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Influenza is a contagious respiratory disease that causes severe illness and death, particularly in elderly populations. Two enhanced formulations of quadrivalent influenza vaccine (QIV) are available in Spain. Adjuvanted QIV (aQIV) is available for those aged 65+ and high-dose QIV (HD-QIV) for those aged 60+. In this study, we used a health economic model to assess the costs and outcomes associated with using aQIV or HD-QIV in subjects aged 65+. Using aQIV instead of HD-QIV to vaccinate an estimated 5,126,343 elderly people results in reductions of 5405 symptomatic cases, 760 primary care visits, 171 emergency room visits, 442 hospitalizations, and 26 deaths in Spain each year. Life-years (LYs) and quality-adjusted LYs (QALYs) increases by 260 and 206, respectively, each year. Savings from a direct medical payer perspective are EUR 63.6 million, driven by the lower aQIV vaccine price and a minor advantage in effectiveness. From a societal perspective, savings increase to EUR 64.2 million. Results are supported by scenario and sensitivity analyses. When vaccine prices are assumed equal, aQIV remains dominant compared to HD-QIV. Potential savings are estimated at over EUR 61 million in vaccine costs alone. Therefore, aQIV provides a highly cost-effective alternative to HD-QIV for people aged 65+ in Spain.

Tubulin is a heterodimer of α and β subunits, both existing as isotypes differing in amino acid sequence encoded by different genes. Specific isotypes of tubulin have associations with cancer that are not well understood. Previous studies found that βII-tubulin is expressed in a number of transformed cells and that this isotype is found in cell nuclei in non-microtubule form. The association of βII expression and its nuclear localization with cancer progression has not previously been addressed. We here used a monoclonal antibody to βII to examine patients with colorectal cancer and found that patients whose tumors over-express βII have a greatly decreased life expectancy which is even shorter in those patients with nuclear βII. Our results suggest that βII-tubulin may facilitate cancer growth and metastasis and, to accomplish this, may not need to be in microtubule form. Furthermore, βII expression and localization could be a useful prognostic marker. We also found that βII appears in the nuclei of otherwise normal cells adjacent to the tumor. It is possible therefore that cancer cells expressing βII influence nearby cells to do the same and to localize βII in their nuclei by an as yet uncharacterized regulatory pathway.

Tasidotin (ILX-651), an orally active synthetic microtubule-targeted derivative of the marine depsipeptide dolastatin-15, is currently undergoing clinical evaluation for cancer treatment. Tasidotin inhibited proliferation of MCF7/GFP breast cancer cells with an IC(50) of 63 nmol/L and inhibited mitosis with an IC(50) of 72 nmol/L in the absence of detectable effects on spindle microtubule polymer mass. Tasidotin inhibited the polymerization of purified tubulin into microtubules weakly (IC(50) approximately 30 micromol/L). However, it strongly suppressed the dynamic instability behavior of the microtubules at their plus ends at concentrations approximately 5 to 10 times below those required to inhibit polymerization. Its major actions were to reduce the shortening rate, the switching frequency from growth to shortening (catastrophe frequency), and the fraction of time the microtubules grew. In contrast with all other microtubule-targeted drugs thus far examined that can inhibit polymerization, tasidotin did not inhibit the growth rate. In contrast to stabilizing plus ends, tasidotin enhanced microtubule dynamic instability at minus ends, increasing the shortening length, the fraction of time the microtubules shortened, and the catastrophe frequency and reducing the rescue frequency. Tasidotin C-carboxylate, the major intracellular metabolite of tasidotin, altered dynamic instability of purified microtubules in a qualitatively similar manner to tasidotin but was 10 to 30 times more potent. The results suggest that the principal mechanism by which tasidotin inhibits cell proliferation is by suppressing spindle microtubule dynamics. Tasidotin may be a relatively weak prodrug for the functionally active tasidotin C-carboxylate.

