bluuebunny/crossref_metadata_2025_split huggingface dataset:
Domain Interactions on the
ntr
Signal Transduction Pathway: Two-Hybrid Analysis of Mutant and Truncated Derivatives of Histidine Kinase NtrB
Authors: Martínez-Argudo Isabel , Salinas Paloma , Maldonado Rafael , Contreras Asunción 
Published: July 2002

ABSTRACT

We have used the yeast two-hybrid system to analyze protein-protein interactions mediated by domains of regulatory proteins of the
ntr
signal transduction system, including interactions among NtrB derivatives and their interactions with NtrC and PII from
Klebsiella pneumoniae
. Interactions took place only between proteins or protein domains belonging to the
ntr
signal transduction system and not between proteins or domains from noncognate regulators. NtrB and its transmitter domain, but not NtrC, CheA, or the cytoplasmic C terminus of EnvZ, interacted with PII. In addition, interaction of NtrB with NtrC, but not with PII, depended on the histidine phosphotransfer domain. Point mutation A129T, diminishing the NtrC phosphatase activity of NtrB, affected the strength of the signals between NtrC and the transmitter module of NtrB but had no impact on PII signals, suggesting that A129T prevents the conformational change needed by NtrB to function as a phosphatase for NtrC, rather than disturbing binding to PII.
Advancing the Quorum in
Pseudomonas aeruginosa
: MvaT and the Regulation of
N
-Acylhomoserine Lactone Production and Virulence Gene Expression
Authors: Diggle Stephen P. , Winzer Klaus , Lazdunski Andrée , Williams Paul , Cámara Miguel 
Published: July 2002

ABSTRACT

Pseudomonas aeruginosa
regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules
N-
(3-oxododecanoyl)-
l
-homoserine lactone (3O-C12-HSL) and
N-
(butanoyl)-
l
-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes
lecA
(coding for PA-IL lectin),
lasB
(coding for elastase), and
rpoS
appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a
P. aeruginosa lecA
::
lux
reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in
lecA
expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as
clpA
and
mvaT
and a putative efflux system, a number of mutations were also mapped to known regulators such as
lasR
,
rhlR
, and
rpoS.
MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in
mvaT
resulted in enhanced
lecA
expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the
mvaT
mutant significantly advanced
lecA
expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the
lecA
gene.
Characterization of theCampylobacter jejuniHeptosyltransferase II Gene,waaF, Provides Genetic Evidence that Extracellular Polysaccharide Is Lipid A Core Independent
Authors: Oldfield Neil J. , Moran Anthony P. , Millar Lorna A. , Prendergast Martina M. , Ketley Julian M. 
Published: July 2002

ABSTRACTCampylobacter jejuniproduces both lipooligosaccharide (LOS) and a higher-molecular-weight polysaccharide that is believed to form a capsule. The role of these surface polysaccharides inC. jejuni-mediated enteric disease is unclear; however, epitopes associated with the LOS are linked to the development of neurological complications. InEscherichia coliandSalmonella entericaserovar Typhimurium thewaaFgene encodes a heptosyltransferase, which catalyzes the transfer of the secondl-glycero-d-manno-heptose residue to the core oligosaccharide moiety of lipopolysaccharide (LPS), and mutation ofwaaFresults in a truncated core oligosaccharide. In this report we confirm experimentally thatC. jejunigene Cj1148 encodes the heptosyltransferase II enzyme, WaaF. TheCampylobacter waaFgene complements anS. entericaserovar TyphimuriumwaaFmutation and restores the ability to produce full-sized lipopolysaccharide. To examine the role of WaaF inC. jejuni,waaFmutants were constructed in strains NCTC 11168 and NCTC 11828. Loss of heptosyltransferase activity resulted in the production of a truncated core oligosaccharide, failure to bind specific ligands, and loss of serum reactive GM1, asialo-GM1, and GM2ganglioside epitopes. The mutation ofwaaFdid not affect the higher-molecular-weight polysaccharide supporting the production of a LOS-independent capsular polysaccharide byC. jejuni. The exact structural basis for the truncation of the core oligosaccharide was verified by comparative chemical analysis. The NCTC 11168 core oligosaccharide differs from that known for HS:2 strain CCUG 10936 in possessing an extra terminal disaccharide of galactose-β(1,3)N-acetylgalactosamine. In comparison, thewaaFmutant possessed a truncated molecule consistent with that observed withwaaFmutants in other bacterial species.
Screening of Active Lyssavirus Infection in Wild Bat Populations by Viral RNA Detection on Oropharyngeal Swabs
Authors: Echevarrı́a Juan E. , Avellón Ana , Juste Javier , Vera Manuel , Ibáñez Carlos 
Published: July 2002

ABSTRACT

Brain analysis cannot be used for the investigation of active lyssavirus infection in healthy bats because most bat species are protected by conservation directives. Consequently, serology remains the only tool for performing virological studies on natural bat populations; however, the presence of antibodies merely reflects past exposure to the virus and is not a valid marker of active infection. This work describes a new nested reverse transcription (RT)-PCR technique specifically designed for the detection of the European bat virus 1 on oropharyngeal swabs obtained from bats but also able to amplify RNA from the remaining rabies-related lyssaviruses in brain samples. The technique was successfully used for surveillance of a serotine bat (
Eptesicus serotinus
) colony involved in a case of human exposure, in which 15 out of 71 oropharyngeal swabs were positive. Lyssavirus infection was detected on 13 oropharyngeal swabs but in only 5 brains out of the 34 animals from which simultaneous brain and oropharyngeal samples had been taken. The lyssavirus involved could be rapidly identified by automatic sequencing of the RT-PCR products obtained from 14 brains and three bat oropharyngeal swabs. In conclusion, RT-PCR using oropharyngeal swabs will permit screening of wild bat populations for active lyssavirus infection, for research or epidemiological purposes, in line not only with conservation policies but also in a more efficient manner than classical detection techniques used on the brain.
Altering Trends in the Dominance of
Shigella flexneri
Serotypes and Emergence of Serologically Atypical
S. flexneri
Strains in Dhaka, Bangladesh
Authors: Talukder Kaisar A. , Dutta Dilip K. , Safa Ashrafus , Ansaruzzaman M. , Hassan Ferdaus , Alam Khorshed , Islam K. M. N. , Carlin N. I. A. , Nair G. B. , Sack David A. 
Published: July 2002

ABSTRACT

Of 469 recently isolated
Shigella flexneri
strains, 452 agglutinated with
Shigella flexneri
-specific monoclonal antibodies. Of these, 396 could be assigned to 10 of the currently recognized 15 serotypes, with
S. flexneri
2b dominating (23.2%). Of the 56 untypeable strains which showed invasive properties, 17 were serologically atypical and the remaining 39 belonged to a new serotype.
Clinical and Environmental Isolates of
Vibrio cholerae
Serogroup O141 Carry the CTX Phage and the Genes Encoding the Toxin-Coregulated Pili
Authors: Dalsgaard A. , Serichantalergs O. , Forslund A. , Lin W. , Mekalanos J. , Mintz E. , Shimada T. , Wells J. G. 
Published: July 2002

ABSTRACT

We report sporadic cases of a severe gastroenteritis associated with
Vibrio cholerae
serogroup O141. Like O1 and O139 serogroup strains of
V. cholerae
isolated from cholera cases, the O141 clinical isolates carry DNA sequences that hybridize to cholera toxin (CT) gene probes. The CT genes of O1 and O139 strains are carried by a filamentous bacteriophage (termed CTX phage) which is known to use toxin-coregulated pili (TCP) as its receptor. In an effort to understand the mechanism of emergence of toxigenic O141
V. cholerae
, we probed a collection of O141 clinical and environmental isolates for genes involved in TCP production, toxigenicity, virulence regulation, and other phylogenetic markers. The collection included strains isolated between 1964 and 1995 from diverse geographical locations, including eight countries and five U.S. states. Information collected about the clinical and environmental sources of these isolates suggests that they had no epidemiological association. All clinical O141 isolates hybridized to probes specific for genes encoding CT (
ctx
), zonula occludens toxin (
zot
), repetitive sequence 1 (RS1), RTX toxin (
rtxA
), the major subunit of TCP (
tcpA
), and the essential regulatory gene that controls expression of both CT and TCP (
toxR
). In contrast, all but one of the nonclinical O141 isolates were negative for
ctx
,
zot
, RS1, and
tcpA
, although these strains were positive for
rtxA
and
toxR
. The one toxigenic environmental O141 isolate was also positive for
tcpA
. Ribotyping and CT typing showed that the O141 clinical isolates were indistinguishable or closely related, while a toxigenic water isolate from Louisiana showed a distantly related ribotype. Nonclinical O141 isolates displayed a variety of unrelated ribotypes. These data support a model for emergence of toxigenic O141 that involves acquisition of the CTX phage sometime after these strains had acquired the pathogenicity island encoding TCP. The clonal nature of toxigenic O141 strains isolated from diverse geographical locations suggests that the emergence is a rare event but that once it occurs, toxigenic O141 strains are capable of regional and perhaps even global dissemination. This study stresses the importance of monitoring
V. cholerae
non-O1, non-O139 serogroup strains for their virulence gene content as a means of assessing their epidemic potential.
Expression of
Babesia equi
Merozoite Antigen 1 in Insect Cells by Recombinant Baculovirus and Evaluation of Its Diagnostic Potential in an Enzyme-Linked Immunosorbent Assay
Authors: Xuan Xuenan , Larsen Alejandra , Ikadai Hiromi , Tanaka Tetsuya , Igarashi Ikuo , Nagasawa Hideyuki , Fujisaki Kozo , Toyoda Yutaka , Suzuki Naoyoshi , Mikami Takeshi 
Published: July 2002

ABSTRACT

The gene encoding the entire
Babesia equi
merozoite antigen 1 (EMA-1) was inserted into a baculovirus transfer vector, and a recombinant virus expressing EMA-1 was isolated. The expressed EMA-1 was transported to the surface of infected insect cells, as judged by an indirect fluorescent-antibody test (IFAT). The expressed EMA-1 was also secreted into the supernatant of a cell culture infected with recombinant baculovirus. Both intracellular and extracellular EMA-1 reacted with a specific antibody in Western blots. The expressed EMA-1 had an apparent molecular mass of 34 kDa that was identical to that of native EMA-1. The secreted EMA-1 was used as an antigen in an enzyme-linked immunosorbent assay (ELISA). The ELISA differentiated
B. equi
-infected horse sera from
Babesia caballi
-infected horse sera or normal horse sera. The ELISA was more sensitive than the complement fixation test and IFAT. These results demonstrated that the recombinant EMA-1 expressed in insect cells might be a useful diagnostic reagent for detection of antibodies to
B. equi
.
Genotypic, Clinical, and Demographic Characteristics of Children Infected with
Helicobacter pylori
Authors: Gold Benjamin D. , van Doorn Leen-Jan , Guarner Jeannette , Owens Marilyn , Pierce-Smith Daphne , Song Qunsheng , Hutwagner Lori , Sherman Philip M. , de Mola Oscar Loret , Czinn Steven J. 
Published: July 2002

ABSTRACT

Helicobacter pylori
isolates vary between geographic regions. Certain
H. pylori
genotypes may be associated with disease outcome. Thirty-eight children underwent diagnostic upper endoscopy at four medical centers and were retrospectively analyzed to determine if
H. pylori
virulence genes were associated with endoscopic disease severity, histologic parameters, and host demographics. The
H. pylori
virulence genotype was analyzed by a reverse hybridization line probe assay and type-specific PCR. Endoscopic ulcers or erosions were found in 17 (45%) patients, with 13 (34%) of these patients having antral nodularity. Histological gastritis, of varying severity, was present in all children. Four patients harbored more than one
H. pylori
strain: one subject had both
cagA
+
and
cagA
-negative strains, while three patients harbored either two different
cagA
-negative strains (two children) or two
cagA
+
strains (one child). There were 28 (74%)
cagA
+
isolates; 19 were associated with the
vacA
s1b genotype, 7 were associated with the
vacA
s1a genotype, 1 was associated with the
vacA
s1c genotype, and 1 was associated with the s2 genotype. Of 14
cagA
-negative isolates, 6 were
vacA
s2 genotype, 4 were
vacA
s1b, 3 were
vacA
s1a, and 1 was
vacA
s1c. Nine of ten (90%) Hispanics had similar
H. pylori
strains (
vacA
s1b,m1), and all Asian-Canadian children were infected by strains with
vacA
s1c genotype. No correlation between
H. pylori
strain and endoscopic or histopathologic abnormalities was found. This study provides a baseline framework of North American children and their
H. pylori
strains, serving as a powerful epidemiological tool for prospective investigations to better understand the transmission and evolution of diverse disease outcomes.
Detection of Microsporidia in Travelers with Diarrhea
Authors: Müller A. , Bialek R. , Kämper A. , Fätkenheuer G. , Salzberger B. , Franzen C. 
Published: July 2002

ABSTRACT
We examined stool specimens of 148 returning travelers from an outpatient department for tropical diseases for the appearence of microsporidia using light microscopy and PCR. Intestinal microsporidiosis was diagnosed for five patients by light microscopy and for nine patients by PCR. Some cases were diagnosed only by PCR, indicating that the true prevalence has to be determined by highly sensitive techniques, such as PCR.
Reaction Difference Rule for Phage Typing of
Staphylococcus aureus
at 100 Times the Routine Test Dilution
Authors: Aucken Hazel M. , Westwell Kate 
Published: July 2002

ABSTRACT

Duplicate phage typing tests of 150 isolates of methicillin-sensitive and -resistant
Staphylococcus aureus
at 100 times the routine test dilution showed that the existing reaction difference rule remains satisfactory when isolates are tested on the same day; i.e., two or more reactions must differ before isolates should be considered distinct. However, if typing is done on different days, a three-reaction-difference rule is needed.
Engineered BGMK Cells for Sensitive and Rapid Detection of Enteroviruses
Authors: Huang Yung T. , Yam Paul , Yan Huimin , Sun Yan 
Published: July 2002

ABSTRACT
Decay-accelerating factor (DAF) has been reported to be a cellular receptor for several enteroviruses. Buffalo green monkey kidney (BGMK) cells expressing human DAF (BGMK-hDAF cells) showed increased susceptibility and sensitivity to several types of enteroviruses compared to wild-type BGMK cells. When 17 frozen positive clinical samples were tested, BGMK cells detected 8 and BGMK-hDAF cells detected 16. Since the CaCo-2 cell line has been documented to support the replication of most enteroviruses, CaCo-2 cells were mixed with BGMK-hDAF cells in order to increase the number of viruses detected. Thirty-four frozen clinical samples that previously had tested positive for enteroviruses were tested, and the following numbers were detected: 33 of 34 by CaCo-2/BGMK-hDAF cells, 29 of 34 by CaCo-2/BGMK cells, 28 of 34 by H292/RD (E-mix A) and A-549/BGMK (E-mix B) cells, and 26 of 34 by MRC-5 and pRhMK cells.
Sequence Analysis of the Ribosomal DNA Intergenic Spacer 1 Regions of
Trichosporon
Species
Authors: Sugita Takashi , Nakajima Masamitsu , Ikeda Reiko , Matsushima Toshiharu , Shinoda Takako 
Published: July 2002

ABSTRACT

We determined the sequence of the intergenic spacer (IGS) 1 region, which is located between the 26S and 5S rRNA genes, in 25 species of the genus
Trichosporon
. IGS 1 sequences varied in length from 195 to 719 bp. Comparative sequence analysis suggested that the divergence of IGS 1 sequences has been greater than that of the internal transcribed spacer regions. We also identified five genotypes of
T
.
asahii
, which is a major causative agent of deep-seated trichosporonosis, based on the IGS 1 sequences of 43 strains. Most of the isolates that originated in Japan were of genotype 1, whereas the American isolates were of genotype 3 or 5. Our results suggest that analysis of IGS regions provides a powerful method to distinguish between phylogenetically closely related species and that a geographic substructure may exist among
T
.
asahii
clinical isolates.
Detection of Antibodies Specific to an Antigenic Cell Wall Galactomannoprotein for Serodiagnosis of
Aspergillus fumigatus
Aspergillosis
Authors: Chan Che-man , Woo Patrick C. Y. , Leung Andy S. P. , Lau Susanna K. P. , Che Xiao-yan , Cao Liang , Yuen Kwok-yung 
Published: July 2002

