bluuebunny/crossref_metadata_2025 huggingface dataset:
P47  Creation of longer‐lasting (substantive) oral care flavours
Authors: Perring KD , Tuck KM , Wilson M
Published: March 2005

Mainstream oral care flavours are primarily designed to provide hedonic (taste) benefits and to promote breath freshness. In particular, a key criterion for a commercially successful toothpaste flavour is a long lasting reduction in the perception of mouth odour. Various additives can help deliver this benefit, in addition to flavours formulated according to patented guidelines. The residence time (substantivity) in the mouth of flavours and additives is clearly critical with respect to the longevity of the breath‐freshening benefit, and little data are available in the literature to guide the selection of substantive components. The aim of this project was to investigate the dynamics of flavour loss from the buccal cavity following brushing using a mass spectrometer equipped with an atmospheric pressure headspace sampler which enabled real‐time determination of flavour components in mouth air. A number of flavour ingredients found in peppermint‐ and spearmint‐based oral care flavours were studied. The in vivo decay kinetics of flavour ingredient loss were quantified and found to be strongly related to the physicochemical properties of ingredients, except in the case of esters where a more complex dependence was observed arising from chemical transformation occurring in addition to physical transportation away from the mouth. Surprisingly, some materials were discovered to undergo rapid degradation with a half‐life of minutes. Confirmatory studies of the decay kinetics of such materials were carried out in vitro, and structural features were identified which were associated with the observed hydrolytic vulnerability. This work has allowed the development of new guidelines to enable the creation of longer‐lasting oral care flavours.
O9  Creation of oral care flavours to deliver breath freshening benefits – an in vitro method
Authors: Cawkill PM , Bradshaw DJ , Richards J , Munroe MJ
Published: March 2005

Flavours conventionally help to deliver breath freshening in oral care products via sensory masking. The aim of this study was to determine whether flavours could additionally inhibit formation of malodorous compounds by bacteria. Oral malodour is in large part related to the production of volatile sulphur compounds (VSCs) by Gram‐negative organisms. We therefore developed a quantitative in vitro assay for hydrogen sulphide production by Klebsiella pneumoniae. The entire Quest dental flavour palette was screened for inhibition of H2S production. A database of flavour ingredient effects was created, and used to create VSC‐inhibitory flavours. These flavours were assessed in the in vitro assay to confirm that the compounded flavour performed as expected. The best performing flavours developed in this way were selected for testing in the Quest Breath Freshness Panel (see poster, P21). Rules based on the performance of dental flavour ingredients, singly or in combination, were also used to develop a patent position. Flavours formulated using these rules (termed Q‐fresh flavours) have been incorporated into a range of oral care products including toothpastes, mouthwashes, chewing gums and breath films. These products have been tested in the Quest Breath freshness panel, and have been shown to deliver significant breath freshening benefits. An in vitro VSC‐inhibition method has allowed reliable development of flavours with breath‐freshening benefits.
A Saga of Wormholes and Anatase
Authors: Fitzhugh William W.
Published: March 2005

Maps, Myths, and Men
The Story of the Vinland Map .
By Kirsten A. Seaver
. Stanford University Press, Palo Alto, CA, 2004. 504 pp. $65, £44.50. ISBN 0-8047-4962-0. Paper, $24.95, £17.50. ISBN: 0-8047-4963-9.

Besides reviewing the controversies surrounding the map and arguing that it is a modern fake, the author discusses our current understanding of medieval Norse culture and voyages to North America.
Residential Biofuels in South Asia: Carbonaceous Aerosol Emissions and Climate Impacts
Authors: Venkataraman C. , Habib G. , Eiguren-Fernandez A. , Miguel A. H. , Friedlander S. K.
Published: March 2005

High concentrations of pollution particles, including “soot” or black carbon, exist over the Indian Ocean, but their sources and geographical origins are not well understood. We measured emissions from the combustion of biofuels, used widely in south Asia for cooking, and found that large amounts of carbonaceous aerosols are emitted per kilogram of fuel burnt. We calculate that biofuel combustion is the largest source of black carbon emissions in India, and we suggest that its control is central to climate change mitigation in the south Asian region.
Impaired Thermosensation in Mice Lacking TRPV3, a Heat and Camphor Sensor in the Skin
Authors: Moqrich Aziz , Hwang Sun Wook , Earley Taryn J. , Petrus Matt J. , Murray Amber N. , Spencer Kathryn S. R. , Andahazy Mary , Story Gina M. , Patapoutian Ardem
Published: March 2005

Environmental temperature is thought to be directly sensed by neurons through their projections in the skin. A subset of the mammalian transient receptor potential (TRP) family of ion channels has been implicated in this process. These “thermoTRPs” are activated at distinct temperature thresholds and are typically expressed in sensory neurons. TRPV3 is activated by heat (>33°C) and, unlike most thermoTRPs, is expressed in mouse keratinocytes. We found that TRPV3 null mice have strong deficits in responses to innocuous and noxious heat but not in other sensory modalities; hence, TRPV3 has a specific role in thermosensation. The natural compound camphor, which modulates sensations of warmth in humans, proved to be a specific activator of TRPV3. Camphor activated cultured primary keratinocytes but not sensory neurons, and this activity was abolished in TRPV3 null mice. Therefore, heat-activated receptors in keratinocytes are important for mammalian thermosensation.
Immobilization ofEscherichia coliCells by Use of the Antimicrobial Peptide Cecropin P1
Authors: Gregory Kalvin , Mello Charlene M.
Published: March 2005

