Mechanism of binding of the new antimitotic drug MDL 27048 to the colchicine site of tubulin: equilibrium studies.

MDL 27048 [trans-1-(2,5-dimethoxyphenyl)-3-[4-(dimethylamino)phenyl]-2- methyl-2-propen-1-one] fluoresces when bound to tubulin but not in solution. This effect has been investigated and found to be mimicked by viscous solvents. Therefore, MDL 27048 appears to be a fluorescent compound whose intramolecular rotational relaxation varies as a function of microenvironment viscosity. The binding parameters of MDL 27048 to tubulin have been firmly established by fluorescence of the ligand, quenching of the protein fluorescence, and gel equilibrium chromatography. The apparent binding equilibrium constant was (2.75 +/- 0.45) x 10(6)M-1, and the binding site number was 0.81 +/- 0.12 (10 mM sodium phosphate-0.1 mM GTP, pH 7.0, at 25 degrees C). The binding is exothermic. The binding of MDL 27048 overlaps the colchicine and podophyllotoxin binding sites. Binding of MDL 27048 to the colchicine site was also measured by competition with MