bluuebunny/crossref_metadata_2025 huggingface dataset:
Prospective evaluation of a cluster program for Finnish forestry and forest industries
Authors: Salo A. , Gustafsson T. , Mild P.
Published: March 2004

In this paper, we report a prospective evaluation process for a major research program for the Finnish forestry and forest industries. To a significant extent, this process was based on fifteen participatory workshops where tools of operational research — most notably multi‐criteria methods embedded in a group support system — were deployed to help representatives from industry, the research community, and public administration in the assessment of socio‐economic impacts and the identification of further research topics. Drawing upon this case study, we also analyze the preconditions, advantages, and limitations of similar kinds of participatory processes in the implementation of formative and summative evaluations.
A contribution to the development of strategic control and planning instruments: An acquisition case study
Authors: Chevalier A. , Kunsch P. L. , Brans J. P.
Published: March 2004

The present paper is part of the efforts made by the authors in recent years to develop strategic control and planning instruments in corporations using OR‐techniques like system dynamics, control theory, and group multicriteria decision aid. A more general framework called ‘adaptive control methodology’ (ACM) combines all these techniques. It has been presented in several papers. The objective of the present analysis is to calibrate this instrument and to tune it to the corporate needs by analysing real‐world applications. More specifically, several case studies have been investigated in large multinational organisations in the food sector. An acquisition case has been used for the calibration purpose. It is analysed in the paper from the ACM perspective to provide additional material for revisiting and improving the methodology.
An AHP/DEA method for measurement of the efficiency of R&D management activities in universities
Authors: Feng Y.J. , Lu H. , Bi K.
Published: March 2004

In order to develop a better tool for the assessment of the management performance of research and development (R&D) activities in research‐oriented universities, a combination of analysis hierarchical process (AHP) and data envelopment analysis (DEA) is proposed for the assessment of the efficiency of R&D management activities in universities. The measure consists of the measurement of a university's previous and present R&D strength by AHP and the assessment of the relative efficiency of its growth in R&D strength against those of other universities by DEA, in which the management basis of the measured universities is taken into consideration. The application of the measure to assess the R&D management efficiency of 29 universities in China indicates the universities which have improved their management work achieved a high efficiency value regardless of whether their original R&D strengths were strong or weak. Such a measure is proved to be helpful for motivating the universities to keep on improving their R&D management.
A surrogate indicator of criticality for stochastic project networks
Authors: Tavares L. V. , Ferreira J. A. , Coelho J. S.
Published: March 2004

The study of the delay that can be caused by any activity of a stochastic project network is a key topic because of the increasing importance of risk and time control in project management. The main concept adopted for this purpose has been the notion of critical activity developed for deterministic project networks but, in this paper, the inadequacy of the concept critical activity for stochastic project networks is shown and a new surrogate indicator of criticality (SIC) is built, using a regression model applied to a large set of generated project networks. This new indicator explains more than 90% of the initial variance estimated for more than 80,000 activities belonging to a wide range of project networks (580 nets), with very different morphological types.
Effect of surface metallization of graphite on the tribological properties of copper hybrid composites
Authors: Zhan Yongzhong , Zhang Guoding , Wu Yuying
Published: March 2004

Copper matrix composites reinforced with SiC particles and graphite with or without copper coating, were fabricated by a powder metallurgy method. The results show that composites with copper‐coated graphite particles exhibit higher densification, electrical conductivity, flexural strength, and ductility than that without copper coating. The addition of graphite particles in the hybrid composites decreases the wear rate of the tribosystem at low‐applied load. At high‐applied load, cracks propagation in graphite‐copper interface results in the dramatic increment of wear rate of the tribosystem and friction coefficient for the composites with uncoated graphite. By improving the Gr–Cu interfacial bonding, a graphite‐rich tribolayer can be formed steadily on the worn surface of the composite till high load, which imparts the hybrid composites with good anti‐friction property and is in favour of increasing the life span of the tribosystem.
Effect of preaging on precipitation behavior in aluminum alloy 6063 during high‐temperature aging
Authors: Ou Bin‐Lung , Shen Chin‐Hui
Published: March 2004

The influence of low‐temperature preaging on the artificial age‐hardening of a aluminum alloy 6063 (Al–0.72Mg–0.42Si) has been investigated in this study. The precipitation behaviors of samples of the alloy were analyzed using differential scanning calorimetry as well as by measuring the electrical resistivity, and confirmed by microstructure observation using transmission electron microscopy. Tensile tests were performed to determine the mechanical properties of the samples. The results indicate that GP zones (I) are formed in the aluminum alloy 6063 at 0–30°C during a long preaging period. The formation of GP zones (I) will retard the precipitation of the intermediate β′ phase during artificial aging and will result in decrease in the strength of the alloy after the artificial aging process. Increasing either the preaging temperature or the holding period will significantly decrease the strength after artificial aging. The results indicate that GP zones (II) are formed in the Al alloy 6063 at a preaging temperature of 40–70°C but this has no influence on age‐hardening after artificial aging, even for a long period. Increasing either the preaging temperature or the holding period will significantly increase the strength after artificial aging. However, for preaging at −10°C, the negative effect on age‐hardening is still not clear and should be investigated in the future.
Antiretroviral Spermicide WHI-07 Prevents Vaginal and Rectal Transmission of Feline Immunodeficiency Virus in Domestic Cats
Authors: D'Cruz Osmond J. , Waurzyniak Barbara , Uckun Fatih M.
Published: March 2004

