bluuebunny/crossref_metadata_2025 huggingface dataset:
Exploratory study into wireless application protocol shopping
Authors: Fenech Tino
Published: September 2002

Using an online survey this paper examines the personality traits and shopping characteristics of high and low intentioned wireless application protocol (WAP) shoppers. Results indicate that individuals with a high intention to use WAP shopping have both similar and contrasting characteristics to previous electronic non‐store adopters, such as television infomercial shoppers. It is recommended that specific marketing efforts be developed to target WAP adopters and these efforts include offering unique items for sale that are offered exclusively to WAP users.
When will they ever learn? The conditions for failure in publicly funded high technology projects: the R101 and Challenger disasters compared
Authors: Weir David
Published: September 2002

Governments of all political complexions seek economic advantage through supporting high profile projects in the field of high technology. “High tech” becomes a mantra, comprising activities with a wide range of possible outcomes, inevitably demanding large investments, and promising high rewards. It is a relative term and every generation defines its “leading edge” projects in a similar way; although the substantive technology differs, building on the learning previously achieved and assimilated into the paradigm of “normal science”. Some of these projects work, but many do not: some end in spectacular and catastrophic failure. The resulting disasters are subsequently ascribed to exogenous causes, such as “Acts of God”, or are explained as due to the inherently risky consequence of working at the frontiers of new technology and of the dangerous but none the less essential choices made at the political level, without which “progress” would not be achieved. Politicians, technologists and managers bear specific and local responsibilities for the failure of systems on their watch but it is arguably the failure of the academic theorists of disaster to contribute in a more socially engaged way that most supports the ability of society not to hear the messages it wishes to evade.
Can European media empires survive? The rise and fall of the House of Kirch
Authors: Fowler Tony , Curwen Peter
Published: September 2002

Although bankruptcies in the TMT sector are flowing thick and fast, the collapse of the media empire built up by Leo Kirch over a 42‐year period is arguably the most dramatic. Protected by its links with German business and financial institutions as well as politicians, the KirchGruppe appeared to be impervious to the periodic downturns in business conditions. However, not only did the “German” way of doing business behind closed doors come under increasing pressure by the end of the 1990s, but the KirchGruppe acquired enemies such as the Springer family as well as shareholders, such as Rupert Murdoch and Silvio Berlusconi, who were intent upon expanding their own empires. Because he assumed that his empire was well‐protected via his associates, Leo Kirch took excessive risks, not least the issuance of put options which, if exercised, could bring down his empire – which in the event was what transpired earlier this year.
Is LoopCo the answer?
Authors: Cave Martin
Published: September 2002

Proposals have been made to separate BT vertically into two parts: an access network (LoopCo) providing services to all operators, and a core network. The aim is to counter discriminatory behaviour by an integrated firm. The paper reviews similar approaches to separation in UK utilities and establishes criteria for evaluating the LoopCo proposal. It suggests that technological developments are moving the dividing line between core and access networks. A separation made on the basis of current technology may lead to difficulties in the future in co‐ordinating investments. It proposes instead more effective means of policing discrimination.
Confined Compression of a Tissue-Equivalent: Collagen Fibril and Cell Alignment in Response to Anisotropic Strain
Authors: Girton T. S. , Barocas V. H. , Tranquillo R. T.
Published: October 2002

A method to impose and measure a one dimensional strain field via confined compression of a tissue-equivalent and measure the resulting cell and collagen fibril alignment was developed. Strain was determined locally by the displacement of polystyrene beads dispersed and entrapped within the network of collagen fibrils along with the cells, and it was correlated to the spatial variation of collagen network birefringence and concentration. Alignment of fibroblasts and smooth muscle cells was determined based on the long axis of elongated cells. Cell and collagen network alignment were observed normal to the direction of compression after a step strain and increased monotonically up to 50% strain. These results were independent of time after straining over 24 hr despite continued cell motility after responding instantly to the step strain with a change in alignment by deforming/convecting with the strained network. Since the time course of cell alignment followed that of strain and not stress which, due to the viscoelastic fluid-like nature of the network relaxes completely within the observation period, these results imply cell alignment in a compacting tissue-equivalent is due to fibril alignment associated with anisotropic network strain. Estimation of a contact guidance sensitivity parameter indicates that both cell types align to a greater extent than the surrounding fibrils.
Innate phonetic boundaries revisited (L)
Authors: Aslin Richard N. , Werker Janet F. , Morgan James L.
Published: October 2002

Nittrouer [J. Acoust. Soc. Am. 110, 1598–1605 (2001)] raised several serious concerns with the so-called Universal Theory of phonetic category development that she characterized as the accepted wisdom in the field of infant speech perception. She then presented data from infants and children that were claimed to be inconsistent with Universal Theory and led her to question the entire notion of phonetic categories. Here we argue that Nittrouer not only misrepresented Universal Theory, but also provided no data to refute either this theory or the existence of phonetic categories.
Modifications of a single saturating non-linearity account for post-onset changes in 2 f1–f2 distortion product otoacoustic emission
Authors: Lukashkin Andrei N. , Russell Ian J.
Published: October 2002

