bluuebunny/crossref_metadata_2025 huggingface dataset:
A role for norepinephrine in the regulation of context‐dependent ZENK expression in male zebra finches (Taeniopygia guttata)
Authors: Castelino Christina B. , Ball Gregory F.
Published: May 2005

Singing drives expression of the immediate‐early gene ZENK in a context‐dependent manner in certain nuclei within the avian song circuit of male zebra finches (Taeniopygia guttata). ZENK mRNA expression is low when males are engaged in female‐ or male‐directed song, but high during solo song. Neurotransmitter systems like catecholamines with diffuse projections to forebrain regions are good candidates for regulation of such context‐dependent brain activity. We investigated whether the noradrenergic system regulates the dramatic switch in ZENK expression across contexts in male zebra finches. We systemically injected a noradrenergic neurotoxin N‐(2‐chloroethyl)‐N‐ethyl‐2‐bromobenzylamine hydrochloride (DSP‐4) and found a marked increase in the resultant ZENK expression in area X of the medial striatum in male zebra finches singing directed song. ZENK protein expression in saline‐treated males across different contexts mirrored the pattern of previously reported ZENK mRNA expression. We corroborated DSP‐4 specificity via immunohistochemical procedures for tyrosine hydroxylase and dopamine‐beta hydroxylase, which revealed decreases in norepinephrine synthesizing nuclei and certain song control nuclei. Based on these results we propose a mechanism by which the noradrenergic system usually downregulates ZENK expression in area X during directed song. By depleting this system we induced a disruption of this regulation and reversion back to the default situation characterized by an increase in motor‐driven ZENK expression in the song circuit. These data demonstrate that the noradrenergic system (probably in concert with other modulatory neurotransmitters) plays an important role in the response of the brain to salient events that occur in the context of a natural behavior − singing.
Opposite effects of tetanic stimulation of the auditory thalamus or auditory cortex on the acoustic startle reflex in awake rats
Authors: Huang Juan , Wu Xihong , Yeomans John , Li Liang
Published: May 2005

The amygdala mediates both emotional learning and fear potentiation of startle. The lateral amygdala nucleus (LA) receives auditory inputs from both the auditory thalamus (medial geniculate nucleus; MGN) and auditory association cortex (AAC), and is critical for auditory fear conditioning. The central amygdala nucleus, which has intra‐amygdaloid connections with LA, enhances startle magnitude via midbrain connections to the startle circuits. Tetanic stimulation of either MGN or AAC in vitro or in vivo can induce long‐term potentiation in LA. In the present study, behavioural consequences of tetanization of these auditory afferents were investigated in awake rats. The acoustic startle reflex of rats was enhanced by tetanic stimulation of MGN, but suppressed by that of AAC. All the tetanization‐induced changes of startle diminished within 24 h. Blockade of GABAB receptors in the LA area reversed the suppressive effect of tetanic stimulation of AAC on startle but did not change the enhancing effect of tetanic stimulation of MGN. Moreover, transient electrical stimulation of MGN enhanced the acoustic startle reflex when it lagged behind acoustic stimulation, but inhibited the acoustic startle reflex when it preceded acoustic stimulation. The results of the present study indicate that MGN and AAC afferents to LA play different roles in emotional modulation of startle, and AAC afferents are more influenced by inhibitory GABAB transmission in LA.
Performance measurement systems in SMEs: A review for a research agenda
Authors: Garengo Patrizia , Biazzo Stefano , Bititci Umit S.
Published: May 2005

In recent years, literature has identified the increasing complexity of small and medium‐sized enterprises (SMEs) and highlighted their sensitivity to differences in managerial culture and management systems. Research has shown that performance measurement systems (PMSs) could play an important role in supporting managerial development in these companies. In this paper, the literature on performance measurement in manufacturing SMEs is reviewed and the diffusion, characteristics and determinants of performance measurement in SMEs are analysed. Shortcomings in the performance measurement systems are highlighted and the many factors that seem to constrain PMSs in manufacturing SMEs are defined, e.g. lack of financial and human resources, wrong perception of the benefits of PMS implementation, short‐term strategic planning. Moreover, using dimensions defined according to the information found in the literature, two PMS models specifically developed for SMEs are compared with generic PMS models. The comparison points out an evolution in PMS models over time; in particular, the models developed in the last 20 years are more horizontal, process‐oriented and focus on stakeholder needs. However, it is not clear whether these changes are due to the evolution of the generic models or an attempt to introduce models suited to the needs of SMEs. To clarify this matter and better to understand PMSs in SMEs, further theoretical and empirical studies are necessary. The main issues still requiring investigation are listed in a research agenda at the end of the paper.
A performance measurement paradigm for integrating strategy formulation: A review of systems and frameworks
Authors: Pun Kit Fai , White Anthony Sydney
Published: May 2005

Measuring organizational performance plays a very important part in translating corporate strategy into results. Various emerging (non‐traditional) performance systems have recently been devised to aid firms in selecting and implementing measures. This paper discusses the strategy/measurement initiatives and compares ten emerging performance measurement systems with respect to a list of performance dimensions, the characteristics of performance measures, and the requirements of development process. Although these systems have constraints borne with their own application domains, they stand by themselves empirically and/or theoretically, and provide guidance about what to measure and how to design performance measures that could be linked to the corporate strategy and objectives of an organization. This paper concludes that there is a need to develop a paradigm for integrating strategy formulation and performance measurement in organizations.
Finance and the Business Cycle: International, Inter‐Industry Evidence
Authors: BRAUN MATÍAS , LARRAIN BORJA
Published: May 2005

