bluuebunny/crossref_metadata_2025 huggingface dataset:
Peripheral chemoreceptor inputs to retrotrapezoid nucleus (RTN) CO2‐sensitive neurons in rats
Authors: Takakura Ana Carolina Thomaz , Moreira Thiago Santos , Colombari Eduardo , West Gavin H. , Stornetta Ruth L. , Guyenet Patrice G.
Published: February 2006

The rat retrotrapezoid nucleus (RTN) contains pH‐sensitive neurons that are putative central chemoreceptors. Here, we examined whether these neurons respond to peripheral chemoreceptor stimulation and whether the input is direct from the solitary tract nucleus (NTS) or indirect via the respiratory network. A dense neuronal projection from commissural NTS (commNTS) to RTN was revealed using the anterograde tracer biotinylated dextran amine (BDA). Within RTN, 51% of BDA‐labelled axonal varicosities contained detectable levels of vesicular glutamate transporter‐2 (VGLUT2) but only 5% contained glutamic acid decarboxylase‐67 (GAD67). Awake rats were exposed to hypoxia (n= 6) or normoxia (n= 5) 1 week after injection of the retrograde tracer cholera toxin B (CTB) into RTN. Hypoxia‐activated neurons were identified by the presence of Fos‐immunoreactive nuclei. CommNTS neurons immunoreactive for both Fos and CTB were found only in hypoxia‐treated rats. VGLUT2 mRNA was detected in 92 ± 13% of these neurons whereas only 12 ± 9% contained GAD67 mRNA. In urethane–chloralose‐anaesthetized rats, bilateral inhibition of the RTN with muscimol eliminated the phrenic nerve discharge (PND) at rest, during hyperoxic hypercapnia (10% CO2), and during peripheral chemoreceptor stimulation (hypoxia and/or i.v. sodium cyanide, NaCN). RTN CO2‐activated neurons were recorded extracellularly in anaesthetized intact or vagotomized rats. These neurons were strongly activated by hypoxia (10–15% O2; 30 s) or by NaCN. Hypoxia and NaCN were ineffective in rats with carotid chemoreceptor denervation. Bilateral injection of muscimol into the ventral respiratory column 1.5 mm caudal to RTN eliminated PND and the respiratory modulation of RTN neurons. Muscimol did not change the threshold and sensitivity of RTN neurons to hyperoxic hypercapnia nor their activation by peripheral chemoreceptor stimulation. In conclusion, RTN neurons respond to brain P presumably via their intrinsic chemosensitivity and to carotid chemoreceptor activation via a direct glutamatergic pathway from commNTS that bypasses the respiratory network. RTN neurons probably contribute a portion of the chemical drive to breathe.
Limited oxygen diffusion accelerates fatigue development in mouse skeletal muscle
Authors: Zhang Shi‐Jin , Bruton Joseph D. , Katz Abram , Westerblad Håkan
Published: February 2006

Isolated whole skeletal muscles fatigue more rapidly than isolated single muscle fibres. We have now employed this difference to study mechanisms of skeletal muscle fatigue. Isolated whole soleus and extensor digitorum longus (EDL) muscles were fatigued by repeated tetanic stimulation while measuring force production. Neither application of 10 mm lactic acid nor increasing the [K+] of the bath solution from 5 to 10 mm had any significant effect on the rate of force decline during fatigue induced by repeated brief tetani. Soleus muscles fatigued slightly faster during continuous tetanic stimulation in 10 mm[K+]. Inhibition of mitochondrial respiration with cyanide resulted in a faster fatigue development in both soleus and EDL muscles. Single soleus muscle fibres were fatigued by repeated tetani while measuring force and myoplasmic free [Ca2+] ([Ca2+]i). Under control conditions, the single fibres were substantially more fatigue resistant than the whole soleus muscles; tetanic force at the end of a series of 100 tetani was reduced by about 10% and 50%, respectively. However, in the presence of cyanide, fatigue developed at a similar rate in whole muscles and single fibres, and tetanic force at the end of fatiguing stimulation was reduced by ∼80%. The force decrease in the presence of cyanide was associated with a ∼50% decrease in tetanic [Ca2+]i, compared with an increase of ∼20% without cyanide. In conclusion, lactic acid or [K+] has little impact on fatigue induced by repeated tetani, whereas hypoxia speeds up fatigue development and this is mainly due to an impaired Ca2+ release from the sarcoplasmic reticulum.
Fetal programming of autonomic and HPA function: do people who were small babies have enhanced stress responses?
Authors: Phillips David I. W. , Jones Alexander
Published: February 2006

Studies in several species have demonstrated that an adverse early environment can influence the development of the autonomic nervous system and hypothalamic–pituitary–adrenal (HPA) axis. The autonomic nervous system and HPA axis are key components of the neuroendocrine response to stress and many of these animal models show altered biological responses to stress. Recent research now suggests that these processes operate in humans. An adverse early environment, as evidenced by reduced birth or infant weight, is associated with enhanced autonomic and HPA responses to experimental psychological stress. However, there appear to be marked sex differences in the mechanisms involved. Epidemiological studies demonstrate that physiological changes in these neuroendocrine systems may predispose to cardiovascular disease through their influence on risk factors such as plasma glucose and lipid concentrations and blood pressure. Thus the combination of enhanced stress susceptibility and the psychosocial stressors to which people are exposed may be an important component of the disease risk in human populations.
Mechanism for bandpass frequency characteristic in distortion product otoacoustic emission generation
Authors: Fahey P. F. , Stagner B. B. , Martin G. K.
Published: February 2006

