bluuebunny/crossref_metadata_2025_split huggingface dataset:
Essential Thioredoxin-Dependent Peroxiredoxin System from
Helicobacter pylori
: Genetic and Kinetic Characterization
Authors: Baker Laura M. S. , Raudonikiene Ausra , Hoffman Paul S. , Poole Leslie B. 
Published: July 2002

ABSTRACT

Helicobacter pylori
, an oxygen-sensitive microaerophile, contains an alkyl hydroperoxide reductase homologue (AhpC, HP1563) that is more closely related to 2-Cys peroxiredoxins of higher organisms than to most other eubacterial AhpC proteins. Allelic replacement mutagenesis revealed
ahpC
to be essential, suggesting a critical role for AhpC in defending
H. pylori
against oxygen toxicity. Characterization of the
ahpC
promoter region divulged two putative regulatory elements and identified the transcription initiation site, which was mapped to 96 and 94 bp upstream of the initiation codon. No homologue of
ahpF
, which encodes the dedicated AhpC reductase in most eubacteria, was found in the
H. pylori
genome. Instead, homologues of
Escherichia coli
thioredoxin (Trx) reductase (TrxR, HP0825) and Trx (Trx1, HP0824) formed a reductase system for
H. pylori
AhpC. A second Trx homologue (Trx2, HP1458) was identified but was incapable of AhpC reduction, although Trx2 exhibited disulfide reductase activity with other substrates [insulin and 5,5′-dithiobis(2-nitrobenzoic acid)]. AhpC interactions with each substrate, Trx1 and hydroperoxide, were bimolecular and nonsaturable (infinite
V
max
and

K
m

values) but rapid enough (at 1 × 10
5
to 2 × 10
5
M
−1
s
−1
) to suggest an important role for AhpC in cellular peroxide metabolism. AhpC also exhibited a wide specificity for hydroperoxide substrates, which, taken together with the above results, suggests a minimal binding site for hydroperoxides composed of little more than the cysteinyl (Cys49) active site.
H. pylori
AhpC was not reduced by
Salmonella typhimurium
AhpF and was slightly more active with
E. coli
TrxR and Trx1 than was
S. typhimurium
AhpC, demonstrating the specialized catalytic properties of this peroxiredoxin.
Molecular Characterization of the PceA Reductive Dehalogenase of
Desulfitobacterium
sp. Strain Y51
Authors: Suyama Akiko , Yamashita Masaki , Yoshino Sadazo , Furukawa Kensuke 
Published: July 2002

ABSTRACT

The tetrachloroethene (PCE) reductive dehalogenase (encoded by the
pceA
gene and designated PceA dehalogenase) of
Desulfitobacterium
sp. strain Y51 was purified and characterized. The expression of the enzyme was highly induced in the presence of PCE and trichloroethene (TCE). The purified enzyme catalyzed the reductive dehalogenation of PCE via TCE to
cis
-1,2-dichloroethene at a specific activity of 113.6 nmol · min
−1
· mg of protein
−1
. The apparent

K
m

values for PCE and TCE were 105.7 and 535.3 μM, respectively. Chlorinated ethenes other than PCE and TCE were not dehalogenated. However, the enzyme exhibited dehalogenation activity for various chlorinated ethanes such as hexachloroethane, pentachloroethane, 1,1,1,2-tetrachloroethane, and 1,1,2,2-tetrachloroethane. The
pceA
gene of
Desulfitobacterium
sp. strain Y51 was identified in a 2.8-kb DNA fragment and used to express the protein in
Escherichia coli
for the preparation of antibodies. Immunoblot analyses located PceA in the periplasm of the cell.
The Enigmatic
Escherichia coli fadE
Gene Is
yafH
Authors: Campbell John W. , Cronan John E. 
Published: July 2002

ABSTRACT

The identity of the gene encoding acyl coenzyme A dehydrogenase is a major remaining mystery of the
Escherichia coli
fatty acid degradation (
fad
) regulon. Our prior genome array analyses showed that transcription of the
yafH
gene is controlled by the FadR regulatory protein. We now report direct experimental proof that
yafH
and
fadE
are the same gene.
Diversity of Strains of
Salmonella enterica
Serotype Enteritidis from English Poultry Farms Assessed by Multiple Genetic Fingerprinting
Authors: Liebana Ernesto , Garcia-Migura Lourdes , Breslin Mark F. , Davies Robert H. , Woodward Martin J. 
Published: July 2002

