bluuebunny/crossref_metadata_2025_split huggingface dataset:
A Sequence-Independent Strategy for Detection and Cloning of Circular DNA Virus Genomes by Using Multiply Primed Rolling-Circle Amplification
Authors: Rector Annabel , Tachezy Ruth , Van Ranst Marc 
Published: April 2004

ABSTRACT

The discovery of novel viruses has often been accomplished by using hybridization-based methods that necessitate the availability of a previously characterized virus genome probe or knowledge of the viral nucleotide sequence to construct consensus or degenerate PCR primers. In their natural replication cycle, certain viruses employ a rolling-circle mechanism to propagate their circular genomes, and multiply primed rolling-circle amplification (RCA) with φ29 DNA polymerase has recently been applied in the amplification of circular plasmid vectors used in cloning. We employed an isothermal RCA protocol that uses random hexamer primers to amplify the complete genomes of papillomaviruses without the need for prior knowledge of their DNA sequences. We optimized this RCA technique with extracted human papillomavirus type 16 (HPV-16) DNA from W12 cells, using a real-time quantitative PCR assay to determine amplification efficiency, and obtained a 2.4 × 10
4
-fold increase in HPV-16 DNA concentration. We were able to clone the complete HPV-16 genome from this multiply primed RCA product. The optimized protocol was subsequently applied to a bovine fibropapillomatous wart tissue sample. Whereas no papillomavirus DNA could be detected by restriction enzyme digestion of the original sample, multiply primed RCA enabled us to obtain a sufficient amount of papillomavirus DNA for restriction enzyme analysis, cloning, and subsequent sequencing of a novel variant of bovine papillomavirus type 1. The multiply primed RCA method allows the discovery of previously unknown papillomaviruses, and possibly also other circular DNA viruses, without a priori sequence information.
Wide Variations in Herpes Simplex Virus Type 1 Inoculum Dose and Latency-Associated Transcript Expression Phenotype Do Not Alter the Establishment of Latency in the Rabbit Eye Model
Authors: O'Neil J. E. , Loutsch J. M. , Aguilar J. S. , Hill J. M. , Wagner E. K. , Bloom D. C. 
Published: April 2004

ABSTRACT
The latency-associated transcript (LAT) is required for efficient reactivation of herpes simplex virus type 1 from latent infection in the rabbit eye model, but LAT's mechanism of action is unknown. In addition to reactivation, the LAT region seems to correspond to multiple functions, with some LAT deletion mutants exhibiting increased virulence, increased neuronal death, and restricted establishment of latency. While a LAT promoter deletion mutant (17ΔPst) seems to be primarily restricted in reactivation in the rabbit, subtle effects on virulence or the establishment of latency cannot be precluded at the normal high levels of virus inoculum used in the rabbit model. Since such additional LAT phenotypes may be more evident with lower doses of virus, we evaluated the influence of initial viral inoculum and LAT expression on the progression of acute infection and the establishment of latency. We have assayed both virus recovery rates and viral genome loads in rabbit corneas and trigeminal ganglia. Our results show that (i) in the corneas and trigeminal ganglia, the maximum amount of virus present during acute infection is independent of the LAT genotype and inoculum dose, although greater viral yields are obtained earlier with higher inoculum doses, and (ii) the range in numbers of latent genomes detected in the ganglia is independent of the inoculum dose and the LAT genotype and therefore no difference in establishment of latency is observed.
Interaction between Human Immunodeficiency Virus Type 1 Reverse Transcriptase and Integrase Proteins
Authors: Hehl Eric A. , Joshi Pheroze , Kalpana Ganjam V. , Prasad Vinayaka R. 
Published: April 2004

