bluuebunny/crossref_metadata_2025 huggingface dataset:
New Family of Regulators in the Environmental Signaling Pathway Which Activates the General Stress Transcription Factor ς
B
of
Bacillus subtilis
Authors: Akbar Samina , Gaidenko Tatiana A. , Kang Choong Min , O'Reilly Mary , Devine Kevin M. , Price Chester W.
Published: July 2002

Expression of the general stress regulon of
Bacillus subtilis
is controlled by the alternative transcription factor ς
B
, which is activated when cells encounter growth-limiting energy or environmental stresses. The RsbT serine-threonine kinase is required to convey environmental stress signals to ς
B
, and this kinase activity is magnified in vitro by the RsbR protein, a positive regulator important for full in vivo response to salt or heat stress. Previous genetic analysis suggested that RsbR function is redundant with other unidentified regulators. A search of the translated
B. subtilis
genome found six paralogous proteins with significant similarity to RsbR: YetI, YezB, YkoB, YojH, YqhA, and YtvA. Their possible regulatory roles were investigated using three different approaches. First, genetic analysis found that null mutations in four of the six paralogous genes have marked effects on the ς
B
environmental signaling pathway, either singly or in combination. The two exceptions were
yetI
and
yezB
, adjacent genes which appear to encode a split paralog. Second, biochemical analysis found that YkoB, YojH, and YqhA are specifically phosphorylated in vitro by the RsbT environmental signaling kinase, as had been previously shown for RsbR, which is phosphorylated on two threonine residues in its C-terminal region. Both residues are conserved in the three phosphorylated paralogs but are absent in the ones that were not substrates of RsbT: YetI and YezB, each of which bears only one of the conserved residues; and YtvA, which lacks both residues and instead possesses an N-terminal PAS domain. Third, analysis in the yeast two-hybrid system suggested that all six paralogs interact with each other and with the RsbR and RsbS environmental regulators. Our data indicate that (i) RsbR, YkoB, YojH, YqhA, and YtvA function in the environmental stress signaling pathway; (ii) YtvA acts as a positive regulator; and (iii) RsbR, YkoB, YojH, and YqhA collectively act as potent negative regulators whose loss increases ς
B
activity more than 400-fold in unstressed cells.
Modulation of Actinorhodin Biosynthesis in
Streptomyces lividans
by Glucose Repression of
afsR2
Gene Transcription
Authors: Kim Eung-Soo , Hong Hee-Jeon , Choi Cha-Yong , Cohen Stanley N.
Published: July 2002

While the biosynthetic gene cluster encoding the pigmented antibiotic actinorhodin (ACT) is present in the two closely related bacterial species,
Streptomyces lividans
and
Streptomyces coelicolor
, it normally is expressed only in
S. coelicolor
—generating the deep-blue colonies responsible for the
S. coelicolor
name. However, multiple copies of the two regulatory genes,
afsR
and
afsR2
, activate ACT production in
S. lividans
, indicating that this streptomycete encodes a functional ACT biosynthetic pathway. Here we report that the occurrence of ACT biosynthesis in
S. lividans
is determined conditionally by the carbon source used for culture. We found that the growth of
S. lividans
on solid media containing glucose prevents ACT production in this species by repressing the synthesis of
afsR2
mRNA; a shift to glycerol as the sole carbon source dramatically relieved this repression, leading to extensive ACT synthesis and obliterating this phenotypic distinction between
S. lividans
and
S. coelicolor
. Transcription from the
afsR2
promoter during growth in glycerol was dependent on
afsR
gene function and was developmentally regulated, occurring specifically at the time of aerial mycelium formation and coinciding temporally with the onset of ACT production. In liquid media, where morphological differentiation does not occur, ACT production in the absence of glucose increased as
S. lividans
cells entered stationary phase, but unlike ACT biosynthesis on solid media, occurred by a mechanism that did not require either
afsR
or
afsR2
. Our results identify parallel medium-dependent pathways that regulate ACT biosynthesis in
S. lividans
and further demonstrate that the production of this antibiotic in
S. lividans
grown on agar can be modulated by carbon source through the regulation of
afsR2
mRNA synthesis.
GcpE Is Involved in the 2-
C
-Methyl-
d
-Erythritol 4-Phosphate Pathway of Isoprenoid Biosynthesis in
Escherichia coli
Authors: Altincicek Boran , Kollas Ann-Kristin , Sanderbrand Silke , Wiesner Jochen , Hintz Martin , Beck Ewald , Jomaa Hassan
Published: July 2002

In a variety of organisms, including plants and several eubacteria, isoprenoids are synthesized by the mevalonate-independent 2-
C
-methyl-
d
-erythritol 4-phosphate (MEP) pathway. Although different enzymes of this pathway have been described, the terminal biosynthetic steps of the MEP pathway have not been fully elucidated. In this work, we demonstrate that the
gcpE
gene of
Escherichia coli
is involved in this pathway.
E. coli
cells were genetically engineered to utilize exogenously provided mevalonate for isoprenoid biosynthesis by the mevalonate pathway. These cells were then deleted for the essential
gcpE
gene and were viable only if the medium was supplemented with mevalonate or the cells were complemented with an episomal copy of
gcpE
.
The ToxR-Mediated Organic Acid Tolerance Response of
Vibrio cholerae
Requires OmpU
Authors: Merrell D. Scott , Bailey Camella , Kaper James B. , Camilli Andrew
Published: July 2002