Microtubule assembly of different beta tubulin isotypes in the presence of 4 M glycerol and 6 mM magnesium ion demonstrates significantly different characteristics. alpha beta II and alpha beta IV assembled faster and to a greater extent than did unfractionated phosphocellulose-purified tubulin (PC-tubulin). Microtubule assembly from alpha beta III showed a distinctive delay in nucleation, proceeded at a slower rate than those of the other beta tubulin isotypes, and had the highest critical concentration. However, treatment of beta tubulin isotypes with subtilisin to remove the C-terminal domain of the tubulin dimer abolished these differences in microtubule assembly pattern and enhanced self-assembly. The kinetic analysis of microtubule elongation of different beta tubulin isotypes also showed significant differences. Elongation of alpha beta III from microtubule seeds had a lower apparent K alpha and a lower apparent Kd than did alpha beta II and alpha beta IV. The dynamic behaviors of different beta tubulin isotypes were qualitatively similar to each other and fit the dynamic instability model. However, microtubules formed from alpha beta III appeared to be less dynamic than microtubules formed from other beta tubulin isotypes. Our results suggest that the beta III isotype might have a different conformation than do the other beta tubulin isotypes. The distinctive nucleation and elongation behaviors of the alpha beta III dimers demonstrated in vitro may have a significant influence on microtubule functions in vivo.

The interaction of beta III-depleted tubulin with taxol was investigated. A monoclonal antibody against the beta III tubulin isotype was immobilized on a sepharose 4B column and used to remove the beta III tubulin isotype from unfractionated tubulin. The assembly of beta III-depleted tubulin in the presence of taxol was enhanced compared to unfractionated tubulin. The critical concentration of unfractionated tubulin in the presence of 10 microM taxol is 0.4 mg/ml, while the critical concentration of beta III-depleted tubulin is 0.16 mg/ml. At different concentration of taxol, the assembly of beta III-depleted tubulin is increased relative to that of unfractionated tubulin and reaches the maximum at about a 1:1 ratio of tubulin and taxol. The assembly of unfractionated tubulin and beta III-depleted tubulin has also been studied by electron microscopy. After 2 minutes at 37 degrees C, unfractionated tubulin assembly in the presence of 10 microM taxol results only in ribbon-like and ring structures; there are no visible microtubules. By 5 minutes, microtubules appear and increase in length. The assembly of beta III-depleted tubulin in the presence of 10 microM taxol occurs more quickly. In contrast to the case with unfractionated tubulin, beta III-depleted tubulin assembles within 2 minutes into microtubules which increase in length with time. At 30 minutes, microtubules assembled from beta III-depleted tubulin are shorter than the microtubules assembled from unfractionated tubulin. There is no visible difference between the microtubules assembled from unfractionated tubulin and beta III-depleted tubulin. Taxol-induced beta III-depleted tubulin assembly is more resistant to the inhibiting effect of podophyllotoxin and colchicine. It is also less sensitive to the inhibiting effect of cold temperature.

Tubulin, the constituent protein of microtubules, is an alpha beta heterodimer; both alpha and beta exist in several isotypic forms whose functional significance is not precisely known. The antimitotic alkaloid colchicine binds to mammalian brain tubulin in a biphasic manner under pseudo-first-order conditions in the presence of a large excess of colchicine (Garland, D. L. (1978) Biochemistry 17, 4266-4272). We have studied the kinetics of colchicine binding to purified beta-tubulin isotypes and find that each of the purified beta-tubulin isotypes binds colchicine in a monophasic manner. The apparent on-rate constants for the binding of colchicine to alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers are respectively 132 +/- 5, 30 +/- 2, and 236 +/- 7 M-1 s-1. When the isotypes are mixed, the kinetics become biphasic. Scatchard analysis revealed that the isotypes differ significantly in their affinity constants (Ka) for binding colchicine. The affinity constants are 0.24 x 10(6), 0.12 x 10(6), and 3.31 x 10(6) M-1, respectively, for alpha beta II-, alpha beta III-, and alpha beta IV-tubulin dimers. Our results are in agreement with the hypothesis that the beta-subunit of tubulin plays a major role in the interaction of colchicine with tubulin. Our binding data raise the possibility that the tubulin isotypes might play important regulatory roles by interacting differently with other non-tubulin proteins in vivo, which in turn, may regulate microtubule-based functions in living cells.

