bluuebunny/crossref_metadata_2025_split huggingface dataset:
Quantitative Flow Cytometric Evaluation of Maximal
Cryptosporidium parvum
Oocyst Infectivity in a Neonate Mouse Model
Authors: Delaunay Agnès , Gargala Gilles , Li Xunde , Favennec Loic , Ballet Jean Jacques 
Published: July 2002

ABSTRACT

The importance of waterborne transmission of
Cryptosporidium parvum
to humans has been highlighted by recent outbreaks of cryptosporidiosis. The first step in a survey of contaminated water currently consists of counting
C. parvum
oocysts. Data suggest that an accurate risk evaluation should include a determination of viability and infectivity of counted oocysts in water. In this study, oocyst infectivity was addressed by using a suckling mouse model. Four-day-old NMRI (Naval Medical Research Institute) mice were inoculated per os with 1 to 1,000 oocysts in saline. Seven days later, the number of oocysts present in the entire small intestine was counted by flow cytometry using a fluorescent, oocyst-specific monoclonal antibody. The number of intestinal oocysts was directly related to the number of inoculated oocysts. For each dose group, infectivity of oocysts, expressed as the percentage of infected animals, was 100% for challenge doses between 25 and 1,000 oocysts and about 70% for doses ranging from 1 to 10 oocysts/animal. Immunofluorescent flow cytometry was useful in enhancing the detection sensitivity in the highly susceptible NMRI suckling mouse model and so was determined to be suitable for the evaluation of maximal infectivity risk.
Field Evaluation of a Semiautomated Method for Rapid and Simple Analysis of Recreational Water Microbiological Quality
Authors: Anglès d'Auriac Marc B. , Roberts Hildegarde , Shaw Terri , Sirevåg Reidun , Hermansen Leonila Fajardo , Berg James D. 
Published: July 2002

ABSTRACT
An early warning system using a rapid enzymatic semiautomated method suitable for fecal coliform detection in recreational waters within 8 h was developed further and evaluated in this study. This rapid method was compared to the standard method followed in the United Kingdom. We used 1,011 samples originating from 206 different locations in Wales. When we assessed the presence or absence of fecal coliforms, targeting very low levels of contamination, we obtained 83.9% agreement between the rapid method and the lauryl sulfate broth-membrane filtration technique, whereas direct confirmation of the samples processed by the rapid method showed 89.3% agreement. Environmental enzymatic background activity was found to be the main limiting factor for this method. Owing to a specific and integrated handling of the results by the software of the instrument, the percentage of false-positive results (a consequence of enzymatic background) was successfully limited to 2.9% by the direct confirmation evaluation. However, 7.8% false-negative results due to “late-growers” had to be accepted in order to produce results within a working day. At present, the method can be used in a more conservative way to assess the environmental threshold of 100 CFU of fecal coliforms per 100 ml in recreational waters. The implications of our findings with regard to the applicability of rapid enzymatic methods are discussed.
Genetic Diversity and Dynamics of
Sinorhizobium meliloti
Populations Nodulating Different Alfalfa Cultivars in Italian Soils
Authors: Carelli Maria , Gnocchi Stefano , Fancelli Silvia , Mengoni Alessio , Paffetti Donatella , Scotti Carla , Bazzicalupo Marco 
Published: July 2002

ABSTRACT

We analyzed the genetic diversity of 531
Sinorhizobium meliloti
strains isolated from nodules of
Medicago sativa
cultivars in two different Italian soils during 4 years of plant growth. The isolates were analyzed for DNA polymorphism with the random amplified polymorphic DNA method. The populations showed a high level of genetic polymorphism distributed throughout all the isolates, with 440 different haplotypes. Analysis of molecular variance allowed us to relate the genetic structure of the symbiotic population to various factors, including soil type, alfalfa cultivar, individual plants within a cultivar, and time. Some of these factors significantly affected the genetic structure of the population, and their relative influence changed with time. At the beginning of the experiment, the soil of origin and, even more, the cultivar significantly influenced the distribution of genetic variability of
S. meliloti
. After 3 years, the rhizobium population was altered; it showed a genetic structure based mainly on differences among plants, while the effects of soil and cultivar were not significant.
Population Structure of Rat-Derived
Pneumocystis carinii
in Danish Wild Rats
Authors: Palmer Robert J. , Settnes Osvald P. , Lodal Jens , Wakefield Ann E. 
Published: July 2002