It was previously demonstrated that the intestinal pathogen
Vibrio cholerae
could undergo an adaptive stress response known as the acid tolerance response (ATR). The ATR is subdivided into two branches, inorganic ATR and organic ATR. The transcriptional regulator ToxR, while not involved in inorganic ATR, is required for organic ATR in a ToxT-independent manner. Herein, we investigate the effect of organic acid stress on global protein synthesis in
V. cholerae
and show by two-dimensional gel electrophoresis that the stress response alters the expression of more than 100 polypeptide species. The expression of more than 20 polypeptide species is altered in a
toxR
strain compared to the wild type. Despite this, ectopic expression of the porin OmpU from an inducible promoter is shown to be sufficient to bypass the
toxR
organic ATR defect. Characterization of the effect of organic acid stress on
ompU
and
ompT
transcription reveals that while
ompU
transcription remains virtually unaffected,
ompT
transcription is repressed in a ToxR-independent manner. These transcript levels are similarly reflected in the extent of accumulation of OmpU and OmpT. Possible roles for OmpU in organic acid resistance are discussed.
Genes Essential to Iron Transport in the Cyanobacterium
Synechocystis
sp. Strain PCC 6803
Authors: Katoh Hirokazu , Hagino Natsu , Grossman Arthur R. , Ogawa Teruo
Published: July 2002

Genes encoding polypeptides of an ATP binding cassette (ABC)-type ferric iron transporter that plays a major role in iron acquisition in
Synechocystis
sp. strain PCC 6803 were identified. These genes are
slr1295, slr0513, slr0327
, and recently reported
sll1878
(Katoh et al., J. Bacteriol. 182:6523–6524, 2000) and were designated
futA1, futA2, futB
, and
futC
, respectively, for their involvement in ferric iron uptake. Inactivation of these genes individually or
futA1
and
futA2
together greatly reduced the activity of ferric iron uptake in cells grown in complete medium or iron-deprived medium. All the
fut
genes are expressed in cells grown in complete medium, and expression was enhanced by iron starvation. The
futA1
and
futA2
genes appear to encode periplasmic proteins that play a redundant role in iron binding. The deduced products of
futB
and
futC
genes contain nucleotide-binding motifs and belong to the ABC transporter family of inner-membrane-bound and membrane-associated proteins, respectively. These results and sequence similarities among the four genes suggest that the Fut system is related to the Sfu/Fbp family of iron transporters. Inactivation of
slr1392
, a homologue of
feoB
in
Escherichia coli
, greatly reduced the activity of ferrous iron transport. This system is induced by intracellular low iron concentrations that are achieved in cells exposed to iron-free medium or in the
fut
-less mutants grown in complete medium.
Caulobacter crescentus
Synthesizes an S-Layer-Editing Metalloprotease Possessing a Domain Sharing Sequence Similarity with Its Paracrystalline S-Layer Protein
Authors: Umelo-Njaka Elizabeth , Bingle Wade H. , Borchani Faten , Le Khai D. , Awram Peter , Blake Theo , Nomellini John F. , Smit John
Published: July 2002

Strains of
Caulobacter crescentus
elaborate an S-layer, a two-dimensional protein latticework which covers the cell surface. The S-layer protein (RsaA) is secreted by a type I mechanism (relying on a C-terminal signal) and is unusual among type I secreted proteins because high levels of protein are produced continuously. In efforts to adapt the S-layer for display of foreign peptides and proteins, we noted a proteolytic activity that affected S-layer monomers with foreign inserts. The cleavage was precise, resulting in fragments with an unambiguous N-terminal sequence. We developed an assay to screen for loss of this activity (i.e., presentation of foreign peptides without degradation), using transposon and traditional mutagenesis. A metalloprotease gene designated
sap
(S-layer-associated protease) was identified which could complement the protease-negative mutants. The N-terminal half of Sap possessed significant similarity to other type I secreted proteases (e.g., alkaline protease of
Pseudomonas aeruginosa
), including the characteristic RTX repeat sequences, but the C-terminal half which normally includes the type I secretion signal exhibited no such similarity. Instead, there was a region of significant similarity to the N-terminal region of RsaA. We hypothesize that Sap evolved by combining the catalytic portion of a type I secreted protease with an S-layer-like protein, perhaps to associate with nascent S-layer monomers to “scan” for modifications.
Characterization of
Streptococcus suis
Genes Encoding Proteins Homologous to Sortase of Gram-Positive Bacteria
Authors: Osaki Makoto , Takamatsu Daisuke , Shimoji Yoshihiro , Sekizaki Tsutomu
Published: July 2002

Many surface proteins which are covalently linked to the cell wall of gram-positive bacteria have a consensus C-terminal motif, Leu-Pro-X-Thr-Gly (LPXTG). This sequence is cleaved, and the processed protein is attached to an amino group of a cross-bridge in the peptideglycan by a specific enzyme called sortase. Using the type strain of
Streptococcus suis
, NCTC 10234, we found five genes encoding proteins that were homologous to sortases of other bacteria and determined the nucleotide sequences of the genetic regions. One gene, designated
srtA
, was linked to
gyrA
, as were the sortase and sortase-like genes of other streptococci. Three genes, designated
srtB
,
srtC
, and
srtD
, were tandemly clustered in a different location, where there were three segments of directly repeated sequences of approximately 110 bp in close vicinity. The remaining gene, designated
srtE
, was located separately on the chromosome with a pseudogene which may encode a transposase. The deduced amino acid sequences of the five Srt proteins showed 18 to 31% identity with the sortases of
Streptococcus gordonii
and
Staphylococcus aureus
, except that SrtA of
S. suis
had 65% identity with that of
S. gordonii
. Isogenic mutants deficient for
srtA
,
srtBCD
, or
srtE
were generated by allelic exchanges. The protein fraction which was released from partially purified cell walls by digestion with
N
-acetylmuramidase was profiled by two-dimensional gel electrophoresis. More than 15 of the protein spots were missing in the profile of the
srtA
mutant compared with that of the parent strain, and this phenotype was completely complemented by
srtA
cloned from
S. suis
. Four genes encoding proteins corresponding to such spots were identified and sequenced. The deduced translational products of the four genes possessed the LPXTG motif in their C-terminal regions. On the other hand, the protein spots that were missing in the
srtA
mutant appeared in the profiles of the
srtBCD
and
srtE
mutants. These results provide evidence that the cell wall sorting system involving
srtA
is also present in
S. suis
.
VanT, a Homologue of
Vibrio harveyi
LuxR, Regulates Serine, Metalloprotease, Pigment, and Biofilm Production in
Vibrio anguillarum
Authors: Croxatto Antony , Chalker Victoria J. , Lauritz Johan , Jass Jana , Hardman Andrea , Williams Paul , Cámara Miguel , Milton Debra L.
Published: July 2002

Vibrio anguillarum
possesses at least two
N
-acylhomoserine lactone (AHL) quorum-sensing circuits, one of which is related to the
luxMN
system of
Vibrio harveyi
. In this study, we have cloned an additional gene of this circuit,
vanT
, encoding a
V. harveyi
LuxR-like transcriptional regulator. A
V. anguillarum ΔvanT
null mutation resulted in a significant decrease in total protease activity due to loss of expression of the metalloprotease EmpA, but no changes in either AHL production or virulence. Additional genes positively regulated by VanT were identified from a plasmid-based gene library fused to a promoterless
lacZ
. Three
lacZ
fusions (
serA
::
lacZ
,
hpdA-hgdA
::
lacZ,
and
sat-vps73
::
lacZ)
were identified which exhibited decreased expression in the
ΔvanT
strain. SerA is similar to 3-phosphoglycerate dehydrogenases and catalyzes the first step in the serine-glycine biosynthesis pathway. HgdA has identity with homogentisate dioxygenases, and HpdA is homologous to 4-hydroxyphenylpyruvate dioxygenases (HPPDs) involved in pigment production.
V. anguillarum
strains require an active VanT to produce high levels of an
l
-tyrosine-induced brown color via HPPD, suggesting that VanT controls pigment production. Vps73 and Sat are related to
Vibrio cholerae
proteins encoded within a DNA locus required for biofilm formation. A
V. anguillarum ΔvanT
mutant and a mutant carrying a polar mutation in the
sat-vps73
DNA locus were shown to produce defective biofilms. Hence, a new member of the
V. harveyi
LuxR transcriptional activator family has been characterized in
V. anguillarum
that positively regulates serine, metalloprotease, pigment, and biofilm production.
Nitrite Reductase of
Nitrosomonas europaea
Is Not Essential for Production of Gaseous Nitrogen Oxides and Confers Tolerance to Nitrite
Authors: Beaumont Hubertus J. E. , Hommes Norman G. , Sayavedra-Soto Luis A. , Arp Daniel J. , Arciero David M. , Hooper Alan B. , Westerhoff Hans V. , van Spanning Rob J. M.
Published: July 2002

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in
Nitrosomonas europaea
. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N
2
O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N
2
O by
N. europaea
. Inactivation of the putative
fnr
gene showed that Fnr is not essential for the expression of
nirK
.
Rapid and Specific Genotyping System for Hepatitis B Virus Corresponding to Six Major Genotypes by PCR Using Type-Specific Primers
Authors: Naito Hideo , Hayashi Shigeki , Abe Kenji
Published: July 2002

A simple and precise genotyping system based on PCR using type-specific primers was developed for the determination of genotypes A through F of hepatitis B virus (HBV). This assay system is considered to be a useful tool for the molecular diagnosis of HBV infection and for large-scale surveys.
Spoligotype Diversity of
Mycobacterium bovis
Strains Isolated in France from 1979 to 2000
Authors: Haddad N. , Ostyn A. , Karoui C. , Masselot M. , Thorel M. F. , Hughes S. L. , Inwald J. , Hewinson R. G. , Durand B.
Published: July 2002

The molecular fingerprints of 1,349 isolates of
Mycobacterium bovis
received between 1979 and August 2000 at Agence Française de Sécurité Sanitaire des Aliments (Afssa) have been obtained by spoligotyping. The majority of the isolates (1,266) were obtained from cattle living in France. An apparently high level of heterogeneity was observed between isolates. One hundred sixty-one spoligotypes were observed in total, of which 153 were from French isolates. The two predominant spoligotypes, designated BCG-like and GB54, accounted for 26 and 12% of the isolates, respectively. In addition, 84% of the spoligotypes were found fewer than 10 times. Analysis of the results by clustering and parsimony-based algorithms revealed that the majority of the spoligotypes were closely related. The predominant spoligotype was identical to that of the vaccine strain
Mycobacterium bovis
BCG, which was isolated in France at the end of the 19th century. Some spoligotypes were closely associated with restricted geographical areas. Interestingly, some spoligotypes, which were frequently observed in France, were also observed in neighboring countries. Conversely, few spoligotypes were common to France and England, and those that were shared were observed at very different frequencies. This last point illustrates the potential role for an international data bank, which could help trace the spread of
M. bovis
across national borders.
Necessity of Quality-Controlled 16S rRNA Gene Sequence Databases: Identifying Nontuberculous
Mycobacterium
Species
Authors: Turenne Christine Y. , Tschetter Lorelee , Wolfe Joyce , Kabani Amin
Published: July 2002

The use of the 16S rRNA gene for identification of nontuberculous mycobacteria (NTM) provides a faster and better ability to accurately identify them in addition to contributing significantly in the discovery of new species. Despite their associated problems, many rely on the use of public sequence databases for sequence comparisons. To best evaluate the taxonomic status of NTM species submitted to our reference laboratory, we have created a 16S rRNA sequence database by sequencing 121 American Type Culture Collection strains encompassing 92 species of mycobacteria, and have also included chosen unique mycobacterial sequences from public sequence repositories. In addition, the Ribosomal Differentiation of Medical Microorganisms (RIDOM) service has made freely available on the Internet mycobacterial identification by 16S rRNA analysis. We have evaluated 122 clinical NTM species using our database, comparing >1,400 bp of the 16S gene, and the RIDOM database, comparing ∼440 bp. The breakdown of analysis was as follows: 61 strains had a sequence with 100% similarity to the type strain of an established species, 19 strains showed a 1- to 5-bp divergence from an established species, 11 strains had sequences corresponding to uncharacterized strain sequences in public databases, and 31 strains represented unique sequences. Our experience with analysis of the 16S rRNA gene of patient strains has shown that clear-cut results are not the rule. As many clinical, research, and environmental laboratories currently employ 16S-based identification of bacteria, including mycobacteria, a freely available quality-controlled database such as that provided by RIDOM is essential to accurately identify species or detect true sequence variations leading to the discovery of new species.
Serotype-Specific Detection of Coxsackievirus A16 in Clinical Specimens by Reverse Transcription-Nested PCR
Authors: Bendig Justin W. A. , O'Brien Patrick S. , Muir Peter
Published: July 2002

We describe the development of a coxsackievirus A16 (CVA16) serotype-specific PCR which correctly differentiated between CVA16 and other enterovirus serotypes of both laboratory isolates and clinical specimens. The assay will be useful for monitoring CVA16 outbreaks and studying the disease association, epidemiology, and evolution of this common enterovirus serotype.
Characterization of
Chlamydia trachomatis omp1
Genotypes among Sexually Transmitted Disease Patients in Sweden
Authors: Jurstrand Margaretha , Falk Lars , Fredlund Hans , Lindberg Margret , Olcén Per , Andersson Sören , Persson Kenneth , Albert Jan , Bäckman Anders
Published: July 2002

A method for detection and genotyping of genital
Chlamydia trachomatis
infections based on
omp1
gene amplification and sequencing was developed. DNA was extracted from urogenital or urine samples using a Chelex-based method, and an approximately 1,100-bp-long fragment from the
omp1
gene was directly amplified and sequenced. Genotyping was performed by BLAST similarity search, and phylogenetic tree analysis was used to illustrate the evolutionary relationships between clinical isolates and reference strains. The method was used to determine the genotypes of
C. trachomatis
in 237 positive urogenital and/or urine specimens collected at a Swedish sexually transmitted disease clinic during 1 year. The most common genotypes corresponded to serotypes E (47%) and F (17%). The
omp1
gene was highly conserved for genotype E (106 of 112 samples without any mutation) and F (41 of 42 samples without any mutation) strains but appear slightly less conserved for genotypes G (
n
= 6) and H (
n
= 6), where the sequences displayed one to four nucleotide substitutions relative to the reference sequence. Genotyping of samples collected at the follow-up visit indicated that two patients had become reinfected, while three other patients suffered treatment failure or reinfection. One woman appeared to have a mixed infection with two different
C. trachomatis
strains. This
omp1
genotyping method had a high reproducibility and could be used for epidemiological characterization of sexually transmitted
Chlamydia
infections.
Transmission of
Borrelia garinii
OspA Serotype 4 to BALB/c Mice by
Ixodes ricinus
Ticks Collected in the Field
Authors: Hu C. M. , Wilske B. , Fingerle V. , Lobet Y. , Gern L.
Published: July 2002

In Europe,
Borrelia garinii
OspA serotype 4 has been isolated from the cerebrospinal fluid of patients but, up to now, has never been identified among culture isolates from
Ixodes ricinus
ticks. This information raises the question of whether OspA serotype 4 is transmitted by
I. ricinus
in nature. In the present study,
I. ricinus
nymphs collected in an area of endemicity in southern Germany were allowed to feed on mice. Cultivation of ear biopsy specimens showed that six of seven
B. garinii
-infected mice were infected by OspA serotype 4. In contrast, very few
B. garinii
OspA serotype 4 organisms were isolated directly from the ticks which infected the mice; most isolates were
B. afzelii
. The infected mice transmitted mainly OspA serotype 4 to xenodiagnostic ticks, preferentially in combination with
B. afzelii
.
Phaeohyphomycosis Caused by
Alternaria infectoria
in a Renal Transplant Recipient
Authors: Halaby Teysir , Boots Hans , Vermeulen Anton , van der Ven André , Beguin Hugues , van Hooff Hans , Jacobs Jan
Published: July 2002

We report on a case of phaeohyphomycosis caused by
Alternaria infectoria
in a renal transplant recipient with pulmonary infiltrates and multiple skin lesions. Diagnosis was based on microscopy and culture of the skin lesions. Treatment consisted of a combination of surgical excision and systemic antifungal therapy, first with itraconazole and subsequently with liposomal amphotericin B, for 39 days. At a 20-month follow-up visit, no recurrence of the skin lesions or the pulmonary infiltrates had occurred.
Phylogeny of
Pneumocystis carinii
from 18 Primate Species Confirms Host Specificity and Suggests Coevolution
Authors: Demanche Christine , Berthelemy Madeleine , Petit Thierry , Polack Bruno , Wakefield Ann E. , Dei-Cas Eduardo , Guillot Jacques
Published: July 2002

Primates are regularly infected by fungal organisms identified as
Pneumocystis carinii
. They constitute a valuable population for the confirmation of
P. carinii
host specificity. In this study, the presence of
P. carinii
was assessed by direct examination and nested PCR at mitochondrial large subunit (mtLSU) rRNA and dihydropteroate synthetase (DHPS) genes in 98 lung tissue samples from captive or wild nonhuman primates. Fifty-nine air samples corresponding to the environment of different primate species in zoological parks were also examined. Cystic forms of
P. carinii
were detected in smears from 7 lung tissue samples corresponding to 5 New World primate species. Amplifications at the mtLSU rRNA gene were positive for 29 lung tissue samples representing 18 different primate species or subspecies and 2 air samples corresponding to the environment of two simian colonies. Amplifications at the DHPS gene were positive for 8 lung tissue samples representing 6 different primate species. Direct sequencing of nested PCR products demonstrated that a specific mtLSU rRNA and DHPS sequence could be attributed to each primate species or subspecies. No nonhuman primate harbored the human type of
P. carinii
(
P. carinii
f. sp.
hominis
). Genetic divergence in primate-derived
P. carinii
organisms varied in terms of the phylogenetic divergence existing among the corresponding host species, suggesting coevolution.
Sampling Variability in the Microbiological Evaluation of Expectorated Sputa and Endotracheal Aspirates
Authors: Nagendra Sanjai , Bourbeau Paul , Brecher Stephen , Dunne Michael , LaRocco Mark , Doern Gary
Published: July 2002

A five-center study was conducted with the aim of determining how reproducibly expectorated sputa and tracheal aspirates could be sampled when preparing Gram-stained smears and inoculating cultures. With both specimen types, excessive variation was noted among Gram stain results obtained from replicate smears. Less variation was noted among culture results, especially with tracheal aspirates.
Reliable Detection of Respiratory Syncytial Virus Infection in Children for Adequate Hospital Infection Control Management
Authors: Abels Susanne , Nadal David , Stroehle Angelika , Bossart Walter
Published: July 2002

By using a rapid test for respiratory syncytial virus (RSV) detection (Abbott TestPack RSV), a number of patients were observed, showing repeatedly positive results over a period of up to 10 weeks. A prospective study was initiated to compare the rapid test with an antigen capture enzyme immunoassay (EIA) and a nested reverse transcriptase PCR (RT-PCR) protocol for detection of RSV serotypes A and B. Only respiratory samples from children exhibiting the prolonged presence of RSV (≥5 days) as determined by the rapid test were considered. A total of 134 specimens from 24 children was investigated by antigen capture EIA and nested RT-PCR. Using RT-PCR as the reference method, we determined the RSV rapid test to have a specificity of 63% and a sensitivity of 66% and the antigen capture EIA to have a specificity of 96% and a sensitivity of 69% for acute-phase samples and the homologous virus serotype A. In 7 (29%) of 24 patients, the positive results of the RSV rapid test could not be confirmed by either nested RT-PCR or antigen capture EIA. In these seven patients a variety of other respiratory viruses were detected. For general screening the RSV rapid test was found to be a reasonable tool to get quick results. However, its lack of specificity in some patients requires confirmation by additional tests to rule out false-positive results and/or detection of other respiratory viruses.
Antibody Responses to Recombinant Epstein-Barr Virus Antigens in Nasopharyngeal Carcinoma Patients: Complementary Test of ZEBRA Protein and Early Antigens p54 and p138
Authors: Dardari R. , Hinderer W. , Lang D. , Benider A. , El Gueddari B. , Joab I. , Benslimane A. , Khyatti M.
Published: July 2002

Serological tests based on the antibodies directed against the Epstein-Barr virus early antigen (EA) and viral capsid antigen (VCA), which have been recognized as tumor markers for nasopharyngeal carcinoma (NPC), are routinely used to help in the diagnosis of this malignancy. The detection of these antibodies reveals very low titers, found only in a small proportion of young compared with older NPC patients. This is a problem for the diagnosis of NPC, especially among Maghrebians, among whom young people are also affected, and emphasizes the necessity to search for more reliable markers. The present study reports results of immunoglobulin G (IgG) and IgA responses of NPC patients to recombinant EA antigens p54 (BMRF1) and p138 (BALF2), VCA complex antigens p18 (BFRF3) and p23 (BLRF2), and EBNA antigen p72 (BKRF1). Our results show that IgA-EA-p54 and -p138 (IgA-EA-p54+138) antibodies have a diagnostic value for detection of NPC (70%), compared with IgA-VCA-p18+23 and IgA-EBNA-p72, which have limited diagnostic value, especially in young patients. It is also noteworthy that IgA-EA-p54+138 can detect a high percentage (64%) of NPC cases negative by immunofluorescence. These results, however, clearly show that a single test cannot achieve the objective of detecting all NPC patients, and it seems advisable to combine different tests for the diagnosis of NPC. The combination of IgG-ZEBRA with IgA-EA-p54+138 improved the sensitivity of detection of NPC to 95% in the overall NPC population. The use of IgA-EA-p54+138 in combination with IgG-ZEBRA will facilitate detailed studies on the pattern of antibody response, which may result in the development of useful serological markers to guide the treatment of NPC.
Characterization of Chloramphenicol Resistance in Beta-Hemolytic
Escherichia coli
Associated with Diarrhea in Neonatal Swine
Authors: Bischoff Kenneth M. , White David G. , McDermott Patrick F. , Zhao Shaohua , Gaines Stuart , Maurer John J. , Nisbet David J.
Published: July 2002

