bluuebunny/crossref_metadata_2025_split huggingface dataset:
Interaction of α-Agglutinin and
a
-Agglutinin,
Saccharomyces cerevisiae
Sexual Cell Adhesion Molecules
Authors: Zhao Hui , Shen Zheng-Ming , Kahn Peter C. , Lipke Peter N. 
Published: July 2002

ABSTRACT

α-Agglutinin and a-agglutinin are complementary cell adhesion glycoproteins active during mating in the yeast
Saccharomyces cerevisiae
. They bind with high affinity and high specificity: cells of opposite mating types are irreversibly bound by a few pairs of agglutinins. Equilibrium and surface plasmon resonance kinetic analyses showed that the purified binding region of α-agglutinin interacted similarly with purified a-agglutinin and with a-agglutinin expressed on cell surfaces. At 20°C, the

K
D

for the interaction was 2 × 10
−9
to 5 × 10
−9
M. This high affinity was a result of a very low dissociation rate (≈ 2.6 × 10
−4
s
−1
) coupled with a low association rate (= 5 × 10
4
M
−1
s
−1
). Circular-dichroism spectroscopy showed that binding of the proteins was accompanied by measurable changes in secondary structure. Furthermore, when binding was assessed at 10°C, the association kinetics were sigmoidal, with a very low initial rate. An induced-fit model of binding with substantial apposition of hydrophobic surfaces on the two ligands can explain the observed affinity, kinetics, and specificity and the conformational effects of the binding reaction.
Mapping Stress-Induced Changes in Autoinducer AI-2 Production in Chemostat-Cultivated
Escherichia coli
K-12
Authors: DeLisa Matthew P. , Valdes James J. , Bentley William E. 
Published: July 2002

ABSTRACT

Numerous gram-negative bacteria employ a cell-to-cell signaling mechanism, termed quorum sensing, for controlling gene expression in response to population density. Recently, this phenomenon has been discovered in
Escherichia coli
, and while pathogenic
E. coli
utilize quorum sensing to regulate pathogenesis (i.e., expression of virulence genes), the role of quorum sensing in nonpathogenic
E. coli
is less clear, and in particular, there is no information regarding the role of quorum sensing during the overexpression of recombinant proteins. The production of autoinducer AI-2, a signaling molecule employed by
E. coli
for intercellular communication, was studied in
E. coli
W3110 chemostat cultures using a
Vibrio harveyi
AI-2 reporter assay (M. G. Surrette and B. L. Bassler, Proc. Natl. Acad. Sci. USA 95:7046–7050, 1998). Chemostat cultures enabled a study of AI-2 regulation through steady-state and transient responses to a variety of environmental stimuli. Results demonstrated that AI-2 levels increased with the steady-state culture growth rate. In addition, AI-2 increased following pulsed addition of glucose, Fe(III), NaCl, and dithiothreitol and decreased following aerobiosis, amino acid starvation, and isopropyl-β-
d
-thiogalactopyranoside-induced expression of human interleukin-2 (hIL-2). In general, the AI-2 responses to several perturbations were indicative of a shift in metabolic activity or state of the cells induced by the individual stress. Because of our interest in the expression of heterologous proteins in
E. coli
, the transcription of four quorum-regulated genes and 20 stress genes was mapped during the transient response to induced expression of hIL-2. Significant regulatory overlap was revealed among several stress and starvation genes and known quorum-sensing genes.
Cell-Surface-Anchoring Role of N-Terminal Surface Layer Homology Domains of
Clostridium cellulovorans
EngE
Authors: Kosugi Akihiko , Murashima Koichiro , Tamaru Yutaka , Doi Roy H. 
Published: July 2002

