circumstances, food shortages and price instability are likely to continue.
Agricultural exports and economic growth in less developed countries
Authors: Dawson P. J.
Published: August 2005

This article examines the contribution of agricultural exports to economic growth in less developed countries (LDCs). A sources‐of‐growth equation is developed from a dual economy model where agricultural and nonagricultural sectors are both divided into export and nonexport subsectors. This is then estimated using panel data for 62 LDCs for 1974–1995. Results provide evidence that there are significant structural differences in economic growth between low, lower‐middle, and upper‐income LDCs. Investment in the agricultural export subsector has a statistically identical impact on economic growth as investment in the nonagricultural export subsector. The marginal productivities in nonexport subsectors are over 30% lower than those in respective export subsectors. From a policy perspective, the results suggest that export‐promotion policies should be balanced.
Does individualization help productivity of transition agriculture?
Authors: Rizov Marian
Published: August 2005

There are large differences across transition countries with respect to agricultural‐sector performance and corresponding scope of farm restructuring and shift to individual farming. In this article we analyze the impact of individualization on productivity growth within an augmented neoclassical growth model framework. This approach allows us to circumvent criticisms on the grounds of lack of theoretical and objective criteria for inclusion of explanatory variables. Furthermore, in the empirical analysis using a panel data covering 15 transition countries over the period 1990–2001 and applying a generalized method of moments with instrumental variable estimator we are able to control for the impact of various factors and the potential endogeneity of variables. Our estimation results are robust and support the view that the shift to individual farming, as well as the overall economic reforms, has positively contributed to the productivity growth in agriculture during the first decade of transition.
Spot water markets and risk in water supply
Authors: Calatrava Javier , Garrido Alberto
Published: August 2005

Water availability patterns in semiarid regions are typically extremely variable. Even in basins with a highly developed infrastructure, users are subject to unreliable water supplies, incurring substantial economic losses during periods of scarcity. More flexible instruments, such as voluntary exchanges of water among users, can help users to reduce risk exposure. This article looks at the effects of spot water markets on the economic risk caused by water availability variations. Our theoretical and empirical risk analyses are based on the random profits of water users. Profit probability density functions are formally and graphically characterized for both water sellers and buyers under several possible market outcomes. We conclude from this analysis that, where water supply is stochastic, water markets unambiguously reduce both parties' risk exposure. The empirical study is conducted on an irrigation district in the Guadalquivir Valley (Southern Spain), where there is a high probability of periods of extreme water scarcity. Water demand functions for the district representative irrigators and a spatial equilibrium model are used to simulate market exchanges and equilibrium. This programming model is combined with statistical simulation techniques. We show that the profit probability distribution of a representative irrigator is modified if water exchanges are authorized, leading to risk reductions. Results also indicate that if the market were extended to several districts and users that are subject to varying hydrological risk exposure, extremely low‐profit events would be less likely to occur. In sum, we show that exchanging water in annual spot markets can reduce farmers' economic vulnerability caused by water supply variability across irrigation seasons. These results support the water policy reform carried out in Spain in 1999 to allow for voluntary water exchanges among right holders.
Treatment‐resistant depression: resistant to definition?
Authors: Malhi G. S. , Parker G. B. , Crawford J. , Wilhelm K. , Mitchell P. B.
Published: August 2005

Objective:  To better define treatment‐resistant depression (TRD) so as to assist clinical management and refine treatment guidelines.

Method:  In this study, we examine a broad range of clinical variables in depressed patients (n = 196) referred to a tertiary referral Mood Disorders Unit (MDU). Information was collected from patients, referrers and assessors over a period of 32 months and included evaluations of treatments, treatment resistance and related variables. Data were analysed across trichotomized ‘high’, ‘low’ and ‘no’ treatment resistance groupings of patients.

Results:  A significantly greater proportion of patients with melancholia were amongst the high TRD group, and this was consistent across different strategies for evaluating melancholia.