WHI-07 [5-bromo-6-methoxy-5,6-dihydro-3′-azidothymidine-5′-(
p
-bromophenyl)-methoxy alaninyl phosphate] is a novel dual-function aryl phosphate derivative of zidovudine with potent anti-human immunodeficiency virus (HIV) and spermicidal activities. WHI-07 was active against the feline immunodeficiency virus (FIV). This study evaluated whether topical application of WHI-07 as a single agent and in combination with an organometallic vanadium complex, vanadocene dithiocarbamate (VDDTC), via a nontoxic gel microemulsion can block vaginal as well as rectal transmission of feline AIDS (FAIDS) by chronically FIV-infected feline T cells in the natural host model. Genital transmission of FIV was monitored in recipient cats by the appearance of viral antibodies to FIV Gag proteins and by virus isolation of blood leukocytes as measured by FIV reverse transcriptase activity and FIV-specific PCR. Microbicidal activity was considered effective when the treated cats did not show evidence of FIV infection for up to 18 weeks postchallenge. An aggregate analysis of 46 specific-pathogen-free cats revealed that a single dose of the infected cell inoculum efficiently transmitted FIV infection when delivered into the vagina (100%) or rectum (66%). Pretreatment of the vagina or rectum with 2% WHI-07 alone or in combination with 0.25% VDDTC significantly (
P
= 0.004) protected cats from genital transmission by the highly infectious inoculum (7 million FIV
Bangston
-infected feline T cells). Collectively, using the vaginal and rectal transmucosal model for FAIDS, our studies demonstrated that WHI-07 either alone or in combination with a vanadocene has clinical potential for the development of a dual-function anti-HIV microbicide for sexually active women.
The
Candida albicans
Lanosterol 14-α-Demethylase (
ERG11
) Gene Promoter Is Maximally Induced after Prolonged Growth with Antifungal Drugs
Authors: Song Jia L. , Harry Jo Beth , Eastman Richard T. , Oliver Brian G. , White Theodore C.
Published: March 2004

The azole antifungal drugs that target lanosterol 14-α-demethylase, encoded by the
ERG11
gene, are used to treat a variety of infections caused by
Candida albicans.
Azoles are known to induce expression of
ERG11
mRNA. The
ERG11
promoter was cloned 5′ of the luciferase-coding region, and the induction of
ERG11
expression by azoles was monitored by luciferase assays. Maximal induction of the
ERG11
promoter by azoles occurs not during logarithmic growth but after the diauxic shift and requires azoles to be present throughout logarithmic growth. The effects of pH, carbon source, and aerobic or anaerobic growth on induction of the
ERG11
promoter by azoles were analyzed. Treatment with terbinafine and fenpropimorph, which target other enzymes in the ergosterol biosynthetic pathway, also resulted in a delayed induction of
ERG11
promoter activity. Nascent sterol synthesis was shown to parallel
ERG11
promoter activity, and total sterols were reduced coincident with the timing of
ERG11
promoter activation. These results as a whole suggest that expression of the
ERG11
promoter is regulated in response to sterol depletion.
Dynamics and Spatial Distribution of β-Lactamase Expression in
Pseudomonas aeruginosa
Biofilms
Authors: Bagge Niels , Hentzer Morten , Andersen Jens Bo , Ciofu Oana , Givskov Michael , Høiby Niels
Published: March 2004

The development of resistance to β-lactam antibiotics is a problem in the treatment of chronic
Pseudomonas aeruginosa
infection in the lungs of patients with cystic fibrosis. The main resistance mechanism is high-level expression of the chromosomally encoded AmpC β-lactamase of
P. aeruginosa
cells growing in biofilms. Several genes have been shown to influence the level of
ampC
expression, but little is known about the regulation of
ampC
expression in
P. aeruginosa
biofilms. To study the expression of
ampC
in
P. aeruginosa
biofilms, we constructed a reporter that consisted of the fusion of the
ampC
promoter to
gfp
(ASV) encoding an unstable version of the green fluorescent protein. In vitro biofilms of
P. aeruginosa
were exposed to the β-lactam antibiotics imipenem and ceftazidime. Sub-MICs of imipenem significantly induced the monitor system of the biofilm bacteria in the peripheries of the microcolonies, but the centers of the microcolonies remained uninduced. However, the centers of the microcolonies were physiologically active, as shown by experiments with another monitor construction consisting of an arabinose-inducible promoter fused to
gfp
(ASV). The whole biofilm was induced in the presence of increased imipenem concentrations. Ceftazidime induced the monitor system of the biofilm bacteria as well, but only bacteria in the peripheries of the microcolonies were induced in the presence of even very high concentrations. The experiments illustrate for the first time the dynamic and spatial distributions of β-lactamase induction in
P. aeruginosa
cells growing in biofilms. Thus, our experiments show that
P. aeruginosa
cells growing in biofilms constitute a heterogeneous population unit which may create different antibiotic-selective environments for the bacteria in the biofilm.
Ambler Class A Extended-Spectrum Beta-Lactamase-Producing
Escherichia coli
and
Klebsiella
spp. in Canadian Hospitals
Authors: Mulvey Michael R. , Bryce Elizabeth , Boyd David , Ofner-Agostini Marianna , Christianson Sara , Simor Andrew E. , Paton Shirley
Published: March 2004

