bluuebunny/crossref_metadata_2025 huggingface dataset:
Amoxicillin Resistance in Helicobacter pylori: Studies from Tokyo, Japan from 1985 to 2003
Authors: Watanabe Kazuhiro , Tanaka Akifumi , Imase Kyoto , Tokunaga Kengo , Sugano Hajime , Kai Akemi , Ishida Hitoshi , Itoh Takeshi , Takahashi Shin’ichi
Published: February 2005

Background.  Previous reports revealed no resistant strains of amoxicillin (AMPC), which is usually used in eradication therapy for H. pylori infection. However, the frequency and evolution of natural AMPC‐resistant strains in the Japanese population remains unknown.

Aim.  To assess the prevalence of H. pylori resistance against AMPC in the Tokyo area, a collection of 648 H. pylori strains isolated from patients with GI diseases from 1985 to 2003 was tested for their sensitivity to AMPC.

Methods.  The susceptibility of the strains was assessed by determination of the minimal inhibitory concentration (MIC) using the E‐test and/or the Dry‐plate method. The susceptibility breakpoints of AMPC for H. pylori were: sensitive (AMPC‐S); MIC < 0.04 µg/ml, intermittent resistance (AMPC‐I); 0.04–1, resistant (AMPC‐R); > 1.

Results.  No AMPC‐R strains were detected in the strains isolated between 1985 and 1996, while the rate of resistance was determined to be 1.1%, 2.1%, 5.4%, 5.6%, 0%, 8.8%, and 1.5% every year, respectively, from 1997 to 2003. The percentage of AMPC‐I strains increased from 2000 to 2003. The total eradication rate of H. pylori in the patients who received triple therapy containing AMPC was 81.4% (214/263). Classified as above, the rates of AMPC‐S, AMPC‐I, and AMPC‐R were 84.6%, 77.8%, 25%, respectively.

Conclusion.  H. pylori resistance to AMPC is still rare in Japan, although the percentage of AMPC‐I strains has increased over the last 4 years. The frequency of isolation of strains showing true resistance to AMPC may increase in the future, along with an increase in the frequency of isolation of AMPC‐I strains.
Effect of Dietary Anti‐Urease Immunoglobulin Y onHelicobacter pyloriInfection in Mongolian Gerbils
Authors: Nomura Sachiko , Suzuki Hidekazu , Masaoka Tatsuhiro , Kurabayashi Kumiko , Ishii Hiromasa , Kitajima Masaki , Nomoto Kikuo , Hibi Toshifumi
Published: February 2005

Background and aim. Helicobacter pyloriis known to be a major pathogenic factor in the development of gastritis, peptic ulcer disease and gastric cancer. Recently, chicken egg yolk immunoglobulin Y (IgY) has been recognized as an inexpensive antibody source for passive immunization against gastrointestinal infections. The present study was designed to investigate the effect of anti‐urease IgY onH. pyloriinfection in Mongolian gerbils.

Methods. H. pylori‐infected Mongolian gerbils were administered a diet containing anti‐urease IgY, with or without famotidine (F). After 10 weeks, bacterial culture and measurement of the gastric mucosal myeloperoxidase (MPO) activity were performed. In a second experiment, another group of gerbils was started on a diet containing F + IgY a week prior toH. pyloriinoculation. After 9 weeks, these animals were examined.

Results. In theH. pylori‐infected gerbils, there were no significant differences in the level ofH. pyloricolonization among the different dietary and control groups. However, the MPO activity was significantly decreased in theH. pylorigroup administered the F + IgY diet compared with that in theH. pylorigroup administered the IgY, F, or control diet. Furthermore, in the gerbils administered the F + IgY diet prior to the bacterial inoculation, inhibition ofH. pyloricolonization and suppression of the elevated gastric mucosal MPO activity were observed.

Conclusions. Oral administration of urease‐specific IgY not only inhibitedH. pyloridisease activity inH. pylori‐infected gerbils, but also preventedH. pyloricolonization in those not yet infected. These encouraging results may pave the way for a novel therapeutic and prophylactic approach in the management ofH. pylori‐associated gastroduodenal disease.
Intranasal CpG‐Oligodeoxynucleotide is a Potent Adjuvant of Vaccine against Helicobacter pylori, and T Helper 1 Type Response and Interferon‐γ Correlate with the Protection
Authors: Shi Tong , Liu Wen‐zhong , Gao Fei , Shi Gui‐ying , Xiao Shu‐dong
Published: February 2005

Background.  Although a series of vaccines against Helicobacter pylori have emerged in the past 10 years, the mechanism involved in their protective effect is yet to be elucidated, and more effective vaccine adjuvants remain to be developed. In this study, CpG‐oligodeoxynucleotide (CpG‐ODN) was investigated as a new candidate for a H. pylori vaccine adjuvant. Furthermore, the role of T helper 1 (Th1) type response and interferon (IFN)‐γ in the protective immunity was explored.

Methods.  C57BL/6 mice and IFN‐γ knockout mice were intranasally or orally immunized with H. pylori whole cell sonicate (WCS)/CpG‐ODN and challenged with different doses [5 × 108 and 5 × 106 colony‐forming units (CFU)] of H. pylori. The protective effect was assessed as the percentage of noninfected mice. The responsive antibodies and cytokines were analyzed using an enzyme‐linked immunosorbent assay (ELISA) and flow cytometry.

Results.  The prevention rates against H. pylori infection in mice intranasally immunized with WCS plus CpG‐ODN were dramatically higher than those in sham‐immunized mice (70% vs. 0%, challenged with 5 × 108 CFU H. pylori; 90% vs. 20%, challenged with 5 × 106 CFU H. pylori). Significantly higher levels of immunoglobulin G2a (IgG2a) and IFN‐γ were detected in the mice immunized with WCS/CpG than in sham‐immunized controls. However, vaccination failed to effectively protect IFN‐γ knockout mice challenged with H. pylori.