By considering yearly production growth rates for several manufacturing industries in more than 100 countries during (roughly) the last 40 years, we show that industries that are more dependent on external finance are hit harder during recessions. The observed difference in the behavior of industries is larger when financial frictions are thought to be more prevalent, linking the result directly to the financial mechanism hypothesis. In particular, more dependent industries are more strongly affected in recessions when they are located in countries with poor financial contractibility, and when their assets are softer or less protective of financiers.
Institutional Holdings and Payout Policy
Authors: GRINSTEIN YANIV , MICHAELY RONI
Published: May 2005

We examine the relation between institutional holdings and payout policy in U.S. public firms. We find that payout policy affects institutional holdings. Institutions avoid firms that do not pay dividends. However, among dividend‐paying firms they prefer firms that pay fewer dividends. Our evidence indicates that institutions prefer firms that repurchase shares, and regular repurchasers over nonregular repurchasers. Higher institutional holdings or a concentration of holdings do not cause firms to increase their dividends, their repurchases, or their total payout. Our results do not support models that predict that high dividends attract institutional clientele, or models that predict that institutions cause firms to increase payout.
Are Firms Underleveraged? An Examination of the Effect of Leverage on Default Probabilities
Authors: MOLINA CARLOS A.
Published: May 2005

A commonly held view in corporate finance is that firms are less leveraged than they should be, given the potentially large tax benefits of debt. In this paper, I study the effect of firms' leverage on default probabilities as represented by the firms' ratings. Using an instrumental variable approach, I find that the leverage's effect on ratings is three times stronger than it is if the endogeneity of leverage is ignored. This stronger effect results in a higher impact of leverage on the ex ante costs of financial distress, which can offset the current estimates of the tax benefits of debt.
A Comparison of Centralized and Fragmented Markets with Costly Search
Authors: YIN XIANGKANG
Published: May 2005

How does quotation transparency affect financial market performance? Biais's irrelevance proposition in 1993 shows that centralized markets yield the same expected bid–ask spreads as fragmented markets, other things equal. However, de Frutos and Manzano demonstrated in 2002 that expected spreads in fragmented markets are smaller and market participants prefer to trade in fragmented markets. This paper introduces liquidity traders' costs of searching for a better quote into the Biais model and derives opposite conclusions to these previous studies: expected spreads in centralized markets are smaller and liquidity traders prefer centralized markets, while market makers prefer fragmented markets.
Reconstruction of Hyman's Second Pacemaker
Authors: FURMAN SEYMOUR , SZARKA GEORGE , LAYVAND DMITRIY
Published: May 2005

Though journal articles and newspaper reports suggest that three Hyman pacemakers existed or were intended; a patent, descriptive photographs and a written description of an earlier model and only a photograph of a later model exist. A replica of the earlier has been made based on the patent and description, and a new replica of the second based on a critical deconstruction and analysis of the photograph of the second with a modern circuit duplicating the function of the earlier circuit. Both replicas were “operational” in that stimuli were mimicked though not delivered and neither could under any circumstances actually resuscitate the heart. The design and construction of the second Hyman pacemaker are presented.
New Simplified Technique for 3D Mapping and Ablation of Right Ventricular Outflow Tract Tachycardia
Authors: SALEEM MOEEN A. , BURKETT SCOTT , PASSMAN ROD , DIBS SAMER , ENGELSTEIN ERICA D. , KADISH ALAN H. , GOLDBERGER JEFFREY J.
Published: May 2005

Objective: To evaluate the safety and efficacy of using a circular multielectrode catheter for mapping and ablation of ventricular tachycardia (VT) or premature ventricular complexes (PVCs) from the right ventricular outflow tract (RVOT).

Background: Three‐dimensional (3D) mapping systems are commonly used for mapping and ablation of RVOT VT and PVCs. Newer catheters that are circular with multiple electrodes, such as the Lasso catheter, are capable of simultaneously recording from multiple points within a circumferential plane. Given the tubular structure of the RVOT, these catheters could be used for mapping tachycardias from the RVOT.

Methods: A retrospective cohort study of patients undergoing radiofrequency (RF) ablation of RVOT VT or PVCs was performed. In group 1 (n = 7), mapping was performed with a single ablation catheter and fluoroscopy. In group 2 (n = 10), 3D mapping using ESI (n = 9) or CARTO (n = 1) was performed. In group 3 (n = 12), mapping was performed with a circular multielectrode catheter (n = 12). All ablations were performed with 4‐mm tip catheters using RF energy.

Results: Catheter ablation for RVOT VT (n = 15) or PVCs (n = 14) was performed on 29 cases in 26 patients, 9 males. Mean age was 35.9 years. In groups 1, 2, and 3, the mean number of lesions was 17.7 ± 7.7, 13.6 ± 7.7, and 18.2 ± 22.7 and the median number of lesions was 20, 13, and 5, respectively. There were no significant differences in the number of lesions, RF time, fluoroscopy time, procedure time, and acute success rate among the three techniques. There were three complications in group 2 and one in group 3.