2 f 1 –f 2 distortion product otoacoustic emissions (DPOAEs) were recorded from guinea pigs. DPOAEs showed complex time dependence at the onset of stimulation. The DPOAE, measured during the first 500 ms, can either decrease or increase at the onset depending on both the frequencies and levels of the primary tones. These changes are closely associated with amplitude minima (notches) of the DPOAE I/O functions. These notches are characteristic of DPOAE growth functions measured from guinea pigs for primary tones of 50–60-dB sound-pressure level (SPL). Apparent changes in the DPOAE amplitude occur because the notch shifts to higher levels of the primaries during the onset of stimulation. This shift of the notch to higher levels increases for lower f2/f1 ratios but does not exceed about 2 dB. DPOAE amplitude increases for a constant level of the primaries if the onset emission is situated at the low-level, falling slope of the notch. If the onset DPOAE is located on the high-level, rising slope of the notch, then the upward shift of the notch causes the emission either to decrease monotonically, or to decrease initially and then increase. By establishing that the 2 f1–f2 onset changes reflect a shift in the growth-function notch, it is possible to predict the temporal behavior of DPOAEs in the two-dimensional space of the amplitude of the primaries and for their different frequency ratios.
Functional Expression of Enterobacterial O-Polysaccharide Biosynthesis Enzymes in
Bacillus subtilis
Authors: Schäffer Christina , Wugeditsch Thomas , Messner Paul , Whitfield Chris
Published: September 2002

The expression of heterologous bacterial glycosyltransferases is of interest for potential application in the emerging field of carbohydrate engineering in gram-positive organisms. To assess the feasibility of using enzymes from gram-negative bacteria, the functional expression of the genes
wbaP
(formerly
rfbP
),
wecA
(formerly
rfe
), and
wbbO
(formerly
rfbF
) from enterobacterial lipopolysaccharide O-polysaccharide biosynthesis pathways was examined in
Bacillus subtilis
. WbaP and WecA are initiation enzymes for O-polysaccharide formation, catalyzing the transfer of galactosyl 1-phosphate from UDP-galactose and
N
-acetylglucosaminyl 1-phosphate from UDP-
N
-acetylglucosamine, respectively, to undecaprenylphosphate. The WecA product (undecaprenylpyrophosphoryl GlcNAc) is used as an acceptor to which the bifunctional
wbbO
gene product sequentially adds a galactopyranose and a galactofuranose residue from the corresponding UDP sugars to form a lipid-linked trisaccharide. Genes were cloned into the shuttle vectors pRB374 and pAW10. In
B. subtilis
hosts, the genes were effectively transcribed under the
vegII
promoter control of pRB374, but the plasmids were susceptible to rearrangements and deletion. In contrast, pAW10-based constructs, in which genes were cloned downstream of the
tet
resistance cassette, were stable but yielded lower levels of enzyme activity. In vitro glycosyltransferase assays were performed in
Escherichia coli
and
B. subtilis
, using membrane preparations as sources of enzymes and endogenous undecaprenylphosphate as an acceptor. Incorporation of radioactivity from UDP-α-
d
-
14
C-sugar into reaction products verified the functionality of WbaP, WecA, and WbbO in either host. Enzyme activities in
B. subtilis
varied between 20 and 75% of those measured in
E. coli.
Isolation and Characterization of a Freeze-Tolerant Diploid Derivative of an Industrial Baker's Yeast Strain and Its Use in Frozen Doughs
Authors: Teunissen Aloys , Dumortier Françoise , Gorwa Marie-Françoise , Bauer Jürgen , Tanghe An , Loïez Annie , Smet Peter , Van Dijck Patrick , Thevelein Johan M.
Published: September 2002

The routine production and storage of frozen doughs are still problematic. Although commercial baker's yeast is highly resistant to environmental stress conditions, it rapidly loses stress resistance during dough preparation due to the initiation of fermentation. As a result, the yeast loses gassing power significantly during storage of frozen doughs. We obtained freeze-tolerant mutants of polyploid industrial strains following screening for survival in doughs prepared with UV-mutagenized yeast and subjected to 200 freeze-thaw cycles. Two strains in the S47 background with a normal growth rate and the best freeze tolerance under laboratory conditions were selected for production in a 20-liter pilot fermentor. Before frozen storage, the AT25 mutant produced on the 20-liter pilot scale had a 10% higher gassing power capacity than the S47 strain, while the opposite was observed for cells produced under laboratory conditions. AT25 also retained more freeze tolerance during the initiation of fermentation in liquid cultures and more gassing power during storage of frozen doughs. Other industrially important properties (yield, growth rate, nitrogen assimilation, and phosphorus content) were very similar. AT25 had only half of the DNA content of S47, and its cell size was much smaller. Several diploid segregants of S47 had freeze tolerances similar to that of AT25 but inferior performance for other properties, while an AT25-derived tetraploid, TAT25, showed only slightly improved freeze tolerance compared to S47. When AT25 was cultured in a 20,000-liter fermentor under industrial conditions, it retained its superior performance and thus appears to be promising for use in frozen dough production. Our results also show that a diploid strain can perform at least as well as a tetraploid strain for commercial baker's yeast production and usage.
Isolation of Elemental Sulfur as a Self-Growth-Inhibiting Substance Produced by
Legionella pneumophila
Authors: Inoue Hiroaki , Kawano Genji , Nagasawa Hiromichi , Sakuda Shohei
Published: September 2002

The addition of HP-20 resin to a medium could enhance the growth of
Legionella
species. Elemental sulfur was isolated as a self-growth-inhibiting substance produced endogenously by
Legionella pneumophila
from methanol extracts of the resins used to culture the bacterium. Elemental sulfur shows strong growth-inhibiting activity toward all
Legionella
species tested.
The Exopolyphosphatase Gene from
Sulfolobus solfataricus
: Characterization of the First Gene Found To Be Involved in Polyphosphate Metabolism in
Archaea
Authors: Cardona Silvia T. , Chávez Francisco P. , Jerez Carlos A.
Published: September 2002