This report describes a study carried out to gain baseline information on the molecular characteristics of extended-spectrum beta-lactamase (ESBL)-producing
Escherichia coli
and
Klebsiella
spp. in Canada. A total of 29,323
E. coli
and 5,156
Klebsiella
sp. isolates were screened at 12 participating sites. Of these, 505 clinically significant, nonrepeat isolates displaying reduced susceptibility to the NCCLS-recommended beta-lactams were submitted to a central laboratory over a 1-year period ending on 30 September 2000. A total of 116 isolates were confirmed to be ESBL producers. PCR and sequence analysis revealed the presence of TEM-11 (
n
= 1), TEM-12 (
n
= 1), TEM-29 (
n
= 1), TEM-52 (
n
= 4), CTX-M-13 (
n
= 1), CTX-M-14 (
n
= 15), CTX-M-15 (
n
= 11), SHV-2 (
n
= 2), SHV-2a (
n
= 12), SHV-5 (
n
= 6), SHV-12 (
n
= 45), and SHV-30 (
n
= 2). Five novel beta-lactamases were identified and designated TEM-115 (
n
= 2), TEM-120 (
n
= 1), SHV-40 (
n
= 2), SHV-41 (
n
= 4), and SHV-42 (
n
= 1). In addition, no molecular mechanism was identified for five isolates displaying an ESBL phenotype. Macrorestriction analysis of all ESBL isolates was conducted, as was restriction fragment length polymorphism analysis of plasmids harboring ESBLs. Although a “clonal” distribution of isolates was observed at some individual sites, there was very little evidence suggesting intrahospital spread. In addition, examples of identical or closely related plasmids that were identified at geographically distinct sites across Canada are given. However, there was considerable diversity with respect to plasmid types observed.
Chemosensitization of Fluconazole Resistance in
Saccharomyces cerevisiae
and Pathogenic Fungi by a
d
-Octapeptide Derivative
Authors: Niimi K. , Harding D. R. K. , Parshot R. , King A. , Lun D. J. , Decottignies A. , Niimi M. , Lin S. , Cannon R. D. , Goffeau A. , Monk B. C.
Published: March 2004

Hyperexpression of the
Saccharomyces cerevisiae
multidrug ATP-binding cassette (ABC) transporter Pdr5p was driven by the
pdr1-3
mutation in the Pdr1p transcriptional regulator in a strain (AD/PDR5
+
) with deletions of five other ABC-type multidrug efflux pumps. The strain had high-level fluconazole (FLC) resistance (MIC, 600 μg ml
−1
), and plasma membrane fractions showed oligomycin-sensitive ATPase activity up to fivefold higher than that shown by fractions from an isogenic
PDR5
-null mutant (FLC MIC, 0.94 μg ml
−1
). In vitro inhibition of the Pdr5p ATPase activity and chemosensitization of cells to FLC allowed the systematic screening of a 1.8-million-member designer
d-
octapeptide combinatorial library for surface-active Pdr5p antagonists with modest toxicity against yeast cells. Library deconvolution identified the 4-methoxy-2,3,6-trimethylbenzensulfonyl-substituted
d-
octapeptide KN20 as a potent Pdr5p ATPase inhibitor (concentration of drug causing 50% inhibition of enzyme activity [IC
50
], 4 μM) which chemosensitized AD/PDR5
+
to FLC, itraconazole, and ketoconazole. It also inhibited the ATPase activity of other ABC transporters, such as
Candida albicans
Cdr1p (IC
50
, 30 μM) and Cdr2p (IC
50
, 2 μM), and chemosensitized clinical isolates of pathogenic
Candida
species and
S. cerevisiae
strains that heterologously hyperexpressed either ABC-type multidrug efflux pumps, the
C. albicans
major facilitator superfamily-type drug transporter Ben
R
p, or the FLC drug target lanosterol 14α-demethylase (Erg11p). Although KN20 also inhibited the
S. cerevisiae
plasma membrane proton pump Pma1p (IC
50
, 1 μM), the peptide concentrations required for chemosensitization made yeast cells permeable to rhodamine 6G. KN20 therefore appears to indirectly chemosensitize cells to FLC by a nonlethal permeabilization of the fungal plasma membrane.
Disposition of Caspofungin, a Novel Antifungal Agent, in Mice, Rats, Rabbits, and Monkeys
Authors: Sandhu Punam , Xu Xin , Bondiskey Peter J. , Balani Suresh K. , Morris Michael L. , Tang Yui S. , Miller Alisha R. , Pearson Paul G.
Published: March 2004

The metabolism, excretion, and pharmacokinetics of caspofungin (Cancidas; Merck & Co., Inc.) were investigated after administration of a single intravenous dose to mice, rats, rabbits, and monkeys. Caspofungin had a low plasma clearance (0.29 to 1.05 ml/min/kg) and a long terminal elimination half-life (11.7 h to 59.7 h) in all preclinical species. The elimination kinetics of caspofungin were multiphasic and displayed an initial distribution phase followed by a dominant β-elimination phase. The presence of low levels of prolonged radioactivity in plasma was observed and was partially attributable to the chemical degradation product M0. Excretion studies with [3H]caspofungin indicated that the hepatic and renal routes play an important role in the elimination of caspofungin, as a large percentage of the radiolabeled dose was recovered in urine and feces. Excretion of radioactivity in all species studied was slow, and low levels of radioactivity were detected in daily urine and fecal samples throughout a prolonged collection period. Although urinary profiles indicated the presence of several metabolites (M0, M1, M2, M3, M4, M5, and M6), the majority of the total radioactivity was associated with the polar metabolites M1 [4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine] and M2 [N-acetyl-4(S)-hydroxy-4-(4-hydroxyphenyl)-l-threonine]. Caspofungin was thus primarily eliminated by metabolic transformation; however, the rate of metabolism was slow. These results suggest that distribution plays a prominent role in determining the plasma pharmacokinetics and disposition of caspofungin, as very little excretion or biotransformation occurred during the early days after dose administration, a period during which concentrations in plasma fell substantially. The disposition of caspofungin in preclinical species was similar to that reported previously in humans.
Antimicrobial and DNA Gyrase-Inhibitory Activities of Novel Clorobiocin Derivatives Produced by Mutasynthesis
Authors: Galm Ute , Heller Stefanie , Shapiro Stuart , Page Malcolm , Li Shu-Ming , Heide Lutz
Published: March 2004

