bluuebunny/crossref_metadata_2025 huggingface dataset:
IPO Failure Risk
Authors: DEMERS ELIZABETH , JOOS PHILIP
Published: March 2007

We explore the factors associated with historical IPO failures by developing an IPO failure prediction model that includes accounting information as well as proxies for the role of information intermediaries and other IPO deal–related characteristics. We document statistically significant differences in failure models applicable to nontech versus high tech IPOs, and these structural differences are largely driven by accounting‐based proxies for firms' investments in intangible assets, operating performance, and financial leverage. We also develop parsimonious, predominantly accounting‐based, strictly out‐of‐sample (i.e., no hindsight) IPO failure forecasting models for each of the two sectors. We find that our forecasts are negatively associated with one‐year post‐IPO abnormal returns. A pseudo‐hedge strategy of going short (long) in high (low) failure risk portfolios yields returns of economically significant magnitudes over the one‐year horizon, and is robust to alternative returns methodologies. Further results suggest that IPO long‐run returns anomalies may persist, but they take different forms for high‐tech and nontech IPOs.
The Book‐to‐Price Effect in Stock Returns: Accounting for Leverage
Authors: PENMAN STEPHEN H. , RICHARDSON SCOTT A. , TUNA İREM
Published: March 2007

This paper lays out a decomposition of book‐to‐price (B/P) that derives from the accounting for book value and that articulates precisely how B/P “absorbs” leverage. The B/P ratio can be decomposed into an enterprise book‐to‐price (that pertains to operations and potentially reflects operating risk) and a leverage component (that reflects financing risk). The empirical analysis shows that the enterprise book‐to‐price ratio is positively related to subsequent stock returns but, conditional upon the enterprise book‐to‐price, the leverage component of B/P is negatively associated with future stock returns. Further, both enterprise book‐to‐price and leverage explain returns over those associated with Fama and French nominated factors—including the book‐to‐price factor—albeit negatively so for leverage. The seemingly perverse finding with respect to the leverage component of B/P survives under controls for size, estimated beta, return volatility, momentum, and default risk.
Neurofuzzy Modeling of Context–Contingent Proximity Relations
Authors: Yao Xiaobai , Thill Jean‐Claude
Published: March 2007

The notion of proximity is one of the foundational elements in humans' understanding and reasoning of the geographical environments. The perception and cognition of distances plays a significant role in many daily human activities. Yet, few studies have thus far provided context–contingent translation mechanisms between linguistic proximity descriptors (e.g., “near,”“far”) and metric distance measures. One problem with previous fuzzy logic proximity modeling studies is that they presume the form of the fuzzy membership functions of proximity relations. Another problem is that previous studies have fundamental weaknesses in considering context factors in proximity models. We argue that statistical approaches are ill suited to proximity modeling because of the inherently fuzzy nature of the relations between linguistic and metric distance measures. In this study, we propose a neurofuzzy system approach to solve this problem. The approach allows for the dynamic construction of context–contingent proximity models based on sample data. An empirical case study with human subject survey data is carried out to test the validity of the approach and to compare it with the previous statistical approach. Interpretation and prediction accuracy of the empirical study are discussed.
Gingival crevicular fluid interleukin‐1β, prostaglandin E2 and periodontal status in a community population
Authors: Zhong Y. , Slade G. D. , Beck J. D. , Offenbacher S.
Published: March 2007

Aim: Interleukin‐1 β (IL‐1β) and prostaglandin E2 (PGE2) are key inflammatory mediators involved in periodontal disease. The purposes of this molecular cross‐sectional epidemiological study were to investigate relationships in a community sample between mean concentrations of IL‐1β and PGE2 in gingival crevicular fluid (GCF) and (1) clinical periodontal signs and (2) risk factors of host inflammatory response and/or periodontal disease.

Material and Methods: The sample comprised 6277 community‐dwelling adults aged 52–74 years enrolled in the Atherosclerosis Risk in Communities (ARIC) study. IL‐1β and PGE2 concentrations were measured using enzyme‐linked immunosorbent assay. Person‐level summary variables were computed for maximum pocket depth (MaxPD), maximum clinical attachment level (MaxCAL) and presence/absence of bleeding on probing (BOP). Mean GCF IL‐1β and PGE2 concentrations were dependent variables in multiple linear regression models with periodontal measures and covariates as explanatory variables.

Results: Both GCF IL‐1β and PGE2 were positively related to MaxPD and BOP in multiple regression models (p<0.01). Increased levels of IL‐1β and PGE2 were associated with body mass index 30 kg/m2.

Conclusion: Higher levels of GCF IL‐1β and PGE2 were significantly associated with clinical signs of periodontal disease and independently related to patient‐based anthropomorphic measures, behaviours and exposures in community‐dwelling adults.
Citation classics in periodontology: a controlled study
Authors: Nieri Michele , Saletta Daniele , Guidi Luisa , Buti Jacopo , Franceschi Debora , Mauro Saverio , Pini‐Prato GiovanPaolo
Published: March 2007

Aim: The aims of this study were to identify the most cited articles in Periodontology published from January 1990 to March 2005; and to analyse the differences between citation Classics and less cited articles.

Material and Methods: The search was carried out in four international periodontal journals: Journal of Periodontology, Journal of Clinical Periodontology, International Journal of Periodontics and Restorative Dentistry and Journal of Periodontal Research. The Classics, that are articles cited at least 100 times, were identified using the Science Citation Index database. From every issue of the journals that contained a Classic, another article was randomly selected and used as a Control.

