bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Complex Zinc Finger Controls the Enzymatic Activities of Nidovirus Helicases
Authors: Seybert Anja , Posthuma Clara C. , van Dinten Leonie C. , Snijder Eric J. , Gorbalenya Alexander E. , Ziebuhr John 
Published: December 2004

ABSTRACT

Nidoviruses (
Coronaviridae
,
Arteriviridae
, and
Roniviridae
) encode a nonstructural protein, called nsp10 in arteriviruses and nsp13 in coronaviruses, that is comprised of a C-terminal superfamily 1 helicase domain and an N-terminal, putative zinc-binding domain (ZBD). Previously, mutations in the equine arteritis virus (EAV) nsp10 ZBD were shown to block arterivirus reproduction by disrupting RNA synthesis and possibly virion biogenesis. Here, we characterized the ATPase and helicase activities of bacterially expressed mutant forms of nsp10 and its human coronavirus 229E ortholog, nsp13, and correlated these in vitro activities with specific virus phenotypes. Replacement of conserved Cys or His residues with Ala proved to be more deleterious than Cys-for-His or His-for-Cys replacements. Furthermore, denaturation-renaturation experiments revealed that, during protein refolding, Zn
2+
is essential for the rescue of the enzymatic activities of nidovirus helicases. Taken together, the data strongly support the zinc-binding function of the N-terminal domain of nidovirus helicases. nsp10 ATPase/helicase deficiency resulting from single-residue substitutions in the ZBD or deletion of the entire domain could not be complemented in
trans
by wild-type ZBD, suggesting a critical function of the ZBD in
cis
. Consistently, no viral RNA synthesis was detected after transfection of EAV full-length RNAs encoding ATPase/helicase-deficient nsp10 into susceptible cells. In contrast, diverse phenotypes were observed for mutants with enzymatically active nsp10, which in a number of cases correlated with the activities measured in vitro. Collectively, our data suggest that the ZBD is critically involved in nidovirus replication and transcription by modulating the enzymatic activities of the helicase domain and other, yet unknown, mechanisms.
In Vitro and In Vivo Relevance of Infectious Laryngotracheitis Virus gJ Proteins That Are Expressed from Spliced and Nonspliced mRNAs
Authors: Fuchs Walter , Wiesner Dorothee , Veits Jutta , Teifke Jens P. , Mettenleiter Thomas C. 
Published: December 2004

ABSTRACT

The positional homologue in the infectious laryngotracheitis virus (ILTV) genome of the glycoprotein gJ gene of herpes simplex virus and the gp2 gene of equine herpesvirus 1 is expressed into four proteins of 85, 115, 160, and 200 kDa (J. Veits, B. Köllner, J. P. Teifke, H. Granzow, T. C. Mettenleiter, and W. Fuchs, Avian Dis.
47:
330-342, 2003). RNA analyses revealed that these proteins are expressed from two different late (γ2) transcripts, an unspliced 5.5-kb and a spliced 4.3-kb mRNA that are translated into proteins of 985 and 611 amino acids, respectively. ILTV gJ is incorporated into virions and is modified by N- and O-linked glycosylation. After cotransfection of chicken cells with genomic DNA of a pathogenic ILTV strain and transfer plasmids, gJ-negative ILTV mutants could be isolated. In vitro growth studies demonstrated that deletion of the gJ gene has only minor effects on direct cell-to-cell spread as measured by plaque size. However, progeny virus titers of ILTV-ΔgJ were significantly reduced in comparison to those of the parental virus and a gJ rescue mutant. After experimental infection of chickens the gJ rescue mutant, like wild-type ILTV, caused severe disease and considerable mortality, whereas ILTV-ΔgJ was significantly attenuated. All immunized animals were protected against subsequent challenge infection with virulent ILTV. In sera collected after immunization with the gJ-rescue mutant or with wild-type ILTV, gJ-specific antibodies were detectable by immunofluorescence on cells that had been transfected with a gJ expression plasmid. As expected, no gJ-specific antibodies were found in sera obtained from chickens immunized with ILTV-ΔgJ. Thus, gJ deletion mutants of ILTV might be usable as attenuated live-virus vaccines. Furthermore, the gJ gene might constitute a reliable marker for serological discrimination between vaccinated and field virus-infected chickens.
Studies of Ebola Virus Glycoprotein-Mediated Entry and Fusion by Using Pseudotyped Human Immunodeficiency Virus Type 1 Virions: Involvement of Cytoskeletal Proteins and Enhancement by Tumor Necrosis Factor Alpha
Authors: Yonezawa Akihito , Cavrois Marielle , Greene Warner C. 
Published: December 2004

