bluuebunny/crossref_metadata_2025 huggingface dataset:
Performance Benefits Using Siemens Advanced Compressor Cleaning System
Authors: Leusden Christoph Pels , Sorgenfrey Christoph , Du¨mmel Lutz
Published: November 2004

Extensive operational performance data from the Siemens Power Generation V64.3 unit in Obernburg, Germany (operated by Kraftwerk Obernburg GmbH) is evaluated. The unit was commissioned in 1996 and has been running continuously in base load operation with fuel gas to supply heat and power to a nearby chemical plant. In rare cases, fuel oil is used as a backup fuel. During the first major outage after approximately 25,000 equivalent operating hours (EOH), the Siemens PG Advanced Compressor Cleaning System (ACCS) was implemented at Obernburg. ACCS features separate nozzle systems for online and offline compressor cleaning accounting for different operating conditions. For online cleaning, the droplet size is optimized for the droplets to remain in the main air flow in order to minimize erosion effects while providing a homogeneous field over the whole air intake. With reduced rotational speed during offline compressor cleaning, erosion is less critical. Offline nozzles therefore provide higher mass flow and larger droplets in order to maximize cleaning performance for all compressor stages. ACCS, in its maximum automated version, features operation from the control room, online-washing at low ambient temperatures (officially released down to −15 °C without GT anti-icing) and minimum use of manpower. The ACCS system in Obernburg was operated according to the recommended online washing procedure. By June 2002, the V64.3 unit in Obernburg reached 50,000 EOH and the second major inspection was carried out. For this paper, operational data from the second inspection intervals (24,350–49,658 EOH) and from three performance tests with calibrated equipment are compared in order to evaluate the effectiveness of the advanced compressor cleaning system. Statistical evaluation of single-wash performance recovery and the evolution of long-term performance are presented. The effects of degradation and fouling are differentiated. It is shown that ACCS has a significant benefit for long-term engine performance.
Advanced Closed-Loop Control on an Atmospheric Gaseous Lean-Premixed Combustor
Authors: Riley A. J. , Park S. , Dowling A. P. , Evesque S. , Annaswamy A. M.
Published: November 2004

Active control of pressure oscillations has been successfully applied to a lean premixed prevaporized (LPP) combustion rig operating at atmospheric conditions. The design of the rig is based on the primary stage of the Rolls-Royce RB211-DLE industrial gas turbine. Control was achieved by modulating the fuel flow rate in response to a measured pressure signal. The feedback control is an adaptive, model-based self-tuning regulator (STR), which only requires the total time delay between actuation and response to achieve control. The STR algorithm achieves a reduction of up to 30 dB on the primary instability frequency. This performance was an improvement of 5–15 dB over an empirical control strategy (simple time-delay controller) specifically tuned to the same operating point. Initial robustness studies have shown that the STR retains control for a 20% change in frequency and a 23% change in air mass flow rate.
A Mutation in
Escherichia coli
DNA Gyrase Conferring Quinolone Resistance Results in Sensitivity to Drugs Targeting Eukaryotic Topoisomerase II
Authors: Gruger Thomas , Nitiss John L. , Maxwell Anthony , Zechiedrich E. Lynn , Heisig Peter , Seeber Siegfried , Pommier Yves , Strumberg Dirk
Published: November 2004

Fluoroquinolones are broad-spectrum antimicrobial agents that target type II topoisomerases. Many fluoroquinolones are highly specific for bacterial type II topoisomerases and act against both DNA gyrase and topoisomerase IV. In
Escherichia coli
, mutations causing quinolone resistance are often found in the gene that encodes the A subunit of DNA gyrase. One common site for resistance-conferring mutations alters Ser
83
, and mutations to Leu or Trp result in high levels of resistance to fluoroquinolones. In the present study we demonstrate that the mutation of Ser
83
to Trp in DNA gyrase (Gyr
S83W
) also results in sensitivity to agents that are potent inhibitors of eukaryotic topoisomerase II but that are normally inactive against prokaryotic enzymes. Epipodophyllotoxins, such as etoposide, teniposide and amino-azatoxin, inhibited the DNA supercoiling activity of Gyr
S83W
, and the enzyme caused elevated levels of DNA cleavage in the presence of these agents. The DNA sequence preference for Gyr
S83W
-induced cleavage sites in the presence of etoposide was similar to that seen with eukaryotic type II topoisomerases. Introduction of the Gyr
S83W
mutation in
E. coli
strain RFM443-242 by site-directed mutagenesis sensitized it to epipodophyllotoxins and amino-azatoxin. Our results demonstrate that sensitivity to agents that target topoisomerase II is conserved between prokaryotic and eukaryotic enzymes, suggesting that drug interaction domains are also well conserved and likely occur in domains important for the biochemical activities of the enzymes.
PnuC and the Utilization of the Nicotinamide Riboside Analog 3-Aminopyridine inHaemophilus influenzae
Authors: Sauer Elizabeta , Merdanovic Melisa , Price Mortimer Anne , Bringmann Gerhard , Reidl Joachim
Published: November 2004

The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized forHaemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC.pnuCofH. influenzaeis only distantly related topnuCofEscherichia coliandSalmonella entericaserovar Typhimurium. WhenE. coliPnuC was expressed in anH. influenzae pnuCmutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory forH. influenzaegrowth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory toH. influenzaeand is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in apnuC+background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistantH. influenzaeisolates were characterized, and mutations innadRcould be detected. We also tested other species of the familyPasteurellaceae,Pasteurella multocidaandActinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.
Detection of CTX-M-Type β-Lactamase Genes in Fecal
Escherichia coli
Isolates from Healthy Children in Bolivia and Peru
Authors: Pallecchi Lucia , Malossi Monica , Mantella Antonia , Gotuzzo Eduardo , Trigoso Christian , Bartoloni Alessandro , Paradisi Franco , Kronvall Göran , Rossolini Gian Maria
Published: November 2004

