bluuebunny/crossref_metadata_2025_split huggingface dataset:
ADP and Other Metabolites Released fromAcanthamoeba castellaniiLead to Human Monocytic Cell Death through Apoptosis and Stimulate the Secretion of Proinflammatory Cytokines
Authors: Mattana A. , Cappai V. , Alberti L. , Serra C. , Fiori P. L. , Cappuccinelli P. 
Published: July 2002

ABSTRACTMonocytes/macrophages are thought to be involved inAcanthamoebainfections. The aim of this work was to study whether soluble metabolites (ADP and other compounds) released byAcanthamoeba castellaniitrophozoites could induce morphological and biochemical changes in human monocytic cells in vitro. We demonstrate here that ADP constitutively released in the medium byA. castellanii, interacting with specific P2y2purinoceptors expressed on the monocytic cell membrane, caused a biphasic rise in [Ca2+]i, morphological changes characteristics of cells undergoing apoptosis, caspase-3 activation, and secretion of tumor necrosis factor alpha (TNF-α). The same results were found in monocytes exposed to purified ADP. Cell damage and TNF-α release induced by amoebic ADP were blocked by the P2y2inhibitor suramin. Other metabolites contained in amoebic cell-free supernatants, with molecular masses of, respectively, >30 kDa and between 30 and 10 kDa, also caused morphological modifications and activation of intracellular caspase-3, characteristics of programmed cell death. Nevertheless, mechanisms by which these molecules trigger cell damage appeared to differ from that of ADP. In addition, other amoebic thermolable metabolites with molecular masses of <10 kDa caused the secretion of interleukin-1β. These findings suggest that pathogenic free-livingA. castellaniiby release of ADP and other metabolites lead to human monocytic cell death through apoptosis and stimulate the secretion of proinflammatory cytokines.
Immunochemical Properties of the Staphylococcal Poly-
N
-Acetylglucosamine Surface Polysaccharide
Authors: Maira-Litrán Tomás , Kropec Andrea , Abeygunawardana C. , Joyce Joseph , Mark George , Goldmann Donald A. , Pier Gerald B. 
Published: July 2002

ABSTRACT

Staphylococcus aureus
and
Staphylococcus epidermidis
often elaborate adherent biofilms, which contain the capsular polysaccharide-adhesin (PS/A) that mediates the initial cell adherence to biomaterials. Biofilm cells produce another antigen, termed polysaccharide intercellular adhesin (PIA), which is composed of a ∼28 kDa soluble linear β(1-6)-linked
N
-acetylglucosamine. We developed a new method to purify PS/A from
S. aureus
MN8m, a strain hyperproducing PS/A. Using multiple analytical techniques, we determined that the chemical structure of PS/A is also β(1-6)-
N
-acetylglucosamine (PNAG). We were unable to find
N
-succinylglucosamine residues in any of our preparations in contrast to previously reported findings (D. McKenney, K. Pouliot, Y. Wang, V. Murthy, M. Ulrich, G. Doring, J. C. Lee, D. A Goldmann, and G. B. Pier, Science 284:1523-1527, 1999). PNAG was produced with a wide range of molecular masses that could be divided into three major fractions with average molecular masses of 460 kDa (PNAG-I), 100 kDa (PNAG-II), and 21 kDa (PNAG-III). The purified antigens were not soluble at neutral pH unless first dissolved in 5 M HCl and then neutralized with 5 M NaOH. PNAG-I was very immunogenic in rabbits, but the responses of individual animals were variable. Immunization of mice with various doses (100, 50, or 10 μg) of PNAG-I, -II, and -III demonstrated that only PNAG-I was able to elicit an immunoglobulin G (IgG) immune response with the highest titers obtained with 100-μg dose. When we purified a small fraction of PNAG with a molecular mass of ∼780 kDa (PNAG-780) from PNAG-I, significantly higher IgG titers than those in mice immunized with the same doses of PNAG-I were obtained, suggesting the importance of the molecular mass of PNAG in the antibody response. These results further clarify the chemical structure of PS/A and help to differentiate it from PIA on the basis of immunogenicity, molecular size, and solubility.
High-Level Expression of the Malaria Blood-Stage Vaccine CandidatePlasmodium falciparumApical Membrane Antigen 1 and Induction of Antibodies That Inhibit Erythrocyte Invasion
Authors: Kocken Clemens H. M. , Withers-Martinez Chrislaine , Dubbeld Martin A. , van der Wel Annemarie , Hackett Fiona , Blackman Michael J. , Thomas Alan W. 
Published: July 2002

