bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification of an Acetoacetyl Coenzyme A Synthetase-Dependent Pathway for Utilization of
l
-(+)-3-Hydroxybutyrate in
Sinorhizobium meliloti
Authors: Aneja Punita , Dziak Renata , Cai Guo-Qin , Charles Trevor C. 
Published: July 2002

ABSTRACT

d
-(−)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of
Sinorhizobium meliloti
. In contrast, wild-type
S. meliloti
is not able to utilize the
l
-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the
S. meliloti acsA2
gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in
S. meliloti
is AcsA2 dependent.
Identification of XcpZ Domains Required for Assembly of the Secreton of
Pseudomonas aeruginosa
Authors: Robert Viviane , Hayes Finbarr , Lazdunski Andrée , Michel Gérard P. F. 
Published: July 2002

ABSTRACT

Most of the exoproteins secreted by
Pseudomonas aeruginosa
are transported via the type II secretion system. This machinery, which is widely conserved in gram-negative bacteria, consists of 12 Xcp proteins organized as a multiprotein complex, also called the secreton. We previously reported that the mutual stabilization of XcpZ and XcpY plays an important role in the assembly of the secreton. In this study, we engineered variant XcpZ proteins by using linker insertion mutagenesis. We identified three distinct regions of XcpZ required for both the stabilization of XcpY and the functionality of the secreton. Interestingly, we also demonstrated that another component of the machinery, XcpP, can modulate the stabilizing activity of XcpZ on XcpY.
Prediction of Gene Function in Methylthioadenosine Recycling from Regulatory Signals
Authors: Murphy Brooke A. , Grundy Frank J. , Henkin Tina M. 
Published: July 2002

ABSTRACT

The S-box transcription termination control system, first identified in
Bacillus subtilis
, is used for regulation of gene expression in response to methionine availability. The presence of the S-box motif provided the first indication that the
ykrTS
and
ykrWXYZ
genes could play a role in recycling of 5′-methylthioadenosine, a by-product of polyamine biosynthesis that can be converted to methionine. In this study we demonstrate a role for the
ykrTS
and
ykrWXYZ
gene products in this pathway.
Detection of Ciprofloxacin-Resistant
Yersinia pestis
by Fluorogenic PCR Using the LightCycler
Authors: Lindler Luther E. , Fan Wei , Jahan Nazma 
Published: July 2002

ABSTRACT

We have developed a fluorescence resonance energy transfer (FRET)-based assay to detect ciprofloxacin resistant (Cp
r
) mutants of the biothreat agent
Yersinia pestis
. We selected spontaneous mutants of the attenuated
Y. pestis
KIM 5 strain that were resistant to a ciprofloxacin (CIP) concentration of at least 1 μg/ml. DNA sequencing of
gyrA
encoded by 65 of these mutants revealed that all isolates contained one of four different point mutations within the quinolone resistance-determining region of
gyrA.
We developed a FRET-based assay that detected all of these mutations by using a single pair of fluorescent probes with sequences complementary to the wild-type
Y. pestis gyrA
sequence. Melting peak analysis revealed that the probe-PCR product hybrid was less stable when amplification occurred from any of the four mutant templates. This instability resulted in the PCR product obtained from the Cp
r
Y. pestis
strains displaying a 4 to 11°C shift in probe melting temperature. Following optimization of the reaction conditions, we were able to detect approximately 10 pg of purified wild-type template DNA or the presence of approximately 4 CFU of wild-type
Y. pestis
KIM 5 or Cp
r
mutants in crude lysates. Taken together, our results demonstrate the utility of FRET-based assays for detection of Cp
r
mutants of
Y. pestis
. This method is both sensitive and rapid.
Isolation of a Lysogenic Bacteriophage Carrying the
stx

1
OX3

Gene, Which Is Closely Associated with Shiga Toxin-Producing
Escherichia coli
Strains from Sheep and Humans
Authors: Koch Claudia , Hertwig Stefan , Lurz Rudi , Appel Bernd , Beutin Lothar 
Published: July 2002

ABSTRACT

A specific PCR for the detection of a variant of the gene encoding Shiga toxin 1 (
stx
1
) called
stx

1
OX3

(GenBank accession no.
Z36901
) was developed. The PCR was used to investigate 148 Stx
1
-producing
Escherichia coli
strains from human patients (
n
= 72), cattle (
n
= 27), sheep (
n
= 48), and a goat (
n
= 1) for the presence of the
stx