Ninety beta-hemolytic
Escherichia coli
isolates associated with diarrhea in neonatal pigs from multiple farms in Oklahoma were investigated for known associated disease serotypes, virulence factors, ribotypes, and antimicrobial susceptibility phenotypes. Fifteen different serotypes were observed, with 58% of isolates belonging to groups that produce one of three major enterotoxins: O149, O147, and O139. Thirty percent of the swine
E. coli
isolates possessed a combination of F4 fimbriae and the heat-labile toxin and heat-stable toxin B enterotoxins. Seventy-three percent of the
E. coli
isolates were resistant to five or more antibiotics. Interestingly, 53% of swine
E. coli
isolates exhibited resistance to chloramphenicol (CHL), an antibiotic whose use in food animals has been prohibited in the United States since the mid-1980s. The
cmlA
gene, which encodes a putative CHL efflux pump, was detected by PCR in 47 of the 48 CHL-resistant isolates, and 4 of these also possessed the
cat2
gene, which encodes a chloramphenicol acetyltransferase. The one CHL-resistant isolate that did not contain either
cmlA
or
cat-2
possessed the
flo
gene, which confers resistance to both florfenicol and CHL. To determine whether CHL-resistant swine
E. coli
isolates represented dissemination of a clonal strain, all 90 isolates were analyzed by ribotyping. Seventeen distinct
E. coli
ribogroups were identified, with CHL resistance observed among the isolates in all except one of the major ribogroups. The identification of the
cmlA
gene among diverse hemolytic enterotoxigenic
E
.
coli
strains demonstrates its broad dissemination in the swine production environment and its persistence even in the absence of CHL selection pressure.
Novel Antigens of
Helicobacter pylori
Correspond to Ulcer-Related Antibody Pattern of Sera from Infected Patients
Authors: Atanassov Christo , Pezennec Leon , d'Alayer Jacques , Grollier Ghislaine , Picard Bertrand , Fauchère Jean-Louis
Published: July 2002

Recently, we reported that the patterns of antibodies to
Helicobacter pylori
protein antigens in serum may be useful for screening patients at high risk for ulcers (P. Aucher et al., J. Clin. Microbiol. 36:931-936, 1998). Here we report the identification, by a combination of electrophoretic, immunochemical, and protein sequencing methods, of five antigens that correspond to this antibody pattern: groEL, catalase A, flagellin A, beta-ketoacyl-acyl carrier protein synthase I (β-ketoacyl-ACP S), and peptidyl prolyl
cis-trans
isomerase (PPiase). β-Ketoacyl-ACP S and PPiase are reported for the first time as antigens of diagnostic interest in infections by
H. pylori
. The antigenicity of the five antigens, together with those of CagA and VacA, was tested in an immunoblot assay with water-soluble protein extracts from two
H. pylori
pathogenic strains (HP 141 and ATCC 43579) and panels of sera from
H. pylori
-positive patients with gastroduodenal ulcers (GDU), nonulcer dyspepsia (NUD), as well as sera from
H. pylori
-negative healthy volunteers. For catalase A, groEL, and flagellin A antigens, no overall statistically important values were found making it possible to discriminate between patients with GDU and NUD. For both
H. pylori
strains, the mean performance indices (MPI) presenting percentages of correctly classified patients with GDU and NUD showed that the most significant antibody patterns were as follows: anti-VacA + anti-β-ketoacyl-ACP S (MPI = 76.1), anti-VacA + anti-PPiase (MPI = 71.8), and anti-CagA + anti-VacA + anti-β-ketoacyl-ACP S (MPI = 70.5). Antibody patterns detected with these antigen profiles may therefore be useful in developing a diagnostic test designed to predict the clinical severity of the
H. pylori
infection within the adult population of France.
Diagnostic Detection of Human Immunodeficiency Virus Type 1 Antibodies in Urine: a Brazilian Study
Authors: Oelemann Walter M. R. , Lowndes Catherine M. , Veríssimo da Costa Giovani C. , Morgado Mariza G. , Castello-Branco Luiz Roberto R. , Grinsztejn Beatriz , Alary Michel , Bastos Francisco I.
Published: July 2002

We evaluated, for the first time in Latin America, the performance of a commercial enzyme immunoassay (EIA) (Calypte Biomedical Corporation, Berkeley, Calif.) that detects human immunodeficiency virus type 1 (HIV-1)-specific antibodies in urine in comparison to standard serological assays (two commercial EIAs and a commercial Western blot [WB] assay). Paired serum and urine specimens were collected from two different groups of Brazilian patients: 225 drug users with unknown HIV status who attended drug treatment centers in Rio de Janeiro, Brazil, and 135 subjects with known HIV status. Patients showing positive results in the serum EIAs and/or in the urine EIA were serologically confirmed by WB assay. For 135 individuals with known HIV status, the urine EIA showed 100% sensitivity (74 positive samples) and 95.1% specificity (58 of 61 negative specimens). For 225 drug users, the test showed 100% sensitivity (2 positive samples) and 98.7% specificity (220 of 223 negative samples) compared to WB-confirmed serological EIA results. Thus, in a total of 360 samples, the urine EIA correctly identified all 76 HIV-positive samples and 278 of 284 negative samples (100% sensitivity and 97.9% specificity). Detailed analysis of the urine EIA results indicates that an increase of the recommended cutoff value might raise the specificity of the assay without affecting its sensitivity. Our results suggest that the HIV-1 urine EIA is a good screening test suitable for developing countries like Brazil. However, as for all other HIV screening tests on the market, it is not specific enough to be used as a one-step test and therefore requires confirmation.
Diversity of Rotavirus Strains among Children with Acute Diarrhea in China: 1998-2000 Surveillance Study
Authors: Fang Zhao-Yin , Yang Hui , Qi Jin , Zhang Jing , Sun Li-Wei , Tang Jing-Yu , Ma Li , Du Zeng-Qing , He Ai-hua , Xie Jian-Ping , Lu Yi-Yu , Ji Zhen-Zhou , Zhu Bao-Quan , Wu Hai-Yan , Lin Si-En , Xie Hua-Ping , Griffin Dixie D. , Ivanoff Bernard , Glass Roger I. , Gentsch Jon R.
Published: July 2002