ABSTRACTApical membrane antigen 1 (AMA-1) is a highly promising malaria blood-stage vaccine candidate that has induced protection in rodent and nonhuman primate models of malaria. Authentic conformation of the protein appears to be essential for the induction of parasite-inhibitory antibody responses. Here we have developed a synthetic gene with adapted codon usage to allow expression ofPlasmodium falciparumFVO strain AMA-1 (PfAMA-1) inPichia pastoris. In addition, potential N-glycosylation sites were changed, exploiting the lack of conservation of these sites inPlasmodium, to obtain high-level secretion of a homogeneous product, suitable for scale-up according to current good manufacturing procedures. Purified PfAMA-1 displayed authentic antigenic properties, indicating that the amino acid changes had no deleterious effect on the conformation of the protein. High-titer antibodies, raised in rabbits, reacted strongly with homologous and heterologousP. falciparumby immunofluorescence. In addition, purified immunoglobulin G from immunized animals strongly inhibited invasion of red blood cells by homologous and, to a somewhat lesser extent, heterologousP. falciparum.
Sequential Expression of the Neuropeptides Substance P and Somatostatin in Granulomas Associated with Murine Cysticercosis
Authors: Robinson Prema , White A. Clinton , Lewis Dorothy E. , Thornby John , David Elliott , Weinstock Joel 
Published: July 2002

ABSTRACTNeurocysticercosis, a parasitic infection of the human central nervous system caused byTaenia solium, is a leading cause of seizures. Seizures associated with neurocysticercosis are caused mainly by the host inflammatory responses to dying parasites in the brain parenchyma. We previously demonstrated sequential expression of Th1 cytokines in early-stage granulomas, followed by expression of Th2 cytokines in later-stage granulomas in murine cysticercosis. However, the mechanism leading to this shift in cytokine response in the granulomas is unknown. Neuropeptides modulate cytokine responses and granuloma formation in murine schistosomiasis. Substance P (SP) induces Th1 cytokine expression and granuloma formation, whereas somatostatin inhibits the granulomatous response. We hypothesized that neuropeptides might play a role in regulation of the granulomatous response in cysticercosis. To test this hypothesis, we compared expression of SP and expression of somatostatin in murine cysticercal granulomas by using in situ hybridization and immunohistochemistry. We also compared expression with granuloma stage. Expression of SP mRNA was more frequent in the early-stage granulomas than in the late-stage granulomas (34 of 35 early-stage granulomas versus 1 of 13 late-stage granulomas). By contrast, somatostatin was expressed primarily in later-stage granulomas (13 of 14 late-stage granulomas versus 2 of 35 early-stage granulomas). The median light microscope grade of SP mRNA expression in the early-stage granulomas was significantly higher than that in the late-stage granulomas (P= 0.008, as determined by the Wilcoxon signed rank test). By contrast, somatostatin mRNA expression was higher at later stages (P= 0.008, as determined by the Wilcoxon signed rank test). SP and somatostatin are therefore temporally expressed in granulomas associated with murine cysticercosis, which may be related to differential expression of Th1 and Th2 cytokines.
Identification ofEscherichia coliOuter Membrane Protein A Receptor on Human Brain Microvascular Endothelial Cells
Authors: Prasadarao Nemani V. 
Published: July 2002

ABSTRACTNeonatalEscherichia colimeningitis continues to be a diagnostic and treatment challenge despite the availability of active antibiotics. Our earlier studies have shown that outer membrane protein A (OmpA) is one of the major factors responsible forEscherichia colitraversal across the blood-brain barrier that constitutes a lining of brain microvascular endothelial cells (BMEC). In this study we showed that OmpA binds to a 95-kDa human BMEC (HBMEC) glycoprotein (Ecgp) forE. coliinvasion. Ecgp was partially purified by wheat germ agglutinin andMaackia amurensislectin (MAL) affinity chromatography. The MAL affinity-purified HBMEC proteins bound to OmpA+E. colibut not to OmpA−E. coli. In addition, the deglycosylated MAL-bound proteins still interact with OmpA+E. coli, indicating the role of protein backbone in mediating the OmpA binding to HBMEC. Interestingly, the MAL affinity-bound fraction showed one more protein, a 65-kDa protein that bound to OmpA+E. coliin addition to Ecgp. Further, the 65-kDa protein was shown to be a cleavage product of Ecgp. Immunocytochemistry of HBMEC infected with OmpA+E. coliby using anti-Ecgp antibody suggests that Ecgp clusters at theE. colientry site. Anti-Ecgp antibody also reacted to microvascular endothelium on human brain tissue sections, indicating the biological relevance of Ecgp inE. colimeningitis. Partial N-terminal amino acid sequence of Ecgp suggested that it has 87% sequence homology to gp96, an endoplasmic reticulum-resident molecular chaperone that is often expressed on the cell surface. In contrast, the 65-kDa protein, which could be the internal portion of Ecgp, showed 70% sequence homology to an S-fimbria-binding sialoglycoprotein reported earlier. These results suggest that OmpA interacts with Ecgp via the carbohydrate epitope, as well as with the protein portion for invading HBMEC.
Effects of Oral Vaccination and Immunomodulation by Cholera Toxin on ExperimentalHelicobacter pyloriInfection, Reinfection, and Gastritis
Authors: Raghavan S. , Svennerholm A.-M. , Holmgren J. 
Published: July 2002

