bluuebunny/crossref_metadata_2025_split huggingface dataset:
Combined Molecular and Biochemical Approach Identifies
Aspergillus japonicus
and
Aspergillus aculeatus
as Two Species
Authors: Pařenicová Lucie , Skouboe Pernille , Frisvad Jens , Samson Robert A. , Rossen Lone , ten Hoor-Suykerbuyk Marjon , Visser Jaap 
Published: July 2002

ABSTRACT

We examined nine
Aspergillus japonicus
isolates and 10
Aspergillus aculeatus
isolates by using molecular and biochemical markers, including DNA sequences of the ITS1-5.8S rRNA gene-ITS2 region, restriction fragment length polymorphisms (RFLP), and secondary-metabolite profiles. The DNA sequence of the internal transcribed spacers (ITS1 and ITS2) and the 5.8S rRNA gene could not be used to distinguish between
A. japonicus
and
A. aculeatus
but did show that these two taxa are more closely related to each other than to other species of black aspergilli.
Aspergillus niger
pyruvate kinase (
pki
A) and pectin lyase A (
pel
A) and
Agaricus bisporus
28S rRNA genes, which were used as probes in the RFLP analysis, revealed clear polymorphism between these two taxa. The
A. niger pki
A and
pel
A probes placed six strains in an
A. japonicus
group and 12 isolates in an
A. aculeatus
group, which exhibited intraspecific variation when they were probed with the
pel
A gene. The secondary-metabolite profiles supported division of the isolates into the two species and differed from those of other black aspergilli. The strains classified as
A. japonicus
produced indole alkaloids and a polar metabolite, while the
A. aculeatus
isolates produced neoxaline, okaramins, paraherquamidelike compounds, and secalonic acid.
A. aculeatus
CBS 114.80 showed specific RFLP patterns for all loci examined. The secondary-metabolite profile of strain CBS 114.80 also differed from those of
A. japonicus
and
A. aculeatus
. Therefore, this strain probably represents a third taxon. This study provides unambiguous criteria for establishing the taxonomic positions of isolates of black aspergilli, which are important in relation to industrial use and legal protection of these organisms.
DNA Extraction from Soils: Old Bias for New Microbial Diversity Analysis Methods
Authors: Martin-Laurent F. , Philippot L. , Hallet S. , Chaussod R. , Germon J. C. , Soulas G. , Catroux G. 
Published: July 2002

ABSTRACT
The impact of three different soil DNA extraction methods on bacterial diversity was evaluated using PCR-based 16S ribosomal DNA analysis. DNA extracted directly from three soils showing contrasting physicochemical properties was subjected to amplified ribosomal DNA restriction analysis and ribosomal intergenic spacer analysis (RISA). The obtained RISA patterns revealed clearly that both the phylotype abundance and the composition of the indigenous bacterial community are dependent on the DNA recovery method used. In addition, this effect was also shown in the context of an experimental study aiming to estimate the impact on soil biodiversity of the application of farmyard manure or sewage sludge onto a monoculture of maize for 15 years.
New Degenerate
Cytophaga
-
Flexibacter
-
Bacteroides
-Specific 16S Ribosomal DNA-Targeted Oligonucleotide Probes Reveal High Bacterial Diversity in River Taff Epilithon
Authors: O’Sullivan Louise A. , Weightman Andrew J. , Fry John C. 
Published: July 2002

