bluuebunny/crossref_metadata_2025 huggingface dataset:
Detection of
Chlamydia trachomatis
and
Neisseria gonorrhoeae
Infections in North American Women by Testing SurePath Liquid-Based Pap Specimens in APTIMA Assays
Authors: Chernesky Max , Freund Gregory G. , Hook Edward , Leone Peter , D'Ascoli Peter , Martens Mark
Published: June 2007

The APTIMA COMBO 2 assay, which detects and amplifies rRNA from
Chlamydia trachomatis
and/or
Neisseria gonorrhoeae
, is approved for use on ThinPrep liquid-based Pap test specimens. The objective was to determine the clinical utility of the APTIMA assays (APTIMA COMBO 2 assay, APTIMA CT assay for
Chlamydia trachomatis
, and APTIMA GC assay for
Neisseria gonorrhoeae
) for screening women during their annual Pap exam, using SurePath liquid-based Pap test specimens. Two cervical samples were collected from 1,615 females attending six clinical sites in North America. A cervical broom sample was processed for cytology, with the residuum aliquoted into an APTIMA specimen transfer kit tube. The second cervical swab sample was put into APTIMA specimen transport medium, and both samples were tested with each APTIMA assay on a direct sampling system. Using a subject-infected status that utilized cervical-swab specimen results from two APTIMA assays, the prevalence was 7.9% for
Chlamydia trachomatis
and 2.5% for
N. gonorrhoeae
. For the liquid-based Pap samples, the sensitivities, specificities, positive predictive values, and negative predictive values for
Chlamydia trachomatis
detection were 85.2%, 99.5%, 93.2%, and 98.7%, respectively, for the APTIMA COMBO 2 assay and 89.1%, 98.7%, 85.7%, and 99.1%, respectively, for the APTIMA CT assay. For
N. gonorrhoeae
detection, the values were 92.5%, 100%, 100%, and 99.8%, respectively, for the APTIMA COMBO 2 assay and 92.5%, 99.9%, 97.4%, and 99.8%, respectively, for the APTIMA GC assay. The high predictive values support the use of the assays with SurePath liquid-based Pap specimens processed with the APTIMA specimen transfer kit.
Evaluation of Methods To Identify the
Klebsiella pneumoniae
Carbapenemase in
Enterobacteriaceae
Authors: Anderson K. F. , Lonsway D. R. , Rasheed J. K. , Biddle J. , Jensen B. , McDougal L. K. , Carey R. B. , Thompson A. , Stocker S. , Limbago B. , Patel J. B.
Published: June 2007

The
Klebsiella pneumoniae
carbapenem (KPC) β-lactamase occurs in
Enterobacteriaceae
and can confer resistance to all β-lactam agents including carbapenems. The enzyme may confer low-level carbapenem resistance, and the failure of susceptibility methods to identify this resistance has been reported. Automated and nonautomated methods for carbapenem susceptibility were evaluated for identification of KPC-mediated resistance. Ertapenem was a more sensitive indicator of KPC resistance than meropenem and imipenem independently of the method used. Carbapenemase production could be confirmed with the modified Hodge test.
Relevance of Routine Use of the Anaerobic Blood Culture Bottle
Authors: Grohs Patrick , Mainardi Jean-Luc , Podglajen Isabelle , Hanras Xavier , Eckert C. , Buu-Hoï A. , Varon E. , Gutmann Laurent
Published: June 2007

Using the BacT/Alert automated system, we conducted a 1-year retrospective study on blood cultures, focusing on the relevance of routine use of the anaerobic bottle. The rate of patients with positive blood cultures was 19.7%. Among these, 13.5% had a positive anaerobic bottle in the absence of any aerobic bottle, and 2/3 of these grew with nonobligate anaerobes. These patients were hospitalized in 20 out of 26 wards of the hospital group. For 65.4% of the monomicrobial-positive blood cultures growing
Enterobacteriaceae
, the anaerobic bottle detected growth earlier than the corresponding aerobic bottle. These data suggest that, in our institution, the use of an anaerobic bottle is still relevant.
The Emergence and Importation of Diverse Genotypes of Methicillin-Resistant
Staphylococcus aureus
(MRSA) Harboring the Panton-Valentine Leukocidin Gene (
pvl
) Reveal that
pvl
Is a Poor Marker for Community-Acquired MRSA Strains in Ireland
Authors: Rossney Angela S. , Shore Anna C. , Morgan Pamela M. , Fitzgibbon Margaret M. , O'Connell Brian , Coleman David C.
Published: June 2007

Community-acquired methicillin-resistant
Staphylococcus aureus
(CA-MRSA) carrying
pvl
is an emerging problem worldwide. CA-MRSA tends to harbor staphylococcal cassette chromosome
mec
type IV (SCC
mec
IV), to be non-multiantibiotic resistant, and to have different genotypes from the local hospital-acquired MRSA (HA-MRSA). However, in Ireland, 80% of HA-MRSA isolates have the non-multiantibiotic-resistant genotype ST22-MRSA-IV. This study investigated MRSA isolates from Ireland (CA-MRSA, health care-associated MRSA, and HA-MRSA) for the carriage of
pvl
and determined the genotypic characteristics of all
pvl
-positive isolates identified. All 1,389 MRSA isolates were investigated by antibiogram-resistogram typing and SmaI DNA macrorestriction analysis.
pvl
-positive isolates were further characterized by multilocus sequence typing and SCC
mec
,
agr
, and toxin gene typing. Twenty-five (1.8%) MRSA isolates belonging to six genotypes (ST30, ST8, ST22, ST80, ST5, and ST154) harbored
pvl
. Nineteen of these (76%) were CA-MRSA isolates, but a prospective study of MRSA isolates from 401 patients showed that only 6.7% (2/30) of patients with CA-MRSA yielded
pvl
-positive isolates. Thus,
pvl
cannot be used as a sole marker for CA-MRSA. Fifty-two percent of
pvl
-positive MRSA isolates were recovered from patients with skin and soft tissue infections; thirty-six percent were from patients of non-Irish ethnic origin, reflecting the increasing heterogeneity of the Irish population due to immigration. All 25
pvl
-positive isolates carried SCC
mec
IV; 14 (56%) harbored SCC
mec
IV.1 or IV.3, and the remaining 11 isolates could not be subtyped. This study demonstrates that
pvl
is not a reliable marker for CA-MRSA in Ireland and reveals the emergence and importation of diverse genotypes of
pvl
-positive MRSA in Ireland.
Changes in Karyotype and Azole Susceptibility of Sequential Bloodstream Isolates from Patients with
Candida glabrata
Candidemia
Authors: Shin Jong Hee , Chae Myung Jong , Song Jeong Won , Jung Sook-In , Cho Duck , Kee Seung Jung , Kim Soo Hyun , Shin Myung Geun , Suh Soon Pal , Ryang Dong Wook
Published: June 2007