A survey was carried out from August to November 2002 to evaluate the antimicrobial susceptibilities of fecal
Escherichia coli
isolates from 3,208 healthy children from four different urban areas of Latin America, two in Bolivia (Camiri and Villa Montes) and two in Peru (Yurimaguas and Moyobamba). Ceftriaxone-resistant
E. coli
isolates were detected in four children, one from each of the areas sampled. The isolates exhibited a multidrug-resistant phenotype, including resistance to oxyimino-cephalosporins and aztreonam, and the MICs of ceftazidime for the isolates were lower than those of cefotaxime. By PCR and sequencing, the
bla
CTX-M-2
determinant was detected in three isolates and the
bla
CTX-M-15
determinant was detected in one isolate (from Peru). The CTX-M-2-producing isolates belonged to three different phylogenetic groups (groups A, B2, and D), while the CTX-M-15-producing isolate belonged to phylogenetic group D. The
bla
CTX-M-2
determinants were transferable to
E. coli
by conjugation, while conjugative transfer of the
bla
CTX-M-15
determinant was not detectable. Plasmids harboring the
bla
CTX-M-2
determinant exhibited similar restriction profiles, and in all of them the gene was located on a 2.2-kb PstI fragment, suggesting a genetic environment similar to that present in In35 and InS21. The findings of the present study confirm the widespread distribution of CTX-M-type β-lactamases and underscore the role that commensal
E. coli
isolates could play as a potential reservoir of these clinically relevant resistance determinants. This is the first report of CTX-M-type enzymes in Bolivia and Peru and also the first report of the detection of CTX-M-15 in Latin America.
Comparative In Vitro Activities of Linezolid, Telithromycin, Clarithromycin, Levofloxacin, Moxifloxacin, and Four Conventional Antimycobacterial Drugs against
Mycobacterium kansasii
Authors: Alcaide Fernando , Calatayud Laura , Santín Miguel , Martín Rogelio
Published: November 2004

Mycobacterium kansasii
is one of the most pathogenic and frequent nontuberculous mycobacteria isolated from humans. Patients with adverse drug reactions, resistant isolates, or suboptimal response require alternative treatment regimens. One hundred forty-eight consecutive clinical isolates of
M. kansasii
were tested for antimicrobial susceptibilities by the BACTEC 460 system (NCCLS) with two different inoculation protocols, one conventional and one alternative. In the alternative protocol, the inoculum 12B vial was incubated until the growth index was between 250 and 500. Four conventional antimycobacterial drugs (isoniazid, rifampin, streptomycin, and ethambutol) were studied with standard critical concentrations. The in vitro activities of linezolid, telithromycin, clarithromycin, levofloxacin, and moxifloxacin were determined by measuring radiometric MICs. All isolates tested were identified as
M. kansasii
genotype I and were resistant to isoniazid at a concentration of 0.4 μg/ml. One hundred twenty isolates (81.1%) were inhibited by 1 μg of isoniazid per ml. A high level of resistance to isoniazid (>10 μg/ml) was observed in six isolates (4.1%). Only five strains (3.4%) were resistant to rifampin (>1 μg/ml). All isolates studied were susceptible to streptomycin and ethambutol. The MICs at which 90% of the isolates were inhibited (in micrograms per milliliter) were as follows: linezolid, 1 (range, ≤0.25 to 2); telithromycin, >16 (range, 4 to >16); clarithromycin, 0.5 (range, ≤0.03 to 1); levofloxacin, 0.12 (range, 0.12 to 0.25); and moxifloxacin, 0.06 (range, ≤0.06 to 0.12). The susceptibility testing results with both inoculation protocols showed perfect correlation. In conclusion, all
M. kansasii
isolates showed decreased susceptibility to isoniazid, but resistance to rifampin was infrequent. Quinolones, especially moxifloxacin, were the most active antimicrobial agents tested, followed by clarithromycin. Linezolid also showed good activity against these microorganisms, but telithromycin's in vitro activity was poor.
Bloodstream Infections Due to Extended-Spectrum β
-
Lactamase-Producing
Escherichia coli
and
Klebsiella pneumoniae
: Risk Factors for Mortality and Treatment Outcome, with Special Emphasis on Antimicrobial Therapy
Authors: Kang Cheol-In , Kim Sung-Han , Park Wan Beom , Lee Ki-Deok , Kim Hong-Bin , Kim Eui-Chong , Oh Myoung-Don , Choe Kang-Won
Published: November 2004