ABSTRACT
The Ebola filoviruses are aggressive pathogens that cause severe and often lethal hemorrhagic fever syndromes in humans and nonhuman primates. To date, no effective therapies have been identified. To analyze the entry and fusion properties of Ebola virus, we adapted a human immunodeficiency virus type 1 (HIV-1) virion-based fusion assay by substituting Ebola virus glycoprotein (GP) for the HIV-1 envelope. Fusion was detected by cleavage of the fluorogenic substrate CCF2 by β-lactamase-Vpr incorporated into virions and released as a result of virion fusion. Entry and fusion induced by the Ebola virus GP occurred with much slower kinetics than with vesicular stomatitis virus G protein (VSV-G) and were blocked by depletion of membrane cholesterol and by inhibition of vesicular acidification with bafilomycin A1. These properties confirmed earlier studies and validated the assay for exploring other properties of Ebola virus GP-mediated entry and fusion. Entry and fusion of Ebola virus GP pseudotypes, but not VSV-G or HIV-1 Env pseudotypes, were impaired in the presence of the microtubule-disrupting agent nocodazole but were enhanced in the presence of the microtubule-stabilizing agent paclitaxel (Taxol). Agents that impaired microfilament function, including cytochalasin B, cytochalasin D, latrunculin A, and jasplakinolide, also inhibited Ebola virus GP-mediated entry and fusion. Together, these findings suggest that both microtubules and microfilaments may play a role in the effective trafficking of vesicles containing Ebola virions from the cell surface to the appropriate acidified vesicular compartment where fusion occurs. In terms of Ebola virus GP-mediated entry and fusion to various target cells, primary macrophages proved highly sensitive, while monocytes from the same donors displayed greatly reduced levels of entry and fusion. We further observed that tumor necrosis factor alpha, which is released by Ebola virus-infected monocytes/macrophages, enhanced Ebola virus GP-mediated entry and fusion to human umbilical vein endothelial cells. Thus, Ebola virus infection of one target cell may induce biological changes that facilitate infection of secondary target cells that play a key role in filovirus pathogenesis. Finally, these studies indicate that pseudotyping in the HIV-1 virion-based fusion assay may be a valuable approach to the study of entry and fusion properties mediated through the envelopes of other viral pathogens.
Transformation of Madin-Darby Canine Kidney Epithelial Cells by Sheep Retrovirus Envelope Proteins
Authors: Liu Shan-Lu , Miller A. Dusty 
Published: December 2004

ABSTRACT
Jaagsiekte sheep retrovirus (JSRV) and enzootic nasal tumor virus (ENTV) induce epithelial tumors in the airways of sheep and goats. In both of these simple retroviruses, the envelope (Env) protein is the active oncogene. Furthermore, JSRV Env can transform cultured cells by two distinct mechanisms. In rat and mouse fibroblasts, the cytoplasmic tail of JSRV Env is essential for transformation, which involves activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, and the virus receptor hyaluronidase 2 (Hyal2) is not involved. In contrast, in the BEAS-2B human bronchial epithelial cell line, transformation is mediated by JSRV Env binding to Hyal2 followed by Hyal2 degradation and activation of the receptor tyrosine kinase RON, the activity of which is normally suppressed by Hyal2. Here we show that JSRV and ENTV Env proteins can also transform Madin-Darby canine kidney (MDCK) epithelial cells, but by a mechanism similar to that observed in fibroblast cell lines. In particular, the cytoplasmic tail of Env is required for transformation, the PI3K/Akt pathway is activated, expression of RON (which is not normally expressed in MDCK cells) does not affect transformation, and canine Hyal2 appears uninvolved. These results show that the JSRV and ENTV Env proteins can transform epithelial cells besides BEAS-2B cells and argue against a model for Env transformation involving different pathways that are uniquely active in fibroblasts or epithelial cells.
Biochemical Fingerprints of Prion Infection: Accumulations of Aberrant Full-Length and N-Terminally Truncated PrP Species Are Common Features in Mouse Prion Disease
Authors: Pan Tao , Wong Poki , Chang Binggong , Li Chaoyang , Li Ruliang , Kang Shin-Chung , Wisniewski Thomas , Sy Man-Sun 
Published: December 2004