As part of a national rotavirus surveillance activity, we collected fecal specimens from 3,177 children with acute diarrhea in 10 regions of China between April 1998 and April 2000 and screened them for rotavirus. Rotavirus was detected in 41% (
n
= 1,305) of specimens, and in these, G1 was the predominant serotype (72.6%), followed by G3 (14.2%), G2 (12.1%), G4 (2.5%), G9 (0.9%), and G untypeable (0.7%). Among 327 G-typed strains tested for P genotype, 14 different P-G combinations were identified, with the globally common strains P[8]G1, P[4]G2, P[8]G3, and P[8]G4 representing 75.6% of all typed rotavirus strains. Among the uncommon strains, 11 were P[6]G9, and others included P[6]G1, P[6]G3, and five novel P-G combinations (P[9]G1, P[4]G1, P[4]G3, P[4]G4, and P[8]G2). Our results indicate that while the common rotavirus strains remain predominant, the diversity of strains is much greater than was previously recognized.
Captive Rhesus Monkeys (
Macaca mulatta
) Are Commonly Infected with
Helicobacter cinaedi
Authors: Fernandez Kathy R. , Hansen Lori M. , Vandamme Peter , Beaman Blaine L. , Solnick Jay V.
Published: July 2002

Helicobacter cinaedi
may cause proctocolitis or bacteremia in homosexual men infected with human immunodeficiency virus or occasionally in other immunocompromised hosts. There are scattered reports of
H. cinaedi
isolated from a variety of animal hosts, but to date only hamsters have been found to be a common natural reservoir. Microaerophillic cultures of feces from 5 of 16 asymptomatic rhesus monkeys (
Macaca mulatta
) (31%) were positive for a curved gram-negative rod. A polyphasic taxonomic approach was used to identify the organism as
H. cinaedi
. These results show that
H. cinaedi
frequently colonizes asymptomatic captive rhesus monkeys, which may serve as another potential reservoir for human infection.
Multilocus Sequence Typing Scheme for
Enterococcus faecium
Authors: Homan Wieger L. , Tribe David , Poznanski Simone , Li Mei , Hogg Geoff , Spalburg Emile , van Embden Jan D. A. , Willems Rob J. L.
Published: July 2002

A multilocus sequence typing (MLST) scheme has been developed for
Enterococcus faecium
. Internal fragments from seven housekeeping genes of 123 epidemiologically unlinked isolates from humans and livestock and 16 human-derived isolates from several outbreaks in the United States, the United Kingdom, Australia, and The Netherlands were analyzed. A total of 62 sequence types were detected in vancomycin-sensitive
E. faecium
(VSEF) and vancomycin-resistant
E. faecium
(VREF) isolates. VSEF isolates were genetically more diverse than VREF isolates. Both VSEF and VREF isolates clustered in host-specific lineages that were similar to the host-specific clustering obtained by amplified fragment length polymorphism analysis. Outbreak isolates from hospitalized humans clustered in a subgroup that was defined by the presence of a unique allele from the housekeeping gene
purK
and the surface protein gene
esp
. The MLST results suggest that epidemic lineages of
E. faecium
emerged recently worldwide, while genetic variation in both VREF and VSEF was created by longer-term recombination. The results show that MLST of
E. faecium
provides an excellent tool for isolate characterization and long-term epidemiologic analysis.
Interleukin-10 Increases Th1 Cytokine Production and Cytotoxic Potential in Human Papillomavirus-Specific CD8+Cytotoxic T Lymphocytes
Authors: Santin Alessandro D. , Hermonat Paul L. , Ravaggi Antonella , Bellone Stefania , Pecorelli Sergio , Roman Juan J. , Parham Groesbeck P. , Cannon Martin J.
Published: July 2002

