bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and Characterization of an Immunodominant 28-Kilodalton
Coxiella burnetii
Outer Membrane Protein Specific to Isolates Associated with Acute Disease
Authors: Zhang Guoquan , To Ho , Russell Kasi E. , Hendrix Laura R. , Yamaguchi Tsuyoshi , Fukushi Hideto , Hirai Katsuya , Samuel James E. 
Published: February 2005

ABSTRACT

Coxiella burnetii
causes acute Q fever in humans and occasional chronic infections that typically manifest as endocarditis or hepatitis. Isolates associated with acute disease were found to be distinct from a group of chronic disease isolates by a variety of biochemical parameters and in a guinea pig fever model of acute disease, suggesting a difference in virulence potential. We compared antigenic polypeptides among
C. burnetii
isolates and found an immunodominant 28-kDa protein in acute group isolates but not in chronic group isolates (T. Ho, A. Hotta, G. Q. Zhang, S. V. Nguyen, M. Ogawa, T. Yamaguchi, H. Fukushi, and K. Hirai, Microbiol. Immunol.
42:
81-85, 1998). In order to clone the
adaA
gene, the N-terminal amino acid sequence of
adaA
was determined and a 59-bp fragment was amplified from Nine Mile phase I DNA by PCR. The putative gene fragment was used to screen a lambda ZAP II genomic DNA library, and an open reading frame expressing a 28-kDa immunoreactive protein was identified. Sequence analysis predicted a gene encoding an ∼28-kDa mature protein with a typical signal sequence. The
adaA
(acute disease antigen A) gene was detected in acute group
C. burnetii
isolates but not identified in chronic group isolates by PCR and Southern blotting. A typical signal peptide was predicted in
adaA
, and specific antibody to
adaA
reacted with the purified membrane fraction of acute group isolates by Western blotting, suggesting that
adaA
is exposed on the outer surface of
C. burnetii. adaA
was overexpressed in pET23a as a fusion protein in
Escherichia coli
to develop anti-recombinant
adaA
(anti-r
adaA
) specific antibody, which recognized a ∼28-kDa band in acute group isolates but not in chronic group isolates. In addition, immunoblotting indicates that r
adaA
reacted with sera derived from animals infected with acute group isolates but did not react with sera from animals infected with chronic group isolates. These results support the idea that an
adaA
gene-targeted PCR assay and an r
adaA
antigen-based serodiagnostic test may be useful for differential diagnosis of acute and chronic Q fever.
Rapid Loss of Motility of
Helicobacter pylori
in the Gastric Lumen In Vivo
Authors: Schreiber Sören , Bücker Roland , Groll Claudia , Azevedo-Vethacke Marina , Garten Désirée , Scheid Peter , Friedrich Susanne , Gatermann Sören , Josenhans Christine , Suerbaum Sebastian 
Published: February 2005

ABSTRACT

The human pathogen
Helicobacter pylori
has infected more than half of the world's population. Nevertheless, the first step of infection, the acute colonization of the gastric mucus, is poorly understood. For successful colonization,
H. pylori
must retain active motility in the gastric lumen until it reaches the safety of the mucus layer. To identify the factors determining the acute colonization, we inserted bacteria into the stomach of anesthetized Mongolian gerbils. We adjusted the gastric juice to defined pH values of between 2.0 and 6.0 by using an autotitrator. Despite the fact that
Helicobacter
spp. are known to survive low pH values for a certain time in vitro, the length of time that
H. pylori
persisted under the assay conditions within the gastric juice in vivo was remarkably shorter. In the anesthetized animal we found
H. pylori
to be irreversibly immotile in less than 1 min at lumen pH values of 2 and 3. At pH 4 motility was lost after 2 min. However, the period of motility increased to more than 15 min at pH 6. Blocking pepsins in the gastric lumen in vivo by using pepstatin significantly increased the period of motility. It was possible to simulate the rapid in vivo immotilization in vitro by adding pepsins. We conclude that pepsin limits the persistence of
H. pylori
in the gastric chymus to only a few minutes by rapidly inhibiting active motility. It is therefore likely that this short period of resistance in the gastric lumen is one of the most critical phases of
Helicobacter
infection.
Monocytes Are Highly Sensitive toClostridium difficileToxin A-Induced Apoptotic and Nonapoptotic Cell Death
Authors: Solomon K. , Webb J. , Ali N. , Robins R. A. , Mahida Y. R. 
Published: February 2005

ABSTRACTIn this study we investigated the in vitro responses of peripheral blood mononuclear preparations and purified monocytes toClostridium difficiletoxin A. In contrast to the responses of T and B cells, exposure to toxin A led to a rapid loss of monocytes in a time- and dose-dependent fashion (the majority of cells were lost within 24 h of exposure to >100 ng of toxin per ml). Transmission electron microscopy, flow cytometry, and fluorescence microscopy after propidium iodide and Hoechst staining showed that cell death in purified preparations of monocytes following exposure to 100 and 1,000 ng of toxin A per ml occurred by apoptosis. Further studies showed that 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazole-carbocyanine iodide aggregates were retained within toxin A-exposed monocyte mitochondria, but cytochromecwas released, suggesting that the apoptotic cascade was triggered in the absence of mitochondrial permeability transition. There was also an increase in caspase-3 activity in toxin A-stimulated monocytes. Following exposure to very high concentrations of toxin A (30 μg/ml), monocyte cell death was predominantly of the necrotic type, with rapid extracellular release of lactate dehydrogenase. These studies demonstrated thatC. difficiletoxin A has a cell-specific effect, in which monocytes exhibit greater susceptibility than lymphocytes and their death is induced in a concentration-dependent manner.
Identification of Enterohemorrhagic
Escherichia coli
O26:H
−
Genes Required for Intestinal Colonization in Calves
Authors: van Diemen Pauline M. , Dziva Francis , Stevens Mark P. , Wallis Timothy S. 
Published: February 2005

ABSTRACT

Enterohemorrhagic
Escherichia coli
(EHEC) infections in humans are an important public health problem and are commonly acquired via contact with ruminant feces. The serogroups that are predominantly associated with human infection in the United States and Europe are O157 and O26. Serotypes O157:H7 and O26:H
−
differ in their virulence and tissue tropism in calves and therefore may colonize calves by distinct mechanisms. The mechanisms underlying EHEC intestinal colonization and pathogenesis are poorly understood. Signature-tagged mutagenesis was used to identify 59 genes of EHEC O26:H
−
that are required for the intestinal colonization of calves. Our results indicate important roles for locus of enterocyte effacement (LEE)-encoded type III secreted proteins in intestinal colonization. In addition, colonization is facilitated by cytotoxins, putative type III secreted proteins unlinked to the LEE, a putative fimbrial operon, and numerous genes involved in central metabolism and transport and genes of unknown function. Our data also imply that the elaboration of type I fimbriae by EHEC O26:H
−
is disadvantageous for persistence within the bovine intestines. These observations have important implications for the design of vaccines to control these important zoonotic pathogens.
Distinct Roles of Pattern Recognition Receptors CD14 and Toll-Like Receptor 4 in Acute Lung Injury
Authors: Jeyaseelan Samithamby , Chu Hong Wei , Young Scott K. , Freeman Mason W. , Worthen G. Scott 
Published: February 2005

ABSTRACTAcute lung injury (ALI) induced by lipopolysaccharide (LPS) is a major cause of mortality among humans. ALI is characterized by microvascular protein leakage, neutrophil influx, and expression of proinflammatory mediators, followed by severe lung damage. LPS binding to its receptors is the crucial step in the causation of these multistep events. LPS binding and signaling involves CD14 and Toll-like receptor 4 (TLR4). However, the relative contributions of CD14 and TLR4 in the induction of ALI and their therapeutic potentials are not clear in vivo. Therefore, the aim of the present study was to compare the roles of CD14 and TLR4 in LPS-induced ALI to determine which of these molecules is the more critical target for attenuating ALI in a mouse model. Our results show that CD14 and TLR4 are necessary for low-dose (300-μg/ml) LPS-induced microvascular leakage, NF-κB activation, neutrophil influx, cytokine and chemokine (KC, macrophage inflammatory protein 2, tumor necrosis factor alpha, interleukin-6) expression, and subsequent lung damage. On the other hand, when a 10-fold-higher dose of LPS (3 mg/ml) was used, these responses were only partially dependent on CD14 and they were totally dependent on TLR4. The CD14-independent LPS response was dependent on CD11b. A TLR4 blocking antibody abolished microvascular leakage, neutrophil accumulation, cytokine responses, and lung pathology with a low dose of LPS but only attenuated the responses with a high dose of LPS. These data are the first to demonstrate that LPS-induced CD14-depdendent and -independent (CD11b-dependent) signaling pathways in the lung are entirely dependent on TLR4 and that blocking TLR4 might be beneficial in lung diseases caused by LPS from gram-negative pathogens.
Role for Flagella but Not Intimin in the Persistent Infection of the Gastrointestinal Tissues of Specific-Pathogen-Free Chicks by Shiga Toxin-Negative
Escherichia coli
O157:H7
Authors: Best Angus , La Ragione Roberto M. , Sayers A. Robin , Woodward Martin J. 
Published: February 2005