ABSTRACTTherapeutic vaccination is an attractive strategy to control infection and disease caused byHelicobacter pylori. In mice infected withH. pyloriwe have studied the protective effect of oral immunization with anH. pylorilysate preparation given together with the mucosal adjuvant cholera toxin (CT), both against the initial infection and against a later reinfection challenge. We have also examined the effects of treatment with the CT adjuvant alone onH. pyloriinfection and reinfection. Specific immunization with lysate was found to result in a sixfold reduction of the extent (bacterial load) of the primary infection and also to provide similar levels of protection against reinfection. However, these effects were associated with severe postimmunization gastritis. In contrast, oral treatment with CT alone at the time of initial infection, while unable to suppress the initial infection, gave rise to a 20-fold reduction in bacterial load upon reinfection without causing any associated gastric inflammation. Both the infected animals that were specifically immunized and those that were treated with CT only displayed increased in vitro proliferative responses of mononuclear cells toH. pyloriantigens. Antibody levels in response toH. pyloriwere on the other hand only marginally increased after treatment with CT, whereas they were markedly elevated after immunization with lysate plus CT, with a rise in both (Th2-driven) immunoglobulin G1 (IgG1) and, especially, (Th1-driven) IgG2a antibodies. The results illustrate the complex balance between protection and harmful inflammation after postinfection vaccination againstH. pylorias studied in a mouse model.
Old Mice Express a Transient Early Resistance to Pulmonary Tuberculosis That Is Mediated by CD8 T Cells
Authors: Turner Joanne , Frank Anthony A. , Orme Ian M. 
Published: July 2002

ABSTRACTDuring the natural aging process the immune system undergoes many alterations. In particular, both the CD4 and CD8 T-cell compartments become compromised, and these changes have serious implications for the capacity of the elderly to control infection. As a result, the elderly are more susceptible to many infectious diseases, including primary infection and reactivation of latent infections. In this study we addressed the capacity of old mice to control an infection withMycobacterium tuberculosisand to characterize the mechanism by which old mice, paradoxically, can express a transient early resistance to infection. This resistance was shown to be associated with the presence of CD8 T cells within the lungs that were capable of secreting gamma interferon, as illustrated by the demonstration that early resistance was lost in aged CD8 gene-disrupted mice. These studies therefore show that, despite a documented decline in general CD8 T-cell responsiveness in the elderly, a subset of CD8 T cells is an important early mediator of protection in the lungs of old mice that have been infected withM. tuberculosis.
Mobilization of Protein Kinase C in Macrophages Induced byListeria monocytogenesAffects Its Internalization and Escape from the Phagosome
Authors: Wadsworth Sandra J. , Goldfine Howard 
Published: July 2002

ABSTRACTListeriolysin O (LLO) and a phosphatidylinositol-specific phospholipase C (PI-PLC) are known virulence factors ofListeria monocytogenesin both tissue cultures and the murine model of infection. LLO is a member of a family of pore-forming cholesterol-dependent cytotoxins and is known to play an essential role in escape from the primary phagocytic vacuole of macrophages. PI-PLC plays an accessory role, in that PI-PLC mutants are partially defective in escape. We have shown that both of these molecules are essential for initiating rapid increases in the calcium level in the J774 murine macrophage cell line (S. J. Wadsworth and H. Goldfine, Infect. Immun.67:1770-1778, 1999). Here we show that both LLO and PI-PLC are required for translocation of protein kinase C δ (PKC δ) to the periphery of J774 cells and for translocation of PKC β II to early endosomes beginning within the first minute after addition of bacteria to the culture medium. Treatment with the calcium channel blocker SK&F 96365 inhibited translocation of PKC β II but not PKC δ. Our findings lead us to propose a host signaling pathway requiring LLO and the formation of diacylglycerol by PI-PLC in which calcium-independent PKC δ is responsible for the initial calcium signal and the subsequent PKC β II translocation. LLO-dependent translocation of PKC β I to early endosomes also occurs between 1 and 4 min after infection, but this occurs in the absence of PI-PLC. All of these signals were observed in cells that had not internalized bacteria. Blocking PKC β translocation with hispidin resulted in more rapid uptake of wild-type bacteria and greatly reduced escape from the primary phagocytic vacuoles of J774 cells.
Natural Resistance to Inhibitors of the Ubiquinol Cytochrome
c
Oxidoreductase of
Rubrivivax gelatinosus
: Sequence and Functional Analysis of the Cytochrome
bc
1
Complex
Authors: Ouchane Soufian , Agalidis Ileana , Astier Chantal 
Published: July 2002