It is commonly observed that the levels of the 2f1-f2 and the other mf1-nf2 (m=n+1=integer) distortion product otoacoustic emissions (DPOAEs) initially increase in level for fixed f2 as f1→f2, starting at f1⪡f2, and then begin to decrease. When these DPOAE levels are plotted as a function of either the distortion product frequency (fdp) or f2∕f1 the curve has an approximate bandpass shape. It has been hypothesized that this effect is due to (1) a second filter, (2) suppression of distortion generation by the primary tones, (3) reemission of distortion products from the distortion product place (on the basilar membrane), (4) the presence of an even order nonlinearity, and (5) cancellation of the DPOAE due to the vector addition of multiple sources of distortion product. In this study distortion products were produced with f1 sweep conditions where there would be minimal vector cancellation of multiple DPOAE sources. It was observed that under this condition, there is no or minimal bandpass shape of the DPOAE generation curve. Therefore, the data support the hypothesis that the bandpass shape obtained with traditional f1 sweeps is due to vector cancellation from multiple sources.
Experimental comparison between speech transmission index, rapid speech transmission index, and speech intelligibility index
Authors: Larm Petra , Hongisto Valtteri
Published: February 2006

During the acoustical design of, e.g., auditoria or open-plan offices, it is important to know how speech can be perceived in various parts of the room. Different objective methods have been developed to measure and predict speech intelligibility, and these have been extensively used in various spaces. In this study, two such methods were compared, the speech transmission index (STI) and the speech intelligibility index (SII). Also the simplification of the STI, the room acoustics speech transmission index (RASTI), was considered. These quantities are all based on determining an apparent speech-to-noise ratio on selected frequency bands and summing them using a specific weighting. For comparison, some data were needed on the possible differences of these methods resulting from the calculation scheme and also measuring equipment. Their prediction accuracy was also of interest. Measurements were made in a laboratory having adjustable noise level and absorption, and in a real auditorium. It was found that the measurement equipment, especially the selection of the loudspeaker, can greatly affect the accuracy of the results. The prediction accuracy of the RASTI was found acceptable, if the input values for the prediction are accurately known, even though the studied space was not ideally diffuse.
A cross-spectrum weighting algorithm for speech enhancement and array processing: Combining phase-shift information and stationary signal properties
Authors: Schwetz Ingo , Gruhler Gerhard , Obermayer Klaus
Published: February 2006

In this paper, a gain function for noise cancellation with a two-channel microphone array is presented. This gain function combines ideas from one- and multichannel algorithms. It is developed using a minimum mean square error estimator for the amplitude of the speech signal from the cross spectrum between two microphone signals. To consider speech pauses and the absence of spectral components of the speech, an extension of this gain function is presented. The performance of the overall gain function is shown in terms of the cancellation of (diffuse) driving noise as well as the cancellation of an interfering speech signal, both recorded in a car.
Nonoptimal propagation of advertisement calls of midwife toads in Iberian habitats
Authors: Penna Mario , Márquez Rafael , Bosch Jaime , Crespo Eduardo G.
Published: February 2006

This study compares the efficiency of transmission of the advertisement calls of two species of midwife toads, Alytes cisternasii and A. obstetricans, in both native and non-native habitats in the Iberian Peninsula. Recorded calls of both species and pure tones were broadcast at ten sites native to either the relatively small A. cisternasii or the larger A. obstetricans. A large variation in the patterns of excess attenuation between localities was observed for calls measured at distances of 0.5to8m from a loudspeaker. However, attenuation rates were higher for calls of both species in habitats of A. obstetricans relative to habitats of A. cisternasii. The calls of A. obstetricans experienced lower attenuation rates than those of A. cisternasii in both conspecific and heterospecific localities. Thus, although A. cisternasii occupies habitats more favorable for sound transmission, its advertisement call spectrum is not optimized for these habitats; the calls of A. obstetricans suffer less attenuation in A. cisternasii habitats. This result argues against the notion that spectral features of the calls are adapted to enhance transmission efficiency in natural habitats, and suggests that differences in call dominant frequency between the two species result from constraints imposed by selection on body size.
Demonstration at sea of the decomposition-of-the-time-reversal-operator technique
Authors: Gaumond Charles F. , Fromm David M. , Lingevitch Joseph F. , Menis Richard , Edelmann Geoffrey F. , Calvo David C. , Kim Elisabeth
Published: February 2006

This paper presents a derivation of the time reversal operator decomposition (DORT) using the sonar equation. DORT is inherently a frequency-domain technique, but the derivation is shown in the time-frequency domain to preserve range resolution. The magnitude of the singular values is related to sonar equation parameters. The time spreading of the time-domain back-propagation image is also related to the sonar equation. Noise-free, noise-only, and signal-plus-noise data are considered theoretically. Contamination of the echo singular component by noise is shown quantitatively to be very small at a signal-to-noise ratio of 0dB. Results are shown from the TREX-04 experiment during April 22 to May 4, 2004 in 94m deep, shallow water southwest of the Hudson Canyon. Rapid transmission of short, 500Hz wide linear frequency modulated beams with center frequencies of 750, 1250, 1750, 2250, 2750, and 3250Hz are used. Degradation caused by a lack of time invariance is found to be small at 750Hz and nearly complete at 3250Hz. A back-propagation image at 750Hz shows focusing on the echo repeater. These results are discussed with comments about further research.
Absolute pitch among American and Chinese conservatory students: Prevalence differences, and evidence for a speech-related critical period
Authors: Deutsch Diana , Henthorn Trevor , Marvin Elizabeth , Xu HongShuai
Published: February 2006