Inorganic polyphosphate (polyP) polymers are widely distributed in all kinds of organisms. Although the presence of polyP in members of the domain
Archaea
has been described, at present nothing is known about the enzymology of polyP metabolism or the genes involved in this domain. We have cloned, sequenced, and overexpressed an exopolyphosphatase (PPX) gene (
ppx
) from thermophilic
Sulfolobus solfataricus
. The gene codes for a functional PPX and possesses an open reading frame for 417 amino acids (calculated mass, 47.9 kDa). The purified recombinant PPX was highly active, degrading long-chain polyP (700 to 800 residues) in vitro at 50 to 60°C. The putative PPXs present in known archaeal genomes showed the highest similarity to yeast PPXs. In contrast, informatic analysis revealed that the deduced amino acid sequence of
S. solfataricus
PPX showed the highest similarity (25 to 45%) to sequences of members of the bacterial PPXs, possessing all of their conserved motifs. To our knowledge, this is the first report of an enzyme characterized to be involved in polyP metabolism in members of the
Archaea
.
Characterization and Implications of the Cell Surface Reactivity of
Calothrix
sp. Strain KC97
Authors: Phoenix V. R. , Martinez R. E. , Konhauser K. O. , Ferris F. G.
Published: September 2002

The cell surface reactivity of the cyanobacterium
Calothrix
sp. strain KC97, an isolate from the Krisuvik hot spring, Iceland, was investigated in terms of its proton binding behavior and charge characteristics by using acid-base titrations, electrophoretic mobility analysis, and transmission electron microscopy. Analysis of titration data with the linear programming optimization method showed that intact filaments were dominated by surface proton binding sites inferred to be carboxyl groups (acid dissociation constants [pK
a
] between 5.0 and 6.2) and amine groups (mean pK
a
of 8.9). Sheath material isolated by using lysozyme and sodium dodecyl sulfate generated pK
a
spectra similarly dominated by carboxyls (pK
a
of 4.6 to 6.1) and amines (pK
a
of 8.1 to 9.2). In both intact filaments and isolated sheath material, the lower ligand concentrations at mid-pK
a
values were ascribed to phosphoryl groups. Whole filaments and isolated sheath material displayed total reactive-site densities of 80.3 × 10
−5
and 12.3 × 10
−5
mol/g (dry mass) of cyanobacteria, respectively, implying that much of the surface reactivity of this microorganism is located on the cell wall and not the sheath. This is corroborated by electrophoretic mobility measurements that showed that the sheath has a net neutral charge at mid-pHs. In contrast, unsheathed cells exhibited a stronger negative-charge characteristic. Additionally, transmission electron microscopy analysis of ultrathin sections stained with heavy metals further demonstrated that most of the reactive binding sites are located upon the cell wall. Thus, the cell surface reactivity of
Calothrix
sp. strain KC97 can be described as a dual layer composed of a highly reactive cell wall enclosed within a poorly reactive sheath.
Bacterial Hemoglobins and Flavohemoglobins for Alleviation of Nitrosative Stress in
Escherichia coli
Authors: Frey Alexander D. , Farrés Judith , Bollinger Christian J. T. , Kallio Pauli T.
Published: September 2002

Escherichia coli
MG1655 cells expressing novel bacterial hemoglobin and flavohemoglobin genes from a medium-copy-number plasmid were grown in shake flask cultures under nitrosative and oxidative stress.
E. coli
cells expressing these proteins display enhanced resistance against the NO
·
releaser sodium nitroprusside (SNP) relative to that of the control strain bearing the parental plasmid. Expression of bacterial hemoglobins originating from
Campylobacter jejuni
(CHb) and
Vitreoscilla
sp. (VHb) conferred resistance on SNP-challenged cells. In addition, it has been shown that NO
·
detoxification is also a common feature of flavohemoglobins originating from different taxonomic groups and can be transferred to a heterologous host. These observations have been confirmed in a specific in vitro NO
·
consumption assay. Protein extracts isolated from
E. coli
strains overexpressing flavohemoglobins consumed authentic NO
·
more readily than protein extracts from the wild-type strain. Oxidative challenge to the cells evoked nonuniform responses from the various cell cultures. Improved oxidative-stress-sustaining properties had also been observed when the flavohemoglobins from
E. coli
,
Klebsiella pneumoniae
,
Deinococcus radiodurans
, and
Pseudomonas aeruginosa
were expressed in
E. coli
.
Isolation and Identification of
Aspergillus fumigatus
Mycotoxins on Growth Medium and Some Building Materials
Authors: Nieminen Susanna M. , Kärki Riikka , Auriola Seppo , Toivola Mika , Laatsch Hartmut , Laatikainen Reino , Hyvärinen Anne , von Wright Atte
Published: September 2002

Genotoxic and cytotoxic compounds were isolated and purified from the culture medium of an indoor air mold,
Aspergillus fumigatus
. One of these compounds was identified as gliotoxin, a known fungal secondary metabolite. Growth of
A. fumigatus
and gliotoxin production on some building materials were also studied. Strong growth of the mold and the presence of gliotoxin were detected on spruce wood, gypsum board, and chipboard under saturation conditions.
Glucose and Nutrient Concentrations Affect the Expression of a 104-Kilodalton
Listeria
Adhesion Protein in
Listeria monocytogenes
Authors: Jaradat Ziad W. , Bhunia Arun K.
Published: September 2002