Results: Fifty‐five Classics and 55 Controls were identified. Classic articles were longer, used more images, had more authors, and contained more self‐references than Controls.

Moreover Classics had on the average a bigger sample size, often dealt with etiopathogenesis and prognosis, but were rarely controlled or randomized studies.

Conclusions: Classic articles play an instructive role, but are often non‐Controlled studies.
Epigenetic events in malignant melanoma
Authors: Rothhammer Tanja , Bosserhoff Anja‐Katrin
Published: March 2007

Irreversible changes in the DNA sequence, including chromosomal deletions or amplification, activating or inactivating mutations in genes, have been implicated in the development and progression of melanoma. However, increasing attention is being turned towards the participation of ‘epigenetic’ events in melanoma progression that do not affect DNA sequence, but which nevertheless may lead to stable inherited changes in gene expression. Epigenetic events including histone modifications and DNA methylation play a key role in normal development and are crucial to establishing the correct program of gene expression. In contrast, mistargeting of such epigenetic modifications can lead to aberrant patterns of gene expression and loss of anti‐cancer checkpoints. Thus, to date at least 50 genes have been reported to be dysregulated in melanoma by aberrant DNA methylation and accumulating evidence also suggests that mistargetting of histone modifications and altered chromatin remodeling activities will play a key role in melanoma. This review gives an overview of the many different types of epigenetic modifications and their involvement in cancer and especially in melanoma development and progression.
Fragmentation of the Golgi Apparatus: An Early Apoptotic Event Independent of the Cytoskeleton
Authors: Mukherjee Shaeri , Chiu Raymond , Leung Som‐Ming , Shields Dennis
Published: March 2007

The Golgi apparatus undergoes irreversible fragmentation during apoptosis, in part as a result of caspase‐mediated cleavage of several Golgi‐associated proteins. However, Golgi structure and orientation is also regulated by the cytoskeleton and cytoskeletal changes have been implicated in inducing apoptosis. Consequently, we have analyzed the role of actin filaments and microtubules in apoptotic Golgi fragmentation. We demonstrate that in Fas receptor‐activated cells, fragmentation of the Golgi apparatus was an early event that coincided with release of cytochrome c from mitochondria. Significantly, Golgi fragmentation preceded major changes in the organization of both the actin cytoskeleton and microtubules. In staurosporine‐treated cells, actin filament organization was rapidly disrupted; however, the Golgi apparatus maintained its juxtanuclear localization and underwent complete fragmentation only at later times. Attempts to stabilize actin filaments with jasplakinolide prior to treatment with staurosporine did not prevent Golgi fragmentation. Finally, in response to Fas receptor activation or staurosporine treatment the levels of β‐actin or α‐tubulin remained unaltered, whereas several Golgi proteins, p115 and golgin‐160, underwent caspase‐mediated cleavage. Our data demonstrate that breakdown of the Golgi apparatus is an early event during apoptosis that occurs independently of major changes to the actin and tubulin cytoskeleton.
A functional Sp1/Egr1‐tandem repeat polymorphism in the 5‐lipoxygenase gene is not associated with myocardial infarction
Authors: González P. , Reguero J. R. , Lozano I. , Morís C. , Coto E.
Published: March 2007

Arachidonate 5‐lipoxygenase is an enzyme encoded by the ALOX5 gene, and plays an important role in the synthesis of leukotrienes. These are inflammatory mediators, and have been involved in atherosclerosis and other pathological processes that require proinflammatory activities. Human and animal studies have suggested a role for the ALOX5 gene in atherosclerosis, including a significant association between a promoter polymorphism and a carotid intimal‐medial thickness in response to dietary fat. This polymorphism was three‐ to six‐tandem repeats of a Sp1/Egr1 binding motif (GGGCGG)n, and the number of repeats has been linked with the amount of gene expression. We hypothesized that this ALOX5 polymorphism could influence the risk for myocardial infarction (MI).

First, we analysed the effect of the four alleles on gene expression by transfecting the HEK‐293 cell line with luciferase reporter‐constructs. We found that luciferase activities are dependent on the number of the Sp1/Egr1 repeats, with the three and six repeats having the lowest and highest values. We genotyped 312 male MI survivors, aged < 55 years, and 376 healthy controls matched with patients for sex, age, and ethnicity. Ninety‐six per cent of the patients were smokers, compared to only 42% among the controls (P < 0.001; OR = 31.84). The 55 + 56 repeat genotypes were less frequent in patients (55 = 56%, 56 = 0.6%) compared to controls (55 = 60%, 56 = 3%). However, these were non‐significantly different frequencies. In addition, no difference in MI‐onset age and biochemical values was found between the allele and genotypes. In conclusion, we confirmed the effect of the ALOX5‐promoter polymorphism on gene expression, but our data did not support a significant effect of this functional variation on MI risk.
A biased Gm haplotype and Gm paraprotein allotype in multiple myeloma suggests a role for the Gm system in myeloma development
Authors: Ilić V. , Milošević‐Jovčić N. , Marković D. , Petrović S. , Stefanović G.
Published: March 2007