ABSTRACT

engE
, coding for endoglucanase E, one of the three major subunits of the
Clostridium cellulovorans
cellulosome, has been cloned and sequenced (Y. Tamaru and R. H. Doi, J. Bacteriol. 181:3270-3276, 1999). The N-terminal-half region of EngE possesses three repeated surface layer homology (SLH) domains, which are homologous to those of some bacterial S-layer proteins. Also, the C-terminal-half region consists of a catalytic domain of glycosyl hydrolase family 5 and a duplicated sequence (dockerin) for binding EngE to scaffolding protein CbpA. Our hypothesis is that the SLH domains serve in the role of anchoring to the cell surface. This model was investigated by using recombinant EngEs (rEngE) with and without SLH domains that were synthesized in
Escherichia coli
and cell wall preparations from
C. cellulovorans
. When rEngE and SLH polypeptides of EngE were incubated with cell wall fragments prepared by sodium dodecyl sulfate treatment, these proteins bound strongly to the cell wall. However, rEngEs without SLH domains lost their ability to bind to cell walls. When rEngE was incubated with mini-CbpA, consisting of two cohesin domains, and cell wall fragments, the mini-CbpA was able to bind to the cell wall with rEngE. However, the binding of mini-CbpA was dramatically inhibited by addition of a chelating reagent, such as EDTA, which prevents cohesin-dockerin interactions. These results suggest not only that the SLH domains of EngE can bind to the cell surface but also that EngE plays an anchoring role for cellulosomes through the interaction of its dockerin domain with a CbpA cohesin.
Site-Directed Mutagenesis Studies of Selected Motif and Charged Residues and of Cysteines of the Multifunctional Tetracycline Efflux Protein Tet(L)
Authors: Jin Jie , Krulwich Terry A. 
Published: July 2002

ABSTRACT

All of the transmembrane glutamates of Tet(L) are essential for tetracycline (TET) resistance, and E397 has been shown to be essential for all catalytic modes, i.e., TET-Me
2+
and Na
+
efflux and K
+
uptake. Loop residues D74 and G70 are essential for TET flux but not for Na
+
or K
+
flux. A cysteineless Tet(L) protein exhibits all activities.
Inferring Genome Trees by Using a Filter To Eliminate Phylogenetically Discordant Sequences and a Distance Matrix Based on Mean Normalized BLASTP Scores
Authors: Clarke G. D. Paul , Beiko Robert G. , Ragan Mark A. , Charlebois Robert L. 
Published: July 2002

ABSTRACTDarwin's paradigm holds that the diversity of present-day organisms has arisen via a process of genetic descent with modification, as on a bifurcating tree. Evidence is accumulating that genes are sometimes transferred not along lineages but rather across lineages. To the extent that this is so, Darwin's paradigm can apply only imperfectly to genomes, potentially complicating or perhaps undermining attempts to reconstruct historical relationships among genomes (i.e., a genome tree). Whether most genes in a genome have arisen via treelike (vertical) descent or by lateral transfer across lineages can be tested if enough complete genome sequences are used. We define a phylogenetically discordant sequence (PDS) as an open reading frame (ORF) that exhibits patterns of similarity relationships statistically distinguishable from those of most other ORFs in the same genome. PDSs represent between 6.0 and 16.8% (mean, 10.8%) of the analyzable ORFs in the genomes of 28 bacteria, eight archaea, and one eukaryote (Saccharomyces cerevisiae). In this study we developed and assessed a distance-based approach, based on mean pairwise sequence similarity, for generating genome trees. Exclusion of PDSs improved bootstrap support for basal nodes but altered few topological features, indicating that there is little systematic bias among PDSs. Many but not all features of the genome tree from which PDSs were excluded are consistent with the 16S rRNA tree.
Global Regulation by
gidA
in
Pseudomonas syringae
Authors: Kinscherf Thomas G. , Willis David K. 
Published: July 2002

