bluuebunny/crossref_metadata_2025 huggingface dataset:
YANKEE BONDS AND CROSS‐BORDER PRIVATE PLACEMENTS: AN UPDATE
Authors: Johnson Greg
Published: April 2005

With U.S. Treasury yields near historical lows and the recent relaxation of U.S. regulatory reporting requirements, the U.S. bond markets are more and more frequently the markets of choice for international issuers. Total crossborder U.S. bond issuance is expected to top $350 billion in 2000, easily surpassing previous issuance levels.

Overseas issuers have three primary forms through which they can participate in the U.S. long‐term debt markets: publicly traded, SEC‐registered bonds (commonly known as “Yankee” bonds); traditional private placements; and underwritten Rule 144A private placements. Each of these three financing methods has distinct benefits and limitations that should be thoroughly evaluated in light of the specific objectives of the issuer. Yankee bonds are typically the most cost efficient vehicle for large, investment grade issuers, and they are expected to account for over 75% of the $350 billion market in 2000. Second in importance is the rule 144A market, which is typically used for complex structures requiring heavy rating‐agency involvement, such as future financial flow transactions and project financings. The 144A market has also become a particular favorite with international issuers because of its less formal disclosure requirements and streamlined execution process. The private placement market continues to be the dominant choice of smaller issuers, companies with complicated “stories,” and firms that do not wish to submit to regular scrutiny by rating agencies. This article provides a detailed analysis of each type of bond issuance and the issues facing a financial officer in trying to determine the most appropriate source of long‐term debt.
A New Analytical Approach for Hole Drilling Residual Stress Analysis by Full Field Method
Authors: Baldi Antonio
Published: April 2005

Most of the full field methods for residual stress investigation combine an optical interferometric technique (grating interferometry, speckle interferometry, holographic interferometry) with the standard hole drilling method. Nowadays many articles describing this kind of approach exist, but most of them focus on the experimental aspects while little attention is devoted to the optimal usage of the huge mass of data that the optical methods make available. This paper, without relying on a specific experimental technique, focuses on the development of a new analytical method that attempts to consistently use all the available data. After a detailed algorithm description, the proposed analysis procedure is tested against numerically generated residual stress fields simulating in-plane speckle interferometry. The results obtained show the reliability and robustness of the algorithm.
Analysis and Control of Transverse Vibrations of Axially Moving Strings
Authors: Chen Li-Qun
Published: April 2005

In this paper, research on transverse vibrations of axially moving strings and their control is thoroughly reviewed. In the last few decades, there have been extensive studies on analysis and control of transverse vibrations of axially moving strings because of the wide applications of many engineering devices that axially moving strings represent. In the investigations adopting linear models of moving strings, the paper summarizes recent studies on modal analysis, complicatedly constrained strings, coupled vibrations, and parametric vibration, as well as some early results. In the investigations adopting nonlinear models of moving strings, the paper presents the governing equations with large amplitude, and reviews progress on discretized or direct approximate analytical analyses and numerical approaches based on the Galerkin method or the finite difference method. Furthermore, investigations are reported on modeling of damping mechanisms as viscoelastic materials, coupled vibration of power transmission systems, and bifurcation and chaos. The state of the art of active control of moving strings is surveyed on controllability and observability, the Laplace transform domain analysis and the energy analysis, nonlinear vibration control and adaptive vibration control. Finally, future research directions are suggested such as nonlinear vibration of moving strings under complex constraints and couplings, energetics of nonlinear and time-varying strings, bifurcation and chaos in transverse motion of moving strings, control of hybrid systems containing moving strings, robust and adaptive controls of nonlinear moving strings, and experimental investigations. In this review article there are 242 references cited.
Mathematical Modeling of Ultra-High-Pressure Waterjet Peening
Authors: Kunaporn S. , Ramulu M. , Hashish M.
Published: April 2005

Waterjet peening is a recent promising method in surface treatment. It has the potential to induce compressive residual stresses that benefit the fatigue life of materials similar to the conventional shot peening process. However, there are no analytical models that incorporate process parameters (i.e., supply pressure, jet exposure time, and nozzle traverse rate, etc) to allow predicting the optimized peening process. Mathematical modeling of high-pressure waterjet peening was developed in this study to describe the relation between the waterjet peening parameters and the resulting material modifications. Results showed the possibility of using the proposed mathematical model to predict an initial range for effective waterjet peening under the variation of waterjet peening conditions. The high cycle fatigue tests were performed to validate the proposed model and fatigue test results showed good agreement with the predictions.
A New Quantitative Sensitivity Analysis of the Flow Stress of 18 Engineering Materials in Machining
Authors: Fang N.
Published: April 2005

It has long been argued about which effect, among the effects of strain hardening, strain-rate hardening, and temperature softening, is predominant in governing the material flow stress in machining. This paper presents a new quantitative sensitivity analysis of the flow stress of 18 engineering materials based on the well-known Johnson-Cook model. It is demonstrated that the first predominant factor governing the material flow stress is either strain hardening or thermal softening, depending on the specific material employed and the varying range of temperatures. Strain-rate hardening is the least important factor governing the material flow stress, especially when machining aluminum alloys.
Mechanical Properties of Kevlar® KM2 Single Fiber
Authors: Cheng Ming , Chen Weinong , Weerasooriya Tusit
Published: April 2005