This study was conducted to evaluate risk factors for mortality and treatment outcome of bloodstream infections due to extended-spectrum beta-lactamase (ESBL)-producing
Escherichia coli
and
Klebsiella pneumoniae
(ESBL-EK). ESBL production in stored
K. pneumoniae
and
E. coli
blood isolates from Jan 1998 to Dec 2002 was phenotypically determined according to NCCLS guidelines and/or the double-disk synergy test. A total of 133 patients with ESBL-EK bacteremia, including 66 patients with ESBL-producing
K. pneumoniae
and 67 with ESBL-producing
E. coli
, were enrolled. The overall 30-day mortality rate was 25.6% (34 of 133). Independent risk factors for mortality were severe sepsis, peritonitis, neutropenia, increasing Acute Physiology and Chronic Health Evaluation II score, and administration of broad-spectrum cephalosporin as definitive antimicrobial therapy (
P
< 0.05 for each of these risk factors). In 117 of the 133 patients, excluding 16 patients who died within 3 days after blood culture sample acquisition, the 30-day mortality rates according to definitive antibiotics were as follows: carbapenem, 12.9% (8 of 62); ciprofloxacin, 10.3% (3 of 29); and others, such as cephalosporin or an aminoglycoside, 26.9% (7 of 26). When patients who received appropriate definitive antibiotics, such as carbapenem or ciprofloxacin, were evaluated, mortality in patients receiving inappropriate empirical antimicrobial therapy was found not to be significantly higher than mortality in those receiving appropriate empirical antimicrobial therapy (18.9 versus 15.5%;
P
= 0.666). Carbapenem and ciprofloxacin were the most effective antibiotics in antimicrobial therapy for ESBL-EK bacteremia. A delay in appropriate definitive antimicrobial therapy was not associated with higher mortality if antimicrobial therapy was adjusted appropriately according to the susceptibility results. Our data suggest that more prudent use of carbapenem as empirical antibiotic may be reasonable.
Effect of Sucralfate on Antibiotic Therapy for
Helicobacter pylori
Infection in Mice
Authors: Watanabe Koichiro , Murakami Kazunari , Sato Ryugo , Kashimura Koji , Miura Masahiro , Ootsu Satoshi , Miyajima Hajime , Nasu Masaru , Okimoto Tadayoshi , Kodama Masaaki , Fujioka Toshio
Published: November 2004

It has been documented that sucralfate, a basic aluminum salt, enhances the efficacies of antibiotics against
Helicobacter pylori
, resulting in eradication rates comparable to those associated with the use of proton pump inhibitors. However, its mechanism of action remains unclear. The aim of the present study was to investigate sucralfate's ability to complement antibiotic treatment of
H. pylori
infection in vivo. Four weeks following induced
H. pylori
infection, clarithromycin (CAM) and amoxicillin (AMPC) were administered orally to C57BL/6 mice for 5 days, both with and without sucralfate or lansoprazole. When sucralfate was concurrently given with CAM and AMPC at the maximum noninhibitory doses for the treatment of
H. pylori
infection, the bacterial clearance rates were comparable to those achieved by treatment with lansoprazole plus those antibiotics. The results of pharmacokinetic studies showed that lansoprazole delayed gastric clearance and accelerated the absorption of CAM, whereas sucralfate suppressed both gastric clearance and absorption. AMPC was undetectable in all samples. Scanning electron microscopy with a microscope to which a energy dispersive spectrometer was attached revealed that aluminum-containing aggregated substances coated the mucosa surrounding
H. pylori
in mice receiving sucralfate plus antibiotics, whereas the gastric surface and pits where
H. pylori
had attached were clearly visible in mice receiving lansoprazole plus antibiotics. The addition of sucralfate to the antibiotic suspension resulted in a more viscous mixture that bound to the
H. pylori
-infected mucosa and that inhibited the loss of CAM bioavailability in the acidic environment. Sucralfate delays gastric clearance of CAM and physically captures
H. pylori
through the creation of an adherent mucus, which leads to bacterial clearance.
Characterization and Molecular Analysis of Macrolide-Resistant
Mycoplasma pneumoniae
Clinical Isolates Obtained in Japan
Authors: Matsuoka Mayumi , Narita Mitsuo , Okazaki Norio , Ohya Hitomi , Yamazaki Tsutomu , Ouchi Kazunobu , Suzuki Isao , Andoh Tomoaki , Kenri Tsuyoshi , Sasaki Yuko , Horino Atsuko , Shintani Miharu , Arakawa Yoshichika , Sasaki Tsuguo
Published: November 2004

In recent years,
Mycoplasma pneumoniae
strains that are clinically resistant to macrolide antibiotics have occasionally been encountered in Japan. Of 76 strains of
M. pneumoniae
isolated in three different areas in Japan during 2000 to 2003, 13 strains were erythromycin (ERY) resistant. Of these 13 strains, 12 were highly ERY resistant (MIC, ≥256 μg/ml) and 1 was weakly resistant (MIC, 8 μg/ml). Nucleotide sequencing of domains II and V of 23S rRNA and ribosomal proteins L4 and L22, which are associated with ERY resistance, showed that 10 strains had an A-to-G transition at position 2063 (corresponding to 2058 in
Escherichia coli
numbering), 1 strain showed A-to-C transversion at position 2063, 1 strain showed an A-to-G transition at position 2064, and the weakly ERY-resistant strain showed C-to-G transversion at position 2617 (corresponding to 2611 in
E. coli
numbering) of domain V. Domain II and ribosomal proteins L4 and L22 were not involved in the ERY resistance of these clinical
M. pneumoniae
strains. In addition, by using our established restriction fragment length polymorphism technique to detect point mutations of PCR products for domain V of the 23S rRNA gene of
M. pneumoniae
, we found that 23 (24%) of 94 PCR-positive oral samples taken from children with respiratory infections showed A2063G mutation. These results suggest that ERY-resistant
M. pneumoniae
infection is not unusual in Japan.
Pharmacokinetics of Telithromycin in Plasma and Soft Tissues after Single-Dose Administration to Healthy Volunteers
Authors: Gattringer Rainer , Urbauer Eleonora , Traunmüller Friederike , Zeitlinger Markus , Dehghanyar Pejman , Zeleny Petra , Graninger Wolfgang , Müller Markus , Joukhadar Christian
Published: November 2004

By use of microdialysis we assessed the concentrations of telithromycin in muscle and adipose tissue to test its ability to penetrate soft tissues. The ratios of the area under the concentration-versus-time curve from 0 to 24 h to the MIC indicated that free concentrations of telithromycin in tissue and plasma might be effective against
Streptococcus pyogenes
but not against staphylococci and human and animal bite pathogens.
Degradation of Human Antimicrobial Peptide LL-37 by
Staphylococcus aureus
-Derived Proteinases
Authors: Sieprawska-Lupa Magdalena , Mydel Piotr , Krawczyk Katarzyna , Wójcik Kinga , Puklo Magdalena , Lupa Boguslaw , Suder Piotr , Silberring Jerzy , Reed Matthew , Pohl Jan , Shafer William , McAleese Fionnuala , Foster Timothy , Travis Jim , Potempa Jan
Published: November 2004