Conclusion:  Melancholia perhaps provides a prototypic TRD subset that perhaps reflects some innate aspects of melancholic depression or factors such as the impact of ageing. Research into TRD is needed to both replicate this finding and perhaps explicate it further.
Space, Place, and the Evidence Base: Part II—Rereading Nursing Environment Through Geographical Research
Authors: Andrews Gavin J. , Moon Graham
Published: August 2005

This, the second and final article in the short health geography series, articulates how, moving beyond the models and assumptions associated with the metaparadigm of Nursing Environment, as a focused subdisciplinary approach, health geography might provide unique insights into nursing. A case study of a fictional yet somewhat typical children's hospital is presented and demonstrates some wide‐ranging geographical issues and research questions (and hence potential geographical data) pertaining to nursing and the allied health professions. Indeed, this broad‐brush approach is purposeful to make as many connections as possible to readers with varied theoretical, methodological, empirical, and practice expertise. In addition to the case study, to indicate further how geographical inquiry might locate quite comfortably in nursing research, the article also makes some initial and tentative connections between geography and an established nursing framework for the uptake of research evidence for practice.

Although it is acknowledged that geographical inquiry should certainly never have the first call on researching the relationships between nurses and their environments, it is argued that its conceptual focus on space and place provides dedicated and detailed attention and a sound basis for a reformed, “spatialized” route to a more comprehensive understanding. Moreover, it is argued that it also demonstrates great versatility in terms of the scales and the subject matter with which it might engage. Some important issues certainly remain with respect to what might be the correct form of engagement between geographical and nursing research, but arguably, as a reformed disciplinary approach, health geography has the potential to provide a wealth of focused evidence for nursing practice.

Worldviews on Evidence‐Based Nursing 2005; 2(3):142‐156
Models to Determine First‐Order Rate Coefficients from Single‐Well Push‐Pull Tests
Authors: Schroth Martin H. , Istok Jonathan D.
Published: August 2005

Push‐pull tests (PPTs) have been successfully employed to quantify various microbially mediated processes in the subsurface. Current models for determining first‐order rate coefficients (k) from PPTs assume complete and instantaneous mixing of injected test solution in the portion of the aquifer investigated by the test, i.e., the system is treated like a well‐mixed reactor. Here we present two alternative models to estimate k that are based on different mixing assumptions, i.e., plug‐flow and variably mixed reactor models. Rate coefficients estimated by the models were compared using a sensitivity analysis and numerical simulations of PPTs. Results indicated that all models yielded reasonably accurate k estimates (errors < 13%), while best accuracy (errors < 1%) was obtained using the variably mixed reactor model. The well‐mixed reactor model generally overestimated true (simulation input) k values, whereas true k values were consistently underestimated by the plug‐flow reactor model. However, estimates of k obtained with the latter models bracketed true k values in all cases. As the variably mixed reactor model is more difficult to apply, we suggest using the well‐mixed and plug‐flow reactor models to obtain intervals for k estimates that will encompass true k values with high certainty. In an example application, we used all models to reanalyze a published PPT data set to obtain k estimates for nitrate consumption in a petroleum‐contaminated aquifer. Similar results were obtained for all three models (relative differences < 10% between k estimates), indicating that all three models are robust tools for estimating k values from PPT experimental data.
Traces of the secondary Geography curriculum
Authors: Chalmers Lex
Published: August 2005

Abstract:  Expressions of dissatisfaction about the post‐compulsory Geography curriculum in the early 1970s were unusually concerted, leading to the creation of a National Geography Curriculum Committee. This essay reviews this history and the resulting Syllabus for Schools: Geography Forms 5–7 as a prelude to a discussion of contemporary curriculum development. The essay argues that curriculum development from 1975 and the ‘education reforms’ of the late 1980s failed to produce conditions in which satisfactory outcomes for a Geography curriculum can be assured in 2005, and that a new and concerted period of participation in curriculum discussion is required. Some goals for this process are outlined in the final section of the paper.
Transformative tans? Gendered and raced bodies on beaches
Authors: Johnston Lynda
Published: August 2005