Kevlar® KM2 fiber is a transversely isotropic material. Its tensile stress-strain response in the axial direction is linear and elastic until failure. However, the overall deformation in the transverse directions is nonlinear and nonelastic, although it can be treated linearly and elastically in infinitesimal strain range. For a linear, elastic, and transversely isotropic material, five material constants are needed to describe its stress-strain response. In this paper, stress-strain behavior obtained from experiments on a single Kevlar KM2 fiber are presented and discussed. The effects of loading rate and the influence of axial loading on transverse and transverse loading on axial stress-strain responses are also discussed.
Numerical Analysis of Electrodeposited Nickel Coating in Multistage Drawing Processes
Authors: Zhou L. Q. , Li Y. P. , Zhou Y. C.
Published: April 2005

The elastic-plastic finite element method of a dynamic explicit algorithm was used to simulate the deep drawing processes of nickel coating electrodeposited on a steel substrate to form an advanced battery shell. The Belytschko-Wong-Chiang shell element was used to mesh the materials, the kinematical work-hardening model was adopted for the components, and the tied-with-failure contact criterion was given to the interfacial combination. The rate-type elastic-plastic constitute law was employed to handle the large deformation, and the central difference method was utilized to solve the finite element equations. The simulations of the materials in the first and final processes illustrated that the steel substrate and the nickel coating were simultaneously deformed and yielded in the die fillet profile and the flange area. The thickness variation of the nickel coating and steel substrate was dependent on the main principal stress, and their variation rule was consistent. In the entire drawing processes, the thinnest region after forming was at the lower part of the cup near the cup bottom, the extent of the coating being thinned after drawing was acceptable, and the material was capable of forming the battery shell. The simulated results were partly compared with tests and other analysis and showed good agreement.
Deformation Behavior of Cold Upset Forming of Sintered Al-Fe Composite Preforms
Authors: Selvakumar N. , Narayanasamy R.
Published: April 2005

Cold upsetting experiments were carried out on sintered Al–Fe preforms in order to evaluate their deformation characteristics. The effects of iron content and initial fractional density of the preforms on deformation behavior have been investigated thoroughly by using graphite as a lubricant. Cylindrical preforms with different initial theoretical density and aspect ratio (0.75) were prepared using a suitable die, a punch and a die bottom insert on a 1.0 MN capacity Universal testing machine. The preforms were well covered with dry fine silica sand and sintered in an electric muffle furnace at 550±10°C for a period of 1 h and then furnace cooled. Cold deformation experiments were carried out in several steps. Dimensions such as height, contact, and bulged diameters and densities were measured for each test. In general, each compact was subjected to an incremental compressive loading in steps of 0.005 MN until fine cracks appeared on its free surface. Analysis of the experimental data has shown that the power law relationship between fractional theoretical density ρf/ρth and eεz−εθ has been established. This remained valid for 0–8% iron content and all initial preform densities. Further it was found that the preforms of higher iron content shows higher values of deformation properties like the axial stress and the Poisson’s ratio than less/without iron preforms provided that the initial fractional density taken is kept constant.
Using Embossing to Create a Fiber Reinforced Honeycomb Composite
Authors: Jordan William
Published: April 2005

This research project used hot embossing to create a strong and tough polymeric based composite structure. A honeycomb type structure was created by pressing small grooves into thin polycarbonate sheets. A trapezoidal die was used to create hexagonal shaped channels in the polymeric sheet. A number of these sheets were then bonded together to form a composite material. Carbon fibers were embedded into the channels in some of the laminates. The embossing process was carried out at an elevated temperature in an environmental chamber attached to an MTS servo hydraulic testing machine. The grooved structure had a 31% to 45% decrease in the apparent density compared to the ungrooved specimens. Bend tests, tensile tests, and Charpy impact tests were performed on laminates made from this material. The specific values of tensile strength, flexural modulus, and Charpy impact toughness were increased. A small percentage of fibers significantly increased both the stiffness and strength of the laminate.
Endophytic Colonization of
Vitis vinifera
L. by Plant Growth-Promoting Bacterium
Burkholderia
sp. Strain PsJN
Authors: Compant Stéphane , Reiter Birgit , Sessitsch Angela , Nowak Jerzy , Clément Christophe , Ait Barka Essaïd
Published: April 2005

Patterns of colonization of
Vitis vinifera
L. cv. Chardonnay plantlets by a plant growth-promoting bacterium,
Burkholderia
sp. strain PsJN, were studied under gnotobiotic conditions. Wild-type strain PsJN and genetically engineered derivatives of this strain tagged with
gfp
(PsJN::
gfp
2x) or
gusA
(PsJN::
gusA
11) genes were used to enumerate and visualize tissue colonization. The rhizospheres of 4- to 5-week-old plantlets with five developed leaves were inoculated with bacterial suspensions. Epiphytic and endophytic colonization patterns were then monitored by dilution plating assays and microscopic observation of organ sections. Bacteria were chronologically detected first on root surfaces, then in root internal tissues, and finally in the fifth internode and the tissues of the fifth leaf. Analysis of the PsJN colonization patterns showed that this strain colonizes grapevine root surfaces, as well as cell walls and the whole surface of some rhizodermal cells. Cells were also abundant at lateral root emergence sites and root tips. Furthermore, cell wall-degrading endoglucanase and endopolygalacturonase secreted by PsJN explained how the bacterium gains entry into root internal tissues. Host defense reactions were observed in the exodermis and in several cortical cell layers. Bacteria were not observed on stem and leaf surfaces but were found in xylem vessels of the fifth internode and the fifth leaf of plantlets. Moreover, bacteria were more abundant in the fifth leaf than in the fifth internode and were found in substomatal chambers. Thus, it seems that
Burkholderia
sp. strain PsJN induces a local host defense reaction and systemically spreads to aerial parts through the transpiration stream.
Proteomic Profiling of RecombinantEscherichia coliin High-Cell-Density Fermentations for Improved Production of an Antibody Fragment Biopharmaceutical
Authors: Aldor Ilana S. , Krawitz Denise C. , Forrest William , Chen Christina , Nishihara Julie C. , Joly John C. , Champion Kathleen M.
Published: April 2005

By using two-dimensional polyacrylamide gel electrophoresis, a proteomic analysis over time was conducted with high-cell-density, industrial, phosphate-limitedEscherichia colifermentations at the 10-liter scale. During production, a recombinant, humanized antibody fragment was secreted and assembled in a soluble form in the periplasm.E. coliprotein changes associated with culture conditions were distinguished from protein changes associated with heterologous protein expression. Protein spots were monitored quantitatively and qualitatively. Differentially expressed proteins were quantitatively assessed by using at-test method with a 1% false discovery rate as a significance criterion. As determined by this criterion, 81 protein spots changed significantly between 14 and 72 h (final time) of the control fermentations (vector only). Qualitative (on-off) comparisons indicated that 20 more protein spots were present only at 14 or 72 h in the control fermentations. These changes reflected physiological responses to the culture conditions. In control and production fermentations at 72 h, 25 protein spots were significantly differentially expressed. In addition, 19 protein spots were present only in control or production fermentations at this time. The quantitative and qualitative changes were attributable to overexpression of recombinant protein. The physiological changes observed during the fermentations included the up-regulation of phosphate starvation proteins and the down-regulation of ribosomal proteins and nucleotide biosynthesis proteins. Synthesis of the stress protein phage shock protein A (PspA) was strongly correlated with synthesis of a recombinant product. This suggested that manipulation of PspA levels might improve the soluble recombinant protein yield in the periplasm for this bioprocess. Indeed, controlled coexpression of PspA during production led to a moderate, but statistically significant, improvement in the yield.
Competitive Interactions in Mixed-Species Biofilms Containing the Marine Bacterium
Pseudoalteromonas tunicata
Authors: Rao Dhana , Webb Jeremy S. , Kjelleberg Staffan
Published: April 2005

Pseudoalteromonas tunicata
is a biofilm-forming marine bacterium that is often found in association with the surface of eukaryotic organisms. It produces a range of extracellular inhibitory compounds, including an antibacterial protein (AlpP) thought to be beneficial for
P. tunicata
during competition for space and nutrients on surfaces. As part of our studies on the interactions between
P. tunicata
and the epiphytic bacterial community on the marine plant
Ulva lactuca
, we investigated the hypothesis that
P. tunicata
is a superior competitor compared with other bacteria isolated from the plant. A number of
U. lactuca
bacterial isolates were (i) identified by 16S rRNA gene sequencing, (ii) characterized for the production of or sensitivity to extracellular antibacterial proteins, and (iii) labeled with a fluorescent color tag (either the red fluorescent protein DsRed or green fluorescent protein). We then grew single- and mixed-species bacterial biofilms containing
P. tunicata
in glass flow cell reactors. In pure culture, all the marine isolates formed biofilms containing microcolony structures within 72 h. However, in mixed-species biofilms,
P. tunicata
removed the competing strain unless its competitor was relatively insensitive to AlpP (
Pseudoalteromonas gracilis
) or produced strong inhibitory activity against
P. tunicata
(
Roseobacter gallaeciensis
). Moreover, biofilm studies conducted with an AlpP
−
mutant of
P. tunicata
indicated that the mutant was less competitive when it was introduced into preestablished biofilms, suggesting that AlpP has a role during competitive biofilm formation. When single-species biofilms were allowed to form microcolonies before the introduction of a competitor, these microcolonies coexisted with
P. tunicata
for extended periods of time before they were removed. Two marine bacteria (
R. gallaeciensis
and
P. tunicata
) were superior competitors in this study. Our data suggest that this dominance can be attributed to the ability of these organisms to rapidly form microcolonies and their ability to produce extracellular antibacterial compounds.
Heat Inactivation of
Mycobacterium avium
subsp.
paratuberculosis
in Milk
Authors: McDonald Wendy L. , O'Riley Kimberly J. , Schroen Christopher J. , Condron Robin J.
Published: April 2005

The effectiveness of pasteurization and the concentration of
Mycobacterium avium
subsp.
paratuberculosis
in raw milk have been identified in quantitative risk analysis as the most critical factors influencing the potential presence of viable
Mycobacterium paratuberculosis
in dairy products. A quantitative assessment of the lethality of pasteurization was undertaken using an industrial pasteurizer designed for research purposes with a validated Reynolds number of 62,112 and flow rates of 3,000 liters/h.
M. paratuberculosis
was artificially added to raw whole milk, which was then homogenized, pasteurized, and cultured, using a sensitive technique capable of detecting one organism per 10 ml of milk. Twenty batches of milk containing 10
3
to 10
4
organisms/ml were processed with combinations of three temperatures of 72, 75, and 78°C and three time intervals of 15, 20, and 25 s. Thirty 50-ml milk samples from each processed batch were cultured, and the logarithmic reduction in
M. paratuberculosis
organisms was determined. In 17 of the 20 runs, no viable
M. paratuberculosis
organisms were detected, which represented >6-log
10
reductions during pasteurization. These experiments were conducted with very heavily artificially contaminated milk to facilitate the measurement of the logarithmic reduction. In three of the 20 runs of milk, pasteurized at 72°C for 15 s, 75°C for 25 s, and 78°C for 15 s, a few viable organisms (0.002 to 0.004 CFU/ml) were detected. Pasteurization at all temperatures and holding times was found to be very effective in killing
M. paratuberculosis
, resulting in a reduction of >6 log
10
in 85% of runs and >4 log
10
in 14% of runs.
Direct Microbial Reduction and Subsequent Preservation of Uranium in Natural Near-Surface Sediment
Authors: Suzuki Yohey , Kelly Shelly D. , Kemner Kenneth M. , Banfield Jillian F.
Published: April 2005

