Electrical responses of the marine ciliate Euplotes vannus (Hypotrichia) to mechanical stimulation at the posterior cell end

Electrical responses upon mechanostimulation at the posterior cell end were investigated in the marine hypotrichous ciliate Euplotes vannus. A new mechanostimulator was developed to mimic stimuli that are identical with those involved in cell-cell collisions. The receptor potential hyperpolarized by 18–35 mV within 12–25 msec, reached a peak value of -62 mV with a delay of 4–9 msec after membrane deformation, and was deactivated after 50–70 msec. Cirri were stimulated to beat accelerated backward. The corresponding receptor current exerted a similar time course with a peak of 2.4 nA. The shift of the reversal potential by 57.6 mV at a tenfold increase of [K⁺] ₀ identifies potassium ions as current carriers within the development of the receptor potential. An intracellular K concentration of 355 mmol/liter was calculated for cells in a medium that was composed similar to sea-water. The mechanically activated potassium current was totally inhibited by extracellular TEA and intracellular Cs⁺, and partially inhibited by extracellular 4-AP. The total inhibition of the current by injected EGTA points to a Ca dependence of the posterior mechanosensitivity. It was confirmed by the increase of the peak current amplitude with rising [Ca²⁺] ₀ . Sodium presumably repolarizes the receptor potential because the repolarization was delayed and after-depolarizations were eliminated in media without sodium. Since deciliation did not affect mechano-sensitivity, the corresponding ion channels reside within the soma membrane.The authors wish to thank Mr. Norbert Spreckelmeier from the electronics workshop and Mr. Herbert Lutter from the fine-mechanical workshop of the department for their excellent work, Mrs. G. Key and Mr. H. Mikoleit for skillful technical assistance and for preparing the figures. This work was supported by Deutsche Forschungsgemeinschaft, SFB 171, C7.     

Proline 21, a residue within the α-helical domain of ΦX174 lysis protein E, is required for its function in Escherichia coli

ΦX174 lysis protein E-mediated lysis of Escherichia coli is characterized by a protein E-specific fusion of the inner and outer membrane and formation of a transmembrane tunnel structure. In order to understand the fusion process, the topology of protein E within the envelope complex of E. coli was investigated. Proteinase K protection studies showed that, during the time course of protein E-mediated lysis process, more of the fusion protein E-FXa-streptavidin gradually became accessible to the protease at the cell surface. These observations postulate a conformational change in protein E during induction of the lysis process by movement of the C-terminal end of the protein throughout the envelope complex from the inner side to the outer side spanning the entire pore and fusing the inner and outer membranes at distinct areas. The initiation mechanism for such a conformational change could be the cis–trans isomerization of proline residues within α-helical membrane-spanning segments. Conversion of proline 21, presumed to be in the membrane-embedded α-helix of protein E, to alanine, glycine, serine and valine, respectively, resulted in lysis-negative E mutant proteins. Proteinase K accessibility studies using streptavidin as a reporter fused to the P21G mutant protein showed that the C-terminal part of the fusion protein is not translocated to the outer side of the membrane, suggesting that this proline residue is essential for the correct folding of protein E within the cell wall complex of E. coli . Oligomerization of protein P21G-StrpA was not disturbed.     

Biotransformation of diphenyl ether by the yeastTrichosporon beigelii SBUG 752

Trichosporon beigelii SBUG 752 was able to transform diphenyl ether. By TLC, HPLC, GC, GC-MS, NMR- and UV-spectroscopy, several oxidation products were identified. The primary attack was initiated by a monooxygenation step, resulting in the formation of 4-hydroxydiphenyl ether, 2-hydroxydiphenyl ether and 3-hydroxydiphenyl ether (48:47:5). Further oxidation led to 3,4-dihydroxydiphenyl ether. As a characteristic product resulting from the cleavage of an aromatic ring, the lactone of 2-hydroxy-4-phenoxymuconic acid was identified. The possible mechanism of ring cleavage to yield this metabolite is discussed.     

Turnover and transport of quinolizidine alkaloids. Diurnal fluctuations of lupanine in the phloem sap,leaves and fruits of Lupinus albus L.