Absolute pitch is extremely rare in the U.S. and Europe; this rarity has so far been unexplained. This paper reports a substantial difference in the prevalence of absolute pitch in two normal populations, in a large-scale study employing an on-site test, without self-selection from within the target populations. Music conservatory students in the U.S. and China were tested. The Chinese subjects spoke the tone language Mandarin, in which pitch is involved in conveying the meaning of words. The American subjects were nontone language speakers. The earlier the age of onset of musical training, the greater the prevalence of absolute pitch; however, its prevalence was far greater among the Chinese than the U.S. students for each level of age of onset of musical training. The findings suggest that the potential for acquiring absolute pitch may be universal, and may be realized by enabling infants to associate pitches with verbal labels during the critical period for acquisition of features of their native language.
A numerical investigation of the influence of windscreens on measurement of sound intensity
Authors: Juhl Peter , Jacobsen Finn
Published: February 2006

Sound intensity probes are often used with windscreens to minimize the effect of noise caused by airflow. A theoretical and experimental study of the effect of windscreens on p-p intensity probes published ten years ago concluded that windscreens give rise to underestimation of the sound intensity at low frequencies in strongly reactive sound fields. The theoretical part of this study was based on the assumption of a windscreen of infinite extent. In this paper windscreens of realistic size and shape are dealt with by means of a coupled boundary element model for the windscreen and the surrounding air. The error of the estimated intensity caused by the windscreen is calculated under a number of sound field conditions of varying reactivity. It is shown that the resulting error can be much larger than the intensity itself in a very reactive sound field. It is also shown that the shape and size of the windscreen has a significant influence on the error.
Phase effects in masking by harmonic complexes in birds
Authors: Lauer Amanda M. , Dooling Robert J. , Leek Marjorie R. , Lentz Jennifer J.
Published: February 2006

Masking by harmonic complexes depends on the frequency content of the masker and its phase spectrum. Harmonic complexes created with negative Schroeder phases (component phases decreasing with increasing frequency) produce more masking than those with positive Schroeder phases (increasing phase) in humans, but not in birds. The masking differences in humans have been attributed to interactions between the masker phase spectrum and the phase characteristic of the basilar membrane. In birds, the similarity in masking by positive and negative Schroeder maskers, and reduced masking by cosine-phase maskers (constant phase), suggests a phase characteristic that does not change much along the basilar papilla. To evaluate this possibility, the rate of phase change across masker bandwidth was varied by systematically altering the Schroeder algorithm. Humans and three species of birds detected tones added in phase to a single component of a harmonic complex. As observed in earlier studies, the minimum amount of masking in humans occurred for positive phase gradients. However, minimum masking in birds occurred for a shallow negative phase gradient. These results suggest a cochlear delay in birds that is reduced compared to that found in humans, probably related to the shorter avian basilar epithelia.
Frequency transposition around dead regions simulated with a noiseband vocoder
Authors: Başkent Deniz , Shannon Robert V.
Published: February 2006

In sensorineural hearing loss, damage to inner hair cells or the auditory nerve may result in dead regions in the cochlea, where the information transmission is disrupted. In cochlear implants, similar dead regions might appear if the spiral ganglia do not function. Shannon et al. [J. Assoc. Res. Otolaryngol. 3, 185–199 (2002)] simulated dead regions of varying size and location using a noiseband vocoder. Phoneme recognition by normal-hearing subjects was measured under two frequency-place mapping conditions: the frequency range corresponding to the dead region was (1) removed or (2) reassigned to bands adjacent to the dead region to simulate the off-frequency stimulation of neurons at the edge of a dead region. The present study extends the results of Shannon et al. by including a frequency transposition mapping condition, where the overall acoustic input frequency range was distributed over the entire remaining nondead region. The frequency transposed map provided more acoustic information when compared to the map with the frequency range corresponding to the dead region removed. However, speech perception did not improve for many simulated dead region conditions, possibly due to the spectral distortions in the frequency-place mapping.
A method for structural vibration characterization in the midfrequency region
Authors: Shepard W. Steve , Liu Yi
Published: February 2006

The ability to characterize elastic structures at higher frequencies in a meaningful and convenient manner has been a topic of much interest. Many analytical or experimental approaches incorporate the use of lumped quantities to represent critical system characteristics at discrete locations. When the excitation frequency increases, the structural wavelength becomes comparable to the dimensions of that contact region and the point-quantity assumption is no longer valid. To provide a similar means for describing structures at higher frequencies, the work described here reformulates the traditional four-pole method in terms of quantities defined over planes. Spatial variations of the important response characteristics across the connecting regions can be considered. As for the four-pole method, the approach has the advantage of providing building blocks for simple structures in order to represent more complex structures. Three application examples are presented. One example involves modeling a simple vibration isolator. In another example, the impact of having spatial functions that extend over only a portion of the structure is studied. Finally, a two-dimensional structure is examined using a finite element approach. In using the approach, additional insight can be gained that cannot be easily found using typical finite element method analysis results.
Spherically focused capacitive-film, air-coupled ultrasonic transducer
Authors: Song Junho , Chimenti D. E. , Holland Stephen D.
Published: February 2006

