Wickerhamiella slavikovae sp. nov. and Wickerhamiella goesii sp. nov., two yeast species isolated from natural substrates.

Two novel yeast species were isolated during three independent studies of yeasts associated with natural substrates in Brazil and Taiwan. Analysis of the sequences of the D1/D2 domains of the large subunit rRNA gene showed that these novel species belong to the Wickerhamiella clade. The first was isolated from freshwater and a leaf of sugar cane (Saccharum officinarum) in Brazil and from leaves of Wedelia biflora in Taiwan. Described here as Wickerhamiella slavikovae sp. nov., it differs by 56 nucleotide substitutions and 19 gaps in the D1/D2 region of the large subunit rRNA gene from Candida sorbophila, the least divergent species. The second species, named Wickerhamiella goesii sp. nov., was isolated from leaves and the rhizosphere of sugar cane collected in Rio de Janeiro, Brazil. The species differs by 54 nucleotide substitutions and nine gaps in the D1/D2 domains from Candida drosophilae, its least divergent relative. The type strains are Wickerhamiella slavikovae sp. nov. IMUFRJ 52096(T) (= CBS 12417(T) = DBVPG 8032(T)) and Wickerhamiella goesii sp. nov. IMUFRJ 52102(T) (= CBS 12419(T) = DBVPG 8034(T)).

Keratinolytic activity of Bacillus subtilis AMR using human hair.

AIMS: To determine the ability of a novel Bacillus subtilis AMR isolated from poultry waste to hydrolyse human hair producing peptidases including keratinases and hair keratin peptides. METHODS AND RESULTS: The Bacillus subtilis AMR was identified using biochemical tests and by analysis of 16S rDNA sequence. The isolate was grown in medium containing human hair as the sole source of carbon and nitrogen. The supplementation of hair medium (HM) with 0.01% yeast extract increased the keratinolytic activity 4.2-fold. B. subtilis AMR presented high keratinase production on the 8th day of fermentation in hair medium (HM) supplemented with 0.01% yeast extract (HMY) at pH 8.0. Keratinase yield was not correlated with increase in biomass. Zymography showed keratin-degrading peptidases migrating at c. 54, 80 and 100 kDa and gelatin-degrading bands at c. 80, 70 63, 54 32 and 15 kDa. Keratinases were optimally active at 50 degrees C and pH 9.0 and was fully inhibited by the serine proteinase inhibitor (PMSF). Scanning electron microscopy showed complete degradation of the hair cuticle after exposure to B. subtilis AMR grown in HMY. MALDI-TOF analysis of culture supernatant containing peptides produced during enzymatic hydrolysis of hair by B. subtilis AMR revealed fragments in a range of 800-2600 Da. CONCLUSIONS: This study showed that B. subtilis AMR was able to hydrolyse human hair producing serine peptidases with keratinase and gelatinase activity as well as hair keratin peptides. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report describing the production and partial characterization of keratinases by a B. subtilis strain grown in a medium containing human hair. These data suggest that peptides obtained from enzymatic hair hydrolysis may be useful for future applications on pharmaceutical and cosmetic formulations.

Diversity of bacteria in the marine sponge Aplysina fulva in Brazilian coastal waters.

Microorganisms can account for up to 60% of the fresh weight of marine sponges. Marine sponges have been hypothesized to serve as accumulation spots of particular microbial communities, but it is unknown to what extent these communities are directed by the organism or the site or occur randomly. To address this question, we assessed the composition of specific bacterial communities associated with Aplysina fulva, one of the prevalent sponge species inhabiting Brazilian waters. Specimens of A. fulva and surrounding seawater were collected in triplicate in shallow water at two sites, Caboclo Island and Tartaruga beach, Búzios, Brazil. Total community DNA was extracted from the samples using "direct" and "indirect" approaches. 16S rRNA-based PCR-denaturing gradient gel electrophoresis (PCR-DGGE) analyses of the total bacterial community and of specific bacterial groups--Pseudomonas and Actinobacteria--revealed that the structure of these assemblages in A. fulva differed drastically from that observed in seawater. The DNA extraction methodology and sampling site were determinative for the composition of actinobacterial communities in A. fulva. However, no such effects could be gleaned from total bacterial and Pseudomonas PCR-DGGE profiles. Bacterial 16S rRNA gene clone libraries constructed from directly and indirectly extracted DNA did not differ significantly with respect to diversity and composition. Altogether, the libraries encompassed 15 bacterial phyla and the candidate division TM7. Clone sequences affiliated with the Cyanobacteria, Chloroflexi, Gamma- and Alphaproteobacteria, Actinobacteria, Bacteroidetes, and Acidobacteria were, in this order, most abundant. The bacterial communities associated with the A. fulva specimens were distinct and differed from those described in studies of sponge-associated microbiota performed with other sponge species.

