bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance of CHROMagar Selective Medium and Oxacillin Resistance Screening Agar Base for Identifying
Staphylococcus aureus
and Detecting Methicillin Resistance
Authors: Kluytmans Jan , Van Griethuysen Arjanne , Willemse Piet , Van Keulen Peter
Published: July 2002

Two new selective media, oxacillin resistance screening agar base (ORSAB) and CHROMagar Staph aureus (CSA), were evaluated for identification of
Staphylococcus aureus
and for screening of methicillin resistance by addition of antimicrobial agents to these media. A well-defined collection consisting of 1,140 staphylococci was used. A total of 624 were
S. aureus
, of which 358 were methicillin susceptible and 266 were methicillin resistant, and 516 were coagulase-negative staphylococci. The methicillin-resistant
S. aureus
(MRSA) strains were selected based on the results of phage typing; 247 different types were included in the analysis. For identification of
S. aureus
, both media performed better after 24 h than after 48 h. The sensitivities at 24 h were comparable (CSA, 98.6%; ORSAB, 97.1%), but the specificity of CSA was significantly higher (CSA, 97.1%; ORSAB, 92.1%). For screening of methicillin resistance, antibiotic supplements were added to both media. The sensitivity was lower after 24 h (CSA, 58.6%; ORSAB, 84.2%) and increased significantly after 48 h (CSA, 77.5%; ORSAB, 91.4%). At both time intervals ORSAB was significantly more sensitive than CSA. However, the specificities of both media were high after 24 h (CSA, 99.1%; ORSAB, 98.3%) and decreased significantly after 48 h of incubation (CSA, 94.7%; ORSAB, 95.5%). In conclusion, for identification of
S. aureus
, CSA is more accurate than ORSAB because of a significantly higher specificity. For screening of MRSA, ORSAB performs better than CSA, but the usefulness in clinical practice is limited because a significant number of strains are not detected.
Phenotypic and Genotypic Characterization of Serologically Atypical Strains ofShigella flexneriType 4 Isolated in Dhaka, Bangladesh
Authors: Talukder Kaisar A. , Islam M. Aminul , Dutta Dilip K. , Hassan Ferdaus , Safa Ashrafus , Nair G. B. , Sack David A.
Published: July 2002

Twenty-one atypicalShigella flexneritype 4 strains isolated from patients attending the Dhaka treatment center of the International Centre for Diarrhoeal Disease Research, Bangladesh, were extensively characterized and compared withS. flexneriserotypes 4a and 4b. The atypical strains agglutinated only with the type antigen factor 4 and did not agglutinate with any group factors, thereby excluding their characterization into serotype 4a or 4b. Of the 21 strains, 85.7% did not ferment mannitol but were able to ferment most of the sugars, whereas the remaining 14.3% strains fermented mannitol but were unable to ferment most of the sugars. Most of the strains were resistant to ampicillin, tetracycline, and trimethoprim-sulfomethoxazole. All of the strains harbored the 140-MDa plasmid, had theipaHgene, had thesengene (encodingShigellaenterotoxin 2), had the ability to bind Congo red, and were positive for keratoconjunctivitis in the guinea pig eye, attesting their invasive properties. All of the strains contained a middle-range plasmid (35 to 62 MDa) as well as a number of stable small plasmids, yielding mainly two plasmid profiles which were different from those of 4a and 4b strains. Conjugation and curing experiments suggested that the middle-range plasmids harbored a self-transferable multiple antibiotic resistance marker. Pulsed-field gel electrophoresis analysis of all of the tested strains yielded two types with numerous subtypes, whereas ribotyping yielded only two types which were completely different from those of types 4a and 4b. This study concluded that two different clones of atypicalS. flexneritype 4 exist and strongly suggests that these are new subserotypes ofS. flexnerithat await further serological classification.
Prevalence of Enterotoxigenic
Escherichia coli
Strains Harboring the Longus Pilus Gene in Brazil
Authors: Nishimura Lucilia S. , Girón Jorge A. , Nunes Solange L. , Guth Beatriz E. C.
Published: July 2002

The longus type IV pilus gene (
lngA
) was highly prevalent (32.8%) among Brazilian enterotoxigenic
Escherichia coli
strains producing both heat-labile and heat-stable enterotoxins and bearing the CFA/I, CS1CS3, or CS6 antigen. Furthermore,
lngA
was more often found in strains isolated from children with diarrhea than in strains isolated from children without diarrhea.
Quantitative Stability of DNA after Extended Storage of Clinical Specimens as Determined by Real-Time PCR
Authors: Jerome Keith R. , Huang Meei-Li , Wald Anna , Selke Stacy , Corey Lawrence
Published: July 2002

Viral DNA stored for extended periods can be amplified by PCR. However, it is unknown whether stored specimens give accurate quantitative results by newer real-time PCR techniques. We therefore compared herpes simplex virus DNA levels in specimens before and after 16 months of storage. The levels of viral DNA remained stable whether the DNA was stored as purified DNA or unextracted DNA in a whole specimen.
Molecular and Seroepidemiological Evidence of Canine Calicivirus Infections in Japan
Authors: Mochizuki Masami , Hashimoto Michiru , Roerink Frank , Tohya Yukinobu , Matsuura Yuichi , Sasaki Nobuo
Published: July 2002

Calicivirus infection of dogs was epidemiologically investigated by using canine calicivirus (CaCV) strain 48 as a reference. Similar RNA polymerase gene sequences and neutralizing antibodies against CaCV were detected in 1.7% of clinical specimens and 57% of serum samples, respectively, suggesting a high prevalence of CaCV in dog populations.
Prevalence of the
fsr
Locus in
Enterococcus faecalis
Infections
Authors: Pillai S. K. , Sakoulas G. , Gold H. S. , Wennersten C. , Eliopoulos G. M. , Moellering R. C. , Inouye R. T.
Published: July 2002