ABSTRACT
Reverse transcriptase (RT) and integrase (IN) are two key catalytic enzymes encoded by all retroviruses. It has been shown that a specific interaction occurs between the human immunodeficiency virus type 1 (HIV-1) RT and IN proteins (X. Wu, H. Liu, H. Xiao, J. A. Conway, E. Hehl, G. V. Kalpana, V. R. Prasad, and J. C. Kappes, J. Virol. 73:2126-2135, 1999). We have now further examined this interaction to map the binding domains and to determine the effects of interaction on enzyme function. Using recombinant purified proteins, we have found that both a HIV-1 RT heterodimer (p66/p51) and its individual subunits, p51 and p66, are able to bind to HIV-1 IN. An oligomerization-defective mutant of IN, V260E, retained the ability to bind to RT, showing that IN oligomerization may not be required for interaction. Furthermore, we report that the C-terminal domain of IN, but not the N-terminal zinc-binding domain or the catalytic core domain, was able to bind to heterodimeric RT. Deletion analysis to map the IN-binding domain on RT revealed two separate IN-interacting domains: the fingers-palm domain and the carboxy-terminal half of the connection subdomain. The carboxy-terminal domain of IN alone retained its interaction with both the fingers-palm and the connection-RNase H fragments of RT, but not with the half connection-RNase H fragment. This interaction was not bridged by nucleic acids, as shown by micrococcal nuclease treatment of the proteins prior to the binding reaction. The influences of IN and RT on each other's activities were investigated by performing RT processivity and IN-mediated 3′ processing and joining reactions in the presence of both proteins. Our results suggest that, while IN had no influence on RT processivity, RT stimulated the IN-mediated strand transfer reaction in a dose-dependent manner up to 155-fold. Thus, a functional interaction between these two viral enzymes may occur during viral replication.
Migration ofAntigen-Specific T Cells Away from CXCR4-Binding Human ImmunodeficiencyVirus Type 1gp120
Authors: Brainard Diana M. , Tharp William G. , Granado Elva , Miller Nicholas , Trocha Alicja K. , Ren Xiang-Hui , Conrad Brian , Terwilliger Ernest F. , Wyatt Richard , Walker Bruce D. , Poznansky Mark C. 
Published: April 2004

ABSTRACTCell-mediated immunity depends in part on appropriate migration and localization of cytotoxic T lymphocytes (CTL), a process regulated by chemokines and adhesion molecules. Many viruses, including human immunodeficiency virus type 1 (HIV-1), encode chemotactically active proteins, suggesting that dysregulation of immune cell trafficking may be a strategy for immune evasion. HIV-1 gp120, a retroviral envelope protein, has been shown to act as a T-cell chemoattractant via binding to the chemokine receptor and HIV-1 coreceptor CXCR4. We have previously shown that T cells move away from the chemokine stromal cell-derived factor 1 (SDF-1) in a concentration-dependent and CXCR4 receptor-mediated manner. Here, we demonstrate that CXCR4-binding HIV-1 X4 gp120 causes the movement of T cells, including HIV-specific CTL, away from high concentrations of the viral protein. This migratory response is CD4 independent and inhibited by anti-CXCR4 antibodies and pertussis toxin. Additionally, the expression of X4 gp120 by target cells reduces CTL efficacy in an in vitro system designed to account for the effect of cell migration on the ability of CTL to kill their target cells. Recombinant X4 gp120 also significantly reduced antigen-specific T-cell infiltration at a site of antigen challenge in vivo. The repellant activity of HIV-1 gp120 on immune cells in vitro and in vivo was shown to be dependent on the V2 and V3 loops of HIV-1 gp120. These data suggest that the active movement of T cells away from CXCR4-binding HIV-1 gp120, which we previously termed fugetaxis, may provide a novel mechanism by which HIV-1 evades challenge by immune effector cells in vivo.
Role of Transmembrane Domain and Cytoplasmic Tail Amino Acid Sequences of Influenza A Virus Neuraminidase in Raft Association and Virus Budding
Authors: Barman Subrata , Adhikary Lopa , Chakrabarti Alok K. , Bernas Carl , Kawaoka Yoshihiro , Nayak Debi P. 
Published: April 2004