The fate of uranium in natural systems is of great environmental importance. X-ray absorption near-edge spectroscopy (XANES) revealed that U(VI) was reduced to U(IV) in shallow freshwater sediment at an open pit in an inactive uranium mine. Geochemical characterization of the sediment showed that nitrate, Fe(III), and sulfate had also been reduced in the sediment. Observations of the sediment particles and microbial cells by scanning and transmission electron microscopy, coupled with elemental analysis by energy dispersive spectroscopy, revealed that uranium was concentrated at microbial cell surfaces. U(IV) was not associated with framboidal pyrite or nanometer-scale iron sulfides, which are presumed to be of microbial origin. Uranium concentrations were not detected in association with algal cells. Phylogenetic analyses of microbial populations in the sediment by the use of 16S rRNA and dissimilatory sulfite reductase gene sequences detected organisms belonging to the families
Geobacteraceae
and
Desulfovibrionaceae
. Cultivated members of these lineages reduce U(VI) and precipitate iron sulfides. The association of uranium with cells, but not with sulfide surfaces, suggests that U(VI) is reduced by the enzymatic activities of microorganisms. Uranium was highly enriched (760 ppm) in a subsurface black layer in unsaturated sediment sampled from a pit which was exposed to seasonal fluctuations in the pond level. XANES analysis showed that the majority of uranium in this layer was U(IV), indicating that uranium is preserved in its reduced form after burial.
Agrobacterium tumefaciens
-Mediated Transformation of
Aspergillus fumigatus
: an Efficient Tool for Insertional Mutagenesis and Targeted Gene Disruption
Authors: Sugui Janyce A. , Chang Yun C. , Kwon-Chung K. J.
Published: April 2005

Agrobacterium tumefaciens
was used to transform
Aspergillus fumigatus
by either random or site-directed integration of transforming DNA (T-DNA). Random mutagenesis via
Agrobacterium tumefaciens
-mediated transformation (ATMT) was accomplished with T-DNA containing a hygromycin resistance cassette. Cocultivation of
A. fumigatus
conidia and
Agrobacterium
(1:10 ratio) for 48 h at 24°C resulted in high frequencies of transformation (>100 transformants/10
7
conidia). The majority of transformants harbored a randomly integrated single copy of T-DNA and were mitotically stable. We chose
alb1
, a polyketide synthase gene, as the target gene for homologous integration because of the clear phenotype difference between the white colonies of Δ
alb1
mutant strains and the bluish-green colonies of wild-type strains. ATMT with a T-DNA-containing
alb1
disruption construct resulted in 66% albino transformants. Southern analysis revealed that 19 of the 20 randomly chosen albino transformants (95%) were disrupted by homologous recombination. These results suggest that ATMT is an efficient tool for transformation, random insertional mutagenesis, and gene disruption in
A. fumigatus
.
High-Temperature-Induced Transposition of Insertion Elements in
Burkholderia multivorans
ATCC 17616
Authors: Ohtsubo Yoshiyuki , Genka Hiroyuki , Komatsu Harunobu , Nagata Yuji , Tsuda Masataka
Published: April 2005

An efficient and quantitative method to analyze the transposition of various insertion sequence (IS) elements in
Burkholderia multivorans
ATCC 17616 was devised. pGEN500, a plasmid carrying a
Bacillus subtilis
-derived
sacB
gene, was introduced into ATCC 17616 cells, and 25% of their sucrose-resistant derivatives were found to carry various IS elements on pGEN500. A PCR-based experimental protocol, in which a mixture of several specific primer pairs was used, revealed that pGEN500 captured, in addition to five previously reported IS elements (IS
401
, IS
402
, IS
406
, IS
407
, and IS
408)
, three novel IS elements, IS
Bmu1
, IS
Bmu2
, and IS
Bmu3
. The global transposition frequency of these IS elements was enhanced more than sevenfold under a high-temperature condition (42°C) but not under oxidative stress or starvation conditions. To our knowledge, this is the first report demonstrating the elevated transposition activities of several IS elements at a high temperature. The efficient experimental protocol developed in this study will be useful in quantitatively and simultaneously investigating various IS elements, as well as in capturing novel functional mobile elements from a wide variety of bacteria.
Potential of the Polyvalent Anti-
Staphylococcus
Bacteriophage K for Control of Antibiotic-Resistant Staphylococci from Hospitals
Authors: O'Flaherty S. , Ross R. P. , Meaney W. , Fitzgerald G. F. , Elbreki M. F. , Coffey A.
Published: April 2005