Conclusion: The use of a circular multielectrode catheter is as effective as the other standard available 3D mapping techniques, both in terms of procedural success and procedural characteristics. Additionally, because of the lower cost associated with using the circular multielectrode catheter approach, further evaluation should be performed to determine whether this is the most cost‐effective approach to 3D mapping and ablation of RVOT tachycardias.
Mechanism of Atrial Flutter Occurring Late After Orthotopic Heart Transplantation with Atrio‐atrial Anastomosis
Authors: MARINE JOSEPH E. , SCHUGER CLAUDIO D. , BOGUN FRANK , KALAHASTY GAUTHAM , ARNALDO FABIAN , CZERSKA BARBARA , KRISHNAN SUBRAMANIAM C.
Published: May 2005

Objective: We sought to better define the electrophysiologic mechanism of atrial flutter in patients after heart transplantation.

Background: Atrial flutter is a recognized problem in the postcardiac transplant population. The electrophysiologic basis of atrial flutter in this patient population is not completely understood.

Methods: Six patients with cardiac allografts and symptoms related to recurrent atrial flutter underwent diagnostic electrophysiologic study with electroanatomic mapping and radiofrequency catheter ablation. Comparison was made with a control nontransplant population of 11 patients with typical counterclockwise right atrial flutter.

Results: In each case, mapping showed typical counterclockwise activation of the donor‐derived portion of the right atrium, with concealed entrainment shown upon pacing in the cavotricuspid isthmus (CTI). The anastomotic suture line of the atrio‐atrial anastomosis formed the posterior barrier of the reentrant circuit. Ablation of the electrically active, donor‐derived portion of the CTI was sufficient to terminate atrial flutter and render it noninducible. Comparison with the control population showed that the electrically active portion of the CTI was significantly shorter in patients with transplant‐associated flutter and that ablation was accomplished with the same or fewer radiofrequency lesions.

Conclusions: Atrial flutter in cardiac transplant recipients is a form of typical counterclockwise, isthmus‐dependent flutter in which the atrio‐atrial anastomotic suture line forms the posterior barrier of the reentrant circuit. Ablation in the donor‐derived portion of the CTI is sufficient to create bidirectional conduction block and eliminate this arrhythmia. Ablation or surgical division of the donor CTI at the time of transplantation could prevent this arrhythmia.
Accidental Transarterial Implantation of Dual Chamber Pacemaker Leads in the Left Ventricle and the Right Coronary Artery
Authors: OVERBECK MATTHIAS , KOLB CHRISTOF , SCHMITT CLAUS , SCHÖMIG ALBERT , LANGE RÜDIGER
Published: May 2005

Implantation of permanent pacemaker systems is associated with a low rate of complications. The case of a 72‐year‐old woman is described who suffered from a cluster of complications related to permanent pacemaker implantation. Besides major bleeding, requiring transfusion and pneumothorax, malposition of both pacemaker leads in the arterial system occurred, with the atrial lead screwn into the right coronary artery, with subsequent occlusion of the vessel and the ventricle lead placed in the left ventricle.
THE BENEFITS AND RISKS OF CHILD CUSTODY EVALUATORS MAKING RECOMMENDATIONS TO THE COURT:
Authors: Stahl Philip M.
Published: May 2005

In their article, Tippins and Wittmann articulate a strong position against custody evaluators making specific custody recommendations to the court. This response will focus on my thoughts about their concepts and my belief that recommendations can be useful and appropriate some of the time, even though there may be significant problems with certain types of recommendations and problems with some evaluators.
Dynamic Modeling and Simulation of Satellite Tethered Systems
Authors: Mankala Kalyan K. , Agrawal Sunil K.
Published: May 2005

The objective of this paper is to study the dynamic simulation of a tether as it is deployed or retrieved by a winch on a satellite orbiting around earth. In an effort to understand the problem incrementally, the following three models were developed: (a) Model 1: A tether with constant length moves on earth in the plane of constant gravity; (b) Model 2: A tether is deployed from a drum on earth in the plane of constant gravity, i.e., length of the cable changes during deployment; (c) Model 3: A tether is deployed from a drum on an orbiting satellite. These models have been chosen to bring different aspects as well as levels of difficulty in the analysis. For example, in Model 1, the length of cable is fixed and the gravity direction is constant during motion. The equations of motion for this model are derived using Newton’s laws and Hamilton’s principle to show the equivalence of the two methods. In Model 2, free length of the cable changes during deployment. The changing length of the cable introduces coupled nonlinearities into the motion. Model 3 includes the orbital effect on the motion of deployed cable. Each of these three dynamic models characterized by partial differential equations are first converted to a finite number of ordinary differential equations using Ritz’s procedure and are then numerically integrated using Matlab ODE solvers.
Energy Conserving Equations of Motion for Gear Systems
Authors: Oh Sejoong , Grosh Karl , Barber James R.
Published: May 2005

A system of two meshing gears exhibits a stiffness that varies with the number of teeth in instantaneous contact and the location of the corresponding contact points. A classical Newtonian statement of the equations of motion leads to a solution that contradicts the fundamental principle of mechanics that the change in total energy in the system is equal to the work done by the external forces, unless the deformation of the teeth is taken into account in defining the direction of the instantaneous tooth interaction force. This paradox is avoided by using a Lagrange’s equations to derive the equations of motion, thus ensuring conservation of energy. This introduces nonlinear terms that are absent in the classical equations of motion. In particular, the step change in stiffness associated with the introduction of an additional tooth to contact implies a step change in strain energy and hence a corresponding step change in kinetic energy and rotational speed. The effect of these additional terms is examined by dynamic simulation, using a system of two involute spur gears as an example. It is shown that the two systems of equations give similar predictions at high rotational speeds, but they differ considerably at lower speeds. The results have implications for gear design, particularly for low speed gear sets.
Combined Expansion and Orthogonalization of Experimental Modeshapes
Authors: Halevi Y. , Morales C. A. , Inman D. J.
Published: May 2005

