bluuebunny/crossref_metadata_2025_split huggingface dataset:
Application of Denaturing Gradient Gel Electrophoresis (DGGE) To Study the Diversity of Marine Picoeukaryotic Assemblages and Comparison of DGGE with Other Molecular Techniques
Authors: Dı́ez Beatriz , Pedrós-Alió Carlos , Marsh Terence L. , Massana Ramon 
Published: July 2002

ABSTRACT
We used denaturing gradient gel electrophoresis (DGGE) to study the diversity of picoeukaryotes in natural marine assemblages. Two eukaryote-specific primer sets targeting different regions of the 18S rRNA gene were tested. Both primer sets gave a single band when used with algal cultures and complex fingerprints when used with natural assemblages. The reproducibility of the fingerprints was estimated by quantifying the intensities of the same bands obtained in independent PCR and DGGE analyses, and the standard error of these estimates was less than 2% on average. DGGE fingerprints were then used to compare the picoeukaryotic diversity in samples obtained at different depths and on different dates from a station in the southwest Mediterranean Sea. Both primer sets revealed significant differences along the vertical profile, whereas temporal differences at the same depths were less marked. The phylogenetic composition of picoeukaryotes from one surface sample was investigated by excising and sequencing DGGE bands. The results were compared with an analysis of a clone library and a terminal restriction fragment length polymorphism fingerprint obtained from the same sample. The three PCR-based methods, performed with three different primer sets, revealed very similar assemblage compositions; the same main phylogenetic groups were present at similar relative levels. Thus, the prasinophyte group appeared to be the most abundant group in the surface Mediterranean samples as determined by our molecular analyses. DGGE bands corresponding to prasinophytes were always found in surface samples but were not present in deep samples. Other groups detected were prymnesiophytes, novel stramenopiles (distantly related to hyphochytrids or labyrinthulids), cryptophytes, dinophytes, and pelagophytes. In conclusion, the DGGE method described here provided a reasonably detailed view of marine picoeukaryotic assemblages and allowed tentative phylogenetic identification of the dominant members.
Detection and Identification of Mammalian Reoviruses in Surface Water by Combined Cell Culture and Reverse Transcription-PCR
Authors: Spinner Michael L. , Di Giovanni George D. 
Published: July 2002

ABSTRACT
Reoviruses are a common class of enteric viruses capable of infecting a broad range of mammalian species, typically with low pathogenicity. Previous studies have shown that reoviruses are common in raw water sources and are often found along with other animal viruses. This suggests that in addition to the commonly monitored enteroviruses, reoviruses might serve as an informative target for monitoring fecal contamination of drinking water sources. Mammalian reoviruses were detected and identified by a combined cell culture–reverse transcription-PCR (RT-PCR) assay with novel primers targeting the L3 gene that encodes the λ3 major core protein. Five of 26 (19.2%) cytopathic effect-positive cell culture lysates inoculated with surface water were positive for reoviruses by RT-PCR. DNA sequence analysis of RT-PCR products revealed significant sequence diversity among isolates, which is consistent with the sequence diversity among previously characterized mammalian reoviruses. Sequence analysis revealed persistence of a reovirus genotype at a single sampling site, while a sample from another site contained two different reovirus genotypes.
Killer Toxin ofKluyveromyces phaffiiDBVPG 6076 as a Biopreservative Agent To Control Apiculate Wine Yeasts
Authors: Ciani M. , Fatichenti F. 
Published: July 2002

ABSTRACTThe use ofKluyveromyces phaffiiDBVPG 6076 killer toxin against apiculate wine yeasts has been investigated. The killer toxin ofK. phaffiiDBVPG 6076 showed extensive anti-Hanseniasporaactivity against strains isolated from grape samples. The proteinaceous killer toxin was found to be active in the pH range of 3 to 5 and at temperatures lower than 40°C. These biochemical properties would allow the use ofK. phaffiikiller toxin in wine making. Fungicidal or fungistatic effects depend on the toxin concentration. Toxin concentrations present in the supernatant during optimal conditions of production (14.3 arbitrary units) exerted a fungicidal effect on a sensitive strain ofHanseniaspora uvarum. At subcritical concentrations (fungistatic effect) the saturation kinetics observed with the increased ratio of killer toxin toH. uvarumcells suggest the presence of a toxin receptor. The inhibitory activity exerted by the killer toxin present in grape juice was comparable to that of sulfur dioxide. The findings presented suggest that theK. phaffiiDBVPG 6076 killer toxin has potential as a biopreservative agent in wine making.
Organization and Regulation of
meta
Cleavage Pathway Genes for Toluene and
o
-Xylene Derivative Degradation in
Pseudomonas stutzeri
OX1
Authors: Arenghi Fabio L. G. , Berlanda Davide , Galli Enrica , Sello Guido , Barbieri Paola 
Published: July 2002