ABSTRACT

River microbial communities play an important role in global nutrient cycles, and aggregated bacteria such as those in epilithic biofilms may be major contributors. In this study the bacterial diversity of River Taff epilithon in South Wales was investigated. A 16S ribosomal DNA (rDNA) clone library was constructed and analyzed by partial sequencing of 76 of 347 clones and hybridization with taxon-specific probes. The epilithon was found to be very diverse, with an estimated 59.6% of the bacterial populations not accounted for by these clones. Members of the
Cytophaga-Flexibacter-Bacteroides
division (CFBs) were most abundant in the library, representing 25% of clones, followed by members of the α subdivision of the division
Proteobacteria
(α-
Proteobacteria
), γ-
Proteobacteria
, gram-positive bacteria,
Cyanobacteria
, β-
Proteobacteria
, δ-
Proteobacteria
, and the
Prosthecobacter
group. This study concentrated on the epilithic CFB populations, and a new set of degenerate 16S rDNA probes was developed to enhance their detection, namely, CFB560, CFB562, and CFB376. The commonly used probe CF319a/b may frequently lead to the underestimation of CFB populations in environmental studies, because it does not fully detect members of the division. CFB560 had exact matches to 95.6% of CFBs listed in the Ribosomal Database Project (release 8.0) small-subunit phylogenetic trees, compared to 60% for CF319a/b. The CFB probes detected 66 of 347 epilithon TAF clones, and 60 of these were partially sequenced. They affiliated with the RDP-designated groups
Cytophaga
,
Sphingobacterium
,
Lewinella
, and
Cytophaga aurantiaca
. CFB560 and CF319a/b detected 94% (62 of 66) and 48.5% (32 of 66) of clones, respectively, and therefore CFB560 is recommended for future use. Probe design in this study illustrated that multiple degenerate positions can greatly increase target range without adversely effecting specificity or experimental performance.
Salmonella enterica
Serovar Typhimurium and
Escherichia coli
Contamination of Root and Leaf Vegetables Grown in Soils with Incorporated Bovine Manure
Authors: Natvig Erin E. , Ingham Steven C. , Ingham Barbara H. , Cooperband Leslie R. , Roper Teryl R. 
Published: July 2002

ABSTRACT

Bovine manure, with or without added
Salmonella enterica
serovar Typhimurium (three strains), was incorporated into silty clay loam (SCL) and loamy sand (LS) soil beds (53- by 114-cm surface area, 17.5 cm deep) and maintained in two controlled-environment chambers. The
S. enterica
serovar Typhimurium inoculum was 4 to 5 log CFU/g in manure-fertilized soil. The conditions in the two environmental chambers, each containing inoculated and uninoculated beds of manure-fertilized soil, simulated daily average Madison, Wis., weather conditions (hourly temperatures, rainfall, daylight, and humidity) for a 1 March or a 1 June manure application and subsequent vegetable growing seasons ending 9 August or 28 September, respectively. Core soil samples were taken biweekly from both inoculated and uninoculated soil beds in each chamber. Radishes, arugula, and carrots were planted in soil beds, thinned, and harvested. Soils, thinned vegetables, and harvested vegetables were analyzed for
S. enterica
serovar Typhimurium and
Escherichia coli
(indigenous in manure). After the 1 March manure application,
S. enterica
serovar Typhimurium was detected at low levels in both soils on 31 May, but not on vegetables planted 1 May and harvested 12 July from either soil. After the 1 June manure application,
S. enterica
serovar Typhimurium was detected in SCL soil on 7 September and on radishes and arugula planted in SCL soil on 15 August and harvested on 27 September. In LS soil,
S. enterica
serovar Typhimurium died at a similar rate (
P
≥ 0.05) after the 1 June manure application and was less often detected on arugula and radishes harvested from this soil compared to the SCL soil. Pathogen levels on vegetables were decreased by washing. Manure application in cool (daily average maximum temperature of <10°C) spring conditions is recommended to ensure that harvested vegetables are not contaminated with
S. enterica
serovar Typhimurium. Manure application under warmer (daily average maximum temperature >20°C) summer conditions is not recommended when vegetable planting is done between the time of manure application and late summer. A late fall manure application will not increase the risk of contaminating vegetables planted the next spring, since further experiments showed that repeated freeze-thaw cycles were detrimental to the survival of
S. enterica
serovar Typhimurium and
E. coli
in manure-fertilized soil. The number of indigenous
E. coli
in soil was never significantly lower (
P
< 0.05) than that of
S. enterica
serovar Typhimurium, suggesting its usefulness as an indicator organism for evaluating the risk of vegetable contamination with manure-borne
S. enterica
serovar Typhimurium.
Immunoglobulin E Antileishmanial Antibody Response in Cutaneous Leishmaniasis
Authors: Sousa-Atta Maria L. B. , Salamé Gregório S. , D’Oliveira Argemiro , Almeida Roque P. , Atta Ajax M. , Carvalho Edgar M. 
Published: July 2002

