bluuebunny/crossref_metadata_2025_split huggingface dataset:
Necrosis of Lung Epithelial Cells during Infection withMycobacterium tuberculosisIs Preceded by Cell Permeation
Authors: Dobos Karen M. , Spotts Ellen A. , Quinn Frederick D. , King C. Harold 
Published: July 2002

ABSTRACTMycobacterium tuberculosisestablishes infection, progresses towards disease, and is transmitted from the alveolus of the lung. However, the role of the alveolar epithelium in any of these pathogenic processes of tuberculosis is unclear. In this study, lung epithelial cells (A549) were used as a model in which to examine cytotoxicity during infection with either virulent or avirulent mycobacteria in order to further establish the role of the lung epithelium during tuberculosis. Infection of A549 cells withM. tuberculosisstrains Erdman and CDC1551 demonstrated significant cell monolayer clearing, whereas infection with eitherMycobacterium bovisBCG orMycobacterium smegmatisLR222 did not. Clearing ofM. tuberculosis-infected A549 cells correlated to necrosis, not apoptosis. Treatment ofM. tuberculosis-infected A549 cells with streptomycin, but not cycloheximide, demonstrated a significant reduction in the necrosis of A549 cell monolayers. This mycobacterium-induced A549 necrosis did not correlate to higher levels of intracellular or extracellular growth by the mycobacteria during infection. Staining of infected cells with propidium iodide demonstrated thatM. tuberculosisinduced increased permeation of A549 cell membranes within 24 h postinfection. Quantitation of lactate dehydrogenase (LDH) release from infected cells further demonstrated that cell permeation was specific toM. tuberculosisinfection and correlated to A549 cellular necrosis. InactivatedM. tuberculosisor its subcellular fractions did not result in A549 necrosis or LDH release. These studies demonstrate that lung epithelial cell cytotoxicity is specific to infection by virulent mycobacteria and is caused by cellular necrosis. This necrosis is not a direct correlate of mycobacterial growth or of the expression of host cell factors, but is preceded by permeation of the A549 cell membrane and requires infection with live bacilli.
Role of Streptolysin O in a Mouse Model of Invasive Group A Streptococcal Disease
Authors: Limbago Brandi , Penumalli Vikram , Weinrick Brian , Scott June R. 
Published: July 2002

ABSTRACTMany of the virulence factors that have been characterized for group A streptococci (GAS) are not expressed in all clinical isolates. One putative virulence factor that is present among most is streptolysin O (Slo), a protein with well-characterized cytolytic activity for many eukaryotic cells types. In other bacterial pathogens, proteins homologous to Slo have been shown to be essential for virulence, but the role of Slo in GAS had not been previously examined. To investigate the role of Slo in GAS virulence, we examined both revertible and stableslomutants in a mouse model of invasive disease. When the revertibleslomutant was used to infect mice, the reversion frequency of bacteria isolated from the wounds and spleens of infected animals was more than 100 times that of the inoculum, indicating that there was selective pressure in the animal for Slo+GAS. Experiments with the stableslomutant demonstrated that Slo was not necessary for the formation of necrotic lesions, nor was it necessary for escape from the lesion to cause disseminated infection. Bacteria were present in the spleens of 50% of the mice that survived infection with the stableslomutant, indicating that dissemination of Slo−GAS does not always cause disease. Finally, mice infected with the stableslomutant exhibited a significant decrease in mortality rates compared to mice infected with wild-type GAS (P< 0.05), indicating that Slo plays an important role in GAS virulence.
Construction of a
Vibrio cholerae
Vaccine Candidate Using Transposon Delivery and FLP Recombinase-Mediated Excision
Authors: Chiang Su L. , Mekalanos John J. 
Published: July 2002

