bluuebunny/crossref_metadata_2025_split huggingface dataset:
Succession of Indigenous
Pseudomonas
spp. and Actinomycetes on Barley Roots Affected by the Antagonistic Strain
Pseudomonas fluorescens
DR54 and the Fungicide Imazalil
Authors: Thirup Laila , Johnsen Kaare , Winding Anne 
Published: July 2002

ABSTRACT

In recent years, the interest in the use of bacteria for biological control of plant-pathogenic fungi has increased. We studied the possible side effects of coating barley seeds with the antagonistic strain
Pseudomonas fluorescens
DR54 or a commercial fungicide, imazalil. This was done by monitoring the number of indigenous
Pseudomonas
organisms and actinomycetes on barley roots during growth in soil, harvest after 50 days, and subsequent decomposition. Bacteria were enumerated by traditional plate spreading on Gould's S1 agar (
Pseudomonas
) and as filamentous colonies on Winogradsky agar (actinomycetes) and by two quantitative competitive PCR assays. For this we developed an assay targeting
Streptomyces
and closely related genera. DR54 constituted more than 75% of the
Pseudomonas
population at the root base during the first 21 days but decreased to less than 10% at day 50. DR54 was not successful in colonizing root tips. Initially, DR54 affected the number of indigenous
Pseudomonas
organisms negatively, whereas imazalil affected
Pseudomonas
numbers positively, but the effects were transient. Although plate counts were considerably lower than the number of DNA copies, the two methods correlated well for
Pseudomonas
during plant growth, but after plant harvest
Pseudomonas
-specific DNA copy numbers decreased while plate counts were in the same magnitude as before. Hence,
Pseudomonas
was 10-fold more culturable in a decomposition environment than in the rhizosphere. The abundance of actinomycetes was unaffected by DR54 or imazalil amendments, and CFU and quantitative PCR results correlated throughout the experiment. The abundance of actinomycetes increased gradually, mostly in numbers of DNA copies, confirming their role in colonizing old roots.
Morphological and Physiological Characterization of
Listeria monocytogenes
Subjected to High Hydrostatic Pressure
Authors: Ritz M. , Tholozan J. L. , Federighi M. , Pilet M. F. 
Published: July 2002

ABSTRACT

High hydrostatic pressure is a new food preservation technology known for its capacity to inactivate spoilage and pathogenic microorganisms. That inactivation is usually assessed by the number of colonies growing on solid media after treatment. Under normal conditions the method does not permit recovery of damaged cells and may underestimate the number of cells that will remain viable and grow after a few days in high-pressure-processed foodstuffs. This study investigated the damage inflicted on
Listeria monocytogenes
cells treated by high pressure for 10 min at 400 MPa in pH 5.6 citrate buffer. Under these conditions, no cell growth occurred after 48 h on plate count agar. Scanning electron microscopy, light scattering by flow cytometry, and cell volume measurements were compared to evaluate the morphological changes in cells after pressurization. All these methods revealed that cellular morphology was not really affected. Esterase activity, as assessed either by enzymatic activity assays or by carboxy fluorescein diacetate fluorescence monitored by flow cytometry, was dramatically lowered, but not totally obliterated, under the effects of treatment. The measurement of propidium iodide uptake followed by flow cytometry demonstrated that membrane integrity was preserved in a small part of the population, although the membrane potential measured by analytical methods or evaluated by oxonol uptake was reduced from −86 to −5 mV. These results showed that such combined methods as fluorescent dyes monitored by flow cytometry and physiological activity measurements provide valuable indications of cellular viability.
Specific Identification and Targeted Characterization of
Bifidobacterium lactis
from Different Environmental Isolates by a Combined Multiplex-PCR Approach
Authors: Ventura Marco , Reniero Roberto , Zink Ralf 
Published: July 2002

ABSTRACT

The species
Bifidobacterium lactis
, with its main representative strain Bb12 (DSM 10140)
,
is a yoghurt isolate used as a probiotic strain and is commercially applied in different types of yoghurts and infant formulas. In order to ensure the genetic identity and safety of this bacterial isolate, species- and strain-specific molecular tools for genetic fingerprinting must be available to identify isolated bifidobacteria or lactic acid bacteria from, e.g., various clinical environments of relevance in medical microbiology. Two opposing rRNA gene-targeted primers have been developed for specific detection of this microorganism by PCR. The specificity of this approach was evaluated and verified with DNA samples isolated from single and mixed cultures of bifidobacteria and lactobacilli (48 isolates, including the type strains of 29
Bifidobacterium
and 9
Lactobacillus
species). Furthermore, we performed a Multiplex-PCR using oligonucleotide primers targeting a specific region of the 16S rRNA gene for the genus
Bifidobacterium
and a conserved eubacterial 16S rDNA sequence. The specificity and sensitivity of this detection with a pure culture of
B. lactis
were, respectively, 100 bacteria/ml after 25 cycles of PCR and 1 to 10 bacteria/ml after a 50-cycle nested-PCR approach.
Single-Base-Pair Discrimination of Terminal Mismatches by Using Oligonucleotide Microarrays and Neural Network Analyses
Authors: Urakawa Hidetoshi , Noble Peter A. , El Fantroussi Said , Kelly John J. , Stahl David A. 
Published: July 2002