Cathelicidin LL-37 is one of the few human bactericidal peptides with potent antistaphylococcal activity. In this study we examined the susceptibility of LL-37 to proteolytic degradation by two major proteinases produced by
Staphylococcus aureus
, a metalloproteinase (aureolysin) and a glutamylendopeptidase (V8 protease). We found that aureolysin cleaved and inactivated LL-37 in a time- and concentration-dependent manner. Analysis of the generated fragments by mass spectroscopy revealed that the initial cleavage of LL-37 by aureolysin occurred between the Arg19-Ile20, Arg23-Ile24, and Leu31-Val32 peptide bonds, instantly annihilating the antibacterial activity of LL-37. In contrast, the V8 proteinase hydrolyzed efficiently only the Glu16-Phe17 peptide bond, rendering the C-terminal fragment refractory to further degradation. This fragment (termed LL-17-37) displayed antibacterial activity against
S. aureus
at a molar level similar to that of the full-length LL-37 peptide, indicating that the antibacterial activity of LL-37 resides in the C-terminal region. In keeping with LL-37 degradation by aureolysin,
S. aureus
strains that produce significant amounts of this metalloprotease were found to be less susceptible to LL-17-37 than strains expressing no aureolysin activity. Taken together, these data suggest that aureolysin production by
S. aureus
contributes to the resistance of this pathogen to the innate immune system of humans mediated by LL-37.
Integron Carrying a Novel Metallo-β-Lactamase Gene,
bla
IMP-16
, and a Fused Form of Aminoglycoside-Resistant Gene
aac(6′)-30/aac(6′)-Ib′
: Report from the SENTRY Antimicrobial Surveillance Program
Authors: Mendes Rodrigo E. , Toleman Mark A. , Ribeiro Julival , Sader Helio S. , Jones Ronald N. , Walsh Timothy R.
Published: November 2004

Since January 2002
Pseudomonas
sp. strains resistant to carbapenems and ceftazidime have been routinely screened as part of the SENTRY Antimicrobial Surveillance Program for metallo-β-lactamase production, and their resistance determinants have been analyzed.
Pseudomonas aeruginosa
index strain 101-4704, which harbors a novel
bla
IMP
variant,
bla
IMP-16
, was isolated in April 2002 from a 60-year-old man in Brasília, Brazil.
bla
IMP-16
was found on the chromosome of the
P. aeruginosa
index strain, and the deduced amino acid sequence (IMP-16) showed the greatest identities to IMP-11 (90.3%) and IMP-8 (89.5%). Sequence analysis revealed that
bla
IMP-16
was associated with a class 1 integron, which also encoded aminoglycoside-modifying enzymes. Downstream of
bla
IMP-16
resided an open reading frame, which consisted of a new aminoglycoside-modifying gene, namely,
aac(6′)-30
, which was fused with
aac(6′)-Ib′
. The amino acid sequence of the
aac(6′)-30
putative protein showed the most identity (52.7%) to the sequence of AAC(6′)-29b described previously. The fourth gene cassette constituted
aadA1
. The steady-state kinetics of IMP-16 demonstrated that the enzyme preferred cephalosporins and carbapenems to penicillins. The main functional difference observed among the kinetic values for IMP-16 compared to those for other IMPs was a lack of cefoxitin hydrolysis and a lower
k
cat
/

K
m

value for imipenem (0.36 μM
−1
 · s
−1
). This report further emphasizes the spread of metallo-β-lactamase genes and their close association with various aminoglycoside resistance genes.
Liposomal Nystatin in Patients with Invasive Aspergillosis Refractory to or Intolerant of Amphotericin B
Authors: Offner Fritz , Krcmery Vladimir , Boogaerts Marc , Doyen Chantal , Engelhard Dan , Ribaud Patricia , Cordonnier Catherine , de Pauw Ben , Durrant Simon , Marie Jean-Pierre , Moreau Philippe , Guiot Harry , Samonis George , Sylvester Richard , Herbrecht Raoul
Published: November 2004

We assessed the activity and safety of liposomal nystatin, a broad-spectrum antifungal agent, for invasive aspergillosis in patients refractory to or intolerant of amphotericin B. Thirty-three patients were enrolled, received at least one dose of the study drug, and were evaluable for safety. Twenty-six patients had confirmed probable or definite aspergillosis and were fully eligible. Most patients had a hematological malignancy (53.8%) or hematopoietic stem cell transplantation (23.0%), were neutropenic (61.5%), and were refractory to previous amphotericin B (92.3%). The median duration of previous amphotericin B treatment was 16.5 days (range, 5 to 64 days). Aspergillosis was definite in 3 cases and probable in 23 cases. Liposomal nystatin was initiated at a dose of 4 mg/kg of body weight/day. Twenty-five patients were evaluable for response: a complete response was achieved for one patient, and a partial response was achieved for six. Thus, the overall response rate is 7 of 25 (28%; 95% confidence interval, 12 to 49%). Seventeen (68.0%) of the 25 evaluable patients died during therapy or within 1 month after the end of therapy. The primary cause of death was invasive aspergillosis for nine patients and underlying malignancy for eight patients. The most frequent side effects included chills, shivering, and fever, leading to discontinuation of therapy for two patients. Grade 1 decline in renal function was seen for 10 (30.3%) patients, and hypokalemia was seen for 13 (39.4%). We conclude that liposomal nystatin can be effective for salvage therapy of invasive aspergillosis. Infusion-related adverse events have been observed frequently.
Novel Nonnucleoside Inhibitor of Hepatitis C Virus RNA-Dependent RNA Polymerase
Authors: Howe Anita Y. M. , Bloom Johnathan , Baldick Carl J. , Benetatos Christopher A. , Cheng Huiming , Christensen Joel S. , Chunduru Srinivas K. , Coburn Glen A. , Feld Boris , Gopalsamy Ariamala , Gorczyca William P. , Herrmann Steve , Johann Stephen , Jiang Xiaoqun , Kimberland Michelle L. , Krisnamurthy Girija , Olson Matthew , Orlowski Mark , Swanberg Steve , Thompson Ian , Thorn Megan , Del Vecchio Alfred , Young Dorothy C. , van Zeijl Marja , Ellingboe John W. , Upeslacis Janis , Collett Marc , Mansour Tarek S. , O'Connell John F.
Published: November 2004