An immobilization scheme for bacterial cells is described, in which the antimicrobial peptide cecropin P1 was used to trapEscherichia coliK-12 and O157:H7 cells on microtiter plate well surfaces. Cecropin P1 was covalently attached to the well surfaces, andE. colicells were allowed to bind to the peptide-coated surface. The immobilized cells were detected colorimetrically with an anti-E. coliantibody-horseradish peroxidase conjugate. Binding curves were obtained in which the signal intensities were dependent upon the cell concentration and upon the amount of peptide attached to the well surface. After normalization for the amount of peptide coupled to the surface and the relative binding affinity of the antibody for each strain, the binding data were compared, which indicated that there was a strong preference forE. coliO157:H7 overE. coliK-12. The cells could be immobilized reproducibly at pH values ranging from 5 to 10 and at ionic strengths up to 0.50 M.
Efficient DNA Fingerprinting of
Clostridium botulinum
Types A, B, E, and F by Amplified Fragment Length Polymorphism Analysis
Authors: Keto-Timonen Riikka , Nevas Mari , Korkeala Hannu
Published: March 2005

Amplified fragment length polymorphism (AFLP) analysis was applied to characterize 33 group I and 37 group II
Clostridium botulinum
strains. Four restriction enzyme and 30 primer combinations were screened to tailor the AFLP technique for optimal characterization of
C. botulinum
. The enzyme combination HindIII and HpyCH4IV, with primers having one selective nucleotide apiece (Hind-C and Hpy-A), was selected. AFLP clearly differentiated between
C. botulinum
groups I and II; group-specific clusters showed <10% similarity between proteolytic and nonproteolytic
C. botulinum
strains. In addition, group-specific fragments were detected in both groups. All strains studied were typeable by AFLP, and a total of 42 AFLP types were identified. Extensive diversity was observed among strains of
C. botulinum
type E, whereas group I had lower genetic biodiversity. These results indicate that AFLP is a fast, highly discriminating, and reproducible DNA fingerprinting method with excellent typeability, which, in addition to its suitability for typing at strain level, can be used for
C. botulinum
group identification.
Species Abundance and Diversity of
Burkholderia cepacia
Complex in the Environment
Authors: Ramette Alban , LiPuma John J. , Tiedje James M.
Published: March 2005

Despite considerable interest in studying
Burkholderia cepacia
complex in the environment, we still do not have efficient methods to detect, isolate, and screen large numbers of
B. cepacia
isolates. To better describe the ecology and diversity of
B. cepacia
complex, a colony hybridization assay was developed to detect specifically all species of the complex based on polymorphism of the variable V3 region of the 16S rRNA sequence. The sensitivity of the assay was dramatically enhanced by using a probe consisting of three repeats of a
B. cepacia
complex-specific probe, each separated by a phosphoramidite spacer. In addition, a duplex PCR targeting
B. cepacia
complex-specific
recA
and 16S rRNA sequences was developed to enable a fast and reliable diagnostic assay for members of the complex. When applied to maize rhizosphere samples, colony hybridization results were in good agreement with those of most-probable-number duplex PCR, both indicating a >100-fold fluctuation of abundance between individual plants. Using restriction analysis of
recA
for a total of 285 confirmed isolates of the
B. cepacia
complex, up to seven
B. cepacia
complex species were identified; however, their diversity and abundance were not evenly distributed among individual plants, and several allelic variants were commonly found from the same rhizosphere sample. These results indicate that not only complex communities of
B. cepacia
complex species and closely related strains of the same species may coexist at high population levels but also species composition and abundance may dramatically vary between individual plants.
Stable-Isotope Probing of Bacteria Capable of Degrading Salicylate, Naphthalene, or Phenanthrene in a Bioreactor Treating Contaminated Soil
Authors: Singleton David R. , Powell Sabrina N. , Sangaiah Ramiah , Gold Avram , Ball Louise M. , Aitken Michael D.
Published: March 2005

[
13
C
6
]salicylate, [U-
13
C]naphthalene, and [U-
13
C]phenanthrene were synthesized and separately added to slurry from a bench-scale, aerobic bioreactor used to treat soil contaminated with polycyclic aromatic hydrocarbons. Incubations were performed for either 2 days (salicylate, naphthalene) or 7 days (naphthalene, phenanthrene). Total DNA was extracted from the incubations, the “heavy” and “light” DNA were separated, and the bacterial populations associated with the heavy fractions were examined by denaturing gradient gel electrophoresis (DGGE) and 16S rRNA gene clone libraries. Unlabeled DNA from
Escherichia coli
K-12 was added to each sample as an internal indicator of separation efficiency. While
E. coli
was not detected in most analyses of heavy DNA, a low number of
E. coli
sequences was recovered in the clone libraries associated with the heavy DNA fraction of [
13
C]phenanthrene incubations. The number of
E. coli
clones recovered proved useful in determining the relative amount of light DNA contamination of the heavy fraction in that sample. Salicylate- and naphthalene-degrading communities displayed similar DGGE profiles and their clone libraries were composed primarily of sequences belonging to the
Pseudomonas
and
Ralstonia
genera. In contrast, heavy DNA from the phenanthrene incubations displayed a markedly different DGGE profile and was composed primarily of sequences related to the
Acidovorax
genus. There was little difference in the DGGE profiles and types of sequences recovered from 2- and 7-day incubations with naphthalene, so secondary utilization of the
13
C during the incubation did not appear to be an issue in this experiment.
Functional Analysis of Three Plasmids from
Lactobacillus plantarum
Authors: van Kranenburg Richard , Golic Natasa , Bongers Roger , Leer Rob J. , de Vos Willem M. , Siezen Roland J. , Kleerebezem Michiel
Published: March 2005

