bluuebunny/crossref_metadata_2025 huggingface dataset:
Human Gingival CD14+Fibroblasts Primed with Gamma Interferon Increase Production of Interleukin-8 in Response to Lipopolysaccharide through Up-Regulation of Membrane CD14 and MyD88 mRNA Expression
Authors: Tamai Riyoko , Sakuta Tetsuya , Matsushita Kenji , Torii Mitsuo , Takeuchi Osamu , Akira Shizuo , Akashi Sachiko , Espevik Terje , Sugawara Shunji , Takada Haruhiko
Published: July 2002

Gamma interferon (IFN-γ)-primed human gingival fibroblasts (HGF) have been shown to produce higher levels of interleukin-8 (IL-8) upon stimulation with bacterial products and inflammatory cytokines than nonprimed controls. In this study, we examined whether priming of HGF with IFN-γ up-regulates IL-8 production by the cells in response to purified lipopolysaccharide (LPS). The priming effect of IFN-γ was clearly observed in the high-CD14-expressing (CD14high) HGF but not in the low-CD14-expressing (CD14low) HGF. The CD14highHGF were most effectively primed with IFN-γ (1,000 IU/ml) for 72 h. To elucidate the mechanism of the priming effects of IFN-γ for the LPS response by HGF, we examined whether IFN-γ regulated expression of CD14, Toll-like receptor 2 (TLR2), TLR4, MD-2, and MyD88, all of which are molecules suggested to be associated with LPS signaling. In CD14highHGF, IFN-γ markedly up-regulated CD14 and MyD88 but not TLR4 protein and MD-2 mRNA expression, while in CD14lowHGF, IFN-γ slightly increased MyD88 and scarcely affected CD14, TLR4 protein, and MD-2 mRNA levels. LPS-induced IL-8 production by IFN-γ-primed CD14highHGF was significantly inhibited by monoclonal antibodies (MAbs) against CD14 and TLR4, but not by an anti-TLR2 MAb. These findings suggested that IFN-γ primed CD14highHGF to enhance production of IL-8 in response to LPS through augmentation of the CD14-TLR system, where the presence of membrane CD14 was indispensable for the response of HGF to LPS.
Analysis of Genes Encoding an Alternative Nitrogenase in the Archaeon
Methanosarcina barkeri
227
Authors: Chien Yueh-Tyng , Auerbuch Victoria , Brabban Andrew D. , Zinder Stephen H.
Published: July 2002

Methanosarcina barkeri
227 possesses two clusters of genes potentially encoding nitrogenases. We have previously demonstrated that one cluster, called
nif2
, is expressed under molybdenum (Mo)-sufficient conditions, and the deduced amino acid sequences for nitrogenase structural genes in that cluster most closely resemble those for the Mo nitrogenase of the gram-positive eubacterium
Clostridium pasteurianum
. The previously cloned
nifH1
from
M. barkeri
shows phylogenetic relationships with genes encoding components of eubacterial Mo-independent eubacterial alternative nitrogenases and other methanogen nitrogenases. In this study, we cloned and sequenced
nifD1
and part of
nifK1
from
M. barkeri
227. The deduced amino acid sequence encoded by
nifD1
from
M. barkeri
showed great similarity with
vnfD
gene products from vanadium (V) nitrogenases, with an 80% identity at the amino acid level with the
vnfD
gene product from
Anabaena variabilis
. Moreover, there was a small open reading frame located between
nifD1
and
nifK1
with clear homology to
vnfG
, a hallmark of eubacterial alternative nitrogenases. Stimulation of diazotrophic growth of
M. barkeri
227 by V in the absence of Mo was demonstrated. The unusual complement of
nif
genes in
M. barkeri
227, with one cluster resembling that from a gram-positive eubacterium and the other resembling a eubacterial V nitrogenase gene cluster, suggests horizontal genetic transfer of those genes.
Characterization of LrpC DNA-Binding Properties and Regulation of
Bacillus subtilis lrpC
Gene Expression
Authors: Beloin Christophe , Exley Rachel , Mahé Anne-Laure , Zouine Mohamed , Cubasch Stephanie , Le Hégarat Françoise
Published: July 2002

The
lrpC
gene was identified during the
Bacillus subtilis
genome sequencing project. Previous experiments suggested that LrpC has a role in sporulation and in the regulation of amino acid metabolism and that it shares features with
Escherichia coli
Lrp, a transcription regulator (C. Beloin, S. Ayora, R. Exley, L. Hirschbein, N. Ogasawara, Y. Kasahara, J. C. Alonso, and F. Le Hégarat, Mol. Gen. Genet. 256:63–71, 1997). To characterize the interactions of LrpC with DNA, the protein was overproduced and purified. We show that LrpC binds to multiple sites in the upstream region of its own gene with a stronger affinity for a region encompassing
P1
, one of the putative promoters identified (
P1
and
P2
). By analyzing
lrpC-lacZ
transcriptional fusions, we demonstrated that
P1
is the major in vivo promoter and that, unlike many members of the
lrp/asnC
family,
lrpC
is not negatively autoregulated but rather slightly positively autoregulated. Production of LrpC in vivo is low in both rich and minimal media (50 to 300 LrpC molecules per cell). In rich medium, the cellular LrpC content is six- to sevenfold lower during the exponentional phase than during the stationary growth phase. Possible determinants and the biological significance of the regulation of
lrpC
expression are discussed.
Properties of Various
RHO1
Mutant Alleles of
Cryptococcus neoformans
Authors: Chang Yun C. , Penoyer Lisa A.
Published: July 2002

The
RHO1
homologue of
Cryptococcus neoformans
complemented
Saccharomyces cerevisiae rho1
mutations. The results of overexpression and site-specific mutagenesis of
CnRHO1
in
C. neoformans
and
S. cerevisiae
indicated that although
CnRHO1
could functionally substitute for the
RHO1
gene of
S. cerevisiae
, mutants of
cnrho1
manifested unique features in certain aspects.
A Second Allele of
eppA
in
Borrelia burgdorferi
Strain B31 Is Located on the Previously Undetected Circular Plasmid cp9-2
Authors: Miller Jennifer C. , Bono James L. , Babb Kelly , El-Hage Nazira , Casjens Sherwood , Stevenson Brian
Published: July 2002

Although sequence analysis of
Borrelia burgdorferi
isolate B31 was recently declared “complete,” we found that cultures of this strain can contain a novel 9-kb circular plasmid, cp9-2. The newly described plasmid contains both sequence similarities with and differences from the previously identified B31 plasmid cp9-1 (formerly cp9). cp9-1 and cp9-2 each encode a unique allele of EppA, a putative membrane protein synthesized by
B. burgdorferi
during mammalian infection.
Identification by Gene Deletion Analysis of a Regulator, VmsR, That Controls Virginiamycin Biosynthesis in
Streptomyces virginiae
Authors: Kawachi Ryu , Wangchaisoonthorn Usamas , Nihira Takuya , Yamada Yasuhiro
Published: July 2002

Virginiae butanolide (VB)-BarA of
Streptomyces virginiae
is one of the newly discovered pairs of a butyrolactone autoregulator and a corresponding receptor protein of
Streptomyces
species and regulates the production of the antibiotic virginiamycin (VM) in
S. virginiae
. The gene
vmsR
was found to be situated 4.7 kbp upstream of the
barA
gene, which encodes the VB-specific receptor. The
vmsR
product was predicted to be a regulator of VM biosynthesis based on its high homology to some
Streptomyces
pathway-specific transcriptional regulators for the biosynthetic gene clusters of polyketide antibiotics, such as
Streptomyces peucetius
DnrI (47.5% identity, 84.3% similarity), which controls daunorubicin biosynthesis. A
vmsR
deletion mutant was created by homologous recombination. Neither virginiamycin M
1
nor virginiamycin S was produced in the
vmsR
mutant, while amounts of VB and BarA similar to those produced in the wild-type strain were detected. Reverse transcription-PCR analyses confirmed that the
vmsR
deletion had no deleterious effects on the transcription of the
vmsR
-surrounding genes, indicating that VmsR is a positive regulator of VM biosynthesis in
S. virginiae
.
The
Sinorhizobium meliloti
Lon Protease Is Involved in Regulating Exopolysaccharide Synthesis and Is Required for Nodulation of Alfalfa
Authors: Summers Michael L. , Botero Lina M. , Busse Scott C. , McDermott Timothy R.
Published: July 2002

While screening for
Sinorhizobium meliloti
Pho regulatory mutants, a transposon mutant was isolated that constitutively expressed higher levels of acid and alkaline phosphatase enzymes. This mutant was also found to form pseudonodules on alfalfa that were delayed in appearance relative to those formed by the wild-type strain, it contained few bacteroids, and it did not fix nitrogen. Sequence analysis of the transposon insertion site revealed the affected gene to have high homology to Lon proteases from a number of organisms. In minimal succinate medium, the mutant strain was found to grow more slowly, reach lower maximal optical density, and produce more extracellular polysaccharide (EPS) than the wild-type strain. The mutant fluoresced brightly on minimal succinate agar containing calcofluor (which binds to EPSI, a constitutively expressed succinoglycan), and gas chromotographic analysis of purified total EPS showed that the glucose-to-galactose ratio in the
lon
mutant total EPS was 5.0 ± 0.2 (mean ± standard error), whereas the glucose-to-galactose ratio in the wild-type strain was 7.1 ± 0.5. These data suggested that in addition to EPSI, the
lon
mutant also constitutively synthesized EPSII, a galactoglucan which is the second major EPS known to be produced by
S. meliloti
, but typically is expressed only under conditions of phosphate limitation.
13
C nuclear magnetic resonance analysis showed no major differences between EPS purified from the mutant and wild-type strains. Normal growth, EPS production, and the symbiotic phenotype were restored in the mutant strain when the wild-type
lon
gene was present in
trans
. The results of this study suggest that the
S. meliloti
Lon protease is important for controlling turnover of a constitutively expressed protein(s) that, when unregulated, disrupts normal nodule formation and normal growth.
Very-Short-Patch Repair in
Escherichia coli
Requires the
dam
Adenine Methylase
Authors: Bell Derek C. , Cupples Claire G.
Published: July 2002

Strains of
Escherichia coli
which lack the
dam
-encoded adenine methylase are mutators due to a reduction in the efficiency of postreplication mismatch repair. In this study, we show that Dam
−
strains are also defective in very-short-patch repair, the system which corrects T/G mismatches arising from the deamination of 5-methylcytosine. This defect is associated with decreased levels of Vsr, the endonuclease which initiates short-patch repair. We also show that production of the
dcm
-encoded cytosine methylase is unaffected in Dam
−
strains. Since the
dcm
and
vsr
genes are cotranscribed, the regulation of Vsr by Dam is probably posttranscriptional.
Coupling of Asymmetric Division to Polar Placement of Replication Origin Regions in
Bacillus subtilis
Authors: Graumann Peter L. , Losick Richard
Published: July 2002

Entry into sporulation in
Bacillus subtilis
is characterized by the formation of a polar septum, which asymmetrically divides the developing cell into forespore (the smaller cell) and mother cell compartments, and by migration of replication origin regions to extreme opposite poles of the cell. Here we show that polar septation is closely correlated with movement of replication origins to the extreme poles of the cell. Replication origin regions were visualized by the use of a cassette of tandem copies of
lacO
that had been inserted in the chromosome near the origin of replication and decorated with green fluorescent protein-LacI. The results showed that extreme polar placement of replication origin regions is not under sporulation control and occurred in stationary phase under conditions under which entry into sporulation was prevented. On the other hand, the formation of a polar septum, which is under sporulation control, was almost invariably associated with the presence of a replication origin region in the forespore. Moreover, cells in which the polar placement of origin regions was perturbed by deletion of the gene (
smc
) for the structural maintenance of chromosomes (SMC) protein were impaired in polar division. A small proportion (≈1%) of the mutant cells were able to undergo asymmetric division, but the forespore compartment of these exceptional cells was generally observed to contain a replication origin region. Immunofluorescence microscopy experiments indicated that the block in polar division caused by the absence of SMC occurred at or prior to the step of bipolar Z-ring formation by the cell division protein FtsZ. A model is discussed in which polar division is under the dual control of sporulation and an event associated with the placement of a replication origin at the cell pole.
DNA-Binding Activity of Amino-Terminal Domains of the
Bacillus subtilis
AbrB Protein
Authors: Xu Ke , Strauch Mark A.
Published: July 2002

Two truncated variants of AbrB, comprising either its first 53 (AbrBN53) or first 55 (AbrBN55) amino acid residues, were constructed and purified. Noncovalently linked homodimers of the truncated variants exhibited very weak DNA-binding activity. Cross-linking AbrBN55 dimers into tetramers and higher-order multimers (via disulfide bonding between penultimate cysteine residues) resulted in proteins having DNA-binding affinity comparable to and DNA-binding specificity identical to those of intact, wild-type AbrB. These results indicate that the DNA recognition and specificity determinants of AbrB binding lie solely within its N-terminal amino acid sequence.
Positive Selection of Mutations Leading to Loss or Reduction of Transcriptional Activity of PrfA, the Central Regulator ofListeria monocytogenesVirulence
Authors: Herler M. , Bubert A. , Goetz M. , Vega Y. , Vazquez-Boland J. A. , Goebel W.
Published: July 2002

Transcription factor PrfA controls the expression of virulence genes essential forListeria monocytogenespathogenesis. To gain insight into the structure-function relationship of PrfA, we devised a positive-selection system to isolate mutations reducing or abolishing transcriptional activity. The system is based on the observation that the listerialiapgene, encoding the p60 protein, is lethal if overexpressed inBacillus subtilis. A plasmid in which theiapgene is placed under the control of the PrfA-dependenthlypromoter was constructed and introduced intoB. subtilis. This strain was rapidly killed when expression ofiapwas induced by introduction of a second plasmid carryingprfA. Two classes ofB. subtilissurvivor mutants were identified: one carried mutations iniap, and the second carried mutations inprfA. Sequence analysis of the defectiveprfAgenes identified mutations in three regions of the PrfA protein: region A, between amino acids 58 and 67 in the β-roll domain of PrfA; region B, between amino acids 169 and 193, which corresponds to the DNA-binding helix-turn-helix motif; and region C, comprising the 38 C-terminal amino acids of PrfA, which form a leucine zipper-like structure. PrfA proteins with mutations in regions B and C were unable to bind to the PrfA-binding site in the target DNA, while mutations in region A resulted in a protein still binding the target DNA but unable to form a stable complex with RNA polymerase and initiate transcription in vitro.
Molecular Characterization of a Secreted Enzyme with Phospholipase B Activity from
Moraxella bovis
Authors: Farn Jacinta L. , Strugnell Richard A. , Hoyne Peter A. , Michalski Wojtek P. , Tennent Jan M.
Published: July 2002

A candidate for a vaccine against infectious bovine keratoconjunctivitis (IBK) has been cloned and characterized from
Moraxella bovis.
The
plb
gene encodes a protein of 616 amino acids (molecular mass of ∼65.8 kDa) that expresses phospholipase B activity. Amino acid sequence analysis revealed that PLB is a new member of the GDSL (Gly-Asp-Ser-Leu) family of lipolytic enzymes.
Construction and Consequences of Directed Mutations Affecting the Hemin Receptor in Pathogenic
Corynebacterium
Species
Authors: Schmitt Michael P. , Drazek E. Susan
Published: July 2002

Genes encoding an ATP-binding cassette transporter system involved in hemin iron utilization from
Corynebacterium ulcerans
were cloned and characterized. The genes are homologous to a hemin transport system previously identified in
Corynebacterium diphtheriae
. Disruption of the
hmuT
gene, which encodes the putative hemin receptor, resulted in greatly reduced ability of
C. ulcerans
to use hemin or hemoglobin as an iron source. Inactivation of
hmuT
in
C. diphtheriae
by site-specific recombination had no effect on hemin utilization, which suggests that
C. diphtheriae
has an additional system for transporting hemin.
Inhibition of Quorum Sensing by a
Pseudomonas aeruginosa dksA
Homologue
Authors: Branny Pavel , Pearson James P. , Pesci Everett C. , Köhler Thilo , Iglewski Barbara H. , Van Delden Christian
Published: July 2002

The
Pseudomonas aeruginosa las (lasR-lasI
) and
rhl (rhlR-rhlI
) quorum-sensing systems regulate the expression of several virulence factors, including elastase and rhamnolipid.
P. aeruginosa
strain PR1-E4 is a
lasR
deletion mutant that contains a second, undefined mutation which allows production of elastase and rhamnolipid despite a nonfunctional
las
system. We have previously shown that this strain accomplishes this by increasing the expression of the autoinducer synthase gene
rhlI
. In this report, we show that the elastolytic phenotype of mutant PR1-E4 can be complemented with a
P. aeruginosa
homologue of the
Escherichia coli dnaK
mutation suppressor gene
dksA
. When supplied in
trans
on a multicopy plasmid, this gene completely suppressed elastase production by mutant PR1-E4. Cloning and Northern blot analysis revealed that
dksA
was neither mutated nor less transcribed in mutant PR1-E4. When overexpressed,
dksA
also reduced rhamnolipid production by both mutant PR1-E4 and the wild type, PAO1. Using Northern blot analysis and
lacZ
reporter fusions, we show that
dksA
inhibits
rhlI, rhlAB
, and
lasB
transcription. Exogenous
N
-butyryl–
l
-homoserine lactone overcame the reduced expression of
rhlI
and restored
rhlAB
and
lasB
expression, as well as elastase production. Our results suggest that the overproduction of the
P. aeruginosa
DksA homologue inhibits quorum-sensing-dependent virulence factor production by downregulating the transcription of the autoinducer synthase gene
rhlI
.
Yersinia pestis
pFra Shows Biovar-Specific Differences and Recent Common Ancestry with a
Salmonella enterica
Serovar Typhi Plasmid
Authors: Prentice Michael B. , James Keith D. , Parkhill Julian , Baker Stephen G. , Stevens Kim , Simmonds Mark N. , Mungall Karen L. , Churcher Carol , Oyston Petra C. F. , Titball Richard W. , Wren Brendan W. , Wain John , Pickard Derek , Hien Tran Tinh , Farrar Jeremy J. , Dougan Gordon
Published: July 2002

Population genetic studies suggest that
Yersinia pestis
, the cause of plague, is a clonal pathogen that has recently emerged from
Yersinia pseudotuberculosis
. Plasmid acquisition is likely to have been a key element in this evolutionary leap from an enteric to a flea-transmitted systemic pathogen. However, the origin of
Y. pestis
-specific plasmids remains obscure. We demonstrate specific plasmid rearrangements in different
Y. pestis
strains which distinguish
Y. pestis
bv. Orientalis strains from other biovars. We also present evidence for plasmid-associated DNA exchange between
Y. pestis
and the exclusively human pathogen
Salmonella enterica
serovar Typhi.
Phage λ Red-Mediated Adaptive Mutation
Authors: Poteete Anthony R. , Wang Hsinju R. , Foster Patricia L.
Published: July 2002

Replacement of the
recBCD
genes of
Escherichia coli
with the
red
recombination genes of bacteriophage lambda results in a strain in which adaptive mutation occurs at an elevated frequency. Like RecBCD-dependent adaptive mutation, Red-mediated adaptive mutation is dependent upon
recA
and
ruvABC
functions.
Regulation and Adaptive Evolution of Lactose Operon Expression in
Lactobacillus delbrueckii
Authors: Lapierre Luciane , Mollet Beat , Germond Jacques-Edouard
Published: July 2002

Lactobacillus delbrueckii
subsp.
bulgaricus
and
L. delbrueckii
subsp.
lactis
are both used in the dairy industry as homofermentative lactic acid bacteria in the production of fermented milk products. After selective pressure for the fast fermentation of milk in the manufacture of yogurts,
L. delbrueckii
subsp.
bulgaricus
loses its ability to regulate
lac
operon expression. A series of mutations led to the constitutive expression of the
lac
genes. A complex of insertion sequence (IS) elements (ISL
4
inside ISL
5
), inserted at the border of the
lac
promoter, induced the loss of the palindromic structure of one of the operators likely involved in the binding of regulatory factors. A
lac
repressor gene was discovered downstream of the β-galactosidase gene of
L. delbrueckii
subsp.
lactis
and was shown to be inactivated by several mutations in
L. delbrueckii
subsp.
bulgaricus
. Regulatory mechanisms of the
lac
gene expression of
L. delbrueckii
subsp.
bulgaricus
and
L. delbrueckii
subsp.
lactis
were compared by heterologous expression in
Lactococcus lactis
of the two
lac
promoters in front of a reporter gene (β-glucuronidase) in the presence or absence of the
lac
repressor gene. Insertion of the complex of IS elements in the
lac
promoter of
L. delbrueckii
subsp.
bulgaricus
increased the promoter's activity but did not prevent repressor binding; rather, it increased the affinity of the repressor for the promoter. Inactivation of the
lac
repressor by mutations was then necessary to induce the constitutive expression of the
lac
genes in
L. delbrueckii
subsp.
bulgaricus
.
Comparison and Evaluation of Antimicrobial Susceptibility Testing of Enterococci Performed in Accordance with Six National Committee Standardized Disk Diffusion Procedures
Authors: Cotter Gary , Adley Catherine C.
Published: July 2002

Studies were conducted to compare and evaluate antimicrobial susceptibility test results for enterococci obtained by six national committee disk diffusion procedures. Variations in the incidence of isolates in resistance categories and errors were associated with the use of ciprofloxacin, gentamicin, nitrofurantoin, rifampin, and teicoplanin in a number of committee procedures. Results indicate that laboratories performing disk diffusion antimicrobial susceptibility testing may have problems correctly identifying resistance in enterococci with agents used to combat infections and that it may be difficult to compare resistance data for surveillance purposes.
Use of Quantitative PCR To Measure Density of
Borrelia burgdorferi
in the Midgut and Salivary Glands of Feeding Tick Vectors
Authors: Piesman Joseph , Schneider Bradley S. , Zeidner Nordin S.
Published: July 2002

Quantitative real-time PCR was used to assay spirochetes in feeding ticks. Spirochetes in tick midguts increased sixfold, from 998 per tick before attachment to 5,884 at 48 h of attachment. Spirochetes in tick salivary glands increased >17-fold, from 1.2 per salivary gland pair before feeding to 20.8 at 72 h postattachment. The period of the most rapid increase in the number of spirochetes in the salivary glands occurred from 48 to 60 h postattachment; this time period coincides with the maximal increase in transmission risk during nymphal tick feeding.
Novel
Ehrlichia
Genotype Detected in Dogs in South Africa
Authors: Allsopp M. T. E. P. , Allsopp B. A.
Published: July 2002

DNA samples from dogs presenting with symptoms suggestive of canine ehrlichiosis, but with no morulae detected on blood smears, frequently failed to give a positive reaction with a North American
Ehrlichia canis
-specific PCR assay targeting the 16S rRNA gene. We suspected the presence of a pathogen genetically different from North American
E
.
canis
, and we performed experiments to test this hypothesis. DNA from one canine blood sample was subjected to PCR with primers designed to amplify
Ehrlichia
(
Cowdria
)
ruminantium
ruminantium
16S and
map1
genes. Amplicon sequencing yielded 16S and
map1
sequences which were more closely related to other
E
.
ruminantium
sequences than to those of any other
Ehrlichia
species. Fifty canine DNA samples were subjected to a PCR assay, previously found to be
Cowdria
-specific, which targets the pCS20 gene. Thirty-seven (74%) gave a positive signal, and 16 (32%) also gave visible amplicons after gel electrophoresis, suggesting that this
E
.
ruminantium
organism is common in the Pretoria-Johannesburg area. The organism has not been isolated in culture, so we cannot definitively state that it was responsible for the canine ehrlichiosis symptoms, although the occurrence of several similar cases suggests this to be so. Most importantly, we also do not yet know whether the organism is infective for, or causes heartwater in, ruminants.
Immunological and PCR Analyses for Borna Disease Virus in Psychiatric Patients and Blood Donors in Japan
Authors: Fukuda Koji , Takahashi Kazuo , Iwata Yasuhide , Mori Norio , Gonda Kenji , Ogawa Tsuguhiro , Osonoe Kouichi , Sato Minako , Ogata Shin-ichi , Horimoto Taisuke , Sawada Takashi , Tashiro Masato , Yamaguchi Kazunari , Niwa Shin-ichi , Shigeta Shiro
Published: July 2002

The involvement of Borna disease virus (BDV) in psychiatric diseases in humans remains controversial. T-cell memory response and seroprevalence of BDV in patients with psychiatric disorders and blood donors in Japan were evaluated collectively by Western blot (WB) analysis with inhibition test, electrochemiluminescence immunoassay, immunofluorescence assay, and T-cell proliferative response as well as detection of BDV p24 RNA in peripheral blood mononuclear cells (PBMCs). Positive proliferative responses to both BDV p40 and p24 proteins were detected in 9% of patients with mood disorders (4 of 45), 4% of schizophrenic patients (2 of 45), and 2% of blood donors (1 of 45). By WB analysis, the antibody to BDV p40 was detected only in 2% of patients with mood disorders (1 of 45). The BDV p24 antibody was detected in 2% of patients with mood disorders (1 of 45) and 9% of schizophrenic patients. (4 of 45) No plasma reacted with both BDV proteins. The finding of a lower seroprevalence than previously reported suggests the presence of false-positive cases in the previous report. BDV RNA was detected only in 2% of patients with mood disorders (1 of 45). In these three serological assays, T-cell responses, and PCR analysis, there was no significant difference in the prevalence among the three groups. However, we found three psychiatric patients who were positive for both BDV antibodies and T-cell proliferative responses and one patient who was positive for BDV RNA in PBMCs. These findings suggest the usefulness of the proliferative T-cell response and that certain individuals are infected with BDV or a BDV-related virus.
Microsatellite Typing as a New Tool for Identification of
Saccharomyces cerevisiae
Strains
Authors: Hennequin C. , Thierry A. , Richard G. F. , Lecointre G. , Nguyen H. V. , Gaillardin C. , Dujon B.
Published: July 2002

Since
Saccharomyces cerevisiae
appears to be an emerging pathogen, there is a need for a valuable molecular marker able to distinguish among strains. In this work, we investigated the potential value of microsatellite length polymorphism with a panel of 91 isolates, including 41 clinical isolates, 14 laboratory strains, and 28 strains with industrial relevance. Testing seven polymorphic regions (five trinucleotide repeats and two dinucleotide repeats) in a subgroup of 58 unrelated strains identified a total of 69 alleles (6 to 13 per locus) giving 52 different patterns with a discriminatory power of 99.03%. We found a cluster of clinical isolates sharing their genotype with a bakery strain, suggesting a digestive colonization following ingestion of this strain with diet. With the exception of this cluster of isolates and isolates collected from the same patient or from patients treated with
Saccharomyces boulardii
, all clinical isolates gave different and unique patterns. The genotypes are stable, and the method is reproducible. The possibility to make the method portable is of great interest for further studies using this technique. This work shows the possibility to readily identify
S. boulardii
(a strain increasingly isolated from invasive infections) using a unique and specific microsatellite allele.
Paratuberculosis Infection of Nonruminant Wildlife in Scotland
Authors: Beard P. M. , Daniels M. J. , Henderson D. , Pirie A. , Rudge K. , Buxton D. , Rhind S. , Greig A. , Hutchings M. R. , McKendrick I. , Stevenson K. , Sharp J. M.
Published: July 2002

Recent reports of natural paratuberculosis (or Johne's disease) in rabbits, foxes, and stoats has focused debate on the presence and importance of wildlife reservoirs in the epidemiology of this disease. This paper describes an extensive study investigating 18 nonruminant wildlife species for evidence of paratuberculosis. Using both culture and histopathological analysis, fox, stoat, weasel, crow, rook, jackdaw, rat, wood mouse, hare, and badger were found to harbor
Mycobacterium avium
subsp.
paratuberculosis
, the causative organism of paratuberculosis, suggesting that the epidemiology of this disease is more complex than previously realized.
Etiology of Children's Diarrhea in Montevideo, Uruguay: Associated Pathogens and Unusual Isolates
Authors: Torres M. E. , Pı́rez M. C. , Schelotto F. , Varela G. , Parodi V. , Allende F. , Falconi E. , Dell'Acqua L. , Gaione P. , Méndez M. V. , Ferrari A. M. , Montano A. , Zanetta E. , Acuña A. M. , Chiparelli H. , Ingold E.
Published: July 2002

We studied microorganisms associated with infant diarrhea in a group of 256 children admitted to a public pediatric hospital in Montevideo, Uruguay. Diagnostic procedures were updated to optimize detection of potential pathogens, which were found in 63.8% of cases, and to be able to define their characteristics down to molecular or antigenic type. Coinfection with two or more agents was detected in more than one-third of positive studies.
Escherichia coli
enteric virotypes, especially enteropathogenic
E. coli
(EPEC), were shown to be prevalent. Rotavirus,
Cryptosporidium
,
Campylobacter
(mainly
Campylobacter jejuni
), and
Shigella flexneri
were also often identified. Enterotoxigenic
E. coli,
Salmonella,
and
Giardia lamblia
were sporadically recognized. Unusual findings included two enteroinvasive
E. coli
strains, one
Shigella dysenteriae
2 isolate, and a non-O:1
Vibrio cholerae
culture. EPEC bacteria and
S. flexneri
(but not
Salmonella
) showed unusually frequent antimicrobial resistance, especially towards beta-lactam antibiotics, which is the subject of ongoing work.
Occurrence of Vancomycin-Resistant Enterococci in Pork and Poultry Products from a Cattle-Rearing Area of France
Authors: Gambarotto Karine , Ploy Marie-Cecile , Dupron Frederic , Giangiobbe Marie , Denis Francois
Published: July 2002