Quinolizidine alkaloids formed in the leaves of Lupinus albus L. are translocated via the phloem to the other plant organs, especially the maturing fruits. Compared with amino-acid transport in the phloem, the alkaloids contribute about 8% to the overall nitrogen being exported from the leaf. Since it is likely that the alkaloids are subsequently degraded in the target tissues a minor role of quinolizidine alkaloids might be nitrogen transport. A marked diurnal fluctuation of alkaloids was observed in the leaves, the phloem sap, the roots and the fruits with an increase during the day and an amplitude of several hundred percent thus providing evidence for a rapid turnover of endogenous alkaloids.Abbreviations QA quinolizidine alkaloids - GLC gas-liquid chromatography     

Die postembryonale Entwicklung und die funktionelle Anatomie des Gnathosoma in der Milbenfamilie Erythraeidae (Acarina,Prostigmata)

Zusammenfassung Das Gnathosoma von sieben Arten der Milbenfamilie Erythraeidae wurde untersucht. Die postembryonale Entwicklung der Cuticula-Strukturen, der Muskulatur und der Drüsen wird beschrieben.Das Gnathosoma der Larven weist Cheliceren auf, die aus Grundglied und klauenförmigem Digitus mobilis bestehen. Sie liegen dem Infracapitulum dorsal auf und inserieren an einem Paar Capitular-Apophysen. Bei den Protound Tritonymphen unterliegt das Gnathosoma der Histolyse und wird anschließend jeweils neu ausgebildet. Bei der Deutonymphe und beim Adultus ist es gegenüber der Larve stark abgewandelt: Die Tracheenstämme sind aus den Capitular-Apophysen der Larve abzuleiten. Die Stech-Cheliceren entsprechen dem Grundglied der larvalen Cheliceren, und ihre medialen Protraktoren gehen aus den Levator-Muskeln der Cheliceren der Larve hervor.Der Weg der Sekrete der podocephalischen und infracapitulären Drüsen über die Dorsalfläche des Infracapitulum erfährt mit dem Erwerb schmaler Stech-Cheliceren eine radikale Umstellung des Schutzes gegen Austrocknung. Während bei der Larve die breiten Cheliceren-Grundglieder den Sekretweg überdecken, und der Raum zwischen den Grundgliedern und dem Infracapi-tulum zusätzlich durch das ölartige Sekret der Intercheliceraldrüse ausgefüllt wird, schließen sich bei der Deutonymphe und beim Adultus die Lateralkiele der Genae über den Stech-Cheliceren zusammen, und der hintere Abschnitt des Cervix wird in das Idiosoma eingesenkt. Dadurch wird der Cervix zu einem internen Cervicalkanal umgebildet.Der Mundraum wird bei den aktiven Stadien durch die lipidartigen Sekrete der Buccal- und Labialdrüsen geschützt.Die phylogenetischen Beziehungen der Parasitengona innerhalb der Prostigmata und der Erythraeidae innerhalb der Parasitengona werden diskutiert.

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Postembryonic development and functional anatomy of the gnathosoma in the family Erythraeidae (Acarina, Prostigmata)

Summary The gnathosoma of seven species of the Erythraeidae was investigated. The postembryonic development of the cuticular structures, the musculature and the glands are described.In the larval gnathosoma, the chelicerae consist of a basal segment and a claw. They rest upon the dorsal surface of the infracapitulum. The basal segments are attached to a pair of capitular apophyses (sigmoid pieces). During the two molts larva to protonymph and deutonymph to tritonymph, the gnathosoma is histolysed. Directly afterwards it is rebuilt. Compared to the larva, in the deutonymph and in the adult it undergoes profound changes: The capitular apophyses are transformed to parts of the tracheae, the basal segment of each chelicera to a styliform chelicera, and the levator muscles of the chelicerae to medial protractors of the styliform chelicerae.The secretions of the podocephalic and infracapitular glands proceed along the dorsal surface of the infracapitulum to the buccal cavity. In the larva, the way of the secretions is protected against desiccation by the broad basal segments of the chelicerae, that cover the infracapitulum. In addition the oily secretion of the intercheliceral gland seals the space between the infracapitulum and the chelicerae. By obtaining styliform chelicerae the protection against desiccation undergoes a radical change: The lateral ridges of the infracapitulum join above the chelicerae, and the posterior part of the cervix is transferred back into the idiosoma. Thus the cervix is transformed into an internal canal.In the active instars, the buccal cavity is protected by the lipid-like secretions of the buccal glands and labial glands.The phylogenetic relationships of the Parasitengona within the Prostigmata, and of the Erythraeidae within the Parasitengona are discussed.