ABSTRACT

Infection with any one of three strains of mouse scrapie prion (PrP
Sc
), 139A, ME7, or 22L, results in the accumulation of two underglycosylated, full-length PrP species and an N-terminally truncated PrP species that are not detectable in uninfected animals. The levels of the N-terminally truncated PrP species vary depending on PrP
Sc
strain. Furthermore, 22L-infected brains consistently have the highest levels of proteinase K (PK)-resistant PrP species, followed by ME7- and 139A-infected brains. The three strains of PrP
Sc
are equally susceptible to PK and proteases papain and chymotrypsin. Their protease resistance patterns are also similar. In sucrose gradient velocity sedimentation, the aberrant PrP species partition with PrP
Sc
aggregates, indicating that they are physically associated with PrP
Sc
. In ME7-infected animals, one of the underglycosylated, full-length PrP species is detected much earlier than the other, before both the onset of clinical disease and the detection of PK-resistant PrP species. In contrast, the appearance of the N-terminally truncated PrP species coincides with the presence of PK-resistant species and the manifestation of clinical symptoms. Therefore, accumulation of the underglycosylated, full-length PrP species is an early biochemical fingerprint of PrP
Sc
infection. Accumulation of the underglycosylated, full-length PrP species and the aberrant N-terminally truncated PrP species may be important in the pathogenesis of prion disease.
Development of a Rotavirus-Shedding Model in Rhesus Macaques, Using a Homologous Wild-Type Rotavirus of a New P Genotype
Authors: McNeal Monica M. , Sestak Karol , Choi Anthony H.-C. , Basu Mitali , Cole Michael J. , Aye Pyone P. , Bohm Rudolf P. , Ward Richard L. 
Published: December 2004

ABSTRACT
Although there are several reports on rotavirus inoculation of nonhuman primates, no reliable model exists. Therefore, this study was designed to develop a rhesus macaque model for rotavirus studies. The goals were to obtain a wild-type macaque rotavirus and evaluate it as a challenge virus for model studies. Once rotavirus was shown to be endemic within the macaque colony at the Tulane National Primate Research Center, stool specimens were collected from juvenile animals (2.6 to 5.9 months of age) without evidence of previous rotavirus infection and examined for rotavirus antigen. Six of 10 animals shed rotavirus during the 10-week collection period, and the electropherotypes of all isolates were identical to each other but distinct from those of prototype simian rotaviruses. These viruses were characterized as serotype G3 and subgroup 1, properties typical of many animal rotaviruses, including simian strains. Nucleotide sequence analysis of the VP4 gene was performed with a culture-grown isolate from the stool of one animal, designated the TUCH strain. Based on both genotypic and phylogenetic comparisons between TUCH VP4 and cognate proteins of representatives of the reported 22 P genotypes, the TUCH virus belongs to a new genotype, P[23]. A pool of wild-type TUCH was prepared and intragastrically administered to eight cesarean section-derived, specific-pathogen-free macaques 14 to 42 days of age. All animals were kept in a biocontainment level 2 facility. Although no diarrhea was observed and the animals remained clinically normal, all animals shed large quantities of rotavirus antigen in their feces after inoculation, which resolved by the end of the 14-day observation period. Therefore, TUCH infection of macaques provides a useful nonhuman primate model for studies on rotavirus protection.
Novel Adeno-Associated Virus Vector Vaccine Restricts Replication of Simian Immunodeficiency Virus in Macaques
Authors: Johnson Philip R. , Schnepp Bruce C. , Connell Mary J. , Rohne Daniela , Robinson Suzanne , Krivulka Georgia R. , Lord Carol I. , Zinn Rebekah , Montefiori David C. , Letvin Norman L. , Clark K. Reed 
Published: December 2004

ABSTRACTGene transfer vectors based on recombinant adeno-associated virus (rAAV) are simple, versatile, and safe. While the conventional applications for rAAV vectors have focused on delivery of therapeutic genes, we have developed the system for delivery of vaccine antigens. In particular, we are interested in generating rAAV vectors for use as a prophylactic human immunodeficiency virus type 1 (HIV-1) vaccine. To that end, we constructed vaccine vectors that expressed genes from the simian immunodeficiency virus (SIV) for evaluation in the monkey SIV model. After a single intramuscular dose, rAAV/SIV vaccines elicited SIV-specific T cells and antibodies in macaques. Furthermore, immunized animals were able to significantly restrict replication of a live, virulent SIV challenge. These data suggest that rAAV vaccine vectors induced biologically relevant immune responses, and thus, warrant continued development as a viable HIV-1 vaccine candidate.
PRODUCTION MANAGEMENT — SIMPLE OR COMPLEX
Authors: HALEVI GIDEON 
Published: December 2004

