bluuebunny/crossref_metadata_2025 huggingface dataset:
Historical biogeography and diversification within the Neotropical parrot genus Pionopsitta (Aves: Psittacidae)
Authors: Ribas Camila C. , Gaban‐Lima Renato , Miyaki Cristina Y. , Cracraft Joel
Published: September 2010

Aim  We investigate spatial and temporal patterns of diversification within the Neotropical avifauna using the phylogenetic history of parrots traditionally belonging to the genus Pionopsitta Bonaparte 1854. This genus has long been of interest for those studying Neotropical biogeography and diversity, as it encompasses species that occur in most Neotropical forest areas of endemism.

Location  The Neotropical lowland forests in South and Central America.

Methods  Phylogenetic relationships were investigated for all species of the genus Pionopsitta and five other short‐tailed parrot genera using complete sequences of the mitochondrial genes cyt b and ND2 as well as 26 plumage characters. The resulting phylogeny was used to test the monophyly of the genus, investigate species limits, and as a framework for reconstructing their historical biogeography and patterns of diversification.

Results  We found that the genus Pionopsitta, as previously defined, is not monophyletic and thus the Chocó, Central American and Amazonian species will now have to be placed in the genus Gypopsitta. The molecular and morphological phylogenies are largely congruent, but disagree on the position of one of the Amazon basin taxa. Using molecular sequence data, we estimate that species within Gypopsitta diversified between 8.7 and 0.6 Ma, with the main divergences occurring between 3.3 and 6.4 Ma. These temporal results are compared to other taxa showing similar vicariance patterns.

Main conclusions  The results suggest that diversification in Gypopsitta was influenced mainly by geotectonic events, marine transgressions and river dynamics, whereas Quaternary glacial cycles of forest change seem to have played a minor role in the origination of the currently recognized species.
Process and pattern in the biogeography of New Zealand – a global microcosm?
Authors: McDowall R. M.
Published: September 2010

Aim  To describe New Zealand’s historical terrestrial biogeography and place this history in a wider Southern Hemisphere context.

Location  New Zealand.

Methods  The analysis is based primarily on literature on the distributions and relationships of New Zealand’s terrestrial flora and fauna.

Results  New Zealand is shown to have a biota that has broad relationships, primarily around the cool Southern Hemisphere, as well as with New Caledonia to the north. There are hints of ancient Gondwanan taxa, although the long‐argued predominance of taxa derived by vicariant processes, driven by plate tectonics and the fragmentation of Gondwana, is no longer accepted as a principal explanation of the biota’s origins and relationships.

Main conclusions  Most of the terrestrial New Zealand flora and fauna has clearly arrived in New Zealand much more recently than the postulated separation of New Zealand from Gondwana, dated at c. 80 Ma. There is a view that New Zealand may have disappeared completely beneath the sea in the early Cenozoic, and acceptance of this would mean derivation of the entire biota by transoceanic dispersal. However, there are elements in the biota that seem to have broad distributions that date back to Gondwanan times, and also some that are thought unlikely to have been able to disperse to New Zealand across ocean gaps, especially freshwater organisms. Very strong connections to the biota of Australia, rather than to South America, are inconsistent with the timing of New Zealand’s ancient and early separation from Gondwana and seem likely to have resulted from dispersal.
Surface imaging of thermally sensitive particulate and fibrous materials with the atomic force microscope: a novel sample preparation method
Authors: BALDWIN P. M. , FRAZIER R. A. , ADLER J. , GLASBEY T. O. , KEANE M. P. , ROBERTS C. J. , TENDLER S. J. B. , DAVIES M. C. , MELIA C. D.
Published: September 2010

High‐resolution surface imaging by atomic force microscopy (AFM) of particulate materials is often problematic, principally as a result of the large height (z) variations in sample topography that either prevent the probe scanning over the particle or cause probe self‐imaging. This paper reports a novel method of embedding thermally sensitive particulate and fibrous materials which overcomes many of these problems and facilitates AFM imaging of these difficult materials. The process involves partial embedding of the sample in a cyanoacrylate film polymerized at room temperature. The sample heating required in currently used methods of particulate embedding is avoided and the method is therefore suitable for thermolabile materials. The cyanoacrylate film provides a flat hard surface which is ideal for AFM imaging, and the method has allowed successful imaging of relatively large particulate and fibrous samples such as starch granules and cellulose fibres. The cyanoacrylate has the added benefit that shrinkage holes in the film allow easy visual identification of areas where the film may have partially covered the sample.
Near‐field scanning optical microscopy studies of l‐α‐dipalmitoylphosphatidylcholine monolayers at the air–liquid interface
Authors: Shiku H. , Dunn R. C.
Published: September 2010

The phase structure in l‐α‐dipalmitoylphosphatidylcholine–2.0 mol% fluorescent 1,1′‐dioctadecyl‐3,3,3′,3′‐tetramethylindocarbocyanine perchlorate Langmuir monolayers dispersed on a 2 m sucrose solution subphase is studied with near‐field scanning optical microscopy (NSOM). Cantilevered NSOM probes operating in a tapping‐mode feedback or an optical interferometric feedback mode are capable of tracking the air–sucrose solution interface. At the micrometre scale, the NSOM fluorescence images reveal lipid domain features similar to those observed previously in supported Langmuir–Blodgett (LB) monolayers. At the submicrometre scale, the small nanometric lipid islands seen in LB films are not observed at the air–sucrose interface. This supports a mechanism in which domain formation in LB films can be induced by means of the transfer process onto the solid support. Progress towards extending these studies to films at the air–water interface using the optical interferometric feedback method is also discussed.
Classification of spatial textures in benign and cancerous glandular tissues by stereology and stochastic geometry using artificial neural networks
Authors: Mattfeldt T. , Gottfried H.‐W. , Schmidt V. , Kestler H. A.
Published: September 2010