The paper describes a method of combined expansion and orthogonalization (CEO) of experimental modeshapes. Most model updating and error localization methods require a set of full length, orthogonal with respect to the mass matrix, eigenvectors. In practically every modal experiment, the number of measurements is less than the order of the model, and hence modeshape expansion, i.e., adding the unmeasured degrees of freedom, is required. This step is then followed by orthogonalization with respect to the mass matrix. Most current methods use two separate steps for expansion and orthogonalization, each one optimal by itself, but their combination is not optimal. The suggested method combines the two steps into one optimization problem for both steps, and minimizes a quadratic criterion. In the case of an equal number of analytical and experimental modeshapes, the problem coincides with the Procrustes problem and has a closed form solution. Otherwise the solution involves nonlinear equations. Several examples show the advantage of CEO, especially in cases where the measurements are limited either in number or in space, i.e., not spanned through the entire structure.
Nonlinear Dynamics of High-Speed Milling—Analyses, Numerics, and Experiments
Authors: Stepan Gabor , Szalai Robert , Mann Brian P. , Bayly Philip V. , Insperger Tamas , Gradisek Janez , Govekar Edvard
Published: May 2005

High-speed milling is often modeled as a kind of highly interrupted machining, when the ratio of time spent cutting to not cutting can be considered as a small parameter. In these cases, the classical regenerative vibration model, playing an essential role in machine tool vibrations, breaks down to a simplified discrete mathematical model. The linear analysis of this discrete model leads to the recognition of the doubling of the so-called instability lobes in the stability charts of the machining parameters. This kind of lobe-doubling is related to the appearance of period doubling vibrations originated in a flip bifurcation. This is a new phenomenon occurring primarily in low-immersion high-speed milling along with the Neimark-Sacker bifurcations related to the classical self-excited vibrations or Hopf bifurcations. The present work investigates the nonlinear vibrations in the case of period doubling and compares this to the well-known subcritical nature of the Hopf bifurcations in turning processes. The identification of the global attractor in the case of unstable cutting leads to contradiction between experiments and theory. This contradiction draws the attention to the limitations of the small parameter approach related to the highly interrupted cutting condition.
Low temperature, fast deposition of metallic titanium nitride films using plasma activated reactive evaporation
Authors: Montes de Oca Valero J. A. , Le Petitcorps Y. , Manaud J. P. , Chollon G. , Carrillo Romo F. J. , López M. A.
Published: May 2005

Titanium and titanium nitride thin films were deposited on silica glass and W substrates at a high coating growth rate by plasma-activated reactive evaporation (ARE). The crystal structure, preferred orientation and grain size of the coatings were determined by x-ray diffraction (XRD) technique using Cu-Kα x rays. The analysis of the coating morphology was performed by field-emission scanning electron microscopy (FE-SEM). The composition of the films was analyzed by Auger electron spectroscopy (AES) and electron-probe microanalysis (EPMA). The titanium and titanium nitride condensates were collected on a carbon-coated collodion film then characterized by transmission electron microscopy (TEM) in order to study the structures of the deposits at very short deposition times. The resistivity of the films was measured by using the four-point-probe method. The titanium coatings were found to consist of very fine particles (40nm in grain size) and to exhibit a strong (002) texture. The titanium nitride coatings were substoichiometric (TiNx,x<1), with an oxygen content ranging from 7 to 15 at. % depending on the deposition conditions. The deposits were found to exhibit a (111) preferred orientation. This behavior became stronger with coating thickness. In spite of the presence of oxygen, all the TiNx coatings obtained at low temperature and a high growth rate in this work exhibited a rather high electrical conductivity.
Role of C2F4, CF2, and ions in C4F8∕Ar plasma discharges under active oxide etch conditions in an inductively coupled GEC cell reactor
Authors: Barela Marcos J. , Anderson Harold M. , Oehrlein Gottlieb S.
Published: May 2005

Utilizing infrared diode-laser absorption spectroscopy (IRDLAS) and UV-Visible absorption spectroscopy (UV-Vis), we show that it is possible to make a near complete mass balance of etch reactants and products in a GEC inductively coupled fluorocarbon discharge while actively etching SiO2 substrates. Langmuir probe measurements were performed to measure the total ion current density. C2F4 and CF2 are shown to be the main dissociation products in a C4F8 plasma discharge. The C2F4 concentration decreases as the SiO2 etching rate increases, along with CF2 and CF radicals, suggesting a role in the SiO2 etching process. The addition of Ar to the C4F8 discharge increased the ion flux at the wafer surface, and the consumption rate of C2F4 relative to CF2. The increased ion flux enhanced the SiO2 etching rate, until at a very high degree of Ar dilution of C4F8∕Ar the etching rate became neutral limited. We also monitored SiF2 using UV-Vis absorption and CO by IRDLAS. In our work we found SiF2 and CO to be the prevalent Si and C gas phase etch products for the SiO2 etching process.
Development of low temperature silicon oxide thin films by photo-CVD for surface passivation
Authors: Mukhopadhyay Sumita , Jana Tapati , Ray Swati
Published: May 2005