The
fsr
locus of
Enterococcus faecalis
confers virulence in animal models. A retrospective analysis of
fsr
prevalence in diverse
E. faecalis
clinical isolates demonstrated
fsr
in all endocarditis isolates versus 53% of stool isolates (
P
= 0.005). This supports a role for
fsr
-mediated virulence in the pathogenesis of enterococcal infections in humans.
Application of RNA Polymerase β-Subunit Gene (
rpoB
) Sequences for the Molecular Differentiation of
Legionella
Species
Authors: Ko Kwan Soo , Lee Hae Kyung , Park Mi-Yeoun , Lee Keun-Hwa , Yun Yeo-Jun , Woo So-Yon , Miyamoto Hiroshi , Kook Yoon-Hoh
Published: July 2002

The nucleotide sequences of the partial
rpoB
gene were determined from 38
Legionella
species, including 15 serogroups of
Legionella pneumophila.
These sequences were then used to infer the phylogenetic relationships among the
Legionella
species in order to establish a molecular differentiation method appropriate for them. The sequences (300 bp) and the phylogenetic tree of
rpoB
were compared to those from analyses using 16S rRNA gene and
mip
sequences. The trees inferred from these three gene sequences revealed significant differences. This sequence incongruence between the
rpoB
tree and the other trees might have originated from the high frequency of synonymous base substitutions and/or from horizontal gene transfer among the
Legionella
species. The nucleotide variation of
rpoB
enabled more evident differentiation among the
Legionella
species than was achievable by the 16S rRNA gene and even by
mip
in some cases. Two subspecies of
L. pneumophila
(
L. pneumophila
subsp.
pneumophila
and subsp.
fraseri
) were clearly distinguished by
rpoB
but not by 16S rRNA gene and
mip
analysis. One hundred and five strains isolated from patient tissues and environments in Korea and Japan could be identified by comparison of
rpoB
sequence similarity and phylogenetic trees. These results suggest that the partial sequences of
rpoB
determined in this study might be applicable to the molecular differentiation of
Legionella
species.
Bacteremia Due to
Moraxella atlantae
in a Cancer Patient
Authors: De Baere Thierry , Muylaert An , Everaert Els , Wauters Georges , Claeys Geert , Verschraegen Gerda , Vaneechoutte Mario
Published: July 2002

A gram-negative alkaline phosphatase- and pyrrolidone peptidase-positive rod-shaped bacterium (CCUG 45702) was isolated from two aerobic blood cultures from a female cancer patient. No identification could be reached using phenotypic techniques. Amplification of the tRNA intergenic spacers revealed fragments with lengths of 116, 133, and 270 bp, but no such pattern was present in our reference library. Sequencing of the 16S rRNA gene revealed its identity as
Moraxella atlantae
, a species isolated only rarely and published only once as causing infection. In retrospect, the phenotypic characteristics fit the identification as
M. atlantae
(formerly known as CDC group M-3). Comparative 16S rRNA sequence analysis indicates that
M. atlantae
,
M. lincolnii
, and
M. osloensis
might constitute three separate genera within the
Moraxellaceae
. After treatment with amoxicillin-clavulanic acid for 2 days, fever subsided and the patient was dismissed.
Selection of 3′-Template Bases and Initiating Nucleotides by Hepatitis C Virus NS5B RNA-Dependent RNA Polymerase
Authors: Shim Jae Hoon , Larson Gary , Wu Jim Zhen , Hong Zhi
Published: July 2002

De novo RNA synthesis by hepatitis C virus (HCV) nonstructural protein 5B (NS5B) RNA-dependent RNA polymerase has been investigated using short RNA templates. Various templates including those derived from the HCV genome were evaluated by examining the early steps of de novo RNA synthesis. NS5B was shown to be able to produce an initiation dinucleotide product from templates as short as 4-mer and from the 3′-terminal sequences of both plus and minus strands of the HCV RNA genome. GMP, GDP, and guanosine were able to act as an initiating nucleotide in de novo RNA synthesis, indicating that the triphosphate moiety is not absolutely required by an initiating nucleotide. Significant amounts of the initiation product accumulated in de novo synthesis, and elongation from the dinucleotide was observed when large amounts of dinucleotide were available. This result suggests that NS5B, a template, and incoming nucleotides are able to form an initiation complex that aborts frequently by releasing the dinucleotide product before transition to an elongation complex. The transition is rate limiting. Furthermore, we discovered that the secondary structure of a template was not essential for de novo initiation and that 3′-terminal bases of a template conferred specificity in selection of an initiation site. Initiation can occur at the +1, +2, or +3 position numbered from the 3′ end of a template depending on base composition. Pyrimidine bases at any of the three positions are able to serve as an initiation site, while purine bases at the +2 and +3 positions do not support initiation. This result implies that HCV possesses an intrinsic ability to ensure that de novo synthesis is initiated from the +1 position and to maintain the integrity of the 3′ end of its genome. This assay system should be an important tool for investigating the detailed mechanism of de novo initiation by HCV NS5B as well as other viral RNA polymerases.
The Broadly Neutralizing Anti-Human Immunodeficiency Virus Type 1 Antibody 2G12 Recognizes a Cluster of α1→2 Mannose Residues on the Outer Face of gp120
Authors: Scanlan Christopher N. , Pantophlet Ralph , Wormald Mark R. , Ollmann Saphire Erica , Stanfield Robyn , Wilson Ian A. , Katinger Hermann , Dwek Raymond A. , Rudd Pauline M. , Burton Dennis R.
Published: July 2002