ABSTRACT

Biochemical analyses of
Rubrivivax gelatinosus
membranes have revealed that the cytochrome
bc
1
complex is highly resistant to classical inhibitors including myxothiazol, stigmatellin, and antimycin. This is the first report of a strain exhibiting resistance to inhibitors of both catalytic Q
0
and Q
i
sites. Because the resistance to cytochrome
bc
1
inhibitors is primarily related to the cytochrome
b
primary structure, the
petABC
operon encoding the subunits of the cytochrome
bc
1
complex of
Rubrivivax gelatinosus
was sequenced. In addition to homologies to the corresponding proteins from other organisms, the deduced amino acid sequence of the cytochrome
b
polypeptide shows (i) an E303V substitution in the highly conserved PEWY loop involved in quinol/stigmatellin binding, (ii) other substitutions that could be involved in resistance to cytochrome
bc
1
inhibitors, and (iii) 14 residues instead of 13 between the histidines in helix IV that likely serve as the second axial ligand to the b
H
and b
L
hemes, respectively. These characteristics imply different functional properties of the cytochrome
bc
1
complex of this bacterium. The consequences of these structural features for the resistance to inhibitors and for the properties of
R. gelatinosus
cytochrome
bc
1
are discussed with reference to the structure and function of the cytochrome
bc
1
complexes from other organisms.
FtsA Mutants ofBacillus subtilisImpaired in Sporulation
Authors: Kemp Jennifer T. , Driks Adam , Losick Richard 
Published: July 2002

ABSTRACTSpore formation inBacillus subtilisinvolves a switch in the site of cell division from the midcell to a polar position. Both medial division and polar division are mediated in part by the actin-like, cytokinetic protein FtsA. We report the isolation of an FtsA mutant (FtsAD265G) that is defective in sporulation but is apparently unimpaired in vegetative growth. Sporulating cells of the mutant reach the stage of asymmetric division but are partially blocked in the subsequent morphological process of engulfment. As judged by fluorescence microscopy and electron microscopy, the FtsAD265Gmutant produces normal-looking medial septa but immature (abnormally thin) polar septa. The mutant was unimpaired in transcription under the control of Spo0A, the master regulator for entry into sporulation, but was defective in transcription under the control of σF, a regulatory protein whose activation is known to depend on polar division. An amino acid substitution at a residue (Y264) adjacent to D265 also caused a defect in sporulation. D265 and Y264 are conserved among endospore-forming bacteria, raising the possibility that these residues are involved in a sporulation-specific protein interaction that facilitates maturation of the sporulation septum and the activation of σF.
Burkholderia thailandensis
E125 Harbors a Temperate Bacteriophage Specific for
Burkholderia mallei
Authors: Woods Donald E. , Jeddeloh Jeffrey A. , Fritz David L. , DeShazer David 
Published: July 2002