A spherically focused (no mirrors) capacitive-film, air-coupled ultrasonic transducer, constructed using a spherically deformed backplate and metalized polymer film, has been designed, fabricated, and its performance characterized. A 1cm diameter device has a center frequency of 805kHz and a 6dB bandwidth of 760kHz. Comparisons of field strength in the focal zone with theoretical calculations for a spherically focused piston show that the device achieves diffraction-limited focusing. The nominal focal point of 25mm lies within 0.01mm of the calculated value for this device.
Two-dimensional problems of diffraction by finite collinear structures
Authors: Budaev Bair V. , Bogy David B.
Published: February 2006

Two closely related problems of diffraction are solved by use of the probabilistic random walk method. The first concerns diffraction by a boundary of a half-space with a piecewise constant boundary impedance, and the second solves the problem of diffraction by a finite segment with different impedances on its sides. The solutions are represented as superpositions of predefined geometric fields with several diffracted fields, which are explicitly represented as mathematical expectations of certain functionals along the trajectories of specified random motions running across multisheet analytic manifolds associated with the boundary conditions. Numerical examples confirm the feasibility of the solutions.
Scattering by quasi-symmetric pipes
Authors: Carley Michael
Published: February 2006

A hypersingular boundary integral method for the prediction of radiation from a straight circular pipe with arbitrary end profile has been developed. The technique represents an extension of established procedures for axisymmetric pipes with the addition of recent advances in special function and quadrature theory to simplify the implementation. The resulting code is applied to two sample problems: first, the prediction of radiation of a plane wave mode from a pipe with its ends cut by an inclined plane, representing the “scarfed intake” proposed for reduction of aircraft engine noise. Second, the method is used to examine scattering of an incident azimuthal mode by a multi-lobed profile, characteristic of the “chevron” nozzles proposed for jet engine exhausts.
Geometric sound propagation through an inhomogeneous and moving ocean: Scattering by small scale internal wave currents
Authors: Colosi John A.
Published: February 2006

Ray equations appropriate for ocean acoustic propagation through an inhomogeneous and moving ocean are put forth with applications to sound scattering by internal waves. The result reveals the important role played by range dependent horizontal current shear. The Garrett–Munk internal wave model and observations of upper ocean shear and sound speed fluctuations suggest that inertial frequency upper ocean shear may play a comparable role to internal wave induced sound speed fluctuations as a source of upper ocean acoustic scattering.
Measuring the critical band for speech
Authors: Healy Eric W. , Bacon Sid P.
Published: February 2006

The current experiments were designed to measure the frequency resolution employed by listeners during the perception of everyday sentences. Speech bands having nearly vertical filter slopes and narrow bandwidths were sharply partitioned into various numbers of equal log- or ERBN-width subbands. The temporal envelope from each partition was used to amplitude modulate a corresponding band of low-noise noise, and the modulated carriers were combined and presented to normal-hearing listeners. Intelligibility increased and reached asymptote as the number of partitions increased. In the mid- and high-frequency regions of the speech spectrum, the partition bandwidth corresponding to asymptotic performance matched current estimates of psychophysical tuning across a number of conditions. These results indicate that, in these regions, the critical band for speech matches the critical band measured using traditional psychoacoustic methods and nonspeech stimuli. However, in the low-frequency region, partition bandwidths at asymptote were somewhat narrower than would be predicted based upon psychophysical tuning. It is concluded that, overall, current estimates of psychophysical tuning represent reasonably well the ability of listeners to extract spectral detail from running speech.
A Cortical Region Consisting Entirely of Face-Selective Cells
Authors: Tsao Doris Y. , Freiwald Winrich A. , Tootell Roger B. H. , Livingstone Margaret S.
Published: February 2006

Face perception is a skill crucial to primates. In both humans and macaque monkeys, functional magnetic resonance imaging (fMRI) reveals a system of cortical regions that show increased blood flow when the subject views images of faces, compared with images of objects. However, the stimulus selectivity of single neurons within these fMRI-identified regions has not been studied. We used fMRI to identify and target the largest face-selective region in two macaques for single-unit recording. Almost all (97%) of the visually responsive neurons in this region were strongly face selective, indicating that a dedicated cortical area exists to support face processing in the macaque.
HIV Decline Associated with Behavior Change in Eastern Zimbabwe
Authors: Gregson Simon , Garnett Geoffrey P. , Nyamukapa Constance A. , Hallett Timothy B. , Lewis James J. C. , Mason Peter R. , Chandiwana Stephen K. , Anderson Roy M.
Published: February 2006

Few sub-Saharan African countries have witnessed declines in HIV prevalence, and only Uganda has compelling evidence for a decline founded on sexual behavior change. We report a decline in HIV prevalence in eastern Zimbabwe between 1998 and 2003 associated with sexual behavior change in four distinct socioeconomic strata. HIV prevalence fell most steeply at young ages—by 23 and 49%, respectively, among men aged 17 to 29 years and women aged 15 to 24 years—and in more educated groups. Sexually experienced men and women reported reductions in casual sex of 49 and 22%, respectively, whereas recent cohorts reported delayed sexual debut. Selective AIDS-induced mortality contributed to the decline in HIV prevalence.
The Global Impact of Scaling Up HIV/AIDS Prevention Programs in Low- and Middle-Income Countries
Authors: Stover John , Bertozzi Stefano , Gutierrez Juan-Pablo , Walker Neff , Stanecki Karen A. , Greener Robert , Gouws Eleanor , Hankins Catherine , Garnett Geoff P. , Salomon Joshua A. , Boerma J. Ties , De Lay Paul , Ghys Peter D.
Published: February 2006