ABSTRACT

The effects of single-base-pair near-terminal and terminal mismatches on the dissociation temperature (
T
d
) and signal intensity of short DNA duplexes were determined by using oligonucleotide microarrays and neural network (NN) analyses. Two perfect-match probes and 29 probes having a single-base-pair mismatch at positions 1 to 5 from the 5′ terminus of the probe were designed to target one of two short sequences representing 16S rRNA. Nonequilibrium dissociation rates (i.e., melting profiles) of all probe-target duplexes were determined simultaneously. Analysis of variance revealed that position of the mismatch, type of mismatch, and formamide concentration significantly affected the
T
d
and signal intensity. Increasing the concentration of formamide in the washing buffer decreased the
T
d
and signal intensity, and it decreased the variability of the signal. Although
T
d
s of probe-target duplexes with mismatches in the first or second position were not significantly different from one another, duplexes with mismatches in the third to fifth positions had significantly lower
T
d
s than those with mismatches in the first or second position. The trained NNs predicted the
T
d
with high accuracies (
R
2
= 0.93). However, the NNs predicted the signal intensity only moderately accurately (
R
2
= 0.67), presumably due to increased noise in the signal intensity at low formamide concentrations. Sensitivity analysis revealed that the concentration of formamide explained most (75%) of the variability in
T
d
s, followed by position of the mismatch (19%) and type of mismatch (6%). The results suggest that position of the mismatch at or near the 5′ terminus plays a greater role in determining the
T
d
and signal intensity of duplexes than the type of mismatch.
Lactobacillus bulgaricus
Proteinase Expressed in
Lactococcus lactis
Is a Powerful Carrier for Cell Wall-Associated and Secreted Bovine β-Lactoglobulin Fusion Proteins
Authors: Bernasconi Eric , Germond Jacques-Edouard , Delley Michèle , Fritsché Rodolphe , Corthésy Blaise 
Published: July 2002

ABSTRACT

Lactic acid bacteria have a good potential as agents for the delivery of heterologous proteins to the gastrointestinal mucosa and thus for the reequilibration of inappropriate immune responses to food antigens. Bovine β-lactoglobulin (BLG) is considered a major allergen in cow's milk allergy. We have designed recombinant
Lactococcus lactis
expressing either full-length BLG or BLG-derived octapeptide T6 (IDALNENK) as fusions with
Lactobacillus bulgaricus
extracellular proteinase (PrtB). In addition to constructs encoding full-length PrtB for the targeting of heterologous proteins to the cell surface, we generated vectors aiming at the release into the medium of truncated PrtB derivatives lacking 100 (PrtB∂, PrtB∂-BLG, and PrtB∂-T6) or 807 (PrtBΔ) C-terminal amino acids. Expression of recombinant products was confirmed using either anti-PrtB, anti-BLG, or anti-peptide T6 antiserum. All forms of the full-length and truncated recombinant products were efficiently translocated, irrespective of the presence of eucaryotic BLG sequences in the fusion proteins.
L. lactis
expressing PrtB∂-BLG yielded up to 170 μg per 10
9
CFU in the culture supernatant and 9 μg per 10
9
CFU at the bacterial cell surface within 14 h. Therefore, protein fusions relying on the use of PrtB gene products are adequate for concomitant cell surface display and secretion by recombinant
L. lactis
and thus may ensure maximal bioavailability of the eucaryotic antigen in the gut-associated lymphoid tissue.
Nonradioactive Techniques for Measurement of In Vitro T-Cell Proliferation: Alternatives to the [3H]Thymidine Incorporation Assay
Authors: Messele Tsehaynesh , Roos Marijke T. L. , Hamann Dorte , Koot Maarten , Fontanet Arnaud L. , Miedema Frank , Schellekens Peter T. A. , Rinke de Wit Tobias F. 
Published: July 2002