Meat products were collected from public retail outlets and tested for the presence of vancomycin-resistant enterococci (VRE) in an area with a high prevalence of VRE reported in human fecal samples. VRE were detected in 66% of the samples, and a predominance of VanC strains was found, which is also true for human fecal samples.
Identification of Two Different 14-α Sterol Demethylase-Related Genes (
cyp51A
and
cyp51B
) in
Aspergillus fumigatus
and Other
Aspergillus
species
Authors: Mellado E. , Diaz-Guerra T. M. , Cuenca-Estrella M. , Rodriguez-Tudela J. L.
Published: July 2002

Two
cyp51
-related genes (
cyp51A
and
cyp51B
) encoding 14-α sterol demethylase-like enzymes were identified in the opportunistic human pathogen
Aspergillus fumigatus
. PCR amplification using degenerate oligonucleotides based on conserved areas of cytochrome P450 demethylases of other filamentous fungi and yeasts allowed the cloning and sequencing of two different homologue genes in
A. fumigatus
. Southern analysis confirmed that both genes hybridized to distinct genomic loci and that both are represented as single copies in the genome. Comparison of the deduced Cyp51A and Cyp51B proteins with the CYP51 proteins from
Penicillium italicum, Aspergillus nidulans, Erysiphe graminis, Uncinula necator, Botrytis cinerea, Ustilago maydis, Cryptococcus neoformans, Candida albicans, Saccharomyces cerevisiae, Candida tropicalis
, and
Candida glabrata
showed that the percentages of identity of the amino acid sequences (range, 40 to 70%) were high enough to consider Cyp51A and Cyp51B to be members of the fungal CYP51 family. Fragments from both genes were also cloned from other
Aspergillus
spp. (
A. flavus, A. nidulans
, and
A. terreus
). Phylogenetic analysis showed that, at least in the most pathogenic species of
Aspergillus
, there are two fungal CYP51 proteins. This is the first report of the existence of two homologue genes coding for 14-α sterol demethylase in the fungal kingdom. This finding could provide insights into the azole resistance mechanisms operating in fungi. The primers used here may be useful molecular tools for facilitating the cloning of novel 14-α sterol demethylase genes in other filamentous fungi.
Serodiagnosis of Human Granulocytic Ehrlichiosis by Using Novel Combinations of Immunoreactive Recombinant Proteins
Authors: Lodes Michael J. , Mohamath Raodoh , Reynolds Lisa D. , McNeill Patricia , Kolbert Christopher P. , Bruinsma Elizabeth S. , Benson Darin R. , Hofmeister Eric , Reed Steven G. , Houghton Raymond L. , Persing David H.
Published: July 2002

A panel of seven recombinant antigens, derived from
Ehrlichia phagocytophila
(the agent of human granulocytic ehrlichiosis), was evaluated by class-specific enzyme-linked immunosorbent assays (ELISAs) for utility in the diagnosis of the infection. Fourteen genomic fragments, obtained by serologic expression screening, contained open reading frames (ORFs) encoding 16 immunodominant antigens. Eleven of these antigens were members of the major surface protein (MSP) multigene family. Alignment of their predicted protein sequences revealed a pattern of conserved sequences, which contained short direct repeats, flanking a variable region. In addition, two genomic clones contained two and three MSP ORFs, respectively, indicating that these genes are clustered in tandem copies. The implications for this pattern of both genomic and protein arrangements in antigenic variations of MSPs and in their utilities in a diagnostic assay are discussed. In addition to two MSP recombinant antigens (rHGE-1 and -3) and a fusion protein of these antigens (rErf-1), five further recombinants were evaluated by ELISA. Two of these antigens (rHGE-14 and -15) were novel, while a third (rHGE-2), with no known function, has been described. The final two recombinant antigens (rHGE-9 and -17) represent overlapping segments of the ankyrin gene (
ank
). The addition of rHGE-9 ELISA data resulted in the detection of 78% (21 of 27) of acute-phase sera. When serologic data for all recombinants are combined, 96.2% (26 of 27) of convalescent-phase patient serum samples and 85.2% (23 of 27) of acute-phase patient serum samples are detected, indicating the potential of these antigens for use in the development of a rapid serologic assay for the detection of
E. phagocytophila
infection.
Efficacy of New Low-Cost Filtration Device for Recovering
Schistosoma haematobium
Eggs from Urine
Authors: Gyorkos Theresa W. , Ramsan Mahdi , Foum Ali , Khamis Iddi Simba
Published: July 2002

A new, inexpensive filtration device for the diagnosis of urinary schistosomiasis was tested against the commonly used Millipore device. The experimental protocol was performed with 25 urine samples known to be positive for
Schistosoma haematobium
. The results suggest that the new device is as effective as the Millipore device for the diagnosis of urinary schistosomiasis. Its low cost will be attractive to schistosomiasis control programs.
Human Infections Due to
Mycobacterium lentiflavum
Authors: Tortoli Enrico , Bartoloni Alessandro , Erba Maria Luigia , Levrè Egle , Lombardi Natalia , Mantella Antonia , Mecocci Lorenzo
Published: July 2002

