bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Trait Loci Controlling Vegetative Growth Rate in the Edible Basidiomycete
Pleurotus ostreatus
Authors: Larraya Luis M. , Idareta Eneko , Arana Dani , Ritter Enrique , Pisabarro Antonio G. , Ramírez Lucia 
Published: July 2002

ABSTRACT

Mycelium growth rate is a quantitative characteristic that exhibits continuous variation. This trait has applied interest, as growth rate is correlated with production yield and increased advantage against competitors. In this work, we studied growth rate variation in the edible basidiomycete
Pleurotus ostreatus
growing as monokaryotic or dikaryotic mycelium on Eger medium or on wheat straw. Our analysis resulted in identification of several genomic regions (quantitative trait loci [QTLs]) involved in the control of growth rate that can be mapped on the genetic linkage map of this fungus. In some cases monokaryotic and dikaryotic QTLs clustered at the same map position, indicating that there are principal genomic areas responsible for growth rate control. The availability of this linkage map of growth rate QTLs can help in the design of rational strain breeding programs based on genomic information.
Development and Validation of a Gamma Interferon ELISPOT Assay for Quantitation of Cellular Immune Responses to Varicella-Zoster Virus
Authors: Smith Jeffrey G. , Liu Xu , Kaufhold Robin M. , Clair James , Caulfield Michael J. 
Published: July 2002

ABSTRACTCell-mediated immunity appears to be critical for the prevention and control of varicella-zoster virus (VZV) infection and complications arising from zoster. Current assays of VZV-specific cell-mediated immunity are cumbersome or lack sensitivity. We have developed a gamma interferon ELISPOT assay that provides a direct measure of the number of T cells secreting a cytokine following stimulation with antigen. This assay is extremely sensitive and specific, with the ability to detect gamma interferon spot-forming cells (SFC) in the range of 10 to 1,000 SFC per million peripheral blood mononuclear cells (PBMCs). This assay has been validated by demonstrating the following: (i) the response detected is mediated almost entirely by CD4+T cells, (ii) ELISPOT responses from fresh-frozen PBMCs are equivalent to those from freshly isolated cells, (iii) frozen PBMCs can be shipped on dry ice for up to 48 h without loss of activity, (iv) frozen PBMC samples can be stored in liquid nitrogen over long periods (>22 months) without any significant change in response, and (v) the numbers of ELISPOTs counted using a computer-based imaging system are equivalent to those counted by humans but have lower variability. The ability to use frozen cells is facilitated by the use of a recombinant nuclease (Benzonase) that can prevent cell clumping when samples are thawed. Frozen PBMC samples can be cycled through multiple changes in storage between liquid nitrogen and dry ice without any change in response being detected. This facilitates collection of samples at one site and testing performed at a remote location. This VZV ELISPOT assay provides a new versatile tool for monitoring cellular immune responses either during a herpes zoster disease outbreak or following vaccination.
Early Events in Macrophage Killing of
Aspergillus fumigatus
Conidia: New Flow Cytometric Viability Assay
Authors: Marr Kieren A. , Koudadoust Michael , Black Michele , Balajee S. Arunmozhi 
Published: July 2002

ABSTRACT

Detailed investigations of macrophage phagocytosis and killing of
Aspergillus fumigatus
conidia have been limited by technical difficulties in quantifying fungal uptake and viability. In order to study early events in cell pathogen ingestion and killing, we developed a new flow cytometry assay that utilizes the fungus-specific viability dye FUN-1. Metabolically active
A. fumigatus
conidia accumulate orange fluorescence in vacuoles, while dormant or dead conidia stain green. After incubation within THP-1 cells, recovered conidia are costained with propidium iodide (PI) to discriminate between dormant and dead cells. Flow cytometric measurements of FUN-1 metabolism and PI uptake provide indicators of conidial viability, dormancy, and death. Conidial phagocytosis and killing are also assessed by measurement of green and orange FUN-1 fluorescence within the THP-1 cell population. Compared to previously described methods, this assay has less error introduced by membrane permeability changes and serial dilution of filamentous fungal forms. Results suggest that the THP-1 cells kill conidia rapidly (within 6 h) after exposure. Conidia that are preexposed to human serum are ingested and killed more quickly than are nonopsonized conidia.
Production of Protective Human Antipneumococcal Antibodies by Transgenic Mice with Human Immunoglobulin Loci
Authors: Russell Nina D. , Corvalan Jose R. F. , Gallo Michael L. , Davis C. Geoffrey , Pirofski Liise-anne 
Published: July 2002