ABSTRACT

Pseudomonas stutzeri
OX1
meta
pathway genes for toluene and
o
-xylene catabolism were analyzed, and loci encoding phenol hydroxylase, catechol 2,3-dioxygenase, 2-hydroxymuconate semialdehyde dehydrogenase, and 2-hydroxymuconate semialdehyde hydrolase were mapped. Phenol hydroxylase converted a broad range of substrates, as it was also able to transform the nongrowth substrates 2,4-dimethylphenol and 2,5-dimethylphenol into 3,5-dimethylcatechol and 3,6-dimethylcatechol, respectively, which, however, were not cleaved by catechol 2,3-dioxygenase. The identified gene cluster displayed a gene order similar to that of the
Pseudomonas
sp. strain CF600
dmp
operon for phenol catabolism and was found to be coregulated by the
tou
operon activator TouR. A hypothesis about the evolution of the toluene and
o
-xylene catabolic pathway in
P. stutzeri
OX1 is discussed.
The
ybiT
Gene of
Erwinia chrysanthemi
Codes for a Putative ABC Transporter and Is Involved in Competitiveness against Endophytic Bacteria during Infection
Authors: Llama-Palacios Arancha , López-Solanilla Emilia , Rodríguez-Palenzuela Pablo 
Published: July 2002

ABSTRACT

We investigated the role in bacterial infection of a putative ABC transporter, designated
ybiT
, of
Erwinia chrysanthemi
AC4150. The deduced sequence of this gene showed amino acid sequence similarity with other putative ABC transporters of gram-negative bacteria, such as
Escherichia coli
and
Pseudomonas aeruginosa
, as well as structural similarity with proteins of
Streptomyces
spp. involved in resistance to macrolide antibiotics. The gene contiguous to
ybiT
, designated as
pab
(putative antibiotic biosynthesis) showed sequence similarity with
Pseudomonas
and
Streptomyces
genes involved in the biosynthesis of antibiotics. A
ybiT
mutant (BT117) was constructed by marker exchange. It retained full virulence in potato tubers and chicory leaves, but it showed reduced ability to compete in planta against the wild-type strain or against selected saprophytic bacteria. These results indicate that the
ybiT
gene plays a role in the in planta fitness of the bacteria.
Denaturation of Either
Manduca sexta
Aminopeptidase N or
Bacillus thuringiensis
Cry1A Toxins Exposes Binding Epitopes Hidden under Nondenaturing Conditions
Authors: Daniel Anu , Sangadala Sreedhara , Dean Donald H. , Adang Michael J. 
Published: July 2002

ABSTRACT

The effect of polypeptide denaturation of
Bacillus thuringiensis
Cry1A toxins or purified
Manduca sexta
120-kDa aminopeptidase N on the specificities of their interactions was investigated. Ligand and dot blotting experiments were conducted with
125
I-labeled Cry1Ac, Cry1Ac mutant
509
QNR-AAA
511
(QNR-AAA), or 120-kDa aminopeptidase N as the probe. Mutant QNR-AAA does not bind the
N
-acetylgalactosamine moiety on the 120-kDa aminopeptidase. Both
125
I-Cry1Ac and
125
I-QNR-AAA bound to 210- and 120-kDa proteins from
M. sexta
brush border membrane vesicles and purified 120-kDa aminopeptidase N on ligand blots. However, on dot blots
125
I-QNR-AAA bound brush border vesicles but did not bind purified aminopeptidase except when aminopeptidase was denatured. In the reciprocal experiment,
125
I-aminopeptidase bound Cry1Ac but did not bind QNR-AAA.
125
I-aminopeptidase bound Cry1Ab to a limited extent but not the Cry1Ab domain I mutant Y153D or Cry1Ca. However, denatured
125
I-aminopeptidase detected each Cry1A toxin and mutant but not Cry1Ca on dot blots. The same pattern of recognition occurred with native (nondenatured)
125
I-aminopeptidase probe and denatured toxins as the targets. The broader pattern of toxin-binding protein interaction is probably due to peptide sequences being exposed upon denaturation. Putative Cry toxin-binding proteins identified by the ligand blot technique need to be investigated under native conditions early in the process of identifying binding proteins that may serve as functional toxin receptors.
Molecular Characterization of the Leucine Cluster in
Buchnera
sp. Strain PSY, a Primary Endosymbiont of the Aphid
Pemphigus spyrothecae
Authors: Sabater-Muñoz Beatriz , Gómez-Valero Laura , van Ham Roeland C. H. J. , Silva Francisco J. , Latorre Amparo 
Published: July 2002