A strong, global commitment to expanded prevention programs targeted at sexual transmission and transmission among injecting drug users, started now, could avert 28 million new HIV infections between 2005 and 2015. This figure is more than half of the new infections that might otherwise occur during that period in 125 low- and middle-income countries. Although preventing these new infections would require investing about U.S.$122 billion over this period, it would reduce future needs for treatment and care. Our analysis suggests that it will cost about U.S.$3900 to prevent each new infection, but that this will produce a savings of U.S.$4700 in forgone treatment and care costs. Thus, greater spending on prevention now would not only prevent more than half the new infections that would occur from 2005 to 2015 but would actually produce a net financial saving as future costs for treatment and care are averted.
Fish Population and Behavior Revealed by Instantaneous Continental Shelf-Scale Imaging
Authors: Makris Nicholas C. , Ratilal Purnima , Symonds Deanelle T. , Jagannathan Srinivasan , Lee Sunwoong , Nero Redwood W.
Published: February 2006

Until now, continental shelf environments have been monitored with highly localized line-transect methods from slow-moving research vessels. These methods significantly undersample fish populations in time and space, leaving an incomplete and ambiguous record of abundance and behavior. We show that fish populations in continental shelf environments can be instantaneously imaged over thousands of square kilometers and continuously monitored by a remote sensing technique in which the ocean acts as an acoustic waveguide. The technique has revealed the instantaneous horizontal structural characteristics and volatile short-term behavior of very large fish shoals, containing tens of millions of fish and stretching for many kilometers.
Exposed Water Ice Deposits on the Surface of Comet 9P/Tempel 1
Authors: Sunshine J. M. , A'Hearn M. F. , Groussin O. , Li J.-Y. , Belton M. J. S. , Delamere W. A. , Kissel J. , Klaasen K. P. , McFadden L. A. , Meech K. J. , Melosh H. J. , Schultz P. H. , Thomas P. C. , Veverka J. , Yeomans D. K. , Busko I. C. , Desnoyer M. , Farnham T. L. , Feaga L. M. , Hampton D. L. , Lindler D. J. , Lisse C. M. , Wellnitz D. D.
Published: February 2006

We report the direct detection of solid water ice deposits exposed on the surface of comet 9P/Tempel 1, as observed by the Deep Impact mission. Three anomalously colored areas are shown to include water ice on the basis of their near-infrared spectra, which include diagnostic water ice absorptions at wavelengths of 1.5 and 2.0 micrometers. These absorptions are well modeled as a mixture of nearby non-ice regions and 3 to 6% water ice particles 10 to 50 micrometers in diameter. These particle sizes are larger than those ejected during the impact experiment, which suggests that the surface deposits are loose aggregates. The total area of exposed water ice is substantially less than that required to support the observed ambient outgassing from the comet, which likely has additional source regions below the surface.
Reading with Selection in Mind
Authors: Fromm Harold
Published: February 2006

The Literary Animal
. Evolution and the Nature of Narrative.
Jonathan Gottschall and David Sloan Wilson, Eds.
. Northwestern University Press, Evanston, IL, 2005. 332 pp. $79.95. ISBN 0-8101-2286-3. Paper, $29.95. ISBN 0-8101-2287-1.

The contributors, who include both literary analysts and evolutionary biologists, offer a Darwinian perspective on the creation, reception, and interpretation of literature.
Bioassay for Nisin in Milk, Processed Cheese, Salad Dressings, Canned Tomatoes, and Liquid Egg Products
Authors: Hakovirta J. , Reunanen J. , Saris P. E. J.
Published: February 2006

A sensitive nisin quantification bioassay was constructed, based onLactococcus lactischromosomally encoding the nisin regulatory proteins NisK and NisR and a plasmid with a green fluorescent protein (GFP) variantgfpuvgene under the control of the nisin-induciblenisApromoter. This strain, LAC275, was capable of transducing the signal from extracellular nisin into measurable GFPuv fluorescence through the NisRK signal transduction system. The LAC275 cells detected nisin concentrations of 10 pg/ml in culture supernatant, 0.2 ng/ml in milk, 3.6 ng/g in processed cheese, 1 ng/g in salad dressings and crushed, canned tomatoes, and 2 ng/g in liquid egg. This method was up to 1,000 times more sensitive than a previously described GFP-based nisin bioassay. This new assay made it possible to detect significantly smaller amounts of nisin than the presently most sensitive published nisin bioassay based on nisin-induced bioluminescence. The major advantage of this sensitivity was that foods could be extensively diluted prior to the assay, avoiding potential inhibitory and interfering substances present in most food products.
In Vivo Emission of Dinitrogen by Earthworms via Denitrifying Bacteria in the Gut
Authors: Horn Marcus A. , Mertel Ralph , Gehre Matthias , Kästner Matthias , Drake Harold L.
Published: February 2006