Fingerprints of partial oxidation of biogenic magnetite from cultivated and natural marine magnetotactic bacteria using synchrotron radiation.

Magnetotactic bacteria are a multi-phyletic group of bacteria that synthesize membrane-bound magnetic minerals. Understanding the preservation of these minerals in various environments (e.g., with varying oxygen concentrations and iron supply) is important for understanding their role as carriers of primary magnetizations in sediments and sedimentary rocks. Here we present X-ray near edge structure (XANES) spectra for Fe in magnetotactic bacteria samples from recent sediments to assess surface oxidation and crystal structure changes in bacterial magnetite during early burial. Our results are compared with a XANES spectrum of cultivated Magnetofaba australis samples, and with magnetic properties, and indicate that oxidation of magnetite to maghemite increases with depth in the sediment due to longer exposure to molecular oxygen. These results are relevant to understanding magnetic signatures carried by magnetofossils in oxic sediments and sedimentary rocks of different ages.

Plasma membrane Ca2+-ATPase isoform 2a is the PMCA of hair bundles.

Mechanoelectrical transduction channels of hair cells allow for the entry of appreciable amounts of Ca(2+), which regulates adaptation and triggers the mechanical activity of hair bundles. Most Ca(2+) that enters transduction channels is extruded by the plasma membrane Ca(2+)-ATPase (PMCA), a Ca(2+) pump that is highly concentrated in hair bundles and may be essential for normal hair cell function. Because PMCA isozymes and splice forms are regulated differentially and have distinct biochemical properties, we determined the identity of hair bundle PMCA in frog and rat hair cells. By screening a bullfrog saccular cDNA library, we identified abundant PMCA1b and PMCA2a clones as well as rare PMCA2b and PMCA2c clones. Using immunocytochemistry and immunoprecipitation experiments, we showed in bullfrog sacculus that PMCA1b is the major isozyme of hair cell and supporting cell basolateral membranes and that PMCA2a is the only PMCA present in hair bundles. This complete segregation of PMCA1 and PMCA2 isozymes holds for rat auditory and vestibular hair cells; PMCA2a is the only PMCA isoform in hair bundles of outer hair cells and vestibular hair cells and is the predominant PMCA of hair bundles of inner hair cells. Our data suggest that hair cells control plasma membrane Ca(2+)-pumping activity by targeting specific PMCA isozymes to distinct subcellular locations. Because PMCA2a is the only Ca(2+) pump present at appreciable levels in hair bundles, the biochemical properties of this pump must account fully for the physiological features of transmembrane Ca(2+) pumping in bundles.

Three-dimensional analysis of the 16 nm urothelial plaque particle: luminal surface exposure, preferential head-to-head interaction, and hinge formation.

The luminal surface of mouse urothelium in contact with the urine is almost entirely covered with plaques consisting of uroplakin-containing particles that form p6 hexagonal crystals with a center-to-center distance of 16 nm. A combination of quick-freeze/deep-etch images and our previous negative staining data indicate that the head domain of the uroplakin particle, which is exposed without an extensive glycocalyx shield, interacts closely with the head domains of the neighboring particles, while the membrane-embedded tail domains are farther apart; and that urothelial particles and plaques are not rigid structures as they can change their configuration in response to mechanical perturbations. Based on these data, we have constructed three-dimensional models depicting the structural organization of urothelial particles and plaques. Our models suggest that the head-to-head interaction may play a key role in determining the shape and size of the urothelial plaques. These models can explain many properties of urothelial plaques including their unique shape, detergent-insolubility, and morphological changes during vesicle maturation.