Theoretical production planning and scheduling is actually very simple task: The plant gets orders which defines the product, the quantity and delivery dates. The resources of the plants are known, the product bill of material is known and the task of production scheduling is to make sure that the orders will be ready on time, that's all. It seems strange that in order to meet this simple task, over 100 complex production planning methods were proposed. Some of the outstanding ones are: PICS; MRP; ERP; GT; TOC; FMS; IMS; CIM; CE; JIT; Kanaban; TQM; Agent…, AGILE etc. Yet the search for "THE" method is carried on. In this paper an attempt to analyze why production planning is regarded as a complex task, and why the search for "THE" production planning method is still an open topic for researchers. Furthermore, to demonstrate how introduction of flexibility will restore the simplicity of production planning.
ARE BIOMEDICAL RESEARCH CLUSTERS EMERGING IN SINGAPORE? AN EXPLORATORY STUDY
Authors: MISHRA ALOK , BHAT SANJAY A. , PALIYATH PRASAD KUMAR , RANGARAJAN HARIBHASKAR 
Published: December 2004

Innovation and new product development in the biomedical device industry is a complex process requiring interface between medical sciences, precision engineering and information technology. Further challenges are added by the presence of stringent regulatory requirements (e.g. FDA), health economics, outcome considerations, intellectual property issues, and complex nature of market where highly influential intermediaries (medical doctors) not only make product selection for end users (patients) but also contribute to product development. As a result, only a few biomedical device innovations succeed in reaching the market and fewer become commercially successful. A unique characteristic of this industry with regards to innovation and new product development is the importance of research collaboration between the clusters of universities, small and medium sized enterprises (SMEs), hospitals and medical doctors (lead users). The evidence of this is seen in the largest and probably the most established biomedical cluster in San Francisco Bay area, which is home to 31 universities and over 700 life sciences companies [California Healthcare Institute and PricewaterhouseCoopers (2000)]. These clusters of innovation are observed to be contributing significantly to regional economies of the US. Hence many nations as well as biomedical device multinationals are recognizing the importance of these new sources of innovation (i.e. universities, SMEs and users) and their collaboration. From this angle, the authors in this exploratory study examined biomedical device innovations taking place in select countries in Asia-Pacific and compared that with the innovation output of Singapore from a sample population of 33 000 patented innovations over a four year period (2000–2003). In addition, an attempt was made to understand the factors that are important for successful commercialization of biomedical device innovations from Singapore universities, through a questionnaire based survey of a sample of 50 researchers, and in-depth interviews with a sample of five venture capitalists and five medical doctors (lead users). The findings based on this study indicate that (1) Singapore university researchers appreciate the need for collaborative research with the industry but are sceptical about the present environment and process in Singapore. (2) Only a third of surveyed researchers have motivation to commercialize their innovations. (3) Medical doctors are more comfortable teaming up with universities than with the industry. (4) Industry has short-term perspective of research collaboration. The authors' recommendations are to (1) Encourage co-invention teams between universities, industry, and hospitals (medical doctors and clinicians), (2) Build a long-term co-invention culture through collaborative leadership, (3) Attract investment in local biomedical device R&D and production, and (4) Help Singapore universities and researchers appreciate Asia-Pacific market opportunities.
Radiative Heat Transfer Analysis within Three-Dimensional Clouds Subjected to Solar and Sky Irradiation
Authors: Nishikawa Toru , Maruyama Shigenao , Sakai Seigo 
Published: December 2004

AbstractA three-dimensional radiative heat transfer analysis of an arbitrary-shaped modeled cloud subjected to solar and sky irradiation has been performed. The Radiation Element Method by Ray Emission Model (REM2) was used for numerical simulation. Nongray, anisotropic scattering, absorbing, and emitting are taken into account in calculating the three-dimensional cloud. The modeled cloud is considered to be a low-level fair-weather cumulus in a tropical atmosphere. The cloud is modeled by unstructured mesh elements in order to investigate the curvature of cloud shape. Radiative cooling occurs in the thin layer below the cloud surface, and the thickness is approximately 20–40 m. Radiative cooling is enhanced at the swelled top of the cloud with a convex shape, which can cause a downward forcing and enhance the entrainment instability. On the other hand, radiative cooling close to the root of the swelled top is relatively weak. The solar heating does not affect the temperature change in the cloud compared with radiative heat transfer by longwave infrared radiation.
Effects of Environmentally Induced Asymmetries on Hurricane Intensity: A Numerical Study
Authors: Wu Liguang , Braun Scott A. 
Published: December 2004