Abstract:  In this paper I examine the intentional act of transforming the colour of one's skin – sun‐tanning – by Pākehā at three New Zealand beaches: Papamoa, Mount Maunganui, and Takapuna. Recent debates about the construction of whiteness are outlined before discussing the historical significance of beaches and sun to New Zealand's leisure culture. This paper seeks to de‐naturalize whiteness through the disclosure of gendered leisure practices of tanning. Empirical material reveals that Pākehā, who actively seek brown skin, negotiate a number of racialized and gendered ‘truths’ as their sun‐tan transforms their bodies. I conclude by arguing that geographers need to be more attentive to the ways in which place constructs particular forms of gendered whiteness.
Changing bodies, spaces, places and politics: Feminist geography at the University of Waikato
Authors: Longhurst Robyn , Johnston Lynda
Published: August 2005

Abstract:  ‘Place matters’ in the production of knowledge. This article examines the production of feminist geographical knowledge at a particular place – the Department of Geography, Tourism and Environmental Planning at the University of Waikato. Feminist geography at Waikato represents a nexus of international, national, regional, and local politics. It represents the efforts, personalities, political and personal motivations of particular individuals, at particular times, in a particular space. While international trends are important, they can also mask the unfolding of events in different ways in ‘other’ places.
Māori women: Bodies, spaces, sacredness and mana
Authors: August Wikitoria
Published: August 2005

Abstract:  This article examines Māori women and the ways in which their bodies are constituted within particular cultural spaces, namely at urupā and sites where food is gathered. Māori bodily rituals, and the impacts of colonization on these bodily rituals, are explored to reveal a ‘nonwestern’ perspective on exclusion. Kaupapa Māori research and postcolonial theory have been combined to produce research that respects and nurtures Māori practices. Colonial notions of blood and menstruation, which inscribe women as ‘dirty’ and ‘unclean’, are critiqued. What is advocated is a Māori perspective that constructs Māori women as connected to Atua, and as powerful, sacred and life‐giving.
Recent changes to conservation of New Zealand's native biodiversity
Authors: Jay Mairi
Published: August 2005

Abstract:  This article examines recent changes to conservation in New Zealand. The argument is influenced by practical experience over the past 15 years, first as conservation planner employed by the Department of Conservation, then as an environmentalist involved in community conservation projects. The development of public‐private partnerships in conservation action over the past 15 years is reviewed. These changes point to the configuration of new landscapes, although the diverse and uncoordinated nature of many contemporary initiatives suggest that future biological communities will comprise a diversity of hybrid mixes from predominantly native to fully non‐native species.
Foreshore and seabed policy: A Māori perspective
Authors: Greensill Angeline
Published: August 2005

Abstract:  The comment provides a Māori perspective on the Foreshore and Seabed Act. In contrast to the western view, Māoris understand their relationship with the coast in ways that are holistic, intertwined and unbounded. The Act disrupts the wairua or spirit of place, impacting on relationships that have been nurtured by whānau and hapū over time.
Human gender differences in fibrinolytic responses to exercise training and their determinants
Authors: Kulaputana Onanong , Macko Richard F. , Ghiu Ioana , Phares Dana A. , Goldberg Andrew P. , Hagberg James M.
Published: August 2005

