bluuebunny/crossref_metadata_2025 huggingface dataset:
Arrhythmogenic Right Ventricular Cardiomyopathy with Left Ventricular Involvement and Aortic Dissection
Authors: MERTEN MICHAEL , WILLEMS STEPHAN , HEINEMANN AXEL , MEINERTZ THOMAS
Published: March 2004

Ten years ago, a 59‐year‐old patient presented with ventricular fibrillations. The resting ECG showed findings typical for ARVC. Echocardiography and ventriculography confirmed the diagnosis of ARVC showing a dilated right ventricle with aneurysms. MRI showed additional fatty replacement of the LV. Furthermore, the diagnosis of a chronic aortic dissection was established. Two years after ICD implantation, the patient died of progressive right heart failure. On autopsy, most of the RV and parts of the LV were replaced by fatty tissue, and the media of the aorta showed degenerative changes. A pathogenetic link between the two diseases remains speculative at this time. (PACE 2004; 27:408–411)
Percutaneous Extraction of a Severed and Frayed Permanent Pacing Lead
Authors: SOCHMAN JAN , BYTESNIK JAN , SKALSKY IVO , PEREGRIN JAN H.
Published: March 2004

From the point of view of established practice, redundant permanent pacing leads have been abandoned or extracted using various catheter‐based procedures. An unusual complication was caused by failed surgical lead extraction resulting in electrode severing and wire unbraiding with subsequent induction of electric interaction between the metallic, noninsulated part of the lead fragment and the defibrillating electrode. This interaction was sensed and detected by the ICD system. A modified catheter‐based extraction procedure is described. (PACE 2004; 27:412–414)
Inappropriate ICD Therapy Due to Proarrhythmic ICD Shocks and Hyperpolarization
Authors: HEALY EDWARD , GOYAL SANJEEV , BROWNING CLIFFORD , ROBOTIS DIONYSSIS , RAMASWAMY KARTHICK , ROFINO‐NADOWORNY KAREN , ROSENTHAL LAWRENCE
Published: March 2004

A 50‐year‐old man with an ischemic cardiomyopathy underwent ICD implantation for inducible ventricular fibrillation (VF). Sixteen months later he experienced inappropriate ICD therapy due to atrial fibrillation with a rapid ventricular response. The initial shock resulted in the initiation of VF (proarrhythmia) and the patient received an additional shock converting his rhythm to an idioventricular rhythm with a cycle length of 490 ms (122 beats/min). Due to lead hyperpolarization, the device oversensed ventricular events and the patient subsequently received additional shocks. (PACE 2004; 27:415–416)
Aortic Perforation by Active‐Fixation Atrial Pacing Lead
Authors: KASHANI AMIR , MEHDIRAD ALI , FREDMAN CAREY , BAROLD S. SERGE
Published: March 2004

This report describes perforation of the aorta related to the implantation of an active‐fixation atrial pacing lead, a previously undocumented complication of pacemaker implantation. The complication was related to excessive tissue penetration by the screw at the tip of the lead or perforation of the lead body by the positioning stylet during manipulation whereupon the stylet traversed the atrial wall and aortic wall. Perforation of the aorta should be part of the differential diagnosis of cardiac tamponade after pacemaker implantation. (PACE 2004; 27:417–418)
Reproducibility and accuracy of automated probe measurements of gingiva and bone levels on stone casts following guided bone regeneration treatment
Authors: Van der Zee Erwin , Vogels Marie‐Faustine , Oosterveld Paul , Van Waas Marinus
Published: March 2004

Objectives: For evidence‐based evaluation of guided bone regeneration (GBR), accurate registration of changes in gingiva and bone levels is needed. A new method is introduced and evaluated.

Methods: In a clinical trial with 30 patients, alginate impressions of the surgical area including the interproximal gingiva and alveolar bone at the adjacent teeth were made in duplicate prior to and during GBR surgery, fixture installation and abutment connection. Poured in hard stone, the casts were used for repeated measurements of the level of the free gingival margin and the alveolar bone with an automated probe (Florida disc‐probe®), using the incisal edge as a fixed reference point. The reproducibility and accuracy of these measurements were evaluated by means of the Intraclass Correlation Coefficients and Generalizability Theory. The effect of treatment was evaluated by multivariate analysis of variance.

Results: Generalizability Theory indicated a high accuracy of the gingiva‐ and bone‐level measurements: the Intraclass Correlation Coefficients for gingiva and bone levels were 0.99 and 0.98, respectively. The intra‐cast reproducibility was 0.09±0.07 mm (mean±SD) and the inter‐cast reproducibility was 0.10±0.09 and 0.20±0.07 mm for gingiva and bone levels, respectively. Clinical applicability is demonstrated by the fact that manova revealed on average a small but highly significant (p=0.001) effect of the staged surgical intervention on the gingiva and bone levels at the adjacent teeth.

Conclusion: It is concluded that the presented method makes it possible to evaluate reproducibly and accurately changes in gingiva and bone levels for GBR studies.
Surface wear on cervical restorations and adjacent enamel and root cementum caused by simulated long‐term maintenance therapy
Authors: Rühling A. , Wulf J. , Schwahn C. , Kocher T.
Published: March 2004

Background: In an in vitro study, the surface wear on cervical restorations and adjacent enamel and root cementum caused by different tooth‐cleaning methods in simulated long‐term therapy was investigated.

Methods: Cervical restorations of amalgam (Oralloy®), modified composite resin (Dyract®), glass‐ionomer cement (ChemFill Superior®), and composite (Tetric®) were instrumented by POL (polishing), CUR+POL (curette and polishing), US+POL (ultrasonic device with polishing) and the polishing agents Cleanic® and Proxyt® in a computer‐controlled test bench. Treatment time corresponding to a real‐time period of 5 or 10 years. Substance loss from instrumented surfaces was measured with a digital gauge. A three‐way anova was used in the statistical evaluation.