Growth media and environmental conditions influence the expression of adhesion and invasion proteins in
Listeria monocytogenes
. Here, the expression of the 104-kDa
Listeria
adhesion protein (LAP) was studied in nutrient-rich media (Trypticase soy broth [TSB] and brain heart infusion [BHI]), minimal medium (Luria-Bertani [LB]), or nutrient-deficient medium (peptone water [PW]) by immunoblotting, enzyme-linked immunosorbent assay (ELISA), and immunoelectron microscopy. Also, the effect of incorporating different concentrations of glucose on LAP expression was studied. Immunoblotting showed that LAP expression was at least twofold higher in LB medium than in TSB or BHI, while PW supported very poor cell growth and LAP expression. ELISA and immunoblotting results showed that higher concentrations of glucose (>1.6 g/liter) lowered the culture pH and suppressed LAP expression by more than 75%; however, the addition of K
2
HPO
4
reduced this effect.
L. monocytogenes
cells grown in LB media with lower concentrations of glucose showed higher adhesion to Caco-2 cells (3,716 and 4,186 cpm of attached bacteria for 0 and 0.2 g of glucose/liter, respectively), while
L. monocytogenes
cells grown in LB with higher glucose concentrations exhibited lower adhesion (2,126 and 2,221 cpm for 1.6 and 3.2 g of glucose/liter, respectively). A LAP-negative
L. monocytogenes
strain (A572) showed low adhesion profiles regardless of the amount of glucose added. Transmission electron microscopy revealed that LAP is localized mainly in the cytoplasm, with only a few molecules located on the cell surface. Growth in LB with high glucose (3.2 g/liter) showed the presence of only a few molecules in the cells, corroborating the results observed with ELISA or immunoblotting. In summary, nutrient-rich media and high concentrations of glucose suppressed LAP expression, which possibly is due to the changes in the pH of the media during growth from the accumulation of sugar fermentation by-products.
Inactivation of the Elongation Factor Tu by Mosquitocidal Toxin-Catalyzed Mono-ADP-Ribosylation
Authors: Schirmer Jörg , Wieden Hans-Joachim , Rodnina Marina V. , Aktories Klaus
Published: September 2002

The mosquitocidal toxin (MTX) produced by
Bacillus sphaericus
strain SSII-1 is an ∼97-kDa single-chain toxin which contains a 27-kDa enzyme domain harboring ADP-ribosyltransferase activity and a 70-kDa putative binding domain. Due to cytotoxicity toward bacterial cells, the 27-kDa enzyme fragment cannot be produced in
Escherichia coli
expression systems. However, a nontoxic 32-kDa N-terminal truncation of MTX can be expressed in
E. coli
and subsequently cleaved to an active 27-kDa enzyme fragment. In vitro the 27-kDa enzyme fragment of MTX ADP-ribosylated numerous proteins in
E. coli
lysates, with dominant labeling of an ∼45-kDa protein. Matrix-assisted laser desorption ionization-time-of-flight mass spectrometry combined with peptide mapping identified this protein as the
E. coli
elongation factor Tu (EF-Tu). ADP ribosylation of purified EF-Tu prevented the formation of the stable ternary EF-Tuaminoacyl-tRNAGTP complex, whereas the binding of GTP to EF-Tu was not altered. The inactivation of EF-Tu by MTX-mediated ADP-ribosylation and the resulting inhibition of bacterial protein synthesis are likely to play important roles in the cytotoxicity of the 27-kDa enzyme fragment of MTX toward
E. coli
.
Diversity and Evolution of Hydrogenase Systems in Rhizobia
Authors: Baginsky Cecilia , Brito Belén , Imperial Juan , Palacios José-Manuel , Ruiz-Argüeso Tomás
Published: September 2002

Uptake hydrogenases allow rhizobia to recycle the hydrogen generated in the nitrogen fixation process within the legume nodule. Hydrogenase (
hup
) systems in
Bradyrhizobium japonicum
and
Rhizobium leguminosarum
bv. viciae show highly conserved sequence and gene organization, but important differences exist in regulation and in the presence of specific genes. We have undertaken the characterization of
hup
gene clusters from
Bradyrhizobium
sp. (
Lupinus
),
Bradyrhizobium
sp. (
Vigna
), and
Rhizobium tropici
and
Azorhizobium caulinodans
strains with the aim of defining the extent of diversity in
hup
gene composition and regulation in endosymbiotic bacteria. Genomic DNA hybridizations using
hupS
,
hupE
,
hupUV
,
hypB
, and
hoxA
probes showed a diversity of intraspecific
hup
profiles within
Bradyrhizobium
sp. (
Lupinus
) and
Bradyrhizobium
sp. (
Vigna
) strains and homogeneous intraspecific patterns within
R. tropici
and
A. caulinodans
strains. The analysis also revealed differences regarding the possession of hydrogenase regulatory genes. Phylogenetic analyses using partial sequences of
hupS
and
hupL
clustered
R. leguminosarum
and
R. tropici hup
sequences together with those from
B. japonicum
and
Bradyrhizobium
sp. (
Lupinus
) strains, suggesting a common origin. In contrast,
Bradyrhizobium
sp. (
Vigna
)
hup
sequences diverged from the rest of rhizobial sequences, which might indicate that those organisms have evolved independently and possibly have acquired the sequences by horizontal transfer from an unidentified source.
Ammonia Production by Ruminal Microorganisms and Enumeration, Isolation, and Characterization of Bacteria Capable of Growth on Peptides and Amino Acids from the Sheep Rumen
Authors: Eschenlauer S. C. P. , McKain N. , Walker N. D. , McEwan N. R. , Newbold C. J. , Wallace R. J.
Published: September 2002

Excessive NH
3
production in the rumen is a major nutritional inefficiency in ruminant animals. Experiments were undertaken to compare the rates of NH
3
production from different substrates in ruminal fluid in vitro and to assess the role of asaccharolytic bacteria in NH
3
production. Ruminal fluid was taken from four rumen-fistulated sheep receiving a mixed hay-concentrate diet. The calculated rate of NH
3
production from Trypticase varied from 1.8 to 19.7 nmol mg of protein
−1
min
−1
depending on the substrate, its concentration, and the method used. Monensin (5 μM) inhibited NH
3
production from proteins, peptides, and amino acids by an average of 28% with substrate at 2 mg/ml, compared to 48% with substrate at 20 mg/ml (
P =
0.011). Of the total bacterial population, 1.4% grew on Trypticase alone, of which 93% was eliminated by 5 μM monensin. Many fewer bacteria (0.002% of the total) grew on amino acids alone. Nineteen isolates capable of growth on Trypticase were obtained from four sheep. 16S ribosomal DNA and traditional identification methods indicated the bacteria fell into six groups. All were sensitive to monensin, and all except one group (group III, similar to
Atopobium minutum
), produced NH
3
at >250 nmol min
−1
mg of protein
−1
, depending on the medium, as determined by a batch culture method. All isolates had exopeptidase activity, but only group III had an apparent dipeptidyl peptidase I activity. Groups I, II, and IV were most closely related to asaccharolytic ruminal and oral
Clostridium
and
Eubacterium
spp. Group V comprised one isolate, similar to
Desulfomonas piger
(formerly
Desulfovibrio pigra
). Group VI was 95% similar to
Acidaminococcus fermentans.
Growth of the
Atopobium
- and
Desulfomonas
-like isolates was enhanced by sugars, while growth of groups I, II, and V was significantly depressed by sugars. This study therefore demonstrates that different methodologies and different substrate concentrations provide an explanation for different apparent rates of ruminal NH
3
production reported in different studies and identifies a diverse range of hyper-ammonia-producing bacteria in the rumen of sheep.
Biofilm Dispersal of
Neisseria subflava
and Other Phylogenetically Diverse Oral Bacteria
Authors: Kaplan Jeffrey B. , Fine Daniel H.
Published: September 2002

Polystyrene petri dishes containing liquid medium were inoculated with single-cell suspensions of a fresh clinical isolate of
Neisseria subflava
and were incubated under conditions of low vibration.
N. subflava
colonies grew firmly attached to the surface of the dish, while the broth remained clear. Growing colonies released cells into the medium, resulting in the appearance of 10
2
to 10
4
small satellite colonies attached to the surface of the dish in an area adjacent to each mature colony after 24 h. Satellite colonies grew in patterns of streamers shaped like jets and flares emanating from mature colonies and pointing toward the center of the dish. This dispersal pattern evidently resulted from the surface translocation of detached biofilm cells by buoyancy-driven convection currents that were generated due to slight temperature gradients in the medium. Streamers of satellite colonies ranged from 2 to >40 mm in length. Satellite colonies in very long streamers were relatively uniform in size regardless of their distance from the mature colony, suggesting that mature colonies released single cells or small clusters of cells into the medium and that the detachment, surface translocation, and subsequent surface reattachment of released cells were a transitory process. Incubation of
N. subflava
single cells in a perfused biofilm fermentor resulted in a large spike of the number of CFU in the perfusate after 9.5 h of growth, consistent with a rapid release of cells into the medium. Biofilm colonies of several other phylogenetically diverse oral bacteria, including
Actinobacillus actinomycetemcomitans
,
Haemophilus aphrophilus
,
Streptococcus mitis
, and a prevalent but previously uncultured oral
Streptococcus
sp., exhibited similar temperature-dependent dispersal patterns in broth culture. This in vitro spreading phenotype could be a useful tool for studying biofilm dispersal in these and other nonflagellated bacteria and may have physiological relevance to biofilm dispersal in the oral cavity.
Mechanisms of Inactivation of Hepatitis A Virus by Chlorine
Authors: Li Jun Wen , Xin Zhong Tao , Wang Xin Wei , Zheng Jin Lai , Chao Fu Huan
Published: September 2002

The study was intended to investigate the feasibility of reverse transcription-PCR (RT-PCR) for evaluation of the efficacy of inactivation of viruses in water and to elucidate the mechanisms of inactivation of hepatitis A virus (HAV) by chlorine. Cell culture, enzyme-linked immunosorbent assay, and long-overlap RT-PCR were used to detect the infectivity, antigenicity, and entire genome of HAV inactivated or destroyed by chlorine. The cell culture results revealed the complete inactivation of infectivity after 30 min of exposure to 10 or 20 mg of chlorine per liter and the highest level of sensitivity in the 5′ nontranslated regions (5′NTR), inactivation of which took as much time as the inactivation of infectivity of HAV by chlorine. However, antigenicity was not completely destroyed under these conditions. Some fractions in the coding region were resistant to chlorine. To determine the specific region of the 5′NTR lost, three segments of primers were redesigned to monitor the region from bp 1 to 1023 across the entire genome. It was shown that the sequence from bp 1 to 671 was the region most sensitive to chlorine. The results suggested that the inactivation of HAV by chlorine was due to the loss of the 5′NTR. It is believed that PCR can be used to assess the efficacy of disinfection of HAV by chlorine as well as to research the mechanisms of inactivation of viruses by disinfectants.
Polyphosphate Kinase from Activated Sludge Performing Enhanced Biological Phosphorus Removal
Authors: McMahon Katherine D. , Dojka Michael A. , Pace Norman R. , Jenkins David , Keasling Jay D.
Published: September 2002

