Abstract. 0t-Tubulin can be posttranslationally modified in that its COOH-terminal amino acid residue, tyrosine, can be selectively removed and replaced again. This reaction cycle involves two enzymes, tubulin carboxypeptidase and tubulin tyrosine ligase. The functional significance of this unusual modification is unclear. The present study demonstrates that posttranslational tyrosinolation of tl-tubulin does occur in the parasitic hemoflagellate Trypanosoma brucei brucei and that posttranslational tyrosinolation can be detected in both a-tubulin isoforms found in this organism. Trypanosomes contain a number of microtubular structures: the flagellar axoneme; the subpellicular layer of singlet microtubules which are closely associated with the cell membrane; the basal bodies; and a cytoplasmic pool of soluble tubulin. Tyrosinolated a-tubulin is present in all these populations. However, immunofluorescence studies demonstrate a distinct localization of tyrosinolated a-tubulin within individual microtubules and organelles. This localization is subject to a temporal modulation that correlates strongly with progress of a cell through the cell cycle. Our results indicate that the presence of tyrosinolated ~t-tubulin is a marker for newly formed microtubules.

Abstract. The cytoskeleton of the parasitic hemoflagellate Trypanosoma brucei brucei essentially consists of two microtubule-based structures: a subpellicular layer of singlet microtubules, which are in close contact with the cell membrane, and the flagellar axoneme. In addition, the cells contain a small pool of soluble tubulin. Two-dimensional gel electrophoretic analysis of the tubulins present in these subcellular compartments revealed two distinct electrophoretic isoforms of a-tubulin, termed al and a3. al-Tubulin most likely represents the primary translation product, while a3-tubulin is a posttmnslationally acetylated derivative of al-tubulin. In the pool of soluble cytoplasmic tubulin, al is the predominant species, while the very stable flagellar microtubules contain almost exclusively the a3-tubulin isoforrn. The subpellicular microtubules contain both isoforms. Neither of the two a-tubulin isoforms is organelle specific, but the a3 isoform is predominantly located in stable microtubules.

A series of saligenin alkoxyalkylphenylsulfonamide beta(2) adrenoceptor agonists were prepared by reacting a protected saligenin oxazolidinone with alkynyloxyalkyl bromides, followed by Sonogashira reaction, hydrogenation, and deprotection. The meta-substituted primary sulfonamide was more potent than the para- and the ortho-analogues. Primary sulfonamides were more potent than the secondary and tertiary analogues. The onset and duration of action in vitro of selected compounds was assessed on isolated superfused guinea pig trachea. Sulfonamide 29b had the best profile of potency, selectivity, onset, and duration of action on both guinea pig trachea and human bronchus. Furthermore, 29b was found to have low oral bioavailability in rat and dog and also to have long duration of action in an in vivo model of bronchodilation. Crystalline salts of 29b were identified that had suitable properties for inhaled administration. A proposed binding mode for 29b to the beta(2)-receptor is presented.

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The detergent-insoluble T. brucei cytoskeleton consists of several morphologically distinct regions and organelles, many of which are detectable only by electron microscopy. We have produced a set of monoclonal antibodies that define each structural component of this highly ordered cytoskeleton. The monoclonal antibodies were selected by cloning of hybridomas produced from mice injected with complex mixtures of proteins of either the cytoskeleton itself or salt extracts thereof. Four antibodies define particular tubulin isotypes and locate the microtubules of the axoneme and sub-pellicular array; two antibodies recognize the flagellum attachment zone; one recognizes the paraflagellar rod and another the basal bodies. Finally, one antibody defines a detergent-insoluble component of the nucleus. The antigens detected by each monoclonal antibody have been analysed by immunofluorescence microscopy, immunogold electron microscopy and Western blotting.