ABSTRACT

Analysis of two virulence mutants of
Pseudomonas syringae
B728a revealed that the Tn
5
sites of insertion were within the
gidA
open reading frame (ORF). These mutations were pleiotropic, affecting diverse phenotypic traits, such as lipodepsipeptide (syringomycin and syringopeptin) antibiotic production, swarming, presence of fluorescent pigment, and virulence. Site-specific recombination of a disrupted
gidA
gene into the chromosome resulted in the same phenotypic pattern as transposon insertion. Mutant phenotypes were restored by the
gidA
ORF on a plasmid. The
salA
gene, a copy number suppressor of the syringomycin-deficient phenotype in
gacS
and
gacA
mutants, was also found to suppress the antibiotic-negative phenotypes of
gidA
mutants, suggesting that
gidA
might play some role in
salA
regulation. Reporter studies with chromosomal
salA
-
lacZ
translational fusions confirmed that
salA
reporter expression decreased approximately fivefold in a
gidA
mutant background, with a concurrent decrease in the expression of the syringomycin biosynthetic reporter fusion
syrB
-
lacZ.
Wild-type levels of reporter expression were restored by supplying an intact
gidA
gene on a plasmid. Often described as being involved in cell division, more recent evidence suggests a role for
gidA
in moderating translational fidelity, suggesting a mechanism by which global regulation might occur. The
gidA
gene is essentially universal in the domains
Bacteria
and
Eucarya
but has no counterparts in
Archaea
, probably reflecting specific differences in the translational machinery between the former and latter domains.
Development of a Canadian Standardized Protocol for Subtyping Methicillin-Resistant
Staphylococcus aureus
Using Pulsed-Field Gel Electrophoresis
Authors: Mulvey M. R. , Chui L. , Ismail J. , Louie L. , Murphy C. , Chang N. , Alfa M. ,   the Canadian Committee for the Standardization of Molecular Methods
Published: July 2002

ABSTRACT

A panel of 24 methicillin-resistant
Staphylococcus aureus
strains was distributed to 15 laboratories in Canada to evaluate their in-house pulsed-field gel electrophoresis (PFGE) protocols and interpretation criteria. Attempts to compare fingerprint images using computer-aided analysis were not successful due to variability in individual laboratory PFGE protocols. In addition, individual site interpretation of the fingerprint patterns was inadequate, as 7 of 13 sites (54%) made at least one error in interpreting the fingerprints from the panel. A 2-day standardized PFGE protocol (culture to gel image) was developed and distributed to all of the sites. Each site was requested to use the standardized protocol on five strains from the original panel. Thirteen sites submitted gel images for comparisons. The protocol demonstrated excellent reproducibility and allowed interlaboratory comparisons with Molecular Analyst DST software (Bio-Rad) and 1.5% band tolerance.
Multiplex PCR Using Internal Transcribed Spacer 1 and 2 Regions for Rapid Detection and Identification of Yeast Strains
Authors: Fujita Shin-Ichi , Senda Yasuko , Nakaguchi Shigeki , Hashimoto Takuma 
Published: July 2002

ABSTRACT

Multiplex PCR amplification followed by either agarose gel electrophoresis (PCR-AGE) or microchip electrophoresis (PCR-ME) was used to test a total of 120 fungal strains. The internal transcribed spacer 1 (ITS1) and ITS2 regions and the 5.8S ribosomal DNA (rDNA) region of the fungi were amplified by using universal primers ITS1 and ITS4. The ITS2 region was simultaneously amplified by using universal primers ITS3 and ITS4. Since
Trichosporon asahi
and
T. asteroides
showed similar lengths for two amplicons, 29 different gel patterns were demonstrated for 30 yeast species tested on the basis of differences in the lengths of one or two amplicons. Of 75 yeast isolates from clinical materials, 5 isolates (6.8%) which were incompletely identified or not identified by the phenotypic method were identified with our PCR-based method (2 isolates as
Candida guilliermondii
, 2 as
C. krusei
, and 1 as
C. zeylanoides
). No differences in discriminating power or sensitivity were observed between the PCR-AGE method and the PCR-ME method. These methods, prospectively applied to 24 yeast-positive blood culture bottles (16 patients), resulted in the correct detection of 24 yeast strains. In conclusion, multiplex PCR followed by electrophoresis seems to be a promising tool for the rapid identification of common and uncommon yeast strains from culture colonies and from yeast-positive blood culture bottles (5.5 h for the PCR-AGE method and 3 h for the PCR-ME method).
Diagnostic Yield of a PCR Assay in Focal Complications of Brucellosis
Authors: Morata P. , Queipo-Ortuño M. I. , Reguera J. M. , Miralles F. , Lopez-Gonzalez J. J. , Colmenero J. D. 
Published: July 2002