ABSTRACT

Shiga toxin (Stx)-positive
Escherichia coli
O157:H7 readily colonize and persist in specific-pathogen-free (SPF) chicks, and we have shown that an Stx-negative
E. coli
O157:H7 isolate (NCTC12900) readily colonizes SPF chicks for up to 169 days after oral inoculation at 1 day of age. However, the role of intimin in the persistent colonization of poultry remains unclear. Thus, to investigate the role of intimin and flagella, which is a known factor in the persistence of non-O157
E. coli
in poultry, isogenic single- and double-intimin and aflagellar mutants were constructed in
E. coli
O157:H7 isolate NCTC12900. These mutants were used to inoculate (10
5
CFU) 1-day-old SPF chicks. In general, significant attenuation of the aflagellate and intimin-aflagellate mutants, but not the intimin mutant, was noted at similar time points between 22 and 92 days after inoculation. The intimin-deficient mutant was still being shed at the end of the experiment, which was 211 days after inoculation, 84 days more than the wild type. Shedding of the aflagellar and intimin-aflagellar mutants ceased 99 and 113 days after inoculation, respectively. Histological analysis of gastrointestinal tissues from inoculated birds gave no evidence for true microcolony formation by NCTC12900 or intimin and aflagellar mutants to epithelial cells. However, NCTC12900 mutant derivatives associated with the mucosa were observed as individual cells and/or as large aggregates. Association with luminal contents was also noted. These data suggest that O157 organisms do not require intimin for the persistent colonization of chickens, whereas flagella do play a role in this process.
Bacterial and Host Factors Implicated in Nasal Carriage of Methicillin-Resistant
Staphylococcus aureus
in Mice
Authors: González-Zorn Bruno , Senna Jose P. M. , Fiette Laurence , Shorte Spencer , Testard Aurélie , Chignard Michel , Courvalin Patrice , Grillot-Courvalin Catherine 
Published: February 2005

ABSTRACT

Nasal carriage is a major risk factor for
Staphylococcus aureus
infection, especially for methicillin-resistant strains (MRSA). Using a mouse model of nasal carriage, we have compared several
S. aureus
strains and demonstrated increased colonization levels by MRSA in cystic fibrosis transmembrane conductance regulator-deficient mice and Toll-like receptor 2 (TLR2)-deficient mice but not TLR4-deficient mice.
Degradation of the Proapoptotic Proteins Bik, Puma, and Bim with Bcl-2 Domain 3 Homology in
Chlamydia trachomatis
-Infected Cells
Authors: Dong Feng , Pirbhai Mustak , Xiao Yangming , Zhong Youmin , Wu Yimou , Zhong Guangming 
Published: February 2005

ABSTRACT

We have previously correlated
Chlamydia trachomatis
antiapoptotic activity with the blockade of mitochondrial cytochrome
c
release and the inhibition of Bax and Bak activation. We now report that
C. trachomatis
infection leads to degradation of Bik, Puma, and Bim, three upstream proapoptotic BH3-only proteins of the Bcl-2 family that can transmit death signals to mitochondria by inhibiting the Bcl-2 antiapoptotic proteins and/or activating the Bcl-2 proapoptotic members, such as Bax and Bak. This observation has provided new information on the chlamydial antiapoptosis mechanisms.
Deletion of Highly Conserved Arginine-Rich RNA Binding Motif in Cowpea Chlorotic Mottle Virus Capsid Protein Results in Virion Structural Alterations and RNA Packaging Constraints
Authors: Annamalai Padmanaban , Apte Swapna , Wilkens Stephan , Rao A. L. N. 
Published: February 2005