Results: The results showed that POL led to slight substance loss, which was greater using Cleanic® (27 μm) than Proxyt® (5 μm). CUR+POL produced a significantly greater substance loss than did US+POL, with 186 μm versus 35 μm on glass‐ionomer cement, respectively, and 123 μm versus 18 μm, respectively, on root cementum, followed by composite (111 μm versus 27 μm, respectively), polyacid modified composite resin/compomer (89 μm versus 36 μm), amalgam (75 μm versus 19 μm), and enamel (32 μm versus 23 μm).

Conclusions: As opposed to the use of US+POL or POL, substance loss on cervical restorations and especially root cementum must be expected to result from tooth‐cleaning during long‐term maintenance treatment using CUR+POL.
Effects of ageing and exercise training on endothelium‐dependent vasodilatation and structure of rat skeletal muscle arterioles
Authors: Spier Scott A. , Delp Michael D. , Meininger Cynthia J. , Donato Anthony J. , Ramsey Michael W. , Muller‐Delp Judy M.
Published: March 2004

Ageing reduces endothelium‐dependent vasodilatation in humans and animals, and in humans, exercise training reverses the ageing‐associated reduction in endothelium‐dependent vasodilatation. The purpose of this study was to determine the mechanism(s) by which 10–12 weeks of treadmill exercise enhances endothelium‐dependent vasodilatation in muscles of differing fibre composition from young and old rats. Three‐ and 22‐month‐old male Fischer 344 rats were assigned to young sedentary, young exercise‐trained, old sedentary, or old exercise‐trained groups. Arterioles were isolated from the soleus and gastrocnemius muscles; luminal diameter changes were determined in response to the endothelium‐dependent vasodilator acetylcholine (ACh, 10−9–10−4 mol l−1) alone and in the presence of the nitric oxide synthase (NOS) inhibitor l‐NAME (10−5 mol l−1) or the combination of l‐NAME and the cyclooxygenase inhibitor indomethacin (10−5 mol l−1). Training ameliorated the ageing‐induced reduction in endothelium‐dependent vasodilatation in soleus muscle arterioles. Treatment with l‐NAME alone and in combination with indomethacin abolished differences in ACh vasodilatation occurring with ageing and training. Expression of endothelial NOS (eNOS) mRNA in soleus arterioles was unaltered by ageing, whereas eNOS protein was increased with age; training elevated both eNOS mRNA and protein. In gastrocnemius muscle arterioles, ageing did not alter maximal vasodilatation, but ageing and training increased maximal arteriolar diameter. These results demonstrate that ageing‐induced reductions and training‐induced enhancement of endothelial vasodilatation both occur through the nitric oxide signalling mechanism in highly oxidative skeletal muscle, but ageing and training do not appear to act on the same portion of the signalling cascade.
3-D Anatomically Based Dynamic Modeling of the Human Knee to Include Tibio-Femoral and Patello-Femoral Joints
Authors: Caruntu Dumitru I. , Hefzy Mohamed Samir
Published: March 2004

An anatomical dynamic model consisting of three body segments, femur, tibia and patella, has been developed in order to determine the three-dimensional dynamic response of the human knee. Deformable contact was allowed at all articular surfaces, which were mathematically represented using Coons’ bicubic surface patches. Nonlinear elastic springs were used to model all ligamentous structures. Two joint coordinate systems were employed to describe the six-degrees-of-freedom tibio-femoral (TF) and patello-femoral (PF) joint motions using twelve kinematic parameters. Two versions of the model were developed to account for wrapping and nonwrapping of the quadriceps tendon around the femur. Model equations consist of twelve nonlinear second-order ordinary differential equations coupled with nonlinear algebraic constraint equations resulting in a Differential-Algebraic Equations (DAE) system that was solved using the D_ifferential/A_lgebraic S_ystem S_ol_ver (DASSL) developed at Lawrence Livermore National Laboratory. Model calculations were performed to simulate the knee extension exercise by applying non-linear forcing functions to the quadriceps tendon. Under the conditions tested, both “screw home mechanism” and patellar flexion lagging were predicted. Throughout the entire range of motion, the medial component of the TF contact force was found to be larger than the lateral one while the lateral component of the PF contact force was found to be larger than the medial one. The anterior and posterior fibers of both anterior and posterior cruciate ligaments, ACL and PCL, respectively, had opposite force patterns: the posterior fibers were most taut at full extension while the anterior fibers were most taut near 90° of flexion. The ACL was found to carry a larger total force than the PCL at full extension, while the PCL carried a larger total force than the ACL in the range of 75° to 90° of flexion.
An Integrated Finite-Element Approach to Mechanics, Transport and Biosynthesis in Tissue Engineering
Authors: Sengers Bram G. , Oomens Cees W. J. , Baaijens Frank P. T.
Published: March 2004

A finite-element approach was formulated, aimed at enabling an integrated study of mechanical and biochemical factors that control the functional development of tissue engineered constructs. A nonlinear biphasic displacement-velocity-pressure description was combined with adjective and diffusive solute transport, uptake and biosynthesis. To illustrate the approach we focused on the synthesis and transport of macromolecules under influence of fluid flow induced by cyclic compression. In order to produce net transport the effect of dispersion was investigated. An abstract representation of biosynthesis was employed, three cases were distinguished: Synthesis dependent on a limited small solute, synthesis dependent on a limited large solute and synthesis independent of solute transport. Results show that a dispersion model can account for augmented solute transport by cyclic compression and indicate the different sensitivity to loading that can be expected depending on the size of the limiting solute.
Experimental and Numerical Modeling of Variable Friction Between Nanoregions in Conventional and Crosslinked UHMWPE
Authors: Ho Sunita P. , Joseph Paul F. , Drews Michael J. , Boland Thomas , LaBerge Martine
Published: March 2004