ABSTRACT

Aspergilloma and invasive aspergillosis are important opportunistic infections caused by
Aspergillus
species, among which
Aspergillus fumigatus
is the most common species associated with human disease. We developed an enzyme-linked immunosorbent assay (ELISA)-based antibody assay with Afmp1p, a purified recombinant antigenic cell wall galactomannoprotein of
A. fumigatus
. Evaluation of the test with guinea pig sera against
A. fumigatus
and other pathogenic fungi indicated that this assay was specific for
A. fumigatus
. Clinical evaluation revealed that the assay was 100% sensitive for patients with aspergilloma and 33.3% sensitive for patients with invasive aspergillosis. No false-positive results were found for serum samples from 80 healthy blood donors, 6 patients with typhoid fever, 4 patients with melioidosis, 20 patients with penicilliosis marneffei, 5 patients with candidiasis, and 4 patients with cryptococcosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Afmp1p antibody can be of significant value as a diagnostic for aspergillosis.
Analysis of Cellular Factors That Mediate Nuclear Export of RNAs Bearing the Mason-Pfizer Monkey Virus Constitutive Transport Element
Authors: Kang Yibin , Bogerd Hal P. , Cullen Bryan R. 
Published: July 2002

ABSTRACT

There is now convincing evidence that the human Tap protein plays a critical role in mediating the nuclear export of mRNAs that contain the Mason-Pfizer monkey virus constitutive transport element (CTE) and significant evidence that Tap also participates in global poly(A)
+
RNA export. Previously, we had mapped carboxy-terminal sequences in Tap that serve as an essential nucleocytoplasmic shuttling domain, while others had defined an overlapping Tap sequence that can bind to the FG repeat domains of certain nucleoporins. Here, we demonstrate that these two biological activities are functionally correlated. Specifically, mutations in Tap that block nucleoporin binding also block both nucleocytoplasmic shuttling and the Tap-dependent nuclear export of CTE-containing RNAs. In contrast, mutations that do not inhibit nucleoporin binding also fail to affect Tap shuttling. Together, these data indicate that Tap belongs to a novel class of RNA export factors that can target bound RNA molecules directly to the nuclear pore without the assistance of an importin β-like cofactor. In addition to nucleoporins, Tap has also been proposed to interact with a cellular cofactor termed p15. Although we were able to confirm that Tap can indeed bind p15 specifically both in vivo and in vitro, a mutation in Tap that blocked p15 binding only modestly inhibited CTE-dependent nuclear RNA export. However, p15 did significantly enhance the affinity of Tap for the CTE in vitro and readily formed a ternary complex with Tap on the CTE. This result suggests that p15 may play a significant role in the recruitment of the Tap nuclear export factor to target RNA molecules in vivo.
Formation and Characterization of the Trimeric Form of the Fusion Protein of Semliki Forest Virus
Authors: Gibbons Don L. , Ahn Anna , Chatterjee Prodyot K. , Kielian Margaret 
Published: July 2002

ABSTRACT
Enveloped animal viruses infect cells via fusion of the viral membrane with a host cell membrane. Fusion is mediated by a viral envelope glycoprotein, which for a number of enveloped animal viruses rearranges itself during fusion to form a trimeric α-helical coiled-coil structure. This conformational change from the metastable, nonfusogenic form of the spike protein to the highly stable form involved in fusion can be induced by physiological activators of virus fusion and also by a variety of destabilizing conditions. The E1 spike protein subunit of Semliki Forest virus (SFV) triggers membrane fusion upon exposure to mildly acidic pH and forms a homotrimer that appears necessary for fusion. We have here demonstrated that formation of the E1 homotrimer was efficiently triggered under low-pH conditions but not by perturbants such as heat or urea, despite their induction of generalized conformational changes in the E1 and E2 subunits and partial exposure of an acid-specific E1 epitope. We used a sensitive fluorescence assay to show that neither heat nor urea treatment triggered SFV-liposome fusion at neutral pH, although either treatment inactivated subsequent low-pH-triggered fusion activity. Once formed, the low-pH-induced E1 homotrimer was very stable and was only dissociated under harsh conditions such as heating in sodium dodecyl sulfate. Taken together, these data, as well as protein structure predictions, suggest a model in which the less stable native E1 subunit specifically responds to low pH to form the more stable E1 homotrimer via conformational changes different from those of the coiled-coil type of fusion proteins.