Normative data of multifrequency tympanometry in rabbits

In an experimental study, we evaluated acoustic immittance in rabbits in order to use these data as normative values for further experimental investigations. This study is the first experimental evaluation of both conventional 226 Hz and multifrequency tympanometry (MFT) in rabbits. For the investigation, we used 33 female New Zealand rabbits weighing 3.2-4.4 kg and aged six months. Bilateral measurements using conventional 226 Hz and MFT were performed under general anaesthetic. A 226 Hz tympanogram was recorded for all animals by conducting an air pressure sweep from +200 to -400 daPa at a rate of 50 daPa/s. Subsequent tympanograms were recorded over a wide frequency range from 250 to 2000 Hz. The acoustic impedance device used in this study provided reproducible and evaluable tympanograms. The applied tone frequency of 226 Hz proved to be especially suitable for determining compliance. Normative data obtained from our study reveal the resonance frequency to be 1368 +/- 205 standard deviation (SD) for the right side and 1413 +/- 216 SD for the left side. The values for physiological acoustic immittance of the middle ear in the rabbit obtained here can serve as normative data in subsequent experimental animal studies.     

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS

To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with (13)C-carbon and (15)N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.     

FeMo cofactor biosynthesis in a nifE- mutant of Rhodobacter capsulatus.

In all diazotrophic micro-organisms investigated so far, mutations in nifE, one of the genes involved in the biosynthesis of the FeMo cofactor (FeMoco), resulted in the accumulation of cofactorless inactive dinitrogenase. In this study, we have found that strains of the phototrophic non-sulfur purple bacterium Rhodobacter capsulatus with mutations in nifE, as well as in the operon harbouring the nifE gene, were capable of reducing acetylene and growing diazotrophically, although at distinctly lower rates than the wild-type strain. The diminished rates of substrate reduction were found to correlate with the decreased amounts of the dinitrogenase component (MoFe protein) expressed in R. capsulatus. The in vivo activity, as measured by the routine acetylene-reduction assay, was strictly Mo-dependent. Maximal activity was achieved under diazotrophic growth conditions and by supplementing the growth medium with molybdate (final concentration 20-50 microM). Moreover, in these strains a high proportion of ethane was produced from acetylene ( approximately 10% of ethylene) in vivo. However, in in vitro measurements with cell-free extracts as well as purified dinitrogenase, ethane production was always found to be less than 1%. The isolation and partial purification of the MoFe protein from the nifE mutant strain by Q-Sepharose chromatography and subsequent analysis by EPR spectroscopy and inductively coupled plasma MS revealed that FeMoco is actually incorporated into the protein (1.7 molecules of FeMoco per tetramer). On the basis of the results presented here, the role of NifNE in the biosynthetic pathway of the FeMoco demands reconsideration. It is shown for the first time that NifNE is not essential for biosynthesis of the cofactor, although its presence guarantees formation of a higher content of intact FeMoco-containing MoFe protein molecules. The implications of our findings for the biosynthesis of the FeMoco will be discussed.     

Über die Pylorusanhänge des Seesterns (Asterias rubens L.), insbesondere ihre Innervation