Conclusions.  CpG‐ODN given intranasally is a potent adjuvant for development of a H. pylori vaccine. Th1‐type response and IFN‐γ are involved in the protection.
Tooth Loss and Helicobacter pylori Seropositivity: the Newcastle Thousand Families Cohort Study at Age 49–51 Years
Authors: Pearce Mark S. , Steele James G. , Campbell David I. , Thomas Julian E.
Published: February 2005

Background.  Helicobacter pylori, one of the commonest chronic bacterial infections of humankind, is an important risk factor for gastric carcinoma. It has also been suggested to be present in dental plaque. This study investigated the potential link between the number of teeth lost and H. pylori seropositivity at age 50 years.

Methods.  H. pylori seropositivity at age 50 years was investigated among 334 individuals born in Newcastle upon Tyne, United Kingdom, in May and June 1947 and related to the number of teeth lost, after adjusting for socioeconomic status.

Results.  The unadjusted risk of being seropositive for H. pylori increased with increasing number of teeth lost (odds ratio per tooth 1.03, 95% confidence interval 1.01–1.06, p = .019). However, after adjustment for socioeconomic status at birth and at age 50 years, the relationship was no longer significant (p = .36).

Conclusions.  Our results, obtained using prospectively collected data, suggest that any relationship between poor oral health and seropositivity to H. pylori may be due to both tooth loss and H. pylori colonization being associated with socioeconomic status and related factors.
Phase relationships in grunerite–garnet‐bearing amphibolites in the system CFMASH, with applications to metamorphic rocks from the Central Zone of the Limpopo Belt, South Africa
Authors: ZEH A. , HOLLAND T. J. B. , KLEMD R.
Published: February 2005

A petrogenetic grid in the model system CaO–FeO–MgO–Al2O3–SiO2–H2O is presented, illustrating the phase relationships among the minerals grunerite, hornblende, garnet, clinopyroxene, chlorite, olivine, anorthite, zoisite and aluminosilicates, with quartz and H2O in excess. The grid was calculated with the computer software thermocalc, using an upgraded version of the internally consistent thermodynamic dataset HP98 and non‐ideal mixing activity models for all solid solutions. From this grid, quantitative phase diagrams (P–T pseudosections) are derived and employed to infer a P–T path for grunerite–garnet‐bearing amphibolites from the Endora Klippe, part of the Venetia Klippen Complex within the Central Zone of the Limpopo Belt. Agreement between calculated and observed mineral assemblages and garnet zonation indicates that this part of the Central Zone underwent a prograde temperature and pressure increase from c. 540 °C/4.5 kbar to 650 °C/6.5 kbar, followed by a post‐peak metamorphic pressure decrease. The inferred P–T path supports a geotectonic model suggesting that the area surrounding the Venetia kimberlite pipes represents the amphibolite‐facies roof zone of migmatitic gneisses and granulites that occur widely within the Central Zone. In addition, the P–T path conforms to an interpretation that the Proterozoic evolution of the Central Zone was controlled by horizontal tectonics, causing stacking and differential heating at c. 2.0 Ga.
Discrete ultrahigh‐pressure domains in the Western Gneiss Region, Norway: implications for formation and exhumation
Authors: ROOT D. B. , HACKER B. R. , GANS P. B. , DUCEA M. N. , EIDE E. A. , MOSENFELDER J. L.
Published: February 2005

New eclogite localities and new 40Ar/39Ar ages within the Western Gneiss Region of Norway define three discrete ultrahigh‐pressure (UHP) domains that are separated by distinctly lower pressure, eclogite facies rocks. The sizes of the UHP domains range from c. 2500 to 100 km2; if the UHP culminations are part of a continuous sheet at depth, the Western Gneiss Region UHP terrane has minimum dimensions of c. 165 × 50 × 5 km. 40Ar/39Ar mica and K‐feldspar ages show that this outcrop pattern is the result of gentle regional‐scale folding younger than 380 Ma, and possibly 335 Ma. The UHP and intervening high‐pressure (HP) domains are composed of eclogite‐bearing orthogneiss basement overlain by eclogite‐bearing allochthons. The allochthons are dominated by garnet amphibolite and pelitic schist with minor quartzite, carbonate, calc‐silicate, peridotite, and eclogite. Sm/Nd core and rim ages of 992 and 894 Ma from a 15‐cm garnet indicate local preservation of Precambrian metamorphism within the allochthons. Metapelites within the allochthons indicate near‐isothermal decompression following (U)HP metamorphism: they record upper amphibolite facies recrystallization at 12–17 kbar and c. 750 °C during exhumation from mantle depths, followed by a low‐pressure sillimanite + cordierite overprint at c. 5 kbar and c. 750 °C. New 40Ar/39Ar hornblende ages of 402 Ma document that this decompression from eclogite‐facies conditions at 410–405 Ma to mid‐crustal depths occurred in a few million years. The short timescale and consistently high temperatures imply adiabatic exhumation of a UHP body with minimum dimensions of 20–30 km. 40Ar/39Ar muscovite ages of 397–380 Ma show that this extreme heat advection was followed by rapid cooling (c. 30 °C Myr−1), perhaps because of continued tectonic unroofing.
Unusual polyclonal anti‐gp91phox peptide antibody interactions with X‐linked chronic granulomatous disease‐derived human neutrophils are not from compensatory expression of Nox proteins 1, 3, or 4
Authors: Baniulis Danas , Nauseef William M. , Burritt James B. , Taylor Ross M. , Heyworth Paul G. , Dinauer Mary C. , Bumelis Vladas A. , Magnusson Karl‐Eric , Jesaitis Algirdas J.
Published: February 2005