Recently, highly crosslinked UHMWPE components have been promoted for their high abrasive wear resistance over conventional UHMWPE (PE) in total joint replacement (TJR) prostheses to minimize osteolysis and consequent implant loosening. This study was aimed at investigating the role of friction gradients induced by localized coefficients of friction at both crystalline and amorphous nanoregions in PE, and crystalline and crosslinked nanoregions in crosslinked UHMWPE (XPE), in submicron wear debris generation. An abrasive wear study performed on both XPE and PE using atomic force microscopy (AFM) illustrated that the onset of plastic deformation for XPE occurred at a normal load that was approximately 3 times higher when compared to PE. Coefficients of friction μd of 0.2, 0.35, and 0.61, experimentally derived using AFM, were used as representative μd for crystalline, amorphous, and crosslinked nanoregions, respectively, in a numerical Hertzian model. An increase in μ (0.2±0.02, 0.35±0.01 and 0.6±0.04) was observed with a decrease in crystallinity and storage modulus at 22°C. Using the Hertzian contact model, it was observed that variability in friction between nanoregions contributed to higher magnitude stresses for XPE (0.2 to 0.61; maximum σeff=2.8) compared to PE (0.2 to 0.35; maximum σeff=1.1) over a negligible thickness of the interfacial zone (IZ) between nanoregions. The experimentally observed increase in abrasive wear resistance of XPE could be attributed to an increase in the thickness of the interfacial zone between nanoregions with μ changing gradually from crystalline to crosslinked nanoregions, a situation that may not be observed with PE. This would cause a decrease in the friction gradient and resulting stresses thereby agreeing with the observed experimental higher abrasive wear resistance for XPE. However, in both PE and XPE, the presence of stress concentrations over a period of time could lead to irreversible damage of the material eventually generating submicron wear debris. Hence, semicrystalline, inhomogenous UHMWPE with several nanoregions (amorphous and crystalline) would be at a disadvantage for bearing application in terms of abrasive wear resistance compared to UHMWPE with relatively lower number of nanoregions and crosslinked nanoregions.
Dynamic Interaction of Stargazin-like TARPs with Cycling AMPA Receptors at Synapses
Authors: Tomita Susumu , Fukata Masaki , Nicoll Roger A. , Bredt David S.
Published: March 2004

Activity-dependent plasticity in the brain arises in part from changes in the number of synaptic AMPA receptors. Synaptic trafficking of AMPA receptors is controlled by stargazin and homologous transmembrane AMPA receptor regulatory proteins (TARPs). We found that TARPs were stable at the plasma membrane, whereas AMPA receptors were internalized in a glutamate-regulated manner. Interaction with AMPA receptors involved both extra- and intracellular determinants of TARPs. Upon binding to glutamate, AMPA receptors detached from TARPs. This did not require ion flux or intracellular second messengers. This allosteric mechanism for AMPA receptor dissociation from TARPs may participate in glutamate-mediated internalization of receptors in synaptic plasticity.
Neutrophil Extracellular Traps Kill Bacteria
Authors: Brinkmann Volker , Reichard Ulrike , Goosmann Christian , Fauler Beatrix , Uhlemann Yvonne , Weiss David S. , Weinrauch Yvette , Zychlinsky Arturo
Published: March 2004

Neutrophils engulf and kill bacteria when their antimicrobial granules fuse with the phagosome. Here, we describe that, upon activation, neutrophils release granule proteins and chromatin that together form extracellular fibers that bind Gram-positive and -negative bacteria. These neutrophil extracellular traps (NETs) degrade virulence factors and kill bacteria. NETs are abundant in vivo in experimental dysentery and spontaneous human appendicitis, two examples of acute inflammation. NETs appear to be a form of innate response that binds microorganisms, prevents them from spreading, and ensures a high local concentration of antimicrobial agents to degrade virulence factors and kill bacteria.
Late Miocene Teeth from Middle Awash, Ethiopia, and Early Hominid Dental Evolution
Authors: Haile-Selassie Yohannes , Suwa Gen , White Tim D.
Published: March 2004

Late Miocene fossil hominid teeth recovered from Ethiopia's Middle Awash are assigned to
Ardipithecus kadabba
. Their primitive morphology and wear pattern demonstrate that
A. kadabba
is distinct from
Ardipithecus ramidus
. These fossils suggest that the last common ancestor of apes and humans had a functionally honing canine–third premolar complex. Comparison with teeth of
Sahelanthropus
and
Orrorin
, the two other named late Miocene hominid genera, implies that these putative taxa are very similar to
A. kadabba
. It is therefore premature to posit extensive late Miocene hominid diversity on the basis of currently available samples.
A Membrane-Anchored Protein Kinase Involved in
Brassica
Self-Incompatibility Signaling
Authors: Murase Kohji , Shiba Hiroshi , Iwano Megumi , Che Fang-Sik , Watanabe Masao , Isogai Akira , Takayama Seiji
Published: March 2004

Self-incompatibility (SI) response in
Brassica
is initiated by haplotype-specific interactions between the pollen-borne ligand
S
locus protein 11/SCR and its stigmatic
S
receptor kinase, SRK. This binding induces autophosphorylation of SRK, which is then thought to trigger a signaling cascade that leads to self-pollen rejection. A recessive mutation of the
modifier
(
m
) gene eliminates the SI response in stigma. Positional cloning of
M
has revealed that it encodes a membrane-anchored cytoplasmic serine/threonine protein kinase, designated
M
locus protein kinase (MLPK). Transient expression of MLPK restores the ability of
mm
papilla cells to reject self-pollen, suggesting that MLPK is a positive mediator of
Brassica
SI signaling.
Convergence of Wnt, ß-Catenin, and Cadherin Pathways
Authors: Nelson W. James , Nusse Roel
Published: March 2004