Low temperature (250°C) silicon oxide (a-SiOx:H) films have been developed for surface passivation as well as antireflection coating in silicon solar cell. Films have been fabricated by ion damage free photochemical vapor decomposition technique using SiH4, N2O, and H2 gas mixture. In this paper we have reported the effect of N2O to SiH4 ratio (R) on optoelectronic and structural properties of the films. The bonding configurations of Si and H were investigated in detail by IR absorption measurement. The Si–H stretching mode supports the presence of H–(Si3-nOn)(n=0–3) structural unit, which is also present in the Si–O stretching mode. Developed silicon oxide films also have been studied on the c-Si solar cell. A substantial enhancement (11.2%) in efficiency has been achieved.
Adsorbate effects on pulsed electron diode anode thermal response
Authors: Statom T. K. , Ruebush M. , Shiffler D. A. , Schamiloglu E. , LaCour M. , Golby K.
Published: May 2005

Anode thermal response is important in determining the operation of repetitively pulsed electron diodes and the systems using them. Thermal response determines desorption, cooling requirements, and power deposition. This article describes experimental results from an electron beam diode fitted with both solid aluminum and two types of carbon-carbon fiber anodes. The temperature response is modeled and is used in conjunction with experimental data to study the effect of adsorbates on the anode. Power to the anode is determined through the material properties and modeled temperature response. Thermal properties of the anode are considered with respect to electron stimulated desorption of adsorbates on the anode as an ion source, leading to plasma development and diode impedance collapse.
Substrate Specificity and Colorimetric Assay for Recombinant TrzN Derived from
Arthrobacter aurescens
TC1
Authors: Shapir Nir , Rosendahl Charlotte , Johnson Gilbert , Andreina Marco , Sadowsky Michael J. , Wackett Lawrence P.
Published: May 2005

The TrzN protein, which is involved in
s
-triazine herbicide catabolism by
Arthrobacter aurescens
TC1, was cloned and expressed in
Escherichia coli
as a His-tagged protein. The recombinant protein was purified via nickel column chromatography. The purified TrzN protein was tested with 31
s
-triazine and pyrimidine ring compounds; 22 of the tested compounds were substrates. TrzN showed high activity with sulfur-substituted
s
-triazines and the highest activity with ametryn sulfoxide. Hydrolysis of ametryn sulfoxide by TrzN, both in vitro and in vivo, yielded a product(s) that reacted with 7-chloro-4-nitrobenz-2-oxa-1,3-diazole (NBD-Cl) to generate a diagnostic blue product. Atrazine chlorohydrolase, AtzA, did not hydrolyze ametryn sulfoxide, and no color was formed by amending those enzyme incubations with NBD-Cl. TrzN and AtzA could also be distinguished by reaction with ametryn. TrzN, but not AtzA, hydrolyzed ametryn to methylmercaptan. Methylmercaptan reacted with NBD-Cl to produce a diagnostic yellow product having an absorption maximum at 420 nm. The yellow color with ametryn was shown to selectively demonstrate the presence of TrzN, but not AtzA or other enzymes, in whole microbial cells. The present study was the first to purify an active TrzN protein in recombinant form and develop a colorimetric test for determining TrzN activity, and it significantly extends the known substrate range for TrzN.
Induction of Oxidative Stress by High Hydrostatic Pressure in
Escherichia coli
Authors: Aertsen Abram , De Spiegeleer Philipp , Vanoirbeek Kristof , Lavilla Maria , Michiels Chris W.
Published: May 2005

Using leaderless alkaline phosphatase as a probe, it was demonstrated that pressure treatment induces endogenous intracellular oxidative stress in
Escherichia coli
MG1655. In stationary-phase cells, this oxidative stress increased with the applied pressure at least up to 400 MPa, which is well beyond the pressure at which the cells started to become inactivated (200 MPa). In exponential-phase cells, in contrast, oxidative stress increased with pressure treatment up to 150 MPa and then decreased again, together with the cell counts. Anaerobic incubation after pressure treatment significantly supported the recovery of MG1655, while mutants with increased intrinsic sensitivity toward oxidative stress (
katE
,
katF
,
oxyR
,
sodAB
, and
soxS
) were found to be more pressure sensitive than wild-type MG1655. Furthermore, mild pressure treatment strongly sensitized
E. coli
toward
t-
butylhydroperoxide and the superoxide generator plumbagin. Finally, previously described pressure-resistant mutants of
E. coli
MG1655 displayed enhanced resistance toward plumbagin. In one of these mutants, the induction of endogenous oxidative stress upon high hydrostatic pressure treatment was also investigated and found to be much lower than in MG1655. These results suggest that, at least under some conditions, the inactivation of
E. coli
by high hydrostatic pressure treatment is the consequence of a suicide mechanism involving the induction of an endogenous oxidative burst.
Identification of Unique Type II Polyketide Synthase Genes in Soil
Authors: Wawrik Boris , Kerkhof Lee , Zylstra Gerben J. , Kukor Jerome J.
Published: May 2005