ABSTRACT

The rat model of
Pneumocystis carinii
pneumonia is frequently used to study human
P. carinii
infection, but there are many differences between the rat and human infections. We studied naturally acquired
P. carinii
in wild rats to examine the relevance of the rat model for human infection.
P. carinii
DNA was detected in 47 of 51 wild rats and in 10 of 12 nonimmunosuppressed laboratory rats. Evidence for three novel formae speciales of rat-derived
P. carinii
was found, and these were provisionally named
Pneumocystis carinii
f. sp.
rattus-secundi
,
Pneumocystis carinii
f. sp.
rattus-tertii
, and
Pneumocystis carinii
f. sp.
rattus-quarti
. Our data suggest that low-level carriage of
P. carinii
in wild rats and nonimmunosuppressed laboratory rats is common and that wild rats are frequently coinfected with more than one forma specialis of
P. carinii
. We also examined the diversity in the internally transcribed spacer (ITS) regions of the nuclear rRNA operon of
Pneumocystis carinii
f. sp.
carinii
by using samples from wild rats and laboratory rats and spore trap samples. We report a lack of variation in the ITS1 and ITS2 regions that is consistent with an evolutionary bottleneck in the
P. carinii
f. sp.
carinii
population. This study shows that human- and rat-derived
P. carinii
organisms are very different, not only in genetic composition but also in population structure and natural history.
Role of Proteolysis in Determining Potency of
Bacillus thuringiensis
Cry1Ac δ-Endotoxin
Authors: Lightwood Daniel J. , Ellar David J. , Jarrett Paul 
Published: July 2002

ABSTRACT

Bacillus thuringiensis
protein δ-endotoxins are toxic to a variety of different insect species. Larvicidal potency depends on the completion of a number of steps in the mode of action of the toxin. Here, we investigated the role of proteolytic processing in determining the potency of the
B. thuringiensis
Cry1Ac δ-endotoxin towards
Pieris brassicae
(family: Pieridae) and
Mamestra brassicae
(family: Noctuidae). In bioassays, Cry1Ac was over 2,000 times more active against
P. brassicae
than against
M. brassicae
larvae. Using gut juice purified from both insects, we processed Cry1Ac to soluble forms that had the same N terminus and the same apparent molecular weight. However, extended proteolysis of Cry1Ac in vitro with proteases from both insects resulted in the formation of an insoluble aggregate. With proteases from
P. brassicae
, the Cry1Ac-susceptible insect, Cry1Ac was processed to an insoluble product with a molecular mass of ∼56 kDa, whereas proteases from
M. brassicae
, the non-susceptible insect, generated products with molecular masses of ∼58, ∼40, and ∼20 kDa. N-terminal sequencing of the insoluble products revealed that both insects cleaved Cry1Ac within domain I, but
M. brassicae
proteases also cleaved the toxin at Arg423 in domain II. A similar pattern of processing was observed in vivo. When Arg423 was replaced with Gln or Ser, the resulting mutant toxins resisted degradation by
M. brassicae
proteases. However, this mutation had little effect on toxicity to
M. brassicae
. Differential processing of membrane-bound Cry1Ac was also observed in qualitative binding experiments performed with brush border membrane vesicles from the two insects and in midguts isolated from toxin-treated insects.
Rapid Dye Decolorization Method for Screening Potential Wood Preservatives
Authors: Borokhov Olga , Rothenburger Stephen 
Published: July 2002

ABSTRACT
We developed a new screening method for potential wood preservatives based on decolorization of the dye Remazol Brilliant Blue R by extracellular oxidative agents produced by wood decay fungi. Oxidative biodegradation of lignin yielded decolorized zones around and under fungal cultures on a dyed agar medium. Inhibitory effects were detected by direct observation and measurement of the decolorized zones.
The
Streptococcus thermophilus
Autolytic Phenotype Results from a Leaky Prophage
Authors: Husson-Kao Clara , Mengaud Jérôme , Cesselin Bénédicte , van Sinderen Douwe , Benbadis Laurent , Chapot-Chartier Marie-Pierre 
Published: July 2002