The specification and proper arrangements of new cell types during tissue differentiation require the coordinated regulation of gene expression and precise interactions between neighboring cells. Of the many growth factors involved in these events, Wnts are particularly interesting regulators, because a key component of their signaling pathway, β-catenin, also functions as a component of the cadherin complex, which controls cell-cell adhesion and influences cell migration. Here, we assemble evidence of possible interrelations between Wnt and other growth factor signaling, β-catenin functions, and cadherin-mediated adhesion.
A Toll-like Receptor That Prevents Infection by Uropathogenic Bacteria
Authors: Zhang Dekai , Zhang Guolong , Hayden Matthew S. , Greenblatt Matthew B. , Bussey Crystal , Flavell Richard A. , Ghosh Sankar
Published: March 2004

Toll-like receptors (TLRs) recognize molecular patterns displayed by microorganisms, and their subsequent activation leads to the transcription of appropriate host-defense genes. Here we report the cloning and characterization of a member of the mammalian TLR family, TLR11, that displays a distinct pattern of expression in macrophages and liver, kidney, and bladder epithelial cells. Cells expressing TLR11 fail to respond to known TLR ligands but instead respond specifically to uropathogenic bacteria. Mice lacking TLR11 are highly susceptible to infection of the kidneys by uropathogenic bacteria, indicating a potentially important role for TLR11 in preventing infection of internal organs of the urogenital system.
Charting Our Extraterrestrial Efforts
Authors: DeVorkin David
Published: March 2004

The Cambridge Encyclopedia of Space
Missions, Applications and Exploration. Fernand Verger, Isabelle Sourbès-Verger, and Raymond Ghirardi. Cambridge University Press, New York, 2003. 428 pp. $50. ISBN 0-521-77300-8.

This profusely illustrated overview of human efforts to explore space surveys the entire spectrum of space applications (scientific, technical, commercial, and military) and the activities of all countries.
Molecular Diversity of Rumen Methanogens from Sheep in Western Australia
Authors: Wright André-Denis G. , Williams Andrew J. , Winder Barbara , Christophersen Claus T. , Rodgers Sharon L. , Smith Kellie D.
Published: March 2004

The molecular diversity of rumen methanogens in sheep in Australia was investigated by using individual 16S rRNA gene libraries prepared from the rumen contents obtained from six merino sheep grazing pasture (326 clones), six sheep fed an oaten hay-based diet (275 clones), and five sheep fed a lucerne hay-based diet (132 clones). A total of 733 clones were examined, and the analysis revealed 65 phylotypes whose sequences (1,260 bp) were similar to those of cultivated methanogens belonging to the order
Methanobacteriales
. Pasture-grazed sheep had more methanogen diversity than sheep fed either the oaten hay or lucerne hay diet.
Methanobrevibacter
strains SM9, M6, and NT7 accounted for over 90% of the total number of clones identified. M6 was more prevalent in grazing sheep, and SM9, despite being found in 16 of the 17 sheep, was more prevalent in sheep fed the lucerne-based diet. Five new species were identified. Two of these species exhibited very little sequence similarity to any cultivated methanogens and were found eight times in two of the six sheep that were grazing pasture. These unique sequences appear to represent a novel group of rumen archaea that are atypical for the rumen environment.
Detection of
Arcobacter
spp. in the Coastal Environment of the Mediterranean Sea
Authors: Fera M. T. , Maugeri T. L. , Gugliandolo C. , Beninati C. , Giannone M. , La Camera E. , Carbone M.
Published: March 2004

The occurrence of
Arcobacter
spp. was studied in seawater and plankton samples collected from the Straits of Messina, Italy, during an annual period of observation by using cultural and molecular techniques. A PCR assay with three pairs of primers targeting the 16S and 23S rRNA genes was used for detection and identification of
Arcobacter butzleri
,
Arcobacter cryaerophilus
, and
Arcobacter skirrowii
in cultures and environmental samples. Only one of the
Arcobacter
species,
A. butzleri
, was isolated from seawater and plankton samples. With some samples the
A. butzleri
PCR assay gave amplified products when cultures were negative.
A. cryaerophilus
and
A. skirrowii
were never detected by culture on selective agar plates; they were detected only by PCR performed directly with environmental samples. Collectively, our data suggest that culturable and nonculturable forms of
Arcobacter
are present in marine environments. The assay was useful for detecting
Arcobacter
spp. both as free forms and intimately associated with plankton. This is the first report showing both direct isolation of
A. butzleri
and the presence of nonculturable
Arcobacter
spp. in the coastal environment of the Mediterranean Sea.
Molecular Diversity of New
Thermococcales
Isolates from a Single Area of Hydrothermal Deep-Sea Vents as Revealed by Randomly Amplified Polymorphic DNA Fingerprinting and 16S rRNA Gene Sequence Analysis
Authors: Lepage Elodie , Marguet Evelyne , Geslin Claire , Matte-Tailliez Oriane , Zillig Wolfram , Forterre Patrick , Tailliez Patrick
Published: March 2004