Endurance exercise training improves fibrinolysis, but this training‐induced adaptation may differ somewhat between men and women. We sought to determine whether the potential gender differences in training‐induced changes in selected fibrinolysis measures were related to changes in adiposity and/or plasma lipoprotein lipid levels. Seventeen men and 28 women, 50–75 years old, who were generally overweight to obese, were assessed for plasminogen activator inhibitor‐1 (PAI‐1) and tissue plasminogen activator (t‐PA) activity, t‐PA antigen and plasma lipoprotein‐lipid levels, and body composition before and after 6 months of endurance exercise training while on a low‐fat diet. At baseline, there were no differences in fibrinolytic measures between the men and women. Baseline levels of these fibrinolytic markers in both men and women were primarily related to other fibrinolytic measures and body composition, with a smaller contribution from plasma high‐density lipoprotein cholesterol (HDL‐C) levels. Exercise training reduced t‐PA antigen levels in both men and women, but the reduction was significantly greater in men (−1.6 ± 0.3 versus−0.5 ± 0.2 ng ml−1, P= 0.007). Exercise training decreased PAI‐1 activity more in men than in women (−2.6 ± 1.4 versus+0.9 ± 0.9 IU ml−1, P= 0.03). Men and women both showed increased t‐PA activity with exercise training to the same extent (+0.38 ± 0.12 versus+0.36 ± 0.24 U ml−1). The changes in fibrinolytic measures with exercise training in men and women were correlated with changes in other fibrinolytic measures, although in men abdominal fat changes were a strong predictor of fibrinolytic changes with training. These findings suggest that training‐induced improvements in endogenous fibrinolysis markers are somewhat greater in men compared to women and may be more strongly associated with abdominal obesity in men.
Neuron-to-Cell Spread of Pseudorabies Virus in a Compartmented Neuronal Culture System
Authors: Ch'ng T. H. , Enquist L.W.
Published: August 2005

Alphaherpesviruses are parasites of the peripheral nervous system in their natural hosts. After the initial infection of peripheral tissues such as mucosal cells, these neurotropic viruses will invade the peripheral nervous system that innervates the site of infection via long-distance axonal transport of the viral genome. In natural hosts, a latent and a nonproductive infection is usually established in the neuronal cell bodies. Upon reactivation, the newly replicated genome will be assembled into capsids and transported back to the site of entry, where a localized infection of the epithelial or mucosal cells will produce infectious virions that can infect naïve hosts. In this paper, we describe an in vitro method for studying neuron-to-cell spread of alphaherpesviruses using a compartmented culture system. Using pseudorabies virus as a model, we infected neuron cell bodies grown in Teflon chambers and observed spread of infection to nonneuronal cells plated in a different compartment. The cells are in contact with the neurons via axons that penetrate the Teflon barrier. We demonstrate that wild-type neuron-to-cell spread requires intact axons and the presence of gE, gI, and Us9 proteins, but does not require gD. We also provide ultrastructural evidence showing that capsids enclosed within vesicles can be found along the entire length of the axon during viral egress.
Biochemical and Immunogenic Characterization of Soluble Human Immunodeficiency Virus Type 1 Envelope Glycoprotein Trimers Expressed by Semliki Forest Virus
Authors: Forsell Mattias N. E. , Li Yuxing , Sundbäck Maria , Svehla Krisha , Liljeström Peter , Mascola John R. , Wyatt Richard , Hedestam Gunilla B. Karlsson
Published: August 2005

The current lack of envelope glycoprotein immunogens that elicit broadly neutralizing antibody responses remains a major challenge for human immunodeficiency virus type 1 (HIV-1) vaccine development. However, the recent design and construction of stable soluble gp140 trimers have shown that some neutralization breadth can be achieved by using immunogens that better mimic the functional viral spike complex. The use of genetic delivery systems to drive the in vivo expression of such immunogens for the stimulation of neutralizing antibodies against HIV-1 may offer advantages by maintaining the quaternary structure of the trimeric envelope glycoproteins. Here, we describe the biochemical and immunogenic properties of soluble HIV-1 envelope glycoprotein trimers expressed by recombinant Semliki Forest virus (rSFV). The results presented here demonstrate that rSFV supports the expression of stable soluble gp140 trimers that retain recognition by conformationally sensitive antibodies. Further, we show that rSFV particle immunizations efficiently primed immune responses as measured after a single boost with purified trimeric gp140 protein, resulting in a Th1-biased antibody response. This differed from the Th2-biased antibody response obtained after repeated immunizations with purified gp140 protein trimers. Despite this difference, both regimens stimulated neutralizing antibody responses of similar potency. This suggests that rSFV may be a useful component of a viral vector prime-protein boost regimen aimed at stimulating both cell-mediated immune responses and neutralizing antibodies against HIV-1.
Envelope Glycoprotein gB of Kaposi's Sarcoma-Associated Herpesvirus Is Essential for Egress from Infected Cells
Authors: Krishnan Harinivas H. , Sharma-Walia Neelam , Zeng Ling , Gao Shou-Jiang , Chandran Bala
Published: August 2005