ABSTRACT

Burkholderia thailandensis
is a nonpathogenic gram-negative bacillus that is closely related to
Burkholderia mallei
and
Burkholderia pseudomallei
. We found that
B. thailandensis
E125 spontaneously produced a bacteriophage, termed φE125, which formed turbid plaques in top agar containing
B. mallei
ATCC 23344. We examined the host range of φE125 and found that it formed plaques on
B. mallei
but not on any other bacterial species tested, including
B. thailandensis
and
B. pseudomallei
. Examination of the bacteriophage by transmission electron microscopy revealed an isometric head and a long noncontractile tail.
B. mallei
NCTC 120 and
B. mallei
DB110795 were resistant to infection with φE125 and did not produce lipopolysaccharide (LPS) O antigen due to IS
407
A insertions in
wbiE
and
wbiG
, respectively.
wbiE
was provided in
trans
on a broad-host-range plasmid to
B. mallei
NCTC 120, and it restored LPS O-antigen production and susceptibility to φE125. The 53,373-bp φE125 genome contained 70 genes, an IS
3
family insertion sequence (IS
Bt
3), and an attachment site (
attP
) encompassing the 3′ end of a proline tRNA (UGG) gene. While the overall genetic organization of the φE125 genome was similar to λ-like bacteriophages and prophages, it also possessed a novel cluster of putative replication and lysogeny genes. The φE125 genome encoded an adenine and a cytosine methyltransferase, and purified bacteriophage DNA contained both N6-methyladenine and N4-methylcytosine. The results presented here demonstrate that φE125 is a new member of the λ supergroup of
Siphoviridae
that may be useful as a diagnostic tool for
B. mallei
.
AmtB Is Necessary for NH
4
+
-Induced Nitrogenase Switch-Off and ADP-Ribosylation in
Rhodobacter capsulatus
Authors: Yakunin Alexander F. , Hallenbeck Patrick C. 
Published: July 2002

ABSTRACT

Rhodobacter capsulatus
possesses two genes potentially coding for ammonia transporters,
amtB
and
amtY
. In order to better understand their role in the physiology of this bacterium and their possible significance in nitrogen fixation, we created single-knockout mutants. Strains mutated in either
amtB
or
amtY
did not show a growth defect under any condition tested and were still capable of taking up ammonia at nearly wild-type rates, but an
amtB
mutant was no longer capable of transporting methylamine. The
amtB
strain but not the
amtY
strain was also totally defective in carrying out ADP-ribosylation of Fe-protein or the switch-off of in vivo nitrogenase activity in response to NH
4
+
addition. ADP-ribosylation in response to darkness was unaffected in
amtB
and
amtBY
strains, and glutamine synthetase activity was normally regulated in these strains in response to ammonium addition, suggesting that one role of AmtB is to function as an ammonia sensor for the processes that regulate nitrogenase activity.
The
Vibrio cholerae vieSAB
Locus Encodes a Pathway Contributing to Cholera Toxin Production
Authors: Tischler Anna D. , Lee Sang Ho , Camilli Andrew 
Published: July 2002

ABSTRACT

The genes encoding cholera toxin (CT),
ctxAB
, are coregulated with those for other
Vibrio cholerae
virulence factors by a cascade of transcriptional activators, including ToxR, TcpP, and ToxT. Additional regulators that modulate expression of
ctxAB
during infection were recently identified in a genetic selection. A transposon insertion in
vieS
, the sensor kinase of the VieSAB three-component signal transduction system, resulted in failure to induce expression of a
ctxA
-recombinase fusion during murine infection. To determine which components of the VieSAB system are essential for CT regulation,
ctxAB
transcript levels were assessed by RNase protection assay in various
vieSAB
in-frame deletion mutants after growth in vitro under virulence gene inducing conditions. A threefold reduction in
ctxAB
transcript levels was observed for the Δ
vieSAB
strain; consistent with this, the Δ
vieSAB
strain produced twofold less CT protein than the wild type, and this defect was complementable in
trans
. These results suggest that the VieSAB three-component system is required for full activation of the
ctxAB
operon during in vitro growth as well as during infection. The VieSAB system may regulate
ctxAB
expression indirectly by affecting production of ToxT, because decreased
toxT
transcript levels were observed in the Δ
vieSAB
strain.
Antigen 43-Mediated Autotransporter Display, a Versatile Bacterial Cell Surface Presentation System
Authors: Kjærgaard Kristian , Hasman Henrik , Schembri Mark A. , Klemm Per 
Published: July 2002

ABSTRACTAntigen 43 (Ag43), a self-recognizing outer membrane protein ofEscherichia coli, has been converted into an efficient and versatile tool for surface display of foreign protein segments. Ag43 is an autotransporter protein characterized by the feature that all information required for transport to the outer membrane and secretion through the cell envelope is contained within the protein itself. Ag43 consists of two subunits (α and β), where the β-subunit forms an integral outer membrane translocator to which the α-subunit is noncovalently attached. The simplicity of the Ag43 system makes it ideally suited as a surface display scaffold. Here we demonstrate that the Ag43 α-module can accommodate and display correctly folded inserts and has the ability to display entire functional protein domains, exemplified by the FimH lectin domain. The presence of heterologous cysteine bridges does not interfere with surface display, and Ag43 chimeras are correctly processed into α- and β-modules, offering optional and easy release of the chimeric α-subunits. Furthermore, Ag43 can be displayed in many gram-negative bacteria. This feature is exploited for display of our chimeras in an attenuatedSalmonellastrain.