Members of the
Thermococcales
are anaerobic
Archaea
belonging to the kingdom
Euryarchaea
that are studied in many laboratories as model organisms for hyperthermophiles. We describe here a molecular analysis of 86 new
Thermococcales
isolates collected from six different chimneys of a single hydrothermal field located in the 13°N 104°W segment of the East Pacific ridge at a depth of 2,330 m. These isolates were sorted by randomly amplified polymorphic DNA (RAPD) fingerprinting into nine groups, and nine unique RAPD profiles were obtained. One RAPD group corresponds to new isolates of
Thermococcus hydrothermalis
, whereas all other groups and isolates with unique profiles are different from the 22 reference strains included in this study. Analysis of 16S rRNA gene sequences of representatives of each RAPD group and unique profiles showed that one group corresponds to
Pyrococcus
strains, whereas all the other isolates are
Thermococcus
strains. We estimated that our collection may contain at least 11 new species. These putative species, isolated from a single area of hydrothermal deep-sea vents, are dispersed in the 16S rRNA tree among the reference strains previously isolated from diverse hot environments (terrestrial, shallow water, hydrothermal vents) located around the world, suggesting that there is a high degree of dispersal of
Thermococcales
. About one-half of our isolates contain extrachromosomal elements that could be used to search for novel replication proteins and to develop genetic tools for hyperthermophiles.
Regulatory Role of
Rhizobium etli
CNPAF512
fnrN
during Symbiosis
Authors: Moris Martine , Dombrecht Bruno , Xi Chuanwu , Vanderleyden Jos , Michiels Jan
Published: March 2004

The
Rhizobium etli
CNPAF512
fnrN
gene was identified in the
fixABCX rpoN

2

region. The corresponding protein contains the hallmark residues characteristic of proteins belonging to the class IB group of Fnr-related proteins. The expression of
R. etli fnrN
is highly induced under free-living microaerobic conditions and during symbiosis. This microaerobic and symbiotic induction of
fnrN
is not controlled by the sigma factor RpoN and the symbiotic regulator
nifA
or
fixLJ
, but it is due to positive autoregulation. Inoculation of
Phaseolus vulgaris
with an
R. etli fnrN
mutant strain resulted in a severe reduction in the bacteroid nitrogen fixation capacity compared to the wild-type capacity, confirming the importance of FnrN during symbiosis. The expression of the
R. etli fixN
,
fixG
, and
arcA
genes is strictly controlled by
fnrN
under free-living microaerobic conditions and in bacteroids during symbiosis with the host. However, there is an additional level of regulation of
fixN
and
fixG
under symbiotic conditions. A phylogenetic analysis of the available rhizobial FnrN and FixK proteins grouped the proteins in three different clusters.
Novel Pathway of Salicylate Degradation by
Streptomyces
sp. Strain WA46
Authors: Ishiyama Daisuke , Vujaklija Dusica , Davies Julian
Published: March 2004

A novel salicylate-degrading
Streptomyces
sp., strain WA46, was identified by UV fluorescence on solid minimal medium containing salicylate; trace amounts of gentisate were detected by high-pressure liquid chromatography when strain WA46 was grown with salicylate. PCR amplification of WA46 DNA with degenerate primers for gentisate 1,2-dioxygenase (GDO) genes produced an amplicon of the expected size. Sequential PCR with nested GDO primers was then used to identify a salicylate degradation gene cluster in a plasmid library of WA46 chromosomal DNA. The nucleotide sequence of a 13.5-kb insert in recombinant plasmid pWD1 (which was sufficient for the complete degradation of salicylate) showed that nine putative open reading frames (ORFs) (
sdgABCDEFGHR
) were involved. Plasmid pWD1 derivatives disrupted in each putative gene were transformed into
Streptomyces lividans
TK64. Disruption of either
sdgA
or
sdgC
blocked salicylate degradation; constructs lacking
sdgD
accumulated gentisate. Cell extracts from
Escherichia coli
DH5α transformants harboring pUC19 that expressed each of the
sdg
ORFs showed that conversions of salicylate to salicylyl-coenzyme A (CoA) and salicylyl-CoA to gentisyl-CoA required SdgA and SdgC, respectively. SdgA required CoA and ATP as cofactors, while NADH was required for SdgC activity; SdgC was identified as salicylyl-CoA 5-hydroxylase. Gentisyl-CoA underwent spontaneous cleavage to gentisate and CoA. SdgA behaved as a salicylyl-CoA ligase despite showing amino acid sequence similarity to an AMP-ligase. SdgD was identified as a GDO. These results suggest that
Streptomyces
sp. strain WA46 degrades salicylate by a novel pathway via a CoA derivative. Two-dimensional polyacrylamide gel electrophoresis and reverse transcriptase-PCR studies indicated that salicylate induced expression of the
sdg
cluster.
Physiology and Nutrition ofTreponema primitia, an H2/ CO2-Acetogenic Spirochete from Termite Hindguts
Authors: Graber Joseph R. , Breznak John A.
Published: March 2004

Treponema primitiastrains ZAS-1 and ZAS-2, the first spirochetes to be isolated from termite hindguts (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science283:686-689, 1999), were examined for nutritional, physiological, and biochemical properties relevant to growth and survival in their natural habitat. In addition to using H2plus CO2as substrates, these strains were capable of homoacetogenic growth on mono- and disaccharides and (in the case of ZAS-2) methoxylated benzenoids. Cells were also capable of mixotrophic growth (i.e., simultaneous utilization of H2and organic substrates). Cell extracts ofT. primitiapossessed enzyme activities of the Wood/Ljungdahl (acetyl coenzyme A) pathway of acetogenesis, including tetrahydrofolate-dependent enzymes of the methyl group-forming branch. However, a folate compound was required in the medium for growth. ZAS-1 and ZAS-2 growing on H2plus CO2displayed H2thresholds of 650 and 490 ppmv, respectively. Anoxic cultures of ZAS-1 and ZAS-2 maintained growth after the addition of as much as 0.5% (vol/vol) O2to the headspace atmosphere. Cell extracts exhibited NADH and NADPH peroxidase and NADH oxidase activities but neither catalase nor superoxide dismutase activity. Results indicate that (i)T. primitiais able to exploit a variety of substrates derived from the food of its termite hosts and in so doing contributes to termite nutrition via acetogenesis, (ii) in situ growth ofT. primitiais likely dependent on secretion of a folate compound(s) by other members of the gut microbiota, and (iii) cells possess enzymatic adaptations to oxidative stress, which is likely to be encountered in peripheral regions of the termite hindgut.
Description of
Treponema azotonutricium
sp. nov. and
Treponema primitia
sp. nov., the First Spirochetes Isolated from Termite Guts
Authors: Graber Joseph R. , Leadbetter Jared R. , Breznak John A.
Published: March 2004