Lactobacillus plantarum
WCFS1 harbors three plasmids, pWCFS101, pWCFS102, and pWCFS103, with sizes of 1,917, 2,365, and 36,069 bp, respectively. The two smaller plasmids are of unknown function and contain replication genes that are likely to function via the rolling-circle replication mechanism. The host range of the pWCFS101 replicon includes
Lactobacillus
species and
Lactococcus lactis
, while that of the pWCFS102 replicon also includes
Carnobacterium maltaromaticum
and
Bacillus subtilis
. The larger plasmid is predicted to replicate via the theta-type mechanism. The host range of its replicon seems restricted to
L. plantarum
. Cloning vectors were constructed based on the replicons of all three plasmids. Plasmid pWCFS103 was demonstrated to be a conjugative plasmid, as it could be transferred to
L. plantarum
NC8. It confers arsenate and arsenite resistance, which can be used as selective markers.
Characterization of the
cro-ori
Region of the
Streptococcus thermophilus
Virulent Bacteriophage DT1
Authors: Lamothe Geneviève , Lévesque Céline , Bissonnette Frédéric , Cochu Armelle , Vadeboncoeur Christian , Frenette Michel , Duplessis Martin , Tremblay Denise , Moineau Sylvain
Published: March 2005

The virulent
cos
-type
Streptococcus thermophilus
phage DT1 was previously isolated from a mozzarella whey sample, and its complete genomic sequence is available. The putative
ori
of phage DT1 is characterized by three inverted and two direct repeats located in a noncoding region between
orf36
and
orf37
. As the replication ability of the putative
ori
and flanking genes could not be established, its ability to confer phage resistance was tested. When
ori
is cloned on a high-copy-number plasmid, it provides protection to
S. thermophilus
strains against phage infection during milk fermentation. This protection is phage specific and strain dependent. Then, a detailed transcriptional map was established for the region located between the
cro
-like gene (
orf29
) and the
ori
. The results of the Northern blots indicated that the transcription of this region started 5 min after the onset of phage infection. Comparative analysis of the expression of the
cro-ori
region in the three
S. thermophilus cos
-type phages DT1, Sfi19 (virulent), and Sfi21 (temperate) reveals significant differences in the number and size of transcripts. The promoter upstream of
orf29
was further investigated by primer extension analysis, and its activity was confirmed by a chloramphenicol acetyltransferase assay, which showed that the phage promoter is more efficient than the constitutive bacterial promoter of the
S. thermophilus
operon encoding the general proteins of the phosphoenolpyruvate:sugar phosphotransferase system. However, the phage promoter is less efficient than the
pts
promoter in
Lactococcus lactis
and in
Escherichia coli
.
Degradation of Wheat Straw by
Fibrobacter succinogenes
S85: a Liquid- and Solid-State Nuclear Magnetic Resonance Study
Authors: Matulova M. , Nouaille R. , Capek P. , Péan M. , Forano E. , Delort A.-M.
Published: March 2005

Wheat straw degradation by
Fibrobacter succinogenes
was monitored by nuclear magnetic resonance (NMR) spectroscopy and chemolytic methods to investigate the activity of an entire fibrolytic system on an intact complex substrate. In situ solid-state NMR with
13
C cross-polarization magic angle spinning was used to monitor the modification of the composition and structure of lignocellulosic fibers (of
13
C-enriched wheat straw) during the growth of bacteria on this substrate. There was no preferential degradation either of amorphous regions of cellulose versus crystalline regions or of cellulose versus hemicelluloses in wheat straw. This suggests either a simultaneous degradation of the amorphous and crystalline parts of cellulose and of cellulose and hemicelluloses by the enzymes or degradation at the surface at a molecular scale that cannot be detected by NMR. Liquid-state two-dimensional NMR experiments and chemolytic methods were used to analyze in detail the various sugars released into the culture medium. An integration of NMR signals enabled the quantification of oligosaccharides produced from wheat straw at various times of culture and showed the sequential activities of some of the fibrolytic enzymes of
F. succinogenes
S85 on wheat straw. In particular, acetylxylan esterase appeared to be more active than arabinofuranosidase, which was more active than α-glucuronidase. Finally, cellodextrins did not accumulate to a great extent in the culture medium.
Enumeration and Diversity of Campylobacters and Bacteriophages Isolated during the Rearing Cycles of Free-Range and Organic Chickens
Authors: El-Shibiny A. , Connerton P. L. , Connerton I. F.
Published: March 2005