Magnetosome size distribution in uncultured rod-shaped bacteria as determined by electron microscopy and electron spectroscopic imaging.

We report uncultured rod-shaped magnetotactic bacteria from natural waters that biomineralize magnetic crystals in two different size ranges. Electron spectroscopic imaging of whole bacteria deposited over formvar-coated grids permitted a better visualization and measurement of the magnetosomes. All magnetosomes of individual bacteria could be observed by this technique. The magnetosomes formed one large chain, composed of three to four columns of crystals, disposed in parallel to the main axis of the bacteria. The magnetosomes ranged from 19 to 136 nm in length and 14 to 112 nm width. Smaller magnetosomes (less than 80 nm in length) localized mostly in extremities of the bacterial body while larger preferentially localized in the middle part of the cell. Electron spectroscopic diffraction and X-ray microanalysis indicate that both types of magnetosomes contain magnetite (Fe3O4). In projection, most magnetosomes seem to present pseudo-hexagonal morphologies described for magnetite. As the aspect ratios for smaller and larger magnetosomes are different, we suggest that different levels of control on biomineralization of magnetosomes may exist.

Imaging faces of shadowed magnetite (Fe(3)O(4)) crystals from magnetotactic bacteria with energy-filtering transmission electron microscopy.

We used energy-filtering transmission electron microscopy to image magnetite crystals isolated from uncultured magnetotactic bacteria. These magnetite crystals were shadowed in high vacuum with platinum at 45 degrees. The shadowed crystals were observed in a Zeiss (Thornwood, NY) CEM902 transmission electron microscope. Imaging shadowed crystals with inelastically scattered electrons provided information of the decoration pattern of small platinum particles over crystal surfaces, and thus information on surface characteristics of crystals. Results were comparable to those obtained from scanning electron microscopy using a field emitter gun. Electron energy loss spectra of the crystals as well as of the supporting film were recorded to evaluate variations of image contrast with energy losses. Results indicated that the contrast is attenuated with inelastic imaging and that the effect of contrast tuning caused a contrast inversion at a given point between 100 and 150 eV. We believe this approach can be useful for studying multilayered materials by transmission electron microscopy.

Structural basis for mechanical transduction in the frog vestibular sensory apparatus: III. The organization of the otoconial mass.

The saccule and the utricle of the vestibular system detect linear acceleration and gravity. Sensory transduction in these organs depends on myriads of calcium carbonate crystals of high specific gravity, called otoconia, embedded in a filament matrix that overlies the sensory epithelium. The coexistence of hard crystals and slender filaments in this complex extracellular matrix makes it difficult to analyze by conventional electron microscopy. We have now examined this structure in the bullfrog saccule using the quick-freeze, deep-etch replica technique. The otoconia in their typical aragonite polymorph shape exhibit smooth surfaces and are embedded in a loose matrix made of two types of filaments. The regular surface of the otoconia forms a natural smooth background against which we could observe with unprecedented detail the network organization and substructure of the filaments. One type of filament is 8 nm in diameter, while the other, which has a characteristic beaded appearance, is 15 nm in diameter. Both types of filaments either make lateral connections with or end directly on the surface of the otoconia. A consistent observation was the presence of short filaments that directly cross-link adjacent otoconia. Very few otoconia were fractured in an orientation that would allow the study of their internal architecture. These otoconia presented a typical conchoidal cleavage of aragonite. Although crystallites were not clearly apparent, thin lamellar microstructures appeared oriented both perpendicularly and longitudinally to the major otoconial axis. This structural study establishes a framework for the identification of the molecular components present in this unique extracellular matrix and may also help elucidate their role in mechanical transduction.

Electron Spectroscopic Imaging of Magnetotactic Bacteria: Magnetosome Morphology and Diversity.