We examined the changes in genotypes and azole susceptibilities among sequential bloodstream isolates of
Candida glabrata
during the course of fungemia and the relationship of these changes to antifungal therapy. Forty-one isolates were obtained from 15 patients (9 patients who received antifungal therapy and 6 patients who did not) over periods of up to 36 days. The isolates were analyzed using pulsed-field gel electrophoresis (PFGE) and multilocus sequence typing (MLST) and tested for antifungal susceptibility to fluconazole, itraconazole, and voriconazole. PFGE typing consisted of electrophoretic karyotyping and restriction endonuclease analysis of genomic DNA by use of NotI (REAG-N). The 41 isolates yielded 23 different karyotypes and 11 different REAG-N patterns but only 3 MLST types. The sequential strains from each patient had identical or similar REAG-N patterns. However, they had two or three different karyotypes in 6 (40%) of 15 patients. The isolates from these six patients exhibited the same or similar azole susceptibilities, and five patients did not receive antifungal therapy. Development of acquired azole resistance in sequential isolates was detected for only one patient. For this patient, an isolate of the same genotype obtained after azole therapy showed three- or fourfold increases in the MICs of all three azole antifungals and exhibited increased expression of the
CgCDR1
efflux pump. This study shows that karyotypic changes can develop rapidly among sequential bloodstream strains of
C. glabrata
from the same patient without antifungal therapy. In addition, we confirmed that
C. glabrata
could acquire azole resistance during the course of fungemia in association with azole therapy.
Evaluation of Gen-Probe APTIMA-Based
Neisseria gonorrhoeae
and
Chlamydia trachomatis
Confirmatory Testing in a Metropolitan Setting of High Disease Prevalence
Authors: Munson Erik , Boyd Vivian , Czarnecka Jolanta , Griep Judy , Lund Brian , Schaal Nancy , Hryciuk Jeanne E.
Published: June 2007

Prompted by reports challenging the validity of the low-positive
Neisseria gonorrhoeae
and
Chlamydia trachomatis
results generated by the APTIMA Combo 2 assay (Gen-Probe, Incorporated) and by a Centers for Disease Control and Prevention recommendation to confirm
N. gonorrhoeae
- or
C. trachomatis
-positive screens by using an alternative amplification target, we report on a comparison of this means of confirmation with an in-house algorithm of repeat testing. Primary clinical specimens yielding
N. gonorrhoeae
- or
C. trachomatis
-specific luminescent values between 100,000 and 1,000,000 were repeat tested in duplicate. A subset of specimens was forwarded for confirmatory assays (Gen-Probe) individualized for alternative
N. gonorrhoeae
or
C. trachomatis
targets. An 18-month audit revealed that 230 of 29,977
C. trachomatis
screens (0.8%) and 41 of 29,064
N. gonorrhoeae
assays (0.1%) yielded low-positive data. When a subset of 40 low-positive
N. gonorrhoeae
screens was repeat tested, 20 (50.0%) remained positive; 22 (55.0%) of the screens remained positive following performance of the confirmatory assay. In contrast, repeat testing of 153 low-positive
C. trachomatis
screens yielded a positive result for fewer specimens (
n
= 97; 63.4%) than when commercial confirmatory testing was used (
n
= 124; 81.0%). However, confirmation of the results for additional
C. trachomatis
screens by use of an alternative target did not translate into significant differences in the calculated overall
C. trachomatis
-positive screen rates (7.39% by repeat testing versus 7.52% by the confirmatory assay;
P
= 0.53). Furthermore, use of the confirmatory assay raised the positive predictive value only 1.8% over that of repeat testing. Molecular confirmatory testing did not significantly enhance the reliability of
C. trachomatis
- or
N. gonorrhoeae
-specific nucleic acid amplification testing in this metropolitan setting compared to the reliability of repeat testing.
Development of a Sensitive and Specific Multiplex PCR Method Combined with DNA Microarray Primer Extension To Detect Betapapillomavirus Types
Authors: Gheit Tarik , Billoud Gaëlle , de Koning Maurits N. C. , Gemignani Federica , Forslund Ola , Sylla Bakary S. , Vaccarella Salvatore , Franceschi Silvia , Landi Stefano , Quint Wim G. V. , Canzian Federico , Tommasino Massimo
Published: June 2007