ABSTRACTT-cell proliferation is an important in vitro parameter of in vivo immune function and has been used as a prognostic marker of human immunodeficiency virus type 1 (HIV-1) disease progression. The proliferative capacity of T cells in response to various stimuli is commonly determined by a radioactive assay based on incorporation of [3H]thymidine ([3H]TdR) into newly generated DNA. In order to assess techniques for application in laboratories where radioactive facilities are not present, two alternative methods were tested and compared to the [3H]TdR assay as a “gold standard.” As an alternative, T-cell proliferation was measured by flow cytometric assessment of CD38 expression on T cells and by an enzyme-linked immunosorbent assay (ELISA) based on bromo-2′-deoxyuridine (BrdU) incorporation. Peripheral blood mononuclear cells (PBMCs), either in whole blood or Ficoll-Isopaque separated, from a total of 26 HIV-1-positive and 18 HIV-1-negative Dutch individuals were stimulated with CD3 monoclonal antibody (MAb) alone, a combination of CD3 and CD28 MAbs, or phytohemagglutinin. BrdU incorporation after 3 days of stimulation with a combination of CD3 and CD28 MAbs correlated excellently with the [3H]TdR incorporation in both study groups (HIV-1 positives,r= 0.96; HIV-1 negatives,r= 0.83). A significant correlation of absolute numbers of T cells expressing CD38 with [3H]TdR incorporation, both in HIV-1-positive (r= 0.96) and HIV-1-negative (r= 0.84) individuals, was also observed under these conditions. The results of this study indicate that determination of both the number of CD38-positive T cells and BrdU incorporation can be used as alternative techniques to measure the in vitro T-cell proliferative capacity. The measurement of CD38 expression on T cells provides the additional possibility to further characterize the proliferating T-cell subsets for expression of other surface markers.
Mycobacterial Di-O-Acyl-Trehalose Inhibits Mitogen- and Antigen-Induced Proliferation of Murine T Cells In Vitro
Authors: Saavedra Rafael , Segura Erika , Leyva Rosario , Esparza Luis A. , López-Marı́n Luz M. 
Published: July 2002

ABSTRACT2,3-Di-O-acyl-trehalose (DAT) is a glycolipid located on the outer layer of theMycobacterium tuberculosiscell envelope. Due to its noncovalent linkage to the mycobacterial peptidoglycan, DAT could easily interact with host cells located in the focus of infection. The aim of the present work was to study the effects of DAT on the proliferation of murine spleen cells. DAT was purified from reference strains ofM. tuberculosis,orM. fortuitumas a surrogate source of the compound, by various chromatography and solvent extraction procedures and then chemically identified. Incubation of mouse spleen cells with DAT inhibited in a dose-dependent manner concanavalin A-stimulated proliferation of the cells. Experiments, including the propidium iodide exclusion test, showed that these effects were not due to death of the cells. Tracking of cell division by labeling with 5,6-carboxyfluorescein diacetate succinimidyl ester revealed that DAT reduces the rounds of cell division. Immunofluorescence with an anti-CD3 monoclonal antibody indicated that T lymphocytes were the population affected in our model. Our experiments also suggest that the extent of the suppressive activity is strongly dependent on the structural composition of the acyl moieties in DATs. Finally, the inhibitory effect was also observed on antigen-induced proliferation of mouse spleen cells specific forToxoplasma gondii. All of these data suggest that DAT could have a role in the T-cell hyporesponsiveness observed in chronic tuberculosis.
Usefulness of Hydatid Cyst Fluid of
Echinococcus granulosus
Developed in Mice with Secondary Infection for Serodiagnosis of Cystic Echinococcosis in Humans
Authors: Mamuti Wulamu , Yamasaki Hiroshi , Sako Yasuhito , Nakaya Kazuhiro , Nakao Minoru , Lightowlers Marshall W. , Ito Akira 
Published: July 2002