ABSTRACT

Buchnera strains from most aphid subfamilies studied to date have been found to carry the leucine gene cluster (
leuA
, -
B
, -
C
, and -
D
) on a plasmid, an organization unique among bacteria. Here, however, we demonstrate a classical chromosomal location of the cluster in
Buchnera
sp. strain PSY from the aphid
Pemphigus spyrothecae
(subfamily Pemphiginae). The genes that flank
leuABCD
in
Buchnera
sp. strain PSY appear to be adjacent in the genome of
Buchnera
sp. strain APS, a strain carrying a leucine plasmid. We propose that the presence of a leucine plasmid predates the diversification of symbiotic
Buchnera
and that the chromosomal location observed in
Buchnera
sp. strain PSY arose by a transfer of the leucine genes from a plasmid to the chromosome.
Antibody to Heat Shock Protein Can Be Used for Early Serological Monitoring of
Helicobacter pylori
Eradication Treatment
Authors: Yunoki Naoko , Yokota Kenji , Mizuno Motowo , Kawahara Yoshiro , Adachi Masayasu , Okada Hiroyuki , Hayashi Shyunji , Hirai Yoshikazu , Oguma Keiji , Tsuji Takao 
Published: July 2002

ABSTRACT

Infection with
Helicobacter pylori
induces humoral immune responses against various antigens of the bacterium. Heat shock proteins (hsps) are immunodominant antigens in various diseases including
H. pylori
infection. In the present study, we measured the anti-hsp antibody titers in 42 patients with
H. pylori
-infected peptic ulcers during a bacterial eradication study. The patients were treated with a proton pump inhibitor and antimicrobial agents to eradicate the organism. Their sera were obtained at pretreatment and at 1 month and 6 months after the eradication therapy. The titers of immunoglobulin G antibodies to the
H. pylori
hsp, whole-cell lysate, and urease (30-kDa subunit) antigens in serum were measured by a capture enzyme-linked immunosorbent assay. The levels of
H. pylori
hsp60 antibodies in sera collected 1 month after treatment had declined significantly, even when changes in the titers of antibodies to whole-cell and urease antigens were not apparent. These results suggest that measurement of antibodies to
H. pylori
hsp60 in serum is useful for the early monitoring of the effectiveness of eradication therapy.
Comparison of Five Serologic Tests for Diagnosis of Acute Infections by
Chlamydia pneumoniae
Authors: Persson Kenneth , Boman Jens 
Published: July 2002

ABSTRACT

Serology is often used to diagnose acute infections by
Chlamydia pneumoniae
. In this study paired sera from patients with acute respiratory tract infection during an epidemic of
C. pneumoniae
infections were examined by five different antibody tests. These tests were the complement fixation (CF) test, the microimmunofluorescence (MIF) test, a recombinant enzyme immunoassay (rEIA) (Medac) based on a recombinant lipopolysaccharide of chlamydia and measuring antibodies to a common chlamydial antigen, and two tests that utilize preparations of
C. pneumoniae
organisms, the SeroCp-EIA (Savyon) (with preserved lipopolysaccharide) and the LOY-EIA (Labsystems) (without this antigen). Both of the last two tests should measure specific antibodies to
C. pneumoniae
, although cross-reacting antibodies may also be detected by the SeroCp-EIA. Acute infection of
C. pneumoniae
was serologically confirmed in 44% of the cases by at least two different tests. Using an expanded “gold standard,” i.e., the presence of significant reactions in at least two tests, the sensitivity of the CF test was 69%, that of the MIF test was 88%, that of the rEIA was 89%, that of the LOY-EIA was 96%, and that of the SeroCp-EIA was 92%. Specificity was high for all methods, but adjustments of diagnostic criteria were made to several of the tests. The basis for these adjustments and supportive data are presented. Infections of
C. pneumoniae
were detected in patients from 8 to 83 years of age. Two peaks in the incidence of such infections were observed: one among young teenagers and a second in adults 30 to 45 years of age, corresponding to parents of young teen-agers. The tests were equally sensitive in different age groups. Reinfections seemed to be rare.
Comparison of a Classical Phagocytosis Assay and a Flow Cytometry Assay for Assessment of the Phagocytic Capacity of Sera from Adults Vaccinated with a Pneumococcal Conjugate Vaccine
Authors: Jansen Wouter T. M. , Väkeväinen-Anttila Merja , Käyhty Helena , Nahm Moon , Bakker N. , Verhoef Jan , Snippe Harm , Verheul André F. M. 
Published: July 2002