ABSTRACTInfections withStreptococcus pneumoniaeremain a significant cause of morbidity and mortality. To gain insight into structure-function relationships for human antibodies to pneumococcal capsular polysaccharide (PPS), we studied the response of transgenic mice reconstituted with human immunoglobulin loci, XenoMouse, to PPS antigens in a pneumococcal vaccine. Enzyme-linked immunosorbent assays of sera from mice vaccinated with a 23-valent pneumococcal vaccine revealed that they produced serotype-specific human antibodies, with the greatest response being to the PPS of serotype 3 (PPS 3). Molecular sequence analysis of three monoclonal antibodies (MAbs) to PPS 3 generated from lymphoid cells from mice vaccinated with a 23-valent pneumococcal vaccine or a PPS 3-bovine serum albumin conjugate revealed that they all used heavy-chain immunoglobulin genes from the VH3 family, two expressed light chain genes from the human Vκ1 family, and one expressed a mouse λ light chain. The protective efficacy of the two MAbs was examined in mice. A 10-μg dose of both, and a 1-μg dose of one, significantly prolonged survival from a lethal serotype 3 infection in CBA/N mice. Our data show that XenoMouse mice produced protective, serotype-specific human antibodies to PPS 3, and they lend support to the proposal that these animals represent a useful model to study the human antibody response to PPS antigens.
Helicobacter pyloriModulates Lymphoepithelial Cell Interactions Leading to Epithelial Cell Damage through Fas/Fas Ligand Interactions
Authors: Wang Jide , Fan Xuejun , Lindholm Catharina , Bennett Michael , O'Connoll Joe , Shanahan Fergus , Brooks Edward G. , Reyes Victor E. , Ernst Peter B. 
Published: July 2002

ABSTRACTHelicobacter pyloricauses a common chronic infection of humans that leads to epithelial cell damage. Studies have shown that apoptosis of the gastric epithelium is increased during infection and this response is associated with an expansion of gastric T-helper type 1 (Th1) cells. We report that gastric T cells contribute to apoptosis of the epithelium by a Fas/Fas ligand (FasL) interaction. Fas receptor expression was detected on freshly isolated gastric epithelial cells by flow cytometry and immunohistochemistry, and this level of expression was increased during infection withH. pylori. The expression of Fas receptor on three gastric epithelial cell lines was increased byH. pylori, either alone or in combination with gamma interferon or tumor necrosis factor alpha. The role of Fas in apoptosis of gastric epithelial cell lines was evidenced by DNA fragmentation after cross-linking of Fas with specific antibodies. FasL expression was detected by immunohistochemistry on mononuclear cells in gastric biopsy specimens of infected but not uninfected subjects. Gastric T-cell lines were also shown to express FasL, as evidenced by reverse transcription-PCR and killing of target cells expressing Fas receptor. Moreover, these T-cell lines were capable of killing cultured gastric epithelial target cells and antibodies that block the interaction between Fas receptor and FasL inhibited this cytotoxic activity. These observations demonstrate that local Th1 cells may contribute to the pathogenesis of gastric disease duringH. pyloriinfection by increasing the expression of Fas on gastric epithelial cells and inducing apoptosis through Fas/FasL interactions.
Depressed Interleukin-12 (IL-12), but not IL-18, Production in Response to a 30- or 32-Kilodalton Mycobacterial Antigen in Patients with Active Pulmonary Tuberculosis
Authors: Song Chang-Hwa , Kim Hwa-Jung , Park Jeong-Kyu , Lim Jae-Hyun , Kim Un-Ok , Kim Jun-Sang , Paik Tae-Hyun , Kim Kyung-Jin , Suhr Ji-Won , Jo Eun-Kyeong 
Published: July 2002

