bluuebunny/crossref_metadata_2025_split huggingface dataset:
Three-Year Study To Assess Human Enteric Viruses in Shellfish
Authors: Le Guyader F. , Haugarreau L. , Miossec L. , Dubois E. , Pommepuy M. 
Published: July 2002

ABSTRACT

The main pathogenic enteric viruses able to persist in the environment, such as hepatitis A virus (HAV), Norwalk-like virus (NLV), enterovirus (EV), rotavirus (RV), and astrovirus (AV), were detected by reverse transcription-PCR and hybridization in shellfish during a 3-year study. Oyster samples (
n
= 108), occasionally containing bacteria, were less frequently contaminated, showing positivity for AV (17%), NLV (23%), EV (19%), and RV (27%), whereas mussel samples, collected in areas routinely impacted by human sewage, were more highly contaminated: AV (50%), HAV (13%), NLV (35%), EV (45%), and RV (52%). Sequences obtained from HAV and NLV amplicons showed a great variety of strains, especially for NLV (strains close to Mexico, Snow Mountain Agent, or Norwalk virus). Viral contamination was mainly observed during winter months, although there were some seasonal differences among the viruses. This first study of virus detection over a fairly long period of time suggests that routine analysis of shellfish by a molecular technique is feasible.
Genotypic Characterization of
Bradyrhizobium
Strains Nodulating Small Senegalese Legumes by 16S-23S rRNA Intergenic Gene Spacers and Amplified Fragment Length Polymorphism Fingerprint Analyses
Authors: Doignon-Bourcier Florence , Willems Anne , Coopman Renata , Laguerre Gisele , Gillis Monique , de Lajudie Philippe 
Published: July 2002

ABSTRACT

We examined the genotypic diversity of 64
Bradyrhizobium
strains isolated from nodules from 27 native leguminous plant species in Senegal (West Africa) belonging to the genera
Abrus
,
Alysicarpus
,
Bryaspis
,
Chamaecrista
,
Cassia
,
Crotalaria
,
Desmodium
,
Eriosema
,
Indigofera
,
Moghania
,
Rhynchosia
,
Sesbania
,
Tephrosia
, and
Zornia
, which play an ecological role and have agronomic potential in arid regions. The strains were characterized by intergenic spacer (between 16S and 23S rRNA genes) PCR and restriction fragment length polymorphism (IGS PCR-RFLP) and amplified fragment length polymorphism (AFLP) fingerprinting analyses. Fifty-three reference strains of the different
Bradyrhizobium
species and described groups were included for comparison. The strains were diverse and formed 27 groups by AFLP and 16 groups by IGS PCR-RFLP. The sizes of the IGS PCR products from the
Bradyrhizobium
strains that were studied varied from 780 to 1,038 bp and were correlated with the IGS PCR-RFLP results. The grouping of strains was consistent by the three methods AFLP, IGS PCR-RFLP, and previously reported 16S amplified ribosomal DNA restriction analysis. For investigating the whole genome, AFLP was the most discriminative technique, thus being of particular interest for future taxonomic studies in
Bradyrhizobium
, for which DNA is difficult to obtain in quantity and quality to perform extensive DNA:DNA hybridizations.
Competitive Exclusion of Heterologous
Campylobacter
spp. in Chicks
Authors: Chen Hui-Cheng , Stern Norman J. 
Published: July 2002

ABSTRACT

Chicken and human isolates of
Campylobacter jejuni
were used to provide oral challenge of day-old broiler chicks. The isolation ratio of the competing challenge strains was monitored and varied, depending upon the isolates used. A PCR-restriction fragment length polymorphism assay of the flagellin gene (
flaA
) was used to discriminate between the chick-colonizing isolates. Our observations indicated that the selected
C. jejuni
colonizers dominated the niche provided by the chicken ceca. Chicken isolates from the
flaA
type 7 grouping generally had numerical superiority over the human isolates when they were administered in our 1-day-old chick model. Our results suggest that it is possible to use combinations of
C. jejuni
chicken isolates as a defined bacterial preparation for the competitive exclusion of human-pathogenic
C. jejuni
in poultry.
Identification ofDekkera bruxellensis(Brettanomyces) from Wine by Fluorescence In Situ Hybridization Using Peptide Nucleic Acid Probes
Authors: Stender Henrik , Kurtzman Cletus , Hyldig-Nielsen Jens J. , Sørensen Ditte , Broomer Adam , Oliveira Kenneth , Perry-O'Keefe Heather , Sage Andrew , Young Barbara , Coull James 
Published: July 2002