Kaposi's sarcoma-associated herpesvirus (KSHV) envelope glycoprotein gB interacts with cell surface heparan sulfate (HS) and α3β1 integrin and plays roles in the initial binding and entry into the target cells and in the induction of preexisting host cell signal pathways. To define gB function further, using a bacterial artificial chromosome (BAC) system carrying the KSHV genome (BAC36wt-KSHV), we constructed a recombinant virus genome with the gB open reading frame (ORF) deleted by replacing a 2-kb gB ORF with a 1.3-kb Kan
r
gene. Stable 293T cells carrying BAC36wt-KSHV and ΔgBBAC36-KSHV genomes were generated. Transcript analyses and immunoprecipitation reactions confirmed the absence of gB in the 293T-ΔgBBAC36 cells. When monolayers of 293T-BAC36wt and 293T-ΔgBBAC36 cells were induced with tetradecanoylphorbol-13-acetate, infectious virus was detected only from the 293T-BAC36wt cell supernatants. No significant amount of DNase I-resistant viral DNA was detected in the supernatants of 293T-ΔgBBAC36 cells. BAC36wt-KSHV infected the target cells, and in contrast, no viral DNA and transcripts could be detected in cells infected with ΔgBBAC36-KSHV. Electron microscopy of 293T-ΔgBBAC36 cells revealed capsids in the nuclei, cytoplasmic vesicles with core-containing capsids, and occasional enveloped virions in the cytoplasm. However, enveloped virus particles were observed in the extracellular compartments of 293T-BAC36wt cells only and not in 293T-ΔgBBAC36 cells. Transfection of 293T-ΔgBBAC36 cells with plasmid expressing full-length gB restored the recovery of infectious KSHV in the supernatant. These results suggest that, besides its role in virus binding and entry into the target cells, KSHV gB also plays a role in the maturation and egress of virus from the infected cells.
A Capsid-Modified Helper-Dependent Adenovirus Vector Containing the β-Globin Locus Control Region Displays a Nonrandom Integration Pattern and Allows Stable, Erythroid-Specific Gene Expression
Authors: Wang Hongjie , Shayakhmetov Dmitry M. , Leege Tobias , Harkey Michael , Li Qiliang , Papayannopoulou Thalia , Stamatoyannopolous George , Lieber André
Published: August 2005

Gene therapy for hemoglobinopathies requires efficient gene transfer into hematopoietic stem cells and high-level erythroid-specific gene expression. Toward this goal, we constructed a helper-dependent adenovirus vector carrying the β-globin locus control region (LCR) to drive green fluorescent protein (GFP) expression, whereby the LCR-GFP cassette is flanked by adeno-associated virus (AAV) inverted terminal repeats (Ad.LCR-β-GFP). This vector possesses the adenovirus type 35 fiber knob that allows efficient infection of hematopoietic cells. Transduction and vector integration studies were performed in MO7e cells, a growth factor-dependent CD34
+
erythroleukemic cell line, and in cord blood-derived human CD34
+
cells. Stable transduction of MO7e cells with Ad.LCR-β-GFP was more efficient and less subject to position effects and silencing than transduction with a vector that did not contain the β-globin LCR. Analysis of integration sites indicated that Ad.LCR-β-GFP integration in MO7e cells was not random but tethered to chromosome 11, specifically to the globin LCR. More than 10% of analyzed integration sites were within the chromosomal β-globin LCR. None of the Ad.LCR-β-GFP integrations occurred in exons. The integration pattern of a helper-dependent vector that contained X-chromosomal stuffer DNA was different from that of the β-globin LCR-containing vector. Infection of primary CD34
+
cells with Ad.LCR-β-GFP did not affect the clonogenic capacity of CD34
+
cells. Transduction of CD34
+
cells with Ad.LCR-β-GFP resulted in vector integration and erythroid lineage-specific GFP expression.
Dengue Virus Nonstructural Protein NS5 Induces Interleukin-8 Transcription and Secretion
Authors: Medin Carey L. , Fitzgerald Katherine A. , Rothman Alan L.
Published: August 2005