Twenty-eight novel clorobiocin derivatives obtained from mutasynthesis experiments were investigated for their inhibitory activity towards
Escherichia coli
DNA gyrase and for their antibacterial activities towards clinically relevant gram-positive and gram-negative bacteria in comparison to novobiocin and clorobiocin. Clorobiocin was the most active compound both against
E. coli
DNA gyrase in vitro and against bacterial growth. All tested modifications of the 3-dimethylallyl-4-hydroxybenzoyl moiety reduced biological activity. The highest activities were shown by compounds containing a hydrophobic alkyl substituent at position 3 of the 4-hydroxybenzoyl moiety. Polar groups in this side chain, especially amide functions, strongly reduced antibacterial activity. Replacement of the alkyl side chain with a halogen atom or a methoxy group at the same position markedly reduced activity. Transfer of the pyrrole carboxylic acid moiety from O-3" to O-2" of
l
-noviose moderately reduced activity, whereas the complete absence of the pyrrole carboxylic acid moiety led to a loss of activity. Desclorobiocin derivatives lacking the chlorine atom at C-8 of the 3-amino-4,7-dihydroxycoumarin moiety also showed low activity. Lack of a methyl group at O-4" of
l
-noviose resulted in an inactive compound. From these findings it appears that clorobiocin represents a “highly evolved” structure optimized for bacterial transport and DNA gyrase inhibition.
Role of Topoisomerase Mutations and Efflux in Fluoroquinolone Resistance of
Bacteroides fragilis
Clinical Isolates and Laboratory Mutants
Authors: Ricci Vito , Peterson Marnie L. , Rotschafer John C. , Wexler Hannah , Piddock Laura J. V.
Published: March 2004

Twelve laboratory mutants and 32 ciprofloxacin-resistant isolates of
Bacteroides fragilis
were examined for the mechanism(s) of fluoroquinolone resistance. Five mutants had mutations in
gyrA
. One mutant and two clinical isolates contained a mutation in
gyrB.
Eight mutants and five clinical isolates accumulated significantly less ciprofloxacin than did wild-type isolates; the mutants and clinical isolates were restored to wild-type characteristics when carbonyl cyanide
m
-chlorophenylhydrazone was used.
Evidence for a Chromosomally Located Third Integron in
Salmonella enterica
Serovar Typhimurium DT104b
Authors: Daly M. , Buckley J. , Power E. , Fanning S.
Published: March 2004

Salmonella enterica
serovar Typhimurium isolates of phage types DT104 and DT104b are frequently associated with multiple antimicrobial resistance. We describe the characterization of a class 1 integron containing
dfrA1
and
aadA1
, genes from two
Salmonella
serovar Typhimurium DT104b isolates. Genetic mapping located the integron to the bacterial chromosome in each case.
Effect of Amoxicillin-Clavulanic Acid on Human Fecal Flora in a Gnotobiotic Mouse Model Assessed with Fluorescence Hybridization Using Group-Specific 16S rRNA Probes in Combination with Flow Cytometry
Authors: Barc Marie Claude , Bourlioux François , Rigottier-Gois Lionel , Charrin-Sarnel Céline , Janoir Claire , Boureau Hélène , Doré Joël , Collignon Anne
Published: March 2004

Predominant groups of bacteria from a human fecal flora-associated mouse model challenged with amoxicillin-clavulanic acid were quantified with fluorescence in situ hybridization combined with flow cytometry using specific 16S rRNA targeted oligonucleotide probes. This approach provides a useful tool with high throughput to evaluate fecal microflora under antibiotic treatment.
Molecular Characterization of the Gene Encoding a New AmpC β-Lactamase in a Clinical Strain of
Acinetobacter
Genomic Species 3
Authors: Beceiro Alejandro , Dominguez Lourdes , Ribera Anna , Vila Jordi , Molina Francisca , Villanueva Rosa , Eiros Jose Maria , Bou German
Published: March 2004

A presumptive chromosomal cephalosporinase (pI, 9.0) from a clinical strain of
Acinetobacter
genomic species 3 (AG3) is reported. The nucleotide sequence of this β-lactamase shows for the first time the gene encoding an AmpC enzyme in AG3. In addition, the biochemical properties of the novel AG3 AmpC β-lactamase are reported
In Vitro Antimicrobial Activity of Doripenem, a New Carbapenem
Authors: Ge Yigong , Wikler Matthew A. , Sahm Daniel F. , Blosser-Middleton Renée S. , Karlowsky James A.
Published: March 2004

The doripenem MICs at which 90% of the tested strains were inhibited ranged from 0.03 to 1 μg/ml for 10 species of
Enterobacteriaceae
(
n
= 351), from 0.03 to 0.12 μg/ml for oxacillin-susceptible staphylococci (
n
= 119), from 4 to 32 μg/ml for oxacillin-resistant staphylococci (
n
= 64), from ≤0.008 to 0.06 μg/ml for penicillin-susceptible streptococci (
n
= 132), and from 1 to 4 μg/ml for penicillin-resistant streptococci (
n
= 51). Overall, doripenem demonstrated in vitro activity similar to that of meropenem against gram-negative pathogens and to that of imipenem against gram-positive pathogens.
Reduced Triclosan Susceptibility in Methicillin-Resistant
Staphylococcus epidermidis
Authors: Schmid Molly B. , Kaplan Nachum
Published: March 2004

Triclosan MIC determination showed that recent
Staphylococcus aureus
clinical isolates (
n
= 100) were highly susceptible to triclosan, with a 50% minimal inhibitory concentration (MIC
50
) of 0.12 μg/ml and a MIC
90
of 0.25 μg/ml.
Staphylococcus epidermidis
isolates (
n
= 96) were less susceptible, with a MIC
50
of 0.12 μg/ml and a MIC
90
of 8 μg/ml. Decreased susceptibility to triclosan was more prevalent among methicillin-resistant
S. epidermidis
than among methicillin-sensitive
S. epidermidis
isolates.
Human Immunodeficiency Virus Type 1 Reverse Transcriptase Mutation Selection during In Vitro Exposure to Tenofovir Alone or Combined with Abacavir or Lamivudine
Authors: Stone Chris , Ait-Khaled Mounir , Craig Charles , Griffin Philip , Tisdale Margaret
Published: March 2004