Magnetotactic bacteria from aquatic environments were analyzed with the electron spectroscopic imaging technique. Rod-shaped bacteria and cocci were present in most of the samples observed. Magnetotactic multicellular aggregates were also observed at some of the sampling sites. The use of electron spectroscopic imaging allowed the observation of magnetosomes inside magnetotactic microorganisms with exceptional clarity. The number, size, and morphology of magnetosomes, as well as their ultrastructural spatial disposition inside the bacterial cell, could be directly observed and associated with the disposition of flagella of the respective cells.This allowed us to examine the structural relationships between magnetosomes and flagella, which are important components in the mechanisms of magnetotaxis. In disrupted magnetotactic multicellular aggregates, connections between cells were also visualized. We believe this technique will be useful in studying not only magnetotactic bacteria but also other uncultured microorganisms from natural environments.

Tracing heme in a living cell: hemoglobin degradation and heme traffic in digest cells of the cattle tick Boophilus microplus.

Heme is present in all cells, acting as a cofactor in essential metabolic pathways such as respiration and photosynthesis. Moreover, both heme and its degradation products, CO, iron and biliverdin, have been ascribed important signaling roles. However, limited knowledge is available on the intracellular pathways involved in the flux of heme between different cell compartments. The cattle tick Boophilus microplus ingests 100 times its own mass in blood. The digest cells of the midgut endocytose blood components and huge amounts of heme are released during hemoglobin digestion. Most of this heme is detoxified by accumulation into a specialized organelle, the hemosome. We followed the fate of hemoglobin and albumin in primary cultures of digest cells by incubation with hemoglobin and albumin labeled with rhodamine. Uptake of hemoglobin by digest cells was inhibited by unlabeled globin, suggesting the presence of receptor-mediated endocytosis. After endocytosis, hemoglobin was observed inside large digestive vesicles. Albumin was exclusively associated with a population of small acidic vesicles, and an excess of unlabeled albumin did not inhibit its uptake. The intracellular pathway of the heme moiety of hemoglobin was specifically monitored using Palladium-mesoporphyrin IX (Pd-mP) as a fluorescent heme analog. When pulse and chase experiments were performed using digest cells incubated with Pd-mP bound to globin (Pd-mP-globin), strong yellow fluorescence was found in large digestive vesicles 4 h after the pulse. By 8 h, the emission of Pd-mP was red-shifted and more evident in the cytoplasm, and at 12 h most of the fluorescence was concentrated inside the hemosomes and had turned green. After 48 h, the Pd-mP signal was exclusively found in hemosomes. In methanol, Pd-mP showed maximal emission at 550 nm, exhibiting a red-shift to 665 nm when bound to proteins in vitro. The red emission in the cytosol and at the boundary of hemosomes suggests the presence of heme-binding proteins, probably involved in transport of heme to the hemosome. The existence of an intracellular heme shuttle from the digestive vesicle to the hemosome acting as a detoxification mechanism should be regarded as a major adaptation of ticks to a blood-feeding way of life. To our knowledge, this is the first direct observation of intracellular transport of heme in a living eukaryotic cell. A similar approach, using Pd-mP fluorescence, could be applied to study heme intracellular metabolism in other cell types.

Amorphous mineral phases in magnetotactic multicellular aggregates.

Magnetotactic multicellular aggregates consist of several bacteria that produce iron sulfide magnetosomes through a complex and poorly understood process. We observed new amorphous mineral particles within the cytoplasm of magnetotactic multicellular aggregates. Elemental mapping and electron energy loss spectroscopy detected iron and oxygen, but not sulfur, in these particles. These amorphous particles were about the same size as mature magnetosomes, around 50-70 nm in diameter. No membranes were observed surrounding the amorphous minerals. Partially crystalline inclusions composed of a crystalline core and an amorphous region around them similar in texture to the amorphous particles were also present. The shape of these amorphous regions followed the shape of the crystalline cores they enveloped. These regions also contained oxygen and iron. The crystalline phase, as previously reported, contained sulfur and iron. The presence of independent amorphous particles has not been reported before in magnetotactic multicellular aggregates.