A novel nonnucleoside inhibitor of hepatitis C virus (HCV) RNA-dependent RNA polymerase (RdRp), [(1
R
)-5-cyano-8-methyl-1-propyl-1,3,4,9-tetrahydropyano[3,4-b]indol-1-yl] acetic acid (HCV-371), was discovered through high-throughput screening followed by chemical optimization. HCV-371 displayed broad inhibitory activities against the NS5B RdRp enzyme, with 50% inhibitory concentrations ranging from 0.3 to 1.8 μM for 90% of the isolates derived from HCV genotypes 1a, 1b, and 3a. HCV-371 showed no inhibitory activity against a panel of human polymerases, including mitochondrial DNA polymerase gamma, and other unrelated viral polymerases, demonstrating its specificity for the HCV polymerase. A single administration of HCV-371 to cells containing the HCV subgenomic replicon for 3 days resulted in a dose-dependent reduction of the steady-state levels of viral RNA and protein. Multiple treatments with HCV-371 for 16 days led to a >3-log
10
reduction in the HCV RNA level. In comparison, multiple treatments with a similar inhibitory dose of alpha interferon resulted in a 2-log
10
reduction of the viral RNA level. In addition, treatment of cells with a combination of HCV-371 and pegylated alpha interferon resulted in an additive antiviral activity. Within the effective antiviral concentrations of HCV-371, there was no effect on cell viability and metabolism. The intracellular antiviral specificity of HCV-371 was demonstrated by its lack of activity in cells infected with several DNA or RNA viruses. Fluorescence binding studies show that HCV-371 binds the NS5B with an apparent dissociation constant of 150 nM, leading to high selectivity and lack of cytotoxicity in the antiviral assays.
Distribution of Antibiotic MICs for
Helicobacter pylori
Strains over a 16-Year Period in Patients from Seoul, South Korea
Authors: Kim Jung Mogg , Kim Joo Sung , Jung Hyun Chae , Kim Nayoung , Kim Young-Jeon , Song In Sung
Published: November 2004

Recently, the development of antibiotic resistance emerged as a significant clinical problem in the eradication of
Helicobacter pylori
. We investigated the MICs of antibiotics for 135
H. pylori
isolates from adults in Seoul, South Korea, over the past 16 years. The MICs of amoxicillin, clarithromycin, metronidazole, tetracycline, azithromycin, and ciprofloxacin increased from 1987 to 2003. Rates of primary resistance to clarithromycin increased from 2.8% in 1994 to 13.8% in 2003. The A2144G mutation was frequently observed in the 23S rRNA gene in clarithromycin-resistant isolates. The increase in resistance to clarithromycin seems to result in a decrease in eradication efficacy for
H. pylori
. These results suggest that the MICs of several antibiotics for
H. pylori
have increased over the past 16 years in Seoul.
Association of a Novel Human Immunodeficiency Virus Type 1 Protease Substrate Cleft Mutation, L23I, with Protease Inhibitor Therapy and In Vitro Drug Resistance
Authors: Johnston Elizabeth , Winters Mark A. , Rhee Soo-Yon , Merigan Thomas C. , Schiffer Celia A. , Shafer Robert W.
Published: November 2004

We observed a previously uncharacterized mutation in the protease substrate cleft, L23I, in 31 of 4,303 persons undergoing human immunodeficiency virus type 1 genotypic resistance testing. In combination with V82I, L23I was associated with a sevenfold reduction in nelfinavir susceptibility and a decrease in replication capacity. In combination with other drug resistance mutations, L23I was associated with multidrug resistance and a compensatory increase in replication capacity.
PCR-Restriction Fragment Length Polymorphism Assay for Detection of
gyrA
Mutations Associated with Fluoroquinolone Resistance in
Campylobacter coli
Authors: Alonso Rodrigo , Mateo Estibaliz , Girbau Cecilia , Churruca Estibaliz , Martinez Irati , Fernández-Astorga Aurora
Published: November 2004

A fragment of the
gyrA
gene was sequenced from 34 isolates of
Campylobacter coli
, including 23 isolates resistant to ciprofloxacin. All ciprofloxacin-resistant isolates examined by DNA sequencing carried a point mutation at position Thr-86 on the
gyrA
gene product, involving the replacement of Thr-86 by Ile. A combined PCR-restriction fragment length polymorphism technique using RsaI was developed to detect this mutation.
The Synthetic N-Terminal Peptide of Human Lactoferrin, hLF(1-11), Is Highly Effective against Experimental Infection Caused by Multidrug-Resistant
Acinetobacter baumannii
Authors: Dijkshoorn Lenie , Brouwer Carlo P. J. M. , Bogaards Sylvia J. P. , Nemec Alexandr , van den Broek Peterhans J. , Nibbering Peter H.
Published: November 2004