Many bacteria, particularly actinomycetes, are known to produce secondary metabolites synthesized by polyketide synthases (PKS). Bacterial polyketides are a particularly rich source of bioactive molecules, many of which are of potential pharmaceutical relevance. To directly access PKS gene diversity from soil, we developed degenerate PCR primers for actinomycete type II KS
α
(ketosynthase) genes. Twenty-one soil samples were collected from diverse sources in New Jersey, and their bacterial communities were compared by terminal restriction fragment length polymorphism (TRFLP) analysis of PCR products generated using bacterial 16S rRNA gene primers (27F and 1525R) as well as an actinomycete-specific forward primer. The distribution of actinomycetes was highly variable but correlated with the overall bacterial species composition as determined by TRFLP. Two samples were identified to contain a particularly rich and unique actinomycete community based on their TRFLP patterns. The same samples also contained the greatest diversity of KS
α
genes as determined by TRFLP analysis of KS
α
PCR products. KS
α
PCR products from these and three additional samples with interesting TRFLP pattern were cloned, and seven novel clades of KS
α
genes were identified. Greatest sequence diversity was observed in a sample containing a moderate number of peaks in its KS
α
TRFLP. The nucleotide sequences were between 74 and 81% identical to known sequences in GenBank. One cluster of sequences was most similar to the KS
α
involved in ardacin (glycopeptide antibiotic) production by
Kibdelosporangium aridum
. The remaining sequences showed greatest similarity to the KS
α
genes in pathways producing the angucycline-derived antibiotics simocyclinone, pradimicin, and jasomycin.
Balamuthia mandrillaris
, Free-Living Ameba and Opportunistic Agent of Encephalitis, Is a Potential Host for
Legionella pneumophila
Bacteria
Authors: Shadrach Winlet Sheba , Rydzewski Kerstin , Laube Ulrike , Holland Gudrun , Özel Muhsin , Kiderlen Albrecht F. , Flieger Antje
Published: May 2005

Balamuthia mandrillaris
is a free-living ameba and an opportunistic agent of granulomatous encephalitis in humans and other mammalian species. Other free-living amebas, such as
Acanthamoeba
and
Hartmannella
, can provide a niche for intracellular survival of bacteria, including the causative agent of Legionnaires' disease,
Legionella pneumophila.
Infection of amebas by
L. pneumophila
enhances the bacterial infectivity for mammalian cells and lung tissues. Likewise, the pathogenicity of amebas may be enhanced when they host bacteria. So far, the colonization of
B. mandrillaris
by bacteria has not been convincingly shown. In this study, we investigated whether this ameba could host
L. pneumophila
bacteria. Our experiments showed that
L. pneumophila
could initiate uptake by
B. mandrillaris
and could replicate within the ameba about 4 to 5 log cycles from 24 to 72 h after infection. On the other hand, a
dotA
mutant, known to be unable to propagate in
Acanthamoeba castellanii
, also did not replicate within
B. mandrillaris
. Approaching completion of the intracellular cycle,
L. pneumophila
wild-type bacteria were able to destroy their ameboid hosts. Observations by light microscopy paralleled our quantitative data and revealed the rounding, collapse, clumping, and complete destruction of the infected amebas. Electron microscopic studies unveiled the replication of the bacteria in a compartment surrounded by a structure resembling rough endoplasmic reticulum. The course of intracellular infection, the degree of bacterial multiplication, and the ultrastructural features of a
L. pneumophila
-infected
B. mandrillaris
ameba resembled those described for other amebas hosting
Legionella
bacteria. We hence speculate that
B. mandrillaris
might serve as a host for bacteria in its natural environment.
Influence of Dietary Zinc Oxide and Copper Sulfate on the Gastrointestinal Ecosystem in Newly Weaned Piglets
Authors: Højberg Ole , Canibe Nuria , Poulsen Hanne Damgaard , Hedemann Mette Skou , Jensen Bent Borg
Published: May 2005

Dietary doses of 2,500 ppm ZnO-Zn reduced bacterial activity (ATP accumulation) in digesta from the gastrointestinal tracts of newly weaned piglets compared to that in animals receiving 100 ppm ZnO-Zn. The amounts of lactic acid bacteria (MRS counts) and lactobacilli (Rogosa counts) were reduced, whereas coliforms (MacConkey counts) and enterococci (Slanetz counts, red colonies) were more numerous in animals receiving the high ZnO dose. Based on 16S rRNA gene sequencing, the colonies on MRS were dominated by three phylotypes, tentatively identified as
Lactobacillus amylovorus
(OTU171),
Lactobacillus reuteri
(OTU173), and
Streptococcus alactolyticus
(OTU180). The colonies on Rogosa plates were dominated by the two
Lactobacillus
phylotypes only. Terminal restriction fragment length polymorphism analysis supported the observations of three phylotypes of lactic acid bacteria dominating in piglets receiving the low ZnO dose and of coliforms and enterococci dominating in piglets receiving the high ZnO dose. Dietary doses of 175 ppm CuSO
4
-Cu also reduced MRS and Rogosa counts of stomach contents, but for these animals, the numbers of coliforms were reduced in the cecum and the colon. The influence of ZnO on the gastrointestinal microbiota resembles the working mechanism suggested for some growth-promoting antibiotics, namely, the suppression of gram-positive commensals rather than potentially pathogenic gram-negative organisms. Reduced fermentation of digestible nutrients in the proximal part of the gastrointestinal tract may render more energy available for the host animal and contribute to the growth-promoting effect of high dietary ZnO doses. Dietary CuSO
4
inhibited the coliforms and thus potential pathogens as well, but overall the observed effect of CuSO
4
was limited compared to that of ZnO.
Quantitative Real-Time PCR Assays To Identify and Quantify Fecal
Bifidobacterium
Species in Infants Receiving a Prebiotic Infant Formula
Authors: Haarman Monique , Knol Jan
Published: May 2005