A novel polyphosphate kinase (PPK) was retrieved from an uncultivated organism in activated sludge carrying out enhanced biological phosphorus removal (EBPR). Acetate-fed laboratory-scale sequencing batch reactors were used to maintain sludge with a high phosphorus content (approximately 11% of the biomass). PCR-based clone libraries of small subunit rRNA genes and fluorescent in situ hybridization (FISH) were used to verify that the sludge was enriched in
Rhodocyclus
-like β-
Proteobacteria
known to be associated with sludges carrying out EBPR. These organisms comprised approximately 80% of total bacteria in the sludge, as assessed by FISH. Degenerate PCR primers were designed to retrieve fragments of putative
ppk
genes from a pure culture of
Rhodocyclus tenuis
and from organisms in the sludge. Four novel
ppk
homologs were found in the sludge, and two of these (types I and II) shared a high degree of amino acid similarity with
R. tenuis
PPK (86 and 87% similarity, respectively). Dot blot analysis of total RNA extracted from sludge demonstrated that the Type I
ppk
mRNA was present, indicating that this gene is expressed during EBPR. Inverse PCR was used to obtain the full Type I sequence from sludge DNA, and a full-length PPK was cloned, overexpressed, and purified to near homogeneity. The purified PPK has a specific activity comparable to that of other PPKs, has a requirement for Mg
2+
, and does not appear to operate in reverse. PPK activity was found mainly in the particulate fraction of lysed sludge microorganisms.
Excretion of Human β-Endorphin into Culture Medium by Using Outer Membrane Protein F as a Fusion Partner in Recombinant
Escherichia coli
Authors: Jeong Ki Jun , Lee Sang Yup
Published: September 2002

Escherichia coli
BL21 strains were found to excrete a large amount of outer membrane protein F (OmpF) into culture medium during high-cell-density cultivation. From this interesting phenomenon, a novel and efficient OmpF fusion system was developed for the excretion of recombinant proteins by
E. coli
. The
ompF
gene of
E. coli
BL21(DE3) was first knocked out by using the
red
operon of bacteriophage λ to construct
E. coli
MBEL-BL101. For the excretion of human β-endorphin as a model protein, the β-endorphin gene was fused to the C terminus of the
E. coli ompF
gene by using a linker containing the Factor Xa recognition site. To develop a fed-batch culture condition that allows efficient production of OmpF-β-endorphin fusion protein, three different feeding strategies, an exponential feeding strategy and two pH-stat strategies with defined and complex nutrient feeding solutions, were examined. Among these, the pH-stat feeding strategy with the complex nutrient feeding solution resulted in the highest productivity (0.33 g of protein per liter per h). Under this condition, up to 5.6 g of OmpF-β-endorphin fusion protein per liter was excreted into culture medium. The fusion protein was purified by anion-exchange chromatography and cleaved by Factor Xa to yield β-endorphin, which was finally purified by reverse-phase chromatography. From 2.7 liters of culture supernatant, 545.4 mg of β-endorphin was obtained.
Culture-Independent Microbial Community Analysis Reveals that Inulin in the Diet Primarily Affects Previously Unknown Bacteria in the Mouse Cecum
Authors: Apajalahti Juha H. A. , Kettunen Hannele , Kettunen Anu , Holben William E. , Nurminen Päivi H. , Rautonen Nina , Mutanen Marja
Published: September 2002

Inulin is a well-known fructose-based prebiotic which has been shown to stimulate the growth of bifidobacteria, a bacterial group generally considered beneficial for intestinal health. In the present study, we analyzed inulin-associated shifts in the total bacterial community of wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene by using DNA-based approaches independent of bacterial culturability. Mice were fed a high-fat, nonfiber diet with or without inulin inclusion at a 10% (wt/wt) concentration. Cecal contents were analyzed after 0, 3, and 9 weeks on the experimental diets. Inulin inclusion significantly affected the total bacterial community structure of the cecum as determined by both a nonselective percent-guanine-plus-cytosine-based profiling analysis and a more specific 16S ribosomal DNA sequence analysis. The shifts included stimulation of bifidobacteria and suppression of clostridia, but sequence comparison revealed that the major shifts were within previously unknown bacterial taxa. Concomitantly, significantly higher bacterial densities, determined by flow cytometry, were observed with the inulin-amended diet, and the metabolism of the cecal bacterial community was altered, as indicated by higher levels of residual short-chain fatty acids, particularly lactic acid. With regard to all of the microbiological parameters measured, the wild-type mice and mice carrying a genetically inactivated adenomatous polyposis coli tumor suppressor gene were essentially identical. Studies of the implications of pre- and probiotics may need to be expanded to include careful analysis of their effects on the entire microbial community, rather than just a few well-known species. Further studies are needed to increase our understanding of the possible roles of currently unknown gastrointestinal bacteria in health and disease.
Bcmfs1
, a Novel Major Facilitator Superfamily Transporter from
Botrytis cinerea
, Provides Tolerance towards the Natural Toxic Compounds Camptothecin and Cercosporin and towards Fungicides
Authors: Hayashi Keisuke , Schoonbeek Henk-jan , De Waard Maarten A.
Published: September 2002

