bluuebunny/crossref_metadata_2025 huggingface dataset:
Assessing Drinking Outcomes in Alcohol Treatment Efficacy Studies: Selecting a Yardstick of Success
Authors: Sobell Linda Carter , Sobell Mark B. , Connors Gerard J. , Agrawal Sangeeta
Published: November 2003

Background: Although the number of alcohol treatment efficacy trials has mushroomed, there is no consensus on how best to measure outcomes. To advance the goal of establishing cross‐trial consistency in measuring outcomes in clinical efficacy studies, the National Institute on Alcohol Abuse and Alcoholism convened a panel of experts and charged them with exploring, debating, and, ultimately, selecting a “sentinel” or “optimal” outcome measure to be used in future alcohol treatment studies. The goal of this article, one in a series of several presented at the National Institute on Alcohol Abuse and Alcoholism conference, is to discuss (1) the rationale underlying selection of an optimal outcome measure, (2) the necessary characteristics of an optimal outcome measure, (3) the utility of selecting an optimal measure, and (4) which drinking assessment methods could be used to collect data to portray the optimal outcome measure.

Methods: At a minimum, the criteria for an “optimal” measure include that it be psychometrically sound. In addition, it should have considerable currency in the field, thereby increasing its prospects for adoption. The measure should also be consistent with the concepts of greatest interest and relevance to the field (e.g., directly reflect the fundamental goal of alcohol treatment). In light of these highly desired features, percent of days heavy drinking was chosen at the conference as a practical and relevant measure of alcohol treatment outcome.

Conclusions: Percent of days heavy drinking should be the optimal measure of alcohol treatment outcome. Currently, daily drinking estimation methods are the most useful for gathering data that can reflect the optimal measure. In addition, data gathered by daily drinking estimation methods can be used to study a variety of other outcome variables of interest to clinical researchers.
Clinical and Methodological Utility of a Composite Outcome Measure for Alcohol Treatment Research
Authors: Zweben Allen , Cisler Ron A.
Published: November 2003

Background: How to capture different response patterns resulting from alcohol treatment has been a troublesome issue for alcohol researchers. A composite measure is one approach to capturing multiple treatment outcomes among diverse client populations. This article provides the rationale, development, and work conducted thus far on the composite outcome index and discusses the clinical utility of the measure.

Methods: With the use of Project MATCH data, the composite outcome measure was examined in relation to self‐reported alcohol consumption and alcohol problems and biological markers as well as in relation to other areas of functioning, such as psychiatric dysfunction and quality of life. Also, for assessing the stability of the measure, different composite outcomes statuses were assessed over time.

Results: Individuals with better scores on composite outcome index had fewer percent days abstinent, consumed more alcohol when they drank, had a greater number of alcohol problems, and evidenced higher blood levels as measured by γ‐glutamyltranspeptidase and carbohydrate‐deficient transferrin. Individuals with poorer composite outcome statuses had poorer social and behavioral role functioning and mental and physical health–related quality of life. Findings on short‐ and long‐term composite outcome statuses revealed that only a minority of clients (30%) sustained a remitted status (i.e., abstinent or moderate drinking without problems) over the 1‐year follow‐up, and fully 70% of the clients had reached a nonremitted status (i.e., heavy drinking and/or problems) on the composite measure at one or more time points during the 12‐month follow‐up.

Conclusions: The composite outcome index could be used usefully along with singular measures of consumption to obtain a more complete picture of what has occurred among clients posttreatment. Future work will involve the testing of the composite outcome index with other client populations and/or different treatments to further our understanding of the varying response patterns found among alcohol‐dependent clients.
Distinct Regions of the slo Subunit Determine Differential BKCa Channel Responses to Ethanol
Authors: Liu Pengchong , Liu Jianxi , Huang Weihua , Li Ming D. , Dopico Alejandro M.
Published: November 2003

Background: Ethanol at clinically relevant concentrations increases BKCa channel activity in dorsal root ganglia neurons, GH3 cells, and neurohypophysial terminals, leading to decreases in cell excitability and peptide release. In contrast, ethanol inhibits BKCa channels from aortic myocytes, which likely contributes to alcohol‐induced aortic constriction. The mechanisms that determine differential BKCa channel responses to ethanol are unknown. We hypothesized that nonconserved regions in the BKCa channel‐forming subunit (slo) are major contributors to the differential alcohol responses of different BKCa channel phenotypes.

Methods: We constructed chimeras by interchanging the core and the tail domains of two BKCa channel‐forming subunits (mslo and bslo) that, after expression, differentially respond to ethanol (activation and inhibition, respectively), and studied ethanol action on these mbslo and bmslo chimeric channels using single‐channel, patch‐clamp techniques.