Abstract:  To obtain topological information about human phagocyte flavocytochrome b558 (Cytb), rabbit anti‐peptide antibodies were raised against synthetic peptides mimicking gp91phox regions: 1–9 (MGN), 30–44 (YRV), 150–159 (ESY), 156–166 (ARK), 247–257 (KIS‐1, KIS‐2). Following affinity purification on immobilized peptide matrices, all antibodies but not prebleed controls recognized purified detergent‐solubilized Cytb by enzyme‐linked immunosorbent assay (ELISA). Affinity‐purified antibodies recognizing KIS, ARK and ESY but not YRV, MGN or prebleed IgG specifically detected gp91phox in immunoblot analysis. Antibodies recognizing MGN, ESY, ARK and KIS but not YRV or the prebleed IgG fraction labeled intact normal neutrophils. Surprisingly, all antibodies, with the exception of YRV and pre‐immune IgG controls, bound both normal and Cytb‐negative neutrophils from the obligate heterozygous mother of a patient with X‐linked chronic granulomatous disease (X‐CGD) and all neutrophils from another patient lacking the gp91phox gene. Further immunochemical examination of membrane fractions derived from nine genetically unrelated patients with X‐CGD, using an antibody that recognizes other Nox protein family members, suggests that the unusual reactivity observed does not reflect the compensatory expression of gp91phox homologs Nox1, 3 or 4. These results suggest that an unusual surface reactivity exists on neutrophils derived from X‐linked chronic granulomatous disease patients that most likely extends to normal neutrophils as well. The study highlights the need for caution in interpreting the binding of rabbit polyclonal antipeptide antibodies to human neutrophils in general and, in the specific case of antibodies directed against Cytb, the need for Cytb‐negative controls.
Thrombotic microangiopathy after combined autografting–allografting for multiple myeloma – report of three cases
Authors: Kornacker M. , Luft T. , Ho A. D. , Schaefer H.‐J.
Published: February 2005

Thrombotic microangiopathy (TMA) has been described as a complication of bone marrow or stem cell transplantation. It is usually associated with high dose therapy for autologous transplantation or myeloablative conditioning in the allogeneic setting. Here we report three cases of TMA after reduced intensity conditioning and allogeneic peripheral blood stem cell transplantation. All three patients had high dose Melphalan therapy with autologous stem cell support preceding the allogeneic transplantation for several weeks, which may have contributed to endothelial damage and subsequent development of TMA.
Nordic Myeloma Study Group, the first 15 years: scientific collaboration and improvement of patient care
Authors: Hippe Erik , Westin Jan , Wislöff Finn
Published: February 2005

Abstract:  The accomplishments of the Nordic Myeloma Study Group (NMSG) during its first 15 yr are briefly surveyed, together with a discussion of principles guiding the group's clinical trials and of problems that need to be addressed in coming years. The group has so far carried out 12 clinical trials, comprising more than 2500 patients, spanning from minor phase II to large randomised phase III trials. At the time of writing, two randomised trials are running (comparing two doses of i.v. pamidronate, and melphalan‐prednisone (MP) vs. MP‐thalidomide to elderly patients). The group has strived for a simple organisation with much responsibility delegated to regional coordinators (Denmark 3, Norway 5, Sweden 5). With regard to trial design, the group has considered it important that studies are based on sound scientific questions, are simple to handle for the participants, population based, investigator initiated, include quality of life and health resources assessment as end‐points, and can be used as basis for diverse scientific spin‐off projects. Like other clinical trial groups, NMSG faces a number of challenges in coming years. The financial situation for independent investigator‐initiated trials is far from satisfactory, especially with regard to the resource‐consuming implementation of more stringent good clinical practice rules and ethical committee demands. NMSG has also encountered increasing difficulties in recruiting patients to recent trials, partly because of problems related to participating physicians (lack of support, laborious paper work, insufficient credit for participation). Solutions to these problems have to be found if industry‐independent clinical trial groups are to survive.
Rapid Formation of Sulfuric Acid Particles at Near-Atmospheric Conditions
Authors: Berndt Torsten , Böge Olaf , Stratmann Frank , Heintzenberg Jost , Kulmala Markku
Published: February 2005

We investigated the formation of new particles in a laboratory study, starting from H
2
SO
4
produced in situ through the reaction of OH radicals with SO
2
. Newly formed particles were observed for H
2
SO
4
concentrations above 7 × 10
6
per cubic centimeter. At 293 kelvin, a rough estimate yielded a nucleation rate of 0.3 to 0.4 particles per cubic centimeter per second for ∼10
7
particles per cubic centimeter of H
2
SO
4
(particle size ≥ 3 nanometers). These findings are in agreement with observations from the atmosphere. The results demonstrate that under laboratory conditions similar to the atmosphere, particle formation occurs at atmospheric H
2
SO
4
concentration levels.
Nod2-Dependent Regulation of Innate and Adaptive Immunity in the Intestinal Tract
Authors: Kobayashi Koichi S. , Chamaillard Mathias , Ogura Yasunori , Henegariu Octavian , Inohara Naohiro , Nuñez Gabriel , Flavell Richard A.
Published: February 2005

The gene encoding the Nod2 protein is frequently mutated in Crohn's disease (CD) patients, although the physiological function of Nod2 in the intestine remains elusive. Here we show that protective immunity mediated by Nod2 recognition of bacterial muramyl dipeptide is abolished in Nod2-deficient mice. These animals are susceptible to bacterial infection via the oral route but not through intravenous or peritoneal delivery. Nod2 is required for the expression of a subgroup of intestinal anti-microbial peptides, known as cryptdins. The Nod2 protein is thus a critical regulator of bacterial immunity within the intestine, providing a possible mechanism for Nod2 mutations in CD.
Simple Foraminifera Flourish at the Ocean's Deepest Point
Authors: Todo Yuko , Kitazato Hiroshi , Hashimoto Jun , Gooday Andrew J.
Published: February 2005