Campylobacters and
Campylobacter
-specific bacteriophages were isolated and enumerated during the rearing cycle of free-range (56 days) and organic chickens (73 days) at 3-day intervals from hatching until slaughter. In both flocks
Campylobacter jejuni
was the initial colonizer but
Campylobacter coli
was detected more frequently from 5 weeks of age. The diversity of the
Campylobacter
isolates was examined by pulsed-field gel electrophoresis of SmaI-digested genomic DNA and antimicrobial resistance typing. Bacteriophages were isolated from 51% (19 of 37 birds) of
Campylobacter
-positive organic birds (log
10
2.5 to log
10
5.7 PFU/g of cecal contents). The bacteriophages were all typical group III
Campylobacter
bacteriophages in terms of genomic size but could be characterized in terms of their host range and placed into five different groups. In contrast to the organic birds, anti-
Campylobacter
activity (bacteriocin-like) was observed in 26% (10 of 38 birds) of
Campylobacter
-positive free-range birds, and only one bacteriophage was isolated. Appearance of either bacteriophages or anti-
Campylobacter
activity was associated with changes in the levels of colonization and the predominant genotypes and species isolated. The frequency and potential influence of naturally occurring bacteriophages and/or inhibitory substances on the diversity and fluctuations of populations of campylobacters have not previously been reported in either free-range or organic chickens.
Poly(A) Polymerase Modification and Reverse Transcriptase PCR Amplification of Environmental RNA
Authors: Botero Lina M. , D'Imperio Seth , Burr Mark , McDermott Timothy R. , Young Mark , Hassett Daniel J.
Published: March 2005

We describe a combination of two established techniques for a novel application for constructing full-length cDNA clone libraries from environmental RNA. The cDNA was cloned without the use of prescribed primers that target specific genes, and the procedure did not involve random priming. Purified RNA was first modified by addition of a poly(A) tail and then was amplified by using a commercially available reverse transcriptase PCR (RT-PCR) cDNA synthesis kit. To demonstrate the feasibility of this approach, a cDNA clone library was constructed from size-fractionated RNA (targeting 16S rRNA) purified from a geothermally heated soil in Yellowstone National Park in Wyoming. The resulting cDNA library contained clones representing
Bacteria
and
Eukarya
taxa and several mRNAs. There was no exact clone match between this library and a separate cDNA library generated from an RT-PCR performed with unmodified rRNA and
Bacteria
-specific forward and universal reverse primers that were designed from cultivated organisms; however, both libraries contained representatives of the
Firmicutes
and the α-
Proteobacteria
. Unexpectedly, there were no
Archaea
clones in the library generated from poly(A)-modified RNA. Additional RT-PCRs performed with universal and
Archaea
-biased primers and unmodified RNA demonstrated the presence of novel
Archaea
in the soil. Experiments with pure cultures of
Sulfolobus solfataricus
and
Halobacterium halobium
revealed that some
Archaea
rRNA may not be a suitable substrate for the poly(A) tail modification step. The protocol described here demonstrates the feasibility of directly accessing prokaryote RNA (rRNA and/or mRNA) in environmental samples, but the results also illustrate potentially important problems.
Novel Activator of Mannose-Specific Phosphotransferase System Permease Expression in
Listeria innocua
, Identified by Screening for Pediocin AcH Resistance
Authors: Xue Junfeng , Hunter Ian , Steinmetz Tori , Peters Adam , Ray Bibek , Miller Kurt W.
Published: March 2005

To identify genes that are important for class IIa bacteriocin interaction and resistance in
Listeria
species, transposon Tn
917
knockout libraries were constructed for
Listeria innocua
strain Lin11 and screened for mutants that are resistant to pediocin AcH. A highly resistant mutant (G7) (MIC > 20 μg/ml; 1,000-fold less susceptible than the wild type), in which the transposon integrated into the putative promoter of the lin0142 gene, was isolated. lin0142 is located immediately upstream of the
mpt
operon (
mptA
/
mptC
/
mptD
) that encodes the mannose-specific phosphoenolpyruvate-dependent phosphotransferase system permease EII
t
Man
, which serves as a docking protein for class IIa bacteriocins. The transcription of the
mpt
operon is known to be positively controlled by σ
54
factor and ManR (a σ
54
-associated activator). Transcripts for lin0142 and
mpt
were undetectable in the G7 mutant, based on quantitative real-time reverse transcriptase PCR analysis. When the wild-type lin0142 gene was expressed at a 7.9-fold-elevated level in the mutant via a multicopy-number plasmid, the level of
mpt
mRNA became 70% higher than that in the wild-type strain. In addition, the complementation strain reverted back to the pediocin AcH-susceptible phenotype. The levels of
manR
and
rpoN
(σ
54
) mRNAs were not directly influenced by the level of lin0142 transcription. lin0142 is the only one of the three
mpt
regulatory genes whose transcription is induced, albeit slightly (1.2-fold), by glucose. The combined results show that the lin0142 gene encodes a novel activator of the
mpt
operon. The Lin0142 protein contains a winged-helix DNA-binding motif and is distantly related to the Crp-Fnr family of transcription regulators.
Diversity of Proteolytic
Clostridium botulinum
Strains, Determined by a Pulsed-Field Gel Electrophoresis Approach
Authors: Nevas Mari , Lindström Miia , Hielm Sebastian , Björkroth K. Johanna , Peck Michael W. , Korkeala Hannu
Published: March 2005

