bluuebunny/crossref_metadata_2025 huggingface dataset:
Cellobiose Uptake in the Hyperthermophilic Archaeon
Pyrococcus furiosus
Is Mediated by an Inducible, High-Affinity ABC Transporter
Authors: Koning Sonja M. , Elferink Marieke G. L. , Konings Wil N. , Driessen Arnold J. M.
Published: July 2002

The hyperthermophilic archaeon
Pyrococcus furiosus
can utilize different β-glucosides, like cellobiose and laminarin. Cellobiose uptake occurs with high affinity (
K

m

= 175 nM) and involves an inducible binding protein-dependent transport system. The cellobiose binding protein (CbtA) was purified from
P. furiosus
membranes to homogeneity as a 70-kDa glycoprotein. CbtA not only binds cellobiose but also cellotriose, cellotetraose, cellopentaose, laminaribiose, laminaritriose, and sophorose. The
cbtA
gene was cloned and functionally expressed in
Escherichia coli. cbtA
belongs to a gene cluster that encodes a transporter that belongs to the Opp family of ABC transporters.
Characterization of PitA and PitB from
Escherichia coli
Authors: Harris Robyn M. , Webb Dianne C. , Howitt Susan M. , Cox Graeme B.
Published: July 2002

Escherichia coli
contains two major systems for transporting inorganic phosphate (P
i
). The low-affinity P
i
transporter (
pitA
) is expressed constitutively and is dependent on the proton motive force, while the high-affinity Pst system (
pstSCAB
) is induced at low external P
i
concentrations by the
pho
regulon and is an ABC transporter. We isolated a third putative P
i
transport gene,
pitB
, from
E. coli
K-12 and present evidence that
pitB
encodes a functional P
i
transporter that may be repressed at low P
i
levels by the
pho
regulon. While a
pitB
+
cosmid clone allowed growth on medium containing 500 μM P
i
,
E. coli
with wild-type genomic
pitB
(
pitA
Δ
pstC345
double mutant) was unable to grow under these conditions, making it indistinguishable from a
pitA pitB
Δ
pstC345
triple mutant. The mutation Δ
pstC345
constitutively activates the
pho
regulon, which is normally induced by phosphate starvation. Removal of
pho
regulation by deleting the
phoB-phoR
operon allowed the
pitB
+
pitA
Δ
pstC345
strain to utilize P
i
, with P
i
uptake rates significantly higher than background levels. In addition, the apparent
K

m

of PitB decreased with increased levels of protein expression, suggesting that there is also regulation of the PitB protein. Strain K-10 contains a nonfunctional
pitA
gene and lacks Pit activity when the Pst system is mutated. The
pitA
mutation was identified as a single base change, causing an aspartic acid to replace glycine 220. This mutation greatly decreased the amount of PitA protein present in cell membranes, indicating that the aspartic acid substitution disrupts protein structure.
Nested Deletions of the SRL Pathogenicity Island of
Shigella flexneri
2a
Authors: Turner Sally A. , Luck Shelley N. , Sakellaris Harry , Rajakumar Kumar , Adler Ben
Published: July 2002

In this study, we determined the boundaries of a 99-kb deletable element of
Shigella flexneri
2a strain YSH6000. The element, designated the multiple-antibiotic resistance deletable element (MRDE), had recently been found to contain a 66-kb pathogenicity island (PAI)-like element (designated the SRL PAI) which carries the
Shigella
resistance locus (SRL), encoding resistance determinants to streptomycin, ampicillin, chloramphenicol, and tetracycline. The YSH6000 MRDE was found to be flanked by two identical IS
91
elements present at the
S. flexneri
homologs of the
Escherichia coli
genes
putA
and
mdoA
on
Not
I fragment D. Sequence data from two YSH6000-derived MRDE deletants, YSH6000T and S2430, revealed that deletion of the MRDE occurred between the two flanking IS
91
elements, resulting in a single IS
91
element spanning the two original IS
91
loci. Selection for the loss of tetracycline resistance confirmed that the MRDE deletion occurred reproducibly from the same chromosomal site and also showed that the SRL PAI and the SRL itself were capable of independent deletion from the chromosome, thus revealing a unique set of nested deletions. The excision frequency of the SRL PAI was estimated to be 10
−5
per cell in the wild type, and mutation of a P4-like integrase gene (
int
) at the left end of the SRL PAI revealed that
int
mediates precise deletion of the PAI.
RpoS-Dependent Transcriptional Control of
sprE
: Regulatory Feedback Loop
Authors: Ruiz Natividad , Peterson Celeste N. , Silhavy Thomas J.
Published: July 2002

The stationary-phase response exhibited by
Escherichia coli
upon nutrient starvation is mainly induced by a decrease of the ClpXP-dependent degradation of the alternate primary ς factor RpoS. Although it is known that the specific regulation of this proteolysis is exercised by the orphan response regulator SprE, it remains unclear how SprE's activity is regulated in vivo. Previous studies have demonstrated that the cellular content of SprE itself is paradoxically increased in stationary-phase cells in an RpoS-dependent fashion. We show here that this RpoS-dependent upregulation of SprE levels is due to increased transcription. Furthermore, we demonstrate that
sprE
is part of the two-gene
rssA-sprE
operon, but it can also be transcribed from an additional RpoS-dependent promoter located in the
rssA-sprE
intergenic region. In addition, by using an in-frame deletion in
rssA
we found that RssA does not regulate either SprE or RpoS under the conditions tested.
Structures of Bacterial Flagellar Motors from Two FliF-FliG Gene Fusion Mutants
Authors: Thomas Dennis , Morgan David Gene , DeRosier David J.
Published: July 2002

Flagella purified from
Salmonella enterica
serovar Typhimurium contain FliG, FliM, and FliN, cytoplasmic proteins that are important in torque generation and switching, and FliF, a transmembrane structural protein. The motor portion of the flagellum (the basal body complex) has a cytoplasmic C ring and a transmembrane M ring. Incubation of purified basal bodies at pH 4.5 removed FliM and FliN but not FliG or FliF. These basal bodies lacked C rings but had intact M rings, suggesting that FliM and FliN are part of the C ring but not a detectable part of the M ring. Incubation of basal bodies at pH 2.5 removed FliG, FliM, and FliN but not FliF. These basal bodies lacked the C ring, and the cytoplasmic face of the M ring was altered, suggesting that FliG makes up at least part of the cytoplasmic face of the M ring. Further insights into FliG were obtained from cells expressing a fusion protein of FliF and FliG. Flagella from these mutants still rotated but cells were not chemotactic. One mutant is a full-length fusion of FliF and FliG; the second mutant has a deletion lacking the last 56 residues of FliF and the first 94 residues of FliG. In the former, C rings appeared complete, but a portion of the M ring was shifted to higher radius. The C-ring–M-ring interaction appeared to be altered. In basal bodies with the fusion-deletion protein, the C ring was smaller in diameter, and one of its domains occupied space vacated by missing portions of FliF and FliG.
Quantification of Expression of
Staphylococcus epidermidis
Housekeeping Genes with Taqman Quantitative PCR during In Vitro Growth and under Different Conditions
Authors: Vandecasteele S. J. , Peetermans W. E. , Merckx R. , Van Eldere J.
Published: July 2002

The aims of the present study were (i) to develop and test a sensitive and reproducible method for the study of gene expression in staphylococci and (ii) to study the expression of five housekeeping genes which are involved in nucleic acid metabolism (
gmk
, guanylate kinase; the dihydrofolate reductase [DHFR] gene), glucose metabolism (
tpi
, triosephosphate isomerase), and protein metabolism (the 16S rRNA gene;
hsp-60
, heat-shock protein 60) during in vitro exponential and stationary growth. A modified method for instant mRNA isolation was combined with gene quantification via Taqman real-time quantitative PCR. The detection limit of our method was 10 copies of RNA. The average intersample variability was 16%. A 10-fold increase in the expression of the
hsp-60
gene was induced by exposure to a 10°C heat shock (37 to 47°C) for 10 min. During in vitro growth, the expression of all five housekeeping genes showed rapid up-regulation after inoculation of the bacteria in brain heart infusion medum and started to decline during the mid-exponential-growth phase. Maximal gene expression was 110- to 300-fold higher than gene expression during stationary phase. This indicates that housekeeping metabolism is a very dynamic process that is extremely capable of adapting to different growth conditions. Expression of the 16S rRNA gene decreases significantly earlier than that of other housekeeping genes. This confirms earlier findings for
Escherichia coli
that a decline in bacterial ribosomal content (measured by 16S rRNA gene expression) precedes the decline in protein synthesis (measured by mRNA expression).
Complex I and Its Involvement in Redox Homeostasis and Carbon and Nitrogen Metabolism in
Rhodobacter capsulatus
Authors: Tichi Mary A. , Meijer Wim G. , Tabita F. Robert
Published: July 2002

A transposon mutant of
Rhodobacter capsulatus
, strain Mal7, that was incapable of photoautotrophic and chemoautotrophic growth and could not grow photoheterotrophically in the absence of an exogenous electron acceptor was isolated. The phenotype of strain Mal7 suggested that the mutation was in some gene(s) not previously shown to be involved in CO
2
fixation control. The site of transposition in strain Mal7 was identified and shown to be in the gene
nuoF
, which encodes one of the 14 subunits for NADH ubiquinone-oxidoreductase, or complex I. To confirm the role of complex I and
nuoF
for CO
2
-dependent growth, a site-directed
nuoF
mutant was constructed (strain SBC1) in wild-type strain SB1003. The complex I-deficient strains Mal7 and SBC1 exhibited identical phenotypes, and the pattern of CO
2
fixation control through the Calvin-Benson-Bassham pathway was the same for both strains. It addition, it was shown that electron transport through complex I led to differential control of the two major
cbb
operons of this organism. Complex I was further shown to be linked to the control of nitrogen metabolism during anaerobic photosynthetic growth of
R. capsulatus
.
Unexpected Presence of Defective
glpR
Alleles in Various Strains of
Escherichia coli
Authors: Holtman C. Kay , Thurlkill Richard , Pettigrew Donald W.
Published: July 2002

Alleles of
glpR
associated with the same GlpR
−
phenotype produce substitutions in different conserved portions of the glycerol 3-phosphate repressor which are not part of the helix-turn-helix motif. Analysis of the effects on growth and enzyme expression show that glucose repression of glycerol utilization is not dependent on a functional repressor.
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

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

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

Campylobacter 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

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

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

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

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

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

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

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

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

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

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

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

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.
In Vitro and In Vivo Infection of Neural Cells by a Recombinant Measles Virus Expressing Enhanced Green Fluorescent Protein
Authors: Duprex W. Paul , Mcquaid Stephen , Roscic-Mrkic Branka , Cattaneo Roberto , Mccallister Cecilia , Rima Bert K.
Published: July 2002

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.
The Infectivities of Turnip Yellow Mosaic Virus Genomes with Altered tRNA Mimicry Are Not Dependent on Compensating Mutations in the Viral Replication Protein
Authors: Filichkin Sergei A. , Bransom Kay L. , Goodwin Joel B. , Dreher Theo W.
Published: July 2002

Five highly infectious turnip yellow mosaic virus (TYMV) genomes with sequence changes in their 3′-terminal regions that result in altered aminoacylation and eEF1A binding have been studied. These genomes were derived from cloned parental RNAs of low infectivity by sequential passaging in plants. Three of these genomes that are incapable of aminoacylation have been reported previously (J. B. Goodwin, J. M. Skuzeski, and T. W. Dreher, Virology 230:113–124, 1997). We now demonstrate by subcloning the 3′ untranslated regions into wild-type TYMV RNA that the high infectivities and replication rates of these genomes compared to their progenitors are mostly due to a small number of mutations acquired in the 3′ tRNA-like structure during passaging. Mutations in other parts of the genome, including the replication protein coding region, are not required for high infectivity but probably do play a role in optimizing viral amplification and spread in plants. Two other TYMV RNA variants of suboptimal infectivities, one that accepts methionine instead of the usual valine and one that interacts less tightly with eEF1A, were sequentially passaged to produce highly infectious genomes. The improved infectivities of these RNAs were not associated with increased replication in protoplasts, and no mutations were acquired in their 3′ tRNA-like structures. Complete sequencing of one genome identified two mutations that result in amino acid changes in the movement protein gene, suggesting that improved infectivity may be a function of improved viral dissemination in plants. Our results show that the wild-type TYMV replication proteins are able to amplify genomes with 3′ termini of variable sequence and tRNA mimicry. These and previous results have led to a model in which the binding of eEF1A to the 3′ end to antagonize minus-strand initiation is a major role of the tRNA-like structure.
Recovery and Characterization of a Chimeric Rinderpest Virus with the Glycoproteins of Peste-des-Petits-Ruminants Virus: Homologous F and H Proteins Are Required for Virus Viability
Authors: Das Subash C. , Baron Michael D. , Barrett Thomas
Published: July 2002

Rinderpest (RP) and peste-des-petits-ruminants (PPR) are two important diseases of domestic ruminants. To improve on currently available vaccines against PPR, we have created cDNA copies of the RP virus genome in which either the fusion (F) or hemagglutinin (H) gene, or both, was replaced with the corresponding gene from PPR virus. It was necessary to develop a modified rescue system in which the T7 RNA polymerase was provided by a recombinant fowlpox virus and the entire rescue procedure took place in Vero cells before we could obtain live virus from these chimeric constructs. No virus was recovered when only one of the glycoprotein genes was changed, but a chimeric virus containing both F and H genes from PPR virus was reproducibly rescued from cDNA, indicating that a virus-specific functional interaction takes place between the F and H proteins. The rescued virus expressing the PPR glycoproteins grew more slowly in tissue culture than either parental virus and formed abnormally large syncytia. Goats infected with the chimera showed no adverse reaction, as assessed by clinical signs, temperature, leukocyte count, virus isolation, and serology, and were protected from subsequent challenge with wild-type PPR virus.
Foreign and Chimeric External Scaffolding Proteins as Inhibitors of
Microviridae
Morphogenesis
Authors: Burch April D. , Fane Bentley A.
Published: July 2002

Viral assembly is an ideal system in which to investigate the transient recognition and interplay between proteins. During morphogenesis, scaffolding proteins temporarily associate with structural proteins, stimulating conformational changes that promote assembly and inhibit off-pathway reactions.
Microviridae
morphogenesis is dependent on two scaffolding proteins, an internal and an external species. The external scaffolding protein is the most conserved protein within the
Microviridae
, whose canonical members are φX174, G4, and α3. However, despite 70% homology on the amino acid level, overexpression of a foreign
Microviridae
external scaffolding protein is a potent cross-species inhibitor of morphogenesis. Mutants that are resistant to the expression of a foreign scaffolding protein cannot be obtained via one mutational step. To define the requirements for and constraints on scaffolding protein interactions, chimeric external scaffolding proteins have been constructed and analyzed for effects on in vivo assembly. The results of these experiments suggest that at least two cross-species inhibitory domains exist within these proteins; one domain most likely blocks procapsid formation, and the other allows procapsid assembly but blocks DNA packaging. A mutation conferring resistance to the expression of a chimeric protein (chiD
r
) that inhibits DNA packaging was isolated. The mutation maps to gene A, which encodes a protein essential for packaging. The
chiD
r
mutation confers resistance only to a chimeric D protein; the mutant is still inhibited by the expression of foreign D proteins. The results presented here demonstrate how closely related proteins could be developed into antiviral agents that specifically target virion morphogenesis.
Diminished Primary and Secondary Influenza Virus-Specific CD8+T-Cell Responses in CD4-Depleted Ig−/−Mice
Authors: Riberdy Janice M. , Christensen Jan P. , Branum Kristen , Doherty Peter C.
Published: July 2002

Optimal expansion of influenza virus nucleoprotein (DbNP366)-specific CD8+T cells following respiratory challenge of naive Ig−/−μMT mice was found to require CD4+T-cell help, and this effect was also observed in primed animals. Absence of the CD4+population was consistently correlated with diminished recruitment of virus-specific CD8+T cells to the infected lung, delayed virus clearance, and increased morbidity. The splenic CD8+set generated during the recall response in Ig−/−mice primed at least 6 months previously showed a normal profile of gamma interferon production subsequent to short-term, in vitro stimulation with viral peptide, irrespective of a concurrent CD4+T-cell response. Both the magnitude and the localization profiles of virus-specific CD8+T cells, though perhaps not their functional characteristics, are thus modified in mice lacking CD4+T cells.
Identification of
Notch1
as a Frequent Target for Provirus Insertional Mutagenesis in T-Cell Lymphomas Induced by Leukemogenic Mutants of Mouse Mammary Tumor Virus
Authors: Yanagawa Shin-ichi , Lee Jong-Seo , Kakimi Kazuhiro , Matsuda Yukihiro , Honjo Tasuku , Ishimoto Akinori
Published: July 2002

In contrast to wild-type mouse mammary tumor virus (MMTV), the MMTV mutants with specific deletions in the U3 region of their long terminal repeats cause T-cell lymphomas. In 30% of T-cell lymphomas arising in BALB/c mice infected with MLA-MMTV, a leukemogenic MMTV mutant, we have found that MMTV proviruses were integrated into a short region of the
Notch1
genome, so that truncated
Notch1
transcripts encoding the transmembrane and the cytoplasmic domains of Notch1 protein could be expressed. Thus,
Notch1
is a major target of provirus insertional mutagenesis in these T-cell lymphomas.
Generation of Subgenomic RNA Directed by a Satellite RNA Associated with Bamboo Mosaic Potexvirus: Analyses of Potexvirus Subgenomic RNA Promoter
Authors: Lee Yun-Shien , Hsu Yau-Heiu , Lin Na-Sheng
Published: July 2002

Satellite RNA of bamboo mosaic potexvirus (satBaMV), a single-stranded positive-sense RNA encoding a nonstructural protein of 20 kDa (P20), depends on bamboo mosaic potexvirus (BaMV) for replication and encapsidation. A full-length cDNA clone of satBaMV was used to examine the sequences required for the synthesis of potexvirus subgenomic RNAs (sgRNAs). Subgenomic promoter-like sequences (SGPs), 107 nucleotides (nt) upstream from the capsid protein (CP) gene of BaMV-V, were inserted upstream of the start codon of the P20 gene of satBaMV. Insertion of SGPs gave rise to the synthesis of sgRNA of satBaMV in protoplasts ofNicotiana benthamianaand leaves ofChenopodium quinoawhen coinoculated with BaMV-V genomic RNA. Moreover, both the satBaMV cassette and its sgRNA were encapsidated. From analysis of the SGPs by deletion mutation, we concluded that an SGP contains one core promoterlike sequence (nt −30 through +16), two upstream enhancers (nt −59 through −31 and −91 through −60), and one downstream enhancer (nt +17 through +52), when the transcription initiation site is taken as +1. Site-directed mutagenesis and compensatory mutation to disrupt and restore potential base pairing in the core promoter-like sequence suggest that the stem-loop structure is important for the function of SGP in vivo. Likewise, the insertion of a putative SGP of the BaMV open reading frame 2 gene or a heterologous SGP of potato virus X resulted in generation of an sgRNA. The satBaMV cassette should be a useful tool to gain insight into sequences required for the synthesis of potexvirus sgRNAs.
TVB Receptors for Cytopathic and Noncytopathic Subgroups of Avian Leukosis Viruses Are Functional Death Receptors
Authors: Brojatsch Jürgen , Naughton John , Adkins Heather B. , Young John A. T.
Published: July 2002

The identification of TVB
S3
, a cellular receptor for the cytopathic subgroups B and D of avian leukosis virus (ALV-B and ALV-D), as a tumor necrosis factor receptor-related death receptor with a cytoplasmic death domain, provides a compelling argument that viral Env-receptor interactions are linked to cell death (4). However, other TVB proteins have been described that appear to have similar death domains but are cellular receptors for the noncytopathic subgroup E of ALV (ALV-E): TVB
T
, a turkey subgroup E-specific ALV receptor, and TVB
S1
, a chicken receptor for subgroups B, D, and E ALV. To begin to understand the role of TVB receptors in the cytopathic effects associated with infection by specific ALV subgroups, we asked whether binding of a soluble ALV-E surface envelope protein (SU) to its receptor can lead to cell death. Here we report that ALV-E SU-receptor interactions can induce apoptosis in quail or turkey cells. We also show directly that TVB
S1
and TVB
T
are functional death receptors that can trigger cell death by apoptosis via a mechanism involving their cytoplasmic death domains and activation of the caspase pathway. These data demonstrate that ALV-B and ALV-E use functional death receptors to enter cells, and it remains to be determined why only subgroups B and D viral infections lead specifically to cell death.
Herpes Simplex Virus Type 1 ICP4 Promotes Transcription Preinitiation Complex Formation by Enhancing the Binding of TFIID to DNA
Authors: Grondin Benoit , DeLuca Neal
Published: July 2002

Infected-cell polypeptide 4 (ICP4) of herpes simplex virus type 1 (HSV-1) activates the expression of many HSV genes during infection. It functions along with the cellular general transcription factors to increase the transcription rates of genes. In this study, an HSV late promoter consisting of only a TATA box and an INR element was immobilized on a magnetic resin and incubated with nuclear extracts or purified TFIID in the presence and absence of ICP4. Analysis of the complexes formed on these promoters revealed that ICP4 increased the formation of transcription preinitiation complexes (PICs) in a TATA box-dependent manner, as determined by the presence of ICP4, TFIID, TFIIB, and polymerase II on the promoter. With both nuclear extract and purified TFIID, it was determined that ICP4 helped TFIID bind to the promoter and the TATA box. These observations differed from those for the activator Gal4-VP16. As previously observed by others, Gal4-VP16 also increased the formation of PICs without helping TFIID bind to the promoter, suggesting that ICP4 and VP16 differ in their mechanism of activation and that ICP4 functions to facilitate PIC formation at an earlier step in the formation of PICs. We also observed that the DNA binding activity of ICP4 was not sufficient to help TFIID bind to the promoter and that the region of ICP4 that was responsible for this activity is located between residues 30 and 274. Taken together these results demonstrate that a specific region of ICP4 helps TFIID bind to the TATA box and that this in turn facilitates the formation of transcription PICs.