Long after their original discovery, termite gut spirochetes were recently isolated in pure culture for the first time. They revealed metabolic capabilities hitherto unknown in the
Spirochaetes
division of the
Bacteria
, i.e., H
2
plus CO
2
acetogenesis (J. R. Leadbetter, T. M. Schmidt, J. R. Graber, and J. A. Breznak, Science
283:
686-689, 1999) and dinitrogen fixation (T. G. Lilburn, K. S. Kim, N. E. Ostrom, K. R. Byzek, J. R. Leadbetter, and J. A. Breznak, Science
292:
2495-2498, 2001). However, application of specific epithets to the strains isolated (
Treponema
strains ZAS-1, ZAS-2, and ZAS-9) was postponed pending a more complete characterization of their phenotypic properties. Here we describe the major properties of strain ZAS-9, which is readily distinguished from strains ZAS-1 and ZAS-2 by its shorter mean cell wavelength or body pitch (1.1 versus 2.3 μm), by its nonhomoacetogenic fermentation of carbohydrates to acetate, ethanol, H
2
, and CO
2
, and by 7 to 8% dissimilarity between its 16S rRNA sequence and those of ZAS-1 and ZAS-2. Strain ZAS-9 is proposed as the type strain of the new species,
Treponema azotonutricium
. Strains ZAS-1 and ZAS-2, which are H
2
-consuming, CO
2
-reducing homoacetogens, are proposed here to be two strains of the new species
Treponema primitia
. Apart from the salient differences mentioned above, the genomes of all three strains were similar in size (3,461 to 3,901 kb), in G+C content (50.0 to 51.0 mol%), and in possession of 2 copies of the gene encoding 16S rRNA (
rrs
). For comparison, the genome of the free-living spirochete
Spirochaeta aurantia
strain J1 was analyzed by the same methods and found to have a size of 3,719 kb, to contain 65.6 mol% G+C, and also to possess 2 copies of the
rrs
gene.
Characterization of the Arginine Deiminase Operon of
Streptococcus rattus
FA-1
Authors: Griswold Ann , Chen Yi-Ywan M. , Snyder Jennifer A. , Burne Robert A.
Published: March 2004

The arginine deiminase system (ADS) is of critical importance in oral biofilm pH homeostasis and microbial ecology. The ADS consists of three enzymes. Arginine is hydrolyzed by AD (ArcA) to generate citrulline and ammonia. Citrulline is then converted to ornithine and carbamoylphosphate via ornithine carbamoyltransferase (ArcB). Finally, carbamate kinase (ArcC) transfers a phosphate from carbamoylphosphate to ADP, yielding ATP. Ammonia production from this pathway protects bacteria from lethal acidification, and ATP production provides a source of energy for the cells. The purpose of this study was to initiate a characterization of the
arc
operon of
Streptococcus rattus
, the least cariogenic and sole ADS-positive member of the mutans streptococci. Using an
arcB
gene fragment obtained by degenerate PCRs, the FA-1
arc
operon was identified in subgenomic DNA libraries and sequence analysis was performed. Results showed that the genes encoding the AD pathway in
S. rattus
FA-1 are organized as an
arcABCDT-adiR
operon gene cluster, including the enzymes of the pathway, an arginine-ornithine antiporter (ArcD) and a putative regulatory protein (AdiR). The
arcA
transcriptional start site was identified by primer extension, and a σ
70
-like promoter was mapped 5′ to
arcA
. Reverse transcriptase PCR was used to establish that
arcABCDT
could be cotranscribed. Reporter gene fusions and AD assays demonstrated that the operon is regulated by substrate induction and catabolite repression, the latter apparently through a CcpA-dependent pathway.
Wood-Destroying Soft Rot Fungi in the Historic Expedition Huts of Antarctica
Authors: Blanchette Robert A. , Held Benjamin W. , Jurgens Joel A. , McNew Douglas L. , Harrington Thomas C. , Duncan Shona M. , Farrell Roberta L.
Published: March 2004