Results and Conclusion: Data from cell‐free membranes patches demonstrate that the activity of channels that share a mslo‐type core‐linker (wt mslo and the mbslo chimera) is consistently and significantly potentiated by acute exposure to ethanol. Thus, a mslo tail is not necessary for ethanol potentiation of slo channels. In contrast, the activity of channels that share a bslo‐type core‐linker (wt bslo and the bmslo chimera) display heterogenous responses to ethanol: inhibition (in the majority of cases), refractoriness, or activation. Overall, our data indicate that the slo core‐linker is a critical region likely contributing to the differential responses of BKCa channels to ethanol.
Creating internal Web tools for collection development
Authors: Level Allison , Myers Sarah
Published: November 2003

Collection development activities are an important and ongoing component of every library. The historically paper‐driven environment of this work is changing. With intranets and Web authoring tools, digitizing your collection development workflow enables libraries to have an increased level of efficiency and accessibility. This article outlines the creation, development, and implementation of Web accessible collection development tools. These tools integrate procedures, forms, policies, and library organization Web sites. Information about project revisions, modifications, and suggestions for future projects is included. A coordinated collection development Web site may consist of online tools, Web resources, work‐flow processes, instructions and other information for selectors and library staff, as well as information for library patrons.
American University of Sharjah Library: a collection development project
Authors: Gyeszly Suzanne D. , Ismail Matthew
Published: November 2003

The American University of Sharjah (AUS) in the United Arab Emirates was established in 1997. The university’s international curriculum and the library holdings have changed with the growth of the student enrollment. Assessment of the library collection was, therefore, necessary to maintain the quality of the materials. The objectives of the assessment were to support the international mission and curriculum of the university and to identify the unique materials to the region and gaps in the collection. Collection development policies were created that were unique to the Gulf region. In addition, the international faculty responded to a collection development survey and participated in a serials evaluation project. As a result of the assessment, an intensive collection development program was undertaken, the approval plan extended, and thousands of new monographs ordered. The project became a model for other newly developed libraries in the Gulf region.
Course‐centered approach to evaluating university library collections for instructional program reviews
Authors: Pancheshnikov Yelena
Published: November 2003

Describes a course‐centered approach to the evaluation of university library collections for instructional program reviews. Summarizes the author’s experience with preparing library reports for systematic program reviews in the agricultural sciences at the University of Saskatchewan. The method is based on the assessment of collections supporting individual courses by comparing the numbers of monographs and journal articles available directly to the library clientele with the numbers of items in the major bibliographic databases. Describes the new elements of the method. The method provides a practical tool for further selection of materials, applicable in similar situations, as well as a background for collaborating with faculty members.
Internet resources on Asian studies: a guide to the best sites of 2003
Authors: Chao Sheau‐yueh J. , Chang Ching
Published: November 2003

The Internet and World Wide Web offer a rapidly increasing quantity of valuable resources on Asia‐specific information. In view of the vast scope of the Asian countries and the fast proliferation of good sites, this article offers only a sampling of valuable Internet resources as starting points for further exploration. It covers meta sites, Asian search engines, library resource pages, and electronic journals and newspapers. The first part of this paper includes the Internet sites of Asian studies, the second part contains selected East Asian country resources from China, Hong Kong, Japan, Korea and Taiwan, and the third part presents the leading Asian electronic journals and newspapers. Preference was given to comprehensive sites on countries or regions that have been the focus of recent academic study and research. All the sources are in English and some of them contain bilingual or multilingual versions.
Six Sigma in health care
Authors: Sehwail Loay , DeYong Camille
Published: November 2003

Six Sigma, originally initiated by Motorola and General Electric, offers a process improvement strategy that has been successfully applied in manufacturing. Service firms have recently trended toward Six Sigma methodology for internal processes and consumer centric. The paper discusses implementing Six Sigma in the health‐care sector. The paper introduces the challenges that face implementing Six Sigma in services, with a special focus on the health‐care industry, and the application of Six Sigma in health care. A case study about Mount Carmel Health System’s successful implementation of Six Sigma is presented. Finally, the paper concludes with the potential of Six Sigma in health care.
Leadership development through action learning: an executive monograph
Authors: Bowerman Jennifer K.
Published: November 2003

This article examines a leadership development program based on action learning principles, in a quasi government health insurance organization, undergoing transformation to a more private and entrepreneurial format and attempting to shake some of the more bureaucratic shackles of the past. The purpose of the program was to develop leadership and build business literacy by bringing managers and supervisors together from across the organization to work on real business projects through an in house action learning program. Using reflective essays written by participants and material provided from interviews some two years after the program ended, the paper examines some of the key leadership themes that emerged. These include an important distinction between leadership of organizations and leadership in organizations and the relationship between the two; the need for managers to understand emergent strategy; the value of a marketing approach for building support around strategic issues, the importance of critical thinking through the interplay of theory and practice; and a different paradigm for understanding learning.
The Eukaryotic Translation Initiation Factor 4GI Is Cleaved by Different Retroviral Proteases
Authors: Álvarez Enrique , Menéndez-Arias Luis , Carrasco Luis
Published: November 2003