Emerging lines of evidence indicate that the cutaneous human papillomavirus (HPV) types that belong to the genus
Betapapillomavirus
(beta HPV) are involved in the development of nonmelanoma skin cancer. Unlike the situation for mucosal HPV types, highly sensitive and reliable methods to identify characterized cutaneous HPV types in a single assay are limited. Here, we describe a novel one-shot method for the detection of all characterized beta HPV types, namely, HPV type 5 (HPV5), 8, 9, 12, 14, 15, 17, 19, 20, 21, 22, 23, 24, 25, 36, 37, 38, 47, 49, 75, 76, 80, 92, 93, and 96. This assay combines two different techniques: multiplex PCR using HPV type-specific primers for amplification of each E7 gene and array primer extension (APEX) for typing. This method has been validated using clinical samples which were analyzed simultaneously for the presence of cutaneous HPV types by two additional methods, i.e., the FAP59/64 PCR protocol and a commercially available PCR-reverse hybridization assay (PM-PCR RHA). Our data show good agreement between the results obtained with the multiplex PCR/APEX assay and the PM-PCR RHA method (overall HPV positivity of 92.2% for multiplex PCR/APEX assay versus 90.6% with the PM-PCR RHA) (kappa value, 50; 95% confidence interval, 13 to 88). In addition, the multiplex PCR/APEX assay showed higher sensitivity than the PM-PCR RHA did. This favorable feature and the high-throughput potential make this assay ideal for large-scale clinical and epidemiological studies aimed at determining the spectrum of cutaneous types in skin cancer.
Characterization of a Catalase-Negative Methicillin-Resistant
Staphylococcus aureus
Strain
Authors: Grüner B. M. , Han S.-R. , Meyer H.-G. , Wulf U. , Bhakdi S. , Siegel E. K.
Published: June 2007

We describe an unusual clinical strain of catalase-negative methicillin-resistant
Staphylococcus aureus
sensu stricto. Sequence analysis of its catalase gene showed 99.60% identities to the catalase genes of the reference strains. A 5-base deletion, however, led to a shift of the nucleotide reading frame and a loss of the enzymatic activity.
Nosocomial Outbreak Due to Extended-Spectrum-Beta-Lactamase- Producing
Enterobacter cloacae
in a Cardiothoracic Intensive Care Unit
Authors: Manzur Adriana , Tubau Fe , Pujol Miquel , Calatayud Laura , Dominguez Maria Angeles , Peña Carmen , Sora Mercedes , Gudiol Francesc , Ariza Javier
Published: June 2007

Enterobacter cloacae
has been associated with several outbreaks, usually involving strains that overproduce chromosomal β-lactamase or, uncommonly, strains expressing extended-spectrum β-lactamases (ESBL). Only sporadic cases of ESBL-producing
E. cloacae
have been identified in our hospital in recent years. We describe the epidemiology and clinical and microbiological characteristics of an outbreak caused by ESBL-producing
E. cloacae
in a cardiothoracic intensive care unit (CT-ICU). Prospective surveillance of patients with infection or colonization by ESBL-producing
E. cloacae
among patients admitted to the CT-ICU was performed during the outbreak. Production of ESBL was determined by decreased susceptibility to expanded-spectrum cephalosporins and a positive double-disk test result. Clone relatedness was determined by pulsed-field gel electrophoresis (PFGE). From July to September 2005, seven patients in the CT-ICU with ESBL-producing
E. cloacae
were identified (four males; median age, 73 years; range, 45 to 76 years); six patients had cardiac surgery. Four patients developed infections; three had primary bacteremia, one had ventilator-associated pneumonia, and one had tracheobronchitis. ESBL-producing
E. cloacae
showed resistance to quinolones and aminoglycosides. PFGE revealed two patterns. Five isolates belonged to clone A; two carried a single ESBL (pI 8.2 and a positive PCR result for the SHV type), and three carried two ESBLs (pIs 8.1 and 8.2 and positive PCR results for the SHV and CTX-M-9 types). Isolates belonging to clone B carried a single ESBL (pI 5.4 and a positive PCR result for the TEM type). Review of antibiotic consumption showed increased use of cefepime and quinolones during June and July 2005. The outbreak was stopped by the implementation of barrier measures and cephalosporin restriction. ESBL production could be increasingly common in nosocomial pathogens other than
Escherichia coli
or
Klebsiella pneumoniae
.
A Membrane Leucine Heptad Contributes to Trafficking, Signaling, and Transformation by Latent Membrane Protein 1
Authors: Lee Jisook , Sugden Bill
Published: June 2007

Latent membrane protein 1 (LMP1) of Epstein Barr virus (EBV) is important for maintaining proliferation of EBV-infected B cells. LMP1, unlike its cellular counterpart, CD40, signals without a ligand and is largely internal to the plasma membrane. In order to understand how LMP1 initiates its ligand-independent signaling, we focused on a leucine heptad in LMP1's first membrane-spanning domain that was shown to be necessary for LMP1's signaling through NF-κB. LZ1EBV, a recombinant EBV genetically altered to express LZ1, a derivative of LMP1 in which a leucine heptad was replaced with alanines, transformed B cells with 56% of wild-type (wt) EBV's efficiency, demonstrating the importance of this heptad. To elucidate the mechanism by which this domain contributes to the functions of LMP1, the properties of the wt and LZ1 were compared in transfected cells. LZ1 failed to home to lipid rafts as efficiently as did wt LMP1. The distribution of tagged derivatives of LZ1 also differed from that of wt LMP1 in transfected cells. LZ1's defect in homing to lipid rafts and altered trafficking likely underlie the defect in transformation of LZ1EBV. While the third and fourth membrane-spanning domains of LMP1 foster its trafficking to the Golgi, the leucine heptad within the first membrane-spanning domain contributes to its trafficking, particularly to internal rafts. B cells that are successfully transformed by LZ1EBV have the same average number of viral genomes and the same fraction of cells with capped LZ1 at the cell surface but express 50% more of the LZ1 allele than wt infected cells.
Baculovirus Caspase Inhibitors P49 and P35 Block Virus-Induced Apoptosis Downstream of Effector Caspase DrICE Activation in
Drosophila melanogaster
Cells
Authors: Lannan Erica , Vandergaast Rianna , Friesen Paul D.
Published: June 2007