Three cases of human disease due to
Mycobacterium lentiflavum
are reported. In the first, the mycobacterium was responsible for chronic pulmonary disease in an elderly woman; in the second, it gave rise to cervical lymphadenitis in a child; and in the third, it caused a liver abscess in a young AIDS patient.
Human Cytomegalovirus and Human Umbilical Vein Endothelial Cells: Restriction of Primary Isolation to Blood Samples and Susceptibilities of Clinical Isolates from Other Sources to Adaptation
Authors: Gerna Giuseppe , Percivalle Elena , Sarasini Antonella , Revello M. Grazia
Published: July 2002

In immunocompromised patients with disseminated infection, human cytomegalovirus (HCMV) is widespread in the microvascular endothelium of multiple organs. Human umbilical vein endothelial cells (HUVEC) were used in parallel to human embryonic lung fibroblasts (HELF) to recover HCMV from blood samples of immunocompromised patients. Using the shell vial technique, comparable median numbers of p72-positive HUVEC and HELF cells were found with the 26 HCMV-positive buffy coat samples out of 150 examined. Analysis of other clinical samples inoculated as controls revealed, in the presence of highly infected HELF monolayers, either the presence of very few infected HUVEC with urine specimens (
n
= 10 samples) or the lack of infected HUVEC with throat washes (
n
= 3) or amniotic fluid samples (
n
= 2). Thus, peripheral blood leukocytes (PBL) appear essential for primary isolation of HCMV in HUVEC. In this respect, HCMV strains, recovered from clinical samples other than buffy coats in HELF only, could be readily adapted to growth in HUVEC by coculturing PBL from healthy blood donors with infected HELF and then inoculating infected PBL onto HUVEC. Recently elucidated mechanisms of interaction of leukocytes and HUVEC with bidirectional transfer of virus seem to provide the basis for the restriction of HCMV primary isolation in HUVEC to blood samples. However, virus strains recovered from only HELF could be adapted to growth in HUVEC when inoculated with HELF-derived (either cell-associated or cell-free) HCMV strains upon primary isolation. In conclusion, due to the in vitro selection of virus variants provided with both PBL tropism and HUVEC tropism, HCMV recovery in HUVEC is PBL mediated and substantially restricted to blood samples. Lack of HCMV recovery in HUVEC from clinical samples other than blood leads to the assumption that epithelial cells, such as urinary, amniotic, or pharyngeal cells, do not possess adequate adhesion molecules to establish close contacts with HUVEC.
Typing and Subtyping of 83 Clinical Isolates Purified from Surgically Implanted Silicone Feeding Tubes by Random Amplified Polymorphic DNA Amplification
Authors: Dautle Melanie P. , Ulrich Ricky L. , Hughes Thomas A.
Published: July 2002

In this study, 83 clinical isolates purified from biofilms colonizing 18 silicone gastrostomy devices (12 “buttons” and six tubes converted to skin level devices) were selected for subtype characterization utilizing genetic analysis. The tubes, previously used for feeding, remained in place for 3 to 47 months (mean, 20.0 months) in children ranging in age from 6 months to 17 years. Classification of specific microbes using random amplified polymorphic DNA (RAPD) analysis revealed genetic similarities and differences among isolates belonging to the same genus. Both gram-positive and -negative bacteria were investigated, including 2 isolates of
Bacillus brevis,
4 isolates of
Bacillus licheniformis
, 2 isolates of
Bacillus pumilus
, 3 isolates of
Enterococcus durans
, 19 isolates of
Enterococcus faecalis
, 8 isolates of
Enterococcus faecium
, 2 isolates of
Enterococcus hirae
, 7 isolates of
Escherichia coli
, 8 isolates of
Lactobacillus plantarum
, 19 isolates of
Staphylococcus aureus
, 2 isolates of
Staphylococcus epidermidis
, and 7 isolates of
Staphylococcus saprophyticus.
Amplified DNA fragments (amplicons) provided species-specific fingerprints for comparison by agarose gel electrophoresis. A total of 62 distinct RAPD types were categorized from the five genera studied. Typing analysis suggested cross acquisition of
E. coli
,
E. faecalis
, and
S. aureus
in three patient pairs. Genomic polymorphism detection proved efficient and reliable for classifying bacterial subtypes isolated from biofilms adhering to various portions of commonly employed enteral access tubes.
Mitochondrial DNA Restriction Fragment Length Polymorphism (RFLP) and 18S Small-Subunit Ribosomal DNA PCR-RFLP Analyses ofAcanthamoebaIsolated from Contact Lens Storage Cases of Residents in Southwestern Korea
Authors: Kong Hyun-Hee , Shin Ji-Yeol , Yu Hak-Sun , Kim Jin , Hahn Tae-Won , Hahn Young-Ho , Chung Dong-Il
Published: July 2002

We applied ribosomal DNA PCR-restriction fragment length polymorphism (RFLP) and mitochondrial DNA (mtDNA) RFLP analyses to 43Acanthamoebaenvironmental isolates (KA/LH1 to KA/LH43) from contact lens storage cases in southwestern Korea. These isolates were compared to American Type Culture Collection strains and clinical isolates (KA/E1 to KA/E12) from patients with keratitis. Seven riboprint patterns were seen. To identify the species of the isolates, a phylogenetic tree was constructed based on the comparison of riboprint patterns with reference strains. Four types accounted for 39 of the isolates belonging to theA. castellaniicomplex. The most predominant (48.8%) type wasA. castellaniiKA/LH2 type, which had identical riboprint and mtDNA RFLP patterns to those ofA. castellaniiCastellani, KA/E3 and KA/E8. The riboprint and mtDNA RFLP patterns of the KA/LH7 (20.9%) type were identical to those ofA. castellaniiMa, a corneal isolate from the United States. The riboprint and mtDNA RFLP patterns of the KA/LH1 (18.6%) type were the same as those ofA. lugdunensisL3a, KA/E2, and KA/E12. The prevalent pattern for each type ofAcanthamoebain southwestern Korea was very different from that from southeastern Korea and Seoul, Korea. It is noteworthy that 38 (88.4%) out of 43 isolates from contact lens storage cases of the residents in southwestern Korea revealed mtDNA RFLP and riboprint patterns identical to those found for clinical isolates in our area. This indicates that most isolates from contact lens storage cases in the surveyed area are potential keratopathogens. More attention should be paid to the disinfection of contact lens storage cases to prevent possible amoebic keratitis.
Comparison of an Automated Ribotyping System to Restriction Endonuclease Analysis and Pulsed-Field Gel Electrophoresis for Differentiating Vancomycin-Resistant
Enterococcus faecium
Isolates
Authors: Price Connie S. , Huynh Holly , Paule Suzanne , Hollis Richard J. , Noskin Gary A. , Pfaller Michael A. , Peterson Lance R.
Published: July 2002