The initiation factor eIF4G plays a central role in the regulation of translation. In picornaviruses, as well as in human immunodeficiency virus type 1 (HIV-1), cleavage of eIF4G by the viral protease leads to inhibition of protein synthesis directed by capped cellular mRNAs. In the present work, cleavage of both eIF4GI and eIF4GII has been analyzed by employing the proteases encoded within the genomes of several members of the family
Retroviridae
, e.g., Moloney murine leukemia virus (MoMLV), mouse mammary tumor virus, human T-cell leukemia virus type 1, HIV-2, and simian immunodeficiency virus. All of the retroviral proteases examined were able to cleave the initiation factor eIF4GI both in intact cells and in cell-free systems, albeit with different efficiencies. The eIF4GI hydrolysis patterns obtained with HIV-1 and HIV-2 proteases were very similar to each other but rather different from those obtained with MoMLV protease. Both eIF4GI and eIF4GII were cleaved very efficiently by the MoMLV protease. However, eIF4GII was a poor substrate for HIV proteases. Proteolytic cleavage of eIF4G led to a profound inhibition of cap-dependent translation, while protein synthesis driven by mRNAs containing internal ribosome entry site elements remained unaffected or was even stimulated in transfected cells.
Transgenic Mouse with the Herpes Simplex Virus Type 1 Latency-Associated Gene: Expression and Function of the Transgene
Authors: Mador Nurith , Braun Efrat , Haim Hillel , Ariel Ilana , Panet Amos , Steiner Israel
Published: November 2003

During herpes simplex virus type 1 (HSV-1) latent infection in human peripheral sensory ganglia, the major viral gene transcribed is the latency-associated transcript (LAT) gene. In order to facilitate the study of this gene, we generated a transgenic mouse that contains the DNA fragment that transcribes the LAT RNAs (2.0 kb and its 1.5-kb spliced transcript) under control of the cytomegalovirus promoter. The tissue distribution of these transcripts and their effects upon HSV-1 replication, latency, and reactivation in the transgenic-mouse model were examined. Different steady-state amounts of both transcripts were found in various tissues. While the highest levels of the 2.0-kb RNA were detected in heart and skeletal muscle, the 1.5-kb transcript was found at elevated levels in the brain and at much higher levels in the trigeminal ganglia (TG). Replication of both the wild-type and a LAT-negative mutant virus was suppressed in primary embryonic fibroblasts obtained from LAT-expressing transgenic mice compared to that in cells obtained from normal mice. HSV-1 DNA amounts in latently infected TG of transgenic mice were similar to those in normal mice. Reactivation of latent HSV-1 LAT-negative mutants by explant cocultivation of TG from transgenic mice was more efficient than reactivation from normal-mouse TG. Considering our present and previous results, we propose that the significantly higher steady-state level of the 1.5-kb RNA in the TG may link this transcript to latency functions and that by inhibition of virus replication, the LAT gene may protect ganglion cells and thereby increase the probability of reactivation.
Methylation Patterns of Papillomavirus DNA, Its Influence on E2 Function, and Implications in Viral Infection
Authors: Kim Kitai , Garner-Hamrick Peggy A. , Fisher Chris , Lee Denis , Lambert Paul F.
Published: November 2003

The biological activities of the papillomavirus E2 protein in transcription, replication, and maintenance of the papillomavirus genome rely on the E2 protein's ability to bind that genome specifically. The E2 binding sites (E2BSs), located within the long control region (LCR) of human papillomavirus (HPV) genomes, contain potential sites for 5′methylation at cytosine (CpG) residues. The E2 protein's capacity to bind E2BS in vitro is inhibited by methylation of these cytosines (59). Herein, we describe experiments to assess the influence of methylation on E2 function in cells. E2's ability to activate transcription was inhibited by the global methylation of CpG dinucleotides in E2-responsive transcriptional templates or when only the CpG dinucleotides within the E2BSs of a transcriptional template were methylated. Thus at least one biological activity of E2 that is dependent on its ability to bind DNA in a site-specific manner is influenced by the methylation status of its cognate binding site. The activity of DNA methylases is influenced by the differentiation status of mammalian cells. The life cycle of HPVs is tied to the differentiation of its host cells within stratified squamous epithelia. To investigate whether methylation of the papillomavirus genomes is influenced by the differentiation status of host epithelial cells, we analyzed HPV16 DNA harvested from a cervical epithelial cell line that was isolated from an HPV16-infected patient. We found, using bisulfite treatment to discriminate between methylated and unmethylated cytosines, that the HPV16 LCR was selectively hypomethylated in highly differentiated cell populations. In contrast, the HPV16 LCR from poorly differentiated, basal cell-like cells contained multiple methylated cytosines and were often methylated at E2BSs, particularly E2BS2. These experiments indicate that the methylation state of the viral genome, and particular that of E2BSs, may vary during the viral life cycle, providing a novel means for modulating E2 function. These studies also uncovered an extensive pattern of methylation at non-CpG dinucleotides indicative of de novo methylation. The potential implications of this de novo methylation pattern are discussed.
Retrospective Analysis of a Local Cessation of Vaccination against Poliomyelitis: a Possible Scenario for the Future
Authors: Korotkova Ekaterina A. , Park Renee , Cherkasova Elena A. , Lipskaya Galina Y. , Chumakov Konstantin M. , Feldman Esfir V. , Kew Olen M. , Agol Vadim I.
Published: November 2003