ABSTRACTA new fluorescence in situ hybridization method using peptide nucleic acid (PNA) probes for identification ofBrettanomycesis described. The test is based on fluorescein-labeled PNA probes targeting a species-specific sequence of the rRNA ofDekkera bruxellensis. The PNA probes were applied to smears of colonies, and results were interpreted by fluorescence microscopy. The results obtained from testing 127 different yeast strains, including 78Brettanomycesisolates from wine, show that the spoilage organismBrettanomycesbelongs to the speciesD. bruxellensisand that the new method is able to identifyBrettanomyces(D. bruxellensis) with 100% sensitivity and 100% specificity.
Purification, Characterization, and Application of a Novel Dye-Linked
l
-Proline Dehydrogenase from a Hyperthermophilic Archaeon,
Thermococcus profundus
Authors: Sakuraba Haruhiko , Takamatsu Yoshinori , Satomura Takenori , Kawakami Ryushi , Ohshima Toshihisa 
Published: July 2002

ABSTRACT

The distribution of dye-linked
l
-amino acid dehydrogenases was investigated in several hyperthermophiles, and the activity of dye-linked
l
-proline dehydrogenase (dye-
l
-proDH,
l
-proline:acceptor oxidoreductase) was found in the crude extract of some
Thermococcales
strains. The enzyme was purified to homogeneity from a hyperthermophilic archaeon,
Thermococcus profundus
DSM 9503, which exhibited the highest specific activity in the crude extract. The molecular mass of the enzyme was about 160 kDa, and the enzyme consisted of heterotetrameric subunits (α
2
β
2
) with two different molecular masses of about 50 and 40 kDa. The N-terminal amino acid sequences of the α-subunit (50-kDa subunit) and the β-subunit (40-kDa subunit) were MRLTEHPILDFSERRGRKVTIHF and XRSEAKTVIIGGGIIGLSIAYNLAK, respectively. Dye-
l
-proDH was extraordinarily stable among the dye-linked dehydrogenases under various conditions: the enzyme retained its full activity upon incubation at 70°C for 10 min, and ca. 40% of the activity still remained after heating at 80°C for 120 min. The enzyme did not lose the activity upon incubation over a wide range of pHs from 4.0 to 10.0 at 50°C for 10 min. The enzyme exclusively catalyzed
l
-proline dehydrogenation using 2,6-dichloroindophenol (Cl2Ind) as an electron acceptor. The Michaelis constants for
l
-proline and Cl2Ind were determined to be 2.05 and 0.073 mM, respectively. The reaction product was identified as Δ
1
-pyrroline-5-carboxylate by thin-layer chromatography. The prosthetic group of the enzyme was identified as flavin adenine dinucleotide by high-pressure liquid chromatography. In addition, the simple and specific determination of
l
-proline at concentrations from 0.10 to 2.5 mM using the stable dye-
l
-proDH was achieved.
Development and Application of a Most-Probable-Number–PCR Assay To Quantify Flagellate Populations in Soil Samples
Authors: Fredslund Line , Ekelund Flemming , Jacobsen Carsten Suhr , Johnsen Kaare 
Published: July 2002

ABSTRACTThis paper reports on the first successful molecular detection and quantification of soil protozoa. Quantification of heterotrophic flagellates and naked amoebae in soil has traditionally relied on dilution culturing techniques, followed by most-probable-number (MPN) calculations. Such methods are biased by differences in the culturability of soil protozoa and are unable to quantify specific taxonomic groups, and the results are highly dependent on the choice of media and the skills of the microscopists. Successful detection of protozoa in soil by DNA techniques requires (i) the development and validation of DNA extraction and quantification protocols and (ii) the collection of sufficient sequence data to find specific protozoan 18S ribosomal DNA sequences. This paper describes the development of an MPN-PCR assay for detection of the common soil flagellateHeteromita globosa, using primers targeting a 700-bp sequence of the small-subunit rRNA gene. The method was tested by use of gnotobiotic laboratory microcosms with sterile tar-contaminated soil inoculated with the bacteriumPseudomonas putidaOUS82 UCB55 as prey. There was satisfactory overall agreement betweenH. globosapopulation estimates obtained by the PCR assay and a conventional MPN assay in the three soils tested.
Novel Approach for Improving the Productivity of Antibiotic-Producing Strains by Inducing Combined Resistant Mutations
Authors: Hu Haifeng , Ochi Kozo 
Published: July 2002