ABSTRACT

Streptococcus thermophilus
autolytic strains are characterized by a typical bell-shaped growth curve when grown under appropriate conditions. The cellular mechanisms involved in the triggering of lysis and the bacteriolytic activities of these strains were investigated in this study. Lactose depletion and organic solvents (ethanol, methanol, and chloroform) were shown to trigger a premature and immediate lysis of M17 exponentially growing cells. These factors and compounds are suspected to act by altering the cell envelope properties, causing either the permeabilization (organic solvents) or the depolarization (lactose depletion) of the cytoplasmic membrane. The autolytic character was shown to be associated with lysogeny. Phage particles, most of which were defective, were observed in the culture supernatants after both mitomycin C-induced and spontaneous lysis. By renaturing sodium dodecyl sulfate-polyacrylamide gel electrophoresis, a bacteriolytic activity was detected at 31 kDa exclusively in the autolytic strains. This enzyme was detected during both growth and spontaneous lysis with the same intensity. We have shown that it was prophage encoded and homologous to the endolysin Lyt51 of the streptococcal temperate bacteriophage φ01205 (M. Sheehan, E. Stanley, G. F. Fitzgerald, and D. van Sinderen, Appl. Environ. Microbiol. 65:569–577, 1999). It appears from our results that the autolytic properties are conferred to the
S. thermophilus
strains by a leaky prophage but do not result from massive prophage induction. More specifically, we propose that phagic genes are constitutively expressed in almost all the cells at a low and nonlethal level and that lysis is controlled and achieved by the prophage-encoded lysis proteins.
Influence of Osmotic and Nutritional Stress on Physiology of
Penicillium fellutanum
in Removal of Phosphocholine and Modification of Phospho-1-
O
-[
N
-Peptidyl-(2-Aminoethanol)] Phosphodiesters of Peptidophosphogalactomannan Species
Authors: Park Yong-Il , Bonetti Sandra J. , Gander John E. 
Published: July 2002

ABSTRACT

Addition of 3 M NaCl to 72-h cultures of
Penicillium fellutanum
in 2 mM phosphate resulted in an increase in percentage of extracellular peptidophosphogalactomannan III (pP
x
GM
iii
) and a decrease in that of pP
x
GM
ii
. The magnitude of
31
P nuclear magnetic resonance signals at 1.47 and 1.33 ppm of phospho-1-
O
-[
N
-peptidyl-(2-aminoethanol)] phosphodiesters pP
x
GM
ii
and pP
x
GM
iii
decreased compared with controls. The data suggest that serine, glycine, and threonine residues from the 3-kDa peptide and from galactofuranosyl-6-
O
-phospho-1′-
O
-[
N
-peptidyl-(2-aminoethanol)] residues were the precursors of the needed choline-derived osmolytes.
Propachlor Removal by
Pseudomonas
Strain GCH1 in an Immobilized-Cell System
Authors: Martín M. , Mengs G. , Plaza E. , Garbi C. , Sánchez M. , Gibello A. , Gutierrez F. , Ferrer E. 
Published: July 2002

ABSTRACT

A bacterial strain capable of growing on propachlor (2-chloro-
N
-isopropylacetanilide) was isolated from soil by using enrichment and isolation techniques. The strain isolated, designated GCH1, was classified as a member of the genus
Pseudomonas
. Washed-cell suspensions of strain GCH1 accumulated
N
-isopropylacetanilide, acetanilide, acetamide, and catechol.
Pseudomonas
strain GCH1 grew on propachlor with a generation time of 4.2 h and a rate of substrate utilization of 1.75 ± 0.15 μmol h
−1
. Gene expression did not require induction but was subject to catabolite expression. Acetanilide was a growth substrate with a yield of 0.56 ± 0.02 mg of protein μmol
−1
. GCH1 strain cells were immobilized by adsorption onto a ceramic support and were used as biocatalysts in an immobilized cell system. Propachlor elimination reached 98%, with a retention time of 3 h and an initial organic load of 0.5 mM propachlor. The viability of immobilized cells increased 34-fold after 120 days of bioreactor operation.
Reductive Cleavage of Demeton-
S
-Methyl by
Corynebacterium glutamicum
in Cometabolism on More Readily Metabolizable Substrates
Authors: Girbal Laurence , Hilaire Didier , Leduc Sébastien , Delery Laure , Rols Jean-Luc , Lindley Nicholas D. 
Published: July 2002

ABSTRACT

Corynebacterium glutamicum
is able to biotransform demeton-
S
-methyl, an organophosphorus compound, during cometabolism with more readily metabolizable substrates. Among the cosubstrates used, fructose is the growth substrate that is most favorable for demeton-
S
-methyl biotransformation. The reaction mechanism of demeton-
S
-methyl biotransformation involves reductive cleavage of an S-C bond, which leads to accumulation of dimethyl thiophosphate in the culture medium.
Transient Growth Requirement in
Bacillus subtilis
following the Cessation of Exponential Growth
Authors: Sung Huang-Mo , Yasbin Ronald E. 
Published: July 2002