Mutations selected or deselected during passage of human immunodeficiency virus strain HXB2 or resistant variants with tenofovir (TFV), abacavir (ABC), and lamivudine (3TC) differed depending on the drug combination and virus genotype. In the wild-type virus, TFV-ABC and TFV-3TC selected K65R (with reduced susceptibility to all three inhibitors) and then Y115F. TFV-containing regimens might increase K65R selection, which confers multiple nucleoside reverse transcriptase inhibitor resistance.
Identification of the
Haemophilus influenzae tolC
Gene by Susceptibility Profiles of Insertionally Inactivated Efflux Pump Mutants
Authors: Trepod Catherine M. , Mott John E.
Published: March 2004

Isogenic strains containing insertional disruptions of 10
Haemophilus influenzae
Rd genes were investigated for their effects on the susceptibility of the organism to various classes of antimicrobial compounds. MIC results show that HI1462, which encodes an
Escherichia coli
TolC homolog, is the third component of the
H. influenzae
AcrAB pump.
2′-Fluoro-2′-Deoxycytidine Inhibits Borna Disease Virus Replication and Spread
Authors: Bajramovic Jeffrey J. , Volmer Romain , Syan Sylvie , Pochet Sylvie , Gonzalez-Dunia Daniel
Published: March 2004

Borna disease virus (BDV) causes neurological diseases in a variety of warm-blooded animal species, possibly including humans. To date, there is no effective treatment against BDV infection. Recently, we reported on the antiviral activity of 1-β-
d
-arabinofuranosylcytosine (Ara-C). However, Ara-C's cytotoxic side effects are a major obstacle for its therapeutic use. Herein, we demonstrate that the nucleoside analog 2′-fluoro-2′-deoxycytidine (2′-FdC) exhibits potent antiviral activity against BDV. Importantly, 2′-FdC-associated cytotoxicity is negligible, indicating 2′-FdC as an excellent candidate for the development of antiviral therapy against BDV.
Genogroup II Noroviruses Efficiently Bind to Heparan Sulfate Proteoglycan Associated with the Cellular Membrane
Authors: Tamura Masaru , Natori Katsuro , Kobayashi Masahiko , Miyamura Tatsuo , Takeda Naokazu
Published: March 2004

Norovirus(NV), a member of the familyCaliciviridae, is one of the important causative agents of acute gastroenteritis. In the present study, we found that virus-like particles (VLPs) derived from genogroup II (GII) NV were bound to cell surface heparan sulfate proteoglycan. Interestingly, the VLPs derived from GII were more than ten times likelier to bind to cells than were those derived from genogroup I (GI). Heparin, a sulfated glycosaminoglycan, and suramin, a highly sulfated derivative of urea, efficiently blocked VLP binding to mammalian cell surfaces. The reagents known to bind to cell surface heparan sulfate, as well as the enzymes that specifically digest heparan sulfate, markedly reduced VLP binding to the cells. Treatment of the cells with chlorate revealed that sulfation of heparan sulfate plays an important role in the NV-heparan sulfate interaction. The binding efficiency of NV to undifferentiated Caco-2 (U-Caco-2) cells differed largely between GI NV and GII NV, whereas the efficiency of binding to differentiated Caco-2 (D-Caco-2) cells did not differ significantly between the two genogroups, although slight differences between strains were observed. Digestion with heparinase I resulted in a reduction of up to 90% in U-Caco-2 cells and a reduction of up to only 50% in D-Caco-2 cells, indicating that heparan sulfate is the major binding molecule for U-Caco-2 cells, while it contributed to only half of the binding in the case of D-Caco-2 cells. The other half of those VLPs was likely to be associated with H-type blood antigen, suggesting that GII NV has two separate binding sites. The present study is the first to address the possible role of cell surface glycosaminoglycans in the binding of recombinant VLPs of NV.
Live, Attenuated Coronavirus Vaccines through the Directed Deletion of Group-Specific Genes Provide Protection against Feline Infectious Peritonitis
Authors: Haijema Bert Jan , Volders Haukeline , Rottier Peter J. M.
Published: March 2004

Feline infectious peritonitis (FIP) is a fatal immunity-mediated disease caused by mutants of a ubiquitous coronavirus. Since previous attempts to protect cats under laboratory and field conditions have been largely unsuccessful, we used our recently developed system of reverse genetics (B. J. Haijema, H. Volders, and P. J. M. Rottier, J. Virol. 77:4528-4538, 2003) for the development of a modified live FIP vaccine. With this objective, we deleted the group-specific gene cluster open reading frame 3abc or 7ab and obtained deletion mutant viruses that not only multiplied well in cell culture but also showed an attenuated phenotype in the cat. At doses at which the wild-type virus would be fatal, the mutants with gene deletions did not cause any clinical symptoms. They still induced an immune response, however, as judged from the high levels of virus-neutralizing antibodies. The FIP virus (FIPV) mutant lacking the 3abc cluster and, to a lesser extent, the mutant missing the 7ab cluster, protected cats against a lethal homologous challenge; no protection was obtained with the mutant devoid of both gene clusters. Our studies show that the deletion of group-specific genes from the coronavirus genome results in live attenuated candidate vaccines against FIPV. More generally, our approach may allow the development of vaccines against infections with other pathogenic coronaviruses, including that causing severe acute respiratory syndrome in humans.
Dissection of Antiviral and Immune Regulatory Functions of Tumor Necrosis Factor Receptors in a Chronic Lymphocytic Choriomeningitis Virus Infection
Authors: Suresh M. , Gao Xiaoyan , Fischer Christopher , Miller Nicole E. , Tewari Kavita
Published: March 2004