The increasing prevalence of antibiotic-resistant staphylococci has prompted the need for antibacterial controls other than antibiotics. In this study, a lytic bacteriophage (phage K) was assessed in vitro for its ability to inhibit emerging drug-resistant
Staphylococcus aureus
strains from hospitals and other species of
Staphylococcus
isolated from bovine infections. In in vitro inhibitory assays, phage K lysed a range of clinically isolated methicillin-resistant
S. aureus
(MRSA) strains,
S. aureus
with heterogeneous vancomycin resistance and vancomycin resistance, and teicoplanin-resistant strains. In these assays, 14 of the MRSA strains were initially only weakly sensitive to this phage. However, propagation of phage K on these less-sensitive strains resulted in all 14 being sensitive to the modified phages. The results enforce the principle that, while certain target bacteria may be relatively insensitive to lytic phage, this can be overcome by obtaining modified phage variants from passage of the phage through the insensitive strains. Model in situ hand wash studies using a phage-enriched wash solution resulted in a 100-fold reduction in staphylococcal numbers on human skin by comparison with numbers remaining after washing in phage-free solution. Infusion of the phage into a nonimmunogenic bismuth-based cream resulted in strong anti-
Staphylococcus
activity from the cream on plates and in broth.
Exposure to Cadmium Elevates Expression of Genes in the OxyR and OhrR Regulons and Induces Cross-Resistance to Peroxide Killing Treatment in
Xanthomonas campestris
Authors: Banjerdkij Peerakan , Vattanaviboon Paiboon , Mongkolsuk Skorn
Published: April 2005

Cadmium is an important heavy metal pollutant. For this study, we investigated the effects of cadmium exposure on the oxidative stress responses of
Xanthomonas campestris
, a soil and plant pathogenic bacterium. The exposure of
X. campestris
to low concentrations of cadmium induces cross-protection against subsequent killing treatments with either H
2
O
2
or the organic hydroperoxide
tert
-butyl hydroperoxide (tBOOH), but not against the superoxide generator menadione. The cadmium-induced resistance to peroxides is due to the metal's ability to induce increased levels of peroxide stress protective enzymes such as alkyl hydroperoxide reductase (AhpC), monofunctional catalase (KatA), and organic hydroperoxide resistance protein (Ohr). Cadmium-induced resistance to H
2
O
2
is dependent on functional OxyR, a peroxide-sensing transcription regulator. Cadmium-induced resistance to tBOOH shows a more complex regulatory pattern. The inactivation of the two major sensor-regulators of organic hydroperoxide, OxyR and OhrR, only partially inhibited cadmium-induced protection against tBOOH, suggesting that these genes do have some role in the process. However, other, as yet unknown mechanisms are involved in inducible organic hydroperoxide protection. Furthermore, we show that the cadmium-induced peroxide stress response is mediated by the metal's ability to predominately cause an increase in intracellular concentrations of organic hydroperoxide and, in part, H
2
O
2
. Analyses of various mutants of peroxide-metabolizing enzymes suggested that this increase in organic hydroperoxide levels is, at least in part, responsible for cadmium toxicity in
Xanthomonas
.
Development of an Immunomagnetic Bead-Immunoliposome Fluorescence Assay for Rapid Detection of
Escherichia coli
O157:H7 in Aqueous Samples and Comparison of the Assay with a Standard Microbiological Method
Authors: DeCory Thomas R. , Durst Richard A. , Zimmerman Scott J. , Garringer Linda A. , Paluca Gary , DeCory Heleen H. , Montagna Richard A.
Published: April 2005

The objective of this study was to develop and optimize a protocol for the rapid detection of
Escherichia coli
O157:H7 in aqueous samples by a combined immunomagnetic bead-immunoliposome (IMB/IL) fluorescence assay. The protocol consisted of the filtration or centrifugation of 30- to 100-ml samples followed by incubation of the filter membranes or pellet with anti-
E. coli
O157:H7 immunomagnetic beads in growth medium specific for
E. coli
O157:H7. The resulting
E. coli
O157:H7-immunomagnetic bead complexes were isolated by magnetic separation, washed, and incubated with sulforhodamine B-containing immunoliposomes specific for
E. coli
O157:H7; the final immunomagnetic bead-
E. coli
O157:H7-immunoliposome complexes were again isolated by magnetic separation, washed, and lysed with a
n
-octyl-β-
d
-glucopyranoside to release sulforhodamine B. The final protocol took less than 8 h to complete and had a detection limit of less than 1 CFU of
E. coli
O157:H7 per ml in various aqueous matrices, including apple juice and cider. To validate the protocol at an independent facility, 100-ml samples of groundwater with and without
E. coli
O157:H7 (15 CFU) were analyzed by a public health laboratory using the optimized protocol and a standard microbiological method. While the IMB/IL fluorescence assay was able to identify
E. coli
O157:H7-containing samples with 100% accuracy, the standard microbiological method was unable to distinguish
E. coli
O157:H7-spiked samples from negative controls without further extensive workup. These results demonstrate the feasibility of using immunomagnetic beads in combination with sulforhodamine B-encapsulating immunoliposomes for the rapid detection of
E. coli
O157:H7 in aqueous samples.
Prevalence and Genetic Diversity ofCampylobacterspp. in Environmental Water Samples from a 100-Square-Kilometer Predominantly Dairy Farming Area
Authors: Kemp R. , Leatherbarrow A. J. H. , Williams N. J. , Hart C. A. , Clough H. E. , Turner J. , Wright E. J. , French N. P.
Published: April 2005

