Differential properties of the two Drosophila gamma-tubulin isotypes.

The functional significance of distinct gamma-tubulins in several unrelated eukaryotes remains an enigma due to the difficulties to investigate this question experimentally. Using specific nucleotidic and immunological probes, we have demonstrated that the two divergent Drosophila gamma-tubulins, gamma-tub23C and gamma-tub37CD, are expressed in cultured cells. Gamma-tub37CD is constantly detected at the centrosome and absent in the mitotic spindle, while gamma-tub23C is extensively recruited to the centrosome during mitosis and relocalizes in the mitotic spindle. The two gamma-tubulins exhibit distinct biochemical properties. Gamma-tub23C is present in the soluble gamma-tubulin small complexes (10S) and gamma-tubulin big complexes (35S) and is loosely associated to the cytoskeleton. In contrast, gamma-tub37CD is undetectable in the soluble fraction and exhibits a tight binding to the centrosome. Syncytial embryos also contain the two gamma-tubulin isotypes, which are differentially recruited at the centrosome. Gamma-tub23C is present in the 10S soluble complexes only, while y-tub37CD is contained in the two soluble complexes and is recruited at the centrosome where it exhibits an heterogeneous binding. These results demonstrated an heterogeneity of the two Drosophila gamma-tubulin isotypes both in the cytoskeletal and the soluble fractions. They suggest the direct implication of the 35S complex in the centrosomal recruitment of gamma-tubulin and a conditional functional redundancy between the two gamma-tubulins.

Lipoprotein access to MHC class I presentation during infection of murine macrophages with live mycobacteria.

Following uptake by macrophages, live mycobacteria initially reside within an immature phagosome that resists acidification and retains access to recycling endosomes. Glycolipids are exported from the mycobacterial phagosome and become available for immune recognition by CD1-restricted T cells. The aim of this study was to explore the possibility that lipoproteins might similarly escape from the phagosome and act as immune targets in cells infected with live mycobacteria. We have focused on a 19-kDa lipoprotein from Mycobacterium tuberculosis that was previously shown to be recognized by CD8(+) T cells. The 19-kDa Ag was found to traffic separately from live mycobacteria within infected macrophages by a pathway that was dependent on acylation of the protein. When expressed as a recombinant protein in rapid-growing mycobacteria, the 19-kDa Ag was able to deliver peptides for recognition by MHC class I-restricted T cells by a TAP-independent mechanism. Entry into the class I pathway was rapid, dependent on acylation, and could be blocked by killing the mycobacteria by heating before infection. Although the pattern of 19-kDa trafficking was similar with different mycobacterial species, preliminary experiments suggest that class I presentation is more efficient during infection with rapid-growing mycobacteria than with the slow-growing bacillus Calmette-Guérin vaccine strain.

Evaluation of human antimycobacterial immunity using recombinant reporter mycobacteria.

A novel in vitro whole blood model was developed to study human antimycobacterial immunity. Recombinant reporter mycobacteria were used to enumerate the bacteria, and interactions between host immune cells and mycobacteria were studied using whole blood rather than cell fractions. The ability of healthy tuberculin-positive and tuberculin-negative individuals to restrict mycobacterial growth was compared. Growth of luminescent mycobacteria was significantly lower in blood samples of tuberculin-positive individuals than in blood samples of tuberculin-negative individuals (P=.005). Restricted mycobacterial growth was associated with significantly higher production of tumor necrosis factor (TNF)-alpha and interferon (IFN)-gamma (P=.01 and.004, respectively). Inhibition of the TNF-alpha and IFN-gamma response pathways by neutralizing monoclonal antibodies increased mycobacterial growth in whole blood. This model is the first functional assay in which individual variations in cell-mediated immunity are shown to correlate with differences in ability to control mycobacterial growth. It provides a new tool for studying human mycobactericidal mechanisms and, potentially, for the evaluation of improved vaccines.

Differential in vitro association of vinca alkaloid-induced tubulin spiral filaments into aggregated spirals.