Bcmfs1
, a novel major facilitator superfamily gene from
Botrytis cinerea
, was cloned, and replacement and overexpression mutants were constructed to study its function. Replacement mutants showed increased sensitivity to the natural toxic compounds camptothecin and cercosporin, produced by the plant
Camptotheca acuminata
and the plant pathogenic fungus
Cercospora kikuchii,
respectively. Overexpression mutants displayed decreased sensitivity to these compounds and to structurally unrelated fungicides, such as sterol demethylation inhibitors (DMIs). A double-replacement mutant of
Bcmfs1
and the ATP-binding cassette (ABC) transporter gene
BcatrD
was more sensitive to DMI fungicides than a single-replacement mutant of
BcatrD
, known to encode an important ABC transporter of DMIs. The sensitivity of the wild-type strain and mutants to DMI fungicides correlated with
Bcmfs1
expression levels and with the initial accumulation of oxpoconazole by germlings of these isolates. The results indicate that Bcmfs1 is a major facilitator superfamily multidrug transporter involved in protection against natural toxins and fungicides and has a substrate specificity that overlaps with the ABC transporter BcatrD. Bcmfs1 may be involved in protection of
B. cinerea
against plant defense compounds during the pathogenic phase of growth on host plants and against fungitoxic antimicrobial metabolites during its saprophytic phase of growth.
Detection and Diversity of Expressed Denitrification Genes in Estuarine Sediments after Reverse Transcription-PCR Amplification from mRNA
Authors: Nogales Balbina , Timmis Kenneth N. , Nedwell David B. , Osborn A. Mark
Published: September 2002

The expression of five denitrification genes coding for two nitrate reductases (
narG
and
napA
), two nitrite reductases (
nirS
and
nirK
), and nitrous oxide reductase (
nosZ
) was analyzed by reverse transcription (RT)-PCR of mRNA extracted from two sediment samples obtained in the River Colne estuary (United Kingdom), which receives high nitrogen inputs and for which high denitrification rates have been observed. The presence of all five genes in both sediment samples was confirmed by PCR amplification from extracted DNA prior to analysis of gene expression. Only
nirS
and
nosZ
mRNAs were detected;
nirS
was detected directly as an RT-PCR amplification product, and
nosZ
was detected following Southern blot hybridization. This indicated that active expression of at least the
nirS
and
nosZ
genes was occurring in the sediments at the time of sampling. Amplified
nirS
RT-PCR products were cloned and analyzed by sequencing, and they were compared with amplified
nirS
gene sequences from isolates obtained from the same sediments. A high diversity of
nirS
sequences was observed. Most of the cloned
nirS
sequences retrieved were specific to one site or the other, which underlines differences in the compositions of the bacterial communities involved in denitrifrification in the two sediments analyzed.
Flow Cytometry Analysis of Changes in the DNA Content of the Polychlorinated Biphenyl Degrader
Comamonas testosteroni
TK102: Effect of Metabolites on Cell-Cell Separation
Authors: Hiraoka Yoshinori , Yamada Tohru , Tone Keiko , Futaesaku Yutaka , Kimbara Kazuhide
Published: September 2002

Flow cytometry was used to monitor changes in the DNA content of the polychlorinated biphenyl (PCB)-degrading bacterium
Comamonas testosteroni
TK102 during growth in the presence or absence of PCBs. In culture medium without PCBs, the majority of stationary-phase cells contained a single chromosome. In the presence of PCBs, the percentage of cells containing two chromosomes increased from 12% to approximately 50%. In contrast, addition of PCBs did not change the DNA contents of three species that are unable to degrade PCBs. In addition, highly chlorinated PCBs that are not degraded by TK102 did not result in a change in the DNA content. These results suggest that PCBs did not affect the DNA content of the cells directly; rather, the intermediate metabolites resulting from the degradation of PCBs caused the increase in DNA content. To study the effect of intermediate metabolites on the DNA content of the cells, four
bph
genes,
bphA1
,
bphB
,
bphC
, and
bphD
, were disrupted by gene replacement. The resulting mutant strains accumulated intermediate metabolites when they were grown in the presence of PCBs or biphenyl (BP). When the
bphB
gene was disrupted, the percentage of cells containing two chromosomes increased in cultures grown with PCBs or BP. When grown with BP, cultures of this mutant accumulated two intermediate metabolites, 2-hydroxybiphenyl (2-OHBP) and 3-OHBP. Addition of 2- or 3-OHBP to a wild-type TK102 and non-PCB-degrading species culture also resulted in an increase in the percentage of cells containing two chromosomes. Electron microscopy revealed that cell-cell separation was inhibited in this culture. This is the first report that hydroxy-BPs can inhibit bacterial cell separation while allowing continued DNA replication.
Mimosine, the Allelochemical from the Leguminous Tree
Leucaena leucocephala
, Selectively Enhances Cell Proliferation in Dinoflagellates
Authors: Yeung Patrick K. K. , Wong Francis T. W. , Wong Joseph T. Y.
Published: September 2002

Mimosine, the allelochemical from the leguminous tree
Leucaena leucocephala
, is toxic to most terrestrial animals and plants. We report here that while mimosine inhibits major phytoplankton groups, it enhances cell proliferation in dinoflagellates. On addition to coastal seawater samples, mimosine is able to confer a growth advantage to dinoflagellates. The use of mimosine will promote the isolation and culture of this group of phytoplankton.
Comparative Inactivation of Enteroviruses and Adenovirus 2 by UV Light
Authors: Gerba Charles P. , Gramos Dawn M. , Nwachuku Nena
Published: September 2002