1
OX3

gene. The
stx

1
OX3

gene was present in 38 Shiga toxin-producing
E. coli
(STEC) strains from sheep belonging to serogroups O5, O125, O128, O146, and OX3 but was absent from Stx
1
-positive ovine STEC O91 strains. The
stx

1
OX3

gene was also detected in 22 STEC strains from humans with nonbloody diarrhea and from asymptomatic excreters. Serotypes O146:H21 and O128:H2 were most frequently associated with
stx

1
OX3

-carrying STEC from sheep and humans. In contrast, Stx
1
-producing STEC strains from cattle and goats and 50 STEC strains from humans were all negative for the
stx

1
OX3

gene. The
stx

1
OX3

-negative strains belonged to 13 serotypes which were different from those of the
stx

1
OX3

-positive STEC strains. Moreover, the
stx

1
OX3

gene was not associated with STEC belonging to enterohemorrhagic
E. coli
(EHEC) serogroups O26, O103, O111, O118, O145, and O157. A bacteriophage carrying the
stx

1
OX3

gene (phage 6220) was isolated from a human STEC O146:H21 strain. The phage was able to lysogenize laboratory
E. coli
K-12 strain C600. Phage 6220 shared a similar morphology and a high degree of DNA homology with Stx
2
-encoding phage 933W, which originates from EHEC O157. In contrast, few similarities were found between phage 6220 and Stx
1
-encoding bacteriophage H-19B from EHEC O26.
Improved Antimicrobial Interventions Have Benefits
Authors: Barenfanger Joan , Short Michael A. , Groesch Alisa A. 
Published: July 2002

ABSTRACT

Studies have shown benefits to patients from improved interventions involving antimicrobial therapy. The purpose of the present study was to evaluate prospectively the impact of improved interventions by (i) the use of TheraTrac 2, a computer software program which electronically links susceptibility testing results immediately to the pharmacy and alerts pharmacists of potential interventions, and (ii) the education of pharmacists involving microbiologic topics. The study group had the new intervention program. The control group had interventions performed the way that they had previously been done by manually reviewing hard copies of susceptibility testing data. In a 5-month period, all inpatients whose last names began with A to K were the study group; inpatients whose last names began with L to Z were controls. Three analyses were done; one analysis (analysis A) involved only patients with interventions, one analysis (analysis B) involved all patients for whom antimicrobial testing was done and who were matched for diagnosis-related groups (DRGs), regardless of whether an intervention occurred, and one analysis (analysis C) involved these DRG-matched patients by using severity-adjusted data. In analysis A, the study group had a 4.8% decreased rate of mortality, an average of a 16.5-day decreased length of stay per patient, and $20,886 decreased variable direct costs per patient. None of these differences was statistically significant. In analysis B, the study patients had a 1.2% higher mortality rate (
P
= 0.741), an average of a 2.7-day decreased length of stay per patient (
P
= 0.035), and $2,626 decreased variable direct costs per patient (
P
= 0.008). In analysis C, the study patients had a 1.4% lower mortality rate, a 1.2-day decreased length of stay per patient, and $1,466 decreased variable direct costs per patient. In conclusion, the institution of this program caused substantial cost savings.
Direct Identification of Mycobacteria from MB/BacT Alert 3D Bottles: Comparative Evaluation of Two Commercial Probe Assays
Authors: Scarparo Claudio , Piccoli Paola , Rigon Alessandra , Ruggiero Giuliana , Nista Domenico , Piersimoni Claudio 
Published: July 2002