2G12 is a broadly neutralizing human monoclonal antibody against human immunodeficiency virus type-1 (HIV-1) that has previously been shown to bind to a carbohydrate-dependent epitope on gp120. Here, site-directed mutagenesis and carbohydrate analysis were used to define further the 2G12 epitope. Extensive alanine scanning mutagenesis showed that elimination of the N-linked carbohydrate attachment sequences associated with residues N295, N332, N339, N386, and N392 by N→A substitution produced significant decreases in 2G12 binding affinity to gp120
JR-CSF
. Further mutagenesis suggested that the glycans at N339 and N386 were not critical for 2G12 binding to gp120
JR-CSF
. Comparison of the sequences of isolates neutralized by 2G12 was also consistent with a lesser role for glycans attached at these positions. The mutagenesis studies provided no convincing evidence for the involvement of gp120 amino acid side chains in 2G12 binding. Antibody binding was inhibited when gp120 was treated with
Aspergillus saitoi
mannosidase, Jack Bean mannosidase, or endoglycosidase H, indicating that Manα1→2Man-linked sugars of oligomannose glycans on gp120 are required for 2G12 binding. Consistent with this finding, the binding of 2G12 to gp120 could be inhibited by monomeric mannose but not by galactose, glucose, or
N
-acetylglucosamine. The inability of 2G12 to bind to gp120 produced in the presence of the glucose analogue
N
-butyl-deoxynojirimycin similarly implicated Manα1→2Man-linked sugars in 2G12 binding. Competition experiments between 2G12 and the lectin cyanovirin for binding to gp120 showed that 2G12 only interacts with a subset of available Manα1→2Man-linked sugars. Consideration of all the data, together with inspection of a molecular model of gp120, suggests that the most likely epitope for 2G12 is formed from mannose residues contributed by the glycans attached to N295 and N332, with the other glycans playing an indirect role in maintaining epitope conformation.
Genetic Evidence that Interhelical Packing Interactions in the gp41 Core Are Critical for Transition of the Human Immunodeficiency Virus Type 1 Envelope Glycoprotein to the Fusion-Active State
Authors: Follis Kathryn E. , Larson Scott J. , Lu Min , Nunberg Jack H.
Published: July 2002

The envelope glycoprotein complex (gp120-gp41) of human immunodeficiency virus type 1 (HIV-1) promotes the fusion of viral and cellular membranes through formation of the fusion-active six-helix bundle in the gp41 ectodomain. This gp41 core structure consists of three C-terminal helices packed in an antiparallel manner into hydrophobic grooves on the surface of the N-terminal trimeric coiled coil. Alanine mutations that destabilize the N- and C-terminal interhelical packing interactions also reduce viral infectivity. Here we show that viruses bearing these mutations exhibit a marked potentiation of inhibition by peptides that make up the gp41 core. By contrast, these viruses are unchanged in their sensitivities to soluble CD4, the CXCR4 coreceptor ligand SDF-1α, and human anti-HIV immunoglobulin, reagents that impact the initial, receptor-induced conformational changes in the envelope glycoprotein. Our results support the notion that these alanine mutations specifically affect the conformational transition to the fusion-active gp41 structure. The mutations also increase viral sensitivity to the gp41-directed monoclonal antibody 2F5, suggesting that this broadly neutralizing antibody may also interfere with this transition. The conformational activation of the HIV-1 envelope glycoprotein likely represents a viable target for vaccine and antiviral drug development.
Analysis of Deletion Mutants Indicates that the 2A Polypeptide of Hepatitis A Virus Participates in Virion Morphogenesis
Authors: Cohen Lisette , Bénichou Danièle , Martin Annette
Published: July 2002

Unlike all other picornaviruses, the primary cleavage of the hepatitis A virus (HAV) polyprotein occurs at the 2A/2B junction and is carried out by the only proteinase encoded by the virus, 3C
pro
. The resulting P1-2A capsid protein precursor is subsequently cleaved by 3C
pro
to generate VP0, VP3, and VP1-2A, which associate as pentamers. An unidentified cellular proteinase acting at the VP1/2A junction releases the mature capsid protein VP1 from VP1-2A later in the morphogenesis process. Although these aspects of polyprotein processing are well characterized, the function of 2A is unknown. To study its role in the viral life cycle, we assessed the infectivity of synthetic, genome-length RNAs containing 11 different in-frame deletions in the 2A region. Deletions in the N-terminal 40% of 2A abolished infectivity, whereas deletions in the C-terminal 60% resulted in viruses with a small-focus replication phenotype. C-terminal deletions in 2A had no effect on RNA replication kinetics under one-step growth conditions, nor did they have an effect on capsid protein synthesis and 3C
pro
-mediated processing. However, C-terminal deletions in 2A altered the VP1/2A cleavage, resulting in accumulation of uncleaved VP1-2A precursor in virions and possibly accounting for a delay in the appearance of infectious particles with these mutants, as well as a fourfold decrease in specific infectivity of the virus particles. When the capsid proteins were expressed from recombinant vaccinia viruses, the N-terminal part of 2A was required for efficient cleavage of the P1-2A precursor by 3C
pro
and assembly of structural precursors into pentamers. These data indicate that the N-terminal domain of 2A must be present as a C-terminal extension of P1 for folding of the capsid protein precursor to allow efficient 3C
pro
-mediated cleavages and to promote pentamer assembly, after which cleavage at the VP1/2A junction releases the mature VP1 protein, a process that appears to be necessary to produce highly infectious particles.
Novel Monoclonal Antibody Directed at the Receptor Binding Site on the Avian Sarcoma and Leukosis Virus Env Complex
Authors: Ochsenbauer-Jambor Christina , Delos Sue E. , Accavitti Mary Ann , White Judith M. , Hunter Eric
Published: July 2002