ABSTRACT

We developed a novel approach for improving the production of antibiotic from
Streptomyces coelicolor
A3(2) by inducing combined drug-resistant mutations. Mutants with enhanced (1.6- to 3-fold-higher) actinorhodin production were detected at a high frequency (5 to 10%) among isolates resistant to streptomycin (Str
r
), gentamicin (Gen
r
), or rifampin (Rif
r
), which developed spontaneously on agar plates which contained one of the three drugs. Construction of double mutants (
str gen
and
str rif
) by introducing gentamicin or rifampin resistance into an
str
mutant resulted in further increased (1.7- to 2.5-fold-higher) actinorhodin productivity. Likewise, triple mutants (
str gen rif
) thus constructed were found to have an even greater ability for producing the antibiotic, eventually generating a mutant able to produce 48 times more actinorhodin than the wild-type strain. Analysis of
str
mutants revealed that a point mutation occurred within the
rpsL
gene, which encodes the ribosomal protein S12.
rif
mutants were found to have a point mutation in the
rpoB
gene, which encodes the β-subunit of RNA polymerase. Mutation points in
gen
mutants still remain unknown. These single, double, and triple mutants displayed in hierarchical order a remarkable increase in the production of ActII-ORF4, a pathway-specific regulatory protein, as determined by Western blotting analysis. This reflects the same hierarchical order observed for the increase in actinorhodin production. The superior ability of the triple mutants was demonstrated by physiological analyses under various cultural conditions. We conclude that by inducing combined drug-resistant mutations we can continuously increase the production of antibiotic in a stepwise manner. This new breeding approach could be especially effective for initially improving the production of antibiotics from wild-type strains.
Zeamatin Inhibits Trypsin and α-Amylase Activities
Authors: Schimoler-O'Rourke Rebecca , Richardson Michael , Selitrennikoff Claude P. 
Published: July 2002

ABSTRACT

Zeamatin is a 22-kDa protein isolated from
Zea mays
that has antifungal activity against human and plant pathogens. Unlike other pathogenesis-related group 5 proteins, zeamatin inhibits insect α-amylase and mammalian trypsin activities. It is of clinical significance that zeamatin did not inhibit human α-amylase activity and inhibited mammalian trypsin activity only at high molar concentrations.
Influence of Effluent Irrigation on Community Composition and Function of Ammonia-Oxidizing Bacteria in Soil
Authors: Oved Tamar , Shaviv Avi , Goldrath Tal , Mandelbaum Raphi T. , Minz Dror 
Published: July 2002

ABSTRACTThe effect of effluent irrigation on community composition and function of ammonia-oxidizing bacteria (AOB) in soil was evaluated, using techniques of molecular biology and analytical soil chemistry. Analyses were conducted on soil sampled from lysimeters and from a grapefruit orchard which had been irrigated with wastewater effluent or fertilizer-amended water (FAW). Specifically, comparisons of AOB community composition were conducted using denaturing gradient gel electrophoresis (DGGE) of PCR-amplified fragments of the gene encoding the α-subunit of the ammonia monooxygenase gene (amoA) recovered from soil samples and subsequent sequencing of relevant bands. A significant and consistent shift in the population composition of AOB was detected in soil irrigated with effluent. This shift was absent in soils irrigated with FAW, despite the fact that the ammonium concentration in the FAW was similar. At the end of the irrigation period,Nitrosospira-like populations were dominant in soils irrigated with FAW, whileNitrosomonas-like populations were dominant in effluent-irrigated soils. Furthermore, DGGE analysis of theamoAgene proved to be a powerful tool in evaluating the soil AOB community population and population shifts therein.
Location Effects of a Reporter Gene on Expression Levels and on Native Protein Synthesis inLactococcus lactisandSaccharomyces cerevisiae
Authors: Thompson Arthur , Gasson Michael J. 
Published: July 2002