Vinblastine, vincristine, vindesine, and vinorelbine, the four vinca alkaloids used in cancer therapy, differ in their antitumoral spectra and toxicities, but not in their inhibitory effects on microtubule assembly in vitro. At higher drug concentrations, vinca alkaloids induce the assembly of spiral filaments of tubulin, which, in turn, can interact laterally and form paracrystals. Using methods that distinguish spiral filaments and paracrystals (aggregated spirals), we found that spiral filament formation was largely independent of the incubation temperature, of the alkaloid used, and of the presence or absence of microtubule-associated proteins (MAPs). In contrast, the formation of aggregated spirals was markedly dependent on the alkaloid used, on the incubation temperature, and on the absence or presence of MAPs. Aggregated spirals failed to assemble in the presence of high concentrations of MAP-1A or MAP-1B, whereas they assembled readily with tau and MAP-2. Differences in patterns of turbidity development using pure tubulin allowed the classification of thirteen cytotoxic vinca alkaloids into five distinct groups, with centrifugal recovery of aggregated spirals in close agreement with the various turbidity patterns. With microtubule protein, i.e. tubulin preparations containing MAPs, only four groups were defined by turbidity patterns, and centrifugal protein recovery was more divergent. Vinblastine, vincristine, vindesine, and vinorelbine fell into distinct groups under both reaction conditions, and thus they appear to have qualitatively distinguishable in vitro interactions with tubulin. These differential effects on spiral filament and aggregated spiral assembly revealed that the four drugs induce different constraints on the tubulin molecule.

Assessment of immunity to mycobacterial infection with luciferase reporter constructs.

Protective immunity to mycobacterial infection is incompletely understood but probably involves the coordinated interaction of multiple cell types and cytokines. With the aim of developing assays that might provide a surrogate measure of protective immunity, we have investigated the use of recombinant mycobacteria carrying luciferase reporter enzymes to assess the effectiveness of antimycobacterial immunity in model systems. Measurement of luminescence was shown to provide a rapid and simple alternative to the counting of CFU as a means of monitoring mycobacterial viability. We describe optimization of a luciferase reporter strain of Mycobacterium tuberculosis and demonstrate its application for the study of mycobacterial interactions with host cells in tissue culture and the rapid assessment of vaccine efficacy in a murine model.

Induction of a type 1 immune response to a recombinant antigen from Mycobacterium tuberculosis expressed in Mycobacterium vaccae.

A 19-kDa lipoprotein from Mycobacterium tuberculosis was expressed as a recombinant antigen in the nonpathogenic mycobacterial host strain M. vaccae. Immunization of mice with the recombinant M. vaccae resulted in induction of a strong type 1 immune response to the 19-kDa antigen, characterized by immunoglobulin G2a (IgG2a) antibodies and gamma interferon (IFN-gamma) production by splenocytes. Immunization with the same antigen in incomplete Freund's adjuvant induced a strong IgG1 response with only low levels of IFN-gamma. Subsequent intravenous and aerosol challenges of immunized mice with virulent M. tuberculosis demonstrated no evidence of protection associated with the response to the 19-kDa antigen; in fact, the presence of the recombinant 19-kDa antigen abrogated the limited protection conferred by M. vaccae (vector control). The recombinant M. vaccae system is a convenient approach to induction of type 1 responses to M. tuberculosis antigens. However, the unexpected reduction in protective efficacy of M. vaccae expressing the 19-kDa antigen highlights the complexity of testing recombinant subunit vaccines and the need for a better understanding of the immune mechanisms required for effective vaccination against tuberculosis.

A single gamma-tubulin gene and mRNA, but two gamma-tubulin polypeptides differing by their binding to the spindle pole organizing centres.