The global eradication of poliomyelitis will require substantial changes in immunization practices. One of the proposed scenarios includes cessation of vaccination with live oral poliovirus vaccine (OPV) and the creation of an OPV stockpile for emergency response in case of the reintroduction of poliovirus into circulation. We describe here a retrospective analysis of the cessation of OPV usage in a region of the Byelorussian Republic of the former Soviet Union in 1963 to 1966. During this period, a widespread circulation and evolution of independent lineages of vaccine-derived polioviruses took place in the region. Some of these lineages appeared to originate from OPV given to 40 children in the community during this period of essentially no vaccinations. The data demonstrate very high risks associated with both the local cessation of OPV vaccination and the proposed use of OPV to control a possible reemergence of poliovirus in the postvaccination period. The high transmissibility of OPV-derived viruses in nonimmune population, documented here, and the known existence of long-term OPV excretors should be also considered in assessing risks of the synchronized global cessation of OPV usage.
Modulation of Minute Virus of Mice Cytotoxic Activities through Site-Directed Mutagenesis within the NS Coding Region
Authors: Daeffler Laurent , Hörlein Rita , Rommelaere Jean , Nüesch Jürg P. F.
Published: November 2003

Late in infection, parvovirus minute virus of mice (MVMp) induces the lysis of mouse A9 fibroblasts. This effect depends on the large nonstructural phosphoprotein NS1, which plays in addition a major role in viral DNA replication and progeny particle production. Since the NS1 C-terminal region is subjected to late phosphorylation events and protein kinase C (PKC) family members regulate NS1 replicative activities, the present study was conducted to determine the impact of PKCs on NS1 cytotoxic functions. To this end, we performed site-directed mutagenesis, substituting alanine residues for two consensus PKC-phosphorylation sites located within the NS1 C-terminal region, T585 and S588. Although these substitutions had no detectable effect on virus multiplication in a single-round infection, the NS1-585A mutant virus was significantly less toxic to A9 cells than wild-type MVMp, whereas the NS1-588A mutant virus was endowed with a higher killing potential. These alterations correlated with specific changes in the late phosphorylation pattern of the mutant NS1 proteins compared to the wild-type polypeptide. Since the mutations introduced in this region of the viral genome also made changes in the minor nonstructural protein NS2, a contribution of this polypeptide to the above-mentioned phenotypes of mutant viruses cannot be excluded at present. However, the involvement of NS1 in these phenotypes was directly supported by the respective reduced and enhanced capacity of NS1-585A and NS1-588A recombinant proteins for inducing morphological alterations and cell detachment in transfected A9 cultures. Altogether, these data suggest that late-occurring phosphorylation of NS1 specifically regulates the cytotoxic functions of the viral product and that residues T585 and S588 contribute to this control in an antagonistic way.
Kinetics of Lymphocyte Proliferation during Primary Immune Response in Macaques Infected with Pathogenic Simian Immunodeficiency Virus SIVmac251: Preliminary Report of the Effect of Early Antiviral Therapy
Authors: Benlhassan-Chahour Kadija , Penit Claude , Dioszeghy Vincent , Vasseur Florence , Janvier Geneviève , Rivière Yves , Dereuddre-Bosquet Nathalie , Dormont Dominique , Le Grand Roger , Vaslin Bruno
Published: November 2003

The aim of this study was to evaluate the kinetics of lymphocyte proliferation during primary infection of macaques with pathogenic simian immunodeficiency virus (SIV) and to study the impact of short-term postexposure highly active antiretroviral therapy (HAART) prophylaxis. Twelve macaques were infected by intravenous route with SIVmac251 and given treatment for 28 days starting 4 h postexposure. Group 1 received a placebo, and groups 2 and 3 received combinations of zidovudine (AZT), lamivudine (3TC), and indinavir. Macaques in group 2 received AZT (4.5 mg/kg of body weight), 3TC (2.5 mg/kg), and indinavir (20 mg/kg) twice per day by the oral route whereas macaques in group 3 were given AZT (4.5 mg/kg) and 3TC (2.5 mg/kg) subcutaneously twice per day, to improve the pharmacokinetic action of these drugs, and a higher dose of indinavir (60 mg/kg). The kinetics of lymphocyte proliferation were analyzed by monitoring 5-bromo-2′-deoxyuridine (BrdU) uptake ex vivo and by fluorescence-activated cell sorting analysis. HAART did not protect against SIV infection but did strongly impact on virus loads: viremia was delayed and lowered during antiviral therapy in group 2, with better control after treatment was stopped, and in group 3, viremia was maintained at lower levels during treatment, with virus even undetectable in the blood of some macaques, but there was no evidence of improved control of the virus after treatment. We provide direct evidence that dividing NK cells are detected earlier than dividing T cells in the blood (mostly in CD45RA−T cells), mirroring plasma viremia. Dividing CD8+T cells were detected earlier than dividing CD4+T cells, and the highest percentages of proliferating T cells coincided with the first evidence of partial control of peak viremia and with an increase in the percentage of circulating gamma interferon-positive CD8+T cells. The level of cell proliferation in the blood during SIV primary infection was clearly associated with viral replication levels because the inhibition of viral replication by postexposure HAART strongly reduced lymphocyte proliferation. The results and conclusions in this study are based on experiments in a small numbers of animals and are thus preliminary.
Bystander Killing during Avian Leukosis Virus Subgroup B Infection Requires TVB
S3
Signaling
Authors: Diaz-Griffero Felipe , Hoschander Steven A. , Brojatsch Jürgen
Published: November 2003