ABSTRACT

Recent efforts to develop a vaccine against the diarrheal disease cholera have focused on the use of live attenuated strains of the causative organism,
Vibrio cholerae
. The Ogawa lipopolysaccharide phenotype is expressed by many epidemic strains, and motility defects reduce the risk of reactive diarrhea in vaccine recipients. We therefore converted a motile Inaba
+
vaccine candidate, Peru-2, to a nonmotile Ogawa
+
phenotype using a
mariner
-based transposon carrying
rfbT
, the gene required for expression of the Ogawa phenotype. Analysis of 22 nonmotile Peru-2 mutants showed that two were Ogawa
+
, and both of these strains had insertions in the
flgE
gene. It was possible to convert these strains to antibiotic sensitivity by introducing a recombinase that acts on sites flanking the antibiotic marker on the transposon. The resulting strains are competent for colonization in infant mice and may therefore be suitable as vaccine candidates for use either independently or in a combination with strains of different biotypes and serotypes.
Involvement of Focal Adhesion Kinase in
Escherichia coli
Invasion of Human Brain Microvascular Endothelial Cells
Authors: Reddy Marpadga A. , Wass Carol A. , Kim Kwang Sik , Schlaepfer David D. , Prasadarao Nemani V. 
Published: July 2002

ABSTRACT

Escherichia coli
K1 traversal across the blood-brain barrier is an essential step in the pathogenesis of neonatal meningitis. We have previously shown that invasive
E. coli
promotes the actin rearrangement of brain microvascular endothelial cells (BMEC), which constitute a lining of the blood-brain barrier, for invasion. However, signal transduction mechanisms involved in
E. coli
invasion are not defined. In this report we show that tyrosine kinases play a major role in
E. coli
invasion of human BMEC (HBMEC).
E. coli
induced tyrosine phosphorylation of HBMEC cytoskeletal proteins, focal adhesion kinase (FAK), and paxillin, with a concomitant increase in the association of paxillin with FAK. Overexpression of a dominant interfering form of the FAK C-terminal domain, FRNK (FAK-related nonkinase), significantly inhibited
E. coli
invasion of HBMEC. Furthermore, we found that FAK kinase activity and the autophosphorylation site (Tyr397) are important in
E. coli
invasion of HBMEC, whereas the Grb2 binding site (Tyr925) is not required. Immunocytochemical studies demonstrated that FAK is recruited to focal plaques at the site of bacterial entry. Consistent with the invasion results, overexpression of FRNK, a kinase-negative mutant (Arg454 FAK), and a Src binding mutant (Phe397 FAK) inhibited the accumulation of FAK at the bacterial entry site. The overexpression of FAK mutants in HBMEC also blocked the
E. coli
-induced tyrosine phosphorylation of FAK and its association with paxillin. These observations provide evidence that FAK tyrosine phosphorylation and its recruitment to the cytoskeleton play a key role in
E. coli
invasion of HBMEC.
Contribution of Plasmid-Encoded Fimbriae and Intimin to Capacity of Rabbit-Specific Enteropathogenic
Escherichia coli
To Attach to and Colonize Rabbit Intestine
Authors: Krejany Efrosinia O. , Grant Travis H. , Bennett-Wood Vicki , Adams Louise M. , Robins-Browne Roy M. 
Published: July 2002

ABSTRACT

Attachment to the intestinal mucosa is an essential step in the pathogenesis of diarrhea caused by enteropathogenic
Escherichia coli
(EPEC). Fimbriae and intimin, the outer membrane protein product of the chromosomal
eae
gene, contribute to this process, but their relative roles and the nature of their interaction are not known. The aim of this study was to determine the relative contribution of plasmid-encoded fimbriae, termed Ral, and intimin to the capacity of rabbit-specific EPEC (REPEC) to attach to the intestinal mucosa of rabbits. To achieve this, we constructed a series of mutants in REPEC strain 83/39 (O15:H−), in which the
ralE
and
eae
genes were insertionally inactivated. These strains were then inoculated into ligated loops of rabbit ileum, which were resected 18 h later and examined by light and electron microscopy. The results showed that intimin, but not Ral, is essential for the elicitation of attaching-effacing lesions by REPEC. Nevertheless, a Δ
eae
Ral-bearing mutant adhered to the intestinal epithelium to the same extent as its
eae
-positive parent and far more extensively than an