ABSTRACT

During an investigation of the parameters controlling mutations in
Bacillus subtilis
we observed that this bacterium exhibits a transient growth requirement for two nonessential amino acids (glutamic acid and isoleucine) during a type of postexponential growth on a minimal medium.
Genetic and Biochemical Approach for Characterization of Resistance to
Bacillus thuringiensis
Toxin Cry1Ac in a Field Population of the Diamondback Moth,
Plutella xylostella
Authors: Sayyed Ali H. , Haward Robert , Herrero Salvador , Ferré Juan , Wright Denis J. 
Published: July 2002

ABSTRACT

Four subpopulations of a
Plutella xylostella
(L.) strain from Malaysia (F
4
to F
8
) were selected with
Bacillus thuringiensis
subsp.
kurstaki
HD-1,
Bacillus thuringiensis
subsp.
aizawai
, Cry1Ab, and Cry1Ac, respectively, while a fifth subpopulation was left as unselected (UNSEL-MEL). Bioassays at F
9
found that selection with Cry1Ac, Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
gave resistance ratios of >95, 10, 7, and 3, respectively, compared with UNSEL-MEL (>10,500, 500, >100, and 26, respectively, compared with a susceptible population, ROTH). Resistance to Cry1Ac, Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
in UNSEL-MEL declined significantly by F
9
. The Cry1Ac-selected population showed very little cross-resistance to Cry1Ab,
B. thuringiensis
subsp.
kurstaki
, and
B. thuringiensis
subsp.
aizawai
(5-, 1-, and 4-fold compared with UNSEL-MEL), whereas the Cry1Ab-,
B. thuringiensis
subsp.
kurstaki
-, and
B. thuringiensis
subsp.
aizawai
-selected populations showed high cross-resistance to Cry1Ac (60-, 100-, and 70-fold). The Cry1Ac-selected population was reselected (F
9
to F
13
) to give a resistance ratio of >2,400 compared with UNSEL-MEL. Binding studies with
125
I-labeled Cry1Ab and Cry1Ac revealed complete lack of binding to brush border membrane vesicles prepared from Cry1Ac-selected larvae (F
15
). Binding was also reduced, although less drastically, in the revertant population, which indicates that a modification in the common binding site of these two toxins was involved in the resistance mechanism in the original population. Reciprocal genetic crosses between Cry1Ac-reselected and ROTH insects indicated that resistance was autosomal and showed incomplete dominance. At the highest dose of Cry1Ac tested, resistance was recessive while at the lowest dose it was almost completely dominant. The F
2
progeny from a backcross of F
1
progeny with ROTH was tested with a concentration of Cry1Ac which would kill 100% of ROTH moths. Eight of the 12 families tested had 60 to 90% mortality, which indicated that more than one allele on separate loci was responsible for resistance to Cry1Ac.
In Situ Reverse Transcription-PCR for Monitoring Gene Expression in Individual
Methanosarcina mazei
S-6 Cells
Authors: Lange Marianne , Tolker-Nielsen Tim , Molin Søren , Ahring Birgitte K. 
Published: July 2002

ABSTRACT

An in situ reverse transcription-PCR protocol for detecting specific mRNA in
Methanosarcina mazei
S-6 is described. This method allowed us to detect heat shock-induced increases in the intracellular levels of the transcript of the universal stress gene
dnaK
. The cell walls of paraformaldehyde-fixed cells were permeabilized by a thermal cycling procedure or by lysozyme treatment, and the cellular DNA was removed with DNase. The cells were subjected to a seminested reverse transcription-PCR protocol in which a digoxigenin-labeled primer was used. Detection of the reporter molecule was based on the 2-hydroxy-3-naphtoic acid-2′-phenylanilide phosphate–Fast Red detection system and binding of anti-digoxigenin–alkaline phosphatase conjugate. Fluorescence in permeabilized cells increased after a heat shock compared to fluorescence in non-heat-shocked cells, and the increase corresponded to an increase in the level of the
dnaK
transcript.
A Novel Amidase (Half-Amidase) for Half-Amide Hydrolysis Involved in the Bacterial Metabolism of Cyclic Imides
Authors: Soong Chee-Leong , Ogawa Jun , Shimizu Sakayu 
Published: July 2002

ABSTRACT

A novel amidase involved in bacterial cyclic imide metabolism was purified from
Blastobacter
sp. strain A17p-4. The enzyme physiologically functions in the second step of cyclic imide degradation, i.e., the hydrolysis of monoamidated dicarboxylates (half-amides) to dicarboxylates and ammonia. Enzyme production was enhanced by cyclic imides such as succinimide and glutarimide but not by amide compounds which are conventional substrates and inducers of known amidases. The purified amidase showed high catalytic efficiency toward half-amides such as succinamic acid (