Baculoviruses induce widespread apoptosis in invertebrates. To better understand the pathways by which these DNA viruses trigger apoptosis, we have used a combination of RNA silencing and overexpression of viral and host apoptotic regulators to identify cell death components in the model system of
Drosophila melanogaster
. Here we report that the principal effector caspase DrICE is required for baculovirus-induced apoptosis of
Drosophila
DL-1 cells as demonstrated by RNA silencing. proDrICE was proteolytically cleaved and activated during infection. Activation was blocked by overexpression of the cellular inhibitor-of-apoptosis proteins DIAP1 and SfIAP but not by the baculovirus caspase inhibitor P49 or P35. Rather, the substrate inhibitors P49 and P35 prevented virus-induced apoptosis by arresting active DrICE through formation of stable inhibitory complexes. Consistent with a two-step activation mechanism, proDrICE was cleaved at the large/small subunit junction TETD
230
-G by a DIAP1-inhibitable, P49/P35-resistant protease and then at the prodomain junction DHTD
28
-A by a P49/P35-sensitive protease. Confirming that P49 targeted DrICE and not the initiator caspase DRONC, depletion of DrICE by RNA silencing suppressed virus-induced cleavage of P49. Collectively, our findings indicate that whereas DIAP1 functions upstream to block DrICE activation, P49 and P35 act downstream by inhibiting active DrICE. Given that P49 has the potential to inhibit both upstream initiator caspases and downstream effector caspases, we conclude that P49 is a broad-spectrum caspase inhibitor that likely provides a selective advantage to baculoviruses in different cellular backgrounds.
Epstein-Barr Virus Latent Membrane Protein 2A Mediates Transformation through Constitutive Activation of the Ras/PI3-K/Akt Pathway
Authors: Fukuda Makoto , Longnecker Richard
Published: June 2007

Epstein-Barr virus (EBV) latent membrane protein 2A (LMP2A) is widely expressed in EBV-infected cells within the infected human host and EBV-associated malignancies, suggesting that LMP2A is important for EBV latency, persistence, and EBV-associated tumorigenesis. Previously, we demonstrated that LMP2A provides an antiapoptotic signal through the activation of phosphatidylinositol 3-kinase (PI3-K)/Akt pathway in vitro. However, the exact function of LMP2A in tumor progression is not well understood. In this study, we found that LMP2A did not induce anchorage-independent cell growth in a human keratinocyte cell line, HaCaT, but did in a human gastric carcinoma cell line, HSC-39. In addition, LMP2A activated the PI3-K/Akt pathway in both HaCaT and HSC-39 cells; however, LMP2A did not activate Ras in HaCaT cells but did in HSC-39 cells. Furthermore, the Ras inhibitors manumycin A and a dominant-negative form of Ras (RasN17) and the PI3-K inhibitor LY294002 blocked LMP2A-mediated Akt phosphorylation and anchorage-independent cell growth in HSC-39 cells. These results suggest that constitutive activation of the Ras/PI3-K/Akt pathway by LMP2A is a key factor for LMP2A-mediated transformation.
Binding-Site Interactions between Epstein-Barr Virus Fusion Proteins gp42 and gH/gL Reveal a Peptide That Inhibits both Epithelial and B-Cell Membrane Fusion
Authors: Kirschner Austin N. , Lowrey Amanda S. , Longnecker Richard , Jardetzky Theodore S.
Published: June 2007

Herpesviruses require membrane-associated glycoproteins gB, gH, and gL for entry into host cells. Epstein-Barr virus (EBV) gp42 is a unique protein also required for viral entry into B cells. Key interactions between EBV gp42 and the EBV gH/gL complex were investigated to further elucidate their roles in membrane fusion. Deletion and point mutants within the N-terminal region of gp42 revealed residues important for gH/gL binding and membrane fusion. Many five-residue deletion mutants in the N-terminal region of gp42 that exhibit reduced membrane fusion activity retain binding with gH/gL but map out two functional stretches between residues 36 and 96. Synthetic peptides derived from the gp42 N-terminal region were studied in in vitro binding experiments with purified gH/gL and in cell-cell fusion assays. A peptide spanning gp42 residues 36 to 81 (peptide 36-81) binds gH/gL with nanomolar affinity, comparable to full-length gp42. Peptide 36-81 efficiently inhibits epithelial cell membrane fusion and competes with soluble gp42 to inhibit B-cell fusion. Additionally, this peptide at low nanomolar concentrations inhibits epithelial cell infection by intact virus. Shorter gp42 peptides spanning the two functional regions identified by deletion mutagenesis had little or no binding to soluble gH/gL and were also unable to inhibit epithelial cell fusion, nor could they complement gp42 deletion mutants in B-cell fusion. These studies identify key residues of gp42 that are essential for gH/gL binding and membrane fusion activation, providing a nanomolar inhibitor of EBV-mediated membrane fusion.
Macrophages Transmit Human Immunodeficiency Virus Type 1 Products to CD4-Negative Cells: Involvement of Matrix Metalloproteinase 9
Authors: Muratori Claudia , Sistigu Antonella , Ruggiero Eliana , Falchi Mario , Bacigalupo Ilaria , Palladino Clelia , Toschi Elena , Federico Maurizio
Published: June 2007

It was previously reported that human immunodeficiency virus type 1 (HIV-1) spreads in CD4 lymphocytes through cell-to-cell transmission. Here we report that HIV-1-infected macrophages, but not lymphocytes, transmit HIV-1 products to CD4-negative cells of either epithelial, neuronal, or endothelial origin in the absence of overt HIV-1 infection. This phenomenon was detectable as early as 1 h after the start of cocultivation and depended on cell-to-cell contact but not on the release of viral particles from donor cells. Transfer of HIV-1 products occurred upon their polarization and colocalization within zones of cell-to-cell contact similar to virological synapses. Neither HIV-1 Env nor Nef expression was required but, interestingly, we found that an HIV-1-dependent increase in matrix metalloproteinase 9 production from donor cells significantly contributed to the cell-to-cell transmission of the viral products. The macrophage-driven transfer of HIV-1 products to diverse CD4-negative cell types may have a significant role in AIDS pathogenesis.
The ORF3 Protein of Porcine Circovirus Type 2 Interacts with Porcine Ubiquitin E3 Ligase Pirh2 and Facilitates p53 Expression in Viral Infection
Authors: Liu Jue , Zhu Yu , Chen Isabelle , Lau Jennifer , He Fang , Lau Adeline , Wang Zhilong , Karuppannan Anbu K. , Kwang Jimmy
Published: June 2007