eae
+
Δral

strain. To examine the contribution of Ral and intimin to colonization of rabbit intestine, we fed these strains to weanling rabbits, which were killed 4 days later, so that the number of bacteria in various regions of the intestine could be determined. The results indicated that strain 83/39 requires both Ral and intimin to colonize the intestine successfully and that a Δ
eae ΔralE
double mutant was incapable of colonizing the intestine. Taken together, these findings indicate that Ral and intimin act independently as adhesion factors of REPEC strain 83/39 and that this strain carries no other significant colonization factor. When both Ral and intimin are present, they appear to act cooperatively, with Ral-mediated adhesion preceding that mediated by intimin.
The Nonfunctional Allele TCRBV6S1B Is Strongly Associated withHelicobacter pyloriInfection
Authors: Kunstmann Erdmute , Hardt Cornelia , Elitok Ercan , Harder Marianne , Suerbaum Sebastian , Peitz Ulrich , Schmiegel Wolff , Epplen Jörg T. 
Published: July 2002

ABSTRACTTo determine genetic susceptibility factors forHelicobacter pyloriinfection, polymorphic T-cell receptor gene elements were investigated in 203H. pylori-infected individuals and 180 uninfected individuals (controls).H. pyloriinfection is highly associated with individuals homozygous for the nonfunctional TCRBV6S1B element (odds ratio = 5.9; χ2= 13;P= 0.00032;Pvalue corrected for multiple comparisons [Bonferroni correction] = 0.00063).
Characterization of the Domain of Fibronectin-Binding Protein I ofStreptococcus pyogenesResponsible for Elicitation of a Protective Immune Response
Authors: Schulze Kai , Medina Eva , Talay Susanne R. , Towers Rebecca J. , Chhatwal Gursharan S. , Guzmán Carlos A. 
Published: July 2002

ABSTRACTFibronectin-binding protein I (SfbI) represents a major adhesin ofStreptococcus pyogenes. Mice were intranasally immunized with recombinant proteins spanning different portions of SfbI to identify the minimal fragment able to elicit a protective response against a lethal challenge withS. pyogenes. The strongest cellular responses and the highest levels of antigen-specific secretory immunoglobulin A (IgA) were detected in mice immunized with the fibronectin-binding region of SfbI. In contrast, animals vaccinated with a polypeptide spanning the aromatic and proline-rich regions showed the highest titers and fastest IgG response in serum. Vaccination with either SfbI without a membrane anchor and signal peptide or a polypeptide encompassing its fibronectin-binding regions resulted in efficient protection against heterologous challenge (60% and 80%, respectively), whereas the use of a polypeptide lacking this region conferred marginal protection (10%) with respect to the control group (0%). These results demonstrate that the fibronectin-binding region of SfbI is a promising candidate antigen for developing anti-S. pyogenesvaccines.
Cyclic Guanosine-3′,5′-Monophosphate and Biopteridine Biosynthesis in
Nocardia
sp
Authors: Son Jong-Keun , Rosazza John P. N. 
Published: July 2002