ABSTRACTHigh levels of antileishmanial immunoglobulin E (IgE) antibodies are associated with disease activity in visceral leishmaniasis. Herein, we report our observations about the relationship between antileishmanial IgE antibodies and clinical aspects of cutaneous leishmaniasis. This study was carried out with 45 patients (29 male and 16 female), with ages ranging from 11 to 48 years. All subjects were from an area to which leishmaniasis is endemic, Corte de Pedra (Bahia, Brazil), and the duration of the illness was ≤30 days. The patients were classified as positive or negative for IgE serology in enzyme-linked immunosorbent assay with leishmanial antigens. IgE antibodies were detected in 18 patients (optical density, 0.421 ± 0.30; 95% confidence interval, 0.27 to 0.57), and only 3 (17%) had more than one ulcer. In this group the diameter of Montenegro’s reaction was 18 ± 12.2 mm. In the group with negative IgE serology, 11 of 27 patients (48%) presented two or more cutaneous ulcers, and the mean of the skin test result was 9 ± 6.9 mm. There was a positive correlation between IgE antibody levels and Montenegro’s reaction size and an inverse correlation between IgE antileishmanial antibodies and the number of skin ulcers. The presence of antileishmanial IgE antibodies in cutaneous leishmaniasis may be a result of immunoregulatory events with clinical implications.
Relevance of Dietary Lipids as Modulators of Immune Functions in Cells Infected with
Listeria monocytogenes
Authors: Puertollano María A. , de Pablo Manuel A. , Álvarez de Cienfuegos Gerardo 
Published: July 2002

ABSTRACT

Nutritional status may have significant importance for the immune system, and particularly, unsaturated fatty acids may serve as modulators of immune functions. Clinical and epidemiological studies have demonstrated that fatty acids are involved in the reduction of the inflammatory processes that occur in diseases characterized by an overactivation of the immune system. At the same time, an increase in susceptibility to infection has also been reported. The importance of immune system modulation by dietary lipids in the presence of an intracellular bacterial pathogen, such as
Listeria monocytogenes
, was evaluated in the present study. BALB/c mice were divided into four groups which were each fed a low-fat (2.5% by weight) diet, an olive oil (OO; 20% by weight) diet, a fish oil (FO; 20% by weight) diet, or a hydrogenated coconut oil (HCO; 20% by weight) diet for 4 weeks. In each group, lymphocye proliferation was measured, and a reduction in the stimulation index was observed in the FO and HCO groups. Cytotoxicity exerted by
L. monocytogenes
was increased in the groups fed diets containing OO and FO after 6 h of incubation with the bacterium. An important increase in the production of reactive oxygen species was found in the groups fed the HCO diet after 12 h of incubation with
L. monocytogenes
. Finally, invasion and adhesion factors were not modified substantially by the action of dietary lipids, although these factors were reduced in cells from mice fed an FO diet. These results underline the importance of several dietary lipids as biological modulators of immune functions and their crucial role in the alteration of host natural resistance.
Elucidation of the Cross-Reactive Immunoglobulin M Response to Human Herpesviruses 6 and 7 on the Basis of Neutralizing Antibodies
Authors: Yoshida Mariko , Torigoe Sadayoshi , Yamada Masao 
Published: July 2002

ABSTRACT
Human herpesvirus 6 (HHV-6) is closely related to HHV-7 in terms of genome organization and sequence. The cross-reactive responses between HHV-6 and HHV-7 have been reported by using immunofluorescent techniques. Recently we have shown that neutralizing (NT) antibody responses are specific to each virus and do not cross-react. We took advantage of this and used the NT antibody response to estimate the time of seroconversion to each virus and examined the pattern of humoral immune response, especially the immunoglobulin M (IgM) response, against each virus antigen in the natural course of infection with HHV-6 and HHV-7. In children who experienced HHV-6 infection first, followed by HHV-7 infection, the IgM response at the first HHV-6 infection was directed only against HHV-6, while no IgM response was directed against HHV-7 at the second HHV-7 infection. In contrast, in children who experienced HHV-7 infection first, followed by HHV-6 infection, the IgM response at the first HHV-7 infection was directed not only against HHV-7 but also against HHV-6. These data suggest that cross-reactive responses to heterologous viruses should be taken into consideration when making a diagnosis based on IgM antibody.
Polyclonal Antibodies to Glutathione
S
-Transferase- Verotoxin Subunit A Fusion Proteins Neutralize Verotoxins
Authors: Leung P. H. M. , Peiris J. S. M. , Ng W. W. S. , Yam W. C. 
Published: July 2002