A healthy intestinal microbiota is considered to be important for priming of the infants' mucosal and systemic immunity. Breast-fed infants typically have an intestinal microbiota dominated by different
Bifidobacterium
species. It has been described that allergic infants have different levels of specific
Bifidobacterium
species than healthy infants. For the accurate quantification of
Bifidobacterium adolescentis
,
Bifidobacterium angulatum
,
Bifidobacterium bifidum
,
Bifidobacterium breve
,
Bifidobacterium catenulatum
,
Bifidobacterium dentium
,
Bifidobacterium infantis
, and
Bifidobacterium longum
in fecal samples, duplex 5′ nuclease assays were developed. The assays, targeting rRNA gene intergenic spacer regions, were validated and compared with conventional PCR and fluorescent in situ hybridization methods. The 5′ nuclease assays were subsequently used to determine the relative amounts of different
Bifidobacterium
species in fecal samples from infants receiving a standard formula or a standard formula supplemented with galacto- and fructo-oligosaccharides (OSF). A breast-fed group was studied in parallel as a reference. The results showed a significant increase in the total amount of fecal bifidobacteria (54.8% ± 9.8% to 73.4% ± 4.0%) in infants receiving the prebiotic formula (OSF), with a diversity of
Bifidobacterium
species similar to breast-fed infants. The intestinal microbiota of infants who received a standard formula seems to resemble a more adult-like distribution of bifidobacteria and contains relatively more
B. catenulatum
and
B. adolescentis
(2.71% ± 1.92% and 8.11% ± 4.12%, respectively, versus 0.15% ± 0.11% and 1.38% ± 0.98% for the OSF group). In conclusion, the specific prebiotic infant formula used induces a fecal microbiota that closely resembles the microbiota of breast-fed infants also at the level of the different
Bifidobacterium
species.
Influence of Top-Down and Bottom-Up Manipulations on the R-BT065 Subcluster of β-Proteobacteria, an Abundant Group in Bacterioplankton of a Freshwater Reservoir
Authors: Šimek Karel , Horňák Karel , Jezbera Jan , Mašín Michal , Nedoma Jiří , Gasol Josep M. , Schauer Michael
Published: May 2005

We studied the effects of nutrient availability and protistan grazing on bacterial dynamics and community composition (BCC) in different parts of the canyon-shaped Římov reservoir (Czech Republic). The effects of protistan grazing on BCC were examined using a size fractionation approach. Water from the dam area with only bacteria (<0.8 μm), bacteria and heterotrophic nanoflagellates (<5 μm), or whole water were incubated in situ inside dialysis bags. Top-down or predator manipulations (size fractionation) were also combined with bottom-up or resource manipulations, i.e., transplantation of samples to the middle and upper inflow parts of the reservoir with increased phosphorus availability. Significant genotypic shifts in BCC occurred with transplantation as indicated by denaturing gradient gel electrophoresis. Using different probes for fluorescence in situ hybridization, we found that 10 to 50% of total bacteria were members of the phylogenetically small cluster of β-proteobacteria (targeted with the probe R-BT065). These rod-shaped cells of very uniform size were vulnerable to predation but very fast growing and responded markedly to the different experimental manipulations. In all the grazer-free treatments, the members of the R-BT065 cluster showed the highest net growth rates of all studied bacterial groups. Moreover, their relative abundance was highly correlated with bacterial bulk parameters and proportions of bacteria with high nucleic acid (HNA) content. In contrast, increasing protistan bacterivory yielded lower proportions of R-BT065-positive and HNA bacteria substituted by increasing proportions of the class
Actinobacteria
, which profited from the enhanced protistan bacterivory.
Potential Role of
Diploscapter
sp. Strain LKC25, a Bacterivorous Nematode from Soil, as a Vector of Food-Borne Pathogenic Bacteria to Preharvest Fruits and Vegetables
Authors: Gibbs Daunte S. , Anderson Gary L. , Beuchat Larry R. , Carta Lynn K. , Williams Phillip L.
Published: May 2005

Diploscapter
, a thermotolerant, free-living soil bacterial-feeding nematode commonly found in compost, sewage, and agricultural soil in the United States, was studied to determine its potential role as a vehicle of
Salmonella enterica
serotype Poona, enterohemorrhagic
Escherichia coli
O157:H7, and
Listeria monocytogenes
in contaminating preharvest fruits and vegetables. The ability of
Diploscapter
sp. strain LKC25 to survive on agar media, in cow manure, and in composted turkey manure and to be attracted to, ingest, and disperse food-borne pathogens inoculated into soil or a mixture of soil and composted turkey manure was investigated.
Diploscapter
sp. strain LKC25 survived and reproduced in lawns of
S. enterica
serotype Poona,
E. coli
O157:H7, and
L. monocytogenes
on agar media and in cow manure and composted turkey manure. Attraction of
Diploscapter
sp. strain LKC25 to colonies of pathogenic bacteria on tryptic soy agar within 10, 20, 30, and 60 min and 24 h was determined. At least 85% of the worms initially placed 0.5 to 1 cm away from bacterial colonies migrated to the colonies within 1 h. Within 24 h, ≥90% of the worms were embedded in colonies. The potential of
Diploscapter
sp. strain LKC25 to shed pathogenic bacteria after exposure to bacteria inoculated into soil or a mixture of soil and composted turkey manure was investigated. Results indicate that
Diploscapter
sp. strain LKC25 can shed pathogenic bacteria after exposure to pathogens in these milieus. They also demonstrate its potential to serve as a vector of food-borne pathogenic bacteria in soil, with or without amendment with compost, to the surface of preharvest fruits and vegetables in contact with soil.
Antimicrobial Resistance Profiles of
Campylobacter jejuni
Isolates from Wild Birds in Sweden
Authors: Waldenström Jonas , Mevius Dik , Veldman Kees , Broman Tina , Hasselquist Dennis , Olsen Björn
Published: May 2005