The association between a particular Gm haplotype and susceptibility to multiple myeloma (MM) is not clear. The reason is probably because no investigations have so far been carried out on the relationship between the Gm haplotype, which represents the inherited combination of IgG Gm allotypes, and the Gm allotype expressed at the IgG paraprotein (M‐component), which reflects the enhanced gene expression within the haplotype in MM. We studied the incidence of Gm allotypic markers present in IgG subclasses in the serum from 52 patients with MM and in parallel with the isolated IgG paraproteins. The results showed that 84.6% of the patients were heterozygous for haplotypes Gm(a; z; n−; g;)/Gm(f; n+/n–; b1; b0; b5) and 15.3% were homozygous for Gm(f; n/n–; b1; b0; b5), while no homozygous Gm(a; z; n–; g) individuals were found among the studied patients. The incidence of these combinations in the healthy population in Serbia is 34%, 66% and < 1%, respectively. Subjects with Gm(a; z; n–; g)/Gm(f; n+/n–; b1; b0; b5) combination are over 10 times [odds ratio (OR) = 10.69; 95% confidence interval 1.67–68] as likely to be affected by the disease as the subjects with homozygous Gm(f; n+/n–; b1; b0; b5) combination (OR = 0.35, 95% confidence interval 0.06–2.23). However, despite the Gm heterozygosity, most of the Gm(a; z; n–; g;)/Gm(f; n+/n–; b1; b0; b5) positive patients with MM (86.3%) had IgG paraprotein with the allotypic marker from the Gm(f; n+/n–; b1; b0; b5) haplotype. Together with patients homozygous for this haplotype, the relative number of patients with serum IgG paraprotein carrying allotypic marker from the Gm(f; n/n–; b1; b0; b5) haplotype was 88.5%. These results suggest that the development of an M‐component could be related to a disturbance on chromosome 14q32 carrying the Gm (f; n+/n–; b1; b0; b5) set of genes.
Making sense of David Cameron
Authors: Finlayson Alan
Published: March 2007

Alan Finlayson examines the emerging phenomenon of ‘Cameronism’ and argues that it could represent a political and intellectual project capable of trumping ‘Blairism’.
Should we raise the age of legal drinking?
Authors: Gerard Jasper
Published: March 2007

In the wake of another glut of reports of binge‐drinking teens, antisocial behaviour and street crime, Jasper Gerard argues that Britain has lost the plot when it comes to regulating alcohol and asks whether we should adopt policies pursued elsewhere.
Discovery of a remarkable subpulse drift pattern in PSR B0818–41
Authors: Bhattacharyya B. , Gupta Y. , Gil J. , Sendyk M.
Published: March 2007

We report the discovery of a remarkable subpulse drift pattern in the relatively less-studied wide profile pulsar B0818–41 using high-sensitivity Giant Metrewave Radio Telescope (GMRT) observations. We find simultaneous occurrences of three drift regions with two different drift rates: an inner region with steeper apparent drift rate flanked on each side by a region of slower apparent drift rate. Furthermore, these closely spaced drift bands always maintain a constant phase relationship. Though these drift regions have significantly different values for the measured P2, the measured P3 value is the same and equal to 18.3P1. We interpret the unique drift pattern of this pulsar as being created by the intersection of our line of sight (LOS) with two conal rings on the polar cap of a fairly aligned rotator (inclination angle α∼ 11°), with an ‘inner’ LOS geometry (impact angle ). We argue that both rings have the same values for the carousel rotation periodicity P4 and the number of sparks Nsp. We find that Nsp is 19–21 and show that it is very likely that P4 is the same as the measured P3, making it a truly unique pulsar. We present results from simulations of the radiation pattern using the inferred parameters, which support our interpretations and reproduce the average profile as well as the observed features in the drift pattern quite well.
Can the soft excess in AGN originate from disc reflection?
Authors: Done Chris , Nayakshin Sergei
Published: March 2007

The soft excess seen in many active galactic nuclei (AGN) is most probably due to partially ionized material moving at relativistic speeds close to the black hole. There are currently two potential geometries for this material, one where it is out of the line of sight, seen via reflection (e.g. the accretion disc), the other where it is in the line of sight, seen in absorption (e.g. a wind above the disc). Both models require apparent fine-tuning of the ionization parameter of this material in order to produce the large jump in opacity at ∼0.7 keV associated with O vii/viii, as required to make the soft excess. However, Chevallier et al. show that these states rather naturally dominate the absorption spectrum for soft X-ray spectra if the illuminated material is (at least approximately) in pressure balance. Here we explore whether hydrostatic pressure equilibrium in a disc can likewise naturally select the required ionization states in reflection. We find the opposite. The soft excess X-ray excess is much weaker in the hydrostatic models than it is in the constant density models. Because even the constant density models cannot fit the largest soft excesses seen without the intrinsic continuum being hidden from view, this means that reflection from a hydrostatic disc cannot realistically match the data. Even if the disc structure is instead more like a constant density atmosphere, the required fine-tuning of the ionization parameter still remains a problem for reflection models.
Feeding behavior, toe count, and the phylogenetic relationships among alcedinine kingfishers (Alcedininae)
Authors: G. Moyle Robert , Fuchs Jérôme , Pasquet Eric , D. Marks Ben
Published: March 2007

The pygmy kingfishers of the Old World tropics (Alcedininae) display a range of feeding behaviors, plumage types, and morphologies that have caused much taxonomic confusion in the group. We sequenced mitochondrial and nuclear genes to reconstruct a phylogenetic hypothesis for the subfamily, which was then used to examine the utility of common taxonomic characters. The phylogenetic hypothesis reveals multiple shifts between insectivory and piscivory, as well as homoplasy in bill color and plumage color. Piscivores and insectivores are interspersed across the tree, as are blue and rufous plumage types. It is apparent that reliance on these characters to delineate genera has resulted in classifications that do not describe natural groups. The sole taxonomic character examined that defines a natural division in the subfamily, toe count, is also the one generally dismissed as non‐informative in the alcedinines, yet all species with a reduction in the number of toes form a clade. It is recommended that four genera be retained to describe the variation within pygmy kingfishers.
Dynamics of the readily releasable pool during post‐tetanic potentiation in the rat calyx of Held synapse
Authors: Habets Ron L. P. , Borst J. Gerard G.
Published: March 2007