We report here on the generation of a mouse monoclonal antibody directed against Rous sarcoma virus (RSV) subgroup A Env that will be useful in functional and structural analysis of RSV Env, as well as in approaches employing the RCAS/Tva system for gene targeting. BALB/c mice were primed and given boosters twice with EnvA-expressing NIH 3T3 cells. Resulting hybridomas were tested by enzyme-linked immunosorbent assay against RCANBP virions and SU-A-immunoglobulin G immunoadhesin. One highly reactive hybridoma clone, mc8C5, was subcloned and tested in immunofluorescence, immunoprecipitation (IP), and Western blotting assays. In all three assays, mc8C5-4 subgroup-specifically recognizes SR-A Env, through the SU domain, expressed from different vectors in both avian and mammalian cells. This multifunctionality is notable for a mouse monoclonal. We furthermore observed a preference for binding to terminally glycosylated Env over core-glycosylated Env precursor in IPs, suggesting that the epitope is at least partially conformational and dependent on glycosylation. Most importantly, we found mc8C5-4 inhibited Env function: in vitro, the monoclonal not only interferes with binding of the EnvA receptor, Tva, but it also blocks the Tva-induced conformational change required for activation of the fusion peptide, without inducing that change itself. Infection of Tva-expressing avian or mammalian cells by avian sarcoma and leukosis virus (ASLV) or EnvA-pseudotyped murine leukemia virus, respectively, is efficiently inhibited by mc8C5-4. The apparent interference of the monoclonal with the EnvA-Tva complex formation suggests that the epitope seen by mc8C5 overlaps with the receptor binding site. This is supported by the observation that mutations of basic residues in hr2 or of the downstream glycosylation site, which both impair Tva-binding to EnvA, have similar effects on the binding of mc8C5. Thus, anti-ASLV-SU-A mc8C5-4 proves to be a unique new immunoreagent that targets the receptor-binding site on a prototypical retroviral envelope.
Kinetics and Frequency of Adeno-Associated Virus Site-Specific Integration into Human Chromosome 19 Monitored by Quantitative Real-Time PCR
Authors: Hüser Daniela , Weger Stefan , Heilbronn Regine
Published: July 2002

Adeno-associated virus type 2 (AAV-2) integrates specifically into a site on human chromosome 19 (chr-19) called AAVS1. To study the kinetics and frequency of chr-19-specific integration after AAV infection, we developed a rapid, sensitive, and quantitative real-time PCR assay for AAV inverted terminal repeat-chr-19-specific junctions. Despite the known variability of junction sites, conditions were established that ensured reliable quantification of integration rates within hours after AAV infection. The overall integration frequency was calculated to peak at between 10 and 20% of AAV-infected, unselected HeLa cells. At least 1 in 1,000 infectious AAV-2 particles was found to integrate site specifically up to day 4 postinfection in the absence of selection. Chromosomal breakpoints within AAVS1 agreed with those found in latently infected clonal cell lines and transgenic animals. Use of this quantitative real-time PCR will greatly facilitate the study of the early steps of wild-type and recombinant AAV vector integration.
Comparison of Predicted Scaffold-Compatible Sequence Variation in the Triple-Hairpin Structure of Human Immunodeficiency Virus Type 1 gp41 with Patient Data
Authors: Boutonnet Nathalie , Janssens Wouter , Boutton Carlo , Verschelde Jean-Luc , Heyndrickx Leo , Beirnaert Els , van der Groen Guido , Lasters Ignace
Published: July 2002

It has been proposed that the ectodomain of human immunodeficiency virus type 1 (HIV-1) gp41 (e-gp41), involved in HIV entry into the target cell, exists in at least two conformations, a pre-hairpin intermediate and a fusion-active hairpin structure. To obtain more information on the structure-sequence relationship in e-gp41, we performed in silico a full single-amino-acid substitution analysis, resulting in a Fold Compatible Database (FCD) for each conformation. The FCD contains for each residue position in a given protein a list of values assessing the energetic compatibility (ECO) of each of the 20 natural amino acids at that position. Our results suggest that FCD predictions are in good agreement with the sequence variation observed for well-validated e-gp41 sequences. The data show that at a minECO threshold value of 5 kcal/mol, about 90% of the observed patient sequence variation is encompassed by the FCD predictions. Some inconsistent FCD predictions at N-helix positions packing against residues of the C helix suggest that packing of both peptides may involve some flexibility and may be attributed to an altered orientation of the C-helical domain versus the N-helical region. The permissiveness of sequence variation in the C helices is in agreement with FCD predictions. Comparison of N-core and triple-hairpin FCDs suggests that the N helices may impose more constraints on sequence variation than the C helices. Although the observed sequences of e-gp41 contain many multiple mutations, our method, which is based on single-point mutations, can predict the natural sequence variability of e-gp41 very well.
Cell Clones Selected from the Huh7 Human Hepatoma Cell Line Support Efficient Replication of a Subgenomic GB Virus B Replicon
Authors: De Tomassi Amedeo , Pizzuti Maura , Graziani Rita , Sbardellati Andrea , Altamura Sergio , Paonessa Giacomo , Traboni Cinzia
Published: July 2002