ABSTRACT

Reliable and sufficiently discriminative methods are needed for differentiating individual strains of
Salmonella enterica
serotype Enteritidis beyond the phenotypic level; however, a consensus has not been reached as to which molecular method is best suited for this purpose. In addition, data are lacking on the molecular fingerprinting of serotype Enteritidis from poultry environments in the United Kingdom. This study evaluated the combined use of classical methods (phage typing) with three well-established molecular methods (ribotyping, macrorestriction analysis of genomic DNA, and plasmid profiling) in the assessment of diversity within 104 isolates of serotype Enteritidis from eight unaffiliated poultry farms in England. The most sensitive technique for identifying polymorphism was
Pst
I-
Sph
I ribotyping, distinguishing a total of 22 patterns, 10 of which were found among phage type 4 isolates. Pulsed-field gel electrophoresis of
Xba
I-digested genomic DNA segregated the isolates into only six types with minor differences between them. In addition, 14 plasmid profiles were found among this population. When all of the typing methods were combined, 54 types of strains were differentiated, and most of the poultry farms presented a variety of strains, which suggests that serotype Enteritidis organisms representing different genomic groups are circulating in England. In conclusion, geographical and animal origins of
Salmonella
serotype Enteritidis isolates may have a considerable influence on selecting the best typing strategy for individual programs, and a single method cannot be relied on for discriminating between strains.
Culture with BACTEC Peds Plus/F Bottle Compared with Conventional Methods for Detection of Bacteria in Synovial Fluid
Authors: Hughes John G. , Vetter Emily A. , Patel Robin , Schleck Cathy D. , Harmsen Scott , Turgeant L. Thomas , Cockerill Franklin R. 
Published: July 2002

ABSTRACT

An evaluation was undertaken to determine the utility of the BACTEC Peds Plus/F bottle and the BACTEC 9240 instrument (Becton Dickinson Diagnostic Instrument Systems, Sparks, Md.) for the detection of clinically significant microorganisms in synovial fluid specimens. The Peds Plus/F bottle was used because in our laboratory the quantity of synovial fluid available for culture is frequently in the range of 0.5 to 3.0 ml. The culture results obtained with the Peds Plus/F bottle were compared to those obtained by a conventional agar plate method for a total of 805 synovial fluid specimens. Microbial growth was produced by 74 cultures (9.2%) from 60 patients, yielding a total of 77 microorganisms. Organisms were classified as pathogens (
n
= 62), contaminants (
n
= 12), or indeterminate (
n
= 3) on the basis of a review of the patients' medical histories. Culture using BACTEC Peds Plus/F bottle detected statistically significantly more pathogens overall (62 versus 51 pathogens [
P
= 0.001]) and statistically fewer contaminants overall (1 versus 11 contaminants [
P
= 0.006]) than culture by the agar plate method. These results indicate the superior performance of the BACTEC Peds Plus/F bottle over the conventional agar plate method for the detection of clinically significant microorganisms from synovial fluid specimens.
Differences in Clinical and Laboratory Diagnostic Characteristics of Penicilliosis Marneffei in Human Immunodeficiency Virus (HIV)- and Non-HIV-Infected Patients
Authors: Wong S. S. Y. , Wong K. H. , Hui W. T. , Lee S. S. , Lo J. Y. C. , Cao L. , Yuen K. Y. 
Published: July 2002

ABSTRACT

We compared the clinical and laboratory features of human immunodeficiency virus (HIV)- and non-HIV-infected patients with penicilliosis marneffei. HIV-infected patients had a higher incidence of fungemia. A total of 85.7% of the HIV-negative patients had underlying diseases including hematologic malignancies or had received therapy with corticosteroids or cytotoxic agents. By a
Penicillium marneffei
-specific mannoprotein Mp1p enzyme-linked immunosorbent assay, serum antigen titers were found to be higher in HIV-positive patients, whereas serum antibody levels were found to be higher in HIV-negative patients.
Consensus and Variable Region PCR Analysis of
Helicobacter pylori
3′ Region of
cagA
Gene in Isolates from Individuals with or without Peptic Ulcer
Authors: Rota Cláudia Augustin , Pereira-Lima Júlio C. , Blaya Carolina , Nardi Nance Beyer 
Published: July 2002