ABSTRACT
Influenza virus neuraminidase (NA), a type II transmembrane glycoprotein, possesses receptor-destroying activity and thereby facilitates virus release from the cell surface. Among the influenza A viruses, both the cytoplasmic tail (CT) and transmembrane domain (TMD) amino acid sequences of NA are highly conserved, yet their function(s) in virus biology remains unknown. To investigate the role of amino acid sequences of the CT and TMD on the virus life cycle, we systematically mutagenized the entire CT and TMD of NA by converting two to five contiguous amino acids to alanine. In addition, we also made two chimeric NA by replacing the CT proximal one-third amino acids of the NA TMD [NA(1T2N)NA] and the entire NA TMD (NATRNA) with that of human transferrin receptor (TR) (a type II transmembrane glycoprotein). We rescued transfectant mutant viruses by reverse genetics and examined their phenotypes. Our results show that all mutated and chimeric NAs could be rescued into transfectant viruses. Different mutants showed pleiotropic effects on virus growth and replication. Some mutants (NA2A5, NA3A7, and NA4A10) had little effect on virus growth while others (NA3A2, NA5A27, and NA5A31) produced about 50- to 100-fold-less infectious virus and still some others (NA5A14, NA4A19, and NA4A23) exhibited an intermediate phenotype. In general, mutations towards the ectodomain-proximal sequences of TMD progressively caused reduction in NA enzyme activity, affected lipid raft association, and attenuated virus growth. Electron microscopic analysis showed that these mutant viruses remained aggregated and bound to infected cell surfaces and could be released from the infected cells by bacterial NA treatment. Moreover, viruses containing mutations in the extreme N terminus of the CT (NA3A2) as well as chimeric NA containing the TMD replaced partially [NA(1T2N)NA] or fully (NATRNA) with TR TMD caused reduction in virus growth and exhibited the morphological phenotype of elongated particles. These results show that although the sequences of NA CT and TMD per se are not absolutely essential for the virus life cycle, specific amino acid sequences play a critical role in providing structural stability, enzyme activity, and lipid raft association of NA. In addition, aberrant morphogenesis including elongated particle formation of some mutant viruses indicates the involvement of NA in virus morphogenesis and budding.
Identification of a Novel Bovine Herpesvirus 1 Transcript Containing a Small Open Reading Frame That Is Expressed in Trigeminal Ganglia of Latently Infected Cattle
Authors: Inman Melissa , Zhou Joe , Webb Heather , Jones Clinton 
Published: April 2004

ABSTRACT

Bovine herpesvirus 1
(BHV-1), like other
Alphaherpesvirinae
subfamily members, establishes latency in sensory neurons. The latency-related (LR) RNA is abundantly expressed during latency, and expression of an LR protein is required for the latency reactivation cycle in cattle. Within LR promoter sequences, a 135-amino-acid open reading frame (ORF) was identified, ORF-E, that is antisense to the LR RNA. ORF-E is also downstream of the gene encoding the major viral transcriptional activator, bICP0. Strand-specific reverse transcription-PCR demonstrated that a transcript containing ORF-E was consistently expressed in trigeminal ganglia (TG) of latently infected calves, productively infected cultured cells, and acutely infected calves. As expected, a late transcript encoding glycoprotein C was not detected in TG of latently infected calves. The ORF-E transcript is polyadenylated and is expressed early when cultured bovine cells are productively infected. Protein coding sequences containing ORF-E were fused to green fluorescent protein (GFP) to examine the cellular localization of the putative protein. In transiently transfected mouse neuroblastoma (neuro-2A) and human neuroblastoma (SK-N-SH) cells, the ORF-E/GFP fusion protein was detected in discreet domains within the nucleus. In contrast, the ORF-E/GFP fusion protein was detected in the cytoplasm and nucleus of rabbit skin cells and bovine kidney cells. As expected, the GFP protein was expressed in the cytoplasm and nucleus of transfected cells. These studies indicate that the ORF-E transcript is consistently expressed during latency. We suggest that the ORF-E gene regulates some aspect of the latency reactivation cycle.
Polymer Nanocomposites for Aerospace Applications: Characterization
Authors: Njuguna J. , Pielichowski K. 
Published: April 2004

AbstractDevelopment of several advanced miscroscopic and sprectroscopic techniques have played a key role in the development of characterization and parametric studies of polymer nanocomposites starting from the molecular level. The main issues affecting the production of good quality composites are fiber alignment, fiber‐matrix adhesion and fiber dispersion which makes characterization so vital. The current work reviews the ongoing investigations and techniques to overcome the challenges posed by polymer nanocomposites characterization.
Taking Snapshots of Photoexcited Molecules in Disordered Media by Using Pulsed Synchrotron X‐rays
Authors: Chen Lin X. 
Published: April 2004