ABSTRACT

Nocardia
sp. strain NRRL 5646 contains a nitric oxide synthase (NOS) enzyme system capable of generating nitric oxide (NO) from arginine and arginine-containing peptides. To explain possible roles of the NOS system in this bacterium, guanylate cyclase (GC) and tetrahydrobiopterin (H
4
B) biosynthetic enzymes were identified in cell extracts and in culture media. Cell extracts contained GC activity, as measured by the conversion of GTP to cyclic guanosine-3′,5′-monophosphate (cGMP) at 9.56 pmol of cGMP h
−1
mg of protein
−1
. Concentrations of extracellular cGMP in culture media were significantly increased, from average control levels of 45 pmol cGMP liter
−1
to a maximum of 315 pmol liter
−1
, in response to additions of GTP,
l
-arginine, H
4
B, and sodium nitroprusside to growing
Nocardia
cultures. On the other hand, the NOS inhibitor
N
G
-nitro-
l
-arginine and the GC inhibitor 1H-[1,2,4]oxadiazole[4,3-a]quinoxalin-1-one both dramatically decreased extracellular cGMP levels. Activities for GTP-cyclohydrase-1,6-pyruvoyltetrahydropterin synthase and sepiapterin reductase, enzymes essential for H
4
B biosynthesis, were present in
Nocardia
culture extracts at 77.5 pmol of neopterin and 45.8 pmol of biopterin h
−1
mg of protein
−1
, respectively. In
Nocardia
spp., as in mammals, GTP is a key intermediate in H
4
B biosynthesis, and GTP is converted to cGMP by a GC enzyme system that is activated by NO.
A Genetic Mechanism for Deletion of the
ser2
Gene Cluster and Formation of Rough Morphological Variants of
Mycobacterium avium
Authors: Eckstein Torsten M. , Inamine Julia M. , Lambert Markus L. , Belisle John T. 
Published: July 2002

ABSTRACT

A major phenotypic trait of the
Mycobacterium avium
complex is the ability to produce rough and smooth colony variants. The chemical basis of this morphological variation is the loss of an antigenic surface structure, termed glycopeptidolipid (GPL), by rough variants. Using
M. avium
serovar 2 strain 2151 as a model system, this laboratory previously reported that rough variants arise via the deletion of large genomic regions encoding GPL biosynthesis. One such deletion encompasses the gene cluster (
ser2
) responsible for production of the serovar 2 GPL haptenic oligosaccharide. In this study, nucleotide sequencing revealed that both ends of the
ser2
gene cluster are flanked by a novel insertion sequence (IS
1601
) oriented as direct repeats. Detailed analyses of the site of deletion in the genome of
M. avium
2151 Rg-1 demonstrated that a single copy of IS
1601
remained and that the
ser2
gene cluster was deleted by homologous recombination. This same deletion pattern was observed for 10 out of 15 rough colony variants tested. Additionally, these studies revealed that IS
1601
contains portions of three independent insertion sequences. This report is the first to define the precise genetic basis of colony variation in
Mycobacterium
spp. and provides further evidence that homologous recombination between insertion sequence elements can be a primary determinant of genome plasticity in these bacteria.
Multiple
N
-Acyl Homoserine Lactone Signals of
Rhizobium leguminosarum
Are Synthesized in a Distinct Temporal Pattern
Authors: Blosser-Middleton Renée S. , Gray Kendall M. 
Published: July 2002