Small organisms (meiofauna) inhabiting sediments in the deepest (>10,000 m) ocean trenches are almost unknown. Through a quantitative study of live foraminifera from an extreme trench environment (10,900 m water depth), we demonstrate that these protists are an abundant meiofaunal component. Foraminifera are organisms of central importance in geology and biology. There is currently great interest in primitive noncalcareous taxa with simple, single-chambered shells. The Challenger Deep assemblages are dominated by these phylogenetically important forms. Molecular evidence suggests that similar taxa are modern representatives of the basal foraminiferal evolutionary radiation that probably occurred in the Precambrian (Neoproterozoic).
Natural Selection and Developmental Constraints in the Evolution of Allometries
Authors: Frankino W. Anthony , Zwaan Bas J. , Stern David L. , Brakefield Paul M.
Published: February 2005

In animals, scaling relationships between appendages and body size exhibit high interspecific variation but low intraspecific variation. This pattern could result from natural selection for specific allometries or from developmental constraints on patterns of differential growth. We performed artificial selection on the allometry between forewing area and body size in a butterfly to test for developmental constraints, and then used the resultant increased range of phenotypic variation to quantify natural selection on the scaling relationship. Our results show that the short-term evolution of allometries is not limited by developmental constraints. Instead, scaling relationships are shaped by strong natural selection.
Voyage to the Bottom of the Tree
Authors: Delwiche Charles Francis
Published: February 2005

The Ancestor's Tale
A Pilgrimage to the Dawn of Life.
By Richard Dawkins
. Weidenfeld and Nicolson, London, 2004. 528 pp. £25. ISBN 0-297-82503-8.

The Ancestor's Tale
A Pilgrimage to the Dawn of Evolution.
By Richard Dawkins
. Houghton Mifflin, Boston, 2004. 687 pp. $28. ISBN 0-618-00583-8.

In this reverse history of life, the author starts with human farmers and works down the phylogenetic tree to Eubacteria, presenting the various taxa in short tales.
Mutualistic Fungi Control Crop Diversity in Fungus-Growing Ants
Authors: Poulsen Michael , Boomsma Jacobus J.
Published: February 2005

Leaf-cutting ants rear clonal fungi for food and transmit the fungi from mother to daughter colonies so that symbiont mixing and conflict, which result from competition between genetically different clones, are avoided. Here we show that despite millions of years of predominantly vertical transmission, the domesticated fungi actively reject mycelial fragments from neighboring colonies, and that the strength of these reactions are in proportion to the overall genetic difference between these symbionts. Fungal incompatibility compounds remain intact during ant digestion, so that fecal droplets, which are used for manuring newly grown fungus, elicit similar hostile reactions when applied to symbionts from other colonies. Symbiont control over new mycelial growth by manurial imprinting prevents the rearing of multiple crops in fungus gardens belonging to the same colony.
End States in One-Dimensional Atom Chains
Authors: Crain J. N. , Pierce D. T.
Published: February 2005

End states—the zero-dimensional analogs of the two-dimensional states that occur at a crystal surface—were observed at the ends of one-dimensional atom chains that were self-assembled by depositing gold on the vicinal Si(553) surface. Scanning tunneling spectroscopy measurements of the differential conductance along the chains revealed quantized states in isolated segments with differentiated states forming over end atoms. A comparison to a tight-binding model demonstrated how the formation of electronic end states transforms the density of states and the energy levels within the chains.
Beringian Paleoecology Inferred from Permafrost-Preserved Fungal DNA
Authors: Lydolph Magnus C. , Jacobsen Jonas , Arctander Peter , Gilbert M. Thomas P. , Gilichinsky David A. , Hansen Anders J. , Willerslev Eske , Lange Lene
Published: February 2005

The diversity of fungi in permanently frozen soil from northeastern Siberia was studied by culture-independent PCR amplification of diverse environmental 18S rRNA genes. Elaborate protocols to avoid contamination during drilling, sampling, and amplification were used. A broad diversity of eukaryotic DNA sequences that were 510 bp long, including sequences of various fungi, plants, and invertebrates, could be obtained reproducibly from samples that were up to 300,000 to 400,000 years old. The sequences revealed that ancient fungal communities included a diversity of cold-adapted yeasts, dark-pigmented fungi, plant-parasitic fungi, and lichen mycobionts. DNA traces of tree-associated macrofungi in a modern tundra sample indicated that there was a shift in fungal diversity following the last ice age and supported recent results showing that there was a severe change in the plant composition in northeastern Siberia during this period. Interestingly, DNA sequences with high homology to sequences of coprophilic and keratinophilic fungi indicated that feces, hair, skin, and nails could have been sources of ancient megafauna DNA recently reported to be present in small amounts of Siberian permafrost sediments.
Utilization of Fluorescent Microspheres and a Green Fluorescent Protein-Marked Strain for Assessment of Microbiological Contamination of Permafrost and Ground Ice Core Samples from the Canadian High Arctic
Authors: Juck D. F. , Whissell G. , Steven B. , Pollard W. , McKay C. P. , Greer C. W. , Whyte L. G.
Published: February 2005