Water samples were taken systematically from a 100-km2area of mainly dairy farmland in northwestern England and examined forCampylobacterspp. Pulsed-field gel electrophoresis-restriction fragment length polymorphism (PFGE-RFLP) andflaAstrain typing ofCampylobacter jejuniandCampylobacter coliisolates were done. Data on the water source and the adjacent environment were recorded and examined as explanatory variables.Campylobacterspp. were isolated from 40.5% (n= 119) of the water samples tested.C. jejuniwas isolated from 14.3%,C. coliwas isolated from 18.5%, andCampylobacter lariwas isolated from 4.2% of the samples.Campylobacter hyointestinaliswas not isolated from any water source. The difference in prevalence between water types (trough, running, and standing) was significant (P= 0.001).C. jejuniwas the species most commonly isolated from trough-water and running-water sources, whileC. coliwas the most frequently isolated from standing water (P< 0.001). No association was found between the presence ofEscherichia coliand that ofCampylobacterspp. The final multivariable logistic regression model forCampylobacterspp. included the following variables: water source, soil type, aspect, and amount of cattle fecal material in the environment (fecal pat count). Strain typing demonstrated a diverse population ofC. jejuniand the presence of a commonC. coli flaAtype that was widely distributed throughout the area. Most of the isolates within the commonflaAtype were discriminated by PFGE-RFLP. These findings suggest a possible role for environmental water in the epidemiology ofCampylobacterspp. in a farming environment.
Biphasic Behavior of Anammox Regulated by Nitrite and Nitrate in an Estuarine Sediment
Authors: Trimmer Mark , Nicholls Joanna C. , Morley Nicholas , Davies Christian A. , Aldridge John
Published: April 2005

The production of N
2
gas via anammox was investigated in sediment slurries at in situ NO
2
−
concentrations in the presence and absence of NO
3
−
. With single enrichment above 10 μM
14
NO
2
−
or
14
NO
3
−
and
15
NH
4
+
, anammox activity was always linear (
P
< 0.05), in agreement with previous findings. In contrast, anammox exhibited a range of activity below 10 μM NO
2
−
or NO
3
−
, including an elevated response at lower concentrations. With 100 μM NO
3
−
, no significant transient accumulation of NO
2
−
could be measured, and the starting concentration of NO
2
−
could therefore be regulated. With dual enrichment (1 to 20 μM NO
2
−
plus 100 μM NO
3
−
), there was a pronounced nonlinear response in anammox activity. Maximal activity occurred between 2 and 5 μM NO
2
−
, but the amplitude of this peak varied across the study (November 2003 to June 2004). Anammox accounted for as much as 82% of the NO
2
−
added at 1 μM in November 2003 but only for 15% in May 2004 and for 26 and 5% of the NO
2
−
added at 5 μM for these two months, respectively. Decreasing the concentration of NO
3
−
but holding NO
2
−
at 5 μM decreased the significance of anammox as a sink for NO
2
−
. The behavior of anammox was explored by use of a simple anammox-denitrification model, and the concept of a biphasic system for anammox in estuarine sediments is proposed. Overall, anammox is likely to be regulated by the availability of NO
3
−
and NO
2
−
and the relative size or activity of the anammox population.
Elimination of Botulinum Neurotoxin (BoNT) Type B from Drinking Water by Small-Scale (Personal-Use) Water Purification Devices and Detection of BoNT in Water Samples
Authors: Hörman Ari , Nevas Mari , Lindström Miia , Hänninen Marja-Liisa , Korkeala Hannu
Published: April 2005

Seven small-scale drinking water purification devices were evaluated for their capacity to eliminate botulinum neurotoxin (BoNT) type B from drinking water. Influent water inoculated with toxic
Clostridium botulinum
cultures and effluent purified water samples were tested for the presence of BoNT by using a standard mouse bioassay and two commercial rapid enzyme immunoassays (EIAs). The water purification devices based on filtration through ceramic or membrane filters with a pore size of 0.2 to 0.4 μm or irradiation from a low-pressure UV-lamp (254 nm) failed to remove BoNT from raw water (reduction of <0.1 log
10
units). A single device based on reverse osmosis was capable of removing the BoNT to a level below the detection limit of the mouse bioassay (reduction of >2.3 log
10
units). The rapid EIAs intended for the detection of BoNT from various types of samples failed to detect BoNT from aqueous samples containing an estimated concentration of BoNT of 396,000 ng/liter.
Efficient Production ofl-Lactic Acid by Metabolically EngineeredSaccharomyces cerevisiaewith a Genome-Integratedl-Lactate Dehydrogenase Gene
Authors: Ishida Nobuhiro , Saitoh Satoshi , Tokuhiro Kenro , Nagamori Eiji , Matsuyama Takashi , Kitamoto Katsuhiko , Takahashi Haruo
Published: April 2005

We developed a metabolically engineered yeast which produces lactic acid efficiently. In this recombinant strain, the coding region for pyruvate decarboxylase 1 (PDC1) on chromosome XII is substituted for that of thel-lactate dehydrogenase gene (LDH) through homologous recombination. The expression of mRNA for the genome-integratedLDHis regulated under the control of the nativePDC1promoter, whilePDC1is completely disrupted. Using this method, we constructed a diploid yeast transformant, with each haploid genome having a single insertion of bovineLDH. Yeast cells expressingLDHwere observed to convert glucose to both lactate (55.6 g/liter) and ethanol (16.9 g/liter), with up to 62.2% of the glucose being transformed into lactic acid under neutralizing conditions. This transgenic strain, which expresses bovineLDHunder the control of thePDC1promoter, also showed high lactic acid production (50.2 g/liter) under nonneutralizing conditions. The differences in lactic acid production were compared among four different recombinants expressing a heterologousLDHgene (i.e., either the bovineLDHgene or theBifidobacterium longum LDHgene): two transgenic strains with 2μm plasmid-based vectors and two genome-integrated strains.
Production of Novel Rapamycin Analogs by Precursor-Directed Biosynthesis
Authors: Ritacco Frank V. , Graziani Edmund I. , Summers Mia Y. , Zabriskie T. Mark , Yu Ker , Bernan Valerie S. , Carter Guy T. , Greenstein Michael
Published: April 2005