Stereology and stochastic geometry can be used as auxiliary tools for diagnostic purposes in tumour pathology. Whether first‐order parameters or stochastic‐geometric functions are more important for the classification of the texture of biological tissues is not known. In the present study, volume and surface area per unit reference volume, the pair correlation function and the centred quadratic contact density function of epithelium were estimated in three case series of benign and malignant lesions of glandular tissues. The information provided by the latter functions was summarized by the total absolute areas between the estimated curves and their horizontal reference lines. These areas are considered as indicators of deviation of the tissue texture from a completely uncorrelated volume process and from the Boolean model with convex grains, respectively. We used both areas and the first‐order parameters for the classification of cases using artificial neural networks (ANNs). Learning vector quantization and multilayer feedforward networks with backpropagation were applied as neural paradigms. Applications included distinction between mastopathy and mammary cancer (40 cases), between benign prostatic hyperplasia and prostatic cancer (70 cases) and between chronic pancreatitis and pancreatic cancer (60 cases). The same data sets were also classified with linear discriminant analysis. The stereological estimates in combination with ANNs or discriminant analysis provided high accuracy in the classification of individual cases. The question of which category of estimator is the most informative cannot be answered globally, but must be explored empirically for each specific data set. Using learning vector quantization, better results could often be obtained than by multilayer feedforward networks with backpropagation.
The continuum normalization method for quantification of X‐ray spectra in biological microanalysis. 1. Generalized bremsstrahlung production cross‐sections and analysis using standards
Authors: Nicholson W. A. P. , Hall T. A.
Published: September 2010

The thin self‐supporting biological specimens used for quantitative X‐ray microanalysis are problematical because the sections are most unlikely to be uniform in thickness or density, so the intensities of the characteristic lines alone are not a good measure of composition. The method developed to overcome these problems was introduced by T. A. Hall in 1971 and uses the bremsstrahlung or continuum intensity recorded in the X‐ray spectrum to normalize each characteristic line, and hence is frequently referred to as the continuum normalization (CN) procedure.

 Reformulating the CN method of quantification in terms of generalized cross‐sections and calculating more accurate values of bremsstrahlung production using a formula allows us a better understanding of the options open to the analyst of biological thin sections by which the errors in the measurement may be reduced. If one chooses to use the original Hall (1971) method using Kramers cross‐sections, the window measuring the continuum for normalization should be set in the 4–7 keV region for typical scanning electron microscope and microprobe beam energies, 20–40 kV, and above 10 keV for transmission electron microscope energies of 80 kV and above. Although it is clear that peak counts must not contribute to the white count, the window should be as wide as possible to reduce statistical errors.
Limited utility of acetoxymethyl (AM)‐based intracellular delivery systems, in vivo: interference by extracellular esterases
Authors: JOBSIS PAUL D. , ROTHSTEIN EMILY C. , BALABAN ROBERT S.
Published: September 2010

The use of acetoxymethyl (AM) groups to deliver and trap exogenous optical probes inside cells is an established tool in cell biology/physiology, however, these probes have not been used extensively in vivo. In this study, the use of the acetoxymethyl delivery system for optical probes was evaluated, in vivo. Initial studies revealed very little trapped probe in intact tissues even when near saturating levels of probe were injected in living animals. We tested the hypothesis that extracellular esterases rapidly cleave the acetoxymethyl groups preventing the probes from entering cells, in vivo. The rates of hydrolysis of 11 acetoxymethyl probes in diluted porcine plasma revealed an essentially first order high rate dye cleavage with half times on the order of minutes or less. Studies on mice and rabbits revealed rates 10‐ to 2‐fold higher, respectively. These plasma studies suggested that the acetoxymethyl probes were being cleaved before having a chance to enter cells in tissues in vivo. This was confirmed using intravital 2‐photon excitation microscopy in muscle tissue where several acetoxymethyl probes were found to rapidly cleave in the vascular space during infusion and not be trapped in the muscle cells. Studies with succinimidyl esters that should quickly bind to proteins on cleavage also failed to enter cells, in vivo, consistent with the notion that the cleavage was occurring in the extracellular space. These data suggest that the high level of plasma and extracellular esterase activity render the classical acetoxymethyl probes ineffective for monitoring intracellular events, in vivo. Different approaches to trapping exogenous probes will need to be explored for physiological studies using intravital microscopy.
Limitation in obtainable surface roughness of hardened cement paste: ‘virtual’ topographic experiment based on focussed ion beam nanotomography datasets
Authors: TRTIK P. , DUAL J. , MUENCH B. , HOLZER L.
Published: September 2010