Pulsed-field gel electrophoresis (PFGE) was applied to the study of the similarity of 55 strains of proteolytic
Clostridium botulinum
(
C. botulinum
group I) types A, AB, B, and F. Rare-cutting restriction enzymes ApaI, AscI, MluI, NruI, PmeI, RsrII, SacII, SmaI, and XhoI were tested for their suitability for the cleavage of DNA of five proteolytic
C. botulinum
strains. Of these enzymes, SacII, followed by SmaI and XhoI, produced the most convenient number of fragments for genetic typing and were selected for analysis of the 55 strains. The proteolytic
C. botulinum
species was found to be heterogeneous. In the majority of cases, PFGE enabled discrimination between individual strains of proteolytic
C. botulinum
types A and B. The different toxin types were discriminated at an 86% similarity level with both SacII and SmaI and at an 83% similarity level with XhoI. Despite the high heterogeneity, three clusters at a 95% similarity level consisting of more than three strains of different origin were noted. The strains of types A and B showed higher diversity than the type F organisms which formed a single cluster. According to this survey, PFGE is to be considered a useful tool for molecular epidemiological analysis of proteolytic
C. botulinum
types A and B. However, epidemiological conclusions based on PFGE data only should be made with discretion, since highly similar PFGE patterns were noticed, especially within the type B strains.
Identification of a New
Borrelia
Species among Small Mammals in Areas of Northern Spain Where Lyme Disease Is Endemic
Authors: Gil Horacio , Barral Marta , Escudero Raquel , García-Pérez Ana L. , Anda Pedro
Published: March 2005

The role of small mammals as reservoir hosts for
Borrelia burgdorferi
was investigated in several areas where Lyme disease is endemic in northern Spain. A low rate of infestation by
Ixodes ricinus
nymphs was found in the small mammal populations studied that correlated with the near-absence of
B. burgdorferi
sensu lato in 184 animals tested and with the lack of transmission of
B. burgdorferi
sensu lato to
I. ricinus
larvae that fed on them. In contrast, questing ticks collected at the same time and in the same areas were found to carry a highly variable
B. burgdorferi
sensu lato repertoire (
B. burgdorferi
sensu stricto,
Borrelia garinii
,
Borrelia valaisiana
, and
Borrelia afzelii
). Interestingly, the only isolate obtained from small mammals (R57, isolated from a bank vole) grouped by phylogenetic analyses with other
Borrelia
species but in a separate clade from the Lyme disease and relapsing fever organisms, suggesting that it is a new species. This new agent was widely distributed among small mammals, with infection rates of 8.5 to 12% by PCR. Moreover, a high seroprevalence to
B. burgdorferi
sensu lato was found in the animal sera, suggesting cross-reactivity between
B. burgdorferi
sensu lato and R57. Although small mammals do not seem to play an important role as reservoirs for
B. burgdorferi
sensu lato in the study area, they seem to be implicated in the maintenance of spirochetes similar to R57.
Patterns of Antimicrobial Resistance Observed in
Escherichia coli
Isolates Obtained from Domestic- and Wild-Animal Fecal Samples, Human Septage, and Surface Water
Authors: Sayah Raida S. , Kaneene John B. , Johnson Yvette , Miller RoseAnn
Published: March 2005

A repeated cross-sectional study was conducted to determine the patterns of antimicrobial resistance in 1,286
Escherichia coli
strains isolated from human septage, wildlife, domestic animals, farm environments, and surface water in the Red Cedar watershed in Michigan. Isolation and identification of
E. coli
were done by using enrichment media, selective media, and biochemical tests. Antimicrobial susceptibility testing by the disk diffusion method was conducted for neomycin, gentamicin, streptomycin, chloramphenicol, ofloxacin, trimethoprim-sulfamethoxazole, tetracycline, ampicillin, nalidixic acid, nitrofurantoin, cephalothin, and sulfisoxazole. Resistance to at least one antimicrobial agent was demonstrated in isolates from livestock, companion animals, human septage, wildlife, and surface water. In general,
E. coli
isolates from domestic species showed resistance to the largest number of antimicrobial agents compared to isolates from human septage, wildlife, and surface water. The agents to which resistance was demonstrated most frequently were tetracycline, cephalothin, sulfisoxazole, and streptomycin. There were similarities in the patterns of resistance in fecal samples and farm environment samples by animal, and the levels of cephalothin-resistant isolates were higher in farm environment samples than in fecal samples. Multidrug resistance was seen in a variety of sources, and the highest levels of multidrug-resistant
E. coli
were observed for swine fecal samples. The fact that water sample isolates were resistant only to cephalothin may suggest that the resistance patterns for farm environment samples may be more representative of the risk of contamination of surface waters with antimicrobial agent-resistant bacteria.
Presence of Noroviruses and Other Enteric Viruses in Sewage and Surface Waters in The Netherlands
Authors: Lodder W. J. , de Roda Husman A. M.
Published: March 2005

Since virus concentrations in drinking waters are generally below the detection limit, the infectious risk from drinking water consumption requires assessment from the virus concentrations in source waters and removal efficiency of treatment processes. In this study, we estimated from reverse transcription-PCR on 10-fold serially diluted RNA that noroviruses, the most prevalent waterborne gastroenteritis agents, were present at 4 (0.2 to 38) to 4,900 (303 to 4.6 × 10
4
) PCR-detectable units (PDU) per liter of river water (ranges are given in parentheses). These virus concentrations are still high compared with 896 to 7,499 PDU/liter of treated sewage and 5,111 to 850,000 PDU/liter in raw sewage. Sequencing analyses designated human norovirus GGII.4 Lordsdale as the most prevalent strain in the sampling period 1998 to 1999 in both sewage and surface waters. Other GGII strains were also very abundant, indicating that the majority of the virus contamination was derived from urban sewage, although very divergent strains and one animal strain were also detected in the surface and sewage waters. Rotaviruses were also detected in two large rivers (the Maas and the Waal) at 57 to 5,386 PDU/liter. The high virus concentrations determined by PCR may in part be explained by the detection of virus RNA instead of infectious particles. Indeed, reoviruses and enteroviruses that can be cultured were present at much lower levels, of 0.3 to 1 and 2 to 10 PFU/liter, respectively. Assuming 1% of the noroviruses and rotaviruses to be infectious, a much higher disease burden than for other viruses can be expected, not only because of the higher levels but also because of these viruses' higher infectivity and attack rates.
Introducing DOTUR, a Computer Program for Defining Operational Taxonomic Units and Estimating Species Richness
Authors: Schloss Patrick D. , Handelsman Jo
Published: March 2005