ABSTRACT

In order to evaluate the diagnostic yield of a PCR assay for patients with focal complications of brucellosis, we studied by PCR and by conventional microbiological techniques 34 nonblood samples from 32 patients with different focal forms of brucellosis. The samples from patients with brucellosis were paired to an equal number of control samples from the same locations of patients whose illnesses had different etiologies. Thirty-three of the 34 nonblood samples (97%) from the brucellosis patients were positive by PCR, whereas
Brucella
spp. were isolated from only 29.4% of the conventional cultures. For 11.4% of the patients, the confirmatory serological tests were either negative or showed titers below the diagnostic range. Two patients (6.2%) from the control group, both with tuberculous vertebral osteomyelitis, had a positive PCR result. The brucella PCR of blood from these two patients was also positive, and the two strains of
Mycobacterium tuberculosis
isolated were analyzed by the brucella PCR, with no evidence of amplification. These results show that the PCR assay is far more sensitive than conventional cultures, and this, coupled with its speed and reduction in risk to laboratory workers, makes this technique a very useful tool for the diagnosis of focal complications of brucellosis.
Rapid Detection of Enterovirus RNA in Cerebrospinal Fluid Specimens with a Novel Single-Tube Real-Time Reverse Transcription-PCR Assay
Authors: Verstrepen Walter A. , Kuhn Sofie , Kockx Mark M. , Van De Vyvere Martine E. , Mertens An H. 
Published: July 2002

ABSTRACT
A single-tube real-time reverse transcription-PCR (RT-PCR) assay for enterovirus detection in cerebrospinal fluid (CSF) was developed based on a fluorogenic probe and primers directed to highly conserved sequences in the 5′ untranslated region of the enterovirus genome. Quantitative detection of enterovirus genome was demonstrated in a linear range spanning at least 5 logs. Endpoint titration experiments revealed that the in-tube detection limit of the assay was 11.8 enterovirus genome equivalents (95% detection rate) corresponding in our current extraction protocol to 592 enterovirus genome equivalents per ml of CSF. Twenty CSF specimens not suspected of viral meningitis were all found to be negative, and no cross-reactivity with herpes simplex virus type 1 and type 2, varicella-zoster virus, rhinovirus type 53, and influenza viruses A and B was observed. Nineteen CSF specimens from 70 patients suspected of viral meningitis were determined to be positive by PCR (27.1%), whereas only 17 were found to be positive by viral culture (24.3%). The sensitivity of the assay was 100% and the specificity was 96.2% compared to viral culture. Data from the real-time RT-PCR assay were available within 4 h. Our data suggest that the novel real-time RT-PCR assay may offer a reliable but significantly faster alternative to viral culture. Owing to the elimination of postamplification detection steps, its conduct required considerably less hands-on time and was associated with a substantially reduced carryover risk compared to previously described PCR-based enterovirus detection assays.
Outbreak of Infection with Multidrug-Resistant
Klebsiella pneumoniae
Carrying
bla
IMP-8
in a University Medical Center in Taiwan
Authors: Yan Jing-Jou , Ko Wen-Chien , Tsai Shu-Huei , Wu Hsiu-Mei , Wu Jiunn-Jong 
Published: July 2002