ABSTRACT

Antibody- and complement-mediated phagocytosis is the main defense mechanism against
Streptococcus pneumoniae
. A standardized, easy to perform phagocytosis assay for pneumococci would be a great asset for the evaluation of the potential efficacy of (experimental) pneumococcal vaccines. Such an assay could replace the laborious phagocytosis assay of viable pneumococci (classical killing assay). Therefore, a newly developed phagocytosis assay based on flow cytometry (flow assay) was compared with the conventional killing assay and enzyme-linked immunosorbent assay (ELISA), using sera obtained from adults pre- and postvaccination with either a bivalent conjugate, a tetravalent conjugate, or the 23-valent polysaccharide vaccine. Highly significant correlations were observed between flow assay phagocytosis titers, killing assay phagocytosis titers, and ELISA antibody titers for serotype 6B and 23F as well. For serotype 19F, strong correlations were only observed between killing assay and ELISA titers. A potential drawback of the flow assay might be the low sensitivity compared with that of the killing assay. The choice of what assay to use, however, will depend on the objectives of the assay. When speed, easy performance, sample throughput, improved worker safety, absence of influence of antibiotics, and absence of false positives are the major criteria, the flow assay is the method of choice. When higher sensitivity is the major requirement, the classical killing assay should be used.
Influence of Patient Age on
Streptococcus pneumoniae
Serotypes Causing Invasive Disease
Authors: Inostroza Jaime , Vinet Ana Maria , Retamal Gloria , Lorca Pedro , Ossa Gonzalo , Facklam Richard R. , Sorensen Ricardo U. 
Published: July 2002

ABSTRACT

All clinical
S. pneumoniae
specimens isolated from patients with invasive or sterile-site infections admitted to one regional general hospital in southern Chile were collected during a 5-year period (February 1994 to September 1999). A total of 247 strains belonging to 50 serotypes were isolated in this survey: 69 in patients under 5 years of age, 129 in patients 5 to 64 years old, and 49 from patients 65 years and older. Eight serotypes were identified in all age groups, while all other serotypes were found exclusively in one age group or in patients over 4 years of age. Serotype 3 was never found in patients under 5 years old, and serotype 14 was not found in patients >64 years of age. There was no difference in the serotypes causing infection in each one of the 5 years of the survey. Our results suggest that both bacterial virulence factors and host factors play an important role in the selection of
S. pneumoniae
serotypes causing invasive infection. Possible host factors include age-related differences in the immune response. Comparative studies with other areas of the world may help to further understanding of our observations in southern Chile.
Induction by a Lactic Acid Bacterium of a Population of CD4+T Cells with Low Proliferative Capacity That Produce Transforming Growth Factor β and Interleukin-10
Authors: von der Weid Thierry , Bulliard Christine , Schiffrin Eduardo J. 
Published: July 2002

ABSTRACTWe investigated whether certain strains of lactic acid bacteria (LAB) could antagonize specific T-helper functions in vitro and thus have the potential to prevent inflammatory intestinal immunopathologies. All strains tested induced various levels of both interleukin-12 (IL-12) and IL-10 in murine splenocytes. In particular,Lactobacillus paracasei(strain NCC2461) induced the highest levels of these cytokines. Since IL-12 and IL-10 have the potential to induce and suppress Th1 functions, respectively, we addressed the impact of this bacterium on the outcome of CD4+T-cell differentiation. For this purpose, bacteria were added to mixed lymphocyte cultures where CD4+T-cells from naive BALB/c mice were stimulated weekly in the presence of irradiated allogeneic splenocytes. In these cultures,L. paracaseiNCC2461 strongly inhibited the proliferative activity of CD4+T cells in a dose-dependent fashion. This was accompanied by a marked decrease of both Th1 and Th2 effector cytokines, including gamma interferon, IL-4, and IL-5. In contrast, IL-10 was maintained and transforming growth factor β (TGF-β) was markedly induced in a dose-dependent manner. The bacteria were not cytotoxic, because cell viability was not affected after two rounds of stimulation. Thus, unidentified bacterial components fromL. paracaseiNCC2461 induced the development of a population of CD4+T cells with low proliferative capacity that produced TGF-β and IL-10, reminiscent of previously described subsets of regulatory cells implicated in oral tolerance and gut homeostasis.
Comparison of Serum and Cell-Specific Cytokines in Humans
Authors: Jason Janine , Archibald Lennox K. , Nwanyanwu Okey C. , Byrd Martha G. , Kazembe Peter N. , Dobbie Hamish , Jarvis William R. 
Published: July 2002