Fluorescent microspheres were applied in a novel fashion during subsurface drilling of permafrost and ground ice in the Canadian High Arctic to monitor the exogenous microbiological contamination of core samples obtained during the drilling process. Prior to each drill run, a concentrated fluorescent microsphere (0.5-μm diameter) solution was applied to the interior surfaces of the drill bit, core catcher, and core tube and allowed to dry. Macroscopic examination in the field demonstrated reliable transfer of the microspheres to core samples, while detailed microscopic examination revealed penetration levels of less than 1 cm from the core exterior. To monitor for microbial contamination during downstream processing of the permafrost and ground ice cores, a
Pseudomonas
strain expressing the green fluorescent protein (GFP) was painted on the core exterior prior to processing. Contamination of the processed core interiors with the GFP-expressing strain was not detected by culturing the samples or by PCR to detect the
gfp
marker gene. These methodologies were quick, were easy to apply, and should help to monitor the exogenous microbiological contamination of pristine permafrost and ground ice samples for downstream culture-dependent and culture-independent microbial analyses.
Nine-Year Study of the Occurrence of Culturable Viruses in Source Water for Two Drinking Water Treatment Plants and the Influent and Effluent of a Wastewater Treatment Plant in Milwaukee, Wisconsin (August 1994 through July 2003)
Authors: Sedmak Gerald , Bina David , MacDonald Jeffrey , Couillard Lon
Published: February 2005

Reoviruses, enteroviruses, and adenoviruses were quantified by culture for various ambient waters in the Milwaukee area. From August 1994 through July 2003, the influent and effluent of a local wastewater treatment plant (WWTP) were tested monthly by a modified U.S. Environmental Protection Agency Information Collection Rule (ICR) organic flocculation cell culture procedure for the detection of culturable viruses. Modification of the ICR procedure included using Caco-2, RD, and HEp-2 cells in addition to BGM cells. Lake Michigan source water for two local drinking water treatment plants (DWTPs) was also tested monthly for culturable viruses by passing 200 liters of source water through a filter and culturing a concentrate representing 100 liters of source water. Reoviruses, enteroviruses, and adenoviruses were detected frequently (105 of 107 samples) and, at times, in high concentration in WWTP influent but were detected less frequently (32 of 107 samples) in plant effluent and at much lower concentrations. Eighteen of 204 samples (8.8%) of source waters for the two DWTPs were positive for virus and exclusively positive for reoviruses at relatively low titers. Both enteroviruses and reoviruses were detected in WWTP influent, most frequently during the second half of the year.
Isolation and Characterization of Temperate Bacteriophages of
Clostridium difficile
Authors: Goh Shan , Riley Thomas V. , Chang Barbara J.
Published: February 2005

The lack of information on bacteriophages of
Clostridium difficile
prompted this study. Three of 56 clinical
C. difficile
isolates yielded double-stranded DNA phages φC2, φC5, φC6, and φC8 upon induction. Superinfection and DNA analyses revealed relatedness between the phages, while partial sequencing of φC2 showed nucleotide homology to the sequenced
C. difficile
strain CD630.
Surface Plasmon Resonance Imaging-Based Protein Array Chip System for Monitoring a Hexahistidine-Tagged Protein during Expression and Purification
Authors: Ro Hyeon-Su , Jung Sun Ok , Kho Byung Hoon , Hong Hyung Pyo , Lee Jae Sung , Shin Yong-Beom , Kim Min Gon , Chung Bong Hyun
Published: February 2005

A surface plasmon resonance imaging-based Ni
2+
-iminodiacetic acid-coated gold chip system was developed to enable specific detection of a hexahistidine-tagged recombinant protein in crude extracts or in column chromatography fractions. This system is especially advantageous for high-throughput analysis of multiple proteins.
Genetic Diversity ofCylindrospermopsisStrains (Cyanobacteria) Isolated from Four Continents
Authors: Gugger Muriel , Molica Renato , Le Berre Brigitte , Dufour Philippe , Bernard Cécile , Humbert Jean-François
Published: February 2005

The genetic diversity ofCylindrospermopsisstrains (cyanobacteria) was examined using mainly the 16S-23S internally transcribed spacer (ITS1) sequences. Strains were grouped in three clusters: (i) America, (ii) Europe, and (iii) Africa and Australia. These results suggested a recent spread ofCylindrospermopsisacross the American and European continents from restricted warm refuge areas instead of exchanges between continents. On the other hand, they also suggested a recent colonization of Australia by African strains.
High-Level Acetaldehyde Production in
Lactococcus lactis
by Metabolic Engineering
Authors: Bongers Roger S. , Hoefnagel Marcel H. N. , Kleerebezem Michiel
Published: February 2005

Efficient conversion of glucose to acetaldehyde is achieved by nisin-controlled overexpression of
Zymomonas mobilis
pyruvate decarboxylase (
pdc
) and
Lactococcus lactis
NADH oxidase (
nox
) in
L. lactis
. In resting cells, almost 50% of the glucose consumed could be redirected towards acetaldehyde by combined overexpression of
pdc
and
nox
under anaerobic conditions.
Efficacy of Removal of Sucrose-Supplemented Interproximal Plaque by Electric Toothbrushes in an In Vitro Model
Authors: Hope C. K. , Petrie A. , Wilson M.
Published: February 2005

Electric toothbrushes were evaluated using a model of plaque removal by fluid shear forces. Sucrose supplementation during plaque development did not affect the removal of bacteria from biofilm exposed to low-energy shear but did increase their resistance to high-energy shear. The toothbrush supplying high-energy shear forces removed significantly more viable bacteria.
Rapid and Simple Quantification of Bacterial Cells by Using a Microfluidic Device
Authors: Sakamoto Chieko , Yamaguchi Nobuyasu , Nasu Masao
Published: February 2005