ABSTRACT

Klebsiella pneumoniae
strains with the transferable carbapenem-hydrolyzing metallo-β-lactamases, which include IMP- and VIM-type enzymes, remain extremely rare. To investigate whether IMP- or VIM-producing
K. pneumoniae
isolates had spread at a university medical center in Taiwan, a total of 3,458 clinical isolates of
K. pneumoniae
consecutively collected in 1999 and 2000 were tested by the agar diffusion method, colony hybridization, PCR, and nucleotide sequencing. A total of 40 isolates (1.2%), or 17 nonrepetitive isolates, from 16 patients were found to carry
bla
IMP-8
, a metallo-β-lactamase gene recently identified from a
K. pneumoniae
strain in Taiwan. Carriage of
bla
VIM
or other
bla
IMP
genes was detected in none of the remaining isolates. Of the 17 nonrepetitive
bla
IMP-8
-positive isolates, 15 isolates (88.2%) appeared susceptible to imipenem (MICs, ≤4 μg/ml) and meropenem (MICs, ≤1 μg/ml), indicating the difficulty in detecting
bla
IMP-8
in
K. pneumoniae
by routine susceptibility tests; 14 isolates (82.4%) produced SHV-12 as well; and 14 isolates (82.4%) were also resistant to fluoroquinolones. The organisms caused wound infections in eight patients and bloodstream infections in three patients. They were not directly associated with the death of nine patients. Before the recovery of the
bla
IMP-8
-positive isolates, all 16 patients had undergone various surgical procedures, and 15 patients had been admitted to the surgical intensive care unit, suggesting a nosocomial outbreak. Two major patterns were observed by pulsed-field gel electrophoresis for 14 of the 17 nonrepetitive isolates, indicating that the clonal spread was mainly responsible for the outbreak.
Evaluation of BACTEC Mycobacteria Growth Indicator Tube (MGIT 960) Automated System for Drug Susceptibility Testing of
Mycobacterium tuberculosis
Authors: Ardito Fausta , Posteraro Brunella , Sanguinetti Maurizio , Zanetti Stefania , Fadda Giovanni 
Published: July 2002

ABSTRACT

The reliability of the BACTEC MGIT 960 system, an automated version of the Mycobacteria Growth Indicator Tube (MGIT), for antimicrobial susceptibility testing of
Mycobacterium tuberculosis
was evaluated on 78 clinical isolates. Rifampin (RMP), isoniazid (INH), streptomycin (SM), and ethambutol (EMB) were tested at the following concentrations: 1.0 μg/ml for RMP, 0.1 and 0.4 μg/ml for INH, 1.0 and 4.0 μg/ml for SM, and 5.0 and 7.5 μg/ml for EMB. Results were compared with those obtained by the BACTEC 460 TB radiometric system. Initially the reproducibility study showed 99.5% agreement on repeat testing with all the four drugs. With susceptibility testing of clinical isolates, excellent agreement between the two systems was found for all the drugs. A total of nine major errors were observed for only three isolates, resistant according to BACTEC MGIT 960 and susceptible according to BACTEC 460 TB, to SM (4.0 μg/ml), INH (0.1 μg/ml), and EMB (5.0 μg/ml) (one isolate) and to SM (1.0 μg/ml), INH (0.4 μg/ml), and EMB (5.0 μg/ml) (two isolates). When these isolates were tested by using the conventional proportion method on Löwenstein-Jensen medium, agreement with BACTEC MGIT 960 was found for five results and with BACTEC 460 TB for the remainder. The time to report results was 7.9 days by MGIT 960 and 7.3 days by BACTEC 460 TB, which was not found statistically significant (
P
> 0.05). In conclusion, the performance of BACTEC MGIT 960 was found similar to that of BACTEC 460 TB and this new system can be considered a good alternative to the radiometric method for routine susceptibility testing of
M. tuberculosis
.
Analysis of Growth Characteristics of Filamentous Fungi in Different Nutrient Media
Authors: Meletiadis Joseph , Meis Jacques F. G. M. , Mouton Johan W. , Verweij Paul E. 
Published: July 2002