The size of the readily releasable pool (RRP) of vesicles was measured in control conditions and during post‐tetanic potentiation (PTP) in a large glutamatergic terminal called the calyx of Held. We measured excitatory postsynaptic currents evoked by a high frequency train of action potentials in slices of 4–11‐day‐old rats. After a tetanus the cumulative release during such a train was enlarged by approximately 50%, indicating that the size of the RRP was increased. The amount of enhancement depended on the duration and frequency of the tetanus and on the age of the rat. After the tetanus, the size of the RRP decayed more slowly (t1/2= 10 versus 3 min) back to control values than the release probability. This difference was mainly due to a very fast initial decay of the release probability, which had a time constant compatible with an augmentation phase (τ≈ 30 s). The overall decay of PTP at physiological temperature was not different from room temperature, but the increase in release probability (Pr) was restricted to the first minute after the tetanus. Thereafter PTP was dominated by an increase in the size of the RRP. We conclude that due to the short lifetime of the increase in release probability, the contribution of the increase in RRP size during post‐tetanic potentiation is more significant at physiological temperature.
Synaptic plasticity in myenteric neurons of the guinea‐pig distal colon: presynaptic mechanisms of inflammation‐induced synaptic facilitation
Authors: Krauter Eric M. , Linden David R. , Sharkey Keith A. , Mawe Gary M.
Published: March 2007

The purpose of this study was to investigate the pre‐ and postsynaptic mechanisms that contribute to synaptic facilitation in the myenteric plexus of the trinitrobenzene sulphonic acid‐inflamed guinea‐pig distal colon. Intracellular recordings of evoked fast excitatory postsynaptic potentials (fEPSPs) in myenteric S neurons were evaluated, and the density of synaptic terminals was morphometrically analysed by transmission electron microscopy. In inflamed tissue, fEPSPs were reduced to control levels by the protein kinase A (PKA) inhibitor, H89, but H89 did not affect the fEPSPs in control tissue. This PKA activation in inflamed tissue did not appear to involve 5‐HT4receptors because the antagonist/inverse agonist, GR 125487, caused comparable decreases of fEPSPs in both tissues. Inhibition of BK channels with iberiotoxin did not alter the fEPSPs in inflamed tissue, but increased the fEPSPs in control tissue to the amplitude detected in inflamed tissue. During trains of stimuli, run‐down of EPSPs was less extensive in inflamed tissue and there was a significant increase in the paired pulse ratio. Depolarizations in response to exogenous neurotransmitters were not altered in inflamed tissue. These inflammation‐induced changes were not accompanied by alterations in the pharmacological profile of EPSPs, and no changes in synaptic density were detected by electron microscopy. Collectively, these data indicate that synaptic facilitation in the inflamed myenteric plexus involves a presynaptic increase in PKA activity, possibly involving an inhibition of BK channels, and an increase in the readily releasable pool of synaptic vesicles.
Charge at the lidocaine binding site residue Phe‐1759 affects permeation in human cardiac voltage‐gated sodium channels
Authors: McNulty Megan M. , Edgerton Gabrielle B. , Shah Ravi D. , Hanck Dorothy A. , Fozzard Harry A. , Lipkind Gregory M.
Published: March 2007

Our homology molecular model of the open/inactivated state of the Na+channel pore predicts, based on extensive mutagenesis data, that the local anaesthetic lidocaine docks eccentrically below the selectivity filter, such that physical occlusion is incomplete. Electrostatic field calculations suggest that the drug's positively charged amine produces an electrostatic barrier to permeation. To test the effect of charge at this pore level on permeation in hNaV1.5 we replaced Phe‐1759 of domain IVS6, the putative binding site for lidocaine's alkylamino end, with positively and negatively charged residues as well as the neutral cysteine and alanine. These mutations eliminated use‐dependent lidocaine block with no effect on tonic/rested state block. Mutant whole cell currents were kinetically similar to wild type (WT). Single channel conductance (γ) was reduced from WT in both F1759K (by 38%) and F1759R (by 18%). The negatively charged mutant F1759E increased γ by 14%, as expected if the charge effect were electrostatic, although F1759D was like WT. None of the charged mutations affected Na+/K+selectivity. Calculation of difference electrostatic fields in the pore model predicted that lidocaine produced the largest positive electrostatic barrier, followed by lysine and arginine, respectively. Negatively charged glutamate and aspartate both lowered the barrier, with glutamate being more effective. Experimental data were in rank order agreement with the predicted changes in the energy profile. These results demonstrate that permeation rate is sensitive to the inner pore electrostatic field, and they are consistent with creation of an electrostatic barrier to ion permeation by lidocaine's charge.
Ryanodine receptor type 2 deficiency changes excitation–contraction coupling and membrane potential in urinary bladder smooth muscle
Authors: Hotta Shingo , Morimura Kozo , Ohya Susumu , Muraki Katsuhiko , Takeshima Hiroshi , Imaizumi Yuji
Published: March 2007