ABSTRACT

The N-proximal region of cowpea chlorotic mottle virus (CCMV) capsid protein (CP) contains an arginine-rich RNA binding motif (ARM) that is also found in the CPs of other members of
Bromoviridae
and in other RNA binding proteins such as the Tat and Rev proteins of human immunodeficiency virus. To assess the critical role played by this motif during encapsidation, a variant of CCMV RNA3 (C3) precisely lacking the ARM region (C3/Δ919) of its CP gene was constructed. The biology and the competence of the matured CP derived in vivo from C3/Δ919 to assemble and package progeny RNA was examined in whole plants. Image analysis and computer-assisted three-dimensional reconstruction of wild-type and mutant virions revealed that the CP subunits bearing the engineered deletion assembled into polymorphic virions with altered surface topology. Northern blot analysis of virion RNA from mutant progeny demonstrated that the engineered mutation down-regulated packaging of all four viral RNAs; however, the packaging effect was more pronounced on genomic RNA1 and RNA2 than genomic RNA3 and its CP mRNA. In vitro assembly assays with mutant CP subunits and RNA transcripts demonstrated that the mutant CP is inherently not defective in packaging genomic RNA1 (53%) and RNA2 (54%), but their incorporation into virions was competitively inhibited by the presence of other viral RNAs. Northern blot analysis of RNA encapsidation in vivo of two distinct bromovirus RNA3 chimeras, constructed by exchanging CPs having the Δ919 deletion, demonstrated that the role of the conserved N-terminal ARM in recognizing and packaging specific RNA is distinct for each virus.
Genetic Analysis of the Human Papillomavirus Type 31 Differentiation-Dependent Late Promoter
Authors: Bodily Jason M. , Meyers Craig 
Published: February 2005

ABSTRACT
Human papillomaviruses infect stratifying squamous epithelia, causing benign and malignant lesions. Upon differentiation of the host keratinocyte, the virus undergoes a dramatic increase in both DNA replication and transcription from the late promoter, leading to expression of late genes and virion morphogenesis. In human papillomavirus type 31 (HPV31), the late promoter is designated p742 and includes multiple start sites embedded within the E7 gene. In this report, we mapped viral DNA elements that control transcriptional activity from p742. Enhancer elements in the viral upstream regulatory region positively regulate this promoter. The region containing the transcriptional start sites is dispensable for activity, and at least two separate elements in the E6/E7 region are capable of supporting transcription. Of these, we mapped one to a 150-bp region of the E7 open reading frame and designate it the core p742 promoter. Using GF109203X, an inhibitor of protein kinase C signaling, we show that p742 activation is independent of viral genome amplification. Finally, we mapped elements in the region of p742 that confer responsiveness to differentiation and show that the upstream regulatory region does not contribute to the differentiation response of p742. These studies are an important step toward understanding the functioning and regulation of this multiple-start promoter.
Identification of Sites in Adenovirus Hexon for Foreign Peptide Incorporation
Authors: Wu Hongju , Han Tie , Belousova Natalya , Krasnykh Victor , Kashentseva Elena , Dmitriev Igor , Kataram Manjula , Mahasreshti Parameshwar J. , Curiel David T. 
Published: February 2005

ABSTRACT

Adenovirus type 5 (Ad5) is one of the most promising vectors for gene therapy applications. Genetic engineering of Ad5 capsid proteins has been employed to redirect vector tropism, to enhance infectivity, or to circumvent preexisting host immunity. As the most abundant capsid protein, hexon modification is particularly attractive. However, genetic modification of hexon often results in failure of rescuing viable viruses. Since hypervariable regions (HVRs) are nonconserved among hexons of different serotypes, we investigated whether the HVRs could be used for genetic modification of hexon by incorporating oligonucleotides encoding six histidine residues (His
6
) into different HVRs in the Ad5 genome. The modified viruses were successfully rescued, and the yields of viral production were similar to that of unmodified Ad5. A thermostability assay suggested the modified viruses were stable. The His
6
epitopes were expressed in all modified hexon proteins as assessed by Western blotting assay, although the intensity of the reactive bands varied. In addition, we examined the binding activity of anti-His tag antibody to the intact virions with the enzyme-linked immunosorbent assay and found the His
6
epitopes incorporated in HVR2 and HVR5 could bind to anti-His tag antibody. This suggested the His
6
epitopes in HVR2 and HVR5 were exposed on virion surfaces. Finally, we examined the infectivities of the modified Ad vectors. The His
6
epitopes did not affect the native infectivity of Ad5 vectors. In addition, the His
6
epitopes did not appear to mediate His
6
-dependent viral infection, as assessed in two His
6
artificial receptor systems. Our study provided valuable information for studies involving hexon modification.
B1 Lymphocytes and Myeloid Dendritic Cells in Lymphoid Organs Are Preferential Extratumoral Sites of Parvovirus Minute Virus of Mice Prototype Strain Expression
Authors: Raykov Zahari , Savelyeva Larissa , Balboni Ginette , Giese Thomas , Rommelaere Jean , Giese Nathalia A. 
Published: February 2005