Although copious qualitative information describes the members of the diverse microbial communities on Earth, statistical approaches for quantifying and comparing the numbers and compositions of lineages in communities are lacking. We present a method that addresses the challenge of assigning sequences to operational taxonomic units (OTUs) based on the genetic distances between sequences. We developed a computer program, DOTUR, which assigns sequences to OTUs by using either the furthest, average, or nearest neighbor algorithm for each distance level. DOTUR uses the frequency at which each OTU is observed to construct rarefaction and collector's curves for various measures of richness and diversity. We analyzed 16S rRNA gene libraries derived from Scottish and Amazonian soils and the Sargasso Sea with DOTUR, which assigned sequences to OTUs rapidly and reliably based on the genetic distances between sequences and identified previous inconsistencies and errors in assigning sequences to OTUs. An analysis of the two 16S rRNA gene libraries from soil demonstrated that they do not contain enough sequences to support a claim that they contain different numbers of bacterial lineages with statistical confidence (
P
> 0.05), nor do they contain enough sequences to provide a robust estimate of species richness when an OTU is defined as containing sequences that are no more than 3% different from each other. In contrast, the richness of OTUs at the 3% level in the Sargasso Sea collection began to plateau after the sampling of 690 sequences. We anticipate that an equivalent extent of sampling for soil would require sampling more than 10,000 sequences, almost 100 times the size of typical sequence collections obtained from soil.
Enhanced Survival of
Salmonella enterica
in Vesicles Released by a Soilborne
Tetrahymena
Species
Authors: Brandl M. T. , Rosenthal B. M. , Haxo A. F. , Berk S. G.
Published: March 2005

Nondestructive ingestion by soilborne protozoa may enhance the environmental resiliency of important bacterial pathogens and may model how such bacteria evade destruction in human macrophages. Here, the interaction of
Salmonella enterica
serovar Thompson with a soilborne
Tetrahymena
sp. isolate was examined using serovar Thompson cells labeled with the green fluorescent protein. The bacteria were mixed in solution with cells of
Tetrahymena
at several ratios. During incubation with serovar Thompson,
Tetrahymena
cells released a large number of vesicles containing green fluorescent serovar Thompson cells. In comparison, grazing on
Listeria monocytogenes
cells resulted in their digestion and thus the infrequent release of this pathogen in vesicles. The number of serovar Thompson cells per vesicle increased significantly as the initial ratio of serovar Thompson to
Tetrahymena
cells increased from 500:1 to 5,000:1. The density of serovar Thompson was as high as 50 cells per vesicle. Staining with propidium iodide revealed that a significantly higher proportion of serovar Thompson cells remained viable when enclosed in vesicles than when free in solution. Enhanced survival rates were observed in vesicles that were secreted by both starved (
F
= 28.3,
P
< 0.001) and unstarved (
F
= 14.09,
P
< 0.005)
Tetrahymena
cells. Sequestration in vesicles also provided greater protection from low concentrations of calcium hypochlorite. Thus, the release of this human pathogen from
Tetrahymena
cells in high-density clusters enclosed in a membrane may have important implications for public health.
Subtle Difference between Benzene and Toluene Dioxygenases of
Pseudomonas putida
Authors: Bagnéris Claire , Cammack Richard , Mason Jeremy R.
Published: March 2005

Benzene dioxygenase and toluene dioxygenase from
Pseudomonas putida
have similar catalytic properties, structures, and gene organizations, but they differ in substrate specificity, with toluene dioxygenase having higher activity toward alkylbenzenes. The catalytic iron-sulfur proteins of these enzymes consist of two dissimilar subunits, α and β; the α subunit contains a [2Fe-2S] cluster involved in electron transfer, the catalytic nonheme iron center, and is also responsible for substrate specificity. The amino acid sequences of the α subunits of benzene and toluene dioxygenases differ at only 33 of 450 amino acids. Chimeric proteins and mutants of the benzene dioxygenase α subunit were constructed to determine which of these residues were primarily responsible for the change in specificity. The protein containing toluene dioxygenase C-terminal region residues 281 to 363 showed greater substrate preference for alkyl benzenes. In addition, we identified four amino acid substitutions in this region, I301V, T305S, I307L, and L309V, that particularly enhanced the preference for ethylbenzene. The positions of these amino acids in the α subunit structure were modeled by comparison with the crystal structure of naphthalene dioxygenase. They were not in the substrate-binding pocket but were adjacent to residues that lined the channel through which substrates were predicted to enter the active site. However, the quadruple mutant also showed a high uncoupled rate of electron transfer without product formation. Finally, the modified proteins showed altered patterns of products formed from toluene and ethylbenzene, including monohydroxylated side chains. We propose that these properties can be explained by a more facile diffusion of the substrate in and out of the substrate cavity.
Signal Transduction by Tga3, a Novel G Protein α Subunit of
Trichoderma atroviride
Authors: Zeilinger Susanne , Reithner Barbara , Scala Valeria , Peissl Isabel , Lorito Matteo , Mach Robert L.
Published: March 2005