ABSTRACTThe engineering of industrially important genetically modified organisms by the integration of heterologous genes into the chromosome is often the method of choice for several reasons concerned with long-term stability, homogeneous population distribution, and the enabling of selection without the addition of antibiotics. However, integration may disrupt endogenous gene expression, giving rise to increased levels of toxic metabolic byproducts or activating otherwise silent genes. The position of integration of a foreign gene in the chromosome can also influence its expression levels, and this effect will be of relevance in terms of optimizing protein production parameters. In this study, we determine how the random integration of a foreign reporter gene might affect expression levels and assess the use of proteome analysis to investigate possible effects on synthesis of endogenous proteins in two important food-relevant microorganisms,Saccharomyces cerevisiaeandLactococcus lactis. ElevenL. lactisintegrants carrying thegusAgene were analyzed, and expression levels were found to vary by a factor of threefold in contrast to expression levels oflacZin 18S. cerevisiaeintegrants, which showed a 14-fold variation. Of relevance to industry is whether any changes in expression levels might occur as a consequence of storage of the modified strains. Here it is also shown that the above differences in expression levels were not significantly affected by storage of frozen cultures over a period of several months. Analysis of the protein composition of the yeast and lactococcal integrant strains by separation on one-dimensional (1D) and 2D gels showed no significant variations in position beyond those observed in control samples.
Isolation and Genotyping of
Helicobacter pylori
from Untreated Municipal Wastewater
Authors: Lu Yingzhi , Redlinger Thomas E. , Avitia Raquel , Galindo Adriana , Goodman Karen 
Published: July 2002

ABSTRACT

For this study, we isolated
Helicobacter pylori
from wastewater by a series of steps beginning with immunomagnetic separation and cell culture. After Gram staining and three standard microbial tests, the 16S rRNA sequences of a total of 23 out of 37 putative
H. pylori
isolates were verified by PCR. Eleven
H. pylori
isolates were genotyped and fell into four
vacA
classes: those with the
vacA
allelic variants
s1a
and
m1
,
s1b
and
m1
,
s2
and
m2
, or
s2
and
m1
. Most
H. pylori
isolates were of the
vacA s1a
/
m1
type, which has been shown to be associated with advanced diseases based on genotyping of
H. pylori
from gastric cancer patients. These results demonstrated that
H. pylori
survives in water and may be a potential source of
H. pylori
transmission, especially where water is not adequately treated.
Incidence ofMycobacterium paratuberculosisin Bulk Raw and Commercially Pasteurized Cows' Milk from Approved Dairy Processing Establishments in the United Kingdom
Authors: Grant Irene R. , Ball Hywel J. , Rowe Michael T. 
Published: July 2002

ABSTRACTOver a 17-month period (March 1999 to July 2000), a total of 814 cows' milk samples, 244 bulk raw and 567 commercially pasteurized (228 whole, 179 semiskim, and 160 skim), from 241 approved dairy processing establishments throughout the United Kingdom were tested for the presence ofMycobacterium paratuberculosisby immunomagnetic PCR (to detect all cells living and dead) and culture (to detect viable cells). Overall,M. paratuberculosisDNA was detected by immunomagnetic PCR in 19 (7.8%; 95% confidence interval, 4.3 to 10.8%) and 67 (11.8%; 95% confidence interval, 9.0 to 14.2%) of the raw and pasteurized milk samples, respectively. ConfirmedM. paratuberculosisisolates were cultured from 4 (1.6%; 95% confidence interval, 0.04 to 3.1%) and 10 (1.8%; 95% confidence interval, 0.7 to 2.8%) of the raw and pasteurized milk samples, respectively, following chemical decontamination with 0.75% (wt/vol) cetylpyridinium chloride for 5 h. The 10 culture-positive pasteurized milk samples were from just 8 (3.3%) of the 241 dairy processing establishments that participated in the survey. Seven of the culture-positive pasteurized milk samples had been heat treated at 72 to 74°C for 15 s; the remainder had been treated at 72 to 75°C for the extended holding time of 25 s. When typed by restriction fragment length polymorphism and pulsed-field gel electrophoresis methods, some of the milk isolates were shown to be types distinct from those of laboratory strains in regular use within the testing laboratory. From information gathered at the time of milk sample collection, all indications were that pasteurization had been carried out effectively at all of the culture-positive dairies. That is, pasteurization time and temperature conditions complied with the legal minimum high-temperature, short-time process; all pasteurized milk samples tested phosphatase negative; and postprocess contamination was considered unlikely to have occurred. It was concluded that viableM. paratuberculosisis occasionally present at low levels in commercially pasteurized cows' milk in the United Kingdom.
Interleukin 7 Can Enhance Antigen-Specific Cytotoxic-T-Lymphocyte and/or Th2-Type Immune Responses In Vivo
Authors: Sin Jeong-Im , Kim Jong , Patchuk Catherine , Weiner David B. 
Published: July 2002