In order to determine the occurrence and frequency of resistant strains of the bacterium
Campylobacter jejuni
and to establish baseline MICs in isolates from an environmental reservoir, the resistance profiles of 10 antimicrobial substances were determined for 137
C. jejuni
isolates from wild birds in Sweden. Observed MICs were generally low, with only low to moderate incidence of resistance to the tested compounds. One isolate, however, was resistant to nalidixic acid and ciprofloxacin, indicating that quinolone-resistant genotypes of
C. jejuni
have the potential to spread to wild bird hosts.
Heterologous Expression of Novobiocin and Clorobiocin Biosynthetic Gene Clusters
Authors: Eustáquio Alessandra S. , Gust Bertolt , Galm Ute , Li Shu-Ming , Chater Keith F. , Heide Lutz
Published: May 2005

A method was developed for the heterologous expression of biosynthetic gene clusters in different
Streptomyces
strains and for the modification of these clusters by single or multiple gene replacements or gene deletions with unprecedented speed and versatility. λ-Red-mediated homologous recombination was used for genetic modification of the gene clusters, and the attachment site and integrase of phage φC31 were employed for the integration of these clusters into the heterologous hosts. This method was used to express the gene clusters of the aminocoumarin antibiotics novobiocin and clorobiocin in the well-studied strains
Streptomyces coelicolor
and
Streptomyces lividans
, which, in contrast to the natural producers, can be easily genetically manipulated.
S. coelicolor
M512 derivatives produced the respective antibiotic in yields comparable to those of natural producer strains, whereas
S. lividans
TK24 derivatives were at least five times less productive. This method could also be used to carry out functional investigations. Shortening of the cosmids' inserts showed which genes are essential for antibiotic production.
Heme-Dependent Rubber Oxygenase RoxA ofXanthomonassp. Cleaves the Carbon Backbone of Poly(cis-1,4-Isoprene) by a Dioxygenase Mechanism
Authors: Braaz Reinhard , Armbruster Wolfgang , Jendrossek Dieter
Published: May 2005

Oxidative cleavage of poly(cis-1,4-isoprene) by rubber oxygenase RoxA purified fromXanthomonassp. was investigated in the presence of different combinations of16O2,18O2, H216O, and H218O. 12-Oxo-4,8-dimethyl-trideca-4,8-diene-1-al (ODTD;m/z236) was the main cleavage product in the absence of18O-compounds. Incorporation of one18O atom in ODTD was found if the cleavage reaction was performed in the presence of18O2and H216O. Incubation of poly(cis-1,4-isoprene) (with RoxA) or of isolated unlabeled ODTD (without RoxA) with H218O in the presence of16O2indicated that the carbonyl oxygen atoms of ODTD significantly exchanged with oxygen atoms derived from water. The isotope exchange was avoided by simultaneous enzymatic reduction of both carbonyl functions of ODTD to the corresponding dialcohol (12-hydroxy-4,8-dimethyl-trideca-4,8-diene-1-ol (HDTD;m/z240) during RoxA-mediated in vitro cleavage of poly(cis-1,4-isoprene). In the presence of18O2, H216O, and alcohol dehydrogenase/NADH, incorporation of two atoms of18O into the reduced metabolite HDTD was found (m/z244), revealing that RoxA cleaves rubber by a dioxygenase mechanism. Based on the labeling results and the presence of two hemes in RoxA, a model of the enzymatic cleavage mechanism of poly(cis-1,4-isoprene) is proposed.
Uptake of CdSe and CdSe/ZnS Quantum Dots into Bacteria via Purine-Dependent Mechanisms
Authors: Kloepfer J. A. , Mielke R. E. , Nadeau J. L.
Published: May 2005

Quantum dots (QDs) rendered water soluble for biological applications are usually passivated by several inorganic and/or organic layers in order to increase fluorescence yield. However, these coatings greatly increase the size of the particle, making uptake by microorganisms impossible. We find that adenine- and AMP-conjugated QDs are able to label bacteria only if the particles are <5 nm in diameter. Labeling is dependent upon purine-processing mechanisms, as mutants lacking single enzymes demonstrate a qualitatively different signal than do wild-type strains. This is shown for two example species, one gram negative and one gram positive. Wild-type
Bacillus subtilis
incubated with QDs conjugated to adenine are strongly fluorescent; very weak signal is seen in mutant cells lacking either adenine deaminase or adenosine phosphoribosyltransferase. Conversely, QD-AMP conjugates label mutant strains more efficiently than the wild type. In
Escherichia coli
, QD conjugates are taken up most strongly by adenine auxotrophs and are extruded from the cells over a time course of hours. No fluorescent labeling is seen in killed bacteria or in the presence of EDTA or an excess of unlabeled adenine, AMP, or hypoxanthine. Spectroscopy and electron microscopy suggest that QDs of <5 nm can enter the cells whole, probably by means of oxidative damage to the cell membrane which is aided by light.