ABSTRACT

The aim of this work was to assess the usefulness of hydatid cyst fluid (HCF) of
Echinococcus granulosus
, obtained from mice experimentally infected with hydatid cyst tissue homogenates, for the serodiagnosis of cystic echinococcosis (CE) in humans. The sensitivity and specificity of HCF obtained from mice for the detection of immunoglobulin G (IgG) antibodies in the sera of CE patients were compared with those of HCF from sheep and/or from a human CE patient by using immunoblotting (IB) and an enzyme-linked immunosorbent assay (ELISA). HCFs obtained from three different host species all were highly useful for immunoblotting, and sera from 19 (95%) of 20 CE patients equally recognized the antigen B subunit (approximately 8 kDa). HCF from mice showed a cross-reaction with 9 of 20 alveolar echinococcosis (AE) sera (45%), whereas HCFs from two other host species cross-reacted with 14 of the AE sera (70%). Although 2 (10%) of 20 sera from neurocysticercosis (NCC) patients were false positive with HCF from both sheep and humans, none of these sera showed a positive reaction with HCF from mouse origin. ELISAs with HCFs from both mouse and sheep origins detected all 20 CE and AE sera; however, these ELISAs showed 45% (9 of 20) and 60% (12 of 20) false-positive reactions with 20 NCC sera, respectively. The presence of nonspecific human IgG in HCF obtained from a CE patient prevented us from applying it to the ELISA. HCF of
E. granulosus
, obtained from laboratory mice with a secondary infection with hydatid cyst tissue homogenates, appears to be highly useful for the serodiagnosis of CE in humans and may be useful in domestic animals.
Identification and Characterization of
Yersinia enterocolitica
Genes Induced during Systemic Infection
Authors: Gort Amy Strohmeier , Miller Virginia L. 
Published: July 2002

ABSTRACT

Yersinia enterocolitica
is one of three pathogenic
Yersinia
species that share a tropism for lymphoid tissues. However, infection of an immunocompromised host is likely to result in a systemic infection, which is often fatal. Little is known about the bacterial proteins needed to establish such an infection. The genes that encode these virulence factors are likely to be active only during systemic infection. A library of random
cat
fusions was used to inoculate BALB/c mice. Fusions expressed during a systemic infection were enriched by the administration of chloramphenicol-succinate.
Y. enterocolitica
isolates recovered from the mice were tested for chloramphenicol resistance in vitro. Fusions that were inactive in vitro were analyzed further and found to represent 31 allelic groups. Each was given a
sif
(for systemic infection factor) designation. Based on homology to known proteins, the
sif
genes are likely to encode proteins important for general physiology, transcription regulation, and other functions. During systemic infections, 13 of the
sif-cat
fusions were able to outcompete the wild type in the presence of chloramphenicol-succinate, confirming that the fusions were active. The in vitro expression of several
sif
genes was determined, showing modest changes in response to various growth conditions. A mutation in
sif15
, which encodes a putative outer membrane protein, caused attenuation during systemic infection but not during colonization of the Peyer's patches. Comparisons between the
Y. enterocolitica sif
genes and the previously identified
hre
genes imply that very different groups of genes are active during a systemic infection and during colonization of the Peyer's patches.
Functional Activities and Immunoglobulin Variable Regions of Human and Murine Monoclonal Antibodies Specific for the P1.7 PorA Protein Loop ofNeisseria meningitidis
Authors: Wang Jianfu , Jarvis Gary A. , Achtman Mark , Rosenqvist Einar , Michaelsen Terje E. , Aase Audun , Griffiss J. McLeod 
Published: July 2002

ABSTRACTThe meningococcal PorA protein is considered a promising vaccine candidate. Although much is understood regarding the structure of PorA proteins, little is known about the structure-function relationships of PorA antibodies. The aim of this study was to compare the functional and molecular characteristics of a human monoclonal antibody (MAb) and three murine MAbs specific for the PorA P1.7 serosubtype. Murine MAbs 207,B-4 (immunoglobulin G2a [IgG2a]) and MN14C11.6 (IgG2a) were both bactericidal and opsonophagocytic for P1.7-expressing meningococci, whereas human MAb SS269 (IgG3) and murine MAb 208,D-5 (IgA) initiated neither effector function. Epitope mapping with synthetic peptides revealed that MAbs 207,B-4 and 208,D-5 recognized the sequence ASGQ, which is the same specificity motif that a previous study had established for SS269 and MN14C11.6. Nucleotide and amino acid sequence analyses of the variable regions of the four MAbs showed that the SS269 VHregion belonged to the VH3 family and was approximately 70% homologous to those of the murine MAbs which were all from the 7183 family, whereas the SS269 VLregion belonged to the Vλ1-b family and was less than 40% homologous to those of the murine MAbs which were all members of the Vκ1 family. The Fab fragment of SS269 was cloned and expressed inEscherichia coliand was shown by enzyme-linked immunosorbent assay analyses to bind as well as intact SS269 MAb to P1.7,16 serosubtype group B strain 44/76. We conclude that distinct differences exist in the effector function activities and variable region gene sequences of human and murine P1.7-specific MAbs despite their recognition of similar epitopes.
Pilot Study of
phoP/phoQ
-Deleted
Salmonella enterica
Serovar Typhimurium Expressing
Helicobacter pylori
Urease in Adult Volunteers
Authors: Angelakopoulos Haroula , Hohmann Elizabeth L. 
Published: July 2002