ABSTRACTThe secreted 30-kDa antigen (Ag) ofMycobacterium tuberculosisdirectly stimulates Th1-type protective cytokine responses in healthy tuberculin reactors but not in patients with active tuberculosis (TB). To examine the cytokine profiles attributable to Th1 suppression associated with active TB, interleukin-12 (IL-12), IL-18, and IL-10 production in response to a 30- or 32-kDa Ag in 16 patients with active pulmonary TB and 24 healthy controls was investigated by enzyme-linked immunosorbent assay. In TB patients, production of IL-12 p40, as well as gamma interferon (IFN-γ), by 30- or 32-kDa Ag-stimulated peripheral blood mononuclear cells (PBMC) was significantly decreased compared with that in healthy tuberculin reactors. There were no significant differences in IL-18 production between patients and controls early during stimulation (16 h). However, PBMC from patients showed significantly enhanced IL-18 proteins after 96 h of stimulation. Similarly, higher IL-10 production was observed in the TB patients than in healthy tuberculin reactors. After 2 months of anti-TB therapy, the mean IFN-γ and IL-12 p40 production and the mean blastogenic responses were significantly increased in PBMC in the 10 TB patients who were followed up. Our findings provide evidence that depressed IL-12 in response to the 30- or 32-kDa Ag is involved in the immunopathogenesis of human active pulmonary TB.
Vacuole Acidification Is Not Required for Survival ofSalmonella entericaSerovar Typhimurium within Cultured Macrophages and Epithelial Cells
Authors: Steele-Mortimer Olivia , St-Louis Maryse , Olivier Martin , Finlay B. Brett 
Published: July 2002

ABSTRACTPhagosome acidification is an important component of the microbicidal response by infected eukaryotic cells. Thus, intracellular pathogens that reside within phagosomes must either block phagosome acidification or be able to survive at low pH. In this work, we studied the effect of phagosomal acidification on the survival of intracellularSalmonella entericaserovar Typhimurium in different cell types. Bafilomycin A1, a specific inhibitor of the vacuolar proton-ATPases, was used to block acidification of salmonella-containing vacuoles. We found that in several epithelial cell lines, treatment with bafilomycin A1 had no effect on intracellular survival or replication. Furthermore, although acidification was essential forSalmonellaintracellular survival in J774 cultured macrophages, as reported previously (13), it is not essential in other macrophage cell lines. These data suggest that vacuolar acidification may play a role in intracellular survival of salmonellae only under certain conditions and in specific cell types.
Shiga Toxin Translocation across Intestinal Epithelial Cells Is Enhanced by Neutrophil Transmigration
Authors: Hurley Bryan P. , Thorpe Cheleste M. , Acheson David W. K. 
Published: July 2002

ABSTRACT

Shiga toxin-producing
E. coli
(STEC) is a food-borne pathogen that causes serious illness, including hemolytic-uremic syndrome (HUS). STEC colonizes the lower intestine and produces Shiga toxins (Stxs). Stxs appear to translocate across intestinal epithelia and affect sensitive endothelial cell beds at various sites. We have previously shown that Stxs cross polarized intestinal epithelial cells (IECs) via a transcellular route and remain biologically active. Since acute inflammatory infiltration of the gut and fecal leukocytes is seen in many STEC-infected patients and since polymorphonuclear leukocyte (PMN) transmigration across polarized IECs diminishes the IEC barrier function in vitro, we hypothesized that PMN transmigration may enhance Stx movement across IECs. We found that basolateral-to-apical transmigration of neutrophils significantly increased the movement of Stx1 and Stx2 across polarized T84 IECs in the opposite direction. The amount of Stx crossing the T84 barrier was proportional to the degree of neutrophil transmigration, and the increase in Stx translocation appears to be due to increases in paracellular permeability caused by migrating PMNs. STEC clinical isolates applied apically induced PMN transmigration across and interleukin-8 (IL-8) secretion from T84 cells. Of the 10 STEC strains tested, three STEC strains lacking
eae
and
espB
(
eae
- and
espB
-negative STEC strains) induced significantly more neutrophil transmigration and significantly greater IL-8 secretion than
eae
- and
espB
-positive STEC or enteropathogenic
E. coli
. This study suggests that STEC interaction with intestinal epithelia induces neutrophil recruitment to the intestinal lumen, resulting in neutrophil extravasation across IECs, and that during this process Stxs may pass in greater amounts into underlying tissues, thereby increasing the risk of HUS.
Characterization of
sarR
, a Modulator of
sar
Expression in
Staphylococcus aureus
Authors: Manna Adhar , Cheung Ambrose L. 
Published: July 2002