AbstractPhotoexcited molecules are quintessential reactants in photochemistry. Structures of these photoexcited molecules in disordered media in which a majority of photochemical reactions take place remained elusive for decades owing to a lack of suitable X‐ray sources, despite their importance in understanding fundamental aspects in photochemistry. As new pulsed X‐ray sources become available, short‐lived excited‐state molecular structures in disordered media can now be captured by using laser‐pulse pump, X‐ray pulse–probe techniques of third‐generation synchrotron sources with time resolutions of 30–100 ps, as demonstrated by examples in this review. These studies provide unprecedented information on structural origins of molecular properties in the excited states. By using other ultrafast X‐ray facilities that will be completed in the near future, time‐resolution for the excited‐state structure measurements should reach the femtosecond timescales, which will make “molecular movies” of bond breaking or formation, and vibrational relaxation, a reality.
Acrylic acid/vinyl acetate suspension copolymerizations. I. Partition coefficients for acrylic acid
Authors: Silva Fabricio Machado , Lima Enrique Luis , Pinto José Carlos 
Published: April 2004

AbstractDuring acrylic acid (AA)/vinyl acetate (VAc) suspension copolymerizations, AA is partitioned between the organic phase and the aqueous phase. For this reason, the AA partition coefficient in suspensions of AA/VAc/water is studied here. Partition experiments were performed at different temperatures and AA concentrations. It was observed that the AA partition coefficient ranged from 0.001 to 1.58, depending strongly on the temperature and on the aqueous phase composition. Therefore, the distribution of AA between the continuous phase and the monomer droplets may exert a significant effect upon the observed kinetic behavior during suspension polymerizations. The UNIFAC‐LLE model was used to describe the observed liquid–liquid equilibrium of the ternary system AA/VAc/water and model predictions were compared to the obtained experimental data. It was observed that the UNIFAC‐LLE model is able to represent AA partition coefficients qualitatively only when AA dissociation effects are taken into consideration. Additional experiments also indicated that AA partition coefficients may be very sensitive to the presence of polymer in the organic phase and to the presence of salt in the aqueous phase, as usual during actual suspension copolymerizations. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1077–1088, 2004
Assessing organo‐clay dispersion in polymer nanocomposites
Authors: Eckel Deborah F. , Balogh Michael P. , Fasulo Paula D. , Rodgers William R. 
Published: April 2004

AbstractOrgano‐clay polymer nanocomposites offer improved material properties at very low filler loadings making them of immediate interest for application in body panels, claddings, and instrument panels. This improvement in properties requires that the organo‐clay be well dispersed if not completely exfoliated. Conventionally, the dispersion and exfoliation of the organo‐clay is evaluated using transmission electron microscopy (TEM) and X‐ray diffraction (XRD). Although both TEM and XRD data were found to correlate with flexural modulus of thermoplastic olefin nanocomposite materials, only TEM proved successful in quantifying the dispersion of the organo‐clay in all nanocomposite materials (exfoliated, tactoid, or agglomerated tactoid). XRD was found to be capable of detecting exfoliation and intercalation but is limited because of clay dilution, preferred orientation, mixed‐layering, and other peak broadening factors. © 2004 Wiley Periodicals, Inc. J Appl Polym Sci 93: 1110–1117, 2004
Automatic generation of domain representations using thesaurus structures
Authors: Lloréns Juan , Velasco Manuel , de Amescua Antonio , Moreiro José A. , Martínez Vicente 
Published: April 2004

AbstractDomain analysis was first used 15 years ago as one of the most important techniques for software reuse. Even today, new techniques appear every year, and different authors propose different domain representation structures to represent and store all the different software components and the relationships among them. These relationships among components are the kernel of the domain semantics. In this report, a set of techniques and tools is presented regarding mathematical, statistical, and neural fields that, when linked together, enable semi‐automatically building domain representations and storing them in a thesaurus structure of software components. Thesaurus structures, widely used in information science, are presented as the domain‐modeling key concept, due to their higher automation possibilities compared with previous structures. New metrics to evaluate the quality, consistency, and completeness of the domain model obtained through this technique are also presented.
Dynamic Chemical Devices: Modulation of Photophysical Properties by Reversible, Ion‐Triggered, and Proton‐Fuelled Nanomechanical Shape‐Flipping Molecular Motions
Authors: Barboiu Mihail , Prodi Luca , Montalti Marco , Zaccheroni Nelsi , Kyritsakas Nathalie , Lehn Jean‐Marie 
Published: April 2004