The lactoferrin-derived peptide hLF(1-11), but not its control peptide, was highly effective against five multidrug-resistant
Acinetobacter baumannii
strains in vitro (3 to 4 log reduction) and against four of these strains in an experimental infection in mice (2 to 3 log reduction). Therefore, this peptide is a promising candidate as a novel agent against infections with multidrug-resistant
A. baumannii
.
Two Amino Acid Mutations in the Capsid Protein of Type 2 Porcine Circovirus (PCV2) Enhanced PCV2 Replication In Vitro and Attenuated the Virus In Vivo
Authors: Fenaux M. , Opriessnig T. , Halbur P. G. , Elvinger F. , Meng X. J.
Published: November 2004

Porcine circovirus type 2 (PCV2) is the primary causative agent of postweaning multisystemic wasting syndrome (PMWS) in pigs. To identify potential genetic determinants for virulence and replication, we serially passaged a PCV2 isolate 120 times in PK-15 cells. The viruses harvested at virus passages 1 (VP1) and 120 (VP120) were biologically, genetically, and experimentally characterized. The PCV2 VP120 virus replicated in PK-15 cells to a titer similar to that of the PK-15 cell line-derived nonpathogenic PCV1 but replicated more efficiently than PCV2 VP1 with a difference of about 1 log unit in the titers. The complete genomic sequences of viruses at passages 0, 30, 60, 90, and 120 were determined. After 120 passages, only two nucleotide mutations were identified in the entire genome, and both were located in the capsid gene: the mutations were located at nucleotide positions 328 (C328G) and 573 (A573C). The C328G mutation, in which a proline at position 110 of the capsid protein changed to an alanine (P110A), occurred at passage 30 and remained in the subsequent passages. The second mutation, A573C, resulting in a change from an arginine to a serine at position 191 (R191S), appeared at passage 120. To experimentally characterize the VP120 virus, 31 specific-pathogen-free pigs were randomly divided into three groups. Ten pigs in group 1 received phosphate-buffered saline as negative controls. Each pig in group 2 (11 pigs) was inoculated intramuscularly and intranasally with 10
4.9
50% tissue culture infective doses (TCID
50
) of PCV2 VP120. Each pig in group 3 (10 pigs) was similarly inoculated with 10
4.9
TCID
50
of PCV2 VP1. Viremia was detected in 9 of 10 pigs in the PCV2 VP1 group with a mean duration of 3 weeks, but in only 4 of 11 pigs in the PCV2 VP120 group with a mean duration of 1.6 weeks. The PCV2 genomic copy numbers in serum in the PCV2 VP1 group were significantly higher than those in the PCV2 VP120 group (
P
< 0.0001). Gross and histopathologic lesions in pigs inoculated with PCV2 VP1 were more severe than those inoculated with PCV2 VP120 at both day 21 and 42 necropsies (
P
= 0.0032 and
P
= 0.0274, respectively). Taken together, the results from this study indicated that the P110A and R191S mutations in the capsid of PCV2 enhanced the growth ability of PCV2 in vitro and attenuated the virus in vivo. This finding has important implications for PCV2 vaccine development.
The Positively Charged Termini of L2 Minor Capsid Protein Required for Bovine Papillomavirus Infection Function Separately in Nuclear Import and DNA Binding
Authors: Fay Alyson , Yutzy William H. , Roden Richard B. S. , Moroianu Junona
Published: November 2004

During the papillomavirus (PV) life cycle, the L2 minor capsid protein enters the nucleus twice: in the initial phase after entry of virions into cells and in the productive phase to mediate encapsidation of the newly replicated viral genome. Therefore, we investigated the interactions of the L2 protein of bovine PV type 1 (BPV1) with the nuclear import machinery and the viral DNA. We found that BPV1 L2 bound to the karyopherin α2 (Kap α2) adapter and formed a complex with Kap α2β1 heterodimers. Previous data have shown that the positively charged termini of BPV1 L2 are required for BPV1 infection after the binding of the virions to the cell surface. We determined that these BPV1 L2 termini function as nuclear localization signals (NLSs). Both the N-terminal NLS (nNLS) and the C-terminal NLS (cNLS) interacted with Kap α2, formed a complex with Kap α2β1 heterodimers, and mediated nuclear import via a Kap α2β1 pathway. Interestingly, the cNLS was also the major DNA binding site of BPV1 L2. Consistent with the promiscuous DNA encapsidation by BPV1 pseudovirions, this DNA binding occurred without nucleotide sequence specificity. Moreover, an L2 mutant encoding a scrambled version of the cNLS, which supports production of virions, rescued the DNA binding but not the Kap α2 interaction. These data support a model in which BPV1 L2 functions as an adapter between the viral DNA via the cNLS and the Kaps via the nNLS and facilitates nuclear import of the DNA during infection.
Immunogenicity Study of Glycoprotein-Deficient Rabies Virus Expressing Simian/Human Immunodeficiency Virus SHIV89.6PEnvelope in a Rhesus Macaque
Authors: McKenna Philip M. , Aye Pyone Pyone , Dietzschold Bernhard , Montefiori David C. , Martin Louis N. , Marx Preston A. , Pomerantz Roger J. , Lackner Andrew , Schnell Matthias J.
Published: November 2004