Porcine circovirus type 2 (PCV2) is the primary causative agent of an emerging swine disease, postweaning multisystemic wasting syndrome. We previously showed that a newly identified protein, ORF3, plays a major role in virus-induced apoptosis and is involved in viral pathogenesis in vitro and in vivo. To characterize the role of the ORF3 protein in modulation of cellular function, a yeast two-hybrid system was used to screen a porcine cDNA library to find its interacting partner. We have isolated and characterized pPirh2 (for “porcine p53-induced RING-H2”), an E3 ubiquitin ligase, which specifically interacts with the ORF3 protein of PCV2. This interaction was further confirmed when the ORF3 protein coimmunoprecipitated with and colocalized to pPirh2 in PK15 cells. The ORF3 protein has been found to interact with the p53 binding domain of pPirh2 in yeast cells. Expression of the protein results in less pPirh2 expression in PCV2-infected cells. Furthermore, increases in p53 expression were observed in PCV2-infected and ORF3 (alone)-transfected cells. Phosphorylation of p53 at Ser-46, which is related to p53-induced apoptosis, was also time-dependently activated in PCV-infected and ORF3-transfected cells. Taken together, our results show that the PCV2 ORF3 protein specifically interacts with pPirh2 and inhibits its stabilization; this may lead to increasing p53 expression, resulting in apoptosis.
A Putative Leucine Zipper within the Herpes Simplex Virus Type 1 UL6 Protein Is Required for Portal Ring Formation
Authors: Nellissery Jacob K. , Szczepaniak Renata , Lamberti Carmela , Weller Sandra K.
Published: June 2007

The herpes simplex virus type 1 UL6 protein forms a 12-subunit ring structure at a unique capsid vertex which functions as a conduit for encapsidation of the viral genome. To characterize UL6 protein domains that are involved in intersubunit interactions and interactions with other capsid proteins, we engineered a set of deletion mutants spanning the entire gene. Three deletion constructs, D-5 (Δ198-295), D-6 (Δ322-416), and D-LZ (Δ409-473, in which a putative leucine zipper was removed), were introduced into the viral genome. All three mutant viruses produced only B capsids, indicating a defect in encapsidation. Western blot analysis showed that the UL6 protein was present in the capsids isolated from two mutants, D-6 and D-LZ. The protein encoded by D-5, on the other hand, was not associated with capsids and was instead localized in the cytoplasm of the infected cells, indicating that this deletion affected the nuclear transport of the portal protein. The UL6 protein from the KOS strain (wild type) and the D-6 mutant were purified from insect cells infected with recombinant baculoviruses and shown to form ring structures as assessed by sucrose gradient centrifugation and electron microscopy. In contrast, the D-LZ mutant protein formed aggregates that sedimented throughout the sucrose gradient as a heterogeneous mixture and did not yield stable ring structures. A mutant (L429E L436E) in which two of the heptad leucines of the putative zipper were replaced with glutamate residues also failed to form stable rings. Our results suggest that the integrity of the leucine zipper region is important for oligomer interactions and stable ring formation, which in turn are required for genome encapsidation.
An RNA Pseudoknot in the 3′ End of the Arterivirus Genome Has a Critical Role in Regulating Viral RNA Synthesis
Authors: Beerens Nancy , Snijder Eric J.
Published: June 2007

In the life cycle of plus-strand RNA viruses, the genome initially serves as the template for both translation of the viral replicase gene and synthesis of minus-strand RNA and is ultimately packaged into progeny virions. These various processes must be properly balanced to ensure efficient viral proliferation. To achieve this, higher-order RNA structures near the termini of a variety of RNA virus genomes are thought to play a key role in regulating the specificity and efficiency of viral RNA synthesis. In this study, we have analyzed the signals for minus-strand RNA synthesis in the prototype of the arterivirus family, equine arteritis virus (EAV). Using site-directed mutagenesis and an EAV reverse genetics system, we have demonstrated that a stem-loop structure near the 3′ terminus of the EAV genome is required for RNA synthesis. We have also obtained evidence for an essential pseudoknot interaction between the loop region of this stem-loop structure and an upstream hairpin residing in the gene encoding the nucleocapsid protein. We propose that the formation of this pseudoknot interaction may constitute a molecular switch that could regulate the specificity or timing of viral RNA synthesis. This hypothesis is supported by the fact that phylogenetic analysis predicted the formation of similar pseudoknot interactions near the 3′ end of all known arterivirus genomes, suggesting that this interaction has been conserved in evolution.
Antibody Recognition of Cell Surface-Associated NS1 Triggers Fc-γ Receptor-Mediated Phagocytosis and Clearance of West Nile Virus-Infected Cells
Authors: Chung Kyung Min , Thompson Bruce S. , Fremont Daved H. , Diamond Michael S.
Published: June 2007

Previous studies have suggested that monoclonal antibodies (MAbs) to flavivirus nonstructural protein-1 (NS-1) protect against infection in mice through an Fc-γ receptor-dependent pathway. To identify a specific mechanism, we evaluated the protective activity of anti-NS1 MAbs to WNV using mice and cells with deficiencies of specific Fc-γ receptors. Our results suggest that only MAbs that recognize cell surface-associated NS1 trigger Fc-γ receptor I- and/or IV-mediated phagocytosis and clearance of WNV-infected cells. These findings may be relevant for generating novel therapeutic MAbs or vaccines against flaviviruses that target the NS1 protein.
Characterization of Chimpanzee/Human Monoclonal Antibodies to Vaccinia Virus A33 Glycoprotein and Its Variola Virus Homolog In Vitro and in a Vaccinia Virus Mouse Protection Model
Authors: Chen Zhaochun , Earl Patricia , Americo Jeffrey , Damon Inger , Smith Scott K. , Yu Fujuan , Sebrell Andrew , Emerson Suzanne , Cohen Gary , Eisenberg Roselyn J. , Gorshkova Inna , Schuck Peter , Satterfield William , Moss Bernard , Purcell Robert
Published: June 2007