This study investigated a microfluidic chip-based system (on-chip flow cytometry) for quantification of bacteria both in culture and in environmental samples. Bacterial numbers determined by this technique were similar to those obtained by direct microscopic count. The time required for this on-chip flow cytometry was only 30 min per 6 samples.
Variation in 16S-23S rRNA Intergenic Spacer Regions in
Photobacterium damselae
: a Mosaic-Like Structure
Authors: Osorio Carlos R. , Collins Matthew D. , Romalde Jesús L. , Toranzo Alicia E.
Published: February 2005

Phenotypically,
Photobacterium damselae
subsp.
piscicida
and
P. damselae
subsp.
damselae
are easily distinguished. However, their 16S rRNA gene sequences are identical, and attempts to discriminate these two subspecies by molecular tools are hampered by their high level of DNA-DNA similarity. The 16S-23S rRNA internal transcribed spacers (ITS) were sequenced in two strains of
Photobacterium damselae
subsp.
piscicida
and two strains of
P. damselae
subsp.
damselae
to determine the level of molecular diversity in this DNA region. A total of 17 different ITS variants, ranging from 803 to 296 bp were found, some of which were subspecies or strain specific. The largest ITS contained four tRNA genes (tDNAs) coding for tRNA
Glu(UUC)
, tRNA
Lys(UUU)
, tRNA
Val(UAC)
, and tRNA
Ala(GGC)
. Five amplicons contained tRNA
Glu(UUC)
combined with two additional tRNA genes, including tRNA
Lys(UUU)
, tRNA
Val(UAC)
, or tRNA
Ala(UGC)
. Five amplicons contained tRNA
Ile(GAU)
and tRNA
Ala(UGC)
. Two amplicons contained tRNA
Glu(UUC)
and tRNA
Ala(UGC)
. Two different isoacceptor tRNA
Ala
genes (GGC and UGC anticodons) were found. The five smallest amplicons contained no tRNA genes. The tRNA-gene combinations tRNA
Glu(UUC)
-tRNA
Val(UAC)
-tRNA
Ala(UGC)
and tRNA
Glu(UUC)
-tRNA
Ala(UGC)
have not been previously reported in bacterial ITS regions. The number of copies of the ribosomal operon (
rrn
) in the
P. damselae
chromosome ranged from at least 9 to 12. For ITS variants coexisting in two strains of different subspecies or in strains of the same subspecies, nucleotide substitution percentages ranged from 0 to 2%. The main source of variation between ITS variants was due to different combinations of DNA sequence blocks, constituting a mosaic-like structure.
Isotope Labeling and Microautoradiography of Active Heterotrophic Bacteria on the Basis of Assimilation of
14
CO
2
Authors: Hesselsoe Martin , Nielsen Jeppe Lund , Roslev Peter , Nielsen Per Halkjær
Published: February 2005

Most heterotrophic bacteria assimilate CO
2
in various carboxylation reactions during biosynthesis. In this study, assimilation of
14
CO
2
by heterotrophic bacteria was used for isotope labeling of active microorganisms in pure cultures and environmental samples. Labeled cells were visualized by microautoradiography (MAR) combined with fluorescence in situ hybridization (FISH) to obtain simultaneous information about activity and identity. Cultures of
Escherichia coli
and
Pseudomonas putida
assimilated sufficient
14
CO
2
during growth on various organic substrates to obtain positive MAR signals. The MAR signals were comparable with the traditional MAR approach based on uptake of
14
C-labeled organic substrates. Experiments with
E. coli
showed that
14
CO
2
was assimilated during both fermentation and aerobic and anaerobic respiration. The new MAR approach, HetCO
2
-MAR, was evaluated by targeting metabolic active filamentous bacteria, including “
Candidatus
Microthrix parvicella” in activated sludge. “
Ca.
Microthrix parvicella” was able to take up oleic acid under anaerobic conditions, as shown by the traditional MAR approach with [
14
C]oleic acid. However, the new HetCO
2
-MAR approach indicated that “
Ca.
Microthrix parvicella,” did not significantly grow on oleic acid under anaerobic conditions with or without addition of NO
2
−
, whereas the addition of O
2
or NO
3
−
initiated growth, as indicated by detectable
14
CO
2
assimilation. This is a metabolic feature that has not been described previously for filamentous bacteria. Such information could not have been derived by using the traditional MAR procedure, whereas the new HetCO
2
-MAR approach differentiates better between substrate uptake and substrate metabolism that result in growth. The HetCO
2
-MAR results were supported by stable isotope analysis of
13
C-labeled phospholipid fatty acids from activated sludge incubated under aerobic and anaerobic conditions in the presence of
13
CO
2
. In conclusion, the novel HetCO
2
-MAR approach expands the possibility for studies of the ecophysiology of uncultivated microorganisms.
Metabolically Independent and Accurately AdjustableAspergillussp. Expression System
Authors: Pachlinger Robert , Mitterbauer Rudolf , Adam Gerhard , Strauss Joseph
Published: February 2005

Filamentous fungi are well-established expression hosts often used to produce extracellular proteins of use in the food and pharmaceutical industries. The expression systems presently used inAspergillusspecies rely on either strong constitutive promoters, e.g., that for glyceraldehyde-3-phosphate dehydrogenase, or inducible systems derived from metabolic pathways, e.g.,glaA(glucoamylase) oralc(alcohol dehydrogenase). We describe forAspergillus nidulansandAspergillus nigera novel expression system that utilizes the transcriptional activation of the human estrogen receptor by estrogenic substances. The system functions independently from metabolic signals and therefore can be used with low-cost, complex media. A combination of positive and negative regulatory elements in the promoter drives the expression of a reporter gene, yielding a linear dose response to the inducer. The off status is completely tight, yet the system responds within minutes to induction and reaches a level of expression of up to 15% of total cell protein after 8 h. BothAspergillusspecies are very sensitive to estrogenic substances, and low-cost inducers function in the picomolar concentration range, at which estrogenic substances also can be found in the environment. Given this high sensitivity to estrogens,Aspergilluscells carrying estrogen-responsive units could be used to detect xenoestrogens in food or in the environment.
Regulation of Resistance to Copper in
Xanthomonas axonopodis
pv. vesicatoria
Authors: Voloudakis Andreas E. , Reignier Therese M. , Cooksey Donald A.
Published: February 2005