ABSTRACT

A microbroth kinetic model based on turbidity measurements was developed in order to analyze the growth characteristics of three species of filamentous fungi (
Rhizopus microsporus, Aspergillus fumigatus
, and
Scedosporium prolificans
) characterized by different growth rates in five nutrient media (antibiotic medium 3, yeast nitrogen base medium, Sabouraud broth, RPMI 1640 alone, and RPMI 1640 with 2% glucose). In general, five distinct phases in the growth of filamentous fungi could be distinguished, namely, the lag phase, the first transition period, the log phase, the second transition period, and the stationary phase. The growth curves were smooth and were characterized by the presence of long transition periods. Among the different growth phases distinguished, the smallest variability in growth rates among the strains of each species was found during the log phase in all nutrient media. The different growth phases of filamentous fungi were barely distinguishable in RPMI 1640, in which the poorest growth was observed for all fungi even when the medium was supplemented with 2% glucose.
R. microsporus
and
A. fumigatus
grew better in Sabouraud and yeast nitrogen base medium than in RPMI 1640, with growth rates three to four times higher. None of the media provided optimal growth of
S. prolificans
. The germination of
Rhizopus
spores and
Aspergillus
and
Scedosporium
conidia commenced after 2 and 5 h of incubation, respectively. The elongation rates ranged from 39.6 to 26.7, 25.4 to 20.2, and 16.9 to 9.9 μm/h for
Rhizopus, Aspergillus
, and
Scedoporium
hyphae, respectively. The germination of conidia and spores and the elongation rates of hyphae were enhanced in antibiotic medium 3 and delayed in yeast nitrogen base medium. In conclusion, the growth curves provide a useful tool to gain insight into the growth characteristics of filamentous fungi in different nutrient media and may help to optimize the methodology for antifungal susceptibility testing.
Epidemiological Patterns of Rotaviruses Causing Severe Gastroenteritis in Young Children throughout Australia from 1993 to 1996
Authors: Bishop Ruth F. , Masendycz Paul J. , Bugg Helen C. , Carlin John B. , Barnes Graeme L. 
Published: July 2002

ABSTRACT
Rotavirus strains that caused severe diarrhea in 4,634 (2,533 male) children aged less than 5 years and admitted to major hospitals in eight centers throughout Australia from 1993 to 1996 were subject to antigenic and genetic analyses. The G serotypes of rotaviruses were identified in 81.9% (3,793 of 4,634) children. They included 67.8% (from 3,143 children) serotype G1 isolates (containing 46 electropherotypes), 11.5% (from 531 children) serotype G2 isolates (27 electropherotypes), 0.8% (from 39 children) serotype G3 isolates (8 electropherotypes), and 1.6% (from 76 children) serotype G4 isolates (9 electropherotypes). G6 (two strains) and G8 (two strains) isolates were identified during the same period. G1 serotypes were predominant in all centers, with intermittent epidemics of G2 serotypes and sporadic detection of G3 and G4 strains. With the exception of two strains (typed as G1P2A[6] and G2P2A[6]) all serotype G1, G3, and G4 strains were P1A[8] and all serotype G2 strains were P1B[4]. Two contrasting epidemiological patterns were identified. In all temperate climates rotavirus incidence peaked during the colder months. The genetic complexity of strains (as judged by electropherotype) was greatest in centers with large populations. Identical electropherotypes appeared each winter in more than one center, apparently indicating the spread of some strains both from west to east and from east to west. Centers caring for children in small aboriginal communities showed unpredictable rotavirus peaks unrelated to climate, with widespread dissemination of a few rotavirus strains over distances of more than 1,000 km. Data from continued comprehensive etiological studies of genetic and antigenic variations in rotaviruses that cause severe disease in young children will serve as baseline data for the study of the effect of vaccination on the incidence of severe rotavirus disease and on the emergence of new strains.