Cell killing by avian leukosis virus subgroup B (ALV-B) in cultures has been extensively studied, but the molecular basis of this process has not been established. Here we show that superinfection, which has been linked to cell killing by ALV-B, plays no crucial role in cell death induction. Instead, we show that signaling by the ALV-B receptor, TVB
S3
, a member of the tumor necrosis factor receptor family, is essential for ALV-B-mediated cell death. TVB
S3
activated caspase-dependent apoptosis during ALV-B infection. Strikingly, apoptosis induction occurred predominantly in uninfected cells, while ALV-B-infected cells were protected against cell death. This bystander killing phenomenon was reproduced in a virus-free system by cocultivating ALV-B Env-expressing cells with TVB
S3
-expressing cells. Taken together, our results indicated that ALV-B-mediated apoptosis is triggered by ALV-B Env-TVB
S3
interactions.
Mutations within the P2 Domain of Norovirus Capsid Affect Binding to Human Histo-Blood Group Antigens: Evidence for a Binding Pocket
Authors: Tan Ming , Huang Pengwei , Meller Jaroslaw , Zhong Weiming , Farkas Tibor , Jiang Xi
Published: November 2003

Noroviruses (NORs) are an important cause of acute gastroenteritis. Recent studies of NOR receptors showed that different NORs bind to different histo-blood group antigens (HBGAs), and at least four distinct binding patterns were observed. To determine the structure-function relationship for NORs and their receptors, two strains representing two of the four binding patterns were studied. Strain VA387 binds to HBGAs of A, B, and O secretors, whereas strain MOH binds to HBGAs of A and B secretors only. Using multiple sequence alignments, homology modeling, and structural analysis of NOR capsids, we identified a plausible “pocket” in the P2 domain that may be responsible for binding to HBGA receptors. This pocket consists of a conserved RGD/K motif surrounded by three strain-specific hot spots (N
302
, T
337
, and Q
375
for VA387 and N
302
, N
338
, and E
378
for MOH). Subsequent mutagenesis experiments demonstrated that all four sites played important roles in binding. A single amino acid mutation at T
337
(to A) in VA387 or a double amino acid mutation at RN
338
(to TT) in MOH abolished binding completely. Change of the entire RGD motif to SAS abolished binding in case of VA387, whereas single amino acid mutations in that motif did not have an apparent effect on binding to A and B antigens but decreased binding to H antigen. Multiple mutations at the RGK motif of MOH (SIRGK to TFRGD) completely knocked out the binding. Mutation of N
302
or Q
375
in VA387 affected binding to type O HBGA only, while switch mutants with three amino acid changes at either site from MOH to VA387 resulted in a weak binding to type O HBGAs. A further switch mutant with three amino acid changes at E
378
from MOH to VA387 diminished the binding to type A HBGA only. Taken together, our data indicate that the binding pocket likely exists on NOR capsids. Direct evidence of this hypothesis requires crystallography studies.
Simian-Human Immunodeficiency Virus Escape from Cytotoxic T-Lymphocyte Recognition at a Structurally Constrained Epitope
Authors: Peyerl Fred W. , Barouch Dan H. , Yeh Wendy W. , Bazick Heidi S. , Kunstman Jennifer , Kunstman Kevin J. , Wolinsky Steven M. , Letvin Norman L.
Published: November 2003

Virus-specific cytotoxic T lymphocytes (CTL) exert intense selection pressure on replicating simian immunodeficiency virus (SIV) and human immunodeficiency virus type 1 (HIV-1) in infected individuals. The immunodominant Mamu-A*01-restricted Gag p11C, C-M epitope is highly conserved among all sequenced isolates of SIV and therefore likely is structurally constrained. The strategies used by virus isolates to mutate away from an immunodominant epitope-specific CTL response are not well defined. Here we demonstrate that the emergence of a position 2 p11C, C-M epitope substitution (T47I) in a simian-human immunodeficiency virus (SHIV) strain 89.6P-infectedMamu-A*01+monkey is temporally correlated with the emergence of a flanking isoleucine-to-valine substitution at position 71 (I71V) of the capsid protein. An analysis of the SIV and HIV-2 sequences from the Los Alamos HIV Sequence Database revealed a significant association between any position 2 p11C, C-M epitope mutation and the I71V mutation. The T47I mutation alone is associated with significant decreases in viral protein expression, infectivity, and replication, and these deficiencies are restored to wild-type levels with the introduction of the flanking I71V mutation. Together, these data suggest that a compensatory mutation is selected for in SHIV strain 89.6P to facilitate the escape of that virus from CTL recognition of the dominant p11C, C-M epitope.
Nonspecific Double-Stranded DNA Binding Activity of Simian Virus 40 Large T Antigen Is Involved in Melting and Unwinding of the Origin
Authors: Jiao Junfang , Simmons Daniel T.
Published: November 2003