Copper-resistant strains of
Xanthomonas axonopodis
pv. vesicatoria were previously shown to carry plasmid-borne copper resistance genes related to the
cop
and
pco
operons of
Pseudomonas syringae
and
Escherichia coli
, respectively. However, instead of the two-component (
copRS
and
pcoRS
) systems determining copper-inducible expression of the operons in
P. syringae
and
E. coli
, a novel open reading frame,
copL
, was found to be required for copper-inducible expression of the downstream multicopper oxidase
copA
in
X. axonopodis. copL
encodes a predicted protein product of 122 amino acids that is rich in histidine and cysteine residues, suggesting a possible direct interaction with copper. Deletions or frameshift mutations within
copL
, as well as an amino acid substitution generated at the putative start codon of
copL
, caused a loss of copper-inducible transcriptional activation of
copA
. A nonpolar insertion of a kanamycin resistance gene in
copL
resulted in copper sensitivity in the wild-type strain. However, repeated attempts to complement
copL
mutations in
trans
failed. Analysis of the genomic sequence databases shows that there are
copL
homologs upstream of
copAB
genes in
X. axonopodis
pv. citri,
X. campestris
pv. campestris, and
Xylella fastidiosa
. The cloned promoter area upstream of
cop
A in
X. axonopodis
pv. vesicatoria did not function in
Pseudomonas syringae
or in
E. coli
, nor did the
P. syringae cop
promoter function in
Xanthomonas
. However, a transcriptional fusion of the
Xanthomonas cop
promoter with the
Pseudomonas copABCDRS
was able to confer resistance to copper in
Xanthomonas
, showing divergence in the mechanisms of regulation of the resistance to copper in phytopathogenic bacteria.
Thio Wax Ester Biosynthesis Utilizing the Unspecific Bifunctional Wax Ester Synthase/Acyl Coenzyme A:Diacylglycerol Acyltransferase of
Acinetobacter
sp. Strain ADP1
Authors: Uthoff Stefan , Stöveken Tim , Weber Nikolaus , Vosmann Klaus , Klein Erika , Kalscheuer Rainer , Steinbüchel Alexander
Published: February 2005

The bifunctional wax ester synthase/acyl coenzyme A (acyl-CoA):diacylglycerol acyltransferase (WS/DGAT) from
Acinetobacter
sp. strain ADP1 (formerly
Acinetobacter calcoaceticus
ADP1) mediating the biosyntheses of wax esters and triacylglycerols was used for the in vivo and in vitro biosynthesis of thio wax esters and dithio wax esters. For in vitro biosynthesis, 5′His
6
WS/DGAT comprising an N-terminal His
6
tag was purified from the soluble protein fraction of
Escherichia coli
Rosetta(DE3)pLysS (pET23a::

5′His
6

atf
). By employing SP-Sepharose high-pressure and Ni-nitrilotriacetic acid fast-protein liquid chromatographies, a 19-fold enrichment with a final specific activity of 165.2 nmol mg of protein
−1
min
−1
was achieved by using 1-hexadecanol and palmitoyl-CoA as substrates. Incubation of purified 5′His
6
WS/DGAT with 1-hexadecanethiol and palmitoyl-CoA as substrates resulted in the formation of palmitic acid hexadecyl thio ester (10.4% relative specific activity of a 1-hexadecanol control). Utilization of 1,8-octanedithiol and palmitoyl-CoA as substrates led to the formation of 1-
S
-monopalmitoyloctanedithiol and minor amounts of 1,8-
S
-dipalmitoyloctanedithiol (59.3% relative specific activity of a 1-hexadecanol control). The latter dithio wax ester was efficiently produced when 1-
S
-monopalmitoyloctanedithiol and palmitoyl-CoA were used as substrates (13.4% specific activity relative to that of a 1-hexadecanol control). For the in vivo biosynthesis of thio wax esters, the knockout mutant
Acinetobacter
sp. strain ADP1
acr1
ΩKm, which is unable to produce fatty alcohols, was used. Cultivation of
Acinetobacter
sp. strain ADP1
acr1
ΩKm in the presence of gluconate, 1-hexadecanethiol, and oleic acid in nitrogen-limited mineral salts medium resulted in the accumulation of unusual thio wax esters that accounted for around 1.19% (wt/wt) of the cellular dry weight and consisted mainly of oleic acid hexadecyl thioester as revealed by gas chromatography-mass spectrometry.
Effects of Growth Medium, Inoculum Size, and Incubation Time on Culturability and Isolation of Soil Bacteria
Authors: Davis Kathryn E. R. , Joseph Shayne J. , Janssen Peter H.
Published: February 2005