Earthworms emit the greenhouse gas nitrous oxide (N2O), and ingested denitrifiers in the gut appear to be the main source of this N2O. The primary goal of this study was to determine if earthworms also emit dinitrogen (N2), the end product of complete denitrification. When [15N]nitrate was injected into the gut, the earthwormsAporrectodea caliginosaandLumbricus terrestrisemitted labeled N2(and also labeled N2O) under in vivo conditions; emission of N2by these two earthworms was relatively linear and approximated 1.2 and 6.6 nmol N2per h per g (fresh weight), respectively. Isolated gut contents also produced [15N]nitrate-derived N2and N2O under anoxic conditions. N2is formed by N2O reductase, and acetylene, an inhibitor of this enzyme, inhibited the emission of [15N]nitrate-derived N2by living earthworms. Standard gas chromatographic analysis demonstrated that the amount of N2O emitted was relatively linear during initial incubation periods and increased in response to acetylene. The calculated rates for the native emissions of N2(i.e., without added nitrate) byA. caliginosaandL. terrestriswere 1.1 and 1.5 nmol N2per h per g (fresh weight), respectively; these emission rates approximated that of N2O. These collective observations indicate that (i) earthworms emit N2concomitant with the emission of N2O via the in situ activity of denitrifying bacteria in the gut and (ii) N2O is quantitatively an important denitrification-derived end product under in situ conditions.
Differences in Fecal Microbiota in Different European Study Populations in Relation to Age, Gender, and Country: a Cross-Sectional Study
Authors: Mueller Susanne , Saunier Katiana , Hanisch Christiana , Norin Elisabeth , Alm Livia , Midtvedt Tore , Cresci Alberto , Silvi Stefania , Orpianesi Carla , Verdenelli Maria Cristina , Clavel Thomas , Koebnick Corinna , Zunft Hans-Joachim Franz , Doré Joël , Blaut Michael
Published: February 2006

A cross-sectional study on intestinal microbiota composition was performed on 230 healthy subjects at four European locations in France, Germany, Italy, and Sweden. The study participants were assigned to two age groups: 20 to 50 years (mean age, 35 years;n= 85) and >60 years (mean age, 75 years;n= 145). A set of 14 group- and species-specific 16S rRNA-targeted oligonucleotide probes was applied to the analysis of fecal samples by fluorescence in situ hybridization coupled with flow cytometry. Marked country-age interactions were observed for the German and Italian study groups. These interactions were inverse for the predominant bacterial groupsEubacterium rectale-Clostridium coccoidesandBacteroides-Prevotella. Differences between European populations were observed for theBifidobacteriumgroup only. Proportions of bifidobacteria were two- to threefold higher in the Italian study population than in any other study group, and this effect was independent of age. Higher proportions of enterobacteria were found in all elderly volunteers independent of the location. Gender effects were observed for theBacteroides-Prevotellagroup, with higher levels in males than in females. In summary, age-related differences in the microbiota makeup were detected but differed between the study populations from the four countries, each showing a characteristic colonization pattern.
Biodegradation of 3-Nitrotyrosine byBurkholderiasp. Strain JS165 andVariovorax paradoxusJS171
Authors: Nishino Shirley F. , Spain Jim C.
Published: February 2006

The cascade of reactive nitrogen species generated from nitric oxide causes modification of proteins, lipids, and nucleic acids in a wide range of organisms. 3-Nitrotyrosine is one of the most common products of the action of reactive nitrogen species on proteins. Although a great deal is known about the formation of 3-nitrotyrosine, the subsequent metabolism of this compound is a mystery.Variovorax paradoxusJS171 andBurkholderiasp. strain JS165 were isolated from soil slurries when 3-nitrotyrosine was provided as the sole carbon, nitrogen, and energy source. During growth on 3-nitrotyrosine stoichiometric amounts of nitrite were released along with approximately one-half of the theoretically available ammonia. The catabolic pathway involving oxidative denitration is distinct from the pathway for tyrosine metabolism. The facile isolation and the specific, regulated pathway for 3-nitrotyrosine degradation in natural ecosystems suggest that there is a significant flux of 3-nitrotyrosine in such environments.
Molecular Cloning and Expression of Genes Encoding a Novel Dioxygenase Involved in Low- and High-Molecular-Weight Polycyclic Aromatic Hydrocarbon Degradation inMycobacterium vanbaaleniiPYR-1
Authors: Kim Seong-Jae , Kweon Ohgew , Freeman James P. , Jones Richard C. , Adjei Michael D. , Jhoo Jin-Woo , Edmondson Ricky D. , Cerniglia Carl E.
Published: February 2006

Mycobacterium vanbaaleniiPYR-1 is able to metabolize a wide range of low- and high-molecular-weight (HMW) polycyclic aromatic hydrocarbons (PAHs). A 20-kDa protein was upregulated in PAH-metabolizingM. vanbaaleniiPYR-1 cells compared to control cultures. The differentially expressed protein was identified as a β subunit of the terminal dioxygenase using mass spectrometry. PCR with degenerate primers designed based on de novo sequenced peptides and a series of plaque hybridizations were done to screen theM. vanbaaleniiPYR-1 genomic library. The genes, designatednidA3B3, encoding the α and β subunits of terminal dioxygenase, were subsequently cloned and sequenced. The deduced enzyme revealed close similarities to the corresponding PAH ring-hydroxylating dioxygenases fromMycobacteriumandRhodococcusspp. but had the highest similarity, 61.9%, to the α subunit fromNocardioidessp. strain KP7. The α subunit also showed 52% sequence homology with the previously reported NidA fromM. vanbaaleniiPYR-1. The genesnidA3B3were subcloned into the expression vector pET-17b, and the enzyme activity inEscherichia colicells was reconstituted through coexpression with the ferredoxin (PhdC) and ferredoxin reductase (PhdD) genes of the phenanthrene dioxygenase fromNocardioidessp. strain KP7. The recombinant PAH dioxygenase appeared to favor the HMW PAH substrates fluoranthene, pyrene, and phenanthrene. Several other PAHs, including naphthalene, anthracene, and benz[a]anthracene, were also converted to their correspondingcis-dihydrodiols. The recombinantE. coli, however, did not show any dioxygenation activity for phthalate and biphenyl. The upregulation ofnidA3B3inM. vanbaaleniiPYR-1 induced by PAHs was confirmed by reverse transcription-PCR analysis.
Identification of Genes Associated with Survival ofSalmonella entericaSerovar Enteritidis in Chicken Egg Albumen
Authors: Clavijo Raul I. , Loui Cindy , Andersen Gary L. , Riley Lee W. , Lu Sangwei
Published: February 2006