ABSTRACT

The clinical outcome of
Helicobacter pylori
infection may be associated with the
cagA
bacterial genotype. To investigate the
cagA
status of
H. pylori
-infected patients and the relationship between
cagA
and peptic ulcer disease, gastric biopsy specimens from 103 Caucasian patients in Brazil were analyzed by PCR. Since allelic variation in
cagA
exists and distinct
H. pylori
subgenotypes may circulate in different regions, PCR using primers for a variable 3′ region of the
cagA
gene according to a Japanese methodology and for a consensus
cagA
3′ region used in Western methods was used for
cagA
detection.
cagA
was present in 53 (71%) of 75
H. pylori
-positive cases when analyzed by the consensus region method and was associated with duodenal ulcer disease (
P
= 0.02), but not with gastric ulcer (
P
= 0.26), when compared to patients with duodenitis or gastritis. The variable region PCR method was able to detect 43 (57%)
cagA
-positive cases within the same group of
H. pylori
-positive patients and showed three subtypes of
cagA
(A, B/D, and C) that were not associated with clinical outcome. However, in 8 (18%) of the cases, more than one subtype was present, and an association between patients with multiple subtypes and disease outcome was observed when compared to patients with isolated subtypes (
P
= 0.048).
cagA
was a marker of
H. pylori
strains for duodenal ulcer disease in our population, and in spite of the differences in the 3′ region of the
cagA
gene, the Japanese methodology was able to detect the
cagA
status in most cases. The presence of multiple subgenotypes of
cagA
was associated with gastric ulcer.
Enzyme-Linked Immunosorbent Assay Using a Virus Type-Specific Peptide Based on a Subdomain of Envelope Protein E
rns
for Serologic Diagnosis of Pestivirus Infections in Swine
Authors: Langedijk J. P. M. , Middel W. G. J. , Meloen R. H. , Kramps J. A. , de Smit J. A. 
Published: July 2002

ABSTRACT

Peptides deduced from the C-terminal end (residues 191 to 227) of pestivirus envelope protein E
rns
were used to develop enzyme-linked immunosorbent assays (ELISAs) to measure specifically antibodies against different types of pestiviruses. The choice of the peptide was based on the modular structure of the E
rns
protein, and the peptide was selected for its probable independent folding and good exposure, which would make it a good candidate for an antigenic peptide to be used in a diagnostic test. A solid-phase peptide ELISA which was cross-reactive for several types of pestivirus antibodies and which can be used for the general detection of pestivirus antibodies was developed. To identify type-specific pestivirus antibodies, a liquid-phase peptide ELISA, with a labeled, specific classical swine fever virus (CSFV) peptide and an unlabeled bovine viral diarrhea virus peptide to block cross-reactivity, was developed. Specificity and sensitivity of the liquid-phase peptide ELISA for CSFV were 98 and 100%, respectively. Because the peptide is a fragment of the E
rns
protein, it can be used to differentiate between infected and vaccinated animals when a vaccine based on the E2 protein, which is another pestivirus envelope protein, is used.
Expression and Self-Assembly in Baculovirus of Porcine Enteric Calicivirus Capsids into Virus-Like Particles and Their Use in an Enzyme-Linked Immunosorbent Assay for Antibody Detection in Swine
Authors: Guo Mingzhang , Qian Yuan , Chang Kyeong-Ok , Saif Linda J. 
Published: July 2002

ABSTRACT
Porcine enteric calicivirus (PEC) causes diarrhea and intestinal lesions in pigs. PEC strain Cowden grows to low to moderate titers in cell culture but only with the addition of intestinal contents from uninfected gnotobiotic pigs (W. T. Flynn and L. J. Saif, J. Clin. Microbiol. 26:206–212, 1988; A. V. Parwani, W. T. Flynn, K. L. Gadfield, and L. J. Saif, Arch. Virol. 120:115–122, 1991). Cloning and sequence analysis of the PEC Cowden full-length genome revealed that it is most closely related genetically to the human Sapporo-like viruses. In this study, the complete PEC capsid gene was subcloned into the plasmid pBlueBac4.5 and the recombinant baculoviruses were identified by plaque assay and PCR. The PEC capsid protein was expressed in insect (Sf9) cells inoculated with the recombinant baculoviruses, and the recombinant capsid proteins self- assembled into virus-like particles (VLPs) that were released into the cell supernatant and purified by CsCl gradient centrifugation. The PEC VLPs had the same molecular mass (58 kDa) as the native virus capsid and reacted with pig hyperimmune and convalescent-phase sera to PEC Cowden in enzyme-linked immunosorbent assay (ELISA) and Western blotting. The PEC capsid VLPs were morphologically and antigenically similar to the native virus by immune electron microscopy. High titers (1:102,400 to 204,800) of PEC-specific antibodies were induced in guinea pigs inoculated with PEC VLPs, suggesting that the VLPs could be useful for future candidate PEC vaccines. A fixed-cell ELISA and VLP ELISA were developed to detect PEC serum antibodies in pigs. For the fixed-cell ELISA, Sf9 cells were infected with recombinant baculoviruses expressing PEC capsids, followed by cell fixation with formalin. For the VLP ELISA, the VLPs were used for the coating antigen. Our data indicate that both tests were rapid, specific, and reproducible and might be used for large-scale serological investigations of PEC antibodies in swine.
Comparative Study Using Amplified Fragment Length Polymorphism Fingerprinting, PCR Genotyping, and Phenotyping To Differentiate
Campylobacter fetus
Strains Isolated from Animals
Authors: Wagenaar Jaap A. , van Bergen Marcel A. P. , Newell Diane G. , Grogono-Thomas Rose , Duim Birgitta 
Published: July 2002