Helicase activity is required for T antigen to unwind the simian virus 40 origin. We previously mapped this activity to residues 131 and 616. In this study, we generated a series of mutants with single-point substitutions in the helicase domain to discover other potential activities required for helicase function. A number of DNA unwinding-defective mutants were generated. Four of these mutants (456RA, 460ED, 462GA, and 499DA) were normal in their ability to hydrolyze ATP and were capable of associating into double hexamers in the presence of origin DNA. Furthermore, they possessed normal ability to bind to single-stranded DNA. However, they were severely impaired in unwinding origin-containing DNA fragments and in carrying out a helicase reaction with an M13 partial duplex DNA substrate. Interestingly, these mutants retained some ability to perform a helicase reaction with artificial replication forks, indicating that their intrinsic helicase activity was functional. Intriguingly, these mutants had almost completely lost their ability to bind to double-stranded DNA nonspecifically. The mutants also failed to melt the early palindrome region of the origin. Taken together, these results indicate that the mutations have destroyed a novel activity required for unwinding of the origin. This activity depends on the ability to bind to DNA nonspecifically, and in its absence, T antigen is unable to structurally distort and subsequently unwind the origin.
Codelivery of CCR7 Ligands as Molecular Adjuvants Enhances the Protective Immune Response against Herpes Simplex Virus Type 1
Authors: Toka Felix N. , Gierynska Malgorzata , Rouse Barry T.
Published: November 2003

Humoral and cellular immunity, associated with long-term protective immunological memory, defines the efficacy of a given vaccine formulation. However, few vaccines achieve this target without the aid of a suitable adjuvant. Molecular adjuvants in vaccination against infectious agents offer a noninvasive means of enhancing the immune response against target antigens. To examine the potency of two β-chemokines as immunomodulators, plasmid DNA encoding β-chemokines CCL19 and CCL21 (CCR7L) was codelivered intranasally with plasmid DNA or recombinant vaccinia virus encoding herpes simplex virus (HSV) gB (HSV-gB) in a prime-and-boost vaccination strategy. This vaccination regimen increased serum and vaginal immunoglobulin G (IgG) and IgA, respectively, as well as the numbers of HSV-gB498-505peptide-specific gamma interferon-producing CD8+T cells. Distinctively, a high number of cytotoxic T lymphocytes was achieved when pCCR7L was applied at both prime and boost as opposed to omission of pCCR7L. A rapid-recall response was induced in the genital tract upon challenge with the HSV McKrae strain, affording a high level of protection and survival of vaccinated mice. Our results demonstrate that high innate immune kinetics and distribution of adaptive response induced in the nasal mucosa appears to be key factors in generating protective memory responses against HSV. Thus CCR7L expressed ectopically may serve as a molecular adjuvant to boost the immune response to a codelivered antigen in mucosal surfaces.
Covalently Linked Human Immunodeficiency Virus Type 1 gp120/gp41 Is Stably Anchored in Rhabdovirus Particles and Exposes Critical Neutralizing Epitopes
Authors: McKenna Philip M. , Pomerantz Roger J. , Dietzschold Bernhard , McGettigan James P. , Schnell Matthias J.
Published: November 2003

Rabies virus (RV) vaccine strain-based vectors show significant promise as potential live-attenuated vaccines against human immunodeficiency virus type 1 (HIV-1). Here we describe a new RV construct that will also likely have applications as a live-attenuated or killed-particle immunogen. We have created a RV containing a chimeric HIV-1 Env protein, which contains introduced cysteine residues that give rise to an intermolecular disulfide bridge between gp120 and the ectodomain of gp41. This covalently linked gp140 (gp140 SOS) is fused in frame to the cytoplasmic domain of RV G glycoprotein and is efficiently incorporated into the RV virion. On the HIV-1 virion, the gp120 and gp41 moieties are noncovalently associated, which leads to extensive shedding of gp120 from virions and virus-infected cells. The ability to use HIV-1 particles as purified, inactivated immunogens has been confounded by the loss of gp120 during preparation. Additionally, monomeric gp120 and uncleaved gp160 molecules have been shown to be poor antigenic representations of virion-associated gp160. Because the gp120 and gp41 portions are covalently attached in the gp140 SOS molecule, the protein is maintained on the surface of the RV virion throughout purification. Surface immunostaining and fluorescence-activated cell sorting analysis with anti-envelope antibodies show that the gp140 SOS protein is stably expressed on the surface of infected cells and maintains CD4 binding capabilities. Furthermore, Western blot and immunoprecipitation experiments with infected-cell lysates and purified virions show that a panel of neutralizing anti-envelope antibodies efficiently recognize the gp140 SOS protein. The antigenic properties of this recombinant RV particle containing covalently attached Env, as well as the ability to present Env in a membrane-bound form, suggest that this approach could be a useful component of a HIV-1 vaccine strategy.
Relationship between RNA Lariat Debranching and Ty1 Element Retrotransposition
Authors: Salem Laura A. , Boucher Christopher L. , Menees Thomas M.
Published: November 2003