     

Selective Staining of Eosinophils and Their Immature Precursors in Tissue Sections and Autoradiographs with Congo Red

The use of Congo red as an elective stain for eosinophilic granulocytes and their precursors in tissue sections and autoradiographs is demonstrated and discussed. The 0.5% alcoholic Congo red solution of Highman, normally used for the detection of amyloid, may also be used with only minor changes. This simple method may aid in the diagnosis of special hematological problems and facilitates the recognition of eosinophil granulocytes as well as proliferating and nonproliferating myelocytes in autoradiographs from paraffin sections.     

Enriched populations of rat retinal ganglion cells: Studies using a cell-type specific surface marker

Cells dissociated from adult and neonatal rat retinas were separated by density gradient centrifugation. Previous work had shown that rat retinal cells labelled by an immunofluorescence assay for the Thy-1 antigen were chiefly or exclusively ganglion cells, and so the proportion of Thy-1 positive cells in the density gradient fractions was used as an index of the enrichment of ganglion cells. The proportion of Thy-1 positive neonatal cells was increased from about 0.4% in the initial dissociate to about 8% in the most enriched fraction of a Percoll step gradient. Amongst adult cells the initial 0.7% Thy-1 positive cells were increased to roughly 2% in the best fraction of a metrizamide step gradient.

The presence of relatively large numbers of Thy-1 positive cells in other fractions suggested that it would be difficult to further increase the proportion of rat ganglion cells by methods based on their sedimentation properties. These results demonstrate the importance of cell-type specific markers in attempts to purify cells from the central nervous system.     

Ubiquitous soluble Mg(2+)-ATPase complex. A structural study.

We have performed a detailed structural analysis of the soluble Mg(2+)-ATPase complex purified from Xenopus laevis ovary, which is an abundant and ubiquitous homo-oligomeric protein complex located in the nucleus and in the cytoplasm, belonging to a novel multigene-family of putative Mg(2+)-ATPases. Enzyme activity staining after non-denaturing polyacrylamide gel electrophoresis revealed that Mg(2+)-ATPase activity of the native protein is dependent on oligomerization and could not be detected in dissociated subunits. For the native protein a sedimentation coefficient of 15.3 S and a corresponding relative molecular mass of 612,000 was determined by analytical ultracentrifugation and a relative molecular mass of 590,000 was estimated from scanning transmission electron microscopy, supporting our previous conclusion that the oligomer comprises six 97,000 Mr subunits. Conventional electron microscopy of negatively stained specimens revealed the Mg(2+)-ATPase complex to be a hexagonal molecule in its favoured "end-on" projection and a double-banded molecule in its "side-on" projection (approx. 12 nm diameter; approx. 9 nm height). In addition, dimerized complexes could be observed in negatively stained specimens, yielding pronounced hexameric images and four-banded images in their end-on and side-on orientations, respectively (approx. 12 nm diameter; approx. 18.5 nm height). Two-dimensional (2D = mono-molecular) crystals have been produced from the dimerized complexes by the negative staining carbon film technique. Hexagonal crystals with a p6 plane group symmetry were obtained from molecules in their end-on orientation and longitudinal arrays with a p2 symmetry from complexes in their side-on orientation. A low-resolution molecular model of the native protein, derived from averages of these two 2D crystals, is presented. From our results we propose oligomerization as an inherent structural principle of organization for this whole newly defined Mg(2+)-ATPase multigene-family, that includes such seemingly diverse functionally defined proteins as mammalian and yeast "vesicle fusion" and "peroxisome assembly" proteins and the product of the yeast cell cycle gene CDC48.