Abstract
The influence of uniform large-scale flow, the beta effect, and vertical shear of the environmental flow on hurricane intensity is investigated in the context of the induced convective or potential vorticity asymmetries in the core region with a hydrostatic primitive equation hurricane model. In agreement with previous studies, imposition of one of these environmental effects weakens the simulated tropical cyclones. In response to the environmental influence, significant wavenumber-1 asymmetries develop. Asymmetric and symmetric tendencies of the mean radial and azimuthal winds and temperature associated with the environment-induced convective asymmetries are evaluated. The inhibiting effects of environmental influences are closely associated with the resulting eddy momentum fluxes, which tend to decelerate tangential and radial winds in the inflow and outflow layers. The corresponding changes in the symmetric circulation tend to counteract the deceleration effect. The net effect is a moderate weakening of the mean tangential and radial winds. The reduced radial wind can be viewed as an anomalous secondary radial circulation with inflow in the upper troposphere and outflow in the lower troposphere, weakening the mean secondary radial circulation.
Effects of Low-Frequency Wave Interactions on Intraseasonal Oscillations
Authors: Roundy Paul E. , Frank William M. 
Published: December 2004

Abstract
Intraseasonal oscillations (ISOs) control much of the large-scale variability of convection in the Tropics on time scales of about 15–100 days. These disturbances are often thought to be dominated by eastward-propagating modes, especially during austral summer, but disturbances that propagate westward are also important.
This work demonstrates by means of a multiple linear regression model and a brief case study that eastward- and westward-moving intraseasonal modes often cooperatively interact with one another to produce many of the characteristics of the observed Southern Hemisphere summer ISO. These interactions appear to be facilitated by topography and/or by the convective anomalies that are cooperatively induced by the eastward- and the westward-moving components of the oscillations. These interactions do not occur during every period of intraseasonal convective activity, but they do commonly occur during periods of high-amplitude convective anomalies. This analysis shows that eastward- and westward-moving intraseasonal modes should not be generally assumed to be linearly independent entities.
Mini-Open Medial Reefing and Arthroscopic Lateral Release for the Treatment of Recurrent Patellar Dislocation
Authors: Nam Ellis K. , Karzel Ronald P. 
Published: December 2004

Background: Although recurrent patellar dislocations are not uncommon, their pathophysiology and treatment are controversial. Hypothesis Stabilization of recurrent patellar dislocations can be successfully managed with a mini-open approach. Study Design Case series; Level of evidence, 4. Methods Twenty-two patients (23 knees) underwent a mini-open medial reefing and arthroscopic lateral release for the treatment of recurrent patellar dislocations with an average follow-up of 4.4 years (range, 1.4-14 years). The average age at the first dislocation was 15 years (range, 5-26 years), and the average age at surgery was 23 years (range, 12-65 years). Results There was 1 postoperative dislocation (4%) and 1 recurrent subluxation (4%). The average Kujala knee score was 88.2 ± 13.5, with overall good scores in each category. The lowest scores involved squatting (5.7), abnormal painful kneecap movements (subluxations) (7.1), and jumping (7.9). Overall, there was a statistically significant improvement in the Tegner score from 3.7 ± 1.8 before surgery to 6.9 ± 2.0 after surgery (P<. 001). Six knees (26%) were rated subjectively as excellent, 15 (65%) as good, 2 (9%) as fair, and 0 (0%) as poor. All 22 patients (100%) stated that the procedure was worthwhile. Radiographically, there was a statistically significant improvement in the congruence angle (normal, –8.0°± 6.0°) from 15.7°± 12.6° (range, 0.0° to +44.0°) before surgery to –11.5°± 8.7° (range, –20.0° to +10.0°) after surgery (P<. 001) and in the lateral patellofemoral angle (normal, > 0°) from –0.2°± 6.4° (range, –10° to +8°) before surgery to 7.9°± 2.6° (range, 0.0° to +11.0°) after surgery (P<. 001). Conclusion Our mini-open technique provides anatomical restoration with limited morbidity and cosmetically appealing results. Furthermore, our redislocation rates compare favorably with traditional, more extensile open approaches.