Minerals of the Radular Apparatus of Falcidens sp. (Caudofoveata) and the Evolutionary Implications for the Phylum Mollusca.

Minerals have been found in the radular teeth of molluscs from the classes Caudofoveata, Polyplacophora, Monoplacophora, and Gastropoda (Patellogastropoda: Acmaeidae, Patellidae). Here we report the discovery of amorphous iron oxide and hydroxyapatite in the highly modified radular apparatus of Falcidens sp. (Caudofoveata). The mineralization process in Falcidens sp. is unique: the components of the radular apparatus, unlike those of other molluscs, are not renewed during the animal's lifetime. We propose that the presence of mineralized teeth among the molluscs is not necessarily connected to their manner of obtaining food and suggest that the molluscan common ancestor had mineralized teeth.

Elemental analysis of uncultured magnetotactic bacteria exposed to heavy metals.

Natural enrichments of magnetotactic bacteria were used to study the sites where heavy metals accumulate in uncultured bacteria. Most bacteria obtained by magnetic concentration from these enrichments contained, in addition to the magnetosomes, large phosphorus-rich granules in the cytoplasm. Metal (Zn, Mn, Sr, Cd, Al, Cr, and Pb) chlorides were added independently to the enrichments, and after 24 h, the elemental composition of the phosphorus-rich granules, magnetosomes, and "soft parts" (cytoplasm plus cell envelope) of whole bacteria was analyzed by energy-dispersive X-ray analysis on a transmission electron microscope. All bacteria contained Mn and Sr in the phosphorus-rich granules; some of them presented Mn peaks also in the soft parts. Zinc accumulation was variable and was found mainly in the phosphorus-rich granules, but also in the soft part of some bacteria. Some analyzed bacteria presented Zn peaks only in the soft parts, and some of them did not present Zn in any structure. Cadmium and Al were found only in the granules of some bacteria. Chromium was found in the soft parts of some bacteria. Lead was not detected in any bacteria. We concluded that the phosphorus-rich granules are major sites for metal accumulation by these bacteria. No conclusive results for magnetosomes were obtained because of the limitations of the analytical techniques particularly when used for whole cell analysis.

The otoconia of the guinea pig utricle: internal structure, surface exposure, and interactions with the filament matrix.

A unique feature of the vertebrate gravity receptor organs, the saccule and utricle, is the mass of biomineral structures, the otoconia, overlying a gelatinous matrix also called "otoconial membrane" on the surface of the sensory epithelium. In mammals, otoconia are deposits of calcium carbonate in the form of composite calcite crystals. We used quick-freezing, deep etching to examine the otoconial mass of the guinea pig utricle. The deep-etching step exposed large expanses of intact and fractured otoconia, showing the fine structure and relationship between their internal crystal structure, their surface components, and the filament matrix in which they are embedded. Each otoconium has a compact central core meshwork of filaments and a composite outer shell of ordered crystallites and macromolecular aggregates. A distinct network of 20-nm beaded filaments covers the surface of the otoconia. The otoconia are interconnected and secured to the gelatinous matrix by surface adhesion and by confinement within a loose interotoconial filament matrix. The gelatinous matrix is a dense network made of yet another type of filament, 22 nm in diameter, which are cross-linked by shorter filaments, characteristically 11 nm in diameter. Our freeze-etching data provide a structural framework for considering the molecular nature of the components of the otoconial complex, their mechanical properties, and the degree of biological versus chemical control of otoconia biosynthesis.

Further studies on the endocytic activity of Tritrichomonas foetus.