Tamarins (
Saguinus
species) infected by GB virus B (GBV-B) have recently been proposed as an acceptable surrogate model for hepatitis C virus (HCV) infection. The availability of infectious genomic molecular clones of both viruses will permit chimeric constructs to be tested for viability in animals. Studies in cells with parental and chimeric constructs would also be very useful for both basic research and drug discovery. For this purpose, a convenient host cell type supporting replication of in vitro-transcribed GBV-B RNA should be identified. We constructed a GBV-B subgenomic selectable replicon based on the sequence of a genomic molecular clone proved to sustain infection in tamarins. The corresponding in vitro-transcribed RNA was used to transfect the Huh7 human hepatoma cell line, and intracellular replication of transfected RNA was shown to occur, even though in a small percentage of transfected cells, giving rise to antibiotic-resistant clones. Sequence analysis of GBV-B RNA from some of those clones showed no adaptive mutations with respect to the input sequence, whereas the host cells sustained higher GBV-B RNA replication than the original Huh7 cells. The enhancement of replication depending on host cell was shown to be a feature common to the majority of clones selected. The replication of GBV-B subgenomic RNA was susceptible to inhibition by known inhibitors of HCV to a level similar to that of HCV subgenomic RNA.
Oligomeric Structure of the Human Immunodeficiency Virus Type 1 Envelope Protein on the Virion Surface
Authors: Center Rob J. , Leapman Richard D. , Lebowitz Jacob , Arthur Larry O. , Earl Patricia L. , Moss Bernard
Published: July 2002

The envelope protein (Env) of human immunodeficiency virus type 1 forms homo-oligomers in the endoplasmic reticulum. The oligomeric structure of Env is maintained after cleavage in a Golgi compartment and transport to the surfaces of infected cells, where incorporation into budding virions takes place. Here, we use biophysical techniques to assess the oligomeric valency of virion-associated Env prior to fusion activation. Virion-associated Env oligomers were stabilized by chemical cross-linking prior to detergent extraction and were purified by immunoaffinity chromatography. Gel filtration revealed a single predominant oligomeric species, and sedimentation equilibrium analysis-derived mass values indicated a trimeric structure. Determination of the masses of individual Env molecules by scanning transmission electron microscopy demonstrated that virion-associated Env was trimeric, and a triangular morphology was observed in 20 to 30% of the molecules. These results, which firmly establish the oligomeric structure of human immunodeficiency virus virion-associated Env, parallel those of our previous analysis of the simian immunodeficiency virus Env.
Murine Cytomegalovirus (CMV) M33 and Human CMV US28 Receptors Exhibit Similar Constitutive Signaling Activities
Authors: Waldhoer Maria , Kledal Thomas N. , Farrell Helen , Schwartz Thue W.
Published: July 2002

Cellular infection by cytomegalovirus (CMV) is associated with very early G-protein-mediated signal transduction and reprogramming of gene expression. Here we investigated the involvement of human CMV (HCMV)-encoded US27, US28, and UL33 receptors as well as murine CMV-encoded M33 transmembrane (7TM) receptors in host cell signaling mechanisms. HCMV-encoded US27 did not show any constitutive activity in any of the studied signaling pathways; in contrast, US28 and M33 displayed ligand-independent, constitutive signaling through the G protein q (Gq)/phospholipase C pathway. In addition, M33 and US28 also activated the transcription factor NF-κB as well as the cyclic AMP response element binding protein (CREB) in a ligand-independent, constitutive manner. The use of specific inhibitors indicated that the p38 mitogen-activated protein (MAP) kinase but not the extracellular signal-regulated kinase 1/2-MAP kinase pathway is involved in M33- and US28-mediated CREB activation but not NF-κB activation. Interestingly, UL33—the HCMV-encoded structural homologue of M33—was only marginally constitutively active in the Gq/phospholipase C turnover and CREB activation assays and did not show any constitutive activity in the NF-κB pathway, where M33 and US28 were highly active. Hence, CMVs appear to have conserved mechanisms for regulating host gene transcription, i.e., constitutive activation of certain kinases and transcription factors through the constitutive activities of 7TM proteins. These data, together with the previous identification of the incorporation of such proteins in the viral envelope, suggest that these proteins could be involved in the very early reprogramming of the host cell during viral infection.
The Long Noncoding Region of the Human Parainfluenza Virus Type 1 F Gene Contributes to the Read-Through Transcription at the M-F Gene Junction
Authors: Bousse Tatiana , Matrosovich Tatyana , Portner Allen , Kato Atsushi , Nagai Yoshiyuki , Takimoto Toru
Published: July 2002

Sendai virus (SV) and human parainfluenza virus type 1 (hPIV1) have genomes consisting of nonsegmented negative-sense RNA in which the six genes are separated by well-conserved intergenic (IG) sequences and transcriptional start (S) and end signals. In hPIV1-infected cells, transcriptional termination at the M-F gene junction is ineffective; a large number of M-F read-through transcripts are produced (T. Bousse, T. Takimoto, K. G. Murti, and A. Portner, Virology
232
:44-52, 1997). In contrast, few M-F read-through transcripts are detected in SV-infected cells. Sequence analysis indicated that the hPIV1 IG and S sequences in the M-F junction differ from those of SV. Furthermore, the hPIV1 F gene contains an unusually long noncoding sequence. To identify the
cis
-acting elements that prevent transcriptional termination at the M-F junction, we rescued recombinant SV (rSVhMFjCG) in which its M-F gene junction was replaced by that of hPIV1. Cells infected with rSVhMFjCG produced an abundance of M-F read-through transcripts; this result indicated that the hPIV1 M-F junction is responsible for inefficient termination. When one or both of the IG and S sites in rSVhMFjCG were replaced by those of SV, the efficiency of transcriptional termination increased but not to the level observed in wild-type SV-infected cells. Deletion of most of the long noncoding region of the hPIV1 F gene in rSVhMFjCG in addition to the mutations in IG and S signals resulted in efficient termination that was equivalent to the level observed in wild-type virus-infected cells. Therefore, the long noncoding sequence of the hPIV1 F gene contains
cis
-acting element(s) that affects transcriptional termination. Our evaluation of the effect of inefficient transcriptional termination on viral replication in culture revealed that cells infected with rSVhMFjCG produced less F protein than cells infected with wild-type SV and that assembly of the recombinant SV in culture was less efficient. These phenotypes seem to be responsible for the extended survival of mice infected with rSVhMFjCG.
Mutations of the RNase H C Helix of the Moloney Murine Leukemia Virus Reverse Transcriptase Reveal Defects in Polypurine Tract Recognition
Authors: Lim David , Orlova Marianna , Goff Stephen P.
Published: July 2002