The effector function of CD8 T cells is mediated via cell-mediated cytotoxicity and production of cytokines like gamma interferon (IFN-γ) and tumor necrosis factor alpha (TNF-α). While the roles of perforin-dependent cytotoxicity, IFN-γ, and TNF-α in controlling acute viral infections are well studied, their relative importance in defense against chronic viral infections is not well understood. Using mice deficient for TNF receptor (TNFR) I and/or II, we show that TNF-TNFR interactions have a dual role in mediating viral clearance and downregulating CD8 and CD4 T-cell responses during a chronic lymphocytic choriomeningitis virus (LCMV) infection. While wild-type (+/+) and TNFR II-deficient (p75−/−) mice cleared LCMV from the liver and lung, mice deficient in TNFR I (p55−/−) or both TNFR I and TNFR II (double knockout [DKO]) exhibited impaired viral clearance. The inability of p55−/−and DKO mice to clear LCMV was not a sequel to either suboptimal activation of virus-specific CD8 or CD4 T cells or impairment in trafficking of LCMV-specific CD8 T cells to the liver and lung. In fact, the expansion of LCMV-specific CD8 and CD4 T cells was significantly higher in DKO mice compared to that in +/+, p55−/−, and p75−/−mice. TNFR deficiency did not preclude the physical deletion of CD8 T cells specific for nucleoprotein 396 to 404 but delayed the contraction of CD8 T-cell responses to the epitopes GP33-41 and GP276-285 in the viral glycoprotein. The antibody response to LCMV was not significantly altered by TNFR deficiency. Taken together, these findings have implications in development of immunotherapy in chronic viral infections of humans.
NFI-Ski Interactions Mediate Transforming Growth Factor β Modulation of Human Papillomavirus Type 16 Early Gene Expression
Authors: Baldwin Amy , Pirisi Lucia , Creek Kim E.
Published: March 2004

Human papillomaviruses (HPVs) are present in virtually all cervical cancers. An important step in the development of malignant disease, including cervical cancer, involves a loss of sensitivity to transforming growth factor β (TGF-β). HPV type 16 (HPV16) early gene expression, including that of the E6 and E7 oncoprotein genes, is under the control of the upstream regulatory region (URR), and E6 and E7 expression in HPV16-immortalized human epithelial cells is inhibited at the transcriptional level by TGF-β. While the URR contains a myriad of transcription factor binding sites, including seven binding sites for nuclear factor I (NFI), the specific sequences within the URR or the transcription factors responsible for TGF-β modulation of the URR remain unknown. To identify potential transcription factors and binding sites involved in TGF-β modulation of the URR, we performed DNase I footprint analysis on the HPV16 URR using nuclear extracts from TGF-β-sensitive HPV16-immortalized human keratinocytes (HKc/HPV16) treated with and without TGF-β. Differentially protected regions were found to be located around NFI binding sites. Electrophoretic mobility shift assays, using the NFI binding sites as probes, showed decreased binding upon TGF-β treatment. This decrease in binding was not due to reduced NFI protein or NFI mRNA levels. Mutational analysis of individual and multiple NFI binding sites in the URR defined their role in TGF-β sensitivity of the promoter. Overexpression of the NFI family members in HKc/HPV16 decreased the ability of TGF-β to inhibit the URR. Since the oncoprotein Ski has been shown to interact with and increase the transcriptional activity of NFI and since cellular Ski levels are decreased by TGF-β treatment, we explored the possibility that Ski may provide a link between TGF-β signaling and NFI activity. Anti-NFI antibodies coimmunoprecipitated endogenous Ski in nuclear extracts from HKc/HPV16, confirming that NFI and Ski interact in these cells. Ski levels dramatically decreased upon TGF-β treatment of HKc/HPV16, and overexpression of Ski eliminated the ability of TGF-β to inhibit the URR. Based on these studies, we propose that TGF-β inhibition of HPV16 early gene expression is mediated by a decrease in Ski levels, which in turn dramatically reduces NFI activity.
Gamma Interferon/Interleukin 10 Balance in Tissue Lymphocytes Correlates with Down Modulation of Mucosal Feline Immunodeficiency Virus Infection
Authors: Avery Paul R. , Hoover Edward A.
Published: March 2004

Understanding the early cytokine response to lentiviral infections may be critical to the design of prevention and treatment strategies. By using the feline immunodeficiency virus (FIV) model, we have documented an interleukin 10 (IL10)-dominated response in lymphoid tissue CD4+and CD8+T lymphocytes within the first 4 weeks after mucosal FIV infection. This profile coincided with the period of high tissue viral replication. By 10 weeks postinfection, tissue viral levels decreased significantly, and gamma interferon (IFNγ) production in CD8+T cells had increased to restore the IL10/IFNγ ratio to control levels. Concurrently, increased production of IL6 and viral RNA was detected in macrophages. These temporal associations of viral replication with cytokine balance in tissues suggest roles for IL10 in the permissive stage of infection and IFNγ in the subsequent down modulation of lentiviral infection.
Protection from Bacterial Infection by a Single Vaccination with Replication-Deficient Mutant Herpes Simplex Virus Type 1
Authors: Lauterbach Henning , Kerksiek Kristen M. , Busch Dirk H. , Berto Elena , Bozac Aleksandra , Mavromara Penelope , Manservigi Roberto , Epstein Alberto L. , Marconi Peggy , Brocker Thomas
Published: March 2004