ABSTRACT

The A1 subunits of verotoxin-1 (VT1) and VT2 genes were cloned into pGEX-4T-2 for the expression of glutathione
S
-transferase (GST) fusion proteins. The N-terminal and the transmembrane regions of the A1 subunits were excluded from the constructs in order to increase the product yields. Polyclonal anti-VT1A1 and anti-VT2A1 antibodies were produced by immunizing rabbits with GST-VT1A1 and GST-VT2A1 fusion proteins, respectively. The antibodies were tested for their ability to neutralize active toxins from 45 VT-producing
Escherichia coli
(VTEC) strains. The antibodies had significantly high neutralizing activities against their homologous toxins. The average percentages of neutralization of VT1 by anti-GST-VT1A1 and anti-GST-VT2A1 were 76.7% ± 7.9% and 3.6% ± 2.3%, respectively, and those of VT2 were 1.7% ± 2.3% and 82.5% ± 13.9%, respectively. VT2 variant toxin was neutralized by anti-GST-VT2A1, with cross neutralization being a possible consequence of sequence homology between VT2 and a VT2 variant. To our knowledge, this is the first report on the production of polyclonal antibodies from GST-VT fusion proteins. The antibodies were shown to exhibit specific toxin neutralizing activities and may be useful for immunological diagnosis of VTEC infections.
Vaccination Strategies for Mucosal Immune Responses
Authors: Ogra Pearay L. , Faden Howard , Welliver Robert C. 
Published: July 2002

SUMMARYMucosal administration of vaccines is an important approach to the induction of appropriate immune responses to microbial and other environmental antigens in systemic sites and peripheral blood as well as in most external mucosal surfaces. The development of specific antibody- or T-cell-mediated immunologic responses and the induction of mucosally induced systemic immunologic hyporesponsiveness (oral or mucosal tolerance) depend on complex sets of immunologic events, including the nature of the antigenic stimulation of specialized lymphoid structures in the host, antigen-induced activation of different populations of regulatory T cells (Th1 versus Th2), and the expression of proinflammatory and immunoregulatory cytokines. Availability of mucosal vaccines will provide a painless approach to deliver large numbers of vaccine antigens for human immunization. Currently, an average infant will receive 20 to 25 percutaneous injections for vaccination against different childhood infections by 18 months of age. It should be possible to develop for human use effective, nonliving, recombinant, replicating, transgenic, and microbial vector- or plant-based mucosal vaccines to prevent infections. Based on the experience with many dietary antigens, it is also possible to manipulate the mucosal immune system to induce systemic tolerance against environmental, dietary, and possibly other autoantigens associated with allergic and autoimmune disorders. Mucosal immunity offers new strategies to induce protective immune responses against a variety of infectious agents. Such immunization may also provide new prophylactic or therapeutic avenues in the control of autoimmune diseases in humans.
Identification of a Fibronectin Binding Protein fromStreptococcus mutans
Authors: Chia Jean-San , Yeh Chiou-Yueh , Chen Jen-Yang 
Published: July 2002

ABSTRACTThe interaction of viridans streptococci with components of the extracellular matrix (ECM) plays an important role in the pathogenesis of infective endocarditis. We have identified a surface protein ofStreptococcus mutanswhich binds the ECM constituent fibronectin (Fn). Initially, we found thatS. mutanscould adsorb soluble Fn in plasma, but with lower efficiency thanStreptococcus pyogenes. In addition,S. mutanscould bind immobilized Fn in a dose-dependent manner when tested using an enzyme-linked immunosorbent assay. Crude extracts of cell wall-associated proteins or extracellular proteins fromS. mutansMT8148 specifically bound Fn through a protein with the molecular mass of ca. 130 kDa, as detected by far-Western immunoblotting. The candidate Fn binding protein (FBP-130) was purified to near homogeneity by using Fn coupled Sepharose 4B affinity column chromatography. A rabbit polyclonal antibody against FBP-130 reacted specifically with a protein of molecular mass of ca. 130 kDa in both cell wall and extracellular fractions, and the abundance of FBP was higher in the former than in the latter fractions. The purified FBP bound specifically to immobilized Fn, whereas the binding of soluble Fn to coated FBP could only be detected in the presence of high concentrations of Fn. The purified FBP, as well as anti-FBP immunoglobulin G, inhibited the adherence ofS. mutansto immobilized Fn and endothelial cells (ECV304) in a dose-dependent manner. These results demonstrated that FBP-130 mediated the adherence ofS. mutansspecifically to Fn and endothelial cells in vitro. The characteristics ofS. mutansand FBP-130 in binding Fn confirmed that viridans streptococci adopt different strategies in their interaction with ECM.
Full Capacity of Recombinant
Escherichia coli
Heat-Stable Enterotoxin Fusion Proteins for Extracellular Secretion, Antigenicity, Disulfide Bond Formation, and Activity
Authors: Batisson Isabelle , Der Vartanian Maurice , Gaillard-Martinie Brigitte , Contrepois Michel 
Published: July 2002