ABSTRACT

The expression of virulence determinants in
Staphylococcus aureus
is controlled by global regulatory loci (e.g.,
sar
and
agr
). The
sar
locus is composed of three overlapping transcripts (
sar
P1, P3, and P2 transcripts from P1, P3, and P2 promoters, respectively), all encoding the 372-bp
sarA
gene. The level of SarA, the major regulatory protein, is partially controlled by the differential activation of
sar
promoters. We previously partially purified a ∼12 kDa protein with a DNA-specific column containing a
sar
P2 promoter fragment. In this study, the putative gene, designated
sarR
, was identified and found to encode a 13.6-kDa protein with homology to SarA. Transcriptional and immunoblot studies revealed the
sarR
gene to be expressed in other staphylococcal strains. Recombinant SarR protein bound
sar
P1, P2, and P3 promoter fragments in gel shift and footprinting assays. A
sarR
mutant expressed a higher level of P1 transcript than the parent, as confirmed by promoter green fluorescent protein fusion assays. As the P1 transcript is the predominant
sar
transcript, we confirmed that the
sarR
mutant expressed more SarA than the parental strain. We thus proposed that SarR is a regulatory protein that binds to the
sar
promoters to down-regulate P1 transcription and the ensuing SarA protein expression.
Human Endothelial Cell Activation and Mediator Release in Response toListeria monocytogenesVirulence Factors
Authors: Rose Frank , Zeller Sven-Arne , Chakraborty Trinad , Domann Eugen , Machleidt Thomas , Kronke Martin , Seeger Werner , Grimminger Friedrich , Sibelius Ulf 
Published: July 2002

ABSTRACTThe interaction ofListeria monocytogeneswith endothelial cells represents a crucial step in the pathogenesis of listeriosis. Incubation of human umbilical vein endothelial cells (HUVEC) with wild-typeL. monocytogenes(EGD) provoked immediate strong NO synthesis, attributable to listerial presentation of listeriolysin O (LLO), as the NO release was missed upon employment of a deletion mutant for LLO (EGDhlymutant) and was reproduced by purified LLO. Studies of conditions lacking extracellular Ca2+suggested LLO-elicited Ca2+flux as the underlying mechanism. In addition, HUVEC incubation with EGD turned out to be a potent stimulus for sustained (>12-h) upregulation of proinflammatory cytokine generation (interleukin 6 [IL-6], IL-8, and granulocyte-macrophage colony-stimulating factor). Use of deletion mutants for LLO (EGDhlymutant), listerial phosphatidylinositol-specific phospholipase C (EGDplcAmutant), broad-spectrum phospholipase C (EGDplcBmutant) and internalin B (EGDinlBmutant), as well as purified LLO, identified LLO as largely responsible for the cytokine response. Endothelial cells responded with diacylglycerole and ceramide generation as well as nuclear translocation of NF-κB to the stimulation with the LLO-producing strains EGD andListeria innocua. The endothelial PC-phospholipase C inhibitor tricyclodecan-9-yl-xanthogenate as well as two independent inhibitors of NF-κB activation, pyrolidine dithiocarbamate and caffeic acid phenethyl ester, suppressed both the NF-κB translocation and the upregulation of cytokine synthesis. We conclude thatL. monocytogenesis a potent stimulus of NO release and sustained upregulation of proinflammatory cytokine synthesis in human endothelial cells, both events being largely attributable to LLO presentation. LLO-induced transmembrane Ca2+flux as well as a sequence of endothelial phospholipase activation and the appearance of diacylglycerole, ceramide, and NF-κB are suggested as underlying host signaling events. These endothelial responses toL. monocytogenesmay well contribute to the pathogenic sequelae in severe listerial infection and sepsis.
Long-Range Mapping of the
Streptococcus agalactiae
Phylogenetic Lineage Restriction Digest Pattern Type III-3 Reveals Clustering of Virulence Genes
Authors: Bohnsack John F. , Whiting April A. , Bradford Russell D. , Van Frank Brenna K. , Takahashi Shinji , Adderson Elisabeth E. 
Published: July 2002

ABSTRACT

Human isolates of serotype III
Streptococcus agalactiae
(group B streptococcus [GBS]) can be divided into three separate phylogenetic lineages based on analysis of the restriction digest patterns (RDPs) of chromosomal DNA. Nine DNA sequences that are present in all isolates of the RDP III-3 phylogenetic lineage, but not in the other lineages, were identified by genomic subtractive hybridization. A complete physical map of a III-3 chromosome was constructed. Six of the nine III-3-specific sequences mapped to a 340-kb
Sse
8387I fragment which contains or is located close to known GBS virulence genes. One of the III-3-specific probes, AW-10, encodes part of GBSi1, a group II intron that is inserted at two sites within the GBS genome. The second chromosomal site for GBSi1 was isolated, sequenced, and mapped to a location near the locus responsible for hemolysin production. These findings suggest that the genetic variation that distinguishes the RDP type III-3 strains from other serotype III strains occurs largely within localized areas of the genome containing known or putative virulence genes.
Insertional Inactivation of
eap
in
Staphylococcus aureus
Strain Newman Confers Reduced Staphylococcal Binding to Fibroblasts
Authors: Hussain Muzaffar , Haggar Axana , Heilmann Christine , Peters Georg , Flock Jan-Ingmar , Herrmann Mathias 
Published: July 2002