Trichoderma
species are used commercially as biocontrol agents against a number of phytopathogenic fungi due to their mycoparasitic characterisitics. The mycoparasitic response is induced when
Trichoderma
specifically recognizes the presence of the host fungus and transduces the host-derived signals to their respective regulatory targets. We made deletion mutants of the
tga3
gene of
Trichoderma atroviride
, which encodes a novel G protein α subunit that belongs to subgroup III of fungal Gα proteins. Δ
tga3
mutants had changes in vegetative growth, conidiation, and conidial germination and reduced intracellular cyclic AMP levels. These mutants were avirulent in direct confrontation assays with
Rhizoctonia solani
or
Botrytis cinerea
, and mycoparasitism-related infection structures were not formed. When induced with colloidal chitin or
N
-acetylglucosamine in liquid culture, the mutants had reduced extracellular chitinase activity even though the chitinase-encoding genes
ech42
and
nag1
were transcribed at a significantly higher rate than they were in the wild type. Addition of exogenous cyclic AMP did not suppress the altered phenotype or restore mycoparasitic overgrowth, although it did restore the ability to produce the infection structures. Thus,
T. atroviride
Tga3 has a general role in vegetative growth and can alter mycoparasitism-related characteristics, such as infection structure formation and chitinase gene expression.
Genomic Analysis of Bacteriophage ΦJL001: Insights into Its Interaction with a Sponge-Associated Alpha-Proteobacterium
Authors: Lohr Jayme E. , Chen Feng , Hill Russell T.
Published: March 2005

Bacteriophage ΦJL001 infects a novel marine bacterium in the α subclass of the
Proteobacteria
isolated from the marine sponge
Ircinia strobilina
. ΦJL001 is a siphovirus and forms turbid plaques on its host. The genome sequence of ΦJL001 was determined in order to better understand the interaction between the marine phage and its sponge-associated host bacterium. The complete genome sequence of ΦJL001 comprised 63,469 bp with an overall G+C content of 62%. The genome has 91 predicted open reading frames (ORFs), and 17 ORFs have been assigned putative functions. ΦJL001 appears to be a temperate phage, and the integrase gene was identified in the genome. DNA hybridization analysis showed that the ΦJL001 genome does not integrate into the host chromosome under the conditions tested. DNA hybridization experiments therefore suggested that ΦJL001 has some pseudolysogenic characteristics. The genome of ΦJL001 contains many putative genes involved in phage DNA replication (e.g., helicase, DNA polymerase, and thymidylate synthase genes) and also contains a putative integrase gene associated with the lysogenic cycle. Phylogeny based on DNA polymerase gene sequences indicates that ΦJL001 is related to a group of siphoviruses that infect mycobacteria. Designation of ΦJL001 as a siphovirus is consistent with the morphology of the phage visualized by transmission electron microscopy. The unique marine phage-host system described here provides a model system for studying the role of phages in sponge microbial communities.
Rose (Rosa rugosa)-flower extract increases the activities of antioxidant enzymes and their gene expression and reduces lipid peroxidation
Authors: Ng T B , Gao W , Li L , Niu S M , Zhao L , Liu J , Shi L S , Fu M , Liu F
Published: March 2005

The effects of rose-flower extract on antioxidant enzymes were studied. The activities of catalase (CAT) and glutathione peroxidase (GPx) in 9-month-old senescence-accelerated mice (SAM mice) were lower than those in 6-month-old SAM mice. Therefore, 9-month-old SAM mice were the most appropriate targets for treatment with the rose-flower extract. The activities of CAT and GPx in SAM mice treated with rose-flower extract showed a marked increase in whole blood and liver. At the same time, the gene-expression level of CAT and GPx was upregulated in the liver, while malondialdehyde content in liver and brain decreased. Male SAM mice were more sensitive than female SAM mice. The mean and the longest lifespan of SAM mice were longer after treatment with rose-flower extract.Key word: anti-aging, CAT, GPx, lipid peroxidation.
Chemokine receptor CXCR4-β1 integrin axis mediates tumorigenesis of osteosarcoma HOS cells
Authors: Miura Kohei , Uniyal Shashi , Leabu Mircea , Oravecz Tamas , Chakrabarti Subrata , Morris Vincent L , Chan Bosco M.C
Published: March 2005

It is known that β1 integrins mediate the migratory response of cells to chemokine stimulation. Also, both β1 integrins and chemokines have roles in tumor development. In the present study, the β1 integrin-chemokine axis is assessed using human osteosarcoma (HOS) transfectant cells expressing the CXCR4 receptor for chemokine SDF-1 (CXCL12). We first identified in vitro the specific β1 integrins that mediated the migratory response to SDF-1 stimulation. Results showed that on collagen type I and laminin, the chemotactic response to SDF-1 was predominantly mediated by α2β1 integrin. On fibronectin, SDF-1-stimulated chemotaxis involved both α4β1 and α5β1 integrins. A comparison of the transfectant clones expressing CXCR4 at low, intermediate, and high levels and the control transfectant revealed that the transfectant clones migratory response in vitro and their ability to form tumors in vivo was related to their levels of CXCR4 expression. In addition, treatment by injection with mAbs to CXCR4, integrin α2β1, or integrin α5β1 effectively inhibited the growth of HOS-CXCR4 transfectant cells in vivo. Therefore, our results show that the β1 integrins that mediated the migratory response were also functionally linked to the enhanced tumor growth of CXCR4-expressing HOS transfectant cells.Key words: integrins, chemokines, chemotaxis, osteosarcoma, tumorigenesis.
Angiostatin and plasminogen share binding to endothelial cell surface actin
Authors: Dudani A K , Ben-Tchavtchavadze M , Porter S , Tackaberry E
Published: March 2005