The possibility that the ryanodine receptor type 2 (RyR2) can function as the major Ca2+‐induced Ca2+release (CICR) channel in excitation–contraction (E‐C) coupling was examined in smooth muscle cells (SMCs) isolated from urinary bladder (UB) of RyR2 heterozygous KO mice (RyR2+/−). RyR2 mRNA expression in UB fromRyR2+/−was much lower than that in wild‐type (RyR2plus;/plus;). In single UBSMCs fromRyR2plus;/+, membrane depolarization under voltage clamp initially induced several local Ca2+transients (hot spots) in peripheral areas of the cell. Then, Ca2+waves spread from Ca2+hot spots to other areas of the myocyte. The number of Ca2+hot spots elicited by a short depolarization (< 20 ms) in UBSMCs ofRyR2+/−was significantly smaller than in those ofRyR2+/+. The force development induced either by direct electrical stimulation or by 10 μmacetylcholine in tissue segments ofRyR2+/−was smaller than and comparable to those inRyR2+/+, respectively. The frequency of spontaneous transient outward currents in single myocytes and the membrane depolarization by 1 μmpaxilline in tissue segments fromRyR2+/−were significantly lower and smaller than those inRyR2+/+, respectively. The urination frequency and volume per voiding inRyR2+/−were significantly increased and reduced, respectively, compared withRyR2+/+. In conclusion, RyR2 plays a crucial role in the regulation of CICR during E‐C coupling and also in the regulation of resting membrane potential, presumably via the modulation of Ca2+‐dependent K+channel activity in UBSMCs and, thereby, has a pivotal role in the control of bladder activity.
Diversity and Abundance of Aerobic and Anaerobic Methane Oxidizers at the Haakon Mosby Mud Volcano, Barents Sea
Authors: Lösekann Tina , Knittel Katrin , Nadalig Thierry , Fuchs Bernhard , Niemann Helge , Boetius Antje , Amann Rudolf
Published: March 2007

Submarine mud volcanoes are formed by expulsions of mud, fluids, and gases from deeply buried subsurface sources. They are highly reduced benthic habitats and often associated with intensive methane seepage. In this study, the microbial diversity and community structure in methane-rich sediments of the Haakon Mosby Mud Volcano (HMMV) were investigated by comparative sequence analysis of 16S rRNA genes and fluorescence in situ hybridization. In the active volcano center, which has a diameter of about 500 m, the main methane-consuming process was bacterial aerobic oxidation. In this zone, aerobic methanotrophs belonging to three bacterial clades closely affiliated with
Methylobacter
and
Methylophaga
species accounted for 56% ± 8% of total cells. In sediments below
Beggiatoa
mats encircling the center of the HMMV, methanotrophic archaea of the ANME-3 clade dominated the zone of anaerobic methane oxidation. ANME-3 archaea form cell aggregates mostly associated with sulfate-reducing bacteria of the
Desulfobulbus
(DBB) branch. These ANME-3/DBB aggregates were highly abundant and accounted for up to 94% ± 2% of total microbial biomass at 2 to 3 cm below the surface. ANME-3/DBB aggregates could be further enriched by flow cytometry to identify their phylogenetic relationships. At the outer rim of the mud volcano, the seafloor was colonized by tubeworms (
Siboglinidae
, formerly known as
Pogonophora
). Here, both aerobic and anaerobic methane oxidizers were found, however, in lower abundances. The level of microbial diversity at this site was higher than that at the central and
Beggiatoa
species-covered part of the HMMV. Analysis of methyl-coenzyme M-reductase alpha subunit (
mcrA
) genes showed a strong dominance of a novel lineage,
mcrA
group f, which could be assigned to ANME-3 archaea. Our results further support the hypothesis of Niemann et al. (54), that high methane availability and different fluid flow regimens at the HMMV provide distinct niches for aerobic and anaerobic methanotrophs.
Proteomic Approach for Characterization of Hop-Inducible Proteins in
Lactobacillus brevis
Authors: Behr Jürgen , Israel Lars , Gänzle Michael G. , Vogel Rudi F.
Published: March 2007

Resistance to hops is a prerequisite for the capability of lactic acid bacteria to grow in beer and thus cause beer spoilage. Bactericidal hop compounds, mainly iso-α-acids, are described as ionophores which exchange H
+
for cellular divalent cations, e.g., Mn
2+
, and thus dissipate ion gradients across the cytoplasmic membrane. The acid stress response of
Lactobacillus brevis
TMW 1.465 and hop adaptation in its variant
L. brevis
TMW 1.465A caused changes at the level of metabolism, membrane physiology, and cell wall composition. To identify the basis for these changes, a proteomic approach was taken. The experimental design allowed the discrimination of acid stress and hop stress. A strategy for improved protein identification enabled the identification of 84% of the proteins investigated despite the lack of genome sequence data for this strain. Hop resistance in
L. brevis
TMW 1.465A implies mechanisms to cope with intracellular acidification, mechanisms for energy generation and economy, genetic information fidelity, and enzyme functionality. Interestingly, the majority of hop-regulated enzymes are described as manganese or divalent cation dependent. Regulation of the manganese level allows fine-tuning of the metabolism, which enables a rapid response to environmental (stress) conditions. The hop stress response indicates adaptations shifting the metabolism into an energy-saving mode by effective substrate conversion and prevention of exhaustive protein de novo synthesis. The findings further demonstrate that hop stress in bacteria not only is associated with proton motive force depletion but obviously implies divalent cation limitation.
Enhancement of 1,4-Dihydroxy-2-Naphthoic Acid Production by
Propionibacterium freudenreichii
ET-3 Fed-Batch Culture
Authors: Furuichi Keisuke , Katakura Yoshio , Ninomiya Kazuaki , Shioya Suteaki
Published: March 2007