Both the RNase H domain of Moloney murine leukemia virus (Mo-MLV) reverse transcriptase (RT) and
Escherichia coli
RNase H possess a positively charged α-helix (C helix) and a loop that are not present in the RNase H domains of human immunodeficiency virus (HIV) RT or avian sarcoma virus RT. Although a mutant Mo-MLV RT lacking the C helix (ΔC RT) retains DNA polymerase activity on homopolymeric substrates and partial RNase H activity, reverse transcription of the viral RNA genome in vivo is defective. To identify the essential features of the C helix, a panel of Mo-MLV RT mutants was generated. Analyses of these mutant viruses revealed the importance of residues H594, I597, R601, and G602. The mutants were tested for their ability to synthesize viral DNA after acute infections and to form proper 5′ and 3′ viral DNA ends. The mutant RTs were tested in vitro for exogenous RT activity, minus-strand strong-stop DNA synthesis in endogenous RT reactions, nonspecific RNase H activity, and finally, proper cleavage at the polypurine tract-U3 junction. The R601A mutant was the most defective mutant both in vivo and in vitro and possessed very little RNase H activity. The H594A, I597A, and G602A mutants had significant reductions in RNase H activity and in their rates of viral replication. Many of the mutants formed improper viral DNA ends and were less efficient in PPT-U3 recognition and cleavage in vitro. The data show that the C helix plays a crucial role for overall RNase H cleavage activity. The data also suggest that the C helix may play an important role in polypurine tract recognition and proper formation of the plus-strand DNA's 5′ end.
Dengue Virus Selectively Induces Human Mast Cell Chemokine Production
Authors: King Christine A. , Anderson Robert , Marshall Jean S.
Published: July 2002

Severe dengue virus infections usually occur in individuals who have preexisting anti-dengue virus antibodies. Mast cells are known to play an important role in host defense against several pathogens, but their role in viral infection has not yet been elucidated. The effects of dengue virus infection on the production of chemokines by human mast cells were examined. Elevated levels of secreted RANTES, MIP-1α, and MIP-1β, but not IL-8 or ENA-78, were observed following infection of KU812 or HMC-1 human mast cell-basophil lines. In some cases a >200-fold increase in RANTES production was observed. Cord blood-derived cultured human mast cells treated with dengue virus in the presence of subneutralizing concentrations of dengue virus-specific antibody also demonstrated significantly (P< 0.05) increased RANTES production, under conditions which did not induce significant degranulation. Chemokine responses were not observed when mast cells were treated with UV-inactivated dengue virus in the presence or absence of human dengue virus-specific antibody. Neither antibody-enhanced dengue virus infection of the highly permissive U937 monocytic cell line nor adenovirus infection of mast cells induced a RANTES, MIP-1α, or MIP-1β response, demonstrating a selective mast cell response to dengue virus. These results suggest a role for mast cells in the initiation of chemokine-dependent host responses to dengue virus infection.
Synthesis, Characterization and Crystal Structure of Complexes of Rare Earth Picrates with N,N,N',N',N",N"-Hexaisopropyl- 2,2',2"-[nitrilotris(ethyleneoxy)]triacetamide (L)
Authors: Fan Jun , Liu Wei-Sheng , Tang Ning , Tang Yu , Tan Min-Yu , Yu Kai-Bei
Published: July 2002

A new tripodal compound, N,N,N',N',N",N"-hexaisopropyl-2,2',2"-[nitrilotris(ethyleneoxy)]triacetamide (L) was synthesized. Its complexes with rare earth picrates, [M(Pic)L](Pic)2·H2O (M = La, Pr, Nd, Sm, Eu, Tb, Er, Yb, Y), have been prepared and characterized by elemental analysis, thermal analysis, conductivity measurements, IR and 1H NMR spectra. The crystal structures of two complexes [M(Pic)L](Pic)2 (M = Sm, Y) have been determined, which reveal that the M(III) ions (M = Sm, Y) are encapsulated by the three chains of the ligand and nine-coordinated by the central nitrogen atom, three ether oxygen atoms and three carbonyl oxygen atoms from the ligand, and two oxygen atoms from the didentate coordinated picrate ion in both cases. The results show that L acts as a heptadentate ligand, forming a cup-like coordination structure to accommodate the rare earth ion. Both rare earth complexes have the similar coordinate modes in spite of different unit cells.
Chemistry on the Rhodacarborane Cluster [9,9-(PPh3)-nido-9,7,8-RhC2B8H11]: Formation of Bidentate Phosphine and Bimetallic Derivatives
Authors: Volkov Oleg , Rath Nigam P. , Barton Lawrence
Published: July 2002