Rabies virus (RV) has recently been developed as a novel vaccine candidate for human immunodeficiency virus type 1 (HIV-1). The RV glycoprotein (G) can be functionally replaced by HIV-1 envelope glycoprotein (Env) if the gp160 cytoplasmic domain (CD) of HIV-1 Env is replaced by that of RV G. Here, we describe a pilot study of the in vivo replication and immunogenicity of an RV with a deletion of G (ΔG) expressing a simian/human immunodeficiency virus SHIV89.6PEnv ectodomain and transmembrane domain fused to the RV G CD (ΔG-89.6P-RVG) in a rhesus macaque. An animal vaccinated with ΔG-89.6P-RVG developed SHIV89.6Pvirus-neutralizing antibodies and SHIV89.6P-specific cellular immune responses after challenge with SHIV89.6P. There was no evidence of CD4+T-cell loss, and plasma viremia was controlled to undetectable levels by 6 weeks postchallenge and has remained suppressed out to 22 weeks postchallenge.
Lytic Cycle Gene Regulation of Epstein-Barr Virus
Authors: Amon Wolfgang , Binné Ulrich K. , Bryant Helen , Jenkins Peter J. , Karstegl Claudio Elgueta , Farrell Paul J.
Published: November 2004

Episomal reporter plasmids containing the Epstein-Barr virus (EBV) oriP sequence stably transfected into Akata Burkitt's lymphoma cells were used to analyze EBV lytic cycle gene regulation. First, we found that the Zp promoter of EBV, but not the Rp promoter, can be activated in the absence of protein synthesis in these oriP plasmids, casting doubt on the immediate early status of Rp. An additional level of regulation of Zp was implied by analysis of a mutation of the ZV element. Second, our analysis of late lytic cycle promoters revealed that the correct relative timing, dependence on ori lyt in
cis
, and sensitivity to inhibitors of DNA replication were reconstituted on the oriP plasmids. Late promoter luciferase activity from oriP plasmids also incorporating replication-competent ori lyt was phosphonoacetic acid sensitive, a hallmark of EBV late genes. A minimal ori lyt, which only replicates weakly, was sufficient to confer late timing of expression specifically on late promoters. Finally, deletion analysis of EBV late promoter sequences upstream of the transcription start site confirmed that sequences between −49 and +30 are sufficient for late gene expression, which is dependent on ori lyt in
cis
. However, the TATT version of the TATA box found in many late genes was not essential for late expression.
Circulation of Type 1 Vaccine-Derived Poliovirus in the Philippines in 2001
Authors: Shimizu Hiroyuki , Thorley Bruce , Paladin Fem Julia , Brussen Kerri Anne , Stambos Vicki , Yuen Lilly , Utama Andi , Tano Yoshio , Arita Minetaro , Yoshida Hiromu , Yoneyama Tetsuo , Benegas Agnes , Roesel Sigrun , Pallansch Mark , Kew Olen , Miyamura Tatsuo
Published: November 2004

In 2001, highly evolved type 1 circulating vaccine-derived poliovirus (cVDPV) was isolated from three acute flaccid paralysis patients and one contact from three separate communities in the Philippines. Complete genomic sequencing of these four cVDPV isolates revealed that the capsid region was derived from the Sabin 1 vaccine strain but most of the noncapsid region was derived from an unidentified enterovirus unrelated to the oral poliovirus vaccine (OPV) strains. The sequences of the cVDPV isolates were closely related to each other, and the isolates had a common recombination site. Most of the genetic and biological properties of the cVDPV isolates were indistinguishable from those of wild polioviruses. However, the most recently identified cVDPV isolate from a healthy contact retained the temperature sensitivity and partial attenuation phenotypes. The sequence relationships among the isolates and Sabin 1 suggested that cVDPV originated from an OPV dose given in 1998 to 1999 and that cVDPV circulated along a narrow chain of transmission. Type 1 cVDPV was last detected in the Philippines in September 2001, and population immunity to polio was raised by extensive OPV campaigns in late 2001 and early 2002.
Coordination of Transcription Factor Phosphorylation and Histone Methylation by the P-TEFb Kinase during Human Immunodeficiency Virus Type 1 Transcription
Authors: Zhou Meisheng , Deng Longwen , Lacoste Vincent , Park Hyeon Ung , Pumfery Anne , Kashanchi Fatah , Brady John N. , Kumar Ajit
Published: November 2004

The human immunodeficiency virus type 1 (HIV-1) Tat protein recruits positive transcription elongation factor b (P-TEFb) to the transactivation response (TAR) RNA structure to facilitate formation of processive transcription elongation complexes (TECs). Here we examine the role of the Tat/TAR-specified cyclin-dependent kinase 9 (CDK9) kinase activity in regulation of HIV-1 transcription elongation and histone methylation. In HIV-1 TECs, P-TEFb phosphorylates the RNA polymerase II (RNAP II) carboxyl-terminal domain (CTD) and the transcription elongation factors SPT5 and Tat-SF1 in a Tat/TAR-dependent manner. Using in vivo chromatin immunoprecipitation analysis, we demonstrate the following distinct properties of the HIV-1 transcription complexes. First, the RNAP II CTD is phosphorylated at Ser 2 and Ser 5 near the promoter and at downstream coding regions. Second, the stable association of SPT5 with the TECs is dependent upon P-TEFb kinase activity. Third, P-TEFb kinase activity is critical for the induction of methylation of histone H3 at lysine 4 and lysine 36 on HIV-1 genes. Flavopiridol, a potent P-TEFb kinase inhibitor, inhibits CTD phosphorylation, stable SPT5 binding, and histone methylation, suggesting that its potent antiviral activity is due to its ability to inhibit several critical and unique steps in HIV-1 transcription elongation.
Role of the VP16-Binding Domain of vhs in Viral Growth, Host Shutoff Activity, and Pathogenesis
Authors: Strand Stephanie S. , Leib David A.
Published: November 2004