Salmonella entericaconsists of over 2,000 serovars that are major causes of morbidity and mortality associated with contaminated food. Despite similarities among serovars ofSalmonella enterica, many demonstrate unique host specificities, epidemiological characteristics, and clinical manifestations. One of the unique epidemiological characteristics of the serovar Enteritidis is that it is the only bacterium routinely transmitted to humans through intact chicken eggs. Therefore,Salmonella entericaserovar Enteritidis must be able to persist inside chicken eggs to be transmitted to humans, and its survival in egg is important for its transmission to the human population. The ability ofSalmonella entericaserovar Enteritidis to survive in and transmit through eggs may have contributed to its drastically increased prevalence in the 1980s and 1990s. In the present study, using transposon-mediated mutagenesis, we have identified genes important for the association ofSalmonella entericaserovar Enteritidis with chicken eggs. Our results indicate that genes involved in cell wall structural and functional integrity, and nucleic acid and amino acid metabolism are important forSalmonella entericaserovar Enteritidis to persist in egg albumen. Two regions unique toSalmonella entericaserovar Enteritidis were also identified, one of which enhanced the survival of aSalmonella entericaserovar Typhimurium isolate in egg albumen. The implication of our results to the serovar specificity ofSalmonella entericais also explored in the present study.
Purification, Characterization, and Gene Cloning of a Novel Maltosyltransferase from anArthrobacter globiformisStrain That Produces an Alternating α-1,4- and α-1,6-Cyclic Tetrasaccharide from Starch
Authors: Mukai Kazuhisa , Watanabe Hikaru , Kubota Michio , Chaen Hiroto , Fukuda Shigeharu , Kurimoto Masashi
Published: February 2006

A glycosyltransferase, involved in the synthesis of cyclic maltosylmaltose [CMM;cyclo-{→6)-α-d-Glcp(1→4)-α-d-Glcp(1→6)-α-d-Glcp(1→4)-α-d-Glcp(1→}] from starch, was purified to homogeneity from the culture supernatant ofArthrobacter globiformisM6. The CMM-forming enzyme had a molecular mass of 71.7 kDa and a pI of 3.6. The enzyme was most active at pH 6.0 and 50°C and was stable from pH 5.0 to 9.0 and up to 30°C. The addition of 1 mM Ca2+enhanced the thermal stability of the enzyme up to 45°C. The enzyme acted on maltooligosaccharides that have degrees of polymerization of ≥3, amylose, and soluble starch to produce CMM but failed to act on cyclomaltodextrins, pullulan, and dextran. The mechanism for the synthesis of CMM from maltotetraose was determined as follows: (i) maltotetraose + maltotetraose → 64-O-α-maltosyl-maltotetraose + maltose and (ii) 64-O-α-maltosyl-maltotetraose → CMM + maltose. Thus, the CMM-forming enzyme was found to be a novel maltosyltransferase (6MT) catalyzing both intermolecular and intramolecular α-1,6-maltosyl transfer reactions. The gene for 6MT, designatedcmmA, was isolated from a genomic library ofA. globiformisM6. ThecmmAgene consisted of 1,872 bp encoding a signal peptide of 40 amino acids and a mature protein of 583 amino acids with a calculated molecular mass of 64,637. The deduced amino acid sequence showed similarities to α-amylase and cyclomaltodextrin glucanotransferase. The four conserved regions common in the α-amylase family enzymes were also found in 6MT, indicating that 6MT should be assigned to this family.
Pure-Culture Growth of Fermentative Bacteria, Facilitated by H
2
Removal: Bioenergetics and H
2
Production
Authors: Adams Cameron J. , Redmond Molly C. , Valentine David L.
Published: February 2006

We used an H
2
-purging culture vessel to replace an H
2
-consuming syntrophic partner, allowing the growth of pure cultures of
Syntrophothermus lipocalidus
on butyrate and
Aminobacterium colombiense
on alanine. By decoupling the syntrophic association, it was possible to manipulate and monitor the single organism's growth environment and determine the change in Gibbs free energy yield (Δ
G
) in response to changes in the concentrations of reactants and products, the purging rate, and the temperature. In each of these situations, H
2
production changed such that Δ
G
remained nearly constant for each organism (−11.1 ± 1.4 kJ mol butyrate
−1
for
S. lipocalidus
and −58.2 ± 1.0 kJ mol alanine
−1
for
A. colombiense
). The cellular maintenance energy, determined from the Δ
G
value and the hydrogen production rate at the point where the cell number was constant, was 4.6 × 10
−13
kJ cell
−1
day
−1
for
S. lipocalidus
at 55°C and 6.2 × 10
−13
kJ cell
−1
day
−1
for
A. colombiense
at 37°C.
S. lipocalidus
, in particular, seems adapted to thrive under conditions of low energy availability.
The Aflatoxin Biosynthesis Cluster Gene,
aflX
, Encodes an Oxidoreductase Involved in Conversion of Versicolorin A to Demethylsterigmatocystin
Authors: Cary Jeffrey W. , Ehrlich Kenneth C. , Bland John M. , Montalbano Beverly G.
Published: February 2006