Previous studies from this laboratory have demonstrated that plasminogen binds to endothelial cell surface-associated actin via its kringles in a dose-dependent and specific manner. The purpose of this study was to determine whether angiostatin, a proteolytic fragment of plasminogen, shares binding properties with plasminogen. Our results indicated that like plasminogen, angiostatin bound to actin in a time-, concentration-, and kringle-dependent manner. Furthermore, this binding was significantly inhibited by excess plasminogen, suggesting that both proteins shared binding motifs on the actin molecule. Fluorescence studies demonstrated that angiostatin bound to intact endothelial cells through its kringles, and this binding was also inhibited by plasminogen but not by unrelated proteins. Ligand blot analyses on endothelial cell lysates indicated that angiostatin interacted with a 42 kDa protein, which was identified as actin. Furthermore, an anti-actin antibody inhibited binding of angiostatin to endothelial cells by approximately 25%. These results suggest that angiostatin and plasminogen share binding to endothelial cell surface actin and, therefore, that angiostatin has the potential to inhibit plasmin-dependent processes such as cell migration–movement.Key words: plasminogen, angiostatin, endothelial cells, actin.
Méthodes de qualification des immobilisations chimiques d'une enzyme sur un support solide
Authors: Belzil Anne , Parent Carmen
Published: March 2005

Carbonic anhydrase was immobilized on a polymer thermoplastic to promote gaseous CO2hydration into bicarbonate ions. Catalyst immobilization was realized through a series of chemical reactions enabling enzyme covalent binding to polyamide support. Different initial enzyme concentrations of 0.25, 0.50, 0.75, 1, and 2 mg/ml were verified. Different techniques were developed to assess enzyme attachment. The amount of bound protein was determined using Bradford dosage of proteins remaining in solution following CA II incubation with solid support. ELISA has given a qualitative evaluation of the protein, enabling a follow up of enzyme binding robustness as a function of time. p-nitrophenyl acetate hydrolysis and CO2hydration were assessed by spectrophotometry and electrometry, respectively. The percentage of active enzyme following immobilization was measured using an esterase linear model. Catalytic transformation rates for gaseous CO2hydration were calculated for each type of immobilization.Key words: immobilization, enzyme, esterase, hydration.[Journal translation]
Influence of mechanical and biological signals on gene expression in human MG-63 cells: evidence for a complex interplay between hydrostatic compression and vitamin D3 or TGF-β1 on MMP-1 and MMP-3 mRNA levels
Authors: Tasevski V , Sorbetti J M , Chiu S S , Shrive N G , Hart D A
Published: March 2005

Biological mediators can influence the activity and differentiation of bone cells. 1,25-dihydroxy-vitamin D3(1,25-(OH)2D3) is known to induce differentiation of precursors into mature osteoblasts, and transforming growth factorβ1 (TGF-β1) can modulate the activity of bone cells leading to alterations in proliferation and gene expression patterns. Bone-derived cells were loaded via intermittent cyclic hydrostatic pressure (icHP) on cells under basal conditions and in the presence of 1,25-(OH)2D3or TGF-β1. Evaluating the effects of loading on the cells allowed for a comparison to be made between responsiveness to biomechanical and biochemical stimuli and their potential interplay. The effects of icHP on mRNA levels for the specific genes involved in bone remodelling and differentiation were measured in MG-63 cells using reverse transcription-polymerase chain reaction (RT-PCR). The mRNA levels for matrix metalloproteinase-1 and -3 (MMP-1 and MMP-3) were significantly, and uniquely, increased (p < 0.001) in cells exposed to icHP under serum-free conditions for 4–12 h. However, mRNA levels for MMP-3, but not MMP-1, were significantly enhanced in cells subjected to static hydrostatic pressure (HP). Treatment of cells with 1,25-(OH)2D3resulted in increased (p < 0.001) mRNA levels for osteocalcin and decreased (p < 0.001) mRNA levels for both MMP-1 and MMP-3. In cells exposed to icHP and 1,25-(OH)2D3, the mRNA levels for both MMP-1 and MMP-3 were elevated (p < 0.001) compared with hormone alone, but not to the same degree (p < 0.01) as cells subjected to icHP alone. Addition of TGF-β1 to cells led to increases in cell proliferation and expression of collagen I, as well as decreases in expression of osteocalcin and MMP-1 and MMP-3. Exposure of cells to icHP and TGF-β1 again led to unique and significant increases in expression of MMP-1 and MMP-3. No changes in mRNA levels for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) or any of the other 9 genes assessed, including those for MMP-2 and MMP-13, were detected under any of the conditions described. Therefore, icHP can induce alterations in mRNA levels for a specific subset of genes in both premature and mature osteoblasts. Such stimuli can modulate the impact of potent biological mediators in defining patterns of gene expression by bone cells and potentially modify function in vivo.Key words: osteoblast, biomechanical loading,1,25-dihydroxy-vitamin D3(1,25-(OH)2D3), mRNA levels, reverse trans cription-polymerase chain reaction (RT-PCR), transforming growth factor-β1 (TGF-β1).