ABSTRACT

A collection of
Campylobacter fetus
strains, including both
C. fetus
subsp.
fetus
and
C. fetus
subsp.
venerealis
, were phenotypically identified to the subspecies level and genotypically typed by PCR and amplified fragment length polymorphism (AFLP) analysis. Phenotypic subspecies determination methods were unreliable. Genotyping of the strains by PCR and AFLP showed a clear discrimination between the two subspecies.
Characterization of Mutations in the
rpoB
Gene Associated with Rifampin Resistance in
Rhodococcus equi
Isolated from Foals
Authors: Fines Marguerite , Pronost Stephane , Maillard Karine , Taouji Said , Leclercq Roland 
Published: July 2002

ABSTRACT

Treatment with a combination of erythromycin and rifampin has considerably improved survival rates of foals and immunocompromised patients suffering from severe pneumonia caused by
Rhodococcus equi
. Frequently, because of monotherapy, emergence of rifampin-resistant strains has been responsible for treatment failure. Using consensus oligonucleotides, we have amplified and sequenced the rifampin resistance (Rif
r
)-determining regions of 12 rifampin-resistant
R. equi
strains isolated from three foals and of mutants selected in vitro from
R. equi
ATCC 3701, a rifampin-susceptible strain. The deduced amino acid sequences compared to those of four rifampin-susceptible
R. equi
strains showed several types of mutations. In 3 of the 10 strains isolated from one foal, His526Asn (
Escherichia coli
numbering) and Asp516Val mutations were associated with low-level resistance (rifampin MIC, 2 to 8 μg/ml), whereas His526Asp conferred high-level resistance (rifampin MIC, 128 μg/ml) in the 7 remaining strains. In strains from the two other foals, His526Asp and Ser531Leu mutations were found to be associated with high-level and low-level resistance, respectively. The in vitro mutants, highly resistant to rifampin, harbored His526Tyr and His526Arg substitutions. As described in other bacterial genera, His526, Ser531, and Asp516 are critical residues for rifampin resistance in
R. equi
, and the resistance levels are dependent on both the location and the nature of the substitution.
Multicenter Comparison of Three Different Analytical Systems for Evaluation of DNA Banding Patterns from
Cryptococcus neoformans
Authors: Cardinali Gianluigi , Martini Alessandro , Preziosi Roberta , Bistoni Francesco , Baldelli Franco 
Published: July 2002

ABSTRACT

The enormous improvement of molecular typing techniques for epidemiological and clinical studies has not always been matched by an equivalent effort in applying optimal criteria for the analysis of both phenotypic and molecular data. In spite of the availability of a large collection of statistical and phylogenetic methods, the vast majority of commercial packages are limited by using only the unweighted pair group method with arithmetic mean algorithm to construct trees and by considering electrophoretic pattern only as migration distances. The latter method has serious drawbacks when different runs (separate gels) of the same molecular analysis are to be compared. This work presents a multicenter comparison of three different systems of banding pattern analysis on random amplified polymorphic DNA, (GACA)
4
, and contour-clamped homogeneous electric field patterns from strains of
Cryptococcus neoformans
var.
neoformans
isolated in different clinical and geographical situations and a standard
Saccharomyces cerevisiae
strain employed as an outgroup. The systems considered were evaluated for their actual ability to(i) recognize identities, (ii) define complete differences (i.e., the ability to place
S. cerevisiae
out of the
C. neoformans
cluster), and (iii) estimate the extent of similarity among different strains. The ability to cluster strains according to the patient from which they were isolated was also evaluated. The results indicate that different algorithms do indeed produce divergent trees, both in overall topology and in clustering of individual strains, thus suggesting that care must be taken by individual investigators to use the most appropriate procedure and by the scientific community in defining a consensus system.
Simian Immunodeficiency Viruses of Diverse Origin Can Use CXCR4 as a Coreceptor for Entry into Human Cells
Authors: Owen Sherry M. , Masciotra Silvina , Novembre Frank , Yee JoAnn , Switzer William M. , Ostyula Moses , Lal Renu B. 
Published: July 2002