ABSTRACT

We have successfully used the major subunit ClpG of
Escherichia coli
CS31A fimbriae as an antigenic and immunogenic exposure-delivery vector for various heterologous peptides varying in nature and length. However, the ability of ClpG as a carrier to maintain in vitro and in vivo the native biological properties of passenger peptide has not yet been reported. To address this possibility, we genetically fused peptides containing all or part of the
E. coli
human heat-stable enterotoxin (STh) sequence to the amino or carboxyl ends of ClpG. Using antibodies to the ClpG and STh portions for detecting the hybrids; AMS (4-acetamido-4′-maleimidylstilbene-2,2′-disulfonate), a potent free thiol-trapping reagent, for determining the redox state of STh in the fusion; and the suckling mouse assay for enterotoxicity, we demonstrated that all ClpG-STh proteins were secreted in vitro and in vivo outside the
E. coli
cells in a heat-stable active oxidized (disulfide-bonded) form. Indeed, in contrast to many earlier studies, blocking the natural NH
2
or COOH extremities of STh had, in all cases, no drastic effect on cell release and toxin activity. Only antigenicity of STh C-terminally extended with ClpG was strongly affected in a conformation-dependent manner. These results suggest that the STh activity was not altered by the chimeric structure, and therefore that, like the natural toxin, STh in the fusion had a spatial structure flexible enough to be compatible with secretion and enterotoxicity (folding and STh receptor recognition). Our study also indicates that disulfide bonds were essential for enterotoxicity but not for release, that spontaneous oxidation by molecular oxygen occurred in vitro in the medium, and that the
E. coli
cell-bound toxin activity in vivo resulted from an effective export processing of hybrids and not cell lysis. None of the ClpG-STh subunits formed hybrid CS31A-STh fimbriae at the cell surface of
E. coli
, and a strong decrease in the toxin activity was observed in the absence of CS31A helper proteins. In fact, chimeras translocated across the outer membrane as a free folded monomer once they were guided into the periplasm by the ClpG leader peptide through the CS31A-dependent secretory pathway. In summary, ClpG appears highly attractive as a carrier reporter protein for basic and applied research through the engineering of
E. coli
for culture supernatant delivery of an active cysteine-containing protein, such as the heat-stable enterotoxin.
Chronic
Helicobacter pylori
Infection Induces an Apoptosis-Resistant Phenotype Associated with Decreased Expression of p27
kip1
Authors: Shirin Haim , Sordillo Emilia Mia , Kolevska Tatiana K. , Hibshoosh Hanina , Kawabata Yuichi , Oh Sung H. , Kuebler Joachim F. , Delohery Thomas , Weghorst Christopher M. , Weinstein I. Bernard , Moss Steven F. 
Published: July 2002

ABSTRACT

Helicobacter pylori
infection is associated with the development of gastric cancer. In short-term coculture with AGS gastric cells,
H. pylori
inhibits cell cycle progression and induces dose-dependent apoptosis. Based on the concept that an imbalance between proliferation and apoptosis may contribute to the emergence of gastric cancer, we chronically exposed AGS cells to
H. pylori
as a model of chronic exposure in humans. The AGS derivatives selected by this process were stably resistant not only to
H. pylori
-induced apoptosis but also to apoptosis induced by other enteric bacteria and by several toxic agents including radiation and cancer chemotherapy. Like the parental AGS cells, the derivatives underwent G
1
/S-phase cell cycle inhibition in response to
H. pylori
. The AGS derivatives displayed a marked decrease in cellular levels of the cell cycle control protein p27
kip1
. We found a similar decrease in epithelial cell p27
kip1
expression in gastric biopsy specimens from
H. pylori
-infected patients. These findings are consistent with observations that link decreases in the p27
kip1
level to increased susceptibility to cancer in mice with p27
kip1
deleted and to a poor prognosis of gastric cancer in humans. This is the first demonstration that bacterial infection can lead to apoptosis resistance and to cross-resistance to other inducers of apoptosis such as bacteria, chemotherapeutic agents, and radiation. The development of apoptosis resistance and downmodulation of p27
kip1
may contribute to the increased risk for gastric cancer observed in humans chronically exposed to
H. pylori
.