Elevated circulating levels of chemokines have been reported in patients with dengue fever and are proposed to contribute to the pathogenesis of dengue disease. To establish in vitro models for chemokine induction by dengue 2 virus (DEN2V), we studied a variety of human cell lines and primary cells. DEN2V infection of HepG2 and primary dendritic cells induced the production of interleukin-8 (IL-8), RANTES, MIP-1α, and MIP-1β, whereas only IL-8 and RANTES were induced following dengue virus infection of HEK293 cells. Chemokine secretion was accompanied by an increase in steady-state mRNA levels. No chemokine induction was observed in HEK293 cells treated with poly(I:C) or alpha interferon, suggesting a direct effect of virus infection. To determine the mechanism(s) involved in the induction of chemokine production by DEN2V, individual dengue virus genes were cloned into plasmids and expressed in HEK293 cells. Transfection of a plasmid expressing NS5 or a dengue virus replicon induced IL-8 gene expression and secretion. RANTES expression was not induced under these conditions, however. Reporter assays showed that IL-8 induction by NS5 was principally through CAAT/enhancer binding protein, whereas DEN2V infection also induced NF-κB. These results indicate a role for the dengue virus NS5 protein in the induction of IL-8 by DEN2V infection. Recruitment and activation of potential target cells to sites of DEN2V replication by virus-induced chemokine production may contribute to viral replication as well as to the inflammatory components of dengue virus disease.
Dipyridamole Reversibly Inhibits Mengovirus RNA Replication
Authors: Fata-Hartley Cori L. , Palmenberg Ann C.
Published: August 2005

Dipyridamole is an effective inhibitor of cardiovirus growth in cell culture. The effects of dipyridamole on mengovirus replication in vivo and in vitro were examined in the hope the drug could be used as an experimental analog of the poliovirus inhibitor guanidine. Guanidine selectively inhibits poliovirus RNA synthesis but not RNA translation, and as such, has been a valuable research tool. Although guanidine does not inhibit cardiovirus infection, a compound with similar discriminatory characteristics would be experimentally useful for parallel work with these viruses. We found that mengovirus plaque formation in HeLa or L cells was inhibited nearly 100% by the presence of 80 μM dipyridamole. The inhibitory effect was reversible and targeted an early step in the replication cycle. Studies with luciferase-expressing mengovirus replicons showed that viral protein synthesis was unaffected by dipyridamole, and rather, RNA synthesis was the step targeted by the drug. This assessment was confirmed by direct analyses of viral translation and RNA synthesis activities in a Krebs-2-derived in vitro system that supported complete, infectious cardiovirus replication. In Krebs extracts, dipyridamole specifically inhibited viral RNA synthesis to more than 95%, with no concomitant effect on viral protein translation or polyprotein processing. The observed inhibition reversibly affected an early step in both minus-strand and plus-strand RNA synthesis, although inhibition of plus-strand synthesis was more profound than that of minus-strand synthesis. We conclude that dipyridamole is a potent experimental tool that readily distinguishes between cardiovirus translation and RNA replication functions.
RNA Interference-Mediated Virus Clearance from Cells both Acutely and Chronically Infected with the Prototypic Arenavirus Lymphocytic Choriomeningitis Virus
Authors: Sánchez Ana B. , Perez Mar , Cornu Tatjana , de la Torre Juan Carlos
Published: August 2005