Adaptive immune responses in which CD8+T cells recognize pathogen-derived peptides in the context of major histocompatibility complex class I molecules play a major role in the host defense against infection with intracellular pathogens. Cells infected with intracellular bacteria such asListeria monocytogenes,Salmonella entericaserovar Typhimurium, orMycobacterium tuberculosisare directly lysed by cytotoxic CD8+T cells. For this reason, current vaccines for intracellular pathogens, such as subunit vaccines or viable bacterial vaccines, aim to generate robust cytotoxic T-cell responses. In order to investigate the capacity of a herpes simplex virus type 1 (HSV-1) vector to induce strong cytotoxic effector cell responses and protection from infection with intracellular pathogens, we developed a replication-deficient, recombinant HSV-1 (rHSV-1) vaccine. We demonstrate in side-by-side comparison with DNA vaccination that rHSV-1 vaccination induces very strong CD8+effector T-cell responses. While both vaccines provided protection from infection withL. monocytogenesat low, but lethal doses, only rHSV-1 vaccines could protect from higher infectious doses; HSV-1 induced potent memory cytotoxic T lymphocytes that, upon challenge by pathogens, efficiently protected the animals. Despite the stimulation of relatively low humoral and CD4-T-cell responses, rHSV-1 vectors are strong candidates for future vaccine strategies that confer efficient protection from subsequent infection with intracellular bacteria.
Impact of Human Cytomegalovirus Latent Infection on Myeloid Progenitor Cell Gene Expression
Authors: Slobedman Barry , Stern J. Lewis , Cunningham Anthony L. , Abendroth Allison , Abate Davide A. , Mocarski Edward S.
Published: March 2004

Herpesviruses establish lifelong latent infections in their hosts. Human cytomegalovirus (CMV) targets a population of bone marrow-derived myeloid lineage progenitor cells that serve as a reservoir for reactivation; however, the mechanisms by which latent CMV infection is maintained are unknown. To gain insights into mechanisms of maintenance and reactivation, we employed microarrays of ∼26,900 sequence-verified human cDNAs to assess global changes in cellular gene expression during experimental CMV latent infection of granulocyte-macrophage progenitors (GM-Ps). This analysis revealed at least 29 host cell genes whose expression was increased and six whose expression was decreased during CMV latency. These changes in transcript levels appeared to be authentic, judging on the basis of further analysis of a subset by semiquantitative reverse transcription-PCR. This study provides a comprehensive snapshot of changes in host cell gene expression that result from latent infection and suggest that CMV regulates genes that encode proteins involved in immunity and host defense, cell growth, signaling, and transcriptional regulation. The host genes whose expression we found altered are likely to contribute to an environment that sustains latent infection.
Role of NF-κB in Cell Survival and Transcription of Latent Membrane Protein 1-Expressing or Epstein-Barr Virus Latency III-Infected Cells
Authors: Cahir-McFarland Ellen D. , Carter Kara , Rosenwald Andreas , Giltnane Jena M. , Henrickson Sarah E. , Staudt Louis M. , Kieff Elliott
Published: March 2004

Epstein-Barr virus (EBV) latency III infection converts B lymphocytes into lymphoblastoid cell lines (LCLs) by expressing EBV nuclear and membrane proteins, EBNAs, and latent membrane proteins (LMPs), which regulate transcription through Notch and tumor necrosis factor receptor pathways. The role of NF-κB in LMP1 and overall EBV latency III transcriptional effects was investigated by treating LCLs with BAY11-7082 (BAY11). BAY11 rapidly and irreversibly inhibited NF-κB, decreased mitochondrial membrane potential, induced apoptosis, and altered LCL gene expression. BAY11 effects were similar to those of an NF-κB inhibitor, ΔN-IκBα, in effecting decreased JNK1 expression and in microarray analyses. More than 80% of array elements that decreased with ΔN-IκBα expression decreased with BAY11 treatment. Newly identified NF-κB-induced, LMP1-induced, and EBV-induced genes included pleckstrin, Jun-B, c-FLIP, CIP4, and IκBε. Of 776 significantly changed array elements, 134 were fourfold upregulated in EBV latency III, and 74 were fourfold upregulated with LMP1 expression alone, whereas only 28 were more than fourfold downregulated by EBV latency III. EBV latency III-regulated gene products mediate cell migration (EBI2, CCR7, RGS1, RANTES, MIP1α, MIP1β, CXCR5, and RGS13), antigen presentation (major histocompatibility complex proteins and JAW1), mitogen-activated protein kinase pathway (DUSP5 and p62Dok), and interferon (IFN) signaling (IFN-γRα, IRF-4, and STAT1). Comparison of EBV latency III LCL gene expression to immunoglobulin M (IgM)-stimulated B cells, germinal-center B cells, and germinal-center-derived lymphomas clustered LCLs with IgM-stimulated B cells separately from germinal-center cells or germinal-center lymphoma cells. Expression of IRF-2, AIM1, ASK1, SNF2L2, and components of IFN signaling pathways further distinguished EBV latency III-infected B cells from IgM-stimulated or germinal-center B cells.
Adenovirus Type 7 Induces Interleukin-8 in a Lung Slice Model and Requires Activation of Erk
Authors: Booth J. L. , Coggeshall K. M. , Gordon B. E. , Metcalf J. P.
Published: March 2004