ABSTRACT

A common form of bacterial quorum sensing involves the production and release of acyl homoserine lactone (AHL) signal metabolites. The nitrogen-fixing symbiont
Rhizobium leguminosarum
reportedly produces at least six different AHLs, but little is known about the regulation of biosynthesis of these molecules. We used a radiolabeling protocol to quantify the relative amounts of AHLs synthesized over time by
R. leguminosarum
cells with and without the symbiosis plasmid pRL1JI. Cells containing pRL1JI were found to produce three predominant signals. In decreasing order of abundance, these were
N
-(3-oxo)octanoyl homoserine lactone [(3-O)C
8
HSL],
N
-octanoyl homoserine lactone, and
N
-hexanoyl homoserine lactone. Cells without pRL1JI produced only two major signals,
N
-(3-hydroxy-7-
cis
)tetradecanoyl homoserine lactone [(3-OH)C
14:1
HSL] and (3-O)C
8
HSL. Each AHL exhibited a distinct temporal pattern of synthesis, suggesting that each AHL is subject to unique regulatory mechanisms. While (3-O)C
8
HSL was produced in both cultures, the patterns of synthesis were different in cells with and without pRL1JI, possibly as a result of redundant gene functions that are present on both the chromosome and the symbiosis plasmid. None of the AHLs appeared to regulate its own biosynthesis, although exogenous (3-OH)C
14:1
HSL did activate synthesis of the three AHLs made by cells containing pRL1JI. These results indicate that the synthesis of multiple AHLs in
R. leguminosarum
is regulated by complex mechanisms that operate independently of quorum sensing itself but that (3-OH)C
14:1
HSL can supersede these controls in pRL1JI-containing cells. This work provides an important global perspective for AHL regulation that both complements and contrasts with the results of previous studies performed with isolated gene systems.
Combination of Reverse Transcriptase PCR Analysis and Immunoglobulin M Detection on Filter Paper Blood Samples Allows Diagnostic and Epidemiological Studies of Measles
Authors: De Swart Rik L. , Nur Yassin , Abdallah Abdallah , Kruining Hans , El Mubarak H. Sittana , Ibrahim Salah A. , Van Den Hoogen Bernadette , Groen Jan , Osterhaus Albert D. M. E. 
Published: July 2002

ABSTRACT
As measles control and elimination campaigns progress, laboratory confirmation of clinically diagnosed measles cases becomes increasingly important. However, in many tropical countries collection and storage of clinical specimens for this purpose are logistically complicated. In this study it is shown that blood samples spotted on filter paper are suitable for the laboratory diagnosis of measles using a combination of reverse transcriptase PCR (RT-PCR) analysis and immunoglobulin M (IgM) detection. First, it was shown that in vitro measles virus (MV)-infected cells diluted in human blood and spotted on filter paper can be detected by RT-PCR. Small amounts of infected cells remained detectable after 25 weeks of storage of the filter paper at room temperature, 4 weeks at 37°C, or 2 weeks at 45°C. Subsequently, this RT-PCR was applied to filter paper blood samples collected from 117 clinically diagnosed measles patients in Sudan in 1997 and 1998. Prior laboratory diagnosis had confirmed 90 cases as acute MV infections, while 27 proved to be nonmeasles rash disease cases. Positive RT-PCR signals were detected in filter paper blood samples of 43 of the 90 confirmed cases (48%) but in none of the 27 nonmeasles cases. In addition, MV-specific IgM levels measured in reconstituted filter paper samples correlated well with those measured in plasma samples. Measles diagnosis based on the combination of filter paper RT-PCR and IgM detection had a sensitivity and specificity of 99 and 96%, respectively. An advantage of this diagnostic approach is that sequencing of RT-PCR products allows phylogenetic analysis of the MV strain involved.
TaqMan 5′-Nuclease Human Immunodeficiency Virus Type 1 PCR Assay with Phage-Packaged Competitive Internal Control for High-Throughput Blood Donor Screening
Authors: Drosten C. , Seifried E. , Roth W. K. 
Published: July 2002

ABSTRACT

Screening of blood donors for human immunodeficiency virus type 1 (HIV-1) infection by PCR permits the earlier diagnosis of HIV-1 infection compared with that by serologic assays. We have established a high-throughput reverse transcription (RT)-PCR assay based on 5′-nuclease PCR. By in-tube detection of HIV-1 RNA with a fluorogenic probe, the 5′-nuclease PCR technology (TaqMan PCR) eliminates the risk of carryover contamination, a major problem in PCR testing. We outline the development and evaluation of the PCR assay from a technical point of view. A one-step RT-PCR that targets the
gag
genes of all known HIV-1 group M isolates was developed. An internal control RNA detectable with a heterologous 5′-nuclease probe was derived from the viral target cDNA and was packaged into MS2 coliphages (Armored RNA). Because the RNA was protected against digestion with RNase, it could be spiked into patient plasma to control the complete sample preparation and amplification process. The assay detected 831 HIV-1 type B genome equivalents per ml of native plasma (95% confidence interval [CI], 759 to 936 HIV-1 B genome equivalents per ml) with a ≥95% probability of a positive result, as determined by probit regression analysis. A detection limit of 1,195 genome equivalents per ml of (individual) donor plasma (95% CI, 1,014 to 1,470 genome equivalents per ml of plasma pooled from individuals) was achieved when 96 samples were pooled and enriched by centrifugation. Up to 4,000 plasma samples per PCR run were tested in a 3-month trial period. Although data from the present pilot feasibility study will have to be complemented by a large clinical validation study, the assay is a promising approach to the high-throughput screening of blood donors and is the first noncommercial test for high-throughput screening for HIV-1.
Pandemic Spread of Cholera: Genetic Diversity and Relationships within the Seventh Pandemic Clone of
Vibrio cholerae
Determined by Amplified Fragment Length Polymorphism
Authors: Lan Ruiting , Reeves Peter R. 
Published: July 2002