The electronically unsaturated rhodacarborane [9,9-(PPh3)2-nido-9,7,8-RhC2B8H11] (1), reacts with Ph2PCH2PPh2 (dppm) to afford [9,9-(dppm-κ2P)-9-(dppm-κP)-nido-9,7,8-RhC2B8H11] (3), in which the Rh-bonded PPh3 ligands have been replaced by two dppm ligands, one in a bidentate mode and the second in a unidentate mode with a free PPh2 end. The structure of 3 is similar to the related, and isoelectronic, species [8,8-(dppm-κ2P)-8-(dppm-κP)-nido-8,7-RhSB9H10] (4), but with a difference in the orientation of the ligands. Reaction of 1 with Ph2P(CH2)2PPh2 (dppe) affords a species tentatively identified as [9,9-(dppe-κ2P)-9-(dppe-κP)-nido-9,7,8-RhC2B8H11] (5). If allowed to react with [Ru(η6-p-cym)Cl2]2, (3) affords [9,9-{Ru(η6-p-cym)dppm-κ2P-(μ-Cl)2}-nido-9,7,8-RhC2B8H11] (6) containing the group [(μ-Cl)2Ru(η6-p-cym)dppm] that coordinates in a multidentate mode to Rh. Compounds 3 and 6 are characterized by 11B, 1H and 31P NMR spectroscopy, elemental analysis and X-ray structure determinations.
Neurological complications of coeliac disease
Authors: Pengiran Tengah D S N A , Wills A J , Holmes G K T
Published: July 2002

A variety of neurological disorders have been reported in association with coeliac disease including epilepsy, ataxia, neuropathy, and myelopathy. The nature of this association is unclear and whether a specific neurological complication occurs in coeliac disease remains unproved. Malabsorption may lead to vitamin and trace element deficiencies. Therefore, patients who develop neurological dysfunction should be carefully screened for these. However, malabsorption does not satisfactorily explain the pathophysiology and clinical course of many of the associated neurological disorders. Other mechanisms proposed include altered autoimmunity, heredity, and gluten toxicity. This review attempts to summarise the literature and suggests directions for future research.
Cullen’s sign in amoebic liver abscess
Authors: Misra A , Agrahari D , Gupta R
Published: July 2002

A 45 year old woman presented with pain in her right upper abdomen and fever. Ultrasound of her abdomen showed a large liver abscess with subhepatic collection. She had Cullen’s sign. The liver abscess was managed by percutaneous catheter drainage.
Holocene deltaic sedimentation along an emerging coast: Nastapoka River delta, eastern Hudson Bay, Quebec
Authors: Lavoie Caroline , Allard Michel , Hill Philip R
Published: July 2002

Eastern Hudson Bay is characterized by falling relative sea level as a result of post-glacial isostatic rebound, which makes the region a natural laboratory for rapid forced regression, where the evolution of deltaic systems and offshore sedimentation patterns can be studied. A multidisciplinary approach involving airphoto analysis, offshore geophysical surveys, sediment coring, and facies and diatom analyses was used in this study of the Nastapoka River delta. The delta has formed as a result of the fluvial erosion of emerged Quaternary sediments but is mainly subaqueous. Offshore, in the prodelta zone, the oldest deposits are glaciomarine, laid down when the ice front of the receding Laurentide ice sheet stood on the Nastapoka hills some 77006800 years BP. Lateral equivalents of this glaciomarine unit are presently exposed on land. The shallow-water platform of the delta shows a thin surficial unit of wave-worked sand that overlies fine-grained, deeper water deposits derived from erosion of clay soils in the river catchment a few centuries ago, probably during periods of intense thermokarst activity. As the isostatic uplift continues, the deltaic platform will gradually emerge and be incised by the river channel.
Detrital zircon geochronology of the Taku terrane, southeast Alaska
Authors: Gehrels George E
Published: July 2002

UPb geochronologic studies have been conducted on 60 detrital zircon grains from Permian(?) and Triassic metasandstones of the Taku terrane in central southeast Alaska. The resulting ages are mainly in the range 349387 Ma, with five additional grains that yield probable ages ranging from ~906 to ~2643 Ma. These ages are similar to the ages of detrital zircons in Carboniferous and older rocks of the YukonTanana terrane, which lies directly east of the Taku terrane. In contrast, these ages are different from the ages of detrital zircon grains in the Alexander terrane to the west. The data are accordingly consistent with models in which the Taku terrane is a western component of the Stikine and YukonTanana terranes, and that this crustal fragment is separated by a fundamental tectonic boundary from rocks of the Alexander and Wrangellia terranes to the west.
Estimation of mortality of juvenile blacktip sharks, Carcharhinus limbatus, within a nursery area using telemetry data
Authors: Heupel M R , Simpfendorfer C A
Published: July 2002

A population of young blacktip sharks (Carcharhinus limbatus) was monitored over three years to determine their mortality rates using a series of acoustic listening stations. Based on these data it was possible to use several mortality estimators, including indirect life history based methods and direct methods such as the Kaplan-Meier and SURVIV methods, to estimate natural, fishing, and total mortality. Kaplan-Meier (6191%) and SURVIV (6292%) methods provided nearly identical total mortality rates during the first six months of life. This agreement suggests that these estimates are accurate for this population. All natural and fishing mortality occurred within the first 15 weeks of the study. This suggests that young sharks are most vulnerable to all types of mortality during this period. Sharks that survived beyond the first 15 weeks successfully left the nursery and were presumed to have migrated southward during fall months. These results provide critical information concerning the early life history of sharks and the importance of nursery areas to the survival of young animals.
Hydromagnetic flow of a dusty fluid in a rectangular channel with Hall current and heat transfer
Authors: Aboul-Hassan A L , Attia H A
Published: July 2002

The transient Hartmann flow of a dusty incompressible fluid under the influence of an applied uniform magnetic field is studied without neglecting the Hall current. The plates of the rectangular channel are assumed to be porous and subjected to a uniform suction from above and injection from below. The fluid is acted upon by a pressure gradient, which is either constant or exponentially decreasing with time. The relevant equations of motion are solved analytically to yield the velocity distribution for both the fluid and dust particles as functions of space and time, while the energy equations, which include the viscous, and Joule dissipation terms, are solved numerically to yield the temperature distributions.