The RiboPrinter Microbial Characterization System was compared with pulsed-field gel electrophoresis (PFGE), restriction endonuclease analysis (REA), and epidemiological data for typing 45 vancomycin-resistant
Enterococcus faecium
(VRE) isolates. In 21 clinically related isolates, 90 to 100% were similar by PFGE and REA, but only 57% were similar by the RiboPrinter. In another eight clinically related isolates, three isolates similar by PFGE and REA were all unique by the RiboPrinter. In contrast, in 16 clinically unrelated isolates, the predominant RiboPrinter ribotype represented 50% of the strains, while the largest PFGE and REA clones represented less than 19% of the strains. These data suggest that the RiboPrinter is not reliable for VRE investigation.
Efficient DNA Transfection Mediated by the C-Terminal Domain of Human Immunodeficiency Virus Type 1 Viral Protein R
Authors: Kichler Antoine , Pages Jean-Christophe , Leborgne Christian , Druillennec Sabine , Lenoir Christine , Coulaud Dominique , Delain Etienne , Le Cam Eric , Roques Bernard P. , Danos Olivier
Published: July 2002

Viral protein R (Vpr) of human immunodeficiency virus type 1 is produced late in the virus life cycle and is assembled into the virion through binding to the Gag protein. It is known to play a significant role early in the viral life cycle by facilitating the nuclear import of the preintegration complex in nondividing cells. Vpr is also able to interact with nucleic acids, and we show here that it induces condensation of plasmid DNA. We have explored the possibility of using these properties in DNA transfection experiments. We report that the C-terminal half of the protein (Vpr
52–96
) mediates DNA transfection in a variety of human and nonhuman cell lines with efficiencies comparable to those of the best-known transfection agents. Compared with polylysine, a standard polycationic transfection reagent, Vpr
52–96
was 10- to 1,000-fold more active. Vpr
52–96
-DNA complexes were able to reach the cell nucleus through a pH-independent mechanism. These observations possibly identify an alternate pathway for DNA transfection.
Cellular and Viral Specificities of Human Immunodeficiency Virus Type 1 Vif Protein
Authors: Madani Navid , Kabat David
Published: July 2002

Thevifgene of human immunodeficiency virus type 1 (HIV-1) greatly enhances the infectivity of HIV-1 virions that are released from cells classified as nonpermissive (e.g., lymphocytes, macrophages, and H9 leukemic T cells) but is irrelevant in permissive cells (e.g., HeLa or COS cells). Recently, it was reported thatvifexpression in nonpermissive cells dramatically increases infectivity not only of HIV-1 but also of other enveloped viruses, including murine leukemia viruses (MLVs). This was surprising in part because MLVs and other murine retroviruses lackvifgenes yet replicate efficiently in T lymphocytes. To investigate these issues, we first developed improved methods for producing substantial quantities of HIV-1 virions withvifdeletions from healthy H9 cells. These virions had approximately the same amounts of major core proteins and envelope glycoproteins as the control wild-type virions but were only approximately 1% as infectious. We then produced H9 cells that contained wild-type orvifdeletion HIV-gptproviruses, which lack a functionalenvgene. After superinfection with either xenotropic or amphotropic MLVs, these cells released HIV-gptvirions pseudotyped with an MLV envelope plus replication-competent MLV. Interestingly, the pseudotyped HIV-gpt(vifdeletion) virions were noninfectious, whereas the MLV virions simultaneously released from the same H9 cells were fully infectious. These results strongly suggest that the Vif protein functions in a manner that is both cell specific and at least substantially specific for HIV-1 and related lentiviruses. In addition, these results confirm thatvifdeletion HIV-1 virions from nonpermissive cells are blocked at a postpenetration stage of the infection pathway.
Pseudotyping of Glycoprotein D-Deficient Herpes Simplex Virus Type 1 with Vesicular Stomatitis Virus Glycoprotein G Enables Mutant Virus Attachment and Entry
Authors: Anderson Dina B. , Laquerre Sylvie , Goins William F. , Cohen Justus B. , Glorioso Joseph C.
Published: July 2002

The use of herpes simplex virus (HSV) vectors for in vivo gene therapy will require the targeting of vector infection to specific cell types in certain in vivo applications. Because HSV glycoprotein D (gD) imparts a broad host range for viral infection through recognition of ubiquitous host cell receptors, vector targeting will require the manipulation of gD to provide new cell recognition specificities in a manner designed to preserve gD's essential role in virus entry. In this study, we have determined whether an entry-incompetent HSV mutant with deletions of all Us glycoproteins, including gD, can be complemented by a foreign attachment/entry protein with a different receptor-binding specificity, the vesicular stomatitis virus glycoprotein G (VSV-G). The results showed that transiently expressed VSV-G was incorporated into gD-deficient HSV envelopes and that the resulting pseudotyped virus formed plaques on gD-expressing VD60 cells, albeit at a 50-fold-reduced level compared to that of wild-type gD. This reduction may be related to differences in the entry pathways used by VSV and HSV or to the observed lower rate of incorporation of VSV-G into virus envelopes than that of gD. The rate of VSV-G incorporation was greatly improved by using recombinant molecules in which the transmembrane domain of HSV glycoprotein B or D was substituted for that of VSV-G, but these recombinant molecules failed to promote virus entry. These results show that foreign glycoproteins can be incorporated into the HSV envelope during replication and that gD can be dispensed with on the condition that a suitable attachment/entry function is provided.