AbstractThe terpy‐derived (terpy=terpyridine) ligand 1 has an extended W shape in which the two appended photoactive pyrenyl groups are held apart. On binding of a zinc(II) ion with a terpy group, ligand 1 is converted into complex 2 whereby it adopts a U shape, thus stacking the aromatic units. This structural modification leads to a very pronounced change in photophysical properties: from a highly fluorescent free ligand to a very weakly emitting complex. The W/U structural switching can be reversibly induced by the addition of a competitive tren ligand, which binds and releases a zinc(II) ion under protonation/deprotonation cycles, thus leading to oscillations in light emission. Therefore, the present system performs periodic modulation of optical output through a nanomechanical shape‐flipping motion, triggered by metal ion binding and fuelled by acid–base neutralisation energy. Overall, it represents an ion‐triggered opto‐mechanical supramolecular device.
Highly Efficient Biocatalytic Resolution of cis‐ and trans‐3‐Aminoindan‐1‐ol: Syntheses of Enantiopure Orthogonally Protected cis‐ and trans‐Indane‐1,3‐diamine
Authors: López‐García Mónica , Alfonso Ignacio , Gotor Vicente 
Published: April 2004

AbstractThe efficient chemoenzymatic synthesis of enantiopure 1,3‐difunctionalized indane derivatives has been achieved. The corresponding cis and trans N‐protected amino alcohols were successfully resolved by acetylation using lipase B, which is a biocatalyst isolated from Candida antarctica. All the possible isomers were obtained in very good chemical yields and ee values (>99 %). The utility of these compounds was subsequently shown by the preparation of orthogonally protected cis‐ and trans‐indane‐1,3‐diamine using a Mitsunobu reaction. Both enantiomers of the trans isomer and a desymmetrized cis diastereomer were prepared in enantiopure form. Complete inversion of configuration during the Mitsunobu reaction was demonstrated by a combination of NMR techniques and molecular modeling. The utility and versatility of the strategy was also demonstrated by the selective deprotection of each nitrogen atom under mild reaction conditions.
Phase II study of oral S‐1 for treatment of metastatic colorectal carcinoma
Authors: Shirao Kuniaki , Ohtsu Atsushi , Takada Hideho , Mitachi Yasushi , Hirakawa Kosei , Horikoshi Noboru , Okamura Takeshi , Hirata Koichi , Saitoh Soh , Isomoto Hiroharu , Satoh Atsushi 
Published: April 2004

AbstractBACKGROUNDThe goal of the current study was to evaluate the objective response rate and toxicity associated with the oral fluoropyrimidine S‐1 (a combination of tegafur, 5‐chloro‐2,4‐dihydroxypyridine, and potassium oxonate) in patients with previously untreated metastatic colorectal carcinoma.METHODSThirty‐eight patients were enrolled in the study. S‐1 was administered orally at a dose of 40 mg/m2 twice daily for 28 days, followed by a 14‐day rest period. Treatment was repeated every 6 weeks unless disease progression was observed.RESULTSA combined total of 173 courses of S‐1 were administered to the 38 enrolled patients. The median number of courses administered to a given patient was 3.5 (range, 1–18). Although no patient exhibited a complete response to treatment, 15 had partial responses (response rate, 39.5%; 95% confidence interval, 24.0–56.6%). In addition, 5 patients had minor responses, and 14 had stable disease. Four patients were found to have progressive disease after two courses of treatment. The median survival time was 358 days (95% confidence interval, 305–490 days), and the 1‐year survival rate was 47.4%. The most common adverse reactions included myelosuppression and gastrointestinal toxicity; most cases involved Grade 1 or 2 toxicity, but Grade 3 toxicities (anemia [7.9% of patients], neutropenia [5.3% of patients], diarrhea [2.6% of patients], and abnormal bilirubin levels [7.9% of patients]) also were noted. Neither Grade 4 toxicity nor treatment‐related death was observed during the study.CONCLUSIONSOrally administered S‐1 is active against metastatic colorectal carcinoma and has an acceptable toxicity profile. This promising agent has the potential to become a valuable chemotherapeutic option. Cancer 2004. © 2004 American Cancer Society.