Interleukin-10 (IL-10) is widely known as an immunosuppressive cytokine by virtue of its ability to inhibit macrophage-dependent antigen presentation, T-cell proliferation, and Th1 cytokine secretion. However, several studies have challenged the perception of IL-10 solely as an immunosuppressive cytokine. As part of an investigation on potentiation of the cytotoxic activity of human papillomavirus E7-specific CD8+cytotoxic T lymphocytes (CTL) for adoptive transfusions to cervical cancer patients, we found that IL-10 in combination with IL-2, unlike several other combinations, including IL-2 with IL-12, gamma interferon (IFN-γ), tumor necrosis factor alpha, and transforming growth factor β, was able to consistently increase cytotoxicity. This augmentation in cytotoxic activity correlated with a significant increase in the cytoplasmic accumulation of perforin as detected by fluorescence-activated cell sorter. Surface expression of both the α and β chains of the CD8 heterodimeric coreceptor and CD56 molecules was increased by exposure of CTL to IL-10. More importantly, we found that administration of IL-10 in combination with IL-2 after antigen stimulation consistently increased the intracellular expression of Th1 cytokines (i.e., IFN-γ and IL-2) compared to results for control CD8+T cells cultured in IL-2 alone. In kinetic studies, proliferation, intracellular perforin levels, cytotoxic activity, and IFN-γ expression were consistently elevated in CTL cultures containing IL-10 compared to control cultures, both at early and late time points following stimulation. In contrast, intracellular IL-2 expression was consistently increased only at early time points following stimulation with autologous tumor cells or solid-phase anti-CD3 antibody. Taken together, these data support the use of IL-10 in combination with IL-2 for the in vitro expansion and potentiation of tumor-specific CTL for clinical use in the therapy of cancer.
The Double-Stranded RNA-Binding Protein Staufen Is Incorporated in Human Immunodeficiency Virus Type 1: Evidence for a Role in Genomic RNA Encapsidation
Authors: Mouland Andrew J. , Mercier Johanne , Luo Ming , Bernier Luc , DesGroseillers Luc , Cohen Éric A.
Published: July 2002

Human Staufen (hStau), a double-stranded RNA (dsRNA)-binding protein that is involved in mRNA transport, is incorporated in human immunodeficiency virus type 1 (HIV-1) and in other retroviruses, including HIV-2 and Moloney murine leukemia virus. Sucrose and Optiprep gradient analyses reveal cosedimentation of hStau with purified HIV-1, while subtilisin assays demonstrate that it is internalized. hStau incorporation in HIV-1 is selective, is dependent on an intact functional dsRNA-binding domain, and quantitatively correlates with levels of encapsidated HIV-1 genomic RNA. By coimmunoprecipitation and reverse transcription-PCR analyses, we demonstrate that hStau is associated with HIV-1 genomic RNA in HIV-1-expressing cells and purified virus. Overexpression of hStau enhances virion incorporation levels, and a corresponding, threefold increase in HIV-1 genomic RNA encapsidation levels. This coordinated increase in hStau and genomic RNA packaging had a significant negative effect on viral infectivity. This study is the first to describe hStau within HIV-1 particles and provides evidence that hStau binds HIV-1 genomic RNA, indicating that it may be implicated in retroviral genome selection and packaging into assembling virions.
Dose-Dependent Changes in Influenza Virus-Infected Dendritic Cells Result in Increased Allogeneic T-Cell Proliferation at Low, but Not High, Doses of Virus
Authors: Oh SangKon , McCaffery J. Michael , Eichelberger Maryna C.
Published: July 2002

During the acute phase of infection with influenza A virus, the degree of lymphopenia correlates with severity of disease. Factors that contribute to T-cell activation during influenza virus infection may contribute to this observation. Since the immune response is initiated when dendritic cells (DC) interact with T cells, we have established an in vitro system to examine the effects of influenza virus infection on DC function. Our results show that allogeneic T-cell proliferation was dependent on the dose of A/PR/8/34 used to infect DC, with enhanced responses at low, but not high, multiplicities of infection. The lack of enhancement at high virus doses was not primarily due to the increased rate of DC apoptosis, but required viral replication and neuraminidase (NA) activity. Clusters that formed between DC or between DC and T cells were also dependent on the viral dose. This change in cellular interaction may oppose T-cell proliferation in response to DC infected with high doses of PR8, since the increased contact between DC resulted in the exclusion of T cells. The enhanced alloreactive T-cell response was restored by neutralization of transforming growth factor β1 (TGF-β1). It is likely that NA present on viral particles released from DC infected with high doses of PR8 activates TGF-β1. Future studies will determine the mechanism by which TGF-β1 modifies the in vitro T-cell response and address the contribution of this cytokine to the lymphopenia observed in severe disease.
Development and Optimization of Herpes Simplex Virus Vectors for Multiple Long-Term Gene Delivery to the Peripheral Nervous System
Authors: Palmer J. A. , Branston R. H. , Lilley C. E. , Robinson M. J. , Groutsi F. , Smith J. , Latchman D. S. , Coffin R. S.
Published: July 2002

Herpes simplex virus (HSV) has often been suggested as a suitable vector for gene delivery to the peripheral nervous system as it naturally infects sensory nerve terminals before retrograde transport to the cell body in the spinal ganglia where latency is established. HSV vectors might therefore be particularly appropriate for the study and treatment of chronic pain following vector administration by relatively noninvasive peripheral routes. However parameters allowing safe and efficient gene delivery to spinal ganglia following peripheral vector inoculation, or the long-term expression of delivered genes, have not been comprehensively studied. We have identified combinations of deletions from the HSV genome which allow highly efficient gene delivery to spinal dorsal root ganglia (DRGs) following either footpad or sciatic nerve injection. These vectors have ICP34.5 deleted and have inactivating mutations in vmw65. We also report that peripheral replication is probably necessary for the efficient establishment of latency in vivo, as fully replication-incompetent HSV vectors allow efficient gene expression in DRGs only after peripheral inoculation at a high virus dose. Very low transduction efficiencies are otherwise achieved. In parallel, promoters have been developed that allow the long-term expression of individual or pairs of genes in DRGs by using elements from the latently active region of the virus to confer a long-term activity onto a number of promoters which otherwise function only in the short term. This work further defines elements and mechanisms within the latently active region that are necessary for long-term gene expression and for the first time allows multiple inserted genes to be expressed from HSV vectors during latency.