A novel fluorescent toxin to detect and investigate Kv1.3 channel up-regulation in chronically activated T lymphocytes

T lymphocytes with unusually high expression of the voltage-gated Kv1.3 channel (Kv1.3(high) cells) have been implicated in the pathogenesis of experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. We have developed a fluoresceinated analog of ShK (ShK-F6CA), the most potent known inhibitor of Kv1.3, for detection of Kv1.3(high) cells by flow cytometry. ShK-F6CA blocked Kv1.3 at picomolar concentrations with a Hill coefficient of 1 and exhibited >80-fold specificity for Kv1.3 over Kv1.1 and other K(V) channels. In flow cytometry experiments, ShK-F6CA specifically stained Kv1.3-expressing cells with a detection limit of approximately 600 channels per cell. Rat and human T cells that had been repeatedly stimulated 7-10 times with antigen were readily distinguished on the basis of their high levels of Kv1.3 channels (>600 channels/cell) and ShK-F6CA staining from resting T cells or cells that had undergone 1-3 rounds of activation. Functional Kv1.3 expression levels increased substantially in a myelin-specific rat T cell line following myelin antigen stimulation, peaking at 15-20 h and then declining to baseline over the next 7 days, in parallel with the acquisition and loss of encephalitogenicity. Both calcium- and protein kinase C-dependent pathways were required for the antigen-induced Kv1.3 up-regulation. ShK-F6CA might be useful for rapid and quantitative detection of Kv1.3(high) expressing cells in normal and diseased tissues, and to visualize the distribution of functional channels in intact cells.     

Delineation of the clotrimazole/TRAM-34 binding site on the intermediate conductance calcium-activated potassium channel, IKCa1.

Selective and potent triarylmethane blockers of the intermediate conductance calcium-activated potassium channel, IKCa1, have therapeutic use in sickle cell disease and secretory diarrhea and as immunosuppressants. Clotrimazole, a membrane-permeant triarylmethane, blocked IKCa1 with equal affinity when applied externally or internally, whereas a membrane-impermeant derivative TRAM-30 blocked the channel only when applied to the cytoplasmic side, indicating an internal drug-binding site. Introduction of the S5-P-S6 region of the triarylmethane-insensitive small conductance calcium-activated potassium channel SKCa3 into IKCa1 rendered the channel resistant to triarylmethanes. Replacement of Thr(250) or Val(275) in IKCa1 with the corresponding SKCa3 residues selectively abolished triarylmethane sensitivity without affecting the affinity of the channel for tetraethylammonium, charybdotoxin, and nifedipine. Introduction of these two residues into SKCa3 rendered the channel sensitive to triarylmethanes. In a molecular model of IKCa1, Thr(250) and Val(275) line a water-filled cavity just below the selectivity filter. Structure-activity studies suggest that the side chain methyl groups of Thr(250) and Val(275) may lock the triarylmethanes in place via hydrophobic interactions with the pi-electron clouds of the phenyl rings. The heterocyclic moiety may project into the selectivity filter and obstruct the ion-conducting pathway from the inside.     

EFFECT OF SEED SIZE ON HETEROBLASTIC DEVELOPMENT IN SEEDLINGS OF DESMODIUM PANICULATUM

Seedlings of Desmodium paniculatum derived from different seed sizes differed in heteroblastic development. The transition from juvenile to adult leaves took place at the fourth node in seedlings derived from larger seeds and at the fifth or sixth node in those derived from smaller ones. Cotyledon removal had no effect on this condition, but growth under reduced irradiance delayed the formation of adult leaves in seedlings from larger seeds. Simple and compound leaves had similar unit area photosynthetic rates and stomatal conductance values, but compound leaves had a 30% higher total area. The earlier production of adult leaves would thus give seedlings from larger seeds an advantage in terms of total carbon gain.     

Referate

Ohne Zusammenfassung     

Cytologische Untersuchungen an einer fertilen triploiden Rose

Ohne ZusammenfassungMit 4 Textabbildungen.     

Beobachtungen an Pollenschlauchkulturen von der Hydrophyllacee Nemophila insignis

Zusammenfassung Es wurden Pollenschläuche vonNemophila insignis untersucht, die auf 15% igem Zuckeragar gewachsen waren.Der vegetative Kern war bei geeigneter Färbung stets sichtbar, und zwar meistens als ein sehr lang ausgezogener, fadenförmiger Körper. Auch nach 72stündiger Kulturdauer waren Degenerationserscheinungen an ihm nicht zu bemerken.Der generative Kern liegt in einer spindelförmigen oder ellipsoidischen Zelle, deren Plasma mit Hämatoxylin nicht gefärbt wird.Die Teilung des generativen Kernes verläuft unregelmäßig: Es tritt wohl eine Teilung der Chromosomen ein, aber die Tochterchromosomen verteilen sich nicht auf zwei Pole, so daß es zur Bildung eines Restitutionskernes kommt.Es wird die Annahme gemacht, daß diese Restitutionskernbildung eine Folge ungünstiger Kulturerscheinungen ist.Mit 5 Textabbildungen.Der letztgenannte Verfasser, der im Sommer 1937 für einige Zeit im Botanischen Institut in Kiel freundliche Aufnahme fand, möchte die Gelegenheit benutzen, um Herrn ProfessorTischler für seine liebenswürdige Gastfreundschaft und die Überlassung eines Arbeitsplatzes auch an dieser Stelle zu danken.     

Chromophore conformation and the evolution of tertiary structural changes in photoactive yellow protein

We use time-resolved crystallography to observe the structural progression of a bacterial blue light photoreceptor throughout its photocycle. Data were collected from 10 ns to 100 ms after photoactivation of the E46Q mutant of photoactive yellow protein. Refinement of transient chromophore conformations shows that the spectroscopically distinct intermediates are formed via progressive disruption of the hydrogen bond network to the chromophore. Although structural change occurs within a few nanoseconds on and around the chromophore, it takes milliseconds for a distinct pattern of tertiary structural change to fully progress through the entire molecule, thus generating the putative signaling state. Remarkably, the coupling between the chromophore conformation and the tertiary structure of this small protein is not tight: there are leads and lags between changes in the conformation of the chromophore and the protein tertiary structure.