The production of 1,4-dihydroxy-2-naphthoic acid (DHNA) was investigated using a fed-batch culture of
Propionibacterium freudenreichii
ET-3. DHNA is a precursor of menaquinone (MK) and is transformed to MK by combination with an isoprenoid unit. We found that ET-3 stopped MK production and increased DHNA production in an anaerobic fed-batch culture by maintaining the lactose concentration at approximately zero. The maximum DHNA concentration observed in the anaerobic fed-batch culture was markedly higher than the maximum DHNA concentration observed in an anaerobic batch culture. Moreover, MK or DHNA production was affected by the lactose feeding rate; this suggests that lactose metabolism participates in the syntheses of these products. On the other hand, accumulation of propionate was found to inhibit DHNA production in the fed-batch culture. Based on the fact that ET-3 increases DHNA production in an aerobic culture by consuming propionate, we carried out a cultivation experiment in which an anaerobic fed-batch culture was switched to an anaerobic batch culture and found that the DHNA production was increased to a greater extent than the DHNA production in an anaerobic fed-batch culture. These results suggest that DHNA production by ET-3 is markedly influenced by carbon source limitation and the oxygen supply.
Characterization of Genetically Matched Isolates of
Campylobacter jejuni
Reveals that Mutations in Genes Involved in Flagellar Biosynthesis Alter the Organism's Virulence Potential
Authors: Malik-Kale Preeti , Raphael Brian H. , Parker Craig T. , Joens Lynn A. , Klena John D. , Quiñones Beatriz , Keech Amy M. , Konkel Michael E.
Published: March 2007

Phenotypic and genotypic evidence suggests that not all
Campylobacter jejuni
isolates are pathogenic for humans. We hypothesized that differences in gene content or gene expression alter the degree of pathogenicity of
C. jejuni
isolates. A
C. jejuni
isolate (Turkey) recovered from a turkey and a second
C. jejuni
isolate (CS) recovered from a chicken differed in their degrees of in vitro and in vivo virulence. The
C. jejuni
Turkey isolate invaded INT 407 human epithelial cells and secreted the Cia (
Campylobacter i
nvasion
a
ntigen) proteins, while the
C. jejuni
CS isolate was noninvasive for human epithelial cells and did not secrete the Cia proteins. Newborn piglets inoculated with the
C. jejuni
Turkey isolate developed more severe clinical signs of campylobacteriosis than piglets inoculated with the
C. jejuni
CS isolate. Additional work revealed that flagellin was not expressed in the
C. jejuni
CS isolate. Microarray and real-time reverse transcription-PCR analyses revealed that all flagellar class II genes were significantly downregulated in the
C. jejuni
CS isolate compared to the
C. jejuni
Turkey isolate. Finally, nucleotide sequencing of the
flgR
gene revealed the presence of a single residue that was different in the FlgR proteins of the
C. jejuni
Turkey and CS isolates. Complementation of the
C. jejuni
CS isolate with a wild-type copy of the
flgR
gene restored the isolate's motility. Collectively, these findings support the hypothesis that critical differences in gene content or gene expression can alter the pathogenic potential of
C. jejuni
isolates.
Effects of Specific Inhibitors on Anammox and Denitrification in Marine Sediments
Authors: Jensen Marlene Mark , Thamdrup Bo , Dalsgaard Tage
Published: March 2007

The effects of three metabolic inhibitors (acetylene, methanol, and allylthiourea [ATU]) on the pathways of N2production were investigated by using short anoxic incubations of marine sediment with a15N isotope technique. Acetylene inhibited ammonium oxidation through the anammox pathway as the oxidation rate decreased exponentially with increasing acetylene concentration; the rate decay constant was 0.10 ± 0.02 μM−1, and there was 95% inhibition at ∼30 μM. Nitrous oxide reduction, the final step of denitrification, was not sensitive to acetylene concentrations below 10 μM. However, nitrous oxide reduction was inhibited by higher concentrations, and the sensitivity was approximately one-half the sensitivity of anammox (decay constant, 0.049 ± 0.004 μM−1; 95% inhibition at ∼70 μM). Methanol specifically inhibited anammox with a decay constant of 0.79 ± 0.12 mM−1, and thus 3 to 4 mM methanol was required for nearly complete inhibition. This level of methanol stimulated denitrification by ∼50%. ATU did not have marked effects on the rates of anammox and denitrification. The profile of inhibitor effects on anammox agreed with the results of studies of the process in wastewater bioreactors, which confirmed the similarity between the anammox bacteria in bioreactors and natural environments. Acetylene and methanol can be used to separate anammox and denitrification, but the effects of these compounds on nitrification limits their use in studies of these processes in systems where nitrification is an important source of nitrate. The observed differential effects of acetylene and methanol on anammox and denitrification support our current understanding of the two main pathways of N2production in marine sediments and the use of15N isotope methods for their quantification.
Inhibition of Quorum Sensing in
Pseudomonas aeruginosa
by
N
-Acyl Cyclopentylamides
Authors: Ishida Takenori , Ikeda Tsukasa , Takiguchi Noboru , Kuroda Akio , Ohtake Hisao , Kato Junichi
Published: March 2007