Several arenaviruses, including Lassa fever virus, cause severe, often lethal hemorrhagic fever in humans. No licensed vaccines are available in the United States, and currently there is no efficacious therapy to treat this viral infection. Therefore the importance of developing effective antiviral approaches to combat pathogenic arenaviruses is clear. Moreover, the prototypic arenavirus lymphocytic choriomeningitis virus (LCMV) is an important model for the study of viral persistence and associated diseases, as well as for exploring therapies to treat viral chronic infections. The use of small interfering RNAs (siRNAs) to downregulate gene expression via RNA interference (RNAi) has emerged as a powerful genetic tool for the study of gene function. In addition, the successful use of siRNAs to target a variety of animal viruses has led us to consider RNAi as a potential novel antiviral strategy. We have investigated the use of RNAi therapy against LCMV. Here, we show that siRNAs targeting sequences within the viral L polymerase and Z mRNAs inhibit LCMV multiplication in cultured cells. Unexpectedly, the antiviral efficacy of RNAi-based therapy against LCMV was highly dependent on the method used to deliver effector siRNA molecules. Thus, transfection of chemically synthesized siRNA pools to L and Z was ineffective in preventing virus multiplication. In contrast, targeting of the same viral L and Z gene products with siRNAs produced inside cells using a replication-deficient recombinant adenovirus expression system inhibited LCMV multiplication very efficiently. Notably, transduction with the replication-deficient recombinant adenovirus expression system to Z and L effectively cured persistently LCMV-infected cells, suggesting the feasibility of using RNAi therapy to combat viral chronic infections by riboviruses.
Monoclonal Antibody AP33 Defines a Broadly Neutralizing Epitope on the Hepatitis C Virus E2 Envelope Glycoprotein
Authors: Owsianka Ania , Tarr Alexander W. , Juttla Vicky S. , Lavillette Dimitri , Bartosch Birke , Cosset François-Loïc , Ball Jonathan K. , Patel Arvind H.
Published: August 2005

Hepatitis C virus (HCV) remains a significant threat to the general health of the world's population, and there is a pressing need for the development of new treatments and preventative vaccines. Here, we describe the generation of retrovirus-based pseudoparticles (HCVpp) incorporating a panel of full-length E1E2 clones representative of the major genotypes 1 through 6, and their application to assess the reactivity and neutralizing capability of antisera and monoclonal antibodies raised against portions of the HCV E2 envelope protein. Rabbit antisera raised against either the first hypervariable region or ectodomain of E2 showed limited and strain specific neutralization. By contrast, the monoclonal antibody (MAb) AP33 demonstrated potent neutralization of infectivity against HCVpp carrying E1E2 representative of all genotypes tested. The concentration of AP33 required to achieve 50% inhibition of infection by HCVpp of diverse genotypes ranged from 0.6 to 32 μg/ml. The epitope recognized by MAb AP33 is linear and highly conserved across different genotypes of HCV. Thus, identification of a broadly neutralizing antibody that recognizes a linear epitope is likely to be of significant benefit to future vaccine and therapeutic antibody development.
Genetic and Molecular In Vivo Analysis of Herpes Simplex Virus Assembly in Murine Visual System Neurons
Authors: LaVail Jennifer H. , Tauscher Andrew N. , Hicks James W. , Harrabi Ons , Melroe Gregory T. , Knipe David M.
Published: August 2005