The doses of UV irradiation necessary to inactivate selected enteric viruses on the U.S. Environmental Protection Agency Contaminant Candidate List were determined. Three-log reductions of echovirus 1, echovirus 11, coxsackievirus B3, coxsackievirus B5, poliovirus 1, and human adenovirus type 2 were effected by doses of 25, 20.5, 24.5, 27, 23, and 119 mW/cm
2
, respectively. Human adenovirus type 2 is the most UV light-resistant enteric virus reported to date.
Carbon and Hydrogen Stable Isotope Fractionation during Aerobic Bacterial Degradation of Aromatic Hydrocarbons
Authors: Morasch Barbara , Richnow Hans H. , Schink Bernhard , Vieth Andrea , Meckenstock Rainer U.
Published: September 2002

13
C/
12
C and D/H stable isotope fractionation during aerobic degradation was determined for
Pseudomonas putida
strain mt-2,
Pseudomonas putida
strain F1,
Ralstonia pickettii
strain PKO1, and
Pseudomonas putida
strain NCIB 9816 grown with toluene, xylenes, and naphthalene. Different types of initial reactions used by the respective bacterial strains could be linked with certain extents of stable isotope fractionation during substrate degradation.
Production of Beauvericin, Moniliformin, Fusaproliferin, and Fumonisins B
1
, B
2
, and B
3
by Fifteen Ex-Type Strains of
Fusarium
Species
Authors: Fotso Joseph , Leslie John F. , Smith J. Scott
Published: September 2002

Fifteen
Fusarium
species were analyzed by high-performance liquid chromatography for the production of six mycotoxins in corn grits cultures. Production of mycotoxins ranged from 66 to 2,500 μg/kg for fumonisin B
1
, 0.6 to 1,500 μg/g for moniliformin, 2.2 to 720 μg/g for beauvericin, and 12 to 130 μg/g for fusaproliferin. Fumonisin B
2
(360 μg/kg) was produced by two species, fumonisin B
3
was not detected in any of the 15 species examined, and
Fusarium bulbicola
produced none of the six mycotoxins that we analyzed.
Significance of Enhanced Morphological Detection of
Cryptosporidium
sp. Oocysts in Water Concentrates Determined by Using 4′,6′-Diamidino-2-Phenylindole and Immunofluorescence Microscopy
Authors: Smith H.V. , Campbell B. M. , Paton C. A. , Nichols R. A. B.
Published: September 2002

Of 2,361 water concentrates analyzed for the presence of
Cryptosporidium
spp. oocysts between January 1992 and May 1998, 269 (11.4%) were positive, of which 235 (87.4%) were raw and 34 were final water concentrates. Of 740 oocysts enumerated in positive samples, 656 oocysts (88.7%) were detected in raw and 84 oocysts (11.3%) were detected in final water concentrates by using a commercially available fluorescein isothiocyanate-labeled anti-
Cryptosporidium
sp. monoclonal antibody and the nuclear fluorogen 4′,6′-diamidino-2-phenylindole (DAPI). Of raw water positive samples, 66.8% had oocysts that contained nuclei, while 58.8% of final water samples had oocysts that contained nuclei. The most frequently identified oocysts had either no DAPI-positive nuclei and no internal morphology according to Nomarski differential interference-contrast microscopy (DIC) or four DAPI-positive nuclei together with internal contents according to DIC (39.5 and 32.8% of raw and 42.9 and 30.9% of final water positives, respectively). By use of the presence of DAPI-stained nuclei to support oocyst identification based upon oocyst wall fluorescence, 56.5% of oocysts were identified when at least one nucleus was present, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 32.8% in raw water concentrates. In final water concentrates, 51% of oocysts were identified using oocyst wall fluorescence and the presence of at least one nucleus, while increasing the number of nuclei necessary for identification to four reduced the percentage identifiable to 30.9%. By consolidating our identification criteria from the presence of at least one nucleus to the presence of four nuclei, we excluded approximately 20% of oocysts in either water type. Approximately 40% of oocysts detected in these United Kingdom samples were empty and could not be detected by alternative methods, including the PCR and fluorescence in situ hybridization.
Socioeconomic variation in participation in colorectal cancer screening
Authors: McCaffery K. , Wardle J. , Nadel M. , Atkin W.
Published: October 2002

OBJECTIVES: To investigate socioeconomic variation in participation in flexible sigmoidoscopy (FS) screening for colorectal cancer. DESIGN: A prospective study nested within a multicentre randomised controlled trial of the efficacy of FS screening for the prevention and early detection of colorectal cancer (the UK flexible sigmoidoscopy trial). SETTING: Glasgow, Scotland. PARTICIPANTS: 55–64 year old adults, registered with general practitioners participating in the FS trial. MAIN OUTCOME MEASURES: Screening participation measured at three levels: questionnaire return; interest in screening; attendance at screening. RESULTS: Socioeconomic deprivation was a strong predictor of participation. Return of the screening questionnaire, expression of interest in screening, and attendance at the test, were all lower in more deprived groups. CONCLUSIONS: These results highlight the need to consider ways to reduce inequalities in screening uptake, in parallel with the introduction of any new screening programmes, to avoid exacerbating social gradients in cancer mortality.