Surface roughness affects the results of nanomechanical tests. The surface roughness values to be measured on a surface of a porous material are dependent on the properties of the naturally occurring pore space. In order to assess the surface roughness of hardened cement paste (HCP) without the actual influence of the usual sample preparation for nanomechanical testing (i.e. grinding and polishing), focussed ion beam nanotomography datasets were utilized for reconstruction of 3D (nanoscale resolution) surface profiles of hardened cement pastes. ‘Virtual topographic experiments' were performed and root mean square surface roughness was then calculated for a large number of such 3D surface profiles. The resulting root mean square (between 115 and 494 nm) is considerably higher than some roughness values (as low as 10 nm) reported in the literature. We suggest that thus‐analysed root mean square values provide an estimate of a ‘hard’ lower limit that can be achieved by ‘artefact‐free’ sample preparation of realistic samples of hardened cement paste. To the best of our knowledge, this ‘hard’ lower limit was quantified for a porous material based on hydraulic cement for the first time. We suggest that the values of RMS below such a limit may indicate sample preparation artefacts. Consequently, for reliable nanomechanical testing of disordered porous materials, such as hardened cement paste, the preparation methods may require further improvement.
Real‐time three‐dimensional imaging of cell division by differential interference contrast microscopy
Authors: TSUNODA M. , ISAILOVIC D. , YEUNG E.S.
Published: September 2010

Differential interference contrast (DIC) microscopy can provide information about subcellular components and organelles inside living cells. Applicability to date, however, has been limited to 2D imaging. Unfortunately, understanding of cellular dynamics is difficult to extract from these single optical sections. We demonstrate here that 3D differential interference contrast microscopy has sub‐diffraction limit resolution both laterally and vertically, and can be used for following Madin Darby canine kidney cell division process in real time. This is made possible by optimization of the microscope optics and by incorporating computer‐controlled vertical scanning of the microscope stage.
New advances in the 3D characterization of mineral coating layers on paper
Authors: CHINGA‐CARRASCO G. , KAUKO H. , MYLLYS M. , TIMONEN J. , WANG B. , ZHOU M. , FOSSUM J. O.
Published: September 2010

The surface characteristics of a large set of commercial lightweight coated paper grades are explored. The quantification of the 3D structure is revealed by atomic force microscopy, laser profilometry and X‐ray microtomography. This comprehensive study demonstrates the suitability of different and modern methods for assessing critical coating layer properties, thus identifying the right tools for specific structural analyses. Based on the assessment of the top and bottom surfaces of 25 commercial lightweight coated samples, three main conclusions can be drawn: (1) the facet orientation polar angle is a function of roughness, (2) skewness did not describe the surface details affecting the gloss of the commercial lightweight coated samples assessed in this study and (3) surface roughness at wavelengths below approximately 1.0 μm does not affect the paper gloss significantly. This is important knowledge for the understanding of lightweight coated paper surface structure and its properties.
Differential aberration correction (DAC) microscopy: a new molecular ruler
Authors: VALLOTTON P.
Published: September 2010

Considerable efforts have been deployed towards measuring molecular range distances in fluorescence microscopy. In the 1–10 nm range, Förster energy transfer microscopy is difficult to beat. Above 300 nm, conventional diffraction limited microscopy is suitable. We introduce a simple experimental technique that allows bridging the gap between those two resolution scales in both 2D and 3D with a resolution of about 20 nm. The method relies on a computational approach to accurately correct optical aberrations over the whole field of view. The method is differential because the probes of interest are affected in exactly the same manner by aberrations as are the reference probes used to construct the aberration deformation field. We expect that this technique will have significant implications for investigating structural and functional questions in bio‐molecular sciences.
Three‐dimensional shapes and spatial distributions of Pt and PtCr catalyst nanoparticles on carbon black
Authors: GONTARD L. CERVERA , DUNIN‐BORKOWSKI R.E. , OZKAYA D.
Published: September 2010

High‐angle annular dark‐field scanning transmission electron microscopy tomography is applied to the study of Pt and PtCr nanoparticles supported on carbon black, which are used as heterogeneous catalysts in the electrodes of proton exchange membrane fuel cells. By using electron tomography, the three‐dimensional architecture of the heterogeneous catalyst system can be determined, providing high‐spatial‐resolution information about the shapes, faceting and crystallographies of 5–20 nm single and multiply twinned catalyst particles, as well as their positions with respect to the carbon support. Approaches that can be used to provide improved information about the distribution and orientation of the particles on their support are proposed and discussed. Our results show that electron tomography provides important information that is complementary to high‐resolution lattice imaging. Both techniques are required to understand fully the nature and role of the surfaces of faceted catalyst particles.
A pixel‐based likelihood framework for analysis of fluorescence recovery after photobleaching data
Authors: JONASSON J.K. , LORÉN N. , OLOFSSON P. , NYDÉN M. , RUDEMO M.
Published: September 2010

A new framework for the estimation of diffusion coefficients from data on fluorescence recovery after photobleaching (FRAP) with confocal laser scanning microscopy (CLSM) is presented. It is a pixel‐based statistical methodology that efficiently utilizes all information about the diffusion process in the available set of images. The likelihood function for a series of images is maximized which gives both an estimate of the diffusion coefficient and a corresponding error. This framework opens up possibilities(1)to obtain localized diffusion coefficient estimates in both homogeneous and heterogeneous materials,(2)to account for time differences between the registrations at the pixels within each image, and(3)to plan experiments optimized with respect to the number of replications, the number of bleached regions for each replicate, pixel size, the number of pixels, the number of images in each series etc. To demonstrate the use of the new framework, we have applied it to a simple system with polyethylene glycol (PEG) and water where we find good agreement with diffusion coefficient estimates from NMR diffusometry. In this experiment, it is also shown that the effect of the point spread function is negligible, and we find fluorochrome‐concentration levels that give a linear response function for the fluorescence intensity.
Scanning electron microscope electron detector with a radial type discrete dynode electron multiplier
Authors: HEJNA J.
Published: September 2010