Three distinct chimpanzee Fabs against the A33 envelope glycoprotein of vaccinia virus were isolated and converted into complete monoclonal antibodies (MAbs) with human γ1 heavy-chain constant regions. The three MAbs (6C, 12C, and 12F) displayed high binding affinities to A33 (

K
d

of 0.14 nM to 20 nM) and may recognize the same epitope, which was determined to be conformational and located within amino acid residues 99 to 185 at the C terminus of A33. One or more of the MAbs were shown to reduce the spread of vaccinia virus as well as variola virus (the causative agent of smallpox) in vitro and to more effectively protect mice when administered before or 2 days after intranasal challenge with virulent vaccinia virus than a previously isolated mouse anti-A33 MAb (1G10) or vaccinia virus immunoglobulin. The protective efficacy afforded by anti-A33 MAb was comparable to that of a previously isolated chimpanzee/human anti-B5 MAb. The combination of anti-A33 MAb and anti-B5 MAb did not synergize the protective efficacy. These chimpanzee/human anti-A33 MAbs may be useful in the prevention and treatment of vaccinia virus-induced complications of vaccination against smallpox and may also be effective in the immunoprophylaxis and immunotherapy of smallpox and other orthopoxvirus diseases.
Cellular Protein Modification by Poliovirus: the Two Faces of Poly(rC)-Binding Protein
Authors: Perera Rushika , Daijogo Sarah , Walter Brandon L. , Nguyen Joseph H. C. , Semler Bert L.
Published: June 2007

During picornavirus infection, several cellular proteins are cleaved by virus-encoded proteinases. Such cleavage events are likely to be involved in the changing dynamics during the intracellular viral life cycle, from viral translation to host shutoff to RNA replication to virion assembly. For example, it has been proposed that there is an active switch from poliovirus translation to RNA replication mediated by changes in RNA-binding protein affinities. This switch could be a mechanism for controlling template selection for translation and negative-strand viral RNA synthesis, two processes that use the same positive-strand RNA as a template but proceed in opposing directions. The cellular protein poly(rC)-binding protein (PCBP) was identified as a primary candidate for regulating such a mechanism. Among the four different isoforms of PCBP in mammalian cells, PCBP2 is required for translation initiation on picornavirus genomes with type I internal ribosome entry site elements and also for RNA replication. Through its three K-homologous (KH) domains, PCPB2 forms functional protein-protein and RNA-protein complexes with components of the viral translation and replication machinery. We have found that the isoforms PCBP1 and -2 are cleaved during the mid-to-late phase of poliovirus infection. On the basis of in vitro cleavage assays, we determined that this cleavage event was mediated by the viral proteinases 3C/3CD. The primary cleavage occurs in the linker between the KH2 and KH3 domains, resulting in truncated PCBP2 lacking the KH3 domain. This cleaved protein, termed PCBP2-ΔKH3, is unable to function in translation but maintains its activity in viral RNA replication. We propose that through the loss of the KH3 domain, and therefore loss of its ability to function in translation, PCBP2 can mediate the switch from viral translation to RNA replication.
DNA-Dependent Oligomerization of Herpes Simplex Virus Type 1 Regulatory Protein ICP4
Authors: Kuddus Ruhul H. , DeLuca Neal A.
Published: June 2007

The human herpes simplex virus type 1 regulatory protein ICP4 binds DNA as a dimer and forms a single protein-DNA complex (A complex) with short DNA probes. ICP4 oligomerized in a DNA-dependent manner, forming two or more protein-DNA complexes with longer DNA fragments containing a single DNA binding site. When resolved electrophoretically, one or more low-mobility DNA-protein complexes follow the fast-moving A complex. The major protein-DNA complex (B complex) formed by ICP4 with long DNA probes migrates just behind the A complex in the electric field, implying the oligomerization of ICP4 on the DNA. Binding experiments with circularly permutated DNA probes containing one ICP4 binding site revealed that about 70 bp of nonspecific DNA downstream of the cognate ICP4 binding site was required for efficient B complex formation. In addition, the C-terminal domain of ICP4 was found to be required for DNA-dependent oligomerization and B complex formation. Gel mobility shift analysis of protein-DNA complexes, combined with supershift analysis using different monoclonal antibodies, indicated that the B complex contained two ICP4 dimers. DNase I footprinting of ICP4-DNA complexes showed that one ICP4 dimer contacts the specific binding site and another ICP4 dimer contacts nonspecific DNA in the B complex. DNA-dependent oligomerization increased the affinity of ICP4 for relatively weak binding sites on large DNA molecules. The results of this study suggest how ICP4 may use multiple weak binding sites to aid in transcription activation.
Rate of Recombinational Deletion among Human Endogenous Retroviruses
Authors: Belshaw Robert , Watson Jason , Katzourakis Aris , Howe Alexis , Woolven-Allen John , Burt Austin , Tristem Michael
Published: June 2007

The fate of most human endogenous retroviruses (HERVs) has been to undergo recombinational deletion. This process involves homologous recombination between the flanking long terminal repeats (LTRs) of a full-length element, leaving a relic structure in the genome termed a solo LTR. We examined loci in one family, HERV-K(HML2), and found that the deletion rate decreased markedly with age: the rate among recently integrated loci was almost 200-fold higher than that among loci whose insertion predated the divergence of humans and chimpanzees (8 × 10
−5
and 4 × 10
−7
recombinational deletion events per locus per generation, respectively). One hypothesis for this finding is that increasing mutational divergence between the flanking LTRs reduces the probability of homologous recombination and thus the rate of solo LTR formation. Consistent with this idea, we were able to replicate the observed rates by a simulation in which the probability of recombinational deletion was reduced 10-fold by a single mutation and 100-fold by any additional mutations. We also discuss the evidence for other factors that may influence the relationship between locus age and the rate of deletion, for example, host recombination rates and selection, and highlight the consequences of recombinational deletion for dating recent HERV integrations.
Genetic Analysis of Varicella-Zoster Virus ORF0 to ORF4 by Use of a Novel Luciferase Bacterial Artificial Chromosome System
Authors: Zhang Zhen , Rowe Jenny , Wang Weijia , Sommer Marvin , Arvin Ann , Moffat Jennifer , Zhu Hua
Published: June 2007