K
m

= 6.2 mM;
k
cat
= 5.76 s
−1
) and glutaramic acid (

K
m

= 2.8 mM;
k
cat
= 2.23 s
−1
). However, the substrates of known amidases such as short-chain (C
2
to C
4
) aliphatic amides, long-chain (above C
16
) aliphatic amides, amino acid amides, aliphatic diamides, α-keto acid amides,
N
-carbamoyl amino acids, and aliphatic ureides were not substrates for the enzyme. Based on its high specificity toward half-amides, the enzyme was named half-amidase. This half-amidase exists as a monomer with an
M
r
of 48,000 and was strongly inhibited by heavy metal ions and sulfhydryl reagents.
Identification of Fluoropyrogallols as New Intermediates in Biotransformation of Monofluorophenols in
Rhodococcus opacus
1cp
Authors: Finkelstein Zoya I. , Baskunov Boris P. , Boersma Marelle G. , Vervoort Jacques , Golovlev Eugene L. , van Berkel Willem J. H. , Golovleva Ludmila A. , Rietjens Ivonne M. C. M. 
Published: July 2002

ABSTRACT

The transformation of monofluorophenols by whole cells of
Rhodococcus opacus
1cp was investigated, with special emphasis on the nature of hydroxylated intermediates formed. Thin-layer chromatography, mass spectrum analysis, and
19
F nuclear magnetic resonance demonstrated the formation of fluorocatechol and trihydroxyfluorobenzene derivatives from each of three monofluorophenols. The
19
F chemical shifts and proton-coupled splitting patterns of the fluorine resonances of the trihydroxyfluorobenzene products established that the trihydroxylated aromatic metabolites contained hydroxyl substituents on three adjacent carbon atoms. Thus, formation of 1,2,3-trihydroxy-4-fluorobenzene (4-fluoropyrogallol) from 2-fluorophenol and formation of 1,2,3-trihydroxy-5-fluorobenzene (5-fluoropyrogallol) from 3-fluorophenol and 4-fluorophenol were observed. These results indicate the involvement of fluoropyrogallols as previously unidentified metabolites in the biotransformation of monofluorophenols in
R. opacus
1cp.
Fortified Sera and Their Use in Environmental Virology
Authors: Hoyt Jonathan L. , Margolin Aaron B. 
Published: July 2002

ABSTRACT
Four commercially available fortified sera were compared to fetal bovine serum (FBS) with regard to their ability to maintain or increase the sensitivity of the Buffalo green monkey (BGM) kidney cell line to viral infection. Nine virus strains and five wastewater samples were used. Fortified sera were comparable to FBS for the enumeration of some viruses by the plaque method and for the detection of virus in wastewater by the most-probable-number assay.
Development of a Novel, Rapid Integrated
Cryptosporidium parvum
Detection Assay
Authors: Kozwich Diane , Johansen Kristine A. , Landau Keli , Roehl Christopher A. , Woronoff Sam , Roehl Patrick A. 
Published: July 2002

ABSTRACT

The aim of this study was to develop a reverse transcription-PCR assay and lateral flow detection protocol for specific identification of
Cryptosporidium parvum
. The method which we developed is sensitive and specific and has a low limit of detection. In our protocol a solid phase material, the Xtra Bind Capture System, was used for extraction and purification of double-stranded RNA (dsRNA) specific for
C. parvum
. The Xtra Bind Capture System interfaced with pellets concentrated from water samples collected with previously developed filtration devices. The pellets were resuspended in reagent water (final volume, 0.5 ml), and an equal amount of rupture buffer and the Xtra Bind Capture System was added to the resuspended pellet mixture. The dsRNA target sequences in a 0.5-ml portion were captured by the solid phase material via hybridization. The debris and potential inhibitors were removed by washing the Xtra Bind material several times with buffer. The Xtra Bind material with its bound dsRNA was added directly to an amplification reaction mixture, and the target was amplified without elution from the Xtra Bind material. A PCR was performed in the presence of the Xtra Bind Capture System, which resulted in robust amplification of the target. The detection system which we used was adapted from lateral flow chromatography methods typically used for antigen-antibody reactions. The result was a colored line that was visible if the organism was present. When this method was used, we were able to reproducibly and correctly identify 10 oocysts added to 0.5 ml of reagent water. When the protocol was evaluated with a small set of environmental samples, the level of detection was as low as 1 oocyst/liter. The total time from resuspension of the pellet to detection was about 3 h, which is considerably less than the 5 h required for immunomagnetic separation followed by an indirect immunofluorescence assay and microscopy.