A discrete dynode electron multiplier with radial flux of electrons was built and tested in the range of low‐voltage scanning electron microscopy as a backscattered electron detector of topographic contrast. The multiplier collects backscattered electron emitted in a specific range of take‐off angles and over the whole azimuth angular range enabling large solid collection angle. Multipliers with different dynode shapes were studied theoretically with the use of the software for particle optics and three assemblies were built and tested experimentally. The gain estimation, assessment of the type of detected electrons (secondary electron or backscattered electron), imaging the spatial collection efficiency and signal‐to‐noise measurements were performed.
Comparison and accuracy of methods to determine the confocal volume for quantitative fluorescence correlation spectroscopy
Authors: RÜTTINGER S. , BUSCHMANN V. , KRÄMER B. , ERDMANN R. , MACDONALD R. , KOBERLING F.
Published: September 2010

Single molecule detection based on fluorescent labels offers the possibility to gain not only qualitative but also quantitative insight into specific functions of complex biological systems. Fluorescence correlation spectroscopy is one of the favourite techniques to determine concentrations and diffusion constants as well as molecular brightness of molecules in the pico‐ to nano‐molar concentration range, with broad applications in biology and chemistry. Although fluorescence correlation spectroscopy in principle has the potential to measure absolute concentrations and diffusion coefficients, the necessity to know the exact size and shape of the confocal volume very often hampers the possibility to obtain quantitative results and restricts fluorescence correlation spectroscopy to relative measurements mainly. The determination of the confocal volume in situ is difficult because it is sensitive to optical alignment and aberrations, optical saturation and variations of the index of refraction as observed in biological specimen. In the present contribution, we compare different techniques to characterize the confocal volume and to obtain the confocal parameters by fluorescence correlation spectroscopy curve fitting, a fluorescence correlation spectroscopy dilution series and confocal scanning of fluorescent beads. The results are compared in the view of quantitative fluorescence correlation spectroscopy measurement and analysis. We investigate how unavoidable artefacts caused by a non‐ideal confocal volume can be experimentally determined and validated.
Electroantennogram responses of a parasitic wasp, Microplitis croceipes, to host‐related volatile and anthropogenic compounds
Authors: Park Kye Chung , Zhu Junwei , Harris Jennifer , Ochieng Samuel A. , Baker Thomas C.
Published: September 2010

The parasitic wasp Microplitis croceipes (Cresson) (Hymenoptera: Braconidae) showed its own characteristic electroantennogram (EAG) response profiles to 13 host‐related (cis‐3‐hexenol, α‐pinene (R)‐(+)‐limonene (S)‐(–)‐limonene, trans‐β‐ocimene (±)‐linalool, (–)‐trans‐caryophyllene, α‐humulene, nerolidol, trans‐nerolidol, cis‐nerolidol, methyl jasmonate and indole) and four anthropogenic (2‐diisopropylaminoethanol, 2,2′‐thiodiethanol, 2‐methyl‐5‐nitroaniline and cyclohexanone) volatile compounds. These profiles were similar between males and females except for 2‐diisopropylaminoethanol, which elicited significantly larger EAG responses in males. Among the compounds tested, cis‐3‐hexenol, linalool and cyclohexanone elicited the largest EAG responses. EAG responses were not influenced by the age of wasps between 1 and 13 days after emergence. EAG responses were dose‐dependent, and highly EAG‐active compounds elicited significant EAG responses with less than 10 μg of the compounds at source. Quantification of compounds released from an odour cartridge indicates that release rate is highly dependent on the chemical nature of stimuli, showing up to 10 000‐fold differences in the amount released between different compounds when the same amount was loaded in the odour cartridge. Wasps having undergone a behavioural training regime to be attracted to either cyclohexanone or methyl jasmonate did not show any differences in EAG responses from those of untrained wasps.
Bactemides gingivalis vesicles mediate attachment of streptococci to serum‐coated hydroxyapatite
Authors: Singh Umadatt , Grenier Daniel , McBride Barry C.
Published: September 2010

Outer membrane vesicles purified from Bacteroides gingivalis culture supernatant bound to serum coated hydroxyapatite (SeHA). The immobilized vesicles served as receptors for a number of species of oral streptococci. The binding of Streptococcus sangnis 12 to SeHA was increased 10 times by the vesicles. Vesicle‐associated binding increased proportionally with an increase in the number of bound vesicles. Arginine and lactose both partially reduced binding of 5. sanguis. Heating the vesicles destroyed their binding ability whereas heating S. sanguis reduced but did not eliminate vesicle‐mediated binding.
Electrophoretic analysis of ureases in Streptococcus salivarius and in saliva
Authors: Sissons C. H. , Loong P. C. , Hancock E. M. , Cutress T. W.
Published: September 2010