N
-Octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in
Pseudomonas aeruginosa
PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in
P. aeruginosa
PAO1. The
lasB
-
lacZ
and
rhlA
-
lacZ
reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing.
N
-Decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for
lasB
-
lacZ
and
rhlA
-
lacZ
expression were 80 and 90 μM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of
P. aeruginosa
PAO1. C10-CPA inhibited induction of both
lasI
-
lacZ
by
N
-(3-oxododecanoyl)-
l
-homoserine lactone (PAI1) and
rhlA
-
lacZ
by
N
-butanoyl-
l
-homoserine lactone (PAI2) in the
lasI rhlI
mutant of
P. aeruginosa
PAO1, indicating that C10-CPA interferes with the
las
and
rhl
quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.
SYBR Green Real-Time PCR Method To Detect
Clostridium botulinum
Type A
Authors: Fenicia Lucia , Anniballi Fabrizio , De Medici Dario , Delibato Elisabetta , Aureli Paolo
Published: March 2007

Botulinum toxins (BoNTs) are classically produced by
Clostridium botulinum
but rarely also from neurotoxigenic strains of
Clostridium baratii
and
Clostridium butyricum
. BoNT type A (BoNT/A), BoNT/B, BoNT/E, and very rarely BoNT/F are mainly responsible for human botulism. Standard microbiological methods take into consideration only the detection of
C. botulinum
. The presumptive identification of the toxigenic strains together with the typing of BoNT has to be performed by mouse bioassay. The development of PCR-based methods for the detection and typing of BoNT-producing clostridia would be an ideal alternative to the mouse bioassay. The objective of this study was to develop a rapid and robust real-time PCR method for detecting
C. botulinum
type A. Four different techniques for the extraction and purification of DNA from cultured samples were initially compared. Of the techniques used, Chelex 100, DNeasy tissue kit, InstaGene matrix DNA, and boiling, the boiling technique was significantly less efficient than the other three. These did not give statistically different results, and Chelex 100 was chosen because it was less expensive than the others. In order to eliminate any false-negative results, an internal amplification control was synthesized and included in the amplification mixture according to ISO 22174. The specificity of the method was tested against 75 strains of
C. botulinum
type A, 4 strains of
C. botulinum
type Ab, and 101 nontarget strains. The detection limit of the reaction was less than 6 × 10
1
copies of
C. botulinum
type A DNA. The robustness of the method was confirmed using naturally contaminated stool specimens to evaluate the tolerance of inhibitor substances. SYBR green real-time PCR showed very high specificity for the detection of
C. botulinum
types A and Ab (inclusivity and exclusivity, 100%).
Impacts of Cultivation of Marine Diatoms on the Associated Bacterial Community
Authors: Sapp Melanie , Wichels Antje , Gerdts Gunnar
Published: March 2007

The composition of bacterial communities associated with four diatom species was monitored during isolation and cultivation of algal cells. Strong shifts in the associated communities, linked with an increase in the numbers of phylotypes belonging to members of the
Gammaproteobacteria
, were observed during cultivation.
Continuous Synthesis and Excretion of the Compatible Solute Ectoine by a Transgenic, Nonhalophilic Bacterium
Authors: Schubert Torsten , Maskow Thomas , Benndorf Dirk , Harms Hauke , Breuer Uta
Published: March 2007

The compatible solute 1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid (ectoine) acts in microorganisms as an osmotic counterweight against halostress and has attracted commercial attention as a protecting agent. Its production and application are restricted by the drawbacks of the discontinuous harvesting procedure involving salt shocks, which reduces volumetric yield, increases reactor corrosion, and complicates downstream processing. In order to synthesize ectoine continuously in less-aggressive media, we introduced the ectoine genes
ectABC
of the halophilic bacterium
Chromohalobacter salexigens
into an
Escherichia coli
strain using the expression vector pASK-IBA7. Under the control of a
tet
promoter, the transgenic
E. coli
synthesized 6 g liter
−1
ectoine with a space-time yield of 40 mg liter
−1
h
−1
, with the vast majority of the ectoine being excreted.
Prevalence of
Campylobacter
spp. in Cattle in Finland and Antimicrobial Susceptibilities of Bovine
Campylobacter jejuni
Strains
Authors: Hakkinen Marjaana , Heiska Helmi , Hänninen Marja-Liisa
Published: March 2007

The study investigated the prevalence of
Campylobacter
spp. in Finnish cattle at slaughter and carcass contamination after slaughter. During the period January to December 2003, bovine rectal fecal samples (
n
= 952) and carcass surface samples (
n
= 948) from 12 out of 15 Finnish slaughterhouses were examined. In total, campylobacters were detected in 31.1% of fecal samples and in 3.5% of carcass surface samples.
Campylobacter jejuni
was isolated from 19.5%,
Campylobacter coli
from 2.2%, and presumptive
Campylobacter hyointestinalis
from 10.8% of fecal samples. Campylobacters were detected in 4.4% and 37.4% of the fecal samples examined both by direct culture and by enrichment (
n
= 730), respectively, suggesting a low level of campylobacters in the intestinal content. A slightly increasing trend was observed in the overall prevalence of campylobacters towards the end of summer and autumn. Seventeen different serotypes were detected among the fecal
C. jejuni
isolates using a set of 25 commercial antisera for serotyping heat-stable antigens (Penner) of
C. jejuni
by passive hemagglutination. The predominant serotypes, Pen2 and Pen4-complex, were isolated from 52% of the fecal samples. Subtyping by pulsed-field gel electrophoresis (SmaI) yielded 56 and 20 subtypes out of 330 fecal and 70 carcass
C. jejuni
isolates, respectively. MICs of ampicillin, enrofloxacin, erythromycin, gentamicin, nalidixic acid, and oxytetracycline for 187
C. jejuni
isolates were determined using a commercial broth microdilution method. Sixteen (9%) of the isolates were resistant to at least one of the antimicrobials tested. Resistance to nalidixic acid was most commonly detected (6%). No multiresistance was observed.
Stable Isotope Probing with
15
N Achieved by Disentangling the Effects of Genome G+C Content and Isotope Enrichment on DNA Density
Authors: Buckley Daniel H. , Huangyutitham Varisa , Hsu Shi-Fang , Nelson Tyrrell A.
Published: March 2007