ABSTRACT

The new INNO-LiPA Mycobacteria (Innogenetics, Ghent, Belgium), a reverse-hybridization-based line probe assay, and the AccuProbe assay (Gen-Probe Inc., San Diego, Calif.) were applied to MB/BacT Alert 3D (MB/BacT) system (Organon Teknika, Boxtel, The Netherlands) culture bottles and evaluated for mycobacterial identification. From 2,532 respiratory and extrapulmonary specimens submitted for culture, 168 were flagged positive by the MB/BacT system and promptly evaluated for identification (within 24 h). Each of 163 vials grew one mycobacterial isolate, including
Mycobacterium tuberculosis
complex (
n
= 73),
M. avium
complex (
n
= 3),
M. avium
(
n
= 8),
M. intracellulare
(
n
= 5),
M. kansasii
(
n
= 15),
M. gordonae
(
n
= 8),
M. malmoense
(
n
= 3),
M. chelonae
(
n
= 13),
M. abscessus
(
n
= 2),
M. xenopi
(
n
= 11),
M. scrofulaceum
(
n
= 2),
M. fortuitum
(
n
= 7),
M. terrae
(
n
= 3),
M. simiae
(
n
= 2),
M. celatum
(
n
= 3),
M. flavescens
(
n
= 1),
M. interjectum
(
n
= 1),
M. bohemicum
(
n
= 1), and
M. pulveris
(
n
= 2). Five cultures yielded mixed growth of two mycobacterial species:
M. tuberculosis
complex plus
M. gordonae
(
n
= 2),
M. tuberculosis
complex plus
M. chelonae
(
n
= 1),
M. tuberculosis
complex plus
M. xenopi
(
n
= 1), and
M. avium
plus
M. chelonae
(
n
= 1). In testing of one-isolate vials, both systems showed excellent sensitivity and specificity for all species and complexes for which they are licensed (nine for INNO-LiPA Mycobacteria versus six for AccuProbe). There were minor discrepancies in results for two isolates identified by INNO-LiPA Mycobacteria as
M. avium - M. intracellulare - M. scrofulaceum
(MAIS) complex and by AccuProbe as
M. intracellulare
. In testing of two-isolate vials, INNO-LiPA Mycobacteria correctly identified all isolates, while the AccuProbe assay failed to identify three
M. tuberculosis
complex isolates and one
M. avium
isolate. The AccuProbe assay was completed within 2 h, while INNO-LiPA Mycobacteria required a 6-h period. In our opinion, INNO-LiPA Mycobacteria offers the following advantages: (i) it contains a genus-specific probe that, in addition to being used in genus identification, may be used as an internal control for both the amplification and hybridization steps; (ii) it simultaneously identifies
M. tuberculosis
complex, MAIS complex, and seven other mycobacterial species, even from mixed cultures; (iii) its mycobacterial DNA amplification ensures reliable results independent from the concentration of viable microorganisms; and (iv) it genotypically identifies
M. kansasii
and
M. chelonae
. In conclusion, even though INNO-LiPA Mycobacteria is considerably less easy to use than AccuProbe, requiring personnel skilled in molecular biology techniques, it represents an excellent approach for routine identification of frequently encountered mycobacteria.
Aspergillus
Galactomannan Antigen in the Cerebrospinal Fluid of Bone Marrow Transplant Recipients with Probable Cerebral Aspergillosis
Authors: Viscoli Claudio , Machetti Marco , Gazzola Paola , De Maria Andrea , Paola Dimitri , Van Lint Maria Teresa , Gualandi Francesca , Truini Mauro , Bacigalupo Andrea 
Published: July 2002

ABSTRACT

The
Aspergillus
galactomannan test was performed on cerebrospinal fluid and serum samples from 5 patients with probable cerebral aspergillosis and from 16 control patients. Cerebrospinal fluid galactomannan levels were significantly higher in aspergillosis patients, and most galactomannan was produced intrathecally. Comparison of serum galactomannan values in pulmonary and cerebral aspergillosis patients showed significant overlapping. Detection of
Aspergillus
galactomannan in cerebrospinal fluid may be diagnostic of cerebral aspergillosis.
Reassessment of a Midwestern Lyme Disease Focus for
Borrelia burgdorferi
and the Human Granulocytic Ehrlichiosis Agent
Authors: Jackson Craig A. , Lovrich Steven D. , Agger William A. , Callister Steven M. 
Published: July 2002

ABSTRACT

Previous studies from the late 1980s defined the risk of human Lyme disease by determining the prevalence of
Borrelia burgdorferi
infection in
Ixodes scapularis
ticks and
Peromyscus
sp. mice captured from areas around La Crosse, Wis. High percentages of
B. burgdorferi
-infected
I. scapularis
ticks and
P. leucopus
mice were common in areas located north of Interstate 90 but were not detected in areas south of this major east-west thoroughfare. In this study, we reevaluated the extent of
B. burgdorferi
infection. High percentages of mice captured from sites north of the interstate were still infected with
B. burgdorferi
. In addition,
B. burgdorferi
was recovered from 12 (67%) of 18 mice captured from a site well south of the highway. However, none of 104 mice or 713
I. scapularis
ticks captured from the study sites were infected with
Ehrlichia
spp. The results confirmed the continued high risk for humans to contract infection with
B. burgdorferi
and the significant southward expansion of the area in which Lyme disease is endemic. In contrast, the risk of acquiring human granulocytic ehrlichiosis remains minimal despite the abundance of appropriate vector ticks and reservoir rodents.
Genotyping Study of
Scedosporium apiospermum
Isolates from Patients with Cystic Fibrosis
Authors: Defontaine Alain , Zouhair Rachid , Cimon Bernard , Carrère Jacqueline , Bailly Eric , Symoens Françoise , Diouri Mohammed , Hallet Jean-Noel , Bouchara Jean-Philippe 
Published: July 2002