ABSTRACT

Primary simian immunodeficiency virus (SIV) isolated from sooty mangabey (SIVsm [
n
= 6]), stumptail (SIVstm [
n
= 1]), mandrill (SIVmnd [
n
= 1]), and African green (SIVagm [
n
= 1]) primates were examined for their ability to infect human cells and for their coreceptor requirements. All isolates infected human peripheral blood mononuclear cells (PBMCs) from a CCR5
+/+
donor, and seven of eight isolates tested also infected CCR5
−/−
PBMCs. Analysis of coreceptor utilization using GHOST and U87 cell lines revealed that all of the isolates tested used CCR5 and the orphan receptors STRL33 and GPR15. Coreceptors such as CCR2b, CCR3, CCR8, and CX3CR1 were also utilized by some primary SIV isolates. More importantly, we found that CXCR4 was used as a coreceptor by the SIVstm, the SIVagm, and four of the SIVsm isolates in GHOST and U87 cells. These data suggest that primary SIV isolates from diverse primate species can utilize CXCR4 for viral entry, similar to what has been described for human immunodeficiency viruses.
Enhanced Binding of Antibodies to Neutralization Epitopes following Thermal and Chemical Inactivation of Human Immunodeficiency Virus Type 1
Authors: Grovit-Ferbas K. , Hsu J. F. , Ferbas J. , Gudeman V. , Chen I. S. Y. 
Published: July 2002

ABSTRACTInactivation of viral particles is the basis for several vaccines currently in use. Initial attempts to use simian immunodeficiency virus to model a killed human immunodeficiency virus type 1 (HIV-1) vaccine were unsuccessful, and limited subsequent effort has been directed toward a systematic study of the requirements for a protective killed HIV-1 vaccine. Recent insights into HIV-1 virion and glycoprotein structure and neutralization epitopes led us to revisit whether inactivated HIV-1 particles could serve as the basis for an HIV-1 vaccine. Our results indicate that relatively simple processes involving thermal and chemical inactivation can inactivate HIV-1 by at least 7 logs. For some HIV-1 strains, significant amounts of envelope glycoproteins are retained in high-molecular-weight fractions. Importantly, we demonstrate retention of each of three conformation-dependent neutralization epitopes. Moreover, reactivity of monoclonal antibodies directed toward these epitopes is increased following treatment, suggesting greater exposure of the epitopes. In contrast, treatment of free envelope under the same conditions leads only to decreased antibody recognition. These inactivated virions can also be presented by human dendritic cells to direct a cell-mediated immune response in vitro. These data indicate that a systematic study of HIV-1 inactivation, gp120 retention, and epitope reactivity with conformation-specific neutralizing antibodies can provide important insights for the development of an effective killed HIV-1 vaccine.
Isolation and Characterization of Monoclonal Antibodies That Inhibit Hepatitis C Virus NS3 Protease
Authors: Ueno Takamasa , Misawa Satoru , Ohba Yoichi , Matsumoto Mitsuhiro , Mizunuma Makiko , Kasai Nobuhiro , Tsumoto Kouhei , Kumagai Izumi , Hayashi Hideya 
Published: July 2002

ABSTRACT

A series of mouse monoclonal antibodies (MAbs) to the nonstructural protein 3 (NS3) of hepatitis C virus was prepared. One of these MAbs, designated 8D4, was found to inhibit NS3 protease activity. This inhibition was competitive with respect to the substrate peptide (

K
i

= 39 nM) but was significantly decreased by the addition of the NS4A peptide, a coactivator of the NS3 protease. 8D4 also showed marked inhibition of the NS3-dependent
cis
processing of the NS3/4A polyprotein but had virtually no effect on the succeeding NS3/4A-dependent
trans
processing of the NS5A/5B polyprotein in vitro. Epitope mapping of 8D4 with a random peptide library revealed a consensus sequence, DxDLV, that matched residues 79 to 83 (DQDLV) of NS3, a region containing the catalytic residue Asp-81. Furthermore, synthetic peptides including this sequence were shown to block the ability of 8D4 to bind to NS3, indicating that 8D4 interacts with the catalytic region of NS3. The data showing decreased inhibition potency of 8D4 against the NS3/4A complex suggest that 8D4 recognizes the conformational state of the protease active site caused by the association of NS4A with the protease.