The endocytic activity of Tritrichomonas foetus was studied at the ultrastructural level using gold-labeled macromolecules (bovine lactoferrin, human and bovine transferrin, bovine albumin, human low-density lipoprotein, horseradish peroxidase, and protein A). All macromolecules were ingested by the protozoan. Binding experiments showed that only bovine lactoferrin bound to the parasite surface in a process that could be inhibited by the unlabeled protein, suggesting that it binds and is internalized via receptors. Label-fracture experiments showed that the receptors were distributed in clusters that did not colocalize with intramembranous particles. Kinetics analysis of the internalization of bovine lactoferrin and horseradish peroxidase, associated with the cytochemical detection of acid phosphatase, revealed that proteins were rapidly ingested through small uncoated vesicles and delivered to acid phosphatase-containing compartments. The colocalization of gold-labeled proteins and reaction product indicative of enzyme activity was confirmed by electron spectroscopic imaging. Simultaneous incubation of cells in the presence of two proteins labeled with gold particles of different diameters showed that they were ingested through the same pathway and were concentrated into cytoplasmic vacuoles corresponding to lysosome-like organelles. These data suggest that the endocytic process in T. foetus is very rapid and that the intracellular pathway for receptor-mediated and fluid-phase endocytosis seems to be the same.

Electron microscopic study of the effect of zinc on Tritrichomonas foetus.

At concentrations of 3.1 to 24 mM, zinc inhibits the multiplication of and kills the pathogenic protozoan Tritrichomonas foetus. Transmission electron microscopy showed that the hydrogenosome, a organelle which is involved in the metabolism of pyruvate and the site of formation of molecular hydrogen, constitutes the main site of the initial effect of zinc. The hydrogenosomal vesicle increases its electron density and dimension. Electron spectroscopy imaging and the electron energy loss spectrum showed the presence of zinc, calcium, and oxygen in the electron-dense areas of the hydrogenosome.

[Counselling versus cognitive group therapy for tinnitus. A retrospective study of their efficacy]. / Counselling vs. Gruppentherapie bei chronischem Tinnitus. Ein retrospektiver Vergleich der Interventionseffizienz.

BACKGROUND: Both counselling and group therapy have been recommended for supporting patients with chronic tinnitus. It is unclear which of these treatments is superior. SCIENTIFIC QUESTION: This retrospective study aimed at comparing relief from tinnitus distress following counselling with that following cognitive group therapy. Distress relief was also compared to the distress level of the waiting group patients. METHOD: Tinnitus distress was assessed through the Tinnitus Questionnaire (TQ, Goebel and Hiller) at three different times: before treatment (in waiting list patients: at initial contact) and at 3 and 6 months after initial assessment. Data from 21 patients per group were included in the analysis. RESULTS: The initial tinnitus distress scores were similar in all groups (about 48 TQ points out of a maximum of 84). After 3 months, both counselling subjects and group therapy participants exhibited a significant distress reduction of 13 TQ points, which remained stable after 6 months. Patients on the waiting list experienced no distress relief over time. CONCLUSION: Results from our data demonstrate the need for a future prospective study on the comparison of efficacy of counselling vs cognitive group therapy.

Structure, morphology, and composition of silicon biocomposites in the palm tree Syagrus coronata (Mart.) Becc.

Syagrus coronata is an economically important palm tree grown as an ornament, for the oil extracted from its seeds, and the wax from its leaves which has several applications in industry. Silicon biocomposites were analyzed in leaves of S. coronata. Silica bodies were found as extracellular silica masses between the hypodermal-layer cell walls and in granules present in the vacuoles of palisade cells. Scanning electron microscopy of the hypodermal layer of cells showed a collection of spherical bodies embedded in enveloping cavities that outlined the general structure of the bodies. Globular subunits with sharp edges formed the spherical bodies that ranged from 6 to 10 microm in diameter (average, 7.8 microm). X-ray microanalysis detected only silicon and oxygen homogeneously distributed throughout the bodies. Vacuoles of palisade cells contained a large number of granules ranging from 20 nm to 1.2 microm in size (average, 300 nm). Transmission electron microscopy associated with electron spectroscopic imaging and electron energy loss spectroscopy were used to determine the elemental composition of the granules. Vacuolar granules were amorphous and composed of silicon and oxygen, suggesting they consist of amorphous silica biominerals. No nitrogen, indicative of organic matter, was detected in the granules.

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes.

Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the alpha-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.