Soils are inhabited by many bacteria from phylogenetic groups that are poorly studied because representatives are rarely isolated in cultivation studies. Part of the reason for the failure to cultivate these bacteria is the low frequency with which bacterial cells in soil form visible colonies when inoculated onto standard microbiological media, resulting in low viable counts. We investigated the effects of three factors on viable counts, assessed as numbers of CFU on solid media, and on the phylogenetic groups to which the isolated colony-forming bacteria belong. These factors were inoculum size, growth medium, and incubation time. Decreasing the inoculum size resulted in significant increases in the viable count but did not appear to affect colony formation by members of rarely isolated groups. Some media that are traditionally used for soil microbiological studies returned low viable counts and did not result in the isolation of members of rarely isolated groups. Newly developed media, in contrast, resulted in high viable counts and in the isolation of many members of rarely isolated groups, regardless of the inoculum size. Increased incubation times of up to 3 months allowed the development of visible colonies of members of rarely isolated groups in conjunction with the use of appropriate media. Once isolated, pure cultures of members of rarely isolated groups took longer to form visible colonies than did members of commonly isolated groups. Using these new media and extended incubation times, we were able to isolate many members of the phyla
Acidobacteria
(subdivisions 1, 2, 3, and 4),
Gemmatimonadetes
,
Chloroflexi
, and
Planctomycetes
(including representatives of the previously uncultured WPS-1 lineage) as well as members of the subclasses
Rubrobacteridae
and
Acidimicrobidae
of the phylum
Actinobacteria
.
Physiological Conditions Conducive to High Cyanophycin Content in Biomass ofAcinetobacter calcoaceticusStrain ADP1
Authors: Elbahloul Yasser , Krehenbrink Martin , Reichelt Rudolf , Steinbüchel Alexander
Published: February 2005

The effects of the inorganic medium components, the initial pH, the incubation temperature, the oxygen supply, the carbon-to-nitrogen ratio, and chloramphenicol on the synthesis of cyanophycin (CGP) byAcinetobacter calcoaceticusstrain ADP1 were studied in a mineral salts medium containing sodium glutamate and ammonium sulfate as carbon and nitrogen sources, respectively. Variation of all these factors resulted in maximum CGP contents of only about 3.5% (wt/wt) of the cell dry matter (CDM), and phosphate depletion triggered CGP accumulation most substantially. However, addition of arginine to the medium as the sole carbon source for growth promoted CGP accumulation most strikingly. This effect was systematically studied, and an optimized phosphate-limited medium containing 75 mM arginine and 10 mM ammonium sulfate yielded a CGP content of 41.4% (wt/wt) of the CDM at 30°C. The CGP content of the cells was further increased to 46.0% (wt/wt) of the CDM by adding 2.5 μg of chloramphenicol per ml of medium in the accumulation phase. These contents are by far the highest CGP contents of bacterial cells ever reported. CGP was easily isolated from the cells by using an acid extraction method, and this CGP contained about equimolar amounts of aspartic acid and arginine and no detectable lysine; the molecular masses ranged from 21 to 29 kDa, and the average molecular mass was about 25 kDa. Transmission electron micrographs of thin sections of cells revealed large CGP granules that frequently had an irregular shape with protuberances at the surface and often severely deformed the cells. AcphI::ΩKm mutant of strain ADP1 with a disrupted putative cyanophycinase gene accumulated significantly less CGP than the wild type accumulated, although the cells expressed cyanophycin synthetase at about the same high level. It is possible that the intact CphI protein is involved in the release of CGP primer molecules from initially synthesized CGP. The resulting lower concentration of primer molecules could explain the observed low rate of accumulation at similar specific activities.
Adaptation of the Yeast
URA3
Selection System to Gram-Negative Bacteria and Generation of a Δ
betCDE Pseudomonas putida
Strain
Authors: Galvão Teca Calcagno , de Lorenzo Víctor
Published: February 2005

A general procedure for efficient generation of gene knockouts in gram-negative bacteria by the adaptation of the
Saccharomyces cerevisiae URA3
selection system is described. A
Pseudomonas putida
strain lacking the
URA3
homolog
pyrF
(encoding orotidine-5′-phosphate decarboxylase) was constructed, allowing the use of a plasmid-borne copy of the gene as the target of selection. The delivery vector pTEC contains the
pyrF
gene and promoter, a conditional origin of replication (
oriR6K
), an origin of transfer (
mobRK2
), and an antibiotic selection marker flanked by multiple sites for cloning appropriate DNA segments. The versatility of
pyrF
as a selection system, allowing both positive and negative selection of the marker, and the robustness of the selection, where
pyrF
is associated with uracil prototrophy and fluoroorotic acid sensitivity, make this setup a powerful tool for efficient homologous gene replacement in gram-negative bacteria. The system has been instrumental for complete deletion of the
P. putida
choline-
O
-sulfate utilization operon
betCDE
, a mutant which could not be produced by any of the other genetic strategies available.
Heat related deaths to young children in parked cars: an analysis of 171 fatalities in the United States, 1995–2002
Authors: Guard A , Gallagher S S
Published: February 2005

Objective:To describe the circumstances surrounding heat related deaths to young children in passenger compartments of motor vehicles.

Methods:Cases of heat related deaths to children aged ⩽5 years confined in parked vehicles were retrospectively identified using online news accounts from 1 January 1995 to 31 December 2002. A standardized data form was developed to collect information related to the victim, the vehicle and its design features, external temperature, length of time confined, and the responsible adults. Cases were categorized by whether the child gained access to the vehicle or was left by adults.

Results:A total of 171 fatalities that met the case criteria were identified. Twenty seven percent (46) were children who gained access to unlocked vehicles and 73% (125) were children who were left by adults. More than a quarter of the adults were aware they were leaving children in the vehicles, while half were unaware or forgot. Forty three percent (54) of deaths to children who were left were associated with childcare: 32 children were left by family members who intended to take them to childcare but forgot and went to work instead; 22 children were left by child care providers or drivers.

Conclusions:Many of the deaths reported in this study may have been prevented by keeping cars locked, educating parents, implementing informed child care transportation policies, passing relevant laws, and working with auto and child safety seat manufacturers to build in warnings and other design features. News sources can be useful for obtaining detailed information not otherwise identifiable through standard sources of fatality data.