Biosynthesis of the toxic and carcinogenic aflatoxins by the fungus
Aspergillus flavus
is a complicated process involving more that 27 enzymes and regulatory factors encoded by a clustered group of genes. Previous studies found that three enzymes, encoded by
verA
,
ver-1
, and
aflY
, are required for conversion of versicolorin A (VA), to demethylsterigmatocystin. We now show that a fourth enzyme, encoded by the previously uncharacterized gene,
aflX
(
ordB
), is also required for this conversion. A homolog of this gene,
stcQ
, is present in the
A. nidulans
sterigmatocystin (ST) biosynthesis cluster. Disruption of
aflX
in
Aspergillus flavus
gave transformants that accumulated ∼4-fold more VA and fourfold less aflatoxin than the untransformed strain. Southern and Northern blot analyses confirmed that
aflX
was the only gene disrupted in these transformants. Feeding ST or
O
-methylsterigmatocystin, but not VA or earlier precursor metabolites, restored normal levels of AF production. The protein encoded by
aflX
is predicted to have domains typical of an NADH-dependent oxidoreductase. It has 27% amino acid identity to a protein encoded by the aflatoxin cluster gene,
aflO
(
avfA
). Some of domains in the protein are similar to those of epoxide hydrolases.
Only a Few Fungal Species Dominate Highly Diverse Mycofloras Associated with the Common Reed
Authors: Neubert Karin , Mendgen Kurt , Brinkmann Henner , Wirsel Stefan G. R.
Published: February 2006

Plants are naturally colonized by many fungal species that produce effects ranging from beneficial to pathogenic. However, how many of these fungi are linked with a single host plant has not been determined. Furthermore, the composition of plant-associated fungal communities has not been rigorously determined. We investigated these essential issues by employing the perennial wetland reed
Phragmites australis
as a model. DNA extracted from roots, rhizomes, stems, and leaves was used for amplification and cloning of internal transcribed spacer rRNA gene fragments originating from reed-associated fungi. A total of 1,991 clones from 15 clone libraries were differentiated by restriction fragment length polymorphism analyses into 345 operational taxonomical units (OTUs). Nonparametric estimators for total richness (Chao1 and ACE) and also a parametric log normal model predicted a total of about 750 OTUs if the libraries were infinite. Sixty-two percent of the OTUs sequenced were novel at a threshold of 3%. Several of these OTUs represented undocumented fungal species, which also included higher taxonomic levels. In spite of the high diversity of the OTUs, the mycofloras of vegetative organs were dominated by just a few typical fungi, which suggested that competition and niche differentiation influence the composition of plant-associated fungal communities. This suggestion was independently supported by the results of nested PCR assays specifically monitoring two OTUs over 3 years, which revealed significant preferences for host habitat and host organ.
Biocontrol of the Food-Borne Pathogens
Listeria monocytogenes
and
Salmonella enterica
Serovar Poona on Fresh-Cut Apples with Naturally Occurring Bacterial and Yeast Antagonists
Authors: Leverentz Britta , Conway William S. , Janisiewicz Wojciech , Abadias Maribel , Kurtzman Cletus P. , Camp Mary J.
Published: February 2006

Fresh-cut apples contaminated with either
Listeria monocytogenes
or
Salmonella enterica
serovar Poona, using strains implicated in outbreaks, were treated with one of 17 antagonists originally selected for their ability to inhibit fungal postharvest decay on fruit. While most of the antagonists increased the growth of the food-borne pathogens, four of them, including
Gluconobacter asaii
(T1-D1), a
Candida
sp. (T4-E4),
Discosphaerina fagi
(ST1-C9), and
Metschnikowia pulcherrima
(T1-E2), proved effective in preventing the growth or survival of food-borne human pathogens on fresh-cut apple tissue. The contaminated apple tissue plugs were stored for up to 7 days at two different temperatures. The four antagonists survived or grew on the apple tissue at 10 or 25°C. These four antagonists reduced the
Listeria monocytogenes
populations and except for the
Candida
sp. (T4-E4), also reduced the
S. enterica
serovar Poona populations. The reduction was higher at 25°C than at 10°C, and the growth of the antagonists, as well as pathogens, increased at the higher temperature.
Determination of Essential and Variable Residues in Pediocin PA-1 by NNK Scanning
Authors: Tominaga Tatsuya , Hatakeyama Yoshinori
Published: February 2006

Pediocin PA-1 is an antimicrobial peptide (called bacteriocin) that shows inhibitory activity against the food-borne pathogen
Listeria monocytogenes
. To elucidate which residue(s) is responsible for this function, the antimicrobial activities of pediocin PA-1 mutants were evaluated and compared. Each of the 44 native codons was replaced with the NNK triplet oligonucleotide in a technique termed NNK scanning, and 35 mutations at each position were examined for antimicrobial activities using a modified colony overlay screening method. As a consequence, the functional responsibility of each residue was estimated by counting the number of active mutants, allowing us to identify candidate essential/variable residues. Activity was abrogated by many of the mutations at residues Y2, G6, C9, C14, C24, W33, G37, and C44, indicating that these residues may be essential. In contrast, activity was retained by almost all versions harboring mutations at K1, T8, G10, S13, G19, N28, and N41, indicating that these are functionally redundant residues. Sequence analysis revealed that only the wild type was active and 14 and 11 substitutions were inactive at G6 and C14, respectively, while 12 and 11 substitutions were active and 2 and 0 substitutions were inactive at T8 and K1, respectively. These findings suggest that NNK scanning is effective for determining essential and variable residues in pediocin PA-1, leading to an elucidation of structure-function relationships and to improvements in the antimicrobial function efficiently by peptide engineering.