TheSaccharomyces cerevisiae DBR1gene encodes a 2′-5′ phosphodiesterase that debranches intron RNA lariats following splicing. Yeastdbr1mutants accumulate intron lariats and are also defective for mobility of the retrotransposons Ty1 and Ty3. We used a mutagenic PCR method to generate a collection ofdbr1mutant alleles to explore the relationship between the roles ofDBR1in transposition and debranching. Eight mutants defective for Ty1 transposition contained single amino acid changes in Dbr1p. Two mutations, G84A and N85D, are in a conserved phosphoesterase motif that is believed to be part of the active site of the enzyme, supporting a connection between enzymatic activity and Ty1 transposition. Two other mutations, Y68F and Y68D, occur at a potential phosphorylation site, and we have shown that Dbr1p is phosphorylated on tyrosine. We have developed an RNase protection assay to quantitate intron RNA accumulation in cells. The assay uses RNA probes that hybridize toACT1intron RNA. Protection patterns confirm that sequences from the 5′ end of the intron to the lariat branch point accumulate indbr1mutants in a branched (lariat) conformation. RNase protection assays indicate that all of the newly generateddbr1mutant alleles are also deficient for debranching, further supporting a role for 2′-5′ phosphodiesterase activity in Ty1 transposition. A Ty1 element lacking most of its internal sequences transposes independently ofDBR1. The existence of Dbr1p-dependent Ty1 sequences raises the possibility that Dbr1p acts on Ty1 RNA.
The Membrane-Proximal Region of Vesicular Stomatitis Virus Glycoprotein G Ectodomain Is Critical for Fusion and Virus Infectivity
Authors: Jeetendra E. , Ghosh Kakoli , Odell Derek , Li Jin , Ghosh Hara P. , Whitt Michael A.
Published: November 2003

The glycoprotein (G) of vesicular stomatitis virus (VSV) is responsible for binding of virus to cells and for mediating virus entry following endocytosis by inducing fusion of the viral envelope with the endosomal membrane. The fusion peptide of G is internal (residues 116 to 137) and exhibits characteristics similar to those of other internal fusion peptides, but recent studies have implicated the region adjacent to the transmembrane domain as also being important for G-mediated membrane fusion. Sequence alignment of the membrane-proximal region of G from several different vesiculoviruses revealed that this domain is highly conserved, suggesting that it is important for G function. Mutational analysis was used to show that this region is not essential for G protein oligomerization, transport to the cell surface, or incorporation into virus particles but that it is essential for acid-induced membrane fusion activity and for virus infectivity. Deletion of the 13 membrane-proximal amino acids (N449 to W461) dramatically reduced cell-cell fusion activity and reduced virus infectivity approximately 100-fold, but mutation of conserved aromatic residues (W457, F458, and W461) either singly or together had only modest effects on cell-cell fusion activity; recombinant virus encoding these mutants replicated as efficiently as wild-type (WT) VSV. Insertion of heterologous sequences in the juxtamembrane region completely abolished membrane fusion activity and virus infectivity, as did deletion of residues F440 to N449. The insertion mutants showed some changes in pH-dependent conformational changes and in virus binding, which could partially explain the defects in membrane fusion activity, but all the other mutants were similar to WT G with respect to conformational changes and virus binding. These data support the hypothesis that the membrane-proximal domain contributes to G-mediated membrane fusion activity, yet the conserved aromatic residues are not essential for membrane fusion or virus infectivity.
In Vitro and In Vivo Interactions between the Hepatitis B Virus Protein P22 and the Cellular Protein gC1qR
Authors: Lainé S. , Thouard A. , Derancourt J. , Kress M. , Sitterlin D. , Rossignol J.-M.
Published: November 2003

gC1qR, a mitochondrial matrix protein, was identified as the main cellular partner of the hepatitis B virus P22 protein. We demonstrated by immunofluorescence studies that some P22 molecules were colocalized with the endogenous gC1qR in both the cytoplasm and the nucleus but never in the mitochondria. We also showed that the last 34 amino acids of P22 were involved in the association with gC1qR.
A Pseudorabies Virus Recombinant Simultaneously Lacking the Major Tegument Proteins Encoded by the UL46, UL47, UL48, and UL49 Genes Is Viable in Cultured Cells
Authors: Fuchs Walter , Granzow Harald , Mettenleiter Thomas C.
Published: November 2003

The UL46, UL47, UL48, and UL49 genes, which encode major tegument proteins, are conserved in most alphaherpesvirus genomes. However, the relative importance of each of these proteins for replication of individual alphaherpesviruses appears to be different. Recently, we demonstrated that single deletions of UL47 or UL48 impair maturation and egress of pseudorabies virus (PrV) particles to different extents, whereas deletions of UL46 or UL49 have no significant effects on virus replication in cell culture (W. Fuchs, H. Granzow, B. G. Klupp, M. Kopp, and T. C. Mettenleiter, J. Virol. 76:6729-6742, 2002; M. Kopp, B. G. Klupp, H. Granzow, W. Fuchs, and T. C. Mettenleiter, J. Virol. 76:8820-8833, 2002). To test for possible functional redundancy between the four tegument proteins, a quadruple gene deletion mutant (PrV-ΔUL46-49) was generated and characterized in vitro. Although plaque formation by this mutant was almost abolished and maximum titers were reduced more than 100-fold compared to those of parental wild-type virus, PrV-ΔUL46-49 could be propagated and serially passaged in noncomplementing porcine and rabbit kidney cells. Electron-microscopic studies revealed that nucleocapsid formation and egress of PrV-ΔUL46-49 from the host cell nucleus were not affected, but secondary envelopment of nucleocapsids in the cytoplasm was only rarely observed. The replication defect of PrV-ΔUL46-49 could be fully corrected by reinsertion of the UL46-to-UL49 gene cluster. Plaque sizes and virus titers were only slightly increased after restoration of only UL47 expression, whereas repair of only UL48 resulted in a significant increase in replication capacity to the level of a UL47 deletion mutant. In conclusion, we show that none of the UL46 to UL49 tegument proteins is absolutely required for productive replication of PrV. Moreover, our data indicate that the UL47 and UL48 proteins function independently during cell-to-cell spread and virus egress.
Potential High-Throughput Assay for Screening Inhibitors of West Nile Virus Replication
Authors: Lo Michael K. , Tilgner Mark , Shi Pei-Yong
Published: November 2003