ABSTRACT

The seventh cholera pandemic started in 1961 and continues today. A collection of 45 seventh pandemic isolates of
V. cholerae
sampled over a 33-year period were analyzed by amplified fragment length polymorphism (AFLP) fingerprinting. All but four pairs and one set of three isolates were distinguished. AFLP revealed far more variation than ribotyping, which was until now the most useful method of revealing variation within the pandemic clone. Unfortunately, the ribotype variation observed is mainly due to recombination between the multiple copies of the
rrn
genes (R. Lan and P. R. Reeves, Microbiology
144:
1213–1221, 1998), which makes changes susceptible to repeat occurrences and reversion. This AFLP study shows that particularly for the common ribotypes G and H, such events have indeed occurred. AFLP grouped most of the 45 isolates into two clusters. Cluster I consists mainly of strains from the 1960s and 1970s, while cluster II contains mainly strains from the 1980s and 1990s, revealing a temporal pattern of change in the clone. This is best seen in the relationships of the strains from Africa, which correlate with the epidemiology of epidemics on that continent. The data confirm independent introductions to Africa during the 1970s outbreak and reveal several other African introductions. In the 1991 cholera upsurge, isolates from the Southern and Eastern African epidemic focus are markedly different from those from the West African epidemic focus. An isolate from 1987 in Algeria was identical to the West epidemic isolates, suggesting that the strain was present in Africa at least 3 years before causing large outbreaks. These observations have major implications for our understanding of cholera epidemiology.
Induction of Cellular Genes Is Mediated by the Bel1 Transactivator in Foamy Virus-Infected Human Cells
Authors: Wagner Andrea , Doerks Anja , Aboud Mordechai , Alonso Angel , Tokino Takashi , Flügel Rolf M. , Löchelt Martin 
Published: July 2002

ABSTRACT
To gain insight into human foamy virus (HFV; also called spumaretrovirus)-induced alterations of cellular genes, the expression profiles of defined genes in HFV-infected primary human cells were analyzed by cDNA array assays. Several distinct cellular genes activated by HFV infection were identified; the identities of the cellular genes were confirmed by RNA blot analyses. Compared with mock-infected controls, the concentrations of cellular Kip2, Egr-1, COUP-TF1, insulin-like growth factor II (IGF-II), and EphB3 mRNAs were significantly increased in HFV-infected cells and showed a gene-specific and time-dependent induction. Immunoblot analyses with antibodies against some of the cellular gene products revealed increased levels of the corresponding proteins. To investigate mechanisms of HFV-induced alterations in cellular gene expression, the capacity of known HFV genes to increase expression of defined cellular genes was analyzed by transient expression experiments. Plasmids that encode the HFV Bel1 transcriptional transactivator were necessary and sufficient to strongly increase expression of p57Kip2, IGF-II, and EphB3 genes in 293T cells. Potential mechanisms and consequences of activation of cellular genes during HFV infection and Bel1 transactivation of the Kip2 gene are discussed.