Stable isotope probing (SIP) of nucleic acids is a powerful tool that can identify the functional capabilities of noncultivated microorganisms as they occur in microbial communities. While it has been suggested previously that nucleic acid SIP can be performed with
15
N, nearly all applications of this technique to date have used
13
C. Successful application of SIP using
15
N-DNA (
15
N-DNA-SIP) has been limited, because the maximum shift in buoyant density that can be achieved in CsCl gradients is approximately 0.016 g ml
−1
for
15
N-labeled DNA, relative to 0.036 g ml
−1
for
13
C-labeled DNA. In contrast, variation in genome G+C content between microorganisms can result in DNA samples that vary in buoyant density by as much as 0.05 g ml
−1
. Thus, natural variation in genome G+C content in complex communities prevents the effective separation of
15
N-labeled DNA from unlabeled DNA. We describe a method which disentangles the effects of isotope incorporation and genome G+C content on DNA buoyant density and makes it possible to isolate
15
N-labeled DNA from heterogeneous mixtures of DNA. This method relies on recovery of “heavy” DNA from primary CsCl density gradients followed by purification of
15
N-labeled DNA from unlabeled high-G+C-content DNA in secondary CsCl density gradients containing bis-benzimide. This technique, by providing a means to enhance separation of isotopically labeled DNA from unlabeled DNA, makes it possible to use
15
N-labeled compounds effectively in DNA-SIP experiments and also will be effective for removing unlabeled DNA from isotopically labeled DNA in
13
C-DNA-SIP applications.
Stable Isotope Probing with
15
N
2
Reveals Novel Noncultivated Diazotrophs in Soil
Authors: Buckley Daniel H. , Huangyutitham Varisa , Hsu Shi-Fang , Nelson Tyrrell A.
Published: March 2007

Biological nitrogen fixation is a fundamental component of the nitrogen cycle and is the dominant natural process through which fixed nitrogen is made available to the biosphere. While the process of nitrogen fixation has been studied extensively with a limited set of cultivated isolates, examinations of
nifH
gene diversity in natural systems reveal the existence of a wide range of noncultivated diazotrophs. These noncultivated diazotrophs remain uncharacterized, as do their contributions to nitrogen fixation in natural systems. We have employed a novel
15
N
2
-DNA stable isotope probing (
5
N
2
-DNA-SIP) method to identify free-living diazotrophs in soil that are responsible for nitrogen fixation in situ. Analyses of 16S rRNA genes from
15
N-labeled DNA provide evidence for nitrogen fixation by three microbial groups, one of which belongs to the
Rhizobiales
while the other two represent deeply divergent lineages of noncultivated bacteria within the
Betaproteobacteria
and
Actinobacteria
, respectively. Analysis of
nifH
genes from
15
N-labeled DNA also revealed three microbial groups, one of which was associated with
Alphaproteobacteria
while the others were associated with two noncultivated groups that are deeply divergent within
nifH
cluster I. These results reveal that noncultivated free-living diazotrophs can mediate nitrogen fixation in soils and that
15
N
2
-DNA-SIP can be used to gain access to DNA from these organisms. In addition, this research provides the first evidence for nitrogen fixation by
Actinobacteria
outside of the order
Actinomycetales
.
Characterization of Cultures Enriched from Acidic Polycyclic Aromatic Hydrocarbon-Contaminated Soil for Growth on Pyrene at Low pH
Authors: Uyttebroek Maarten , Vermeir Steven , Wattiau Pierre , Ryngaert Annemie , Springael Dirk
Published: March 2007

Two polycyclic aromatic hydrocarbon (PAH)-contaminated soils of pH 2 were successfully used as inoculum to enrich cultures growing on phenanthrene and pyrene at different pHs, including pH 3. Selected pyrene-utilizing cultures obtained at pH 3, pH 5, and pH 7 were further characterized. All showed rapid [
14
C]pyrene mineralization at pH 3 and pH 5 and grew on pyrene at pH values ranging from 2 to 6. Eubacterial and mycobacterial 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and sequencing indicated that the cultures were dominated by a single bacterium closely related to
Mycobacterium montefiorense
, belonging to the slow-growing
Mycobacterium
sp. In contrast, a culture enriched on pyrene at pH 7 from a slightly alkaline soil sampled at the same site was dominated by
Pseudomonas putida
and a fast-growing
Mycobacterium
sp. The
M. montefiorense
-related species dominating the pyrene-utilizing cultures enriched from the acidic soils was also the dominant
Mycobacterium
species in the acidic soils. Our data indicate that a slow-growing
Mycobacterium
species is involved in PAH degradation in that culture and show that bacteria able to degrade high-molecular-weight PAHs at low pH are present in acidic PAH-contaminated soil.