The natural product rapamycin, produced during fermentation byStreptomyces hygroscopicus, is known for its potent antifungal, immunosuppressive, and anticancer activities. During rapamycin biosynthesis, the amino acidl-pipecolate is incorporated into the rapamycin molecule. We investigated the use of precursor-directed biosynthesis to create new rapamycin analogs by substitution of unusuall-pipecolate analogs in place of the normal amino acid. Our results suggest that thel-pipecolate analog (±)-nipecotic acid inhibits the biosynthesis ofl-pipecolate, thereby limiting the availability of this molecule for rapamycin biosynthesis. We used (±)-nipecotic acid in our precursor-directed biosynthesis studies to reducel-pipecolate availability and thereby enhance the incorporation of other pipecolate analogs into the rapamycin molecule. We describe here the use of this method for production of two new sulfur-containing rapamycin analogs, 20-thiarapamycin and 15-deoxo-19-sulfoxylrapamycin, and report measurement of their binding to FKBP12.
Expression of
abrB310
and
sinR
, and Effects of Decreased
abrB310
Expression on the Transition from Acidogenesis to Solventogenesis, in
Clostridium acetobutylicum
ATCC 824
Authors: Scotcher Miles C. , Rudolph Frederick B. , Bennett George N.
Published: April 2005

The transcription factors
sinR
and
abrB
are involved in the control of sporulation initiation in
Bacillus subtilis
. We identified a single homologue to
sinR
and three highly similar homologues to
abrB
, designated
abrB310
,
abrB1941
, and
abrB3647
, in
Clostridium acetobutylicum
ATCC 824. Using reporter vectors, we showed that the promoters of
abrB1941
and
abrB3647
were not active under the growth conditions tested. The
abrB310
promoter was strongly active throughout growth and exhibited a transient elevation of expression at the onset of solventogenesis. Primer extension assays showed that two transcripts of
abrB310
and a single, extremely weak transcript for
sinR
are expressed. Potential −35 and −10 consensus motifs are readily identifiable surrounding the transcription start sites of
abrB310
and
sinR
, with a single putative 0A box present within the promoter of
abrB310
. In strains of
C. acetobutylicum
transformed with plasmids to elevate
sinR
expression or decrease
sinR
expression, no significant differences in growth or in acid or solvent production were observed compared to the control strains. In
C. acetobutylicum
strain 824(pAS310), which expressed an antisense RNA construct targeted against
abrB310
, the acids acetate and butyrate accumulated to approximately twice the normal concentration. This accumulation corresponded to a delay and decrease in acetone and butanol production. It was also found that sporulation in strain 824(pAS310) was delayed but that the morphology of sporulating cells and spores was normal. Based upon these observations, we propose that
abrB310
may act as a regulator at the transition between acidogenic and solventogenic growth.
Accumulation of Polyhydroxyalkanoate from Styrene and Phenylacetic Acid by
Pseudomonas putida
CA-3
Authors: Ward Patrick G. , de Roo Guy , O'Connor Kevin E.
Published: April 2005

Pseudomonas putida
CA-3 is capable of converting the aromatic hydrocarbon styrene, its metabolite phenylacetic acid, and glucose into polyhydroxyalkanoate (PHA) when a limiting concentration of nitrogen (as sodium ammonium phosphate) is supplied to the growth medium. PHA accumulation occurs to a low level when the nitrogen concentration drops below 26.8 mg/liter and increases rapidly once the nitrogen is no longer detectable in the growth medium. The depletion of nitrogen and the onset of PHA accumulation coincided with a decrease in the rate of substrate utilization and biochemical activity of whole cells grown on styrene, phenylacetic acid, and glucose. However, the efficiency of carbon conversion to PHA dramatically increased once the nitrogen concentration dropped below 26.8 mg/liter in the growth medium. When supplied with 67 mg of nitrogen/liter, the carbon-to-nitrogen (C:N) ratios that result in a maximum yield of PHA (grams of PHA per gram of carbon) for styrene, phenylacetic acid, and glucose are 28:1, 21:1, and 18:1, respectively. In cells grown on styrene and phenylacetic acid, decreasing the carbon-to-nitrogen ratio below 28:1 and 21:1, respectively, by increasing the nitrogen concentration and using a fixed carbon concentration leads to lower levels of PHA per cell and lower levels of PHA per batch of cells. Increasing the carbon-to-nitrogen ratio above 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, by decreasing the nitrogen concentration and using a fixed carbon concentration increases the level of PHA per cell but results in a lower level of PHA per batch of cells. Increasing the carbon and nitrogen concentrations but maintaining the carbon-to-nitrogen ratio of 28:1 and 21:1 for cells grown on styrene and phenylacetic acid, respectively, results in an increase in the total PHA per batch of cells. The maximum yields for PHA from styrene, phenylacetic acid, and glucose are 0.11, 0.17, and 0.22 g of PHA per g of carbon, respectively.
Characterization of a MobileclpLGene fromLactobacillus rhamnosus
Authors: Suokko Aki , Savijoki Kirsi , Malinen Erja , Palva Airi , Varmanen Pekka
Published: April 2005