Three expedition huts in the Ross Sea region of Antarctica, built between 1901 and 1911 by Robert F. Scott and Ernest Shackleton, sheltered and stored the supplies for up to 48 men for 3 years during their explorations and scientific investigation in the South Pole region. The huts, built with wood taken to Antarctica by the early explorers, have deteriorated over the past decades. Although Antarctica has one of the coldest and driest environments on earth, microbes have colonized the wood and limited decay has occurred. Some wood in contact with the ground contained distinct microscopic cavities within secondary cell walls caused by soft rot fungi.
Cadophora
spp. could be cultured from decayed wood and other woods sampled from the huts and artifacts and were commonly associated with the soft rot attack. By using internal transcribed spacer sequences of ribosomal DNA and morphological characteristics, several species of
Cadophora
were identified, including
C. malorum
,
C. luteo-olivacea
, and
C. fastigiata
. Several previously undescribed
Cadophora
spp. also were found. At the Cape Evans and Cape Royds huts,
Cadophora
spp. commonly were isolated from wood in contact with the ground but were not always associated with soft rot decay. Pure cultures of
Cadophora
used in laboratory decay studies caused dark staining of all woods tested and extensive soft rot in
Betula
and
Populus
wood. The presence of
Cadophora
species, but only limited decay, suggests there is no immediate threat to the structural integrity of the huts. These fungi, however, are widely found in wood from the historic huts and have the capacity to cause extensive soft rot if conditions that are more conducive to decay become common.
Heat Shock Response in
Lactobacillus plantarum
Authors: De Angelis Maria , Di Cagno Raffaella , Huet Claude , Crecchio Carmine , Fox Patrick F. , Gobbetti Marco
Published: March 2004

Heat stress resistance and response were studied in strains of
Lactobacillus plantarum
. Stationary-phase cells of
L. plantarum
DPC2739 had decimal reduction times (
D
values) (
D
value was the time that it took to reduce the number of cells by 1 log cycle) in sterile milk of 32.9, 14.7, and 7.14 s at 60, 72, and 75°C, respectively. When mid-exponential-phase cells were used, the
D
values decreased. The temperature increases which caused a 10-fold reduction in the
D
value ranged from 9 to 20°C, depending on the strain. Part of the cell population treated at 72°C for 90 s recovered viability during incubation at 7°C in sterile milk for 20 days. When mid-exponential- or stationary-phase cells of
L. plantarum
DPC2739 were adapted to 42°C for 1 h, the heat resistance at 72°C for 90 s increased ca. 3 and 2 log cycles, respectively. Heat-adapted cells also showed increased growth at pH 5 and in the presence of 6% NaCl. Two-dimensional gel electrophoresis of proteins expressed by control and heat-adapted cells revealed changes in the levels of expression of 31 and 18 proteins in mid-exponential- and stationary-phase cells, respectively. Twelve proteins were commonly induced. Nine proteins induced in the heat-adapted mid-exponential- and/or stationary-phase cells of
L. plantarum
DPC2739 were subjected to N-terminal sequencing. These proteins were identified as DnaK, GroEL, trigger factor, ribosomal proteins L1, L11, L31, and S6, DNA-binding protein II HlbA, and CspC. All of these proteins have been found to play a role in the mechanisms of stress adaptation in other bacteria. Antibodies against GroES detected a protein which was induced moderately, while antibodies against DnaJ and GrpE reacted with proteins whose level of expression did not vary after heat adaptation. This study showed that the heat resistance of
L. plantarum
is a complex process involving proteins with various roles in cell physiology, including chaperone activity, ribosome stability, stringent response mediation, temperature sensing, and control of ribosomal function. The physiological mechanisms of response to pasteurization in
L. plantarum
are fundamental for survival in cheese during manufacture.
Molecular Detection and Identification ofBrettanomyces/Dekkera bruxellensisandBrettanomyces/Dekkera anomalusin Spoiled Wines
Authors: Cocolin Luca , Rantsiou Kalliopi , Iacumin Lucilla , Zironi Roberto , Comi Giuseppe
Published: March 2004

In this paper we describe the development of a PCR protocol to specifically detectBrettanomyces bruxellensisandB. anomalus.Primers DB90F and DB394R, targeting the D1-D2 loop of the 26S rRNA gene, were able to produce amplicons only when the DNA from these two species were used. No amplification product was obtained when DNA from otherBrettanomycesspp. or wine yeasts were used as the templates. The 305-bp product was subjected to restriction enzyme analysis withDdeI to differentiate betweenB. bruxellensisandB. anomalus, and each species could be identified on the basis of the different restriction profiles. After optimization of the method by using strains from international collections, wine isolates were tested with the method proposed. Total agreement between traditional identification and molecular identification was observed. The protocol developed was also used for direct detection ofB. bruxellensisandB. anomalusin wines suspected to be spoiled byBrettanomycesspp. Application of culture-based and molecular methods led us to the conclusion that 8 of 12 samples were spoiled byB. bruxellensis. Results based on the application of molecular methods suggested that two of the eight positive samples had been infected more recently, since specific signals were obtained at both the DNA and RNA levels.
Inoculum Size Effect in Dimorphic Fungi: Extracellular Control of Yeast-Mycelium Dimorphism in
Ceratocystis ulmi
Authors: Hornby Jacob M. , Jacobitz-Kizzier Sarah M. , McNeel Donna J. , Jensen Ellen C. , Treves David S. , Nickerson Kenneth W.
Published: March 2004

We studied the inoculum size effect in
Ceratocystis ulmi
, the dimorphic fungus that causes Dutch elm disease. In a defined glucose-proline-salts medium, cells develop as budding yeasts when inoculated at ≥10
6
spores per ml and as mycelia when inoculated at <10
6
spores per ml. The inoculum size effect was not influenced by inoculum spore type, age of the spores, temperature, pH, oxygen availability, trace metals, sulfur source, phosphorous source, or the concentration of glucose or proline. Similarly, it was not influenced by added adenosine, reducing agents, methyl donors, amino sugars, fatty acids, or carbon dioxide. Instead, growing cells excreted an unknown quorum-sensing factor that caused a morphological shift from mycelia to budding yeasts. This yeast-promoting effect is abolished if it is extracted with an organic solvent such as ethyl acetate. The quorum-sensing activity acquired by the organic solvent could be added back to fresh medium in a dose-dependent fashion. The quorum-sensing activity in
C. ulmi
spent medium was specific for
C. ulmi
and had no effect on the dimorphic fungus
Candida albicans
or the photomorphogenic fungus
Penicillium isariaeforme
. In addition, farnesol, the quorum-sensing molecule produced by
C. albicans
, did not inhibit mycelial development of
C. ulmi
when present at concentrations of up to 100 μM. We conclude that the inoculum size effect is a manifestation of a quorum-sensing system that is mediated by an excreted extracellular molecule, and we suggest that quorum sensing is a general phenomenon in dimorphic fungi.
Development and Use of Flow Cytometry for Detection of Airborne Fungi
Authors: Prigione Valeria , Lingua Guido , Marchisio Valeria Filipello
Published: March 2004