ABSTRACTCytokines function at the cellular, microenvironmental level, but human cytokine assessment is most commonly done at the macro level, by measuring serum cytokines. The relationships between serum and cellular cytokines, if there are any, are undefined. In a study of hospitalized patients in Malawi, we compared cytometrically assessed, cell-specific cytokine data to serum interleukin 2 (IL-2), IL-4, IL-6, IL-8, IL-10, gamma interferon (IFN-γ), and tumor necrosis factor alpha (TNF-α) levels in 16 children and 71 (IL-2, -4, -6, -10) or 159 (IL-8, IFN-γ, and TNF-α) adults, using Wilcoxon rank sum tests and Pearson's (rp) and Spearman's (rs) rank correlations. For the entire study group, correlations between identical serum and cellular cytokines mainly involved IL-8 and IFN-γ, were few, and were weakly positive (r< 0.40). Blood culture-positive persons had the most and strongest correlations, including those between serum IL-2 levels and the percentages of lymphocytes spontaneously making IL-2 (rs= +0.74), serum IL-8 levels and the percentages of lymphocytes spontaneously making IL-8 (rp= +0.66), and serum IL-10 levels and the percentages of CD8+T cells making TNF-α (rp= +0.89). Human immunodeficiency virus (HIV)-positive persons had the next largest number of correlations, including several serum IL-8 level correlations, correlation of serum IL-10 levels with the percentages of lymphocytes producing induced IL-10 (rs= +0.36), and correlation of serum IFN-γ levels and the percentages of lymphocytes spontaneously making both IL-6 and IFN-γ in the same cell (rp= +0.59). HIV-negative, malaria smear-positive, and pediatric patients had few significant correlations; for the second and third of these subgroups, serum IL-8 level was correlated with the percentage of CD8−T cells producing induced IL-8 (rs= +0.40 andrs= +0.56, respectively). Thus, the strength of associations between serum and cellular cytokines varied with the presence or absence of bloodstream infection, HIV status, and perhaps other factors we did not assess. These results strongly suggest that serum cytokines at best only weakly reflect peripheral blood cell cytokine production and balances.
Antibodies to 60-Kilodalton Heat Shock Protein and Outer Membrane Protein 2 of
Chlamydia pneumoniae
in Patients with Coronary Heart Disease
Authors: Ciervo Alessandra , Visca Paolo , Petrucca Andrea , Biasucci Luigi Maria , Maseri Attilio , Cassone Antonio 
Published: July 2002

ABSTRACT

Evidence linking
Chlamydia pneumoniae
infection to atherosclerosis and to atherothrombotic events has recently emerged. A primary candidate implicated in these pathogenetic events is the 60-kDa chlamydial heat shock protein (HSP60). Another putative candidate to activate a potential proinflammatory mechanism is the chlamydial outer membrane protein 2 (OMP2). We have generated both HSP60 and OMP2 recombinant antigens in a nondenatured form and shown that (i) the two antigens were highly immunogenic in mice and (ii) murine antisera thus generated recognized the native
C. pneumoniae
proteins. We measured by enzyme linked immunosorbent assay (ELISA) and immunoblot assay antibody titers to the recombinant antigens in samples from 219 patients with coronary heart disease (CHD), 179 patients with unstable angina (UA), 40 patients with acute myocardial infarction (AMI), and 100 age-, sex-, and risk factor-matched healthy controls. We also examined whether anti-HSP60 and/or anti-OMP2 antibodies correlated with anti-
C. pneumoniae
antibodies assessed by a commercial microimmunofluorescence (MIF) assay. Immunoglobulin G (IgG), but neither IgA nor IgM, antibodies against the two recombinant proteins were detected by ELISA. In particular, anti-HSP60 antibodies were detected in >99% of CHD patients versus 0% of the controls, whereas the proportions of anti-OMP2 positive subjects were >70 and 27%, respectively. Nonetheless, among CHD patients, similar frequencies of positive subjects and titers of anti-HSP60 or anti-OMP2 antibodies were present in UA and AMI subjects. The anti-OMP2, but not the anti-HSP60, antibodies showed high specificity. Consistently, high serological correlation was observed between IgG MIF titers and IgG ELISA reactivity to OMP2 but not to HSP60. Overall, the results of this study demonstrate a strong correlation between CHD and anti-HSP60 IgG levels, as measured by our in-house ELISA. They also suggest that recombinant OMP2 ELISA, because of its high specificity and strong correlation with MIF assay, could be a candidate diagnostic marker for
C. pneumoniae
infection, which would be of potential usefulness for its specificity and nonsubjective nature.