Two genes encoding ClpL ATPase proteins were identified in a probioticLactobacillus rhamnosusstrain, E-97800. Sequence analyses revealed that the genes, designatedclpL1andclpL2, share 80% identity. TheclpL2gene showed the highest degree of identity (98.5%) to aclpLgene fromLactobacillus plantarumWCFSI, while it was not detected in three otherL. rhamnosusstrains studied. According to Northern analyses, the expression ofclpL1and theclpL2were induced during heat shock by >20- and 3-fold, respectively. The functional promoter regions were determined by primer extension analyses, and theclpL1promoter was found to be overlapped by an inverted repeat structure identical to the conserved CIRCE element, indicating thatclpL1belongs to the HrcA regulon inL. rhamnosus. No consensus binding sites for HrcA or CtsR could be identified in theclpL2promoter region. Interestingly, theclpL2gene was found to be surrounded by truncated transposase genes and flanked by inverted repeat structures nearly identical to the terminal repeats of the ISLpl1fromL. plantarumHN38. Furthermore,clpL2was shown to be mobilized during prolonged cultivation at elevated temperature. The presence of a gene almost identical toclpL2inL. plantarumand its absence in otherL. rhamnosusstrains suggest that theL. rhamnosusE-97800 has acquired theclpL2gene via horizontal transfer. No change in the stress tolerance of the ClpL2-deficient derivative of E-97800 compared to the parental strain was observed.
Spatial Distribution and Transcriptional Activity of an Uncultured Clade of Planktonic Diazotrophic γ-Proteobacteria in the Arabian Sea
Authors: Bird Clare , Martinez Martinez Joaquín , O'Donnell Anthony G. , Wyman Michael
Published: April 2005

The spatial distribution of an uncultured clade of marine diazotrophic γ-proteobacteria in the Arabian Sea was investigated by the development of a specific primer pair to amplify an internal fragment of
nifH
by PCR. These organisms were most readily detected in highly oligotrophic surface waters but could also be found in deeper waters below the nutricline.
nifH
transcripts originating from this clade were detected in oligotrophic surface waters and, in addition, in the deeper and the more productive near-coastal waters. The
nifH
sequences most closely related to the unidentified marine bacterial group are from environmental clones amplified from the Atlantic and Pacific Oceans. These findings suggest that these γ-proteobacteria are widespread and likely to be an important component of the heterotrophic diazotrophic microbial community of the tropical and subtropical oceans.
Rehydration of the LichenRamalina laceraResults in Production of Reactive Oxygen Species and Nitric Oxide and a Decrease in Antioxidants
Authors: Weissman Lior , Garty Jacob , Hochman Ayala
Published: April 2005

Lichens are slow-growing associations of fungi and unicellular green algae or cyanobacteria. They are poikilohydric organisms whose lifestyle in many cases consists of alternating periods of desiccation, with low metabolic activity, and hydration, which induces increase in their metabolism. Lichens have apparently adapted to such extreme transitions between desiccation and rehydration, but the mechanisms that govern these adaptations are still poorly understood. In this study, the effect of rehydration on the production of reactive oxygen species and nitric oxide as well as low-molecular-weight antioxidants was investigated with the lichenRamalina lacera. Rehydration ofR. laceraresulted in the initiation of and a rapid increase in photosynthetic activity. Recovery of photosynthesis was accompanied by bursts of intracellular production of reactive oxygen species and nitric oxide. Laser-scanning confocal microscopy using dichlorofluorescein fluorescence revealed that formation of reactive oxygen species following rehydration was associated with both symbiotic partners of the lichen. The rate and extent of reactive oxygen species production were similar in the light and in the dark, suggesting a minor contribution of photosynthesis. Diaminofluorescein fluorescence, indicating nitric oxide formation, was detected only in fungal hyphae. Activities associated with rehydration did not have a deleterious effect on membrane integrity as assessed by measurement of electrolyte leakage, but water-soluble low-molecular-weight antioxidants decreased significantly.
Differential Sunlight Sensitivity of Picophytoplankton from Surface Mediterranean Coastal Waters
Authors: Sommaruga Ruben , Hofer Julia S. , Alonso-Sáez Laura , Gasol Josep M.
Published: April 2005

We tested the sensitivity of coastal picophytoplankton exposed to natural sunlight in short-term experiments. Cell abundance and cell-specific chlorophyll fluorescence were significantly reduced in
Prochlorococcus
spp. but not in
Synechococcus
, whereas picoeukaryotes had an intermediate response. These results are the first direct evidence of a differential sensitivity to sunlight of these ubiquitous marine members of unicellular phytoplankton.
Anaerobic, Nitrate-Dependent Oxidation of U(IV) Oxide Minerals by the Chemolithoautotrophic Bacterium
Thiobacillus denitrificans
Authors: Beller Harry R.
Published: April 2005

Under anaerobic conditions and at circumneutral pH, cells of the widely distributed, obligate chemolithoautotrophic bacterium
Thiobacillus denitrificans
oxidatively dissolved synthetic and biogenic U(IV) oxides (uraninite) in nitrate-dependent fashion: U(IV) oxidation required the presence of nitrate and was strongly correlated with nitrate consumption. This is the first report of anaerobic U(IV) oxidation by an autotrophic bacterium.