To efficiently generate varicella-zoster virus (VZV) mutants, we inserted a bacterial artificial chromosome (BAC) vector in the pOka genome. We showed that the recombinant VZV (VZV
BAC
) strain was produced efficiently from the BAC DNA and behaved indistinguishably from wild-type virus. Moreover, VZV's cell-associated nature makes characterizing VZV mutant growth kinetics difficult, especially when attempts are made to monitor viral replication in vivo. To overcome this problem, we then created a VZV strain carrying the luciferase gene (VZV
Luc
). This virus grew like the wild-type virus, and the resulting luciferase activity could be quantified both in vitro and in vivo. Using PCR-based mutagenesis, open reading frames (ORF) 0 to 4 were individually deleted from VZV
Luc
genomes. The deletion mutant viruses appeared after transfection into MeWo cells, except for ORF4, which was essential. Growth curve analysis using MeWo cells and SCID-hu mice indicated that ORF1, ORF2, and ORF3 were dispensable for VZV replication both in vitro and in vivo. Interestingly, the ORF0 deletion virus showed severely retarded growth both in vitro and in vivo. The growth defects of the ORF0 and ORF4 mutants could be fully rescued by introducing wild-type copies of these genes back into their native genome loci. This work has validated and justified the use of the novel luciferase VZV BAC system to efficiently generate recombinant VZV variants and ease subsequent viral growth kinetic analysis both in vitro and in vivo.
Inhibition of T-Cell Receptor Signal Transduction and Viral Expression by the Linker for Activation of T Cells-Interacting p12
I
Protein of Human T-Cell Leukemia/Lymphoma Virus Type 1
Authors: Fukumoto Risaku , Dundr Miroslav , Nicot Christophe , Adams Anthony , Valeri Valerio W. , Samelson Lawrence E. , Franchini Genoveffa
Published: June 2007

The p12
I
protein of human T-cell leukemia/lymphoma virus type 1 (HTLV-1) is a small oncoprotein that increases calcium release following protein kinase C activation by phorbol myristate acetate, and importantly, this effect is linker for activation of T cells (LAT) independent. Here, we demonstrate that p12
I
inhibits the phosphorylation of LAT, Vav, and phospholipase C-γ1 and decreases NFAT (nuclear factor of activated T cells) activation upon engagement of the T-cell receptor (TCR) with anti-CD3 antibody. Furthermore, we demonstrate that p12
I
localizes to membrane lipid rafts and, upon engagement of the TCR, relocalizes to the interface between T cells and antigen-presenting cells, defined as the immunological synapse. A p12
I
knockout molecular clone of HTLV-1 expresses more virus upon antigen stimulation than the isogenic wild type, suggesting that, by decreasing T-cell responsiveness, p12
I
curtails viral expression. Thus, p12
I
has contrasting effects on TCR signaling: it down-regulates TCR in a LAT-dependent manner on one hand, and on the other, it increases calcium release in a LAT-independent manner. The negative regulation of T-cell activation by p12
I
may have evolved to minimize immune recognition of infected CD4
+
T cells, to impair the function of infected cytotoxic CD8
+
T cells, and to favor viral persistence in the infected host.
Ethene/Norbornene Copolymerization with (Isodicyclopentadienyl)titanium Complex-MAO Catalyst
Authors: Gentil Sébastien , Pirio Nadine , Meunier Philippe , Gallou Fabrice , Paquette Leo A.
Published: June 2007

Norbornene (NB) homopolymerization and ethene/NB copolymerizations with a silylene-bridged (isodicyclopentadienyl)(tert-butylamido)titanium dichloride-methylalumoxane (MAO) catalyst system were investigated. This catalytic system shows no efficiency towards NB homopolymerization but produces poly(ethene-co-norbornene)s. An increase in the initial NB feed content leads to a loss of copolymerization activity as well as NB copolymer incorporation. The structure of the isodicyclopentadienyl fragment (IsodiCp) has a strong limiting effect on comonomer incorporation possibilities.
The Mechanism of the Permanganate-Promoted Oxidative Cyclization of 1,5-Dienes - a DFT Study
Authors: Poethig Alexander , Strassner Thomas
Published: June 2007

The mechanism of the permanganate oxidation of 1,5-dienes was investigated by density functional theory calculations (B3LYP/6-311+G*). The experimentally observed (unexpected) products, 2,5-bis(hydroxymethyl) tetrahydrofurans, are formed in a concerted reaction, which explains the observed stereoselectivity of the reaction. Water plays an important role in the reaction mechanism by significantly lowering the activation energies.
The Effect of Type of Acid Sites in Molecular Sieves on Activity and Selectivity in Acylation Reactions
Authors: Bejblová Martina , Vlk Josef , Procházková Dana , Šiklová Helena , Čejka Jiří
Published: June 2007

The role of the type of acid site (Broensted vs. Lewis) on the activity and selectivity of molecular sieve catalysts was investigated in ferrocene and toluene acylation. H-, Zn-, Fe-, Al- and La-forms of zeolite Beta, USY and mesoporous molecular sieves (Al)MCM-41, (Al)SBA-15 were tested. It was observed that addition of metal cations acting as Lewis acid sites can increase the acidity of various molecular sieve catalysts. No general relationship between the type of cation and conversion of individual substrate was found. While the highest activity in ferrocene acylation was observed after addition of Zn, in the case of toluene acylation Al-forms of catalysts were the most active. The results indicate that the acid strength of cationic Lewis sites controls their activity in acylation reactions.
Synthesis of Bulky Zirconocene Dichloride Compounds and Their Applications in Olefin Polymerization
Authors: Polo-Cerón Dorian , Gómez-Ruiz Santiago , Prashar Sanjiv , Fajardo Mariano , Antiñolo Antonio , Otero Antonio
Published: June 2007