ABSTRACTDue to their oncolytic properties and apathogenicity, autonomous parvoviruses have attracted significant interest as possible anticancer agents. Recent preclinical studies provided evidence of the therapeutic potential of minute virus of mice prototype strain (MVMp) and its recombinant derivatives. In a murine model of hemangiosarcoma, positive therapeutic outcome correlated with high intratumoral expression of MVMp-encoded genes in tumors and lymphoid organs, especially in tumor-draining lymph nodes. The source and relevance of this extratumoral expression, which came as a surprise because of the known fibrotropism of MVMp, remained unclear. In the present study, we investigated (i) whether the observed expression pattern occurs in different tumor models, (ii) which cell population is targeted by the virus, and (iii) the immunological consequences of this infection. Significant MVMp gene expression was detected in lymphoid tissues from infected tumor-free as well as melanoma-, lymphoma-, and hemangiosarcoma-bearing mice. This expression was especially marked in lymph nodes draining virus-injected tumors. Fluorescent in situ hybridization analysis, multicolor fluorescence-activated cell sorting, and quantitative reverse transcription-PCR revealed that MVMp was expressed in rare subpopulations of CD11b (Mac1)-positive cells displaying CD11c+(myeloid dendritic cells [MDC]) or CD45B (B220+[B1 lymphocytes]) markers. Apart from the late deletion of cytotoxic memory cells (CD8+CD44+CD62L−), this infection did not lead to significant alteration of the immunological profile of cells populating lymphoid organs. However, subtle changes were detected in the production of specific proinflammatory cytokines in lymph nodes from virus-treated animals. Considering the role of B1 lymphocytes and MDC in cancer and immunological surveillance, the specific ability of these cell types to sustain parvovirus-driven gene expression may be exploited in gene therapy protocols.
3′-Terminal Sequence in Poliovirus Negative-Strand Templates Is the Primary
cis
-Acting Element Required for VPgpUpU-Primed Positive-Strand Initiation
Authors: Sharma Nidhi , O'Donnell Brian J. , Flanegan James B. 
Published: February 2005

ABSTRACT

The 5′ cloverleaf in poliovirus RNA has a direct role in regulating the stability, translation, and replication of viral RNA. In this study, we investigated the role of stem a in the 5′ cloverleaf in regulating the stability and replication of poliovirus RNA in HeLa S10 translation-replication reactions. Our results showed that disrupting the duplex structure of stem a destabilized viral RNA and inhibited efficient negative-strand synthesis. Surprisingly, the duplex structure of stem a was not required for positive-strand synthesis. In contrast, altering the primary sequence at the 5′-terminal end of stem a had little or no effect on negative-strand synthesis but dramatically reduced positive-strand initiation and the formation of infectious virus. The inhibition of positive-strand synthesis observed in these reactions was most likely a consequence of nucleotide alterations in the conserved sequence at the 3′ ends of negative-strand RNA templates. Previous studies suggested that VPgpUpU synthesized on the
cre
(2C) hairpin was required for positive-strand synthesis. Therefore, these results are consistent with a model in which preformed VPgpUpU serves as the primer for positive-strand initiation on the 3′AAUUUUGUC5′ sequence at the 3′ ends of negative-strand templates. Our results suggest that this sequence is the primary
cis
-acting element that is required for efficient VPgpUpU-primed positive-strand initiation.
Functional Domains within the Human Immunodeficiency Virus Type 2 Envelope Protein Required To Enhance Virus Production
Authors: Abada Paolo , Noble Beth , Cannon Paula M. 
Published: February 2005

ABSTRACTPrimate lentiviruses code for a protein that stimulates virus production. In human immunodeficiency virus type 1 (HIV-1), the activity is provided by the accessory protein, Vpu, while in HIV-2 and simian immunodeficiency virus it is a property of the envelope (Env) glycoprotein. Using a group of diverse retroviruses and cell types, we have confirmed the functional equivalence of the two proteins. However, despite these similarities, the two proteins have markedly different functional domains. While the Vpu activity is associated primarily with its membrane-spanning region, we have determined that the HIV-2 Env activity requires both the cytoplasmic tail and ectodomain of the protein, with the membrane-spanning domain being less important. Within the Env cytoplasmic tail, we further defined the necessary sequence as a membrane-proximal tyrosine-based motif. Providing the two Env regions separately as distinct CD8 chimeric proteins did not increase virus release. This suggests that the two domains must be either contained within a single protein or closely associated within a multiprotein oligomer, such as the Env trimer, in order to function. Finally, we observed that wild-type levels of incorporation of the HIV-2 Env into budding viruses were not required for this activity.