Zusammenfassung Die Pylorusanhänge (P. A.) von Asterias rubens L. wurden licht- und elektronenmikroskopisch untersucht. — Die Wandung der P. A. gliedert sich in eine hohe Epithelschicht, in deren basalen Abschnitten zahlreiche Nervenfasern verlaufen, eine bindegewebige Basallamelle und ein Coelomepithel, dessen Zellen eine lockere Lage von glatten Muskelzellen und Axonen bedecken.Die Zellen des Epithels der P. A. sind mit einem dichten, gleichmäßig ausgebildeten Bürstensaum ausgestattet, ferner mit je einer langen, mit einer Wimperwurzel verbundenen Cilie (9+2-Typus), die sich in die Lichtung der Caeca erhebt. Die Wimperwurzeln besitzen eine periodische Gliederung. Vereinzelte Epithelzellen tragen ein plumperes, unregelmäßig gestaltetes Geäst von Mikrovilli. Die bereits lichtmikroskopisch wahrnehmbare zarte Streifung der Zellapices beruht auf dem Vorhandensein von parallelisierten Mikrotubuli. Starke Vesikulation der Saumzellen und das Vorhandensein pinozytotischer Bläschen an der Basis der Mikrovilli ist als Ausdruck lebhafter resorptiver Tätigkeit des Epithels der P. A. anzusehen. Vor allem in den basalen Abschnitten der Epithelzellen liegen kugelige Lipideinschlüsse, in anderen Zellen Mukopolysaccharidgranula. An der Oberfläche der Epithelzellen werden Mukopolysaccharide ausgestoßen, teilweise in Form zusammengesinterter, unregelmäßig gestalteter Bildungen. Das morphologische Äquivalent der von anderen Autoren beschriebenen sog. Zymogenkörnchen konnte mit Sicherheit nicht ermittelt werden. Intraepitheliale Sinneszellen wurden nicht beobachtet.Die nackten Axone innerhalb des Epithels der P. A. lagern sich den Basalteilen der Saumzellen an, bilden jedoch mit ihnen keine typischen, d. h. durch Membranverdickungen ausgezeichneten Synapsen. Ihr Axoplasma enthält außer Neurotubuli Bläschen mit massendichtem Inhalt, die Granula aminerger oder peptiderger Nervenfasern ähneln. Möglicherweise sind bei Asterias Nervenfasern jeweils verschiedenen Inhalts ausgebildet, da Profile von Axonen mit kleineren (1000–1200 Å Durchmesser) und größeren Granula (1200–1600 Å) nachzuweisen sind. Das Vorkommen von Ergastoplasmastrukturen und Golgimembranen in den Axonen sowie die Abschnürung von Vesikeln mit massendichtem Inhalt vom Golgiapparat spricht für eine Entstehung von Elementargranula in der Peripherie der entsprechenden Neurone. Das Bild synaptischer Bläschen kann durch entleerte Elementargranula vorgetäuscht werden.Die glatten Muskelzellen unter dem Coelomepithel stehen mit Nervenendigungen, die dense-cored vesicles enthalten, in Berührung, doch sind Synapsen mit Membranverdickungen, wie sie bei den Vertebraten angetroffen werden, nicht ausgebildet.

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Summary The pyloric ceca (p. c.) of the starfish, Asterias rubens L., are investigated light- and electronmicroscopically.The wall of the p. c. consists of 1. a high columnar epithelium the basal part of which is permeated by many axons, 2. a basal lamina, 3. a loose layer of smooth muscle cells intermingled with thin nerve fibres, and 4. the coelomic epithelium. Intraepithelial sensory elements are lacking in the epithelium.The epithelial cells of the p. c. are provided with a very well developed brush border built up by microvilli of equal length and diameter. In addition each cell bears a long cilium (9 + 2 pattern) connected with a periodically structured rootlet. Only single cells are characterized by a more or less irregular seam of plump ramified microvilli. The parallel orientation of microtubules in the apical parts of the cells causes the striation already to be observed under the light microscope.The intense vesiculation of the cytoplasm and the occurrence of many pinocytotic invaginations on the basis of the brush border is considered to be the equivalent of the high absorption activity of the epithelial cells.Spheroidal lipid inclusions, mucopolysaccharide granules and large masses of unknown nature are embedded in the cytoplasm of the epithelial cells. Irregularly shaped mucopolysaccharide substances are extruded into the lumen of the p. c. The occurrence of so-called zymogen granules has not been observed in our material.The intraepithelial axons are closely attached to the basal parts of the epithelial cells without forming typical synapses with membrane thickenings. Apart from neurotubules, the axoplasm of these tiny fibres contains dense-cored vesicles (diameter 1000–1200 Å, 1200–1600 Å) resembling the elementary granules of aminergic and peptidergic nerve fibres. The presence of ergastoplasmic structures and Golgi membranes within the axoplasm and the gemmation of dense-cored vesicles from the Golgi apparatus speaks in favour of a peripheral elaboration of elementary granules in the axons of the starfish.The smooth muscle cells covered by the coelomic epithelium of the p. c. are in contact with axonal terminals containing dense-cored and empty vesicles. Typical synaptic structures as described for vertebrates apparently do not exist in the starfish.

     

Role of nucleoside transport in glucocorticoid-induced regression of mouse lymphoma P1798

Exposure of corticoid-sensitive P1798 lymphocytes to cortisol results in inhibition of uridine uptake and incorporation with no effect on 2-deoxyglucose transport. Nucleoside uptake by corticoid-resistant cells is not affected by the hormone. Inhibition of uridine transport may play a key role in tumor regression and does not depend on reduced availability of glucose.