The virion host shutoff (vhs) protein of herpes simplex virus type 1 causes the degradation of host and viral mRNA immediately upon infection of permissive cells. vhs can interact with VP16 through a 20-amino-acid binding domain, and viruses containing a deletion of this VP16-binding domain of vhs (Δ20) and a corresponding marker rescue (Δ20R) were constructed and characterized. Transient-transfection assays showed that this domain was dispensable for vhs activity. The Δ20 recombinant virus, however, was unable to induce mRNA degradation in the presence of actinomycin D, while degradation induced by Δ20R was equivalent to that for wild-type virus. Δ20, Δ20R, and KOS caused comparable RNA degradation in the absence of actinomycin D. Western blot analysis of infected cells indicated that comparable levels of vhs were expressed by Δ20, Δ20R, and KOS, and there was only a modest reduction of vhs packaging in Δ20. Immunoprecipitation of protein from cells infected with Δ20 and Δ20R showed equivalent coprecipitation of vhs and VP16. Pathogenesis studies with Δ20 showed a significant decrease in replication in the corneas, trigeminal ganglia, and brains, as well as a significant reduction in clinical disease and lethality, but no significant difference in the establishment of, or reactivation from, latency compared to results with KOS and Δ20R. These results suggest that the previously described VP16-binding domain is not required for vhs packaging or for binding to VP16. It is required, however, for RNA degradation activity of tegument-derived vhs and wild-type replication and virulence in mice.
Molecular Characterization of Human Immunodeficiency Virus Type 1 and Hepatitis C Virus in Paid Blood Donors and Injection Drug Users in China
Authors: Zhang Linqi , Chen Zhiwei , Cao Yunzhen , Yu Jian , Li Guanghan , Yu Wenjie , Yin Ning , Mei Shan , Li Li , Balfe Peter , He Tian , Ba Lei , Zhang Fengwen , Lin His-Hsun , Yuen Man-Fung , Lai Ching-Lung , Ho David D.
Published: November 2004

China is facing a rapid upsurge in cases of human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infection due to large numbers of paid blood donors (PBD), injection drug users (IDU), and sexual partners of infected individuals. In this report, a total of 236 HIV-1-positive blood samples were collected from PBD, IDU, and their sexual partners in the most severely affected provinces, such as Henan, Yunnan, Guangxi, and Xinjiang. PCR was used to amplify the p17 region of
gag
and the C2-V3 region of
env
of HIV-1 and the 5′ noncoding region and a region of E1/E2 of HCV. Genetic characterization of viral sequences indicated that there are two major epidemics of HIV-1 and multiple HCV epidemics in China. The PBD and transfusion recipients in Henan harbored HIV-1 subtype B′, which is similar to the virus found in Thailand, and HCV genotypes 1b and 2a, whereas the IDU in Yunnan, Guangxi, and Xinjiang carried HIV-1 circulating recombinant forms 07 and 08, which resemble those in India, and HCV genotypes 1b, 3a, and 3b. Our findings show that the epidemics of HIV-1 and HCV infection in China are the consequences of multiple introductions. The distinct distribution patterns of both the HIV-1 and HCV genotypes in the different high-risk groups are tightly linked to the mode of transmission rather than geographic proximity. These findings provide information relevant to antiviral therapy and vaccine development in China and should assist public health workers in implementing measures to reduce the further dissemination of these viruses in the world's most populous nation.
Mucosal Human Papillomaviruses Encode Four Different E5 Proteins Whose Chemistry and Phylogeny Correlate with Malignant or Benign Growth
Authors: Bravo Ignacio G. , Alonso Ángel
Published: November 2004

We performed a phylogenetic study of the E2-L2 region of human mucosal papillomaviruses (PVs) and of the proteins therein encoded. Hitherto, proteins codified in this region were known as E5 proteins. We show that many of these proteins could be spurious translations, according to phylogenetic and chemical coherence criteria between similar protein sequences. We show that there are four separate families of E5 proteins, with different characteristics of phylogeny, chemistry, and rate of evolution. For the sake of clarity, we propose a change in the present nomenclature. E5α is present in groups A5, A6, A7, A9, and A11, PVs highly associated with malignant carcinomas of the cervix and penis. E5β is present in groups A2, A3, A4, and A12, i.e., viruses associated with certain warts. E5γ is present in group A10, and E5δ is encoded in groups A1, A8, and A10, which are associated with benign transformations. The phylogenetic relationships between mucosal human PVs are the same when considering the oncoproteins E6 and E7 and the E5 proteins and differ from the phylogeny estimated for the structural proteins L1 and L2. Besides, the protein divergence rate is higher in early proteins than in late proteins, increasing in the order L1 < L2 < E6 ≈ E7 < E5. Moreover, the same proteins have diverged more rapidly in viruses associated with malignant transformations than in viruses associated with benign transformations. The E5 proteins display, therefore, evolutionary characteristics similar to those of the E6 and E7 oncoproteins. This could reflect a differential involvement of the E5 types in the transformation processes.
Protein Kinase C-α Activity Is Required for Respiratory Syncytial Virus Fusion to Human Bronchial Epithelial Cells
Authors: San-Juan-Vergara Homero , Peeples Mark E. , Lockey Richard F. , Mohapatra Shyam S.
Published: November 2004

Respiratory syncytial virus (RSV) infection activates protein kinase C (PKC), but the precise PKC isoform(s) involved and its role(s) remain to be elucidated. On the basis of the activation kinetics of different signaling pathways and the effect of various PKC inhibitors, it was reasoned that PKC activation is important in the early stages of RSV infection, especially RSV fusion and/or replication. Herein, the role of PKC-α during the early stages of RSV infection in normal human bronchial epithelial cells is determined. The results show that the blocking of PKC-α activation by classical inhibitors, pseudosubstrate peptides, or the overexpression of dominant-negative mutants of PKC-α in these cells leads to significantly decreased RSV infection. RSV induces phosphorylation, activation, and cytoplasm-to-membrane translocation of PKC-α. Also, PKC-α colocalizes with virus particles and is required for RSV fusion to the cell membrane. Thus, PKC-α could provide a new pharmacological target for controlling RSV infection.