Experimental conditions were established for the extraction, electrophoresis and detection of urease isoenzymes from Streptococcus salivarius. Thiol concentrations were critical and ureases from different strains varied in ease of dissociation. A characteristic pattern was obtained for 30 ureolytic S. salivarius strains isolated from; saliva (6), dental plaque (12), artificial dental plaque (6) and non‐oral sources (6), and also for a ureolytic Streptococcus bovis from artificial plaque. One non‐oral S. salivarius strain had ureases with slightly slower mobility. The electrophoretic pattern and mobility of ureases extracted from human mixed salivary bacteria were identical to those from S. salivarius except for an additional set of urease bands from unknown species of bacteria. There were no ureases from saliva matching those from Staphylococcus epidermidis– a contributor to ureolysis in artificial plaque. We conclude that there is considerable biochemical homogeneity among S. salivarius ureases and possibly other ureolytic streptococci. In saliva. urea is metabolized mainly by streptococcal ureases.
Purification and characterization of glycylprolyl aminopeptidase from Bacteroides gingivalis
Authors: Miyauchi T. , Hayakawa M. , Abiko Y.
Published: September 2010

A glycylprocyl aminopeptidase from cell extracts of Bacteriodes gingivalis 381 was purified 1058‐fold by hydrophobic adsorbent, HPLC anion exchange, and HPLC gel filtration column chromatography. The final enzyme preparation was homogeneous with a molecular weight of 75,000 daltons by SDS‐PAGE, and the isoelectric point was 6.2. The optimum pH of the enzyme was 8.0, and the enzyme activity was inhibited by DFP Ni2+ and Hg2+.
Salivary levels of putative cariogenic organisms in patients with eating disorders
Authors: Bretz W. A. , Krahn D. D. , Drewnowski A. , Loesche W. J.
Published: September 2010

The present study examined the hypothesis that women with eating disorders associated with a history of chronic vomiting can be characterized by a salivary flora with high levels of aciduric organisms, such as, mutans strepotococci, lactobacilli and yeast. Three groups of female subjects were studied: vomiting bulimics (G1; n = 14), and comparison groups selected for high Streptococcus mutans (G2; n = 13), and low S. mutans levels (G3; n = 12). The prevalence and levels of mutans streptococci, lactobacilli and yeast tended to be higher in bulimics than in non‐bulimics. The bulimics had significantly higher levels and higher prevalence of Streptococcus sobrinus than the non‐bulimics. A high S. sobrinus colonization may be a marker for a history of vomiting in bulimia.
The occurrence of Actinobacillus actinomycetemcomitans, Bacteroides gingivalis, Bacteroides intermedius and spirochaetes in the subgingival microflora in relation to the early onset of periodontitis in a group of adolescents
Authors: Ashley F. P. , Gallagher J. , Wilson R. F.
Published: September 2010

Subgingival samples were obtained from mesial and distal surfaces of first molars in 100 subjects aged 14 years at baseline. Sites were monitored at 6‐monthly intervals for clinical loss of attachment. Six subjects showed greater than 1 mm loss of attachment at one site 18‐24 months later. Further samples were obtained from these sites together witha comparison sample from the contralateral site and equivalent sites in a matched control subject. Actinobacillus actinomycetemcomitans was more fequently isolated from sites following loss of attachment, but it was also found in one comparison site and one control subject. Bacteroides gingivalis was found in only one subject at a site which subsequently experienced loss of attachment. A higher median proportion of spirochaetes was observed in sites after diagnosis of loss of attachment. The median proportion of Bacteroides intermedius was higher at both the baseline and 12‐month examinations in subjects who subsequently lost attachment. Further work is required to substantiate the differences which preceded diagnosis of loss of attachment.
Lipopolysaccharide from Actinobacillus actinomycetemcomitans stimulates macrophages to produce interleukin‐1 and tumor necrosis factor mRNA and protein
Authors: Saglie F. Reinaldo , Simon Ken , Merrill Jean , Koeffler H. Phillip
Published: September 2010

Actinobacillus actinomycetemcomitans is associated with periodontal disease in children and adults. We report that low concentrations of lipopolysaccharide (LPS) from A. actinomycetemcomitans stimulated human macrophages to increase dramatically their accumulation of mRNA coding for interleukin‐1α (IL‐1α), IL‐1β as well as tumor necrosis factor (TNF). Protein levels of IL‐1 and TNFa also increased. Levels of these mRNAs increased by 4–5 fold as compared with unstimulated macrophages when these cells were cultured with as little as 2 ng/ ml LPS from A. actinomycetemcomitans. Polymyxin binds and blocks the action of LPS; polymyxin inhibited the ability of LPS from A. actinomycetemcomitans to increase levels of IL‐1β mRNA. The LPS of A. actinomycetemcomitans stimulated increased levels of IL‐1β mRNA in the presence of cycloheximide, showing that stimulation by this LPS did not require new synthesis of protein. Furthermore, dexamethasone inhibited the ability of LPS from A. actinomycetemcomitans to stimulate the accumulation of mRNA coding for IL‐1βA. actinomycetemcomitans is an invasive microorganism of the gingiva; high intragingival numbers correlate with sites undergoing local destruction of the periodontium. IL‐1α, IL‐1β, and TNF are potent monokines that mediate inflammation and resorption of bone. Out studies suggest that macrophages migrating to these gingival sites of A. actinomycetemcomitans infection will be stimulated by LPS of A. actinomycetemcomitans to produce IL‐1α, IL‐1β and TNF. These cytokines will mediate gingival inflammation and stimulate resorption of alveolar bone.
Haemagglutinating and haemolytic activity of the extracellular vesicles of Bacteroides gingivalis W50
Authors: Kay Helen M. , Birss Andrew J. , Smalley John W.
Published: September 2010