The bulky substituted cyclopentadienyllithium derivatives, LiC5H4(CHMeR) (R = C6H5 (1), 1-naphthyl (2), 9-anthryl (3)), were synthesized from the reaction of 6-phenylfulvene, 6-(1-naphthyl)fulvene or 6-(9-anthryl)fulvene with LiMe. The ansa-bis(cyclopentadiene) ligands Me2Si(C5HMe4){C5H4(CHMeR)} (R = C6H5 (4), 1-naphthyl (5), 9-anthryl (6)), and their lithium derivatives Li2(Me2Si(C5Me4){C5H3(CHMeR)}) (R = C6H5 (7), 1-naphthyl (8), 9-anthryl (9)) have been prepared. The zirconocene complexes, [Zr(η5-C5H5){η5-C5H4- (CHMeR)}Cl2] (R = C6H5 (10), 1-naphthyl (11), 9-anthryl (12)) and [Zr{η5-C5H4(CHMeR)}2Cl2] (R = C6H5 (13), 1-naphthyl (14), 9-anthryl (15)), were synthesized by the reaction of lithium derivatives 1-3 and [Zr(η5-C5H5)Cl3] or ZrCl4, respectively. The reaction of the lithium ansa-derivatives 7-9 and zirconium tetrachloride yielded the ansa-zirconocene complexes, [Zr(Me2Si(η5-C5Me4){η5-C5H3(CHMeR)})Cl2] (R = C6H5 (16), 1-naphthyl (17), 9-anthryl (18)). The zirconocene complexes have been tested in the polymerization of ethene and propene. The polymerization of propene with the ansa-zirconocene catalysts 16-18 gave polypropylene with 70% mmmm pentads and the symmetric zirconocene catalysts 13-15 30-60% mmmm pentads.
Platinum(II) Complexes with Bidentate NN and Tridentate NNO Ligands for C-H Bond Activation
Authors: Duin Marcel A. , Ernsting Jan M. , Elsevier Cornelis J.
Published: June 2007

A series of neutral and cationic methylplatinum(II) complexes with bidentate NN and tridentate NNO ligands has been prepared. The complexes involving tridentate NNO ligands were expected to be easier to handle than those with NN ligands, which has indeed been confirmed by the experiments. Nevertheless, the neutral [Pt(Me)(NNO)] and ionic [Pt(Me)(NNO)]+BF4- complexes retain their (non-selective) reactivity to hydrocarbon C-H bonds.
Pd-Catalyzed Amination of 2,6-Dihalopyridines with Polyamines
Authors: Averin Alexei D. , Ulanovskaya Olesya A. , Pleshkova Nataliya A. , Borisenko Anatolii A. , Beletskaya Irina P.
Published: June 2007

The Pd-catalyzed amination of 2,6-dibromopyridine with various linear polyamines and oxapolyamines was studied. In this way polyazamacrocycles 3 containing one pyridine and one polyamine moiety were synthesized using equimolar amounts of starting compounds. Two alternative approaches were elaborated and compared for synthesis of macrocycles 11 comprising two pyridine and two polyamine fragments: via intermediate formation of Nα,Nω-bis(6-halopyridin-2-yl)polyamines 7, 8 or via 2,6-bis(polyamino)-substituted pyridines 10. A series of N-(6-tert-butoxypyridin-2-yl)-substituted polyamines 12 and Nα,Nωbis(6-tert-butoxypyridin-2-yl)-substituted polyamines 13 were obtained by similar procedures, and the possibilities of N,N-dihetarylation of such compounds using 2-bromopyridine and 2-bromo-6-tert-butoxypyridine were studied. The yields of linear and cyclic products were shown to be strongly dependent on the nature of starting polyamines and of the halogen atom.
GHOST COHOMOLOGIES AND HIDDEN SPACE–TIME SYMMETRIES
Authors: POLYAKOV DIMITRI
Published: June 2007

We observe and study new nonlinear global space–time symmetries of the full ghost + matter action of RNS superstring theory. We show that these surprising new symmetries are generated by the special worldsheet currents (physical vertex operators) of RNS superstring theory, violating the equivalence of superconformal ghost pictures. We review the questions of BRST-invariance and nontriviality of picture-dependent vertex operators and show their relation to hidden space–time symmetries and hidden space–time dimensions. In particular, we relate the space–time transformations, induced by picture-dependent currents, to the symmetries observed in the 2T physics approach.
THE LAYERED NET SURFACE PROBLEMS IN DISCRETE GEOMETRY AND MEDICAL IMAGE SEGMENTATION
Authors: WU XIAODONG , CHEN DANNY Z. , LI KANG , SONKA MILAN
Published: June 2007

Efficient detection of multiple inter-related surfaces representing the boundaries of objects of interest in d-D images (d ≥ 3) is important and remains challenging in many medical image analysis applications. In this paper, we study several layered net surface (LNS) problems captured by an interesting type of geometric graphs called ordered multi-column graphs in the d-D discrete space (d ≥ 3 is any constant integer). The LNS problems model the simultaneous detection of multiple mutually related surfaces in three or higher dimensional medical images. Although we prove that the d-D LNS problem (d ≥ 3) on a general ordered multi-column graph is NP-hard, the (special) ordered multi-column graphs that model medical image segmentation have the self-closure structures and thus admit polynomial time exact algorithms for solving the LNS problems. Our techniques also solve the related net surface volume (NSV) problems of computing well-shaped geometric regions of an optimal total volume in a d-D weighted voxel grid. The NSV problems find applications in medical image segmentation and data mining. Our techniques yield the first polynomial time exact algorithms for several high dimensional medical image segmentation problems. Experiments and comparisons based on real medical data showed that our LNS algorithms and software are computationally efficient and produce highly accurate and consistent segmentation results.