Cells of eukaryotic organisms exhibit microtubules with various functions during the different developmental stages. The identification of multiple forms of alpha- and beta-tubulins had raised the question of their possible physiological roles. In the myxomycete Physarum polycephalum a complex polymorphism for alpha- and beta-tubulins has been correlated with a specific developmental expression pattern. Here, we have investigated the potential heterogeneity of gamma-tubulin in this organism. A single gene, with 3 introns and 4 exons, and a single mRNA coding for gamma-tubulin were detected. They coded for a polypeptide of 454 amino acids, with a predicted molecular mass of 50,674, which presented 64-76% identity with other gamma-tubulins. However, immunological studies identified two gamma-tubulin polypeptides, both present in the two developmental stages of the organism, uninucleate amoebae and multinucleate plasmodia. The two gamma-tubulins, called gamma s- and gamma f-tubulin for slow and fast electrophoretic mobility, exhibited apparent molecular masses of 52,000 and 50,000, respectively. They were recognized by two antibodies (R70 and JH46) raised against two distinct conserved sequences of gamma-tubulins. They were present both in the preparations of amoebal centrosomes possessing two centrioles and in the preparations of plasmodial nuclear metaphases devoid of structurally distinct polar structures. These two gamma-tubulins exhibited different sedimentation properties as shown by ultracentrifugation and sedimentation in sucrose gradients. Moreover, gamma s-tubulin was tightly bound to microtubule organizing centers (MTOCs) while gamma f-tubulin was loosely associated with these structures. This first demonstration of the presence of two gamma-tubulins with distinct properties in the same MTOC suggests a more complex physiological role than previously assumed.

Differential in vitro action of S-12363, a new vinblastine derivative, and of its epimer on microtubule proteins.

The action of two epimers of a new vinblastine derivative that differ in their in vivo antitumor activity and their cytotoxicity was studied in vitro in brain microtubule proteins. These two compounds, called S-12363 and S-12362, could not be distinguished from one another or from other active vinca alkaloids by their ability to prevent microtubule assembly. However, they differed strongly both from one another and from vincristine and vinblastine in their ability to induce the formation of tubulin paracrystals and in the stability of the paracrystals following temperature shifts from 0 degree to 37 degrees C and vice versa. The most potent drug, S-12363, induced considerable tubulin aggregation, which was even more pronounced than that observed in the presence of vincristine. Previous results have shown that S-12363, in contrast to vincristine, induces no neurotoxic effects. This observation is in disagreement with a direct relationship between tubulin aggregation and neurotoxicity.

Taxol metabolism. Isolation and identification of three major metabolites of taxol in rat bile.

The elimination of nonradioactive taxol in bile and urine was investigated in the rat after administration via the caudal vein (10 mg/kg). As in humans, no metabolites of taxol were detected by HPLC in rat urine, and only 10% of the injected taxol was recovered in urine over a 24-hr period. In contrast, 11.5% and 29% of the injected taxol was recovered in rat bile as unchanged taxol and metabolites, respectively. Among the nine taxol metabolites detected by HPLC, the side chain at C13, which is required for pharmacological activity, had been removed in only one minor metabolite, baccatin III. The chemical structures of the two major hydroxylated metabolites were determined by mass spectrometry (fast atom bombardment and desorption chemical ionization) and 1H-NMR spectroscopy. One was a taxol derivative hydroxylated on the phenyl group at C3' of the side chain at C13, while the other corresponded to a taxol derivative hydroxylated in the m-position on the benzoate of the side chain at C2. Although these two major taxol metabolites were as active as taxol in preventing cold microtubule disassembly, they were, respectively, 9 and 39 times less cytotoxic as taxol on in vitro L1210 leukemia growth. These results show for the first time that there is a significant hepatic metabolism of taxol.

Cell cycle regulation of p34cdc2 kinase activity in Physarum polycephalum.

The regulation of the mitotic histone H1 kinase activity has been analyzed during the naturally synchronous cell cycle of Physarum polycephalum plasmodia. The universal binding property of the p13suc1 Schizosaccharomyces pombe gene product was used to precipitate and assay the cdc2 histone H1 kinase activity. The kinase activity peaks at the beginning of metaphase and its decline, which requires protein synthesis, appears to be an early event during the metaphase process. Microtubular poisons, temperature shifts and DNA synthesis inhibitors were used to perturb cell cycle regulatory pathways and characterize their effects on cdc2 kinase activation. Our results suggest that the full activation of the mitotic kinase requires at least two successive triggering signals involving microtubular components and DNA synthesis.