ABSTRACTInterleukin 7 (IL-7) protein has been reported to be important in the development of cytotoxic-T-lymphocyte (CTL) responses. However, other studies also support a partial Th2 phenotype for this cytokine. In an effort to clarify this unusual conflict, we compared IL-7 along with IL-12 (Th1 control) and IL-10 (Th2 control) for its ability to induce antigen (Ag)-specific CTL and Th1- versus Th2-type immune responses using a well established DNA vaccine model. In particular, IL-7 codelivery showed a significant increase in immunoglobulin G1 (IgG1) levels compared to IgG2a levels. IL-7 coinjection also decreased production of Th1-type cytokine IL-2, gamma interferon, and the chemokine RANTES but increased production of the Th2-type cytokine IL-10 and the similarly biased chemokine MCP-1. In herpes simplex virus (HSV) challenge studies, IL-7 coinjection decreased the survival rate after lethal HSV type 2 (HSV-2) challenge compared with gD plasmid vaccine alone in a manner similar to IL-10 coinjection, whereas IL-12 coinjection enhanced the protection, further supporting that IL-7 drives immune responses to the Th2 type, resulting in reduced protection against HSV-2 challenge. Moreover, coinjection with human immunodeficiency virus type 1envandgag/polgenes plus IL-12 or IL-7 cDNA enhanced Ag-specific CTLs, while coinjection with IL-10 cDNA failed to influence CTL induction. Thus, IL-7 could drive Ag-specific Th2-type cellular responses and/or CTL responses. These results support that CTLs could be induced by IL-7 in a Th2-type cytokine and chemokine environment in vivo. This property of IL-7 allows for an alternative pathway for CTL development which has important implications for host-pathogen responses.
Antibody Responses of Cattle Immunized with the Tf190 Adhesin ofTritrichomonas foetus
Authors: Voyich Jovanka M. , Ansotegui Raymond , Swenson Connie , Bailey John , Burgess Donald E. 
Published: July 2002

ABSTRACTThe antibody response patterns of cattle after subcutaneous and intranasal immunizations with adhesin Tf190 ofTritrichomonas foetuswere investigated. Reactions of antibody from cattle parenterally immunized with Tf190 revealed antigen specificity and Tf190 sensitization in the majority of the animals, as determined by Western blotting. The results also demonstrated strong preimmune immunoglobulin G2 (IgG2) binding toT. foetusantigens not seen in IgG1 profiles. Subcutaneous injections of Tf190 resulted in significant (P< 0.05) increases in serum IgG1 and IgG2 titers over time, as determined by parasite specific enzyme-linked immunosorbent assay. Immune sera also significantly inhibited parasite adhesion to mammalian cell lines compared to the level of inhibition obtained with preimmune sera (P< 0.05). Intranasal immunization with Tf190 failed to produce measurable parasite-specific antibody in serum; however, this immunization route did result in significant (P< 0.05) increases in parasite-specific IgA titers in cervical mucus secretions from immunized animals that were more resistant to intravaginal challenge withT. foetusthan controls. These results suggest that systemic immunization with Tf190 results in serum antibody production and antiparasitic adhesin antibodies. Additionally, the results of challenge experiments with intranasally immunized animals suggests that Tf190 primes protective immune responses that lead to lower rates of infection among these animals.