Tubulin, the 100-kDa subunit protein of microtubules, is a heterodimer of two 50-kDa subunits, alpha and beta. Both alpha and beta subunits exist as numerous isotypic forms. There are four isotypes of beta-tubulin in bovine brain tubulin preparations; their designations and relative abundances in these preparations are as follows: beta I, 3%; beta II, 58%; beta III, 25%; and beta IV, 13%. We have previously reported the preparation of monoclonal antibodies specific for beta II and beta III (Banerjee, A., Roach, M. C., Wall, K. A., Lopata, M. A., Cleveland, D. W., and Luduena, R. F. (1988) J. Biol. Chem. 263, 3029-3034; Banerjee, A., Roach, M. C., Trcka, P., and Luduena, R. F. (1990) J. Biol. Chem. 265, 1794-1799). We here report the preparation of a monoclonal antibody specific for beta IV. By using this antibody together with those specific for beta II and beta III, we have prepared isotypically pure tubulin dimers with the composition alpha beta II, alpha beta III, and alpha beta IV. We have found that, in the presence of microtubule-associated proteins, all three dimers assemble into microtubules considerably faster and to a greater extent than does unfractionated tubulin. More assembly was noted with alpha beta II and alpha beta III than with alpha beta IV. When assembly is measured in the presence of taxol (10 microM), little difference is seen among the isotypically purified dimers or between them and unfractionated tubulin. These results indicate that the assembly properties of a tubulin preparation are influenced by its isotypic composition and raise the possibility that the structural differences among tubulin isotypes may have functional significance.

Microtubule assembly in vitro is sensitive to a variety of non-physiological sulfhydryl-oxidizing agents, but the physiological significance of this phenomenon is unknown, since no physiological sulfhydryl-oxidizing agent has been shown to affect microtubule assembly in vitro. We have accordingly investigated the interaction of tubulin with cystamine. We have found that millimolar concentrations of cystamine inhibit microtubule assembly and induce an abnormal form of tubulin polymerization. Cystamine-induced polymerization does not occur at cold temperature. Formation of the polymer requires reaction of cystamine with two sulfhydryls which become available at 37 degrees C. In addition, cystamine reacts with about three sulfhydryls at 0 degrees C without inducing polymerization. This latter set of sulfhydryls appear to include one or both of the previously defined beta s sulfhydryls whose reaction with N, N'-ethylene-bis(iodoacetamide) is markedly inhibited by GTP, maytansine and vinblastine [Roach, M. C. & Luduena, R. F. (1984) J. Biol. Chem. 259, 12063-12071]. Cystamine's specific manner of interacting with tubulin suggests that it may mimic an endogenous sulfhydryl-directed regulator of microtubule assembly.

The kinetics of colchicine binding to bovine brain tubulin have been reported to be biphasic under pseudo first order conditions [(1978) Biochemistry 17, 4466-4472]. Unlike brain tubulin, the kinetics of colchicine binding to bovine renal tubulin are monophasic. The apparent on-rate constant for the binding of colchicine to renal tubulin is found to be very close to that of the faster binding component in brain tubulin. Similarly, the dissociation of colchicine-tubulin complex in the presence of iodide is biphasic for brain tubulin but monophasic for renal tubulin. Since brain and renal tubulin apparently differ in beta-tubulin, our results suggest that the biphasic nature of the kinetics for bovine brain tubulin could possibly originate from the existence of multiple isotypes of tubulin differing in drug binding affinity.

Two kinds of tubulin (alpha and beta) have been described in microtubules from many different systems. In this study a discontinuous acrylamide-gel system containing sodium dodecyl sulfate was used to separate milligram quantities of alpha- and beta-tubulin from microtubules of chick-embryo brain and from outer doublets of sea-urchin sperm. The isolated tubulins were characterized by peptide mapping and automated sequencing of the first 25 NH(2)-terminal amino acids. Our results show that alpha- and beta-tubulin are related but distinctly different proteins and that each one has been highly conserved in the course of evolution.