ABSTRACT

To initiate invasive infection,
Staphylococcus aureus
must adhere to host substrates, such as the extracellular matrix or eukaryotic cells, by virtue of different surface proteins (adhesins). Recently, we identified a 60-kDa cell-secreted extracellular adherence protein (Eap) of
S. aureus
strain Newman with broad-spectrum binding characteristics (M. Palma, A. Haggar, and J. I. Flock, J. Bacteriol. 181:2840-2845, 1999), and we have molecularly confirmed Eap to be an analogue of the previously identified major histocompatibility complex class II analog protein (Map) (M. Hussain, K. Becker, C. von Eiff, G. Peter, and M. Herrmann, Clin. Diagn. Lab. Immunol. 8:1281-1286, 2001). Previous analyses of the Eap/Map function performed with purified protein did not allow dissection of its precise role in the complex situation of the staphylococcal whole cell presenting several secreted and wall-bound adhesins. Therefore, the role of Eap was investigated by constructing a stable
eap
::
ermB
deletion in strain Newman and by complementation of the mutant. Patterns of extracted cell surface proteins analyzed both by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by Western ligand assays with various adhesive matrix molecules clearly confirmed the absence of Eap in the mutant. However, binding and adhesion tests using whole staphylococcal cells demonstrated that both the parent and mutant strains bound equally well to fibronectin- and fibrinogen-coated surfaces, possibly due to their recognition by other staphylococcal adhesins. Furthermore, Eap mediated staphylococcal agglutination of both wild-type and mutant cells. In contrast, the mutant adhered to a significantly lesser extent to cultured fibroblasts (
P
< 0.001) than did the wild type, while adherence was restorable upon complementation. Furthermore, adherence to both epithelial cells (
P
< 0.05) and fibroblasts (not significant) could be blocked with antibodies against Eap, whereas preimmune serum was not active. In conclusion, Eap may contribute to pathogenicity by promoting adhesion of whole staphylococcal cells to complex eukaryotic substrates.
Purification, Characterization, and Immunogenicity of the Refolded Ectodomain of the
Plasmodium falciparum
Apical Membrane Antigen 1 Expressed in
Escherichia coli
Authors: Dutta Sheetij , Lalitha P. V. , Ware Lisa A. , Barbosa Arnoldo , Moch J. Kathleen , Vassell Meredith A. , Fileta Bader B. , Kitov Svetlana , Kolodny Nelly , Heppner D. Gray , Haynes J. David , Lanar David E. 
Published: July 2002

ABSTRACT

The apical membrane antigen 1 (AMA1) has emerged as a promising vaccine candidate against malaria. Advanced evaluation of its protective efficacy in humans requires the production of highly purified and correctly folded protein. We describe here a process for the expression, fermentation, refolding, and purification of the recombinant ectodomain of AMA1 (amino acids 83
Gly
to 531
Glu
) of
Plasmodium falciparum
(3D7) produced in
Escherichia coli.
A synthetic gene containing an
E. coli
codon bias was cloned into a modified pET32 plasmid, and the recombinant protein was produced by using a redox-modified
E. coli
strain, Origami (DE3). A purification process was developed that included Sarkosyl extraction followed by affinity purification on a Ni-nitrilotriacetic acid column. The recombinant AMA1 was refolded in the presence of reduced and oxidized glutathione and further purified by using two ion-exchange chromatographic steps. The final product, designated AMA1/E, was homogeneous, monomeric, and >99% pure and had low endotoxin content and low host cell contamination. Analysis of AMA1/E showed that it had the predicted primary sequence, and tertiary structure analysis confirmed its compact disulfide-bonded nature. Rabbit antibodies made to the protein recognized the native parasite AMA1 and inhibited the growth of the
P. falciparum
homologous 3D7 clone in an in vitro assay. Reduction-sensitive epitopes on AMA1/E were shown to be necessary for the production of inhibitory anti-AMA1 antibodies. AMA1/E was recognized by a conformation-dependent, growth-inhibitory monoclonal antibody, 4G2dc1. The process described here was successfully scaled up to produce AMA1/E protein under GMP conditions, and the product was found to induce highly inhibitory antibodies in rabbits.