PACS No.: 47.60+i
Ground-based Ring-effect measurements with the OSIRIS development model
Authors: Sioris C E , Evans W FJ , Gattinger R L , McDade I C , Degenstein D A , Llewellyn E J
Published: July 2002

The Ring effect is measured using the UVvisible spectrometer (~1 nm spectral resolution) of OSIRIS, the optical spectrograph and infrared imaging system. The observed filling in of Ca(II) H and K (~395 nm), the two largest Fraunhofer lines in the solar spectrum, are compared with the filling in simulated with a new model that includes rotational Raman scattering (RRS) by N2 and O2. The filling in is (1.06 ± 0.60)% at Ca(II) K and (1.40 ± 0.50)% at Ca(II) H for blue-sky observations at a solar zenith angle of 37°. The measured filling in agrees with the modelled filling in within the uncertainties.

PACS Nos.: 07.60Rd, 33.20Fb, 42.68Ay, 42.68Ca, 42.68Ge, 42.68Mj
Variational formulation of relativistic four-body systems in quantum field theory: scalar quadronium
Authors: Ding B , Darewych J W
Published: July 2002

We discuss a variational method for describing relativistic four-body systems within the Hamiltonian formalism of quantum field theory. The scalar Yukawa (or WickCutkosky) model, in which scalar particles and antiparticles interact via a massive or massless scalar field, is used to illustrate the method. A Fock-space variational trial state is used to describe the stationary states of scalar quadronium (two particles and two antiparticles) interacting via one-quantum exchange and virtual annihilation pairwise interactions. Numerical results for the ground-state mass and approximate wave functions of quadronium are presented for various strengths of the coupling, for the massive and massless quantum exchange cases.

PACS Nos.: 11.10Ef, 11.10St, 03.70+k, 03.65Pm
Novel esterase activity of dihydrolipoamide acetyltransferase AcoC of Pseudomonas putida identified by mutation of the acoR regulator
Authors: Pedroni Paola , Friedrich Thomas , Breuer Michael , McBeth Dani , Hauer Bernhard
Published: July 2002

A mutant strain of Pseudomonas putida (LU2201) expressing a novel ethylphenylacetate (EPA) esterase was isolated after mutagenesis of P. putida LU6456. The DNA fragment conferring the EPA+ phenotype was cloned and characterized. It included the structural genes of the 2,3-butanediol catabolic pathway, including acoC, which catalyzes the acetyl transfer step (i.e., dihydrolipoamide acetyltransferase). The acoC gene product from LU2201 was expressed in E. coli and shown to be responsible for the novel EPA-esterase activity. Biochemical characterization revealed that the enzyme still retained the natural dihydrolipoamide acetyltransferase activity. Sequence comparison with the corresponding wild-type gene indicated a single-base pair change leading to a I238V replacement in the respective protein. Biochemical characterization of wild-type AcoC enzyme showed that it too was able to catalyze EPA hydrolysis despite the apparent absence of this activity in cells grown in the presence of EPA. Unlike the wild type, EPA serves as an inducer in the mutant strain. Inverse PCR was used to clone the putative regulator acoR from the aco operon in both wild type and mutant strains. Sequence analyses indicated an S14R exchange in the AcoR from the P. putida mutant. This mutation was located in the N-terminal region responsible for regulatory functions, presumably by interaction with signal molecules. This may account for the ability of EPA to induce expression of acoC only in the mutant strain.Key words: Pseudomonas putida, ethylphenylacetate esterase, acetoin cleaving system, acoC gene, dihydrolipoamide acetyl-transferase, E2 component, acoR gene.
Effect of pH on the mechanism of OClO· oxidation of aromatic compounds
Authors: Svenson Doug , Kadla John F , Chang Hou-min , Jameel Hasan
Published: July 2002

Contrary to previous reports, the reaction mechanism of chlorine dioxide (OClO·) with benzyl alcohols involves both radical cation and benzyl radical mechanisms dependent on pH. The primary reaction product between OClO· and 1-(3,4-dimethoxy-phenyl) ethanol at pH 8 is 3,4-dimethoxyacetophenone. At pH 4 no acetophenone was observed; the majority of the degradation products were chlorinated and aromatic ring-oxidized compounds. A primary kinetic isotope effect (kH/kD = 2.05) was observed in the reaction of OClO· with 1-(3,4-dimethoxy-phenyl)-(1-2H) ethanol at pH 8, but was absent at pH 4 (kH/kD [Formula: see text] 1). Similarly, the corresponding methyl ether (4-(1-methoxy)ethyl-1,2-dimethoxybenzene) was substantially less reactive at pH > 6. On the basis of these results, competing pH-dependent reaction mechanisms have been proposed, where at high pH OClO· reacts with benzyl alcohols via a OClO·benzyl alcohol complex.Key words: chlorine dioxide, mechanism, kinetic isotope effect, aromatic radical cation, benzyl radical.
Kinetic resolution of oxime esters with lipases  synthesis of enantiomerically enriched building blocks with quaternary carbon centers and formal total synthesis of perhydro histrionicotoxin
Authors: Diedrichs Nicole , Krelaus Ralf , Gedrath Ina , Westermann Bernhard
Published: July 2002

Enantiomerically enriched oximes bearing stereogenic quaternary carbon centers can be obtained by lipase-catalyzed kinetic resolution of oxime esters. Substrate specificity, solvent effects, and the use of different lipases are discussed. Kinetic resolution of butyrylated oximes by lipase PS in the presence of n-butanol gave the best ee-values of both the saponified oxime and the residual oxime ester. Subsequent stereospecific Beckmann rearrangement of an enantiomerically enriched oxime provided lactams, which could be employed for the synthesis of optically active perhydro histrionicotoxin.Key words: oxime, lipase, kinetic resolution, Beckmann rearrangement, perhydro histrionicotoxin.