Adenovirus (Ad), particularly Ad type 7 (Ad7), causes severe lung infection and pneumonia. Initially, Ad causes neutrophilic inflammation of the distal airways and alveoli. Interleukin-8 (IL-8) is the major lung neutrophil chemotaxin, and we have shown that Ad7 induces IL-8 release from the A549 alveolar epithelial cell line. We sought to determine whether ex vivo human and bovine lung tissue containing primary pneumocytes could be used as a more accurate and relevant model to study Ad acute inflammation. We found that cultured lung tissue preserved normal lung architecture for more than 10 days. IL-8 was generated upon exposure of the lung organ culture to Ad7. IL-8 production required activation of the Ras/Erk pathway, since a pharmacological inhibitor blocked the appearance of IL-8 in the medium. Both human and bovine lung explants supported replication of Ad7, and immunohistochemistry experiments demonstrated the presence of the Ad hexon antigen within alveolar epithelial cells. These findings show that our novel human lung organ culture accurately reproduces the in vivo infectious disease process. Thus, this organ culture model represents a valuable tool for studying the acute innate immune response to respiratory infections.
Fatty Acid Synthase Expression Is Induced by the Epstein-Barr Virus Immediate-Early Protein BRLF1 and Is Required for Lytic Viral Gene Expression
Authors: Li Yuling , Webster-Cyriaque Jennifer , Tomlinson Christine C. , Yohe Marielle , Kenney Shannon
Published: March 2004

The Epstein-Barr virus (EBV) immediate-early (IE) protein BRLF1 (R) is a transcription factor that induces the lytic form of EBV infection. R activates certain early viral promoters through a direct binding mechanism but induces transcription of the other EBV IE gene, BZLF1 (Z), indirectly through cellular factors binding to a CRE motif in the Z promoter (Zp). Here we demonstrate that R activates expression of the fatty acid synthase (FAS) cellular gene through a p38 stress mitogen-activated protein kinase-dependent mechanism. B-cell receptor engagement of Akata cells also increases FAS expression. The FAS gene product is required for de novo synthesis of the palmitate fatty acid, and high-level FAS expression is normally limited to liver, brain, lung, and adipose tissue. We show that human epithelial tongue cells lytically infected with EBV (from oral hairy leukoplakia lesions) express much more FAS than uninfected cells. Two specific FAS inhibitors, cerulenin and C75, prevent R activation of IE (Z) and early (BMRF1) lytic EBV proteins in Jijoye cells. In addition, cerulenin and C75 dramatically attenuate IE and early lytic gene expression after B-cell receptor engagement in Akata cells and constitutive lytic viral gene expression in EBV-positive AGS cells. However, FAS inhibitors do not reduce lytic viral gene expression induced by a vector in which the Z gene product is driven by a strong heterologous promoter. In addition, FAS inhibitors do not reduce R activation of a naked DNA reporter gene construct driven by the Z promoter (Zp). These results suggest that cellular FAS activity is important for induction of Z transcription from the intact latent EBV genome, perhaps reflecting the involvement of lipid-derived signaling pathways or palmitoylated proteins. Furthermore, using FAS inhibitors may be a completely novel approach for blocking the lytic form of EBV replication.
Bovine Herpesvirus Tegument Protein VP22 Enhances Thymidine Kinase/Ganciclovir Suicide Gene Therapy for Neuroblastomas Compared to Herpes Simplex Virus VP22
Authors: Qiu Zhaohua , Harms Jerome S. , Zhu Jun , Splitter Gary A.
Published: March 2004

Herpesvirus tegument protein VP22 can enhance the effect of therapeutic proteins in gene therapy, such as thymidine kinase (tk) and p53; however, the mechanism is unclear or controversial. In this study, mammalian expression vectors carrying bovine herpesvirus 1 (BHV-1) VP22 (BVP22) or herpes simplex virus type 1 (HSV-1) VP22 (HVP22) and equine herpesvirus type 4 (EHV-4) tk (Etk) were constructed in order to evaluate and compare the therapeutic potentials of BVP22 and HVP22 to enhance Etk/ganciclovir (Etk/GCV) suicide gene therapy for neuroblastomas by GCV cytotoxicity assays and noninvasive bioluminescent imaging in vitro and in vivo. BVP22 enhanced Etk/GCV cytotoxicity compared to that with HVP22 both in vitro and in vivo. However, assays utilizing a mixture of parental and stably transfected cells indicated that the enhancement was detected only in transfected cells. Thus, the therapeutic potential of BVP22 and HVP22 in Etk/GCV suicide gene therapy in this tumor system is not due to VP22 delivery of Etk into surrounding cells but rather is likely due to an enhanced intracellular effect.
Detection of Template Strand Switching during Initiation and Termination of DNA Replication of Porcine Circovirus
Authors: Cheung Andrew K.
Published: March 2004

Nucleotide substitution mutagenesis was conducted to investigate the importance of the inverted repeats (palindrome) at the origin of DNA replication (Ori) of porcine circovirus type 1 (PCV1). Viral genomes with engineered mutations on either arm or both arms of the palindrome were not impaired in protein synthesis and yielded infectious progeny viruses with restored or new palindromes. Thus, a flanking palindrome at the Ori was not essential for initiation of DNA replication, but one was generated inevitably at termination. Among the 26 viruses recovered, 16 showed evidence of template strand switching, from minus-strand genome DNA to palindromic strand DNA, during biosynthesis of the Ori. Here I propose a novel rolling-circle “melting-pot” model for PCV1 DNA replication. In this model, the replicator Rep protein complex binds, destabilizes, and nicks the Ori sequence to initiate leading-strand DNA synthesis. All four strands of the destabilized inverted repeats exist in a “melted” configuration, and the minus-strand viral genome and a palindromic strand are available as templates, simultaneously, during initiation or termination of DNA replication. Inherent in this model is a “gene correction” or “terminal repeat correction” mechanism that can restore mutilated inverted-repeat sequences to a palindrome at the Ori of circular DNAs or at the termini of circularized linear DNAs. Potentially, the melted state of the inverted repeats increases the rate of noncomplementary or illegitimate nucleotide incorporation into the palindrome. Thus, this melting-pot model provides insight into the mechanisms of DNA replication, gene correction, and illegitimate recombination at the Ori of PCV1, and it may be applicable to the replication of other circular DNA molecules.