Traditional methods for the enumeration of airborne fungi are slow, tedious, and rather imprecise. In this study, the possibility of using flow cytometry (FCM) for the assessment of exposure to the fungus aerosol was evaluated. Epifluorescence microscopy direct counting was adopted as the standard for comparison. Setting up of the method was achieved with pure suspensions of
Aspergillus fumigatus
and
Penicillium brevicompactum
conidia at different concentrations, and then analyses were extended to field samples collected by an impinger device. Detection and quantification of airborne fungi by FCM was obtained combining light scatter and propidium iodide red fluorescence parameters. Since inorganic debris are unstainable with propidium iodide, the biotic component could be recognized, whereas the preanalysis of pure conidia suspensions of some species allowed us to select the area corresponding to the expected fungal population. A close agreement between FCM and epifluorescence microscopy counts was found. Moreover, data processing showed that FCM can be considered more precise and reliable at any of the tested concentrations.
Quantitative Detection of
Listeria monocytogenes
and
Listeria innocua
by Real-Time PCR: Assessment of
hly
,
iap
, and
lin02483
Targets and AmpliFluor Technology
Authors: Rodríguez-Lázaro David , Hernández Marta , Scortti Mariela , Esteve Teresa , Vázquez-Boland José A. , Pla Maria
Published: March 2004

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen
Listeria monocytogenes
and the closely related nonpathogenic species
L. innocua
. The target genes were
hly
and
iap
for
L. monocytogenes
and
lin02483
for
L. innocua
. The assays were 100% specific, as determined with 100
Listeria
strains and 45 non-
Listeria
strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and
R
2
values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The
hly
-based assay accurately quantified
L. monocytogenes
in all of the samples tested. The
iap
-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify
L. monocytogenes
isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of
L. monocytogenes
by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).
Interaction ofBacillus thuringiensisToxins with Larval Midgut Binding Sites ofHelicoverpa armigera(Lepidoptera: Noctuidae)
Authors: Estela Anna , Escriche Baltasar , Ferré Juan
Published: March 2004

In 1996, Bt-cotton (cotton expressing aBacillus thuringiensistoxin gene) expressing the Cry1Ac protein was commercially introduced to control cotton pests. A threat to this first generation of transgenic cotton is the evolution of resistance by the insects. Second-generation Bt-cotton has been developed with either newB. thuringiensisgenes or with a combination ofcrygenes. However, one requirement for the “stacked” gene strategy to work is that the stacked toxins bind to different binding sites. In the present study, the binding of125I-labeled Cry1Ab protein (125I-Cry1Ab) and125I-Cry1Ac to brush border membrane vesicles (BBMV) ofHelicoverpa armigerawas analyzed in competition experiments with 11 nonlabeled Cry proteins. The results indicate that Cry1Aa, Cry1Ab, and Cry1Ac competed for common binding sites. No other Cry proteins tested competed for either125I-Cry1Ab or125I-Cry1Ac binding, except Cry1Ja, which competed only at the highest concentrations used. Furthermore, BBMV from fourH. armigerapopulations were also tested with125I-Cry1Ac and Cry1Ab to check the influence of the insect population on the binding results. Finally, the inhibitory effect of selected sugars and lectins was also determined.125I-Cry1Ac binding was strongly inhibited byN-acetylgalactosamine, sialic acid, and concanavalin A and moderately inhibited by soybean agglutinin. In contrast,125I-Cry1Ab binding was only significantly inhibited by concanavalin A. These results show that Cry1Ac and Cry1Ab use different epitopes for binding to BBMV.
Aerobic and Anaerobic Toluene Degradation by a Newly Isolated Denitrifying Bacterium,
Thauera
sp. Strain DNT-1
Authors: Shinoda Yoshifumi , Sakai Yasuyoshi , Uenishi Hiroshi , Uchihashi Yasumitsu , Hiraishi Akira , Yukawa Hideaki , Yurimoto Hiroya , Kato Nobuo
Published: March 2004

A newly isolated denitrifying bacterium,
Thauera
sp. strain DNT-1, grew on toluene as the sole carbon and energy source under both aerobic and anaerobic conditions. When this strain was cultivated under oxygen-limiting conditions with nitrate, first toluene was degraded as oxygen was consumed, while later toluene was degraded as nitrate was reduced. Biochemical observations indicated that initial degradation of toluene occurred through a dioxygenase-mediated pathway and the benzylsuccinate pathway under aerobic and denitrifying conditions, respectively. Homologous genes for toluene dioxygenase (
tod
) and benzylsuccinate synthase (
bss
), which are the key enzymes in aerobic and anaerobic toluene degradation, respectively, were cloned from genomic DNA of strain DNT-1. The results of Northern blot analyses and real-time quantitative reverse transcriptase PCR suggested that transcription of both sets of genes was induced by toluene. In addition, the
tod
genes were induced under aerobic conditions, whereas the
bss
genes were induced under both aerobic and anaerobic conditions. On the basis of these results, it is concluded that strain DNT-1 modulates the expression of two different initial pathways of toluene degradation according to the availability of oxygen in the environment.