The extracellular vesicles (ECV) and extracellular protein (EP) fractions of Bacteroides gingivalis W50 showed haemagglutinating (HA) activity towards sheep erythrocytes. Similar fractions from the nonpathogenic strain W50/BE1 did not haemagglutinate. W50 ECV HA activity was not inhibited by various glycosidase, phospholipase or protease pretreatments, sugars or amino acids, including arginine or lysine. The haemagglutinating activity of ECV was associated only with the extracellular vesicle membrane. The EP and ECV of both strains displayed haemolytic activity. This activity was apparently depressed in the presence of 10 mM dithiothreitol (DTT). All EP and ECV fractions degraded certain structural sheep erythrocyte membrane proteins. The greatest activity was displayed by W50 ECV and W50/BE1 EP and was enhanced by DTT. In the Presence of DTT, the ECV of both strains degraded purified human haemoglobin but this activity was greatly reduced in its absence.
Maintenance of proton motive force by Streptococcus mutans and Streptococcus sobrinus during growth in continuous culture
Authors: Hamilton I. R.
Published: September 2010

The components of the transmembrane electrochemical proton gradient, or proton motive force (PMF, Δp), were determined in cells of Streptococcus mutans Ingbritt and Streptococcus sobrinus ATCC 27352 growing in continuous culture under conditions of changing glucose concentration, growth rate and growth pH. The pH gradient (ΔpH) and membrane electrical potential (ΔΨ) were assayed with the weak acid, salicylic acid, and the lipophilic cation, methyltriphenylphosphonium iodide, respectively. S. mutans Ingbritt growing in continuous culture (pH 7.0, dilution rate (D) = 0.1 h−1) at 8 glucose concentrations ranging from 2.8 to 288 mM maintained a relatively constant Δp of 58.3 mV (SD ± 5.8) in spite of a transition from glucose to nitrogen‐limited growth and significant changes in cell physiology. Changes included a decreasing yield constant, increasing glucose uptake rates in the ehemostat, repression of Ellglc of the PEP phosphotransferase sugar transport system and decreasing glycolytic capacity of the cells as the medium glucose concentration increased. Changes in the dilution or growth rate of S. mutans Ingbritt from 0.1 to 1.0 h−1 and S. sobrinus from 0.1 to 0.8 h−1, when growing at pH 7.0 with limited glucose and lactose, respectively, resulted in significantly lower Δp values due to the dissipation of the ΔΨ. When the cells of S. mutans Ingbritt were grown with excess glucose (nitrogen limitation), lower Δp values were observed at pH 5.5, but not at pH 7.0. These results indicate that the generation of proton motive force is maintained by S. mutans and S. sobrinus during growth in continuous culture, except when the organisms are stressed at high growth rates and with excess glucose at low pH. Furthermore, the data indicate that, unlike other bacteria, these oral streptococci do not maintain significant pH gradients (i.e., < 0.74) even when growing in continuous culture at pH values as low as 5.0.
In vitro antimicrobial sensitivity of enteric rods and pseudomonads from advanced adult periodontitis
Authors: Slots Jørgen , Feik Diane , Rams Thomas E.
Published: September 2010

The prevalence and in vitro antimicrobial sensitivity of isolates of enteric rods and pseudomonads was examined in 844 adult periodontitis patients. These organisms were recovered from 13.5% of the study subjects. Ciprofloxacin exhibited the highest inhibitory activity of the 14 oral antimicrobial agents tested. Beta‐lactam antibiotics were largely ineffective, and tetracycline failed to inhibit most Pseudomonas species. In vitro sensitivity data suggest that a subgingival microbiota comprising mainly streptococci would result from therapy that combined ciprofloxacin and metronidazole. Since streptococci may inhibit the growth of several putative periodontal pathogens, populations of “beneficial” streptococci in the periodontal pocket might constitute a very attractive therapeutic outcome. Controlled clinical studies are needed to clarify the possible role of ciprofloxacin in the treatment of destructive periodontitis.
Oral and vaginal epithelial cell anti‐Candida activity is acid labile and does not require live epithelial cells
Authors: Yano J. , Lilly E. A. , Steele C. , Fortenberry D. , Fidel P. L.
Published: September 2010

Background:  Candida albicans is the causative agent of oral and vaginal candidiasis. Innate host defenses against C. albicans are important against each infection. Among these are oral and vaginal epithelial cells that have anti‐Candida activity. The mechanism of action includes a requirement for cell contact with no role for soluble factors, and a putative role for carbohydrates based on the sensitivity of the activity to periodic acid.

Methods:  Periodic acid treatment of epithelial cells as well as the property of partial resistance of antifungal activity to fixation was used to further dissect the mechanism of action.