Herpes simplex virus (HSV) infects both epithelial cells and neuronal cells of the human host. Although HSV assembly has been studied extensively for cultured epithelial and neuronal cells, cultured neurons are biochemically, physiologically, and anatomically significantly different than mature neurons in vivo. Therefore, it is imperative that viral maturation and assembly be studied in vivo. To study viral assembly in vivo, we inoculated wild-type and replication-defective viruses into the posterior chamber of mouse eyes and followed infection in retinal ganglion cell bodies and axons. We used PCR techniques to detect viral DNA and RNA and electron microscopy immunohistochemistry and Western blotting to detect viral proteins in specific portions of the optic tract. This approach has shown that viral DNA replication is necessary for viral DNA movement into axons. Movement of viral DNA along ganglion cell axons occurs within capsid-like structures at the speed of fast axonal transport. These studies show that the combined use of intravitreal injections of replication-defective viruses and molecular probes allows the genetic analysis of essential viral replication and maturation processes in neurons in vivo. The studies also provide novel direct evidence for the axonal transport of viral DNA and support for the subassembly hypothesis of viral maturation in situ.
Differential Pathogenesis of Primary CCR5-Using Human Immunodeficiency Virus Type 1 Isolates in Ex Vivo Human Lymphoid Tissue
Authors: Karlsson Ingrid , Grivel Jean-Charles , Chen Silvia Sihui , Karlsson Anders , Albert Jan , Fenyö Eva Maria , Margolis Leonid B.
Published: August 2005

In the course of human immunodeficiency virus (HIV) disease, CCR5-utilizing HIV type 1 (HIV-1) variants (R5), which typically transmit infection and dominate its early stages, persist in approximately half of the infected individuals (nonswitch virus patients), while in the other half (switch virus patients), viruses using CXCR4 (X4 or R5X4) emerge, leading to rapid disease progression. Here, we used a system of ex vivo tonsillar tissue to compare the pathogeneses of sequential primary R5 HIV-1 isolates from patients in these two categories. The absolute replicative capacities of HIV-1 isolates seemed to be controlled by tissue factors. In contrast, the replication level hierarchy among sequential isolates and the levels of CCR5+CD4+T-cell depletion caused by the R5 isolates seemed to be controlled by viral factors. R5 viruses isolated from nonswitch virus patients depleted more target cells than R5 viruses isolated from switch virus patients. The high depletion of CCR5+cells by HIV-1 isolates from nonswitch virus patients may explain the steady decline of CD4+T cells in patients with continuous dominance of R5 HIV-1. The level of R5 pathogenicity, as measured in ex vivo lymphoid tissue, may have a predictive value reflecting whether, in an infected individual, X4 HIV-1 will eventually dominate.
The Nonnucleoside Reverse Transcriptase Inhibitor UC-781 Inhibits Human Immunodeficiency Virus Type 1 Infection of Human Cervical Tissue and Dissemination by Migratory Cells
Authors: Fletcher Patricia , Kiselyeva Yana , Wallace Greg , Romano Joseph , Griffin George , Margolis Leonid , Shattock Robin
Published: August 2005

Heterosexual transmission of human immunodeficiency virus remains the major route of transmission worldwide; thus, there is an urgent need for additional prevention strategies, particularly those that could be controlled by women. Using cellular and tissue explant models, we have evaluated the potential activity of thiocarboxanilide nonnucleoside analogue reverse transcriptase inhibitor UC-781 as a vaginal microbicide. We were able to demonstrate a potent dose-dependent effect against R5 and X4 infections of T cells. In human cervical explant cultures, UC-781 was not only able to inhibit direct infection of mucosal tissue but was able to prevent dissemination of virus by migratory cells. UC-781 formulated into a carbopol gel (0.1%) retained significant activity against both direct tissue infection and transinfection mediated by migratory cells. Furthermore, UC-781 demonstrated prolonged inhibitory effects able to prevent both localized and disseminated infections up to 6 days post compound treatment. Additional studies were carried out to determine the concentration of compound that might be required to block a primary infection within draining lymph nodes. While a greater dose of compound was required to inhibit both X4 and R5 infections of lymphoid versus cervical explants, this was equivalent to a 1:3,000 dilution of the 0.1% formulation. Furthermore, a 2-h exposure to the compound prevented infection of lymphoid tissue when challenged up to 2 days later. The prolonged protection observed following pretreatment of both genital and lymphoid tissues with UC-781 suggests that this class of inhibitors may have unique advantages over other classes of potential microbicide candidates.