ABSTRACT

Attenuated
Salmonella enterica
serovar Typhi has been studied as an oral vaccine vector. Despite success with attenuated
S. enterica
serovar Typhimurium vectors in animals, early clinical trials of
S. enterica
serovar Typhi expressing heterologous antigens have shown that few subjects have detectable immune responses to vectored antigens. A previous clinical study of
phoP/phoQ
-deleted
S. enterica
serovar Typhi expressing
Helicobacter pylori
urease from a multicopy plasmid showed that none of eight subjects had detectable immune responses to the vectored antigen. In an attempt to further define the variables important for engendering immune responses to vectored antigens in humans, six volunteers were inoculated with 5 × 10
7
to 8 × 10
7
CFU of
phoP/phoQ
-deleted
S. enterica
serovar Typhimurium expressing the same antigen. Two of the six volunteers had fever; none had diarrhea, bacteremia, or other serious side effects. The volunteers were more durably colonized than in previous studies of
phoP/phoQ
-deleted
S. enterica
serovar Typhi. Five of the six volunteers seroconverted to
S. enterica
serovar Typhimurium antigens and had strong evidence of anti-
Salmonella
mucosal immune responses by enzyme-linked immunospot studies. Three of six (three of five who seroconverted to
Salmonella
) had immune responses in the most sensitive assay of urease-specific immunoglobulin production by blood mononuclear cells in vitro. One of these had a fourfold or greater increase in end-point immunoglobulin titer in serum versus urease. Attenuated
S. enterica
serovar Typhimurium appears to be more effective than
S. enterica
serovar Typhi for engendering immune responses to urease. Data suggest that this may be related to a greater stability of antigen-expressing plasmid in
S. enterica
serovar Typhimurium and/or prolonged intestinal colonization. Specific factors unique to nontyphoidal salmonellae may also be important for stimulation of the gastrointestinal immune system.
Potentiality of Interleukin-18 as a Useful Reagent for Treatment and Prevention ofLeishmania majorInfection
Authors: Ohkusu Kazunobu , Yoshimoto Tomohiro , Takeda Kiyoshi , Ogura Takeharu , Kashiwamura Shin-ichiro , Iwakura Yoichiro , Akira Shizuo , Okamura Haruki , Nakanishi Kenji 
Published: July 2002

ABSTRACTInterleukin-18 (IL-18) is a proinflammatory cytokine that plays an important role in natural killer cell activation and the T helper 1 (Th1) cell response, particularly in collaboration with IL-12. Since Th1 cells play a pivotal role in the host defense against infection with intracellular microbes, such asLeishmania major, we investigated whether IL-18 is critically involved in protection againstL. majorinfection by activation of Th1 cells. We administered IL-12 and/or IL-18 daily toL. major-susceptible BALB/c mice. Neither IL-12 (10 ng/mouse) nor IL-18 (1,000 ng/mouse) induced wound healing, while daily injection of IL-12 and IL-18 during the first week after infection strongly protected the mice from footpad swelling by induction and activation of Th1 cells. Furthermore, these mice acquired protective immunity. We also investigated a protective role of endogenous IL-18 by using anti-IL-18 antibody-treated C3H/HeN mice (anL. major-resistant strain) or IL-18 deficient (IL-18−/−) mice with a resistant background (C57BL/6). We found that in the absence of endogenous IL-18, these mice showed prolonged footpad swelling as well as diminished nitric oxide production. However, daily injection of IL-18 into IL-18−/−mice corrected their deficiencies, suggesting that these mice have Th1 cells that produce gamma interferon (IFN-γ) in response to IL-18. Indeed, these mice had normal levels of Th1 cells. Thus, IL-18 is not responsible for inducing Th1 cells but participates in host resistance by its action in stimulating Th1 cells to produce IFN-γ. Our results also indicate the high potentiality of IL-18 as a useful reagent for treatment as well as prevention against reinfection.
Granulocytic Ehrlichiosis in Mice Deficient in Phagocyte Oxidase or Inducible Nitric Oxide Synthase
Authors: Banerjee Rila , Anguita Juan , Fikrig Erol 
Published: July 2002

ABSTRACTMice deficient in phox (gp91phox−/−) or NOS2 (NOS2−/−) were infected with the agent of human granulocytic ehrlichiosis (HGE) to evaluate the importance of these pathways in the eradication of HGE bacteria. NOS2−/−mice had delayed clearance of the HGE agent in comparison to control or gp91phox−/−mice, suggesting that reactive nitrogen intermediates play a role in the early control of HGE.