Results:  The results herein effectively now challenge a role for carbohydrates alone. Firstly, the putative carbohydrate(s) released into supernatants of periodic acid‐treated epithelial cells could not compete with fresh epithelial cells for activity, and equivalent abrogation of activity was observed by periodic acid‐treated cells irrespective of the amount of carbohydrate released. Instead, the similar abrogation of activity following treatment with other acids or when cocultured under acidic conditions suggests that the activity is acid‐labile. Finally, while activity requires intact epithelial cells, it does not require live cells; activity was minimally affected by fixing epithelial cells prior to coculture where the majority of cells remained impermeable to Trypan blue but were defined as non–viable by positive nuclear staining with propidium iodide.

Conclusion:  These results suggest that antifungal activity is dependent on contact by intact, but not necessarily live, epithelial cells through an acid‐labile mechanism.
Microbicidal efficacy of ozonated water against Candida albicans adhering to acrylic denture plates
Authors: Arita M. , Nagayoshi M. , Fukuizumi T. , Okinaga T. , Masumi S. , Morikawa M. , Kakinoki Y. , Nishihara T.
Published: September 2010

Background/aims:  Ozone is known to act as a strong antimicrobial agent against bacteria, fungi, and viruses. We examined the effect of ozonated water on Candida albicans on acrylic denture plate.

Methods:  The heat‐cured acrylic resins were cultured with C. albicans. After treatment of flowing ozonated water, the number of attached C. albicans was counted. In some experiments, the test samples were treated with ozonated water in combination with ultrasonication.

Results:  After exposure to flowing ozonated water (2 or 4 mg/l) for 1 min, viable C. albicans cells were nearly nonexistent. The combination of ozonated water and ultrasonication had a strong effect on the viability of C. albicans adhering to the acrylic resin plates. There were no significant differences in antimicrobial activity against C. albicans between plates immersed in ozonated water with ultrasonication and those treated with commercially available denture cleaners. In addition, electron microscopic analysis revealed that small amounts of C. albicans remained on the plate after exposure to flowing ozonated water or immersion in ozonated water with ultrasonication.

Conclusion:  Our results suggest that application of ozonated water may be useful in reducing the number of C. albicans on denture plates.
Commensal oral bacteria antigens prime human dendritic cells to induce Th1, Th2 or Treg differentiation
Authors: Kopitar A. N. , Ihan Hren N. , Ihan A.
Published: September 2010

In various immunopathologic conditions, bacterial flora induce an immune response which results in inflammatory manifestations, e.g. periapical granuloma. Dendritic cells provide the main orchestration of specific immune responses. The aim of our study was to test the capacity of distinct oral bacterial antigens (prepared from Streptococcus mitis, Propionibacterium acnes, and Bacteroides spp.) to prime human dendritic cells for stimulation of the T‐lymphocyte response. To assess the T‐lymphocyte response, the expression of CD25, CD69, intracellular interferon γ (cIFN‐γ), and intracellular interleukin 4 (cIL‐4) was determined. Dendritic cells were prepared from leukocyte buffy coat from healthy blood donors. Monocytes were stimulated with IL‐4 and GM‐CSF and dendritic cells activated with bacterial lysates. Cell suspensions contained up to 90% dendritic cells, which represented 2–12% of the initial number of mononuclear cells. Lymphocyte subsets that developed in lymphocyte cultures after 1 week of stimulation were analyzed by flow cytometry. Dendritic cells, primed with antigens of Bacteroides fragilis have shown significantly higher activation and expression of intercellular IFN‐γ by T lymphocytes compared to negative controls. The dendritic cells primed with antigens of P. acnes had no effect on T‐lymphocyte activation or cytokine production; instead they induced differentiation of T lymphocytes into CD25bright cells (regulatory T cells) with a potentially inhibitory effect on immune response. Dendritic cells primed with antigens of S. mitis induced increased expression of cIL‐4. We conclude that commensal oral bacteria antigens prepared from B. fragilis, S. mitis, and P. acnes prime human dendritic cells to induce Th1, Th2, and Treg differentiation, respectively. This may advance our understanding of immunopathologic manifestations in the oral cavity and offer new possibilities for redirecting immune responses in mucosal vaccination.
A new checkerboard panel for testing bacterial markers in periodontal disease
Authors: Dahlén G. , Leonhardt Å.
Published: September 2010

Background/aims:  Various microbiological methods have been used for testing bacterial markers for periodontitis and periodontal disease progression. Most studies have used only a limited number of well recognized bacterial species. The purpose of the present study was to evaluate the association of 13 more recently identified bacterial species in a new panel in comparison with 12 previously more recognized periodontotopathogens (‘old panel’) using the ‘checkerboard’ DNA–DNA hybridization method.

Methods:  Fifty individuals were chosen who showed at least one site with a probing pocket depth of 6 mm or more (disease) and bleeding on probing and at least one site with a probing pocket depth of 3 mm and without bleeding on probing (health). One diseased and one healthy site on each individual were sampled with the paperpoint technique and the samples were processed in the checkerboard technique against deoxigenin‐labeled whole genomic probes to 25 subgingival species representing 12 well recognized and 13 newly identified periodontitis associated species.

Results:  Twenty‐four (out of 25) species were detected more frequently in the subgingival plaque of diseased than healthy sites both at score 1 (> 104) and score 3 (> 105). A significant difference at the higher score (score 3) was noticed for all species of the old panel except for three (Streptococcus intermedius, Selenomonas noxia, and Eikenella corrodens). Of the species in the new panel only Prevotella tannerae, Filifactor alocis, and Porphyromonas endodontalis showed a statistical significant difference between diseased and healthy sites.