Prevention and treatment of infection by West Nile virus (WNV) and other flaviviruses are public health priorities. We describe a reporting cell line that can be used for high-throughput screening of inhibitors against all targets involved in WNV replication. Dual reporter genes, encodingRenillaluciferase (Rluc) and neomycin phosphotransferase (Neo), were engineered into a WNV subgenomic replicon, resulting in Rluc/NeoRep. Geneticin selection of BHK-21 cells transfected with Rluc/NeoRep yielded a stable cell line that contains persistently replicating replicons. Incubation of the reporting cells with known WNV inhibitors decreased Rluc activity, as well as the replicon RNA level. The efficacies of the inhibitors, as measured by the depression of Rluc activity in the reporting cells, are comparable to those derived from authentic viral infection assays. Therefore, the WNV reporting cell line can be used as a high-throughput assay for anti-WNV drug discovery. A similar approach should be applicable to development of genetics-based antiviral assays for other flaviviruses.
EFFECTIVE POTENTIALS AND QUANTUM FLUID MODELS: A THERMODYNAMIC APPROACH
Authors: RINGHOFER CHRISTIAN , GARDNER CARL , VASILESKA DRAGICA
Published: November 2003

We present a thermodynamic approach to introducing quantum corrections to the classical transport picture in semiconductor device simulation. This approach leads to a modified Boltzmann equation with a quantum corrected force term and to quantum corrected fluid, or quantum hydrodynamic models. We present the quantum interaction of electrons with a gate oxide barrier potential and quantum hydrodynamic simulations of a resonant tunneling diode as application examples.
HOT CARRIER EFFECTS WITHIN MACROSCOPIC TRANSPORT MODELS
Authors: GRASSER TIBOR , KOSINA HANS , SELBERHERR SIEGFRIED
Published: November 2003

The distribution function of hot carriers in state-of-the-art devices is insufficiently described using just the electric field or the average carrier energy as parameters. Still, the standard models to describe carrier transport in semiconductor devices, namely the drift-diffusion model and the energy-transport model rely on these assumptions. In this article we summarize our work on six moments transport models which allow an accurate characterization of the distribution function. Within this framework it is possible to selfconsistently model the scattering integral without resorting to the relaxation time approximation. In addition, hot electron processes such as impact ionization, which are difficult to model in lower order transport models, can be described accurately.
MAPPING, PROGRAMMABILITY AND SCALABILITY OF PROBLEMS FOR QUANTUM SPEED-UP
Authors: KRISHNAMURTHY E. V.
Published: November 2003

This paper explores the reasons as to why the quantum paradigm is not so easy to extend to all of the classical computational algorithms. We also explain the failure of programmability and scalability in quantum speed-up. Due to the presence of quantum entropy, quantum algorithm cannot obviate the curse of dimensionality encountered in solving many complex numerical and optimization problems. Finally, the stringent condition that quantum computers have to be interaction-free, leave them with little versatility and practical utility.
Applied Studies of Plant Meridian System I.
Authors: Hou T. Z. , Mooneyham R. E.
Published: November 2003

Agri-wave technology is a new agricultural technology based on the plant meridian system, that focuses on measurement of plant sound characteristics. The basic principle of agri-wave technology is to improve the yield and quality of plants such as vegetables, flowers, and fruit trees by broadcasting sound waves of certain frequencies and spraying a compound microelement fertilizer on the leaves. The application of agri-wave technology on tomatoes remarkably stimulates growth of their seedlings. Fresh weight of the branch, stems, and leaves of the treated tomatoes is significantly (59.53%, P < 0.0001) higher than that of the control group. Sampling survey results indicate that agri-wave technology accelerates the ripeness of tomatoes. The fresh weight of ripe tomatoes treated with this technique is 30.73% higher than that of the untreated (P = 0.0018), while the fresh weight of the treated unripe tomatoes is 27.29% lower than that of the untreated unripe group (P = 0.0020). Yield surveys show that the yield of treated plants is 13.89% (p < 0.0001) higher than that of the control group. Moreover, with agri-wave technology treatment the storage period of tomatoes is almost doubled. Analysis of tomato nutrition shows that agri-wave technology has increased their sugar content by 26.19%, vitamin A and niacin (an antifavours vitamin) by 55.39% and 92.31% respectively. There is no difference concerning vitamin B1, B2, and D content between the two groups, and vitamin C and E contents decreased by 2.10% and 12.69%, respectively. Among the analyzed 33 minerals of tomatoes, 26 increased in content, while 7 decreased. In conclusion, agri-wave technology has promoted the growth of the tomato, increased its yield, and improved its quality.