ABSTRACT

Usually a saprophyte,
Scedosporium apiospermum
often colonizes the respiratory tracts of patients with cystic fibrosis (CF). In order to improve our understanding of the molecular epidemiology of the airway colonization, 129 sequential and multiple isolates collected from January 1998 to March 1999 from nine CF patients monitored in three hospitals in France were typed by random amplification of polymorphic DNA with primers GC70, UBC-701, and UBC-703. Among these primers, UBC-703 was the most discriminating, allowing the differentiation of 14 genotypes. Combining the results obtained with this three-primer set resulted in the differentiation of 16 genotypes. No common genotype was found among the different patients, and no clustering according to geographic origin of the isolates was seen. In addition, five of the patients were colonized by a single genotype. The others usually exhibited a predominant genotype accompanied by one or two others, which were found occasionally and were genetically close to the predominant genotype. Thus, our study demonstrates the persistence of the fungus despite antifungal treatments and therefore reinforces the need for the development of new antifungals that are more efficient against this species.
Isolation and Characterization of a New Subtype of Borna Disease Virus
Authors: Nowotny Norbert , Kolodziejek Jolanta , Jehle Christian O. , Suchy Angelika , Staeheli Peter , Schwemmle Martin 
Published: July 2002

ABSTRACT

Borna disease virus (BDV), the causative agent of severe meningoencephalitis in a wide variety of animal species, has been considered to be genetically invariable and to form a single type within the genus
Bornavirus
of the family
Bornaviridae
. BDV infections are of particular interest, because for the first time a virus infection appears to be linked to human psychiatric disorders. We now describe a new subtype of BDV isolated from a horse which was euthanatized due to severe, incurable neurological disease. The nucleotide sequence of this new strain, named No/98, differs from the reference strains by more than 15%, and the subtype is difficult to detect by standard reverse transcriptase PCR protocols. The nucleotide exchanges of the novel BDV isolate have surprisingly little effect on the primary structures of most viral proteins, with the notable exception of the X protein (p10), which is only 81% identical to its counterpart in reference strains. Our data indicate that the genome of BDV is far more variable than previously assumed and that naturally occurring subtypes may escape detection by currently used diagnostic assays.
Selective Membrane Permeabilization by the Rotavirus VP5* Protein Is Abrogated by Mutations in an Internal Hydrophobic Domain
Authors: Dowling William , Denisova Evgeniya , LaMonica Rachel , Mackow Erich R. 
Published: July 2002

ABSTRACT

Rotavirus infectivity is dependent on the proteolytic cleavage of the VP4 spike protein into VP8* and VP5* proteins. Proteolytically activated virus, as well as expressed VP5*, permeabilizes membranes, suggesting that cleavage exposes a membrane-interactive domain of VP5* which effects rapid viral entry. The VP5* protein contains a single long hydrophobic domain (VP5*-HD, residues 385 to 404) at an internal site. In order to address the role of the VP5*-HD in permeabilizing cellular membranes, we analyzed the entry of
o
-nitrophenyl-β-
d
-galactopyranoside (ONPG) into cells induced to express VP5* or mutated VP5* polypeptides. Following IPTG (isopropyl-β-
d
-thiogalactopyranoside) induction, VP5* and VP5* truncations containing the VP5*-HD permeabilized cells to the entry and cleavage of ONPG, while VP8* and control proteins had no effect on cellular permeability. Expression of VP5* deletions containing residues 265 to 474 or 265 to 404 permeabilized cells; however, C-terminal truncations which remove the conserved GGA (residues 399 to 401) within the HD abolished membrane permeability. Site-directed mutagenesis of the VP5-HD further demonstrated a requirement for residues within the HD for VP5*-induced membrane permeability. Functional analysis of mutant VP5*s indicate that conserved glycines within the HD are required and suggest that a random coiled structure rather than the strictly hydrophobic character of the domain is required for permeability. Expressed VP5* did not alter bacterial growth kinetics or lyse bacteria following induction. Instead, VP5*-mediated size-selective membrane permeability, releasing 376-Da carboxyfluorescein but not 4-kDa fluorescein isothiocyanate-dextran from preloaded liposomes. These findings suggest that the fundamental role for VP5* in the rotavirus entry process may be to expose triple-layered particles to low [Ca]
i
, which uncoats the virus, rather than to effect the detergent-like lysis of early endosomal membranes.
Functional Dissection of the Major Structural Protein of Bluetongue Virus: Identification of Key Residues within VP7 Essential for Capsid Assembly
Authors: Limn Chang-Kwang , Staeuber Norbert , Monastyrskaya Katherine , Gouet Patrice , Roy Polly 
Published: July 2002

ABSTRACT
A lattice of VP7 trimers forms the surface of the icosahedral bluetongue virus (BTV) core. To investigate the role of VP7 oligomerization in core assembly, a series of residues for substitution were predicted based on crystal structures of BTV type 10 VP7 molecule targeting the monomer-monomer contacts within the trimer. Seven site-specific substitution mutations of VP7 have been created using cDNA clones and were employed to produce seven recombinant baculoviruses. The effects of these mutations on VP7 solubility, ability to trimerize and formation of core-like particles (CLPs) in the presence of the scaffolding VP3 protein, were investigated. Of the seven VP7 mutants examined, three severely affected the stability of CLP, while two other mutants had lesser effect on CLP stability. Only one mutant had no apparent effect on the formation of the stable capsid. One mutant in which the conserved tyrosine at residue 271 (lower domain helix 6) was replaced by arginine formed insoluble aggregates, implying an effect in the folding of the molecule despite the prediction that such a change would be accommodated. All six soluble VP7 mutants were purified, and their ability to trimerize was examined. All mutants, including those that did not form stable CLPs, assembled into stable trimers, implying that single substitution may not be sufficient to perturb the complex monomer-monomer contacts, although subtle changes within the VP7 trimer could destabilize the core. The study highlights some of the key residues that are crucial for BTV core assembly and illustrates how the structure of VP7 in isolation underrepresents the dynamic nature of the assembly process at the biological level.
A Putative G Protein-Coupled Receptor, RDC1, Is a Novel Coreceptor for Human and Simian Immunodeficiency Viruses
Authors: Shimizu Nobuaki , Soda Yasushi , Kanbe Katsuaki , Liu Hui-yu , Mukai Ryozaburo , Kitamura Toshio , Hoshino Hiroo 
Published: July 2002

ABSTRACT

More than 10 G protein-coupled receptors (GPCRs) have been shown to act as coreceptors for infection of human immunodeficiency virus type 1 (HIV-1), HIV-2, and simian immunodeficiency virus (SIV). We have isolated HIV-1 variants infectious to primary brain-derived CD4-positive cells (BT-3 and BT-20/N) and U87/CD4 glioma cells that are resistant to T-cell line-tropic (T-tropic), macrophage-tropic (M-tropic), and T- and M-tropic (dualtropic) (X4, R5, and R5X4) HIV-1 strains. These primary brain-derived cells were also highly susceptible to HIV-2
ROD
, HIV-2
SBL6669
, and SIV
mndGB-1
. A factor or coreceptor that determines the susceptibility of these brain-derived cells to these HIV and SIV strains has not been fully identified. To identify this coreceptor, we examined amino acid sequences of all known HIV and SIV coreceptors and noticed that tyrosine residues are well conserved in their extracellular amino-terminal domains. By this criterion, we selected 18 GPCRs as candidates of coreceptors for HIV and SIV strains infectious to these brain-derived cells. mRNA expression of an orphan GPCR, RDC1, was detected in the brain-derived cells, the C8166 T-cell line, and peripheral blood lymphocytes, all of which are susceptible to HIV-1 variants, but not in macrophages, which are resistant to them. When a CD4-expressing cell line, NP-2/CD4, which shows strict resistance to infection not only with HIV-1 but also with HIV-2 or SIV, was transduced with the RDC1 gene, the cells became highly susceptible to HIV-2 and SIV
mnd
strains but to neither M- nor T-tropic HIV-1 strains. The cells also acquired a low susceptibility to the HIV-1 variants. These findings indicate that RDC1 is a novel coreceptor for several HIV-1, HIV-2, and SIV strains which infect brain-derived cells.