Conclusion:  It was concluded that P. tannerae, F. alocis, and P. endodontalis should be added to the 12 species used for routine diagnostics of periodontitis‐associated bacterial flora.
Cytokine pattern determines the progression of experimental periodontal disease induced by Actinobacillus actinomycetemcomitans through the modulation of MMPs, RANKL, and their physiological inhibitors
Authors: Garlet G. P. , Cardoso C. R. , Silva T. A. , Ferreira B. R. , Ávila‐Campos M. J. , Cunha F. Q. , Silva J. S.
Published: September 2010

Objective:  Inflammatory and immune reactions raised in response to periodontopathogens are thought to trigger periodontal tissue destruction. We therefore investigated the expression of matrix metalloproteinases (MMPs) and the osteoclastogenic factor RANKL (receptor activator of nuclear factor‐κB ligand), their respective inhibitors TIMPs (tissue inhibitors of metalloproteinases) and OPG (osteoprotegerin) and their possible correlation with the expression of inflammatory and regulatory cytokines in the course of experimental periodontal disease in mice.

Methods:  We characterized the time course of leukocyte migration and alveolar bone loss in C57BL/6 mice infected with Actinobacillus actinomycetemcomitans. Quantitative polymerase chain reaction (RealTime PCR) and ELISA were performed to determine the expression of MMPs, TIMPs, RANKL, OPG and cathepsin K, interleukin‐1β, tumor necrosis factor‐α, interferon‐γ, interleukin‐12, interleukin‐4 and interleukin‐10 in periodontal tissue samples harvested throughout the course of experimental disease.

Results:  Oral inoculation of A. actinomycetemcomitans results in an intense and widespread migration of leukocytes to the gingival tissues, besides marked alveolar bone resorption. Our data also demonstrate two distinct patterns of MMP/TIMP and RANKL/OPG expression in the course of experimental periodontal disease. The expression of MMPs (MMP‐1, 2 and 9) and RANKL was correlated with the expression of interleukin‐1β, tumor necrosis factor‐α and interferon‐γ, in a time period characterized by the intense increase of inflammatory reaction and alveolar bone loss. On the other hand, interleukin‐4 and interleukin‐10 were associated with higher expression of TIMPs (TIMP 1, 2 and 3) and OPG, with a lower expression of MMPs and RANKL, and with reduced rates of increase of cellular infiltration in periodontal tissues and alveolar bone loss.

Conclusions:  It is possible that the pattern of cytokines produced in periodontal tissues determines the progression and the severity of experimental periodontal disease, controlling the breakdown of soft and bone tissues through the balance between MMPs/TIMP and RANKL/OPG expression in gingival tissues.
Genomic variation in Streptococcus mutans: deletions affecting the multiple pathways of β‐glucoside metabolism
Authors: Old L. A. , Lowes S. , Russell R. R. B.
Published: September 2010

The genome of Streptococcus mutans UA159 contains two phospho‐β‐glucosidase genes, bglA and celA, which occur in operon‐like arrangements along with genes for components of phosphotransferase transport systems and a third phospho‐β‐glucosidase encoded by the arb gene, which does not have its own associated transport system but relies on uptake by the bgl or cel systems. Targeted inactivation of each of the phospho‐β‐glucosidase genes revealed that bglA is involved in aesculin hydrolysis, celA is essential for utilisation of cellobiose, amygdalin, gentobiose and salicin, and arb is required for utilisation of arbutin. Inactivation of genes for the phosphotransferase systems revealed an overlap of specificity for transport of β‐glucosides and also indicated that further, unidentified transport systems exist. The cel and arb genes are subject to catabolite repression by glucose, but the regM gene is not essential for catabolite repression. Screening a collection of isolates of S. mutans revealed strains with deletions affecting the msm, bgl and/or cel operons. The phenotypes of these strains could largely be explained on the basis of the results obtained from the knockout mutants of S. mutans UA159 but also indicated the existence of other pathways apparently absent from UA159. The extensive genetic and phenotypic variation found in β‐glucoside metabolism indicates that there may be extensive heterogeneity in the species.
Binding of glucan‐binding protein C to GTFD‐synthesized soluble glucan in sucrose‐dependent adhesion of Streptococcus mutans
Authors: Matsumoto M. , Fujita K. , Ooshima T.
Published: September 2010

Streptococcus mutans produces glucan‐binding proteins (Gbp proteins) which promote the adhesion of the organism to teeth. Three Gbp proteins, GbpA protein, GbpB protein, and GbpC protein have been identified; however, the mechanism of adhesion between glucans and bacterial cell surfaces is unknown. We used glucosyltransferase (GTF)‐ and/or Gbp‐deficient mutants to examine the role of GbpC protein in the sucrose‐dependent cellular adhesion of S. mutans to glass surfaces. The wild‐type strain MT8148 and a GbpA‐deficient mutant strain displayed increased sucrose‐dependent adhesion following the addition of rGTFD. However, a GbpC‐deficient mutant strain demonstrated no changes in the level of sucrose‐dependent adhesion in spite of the addition of rGTFD. Further, the binding of rGbpC protein to the glucan synthesized by rGTFD was significantly higher than that to the glucan synthesized by either rGTFB or rGTFC. These results suggest that GbpC protein may play an important role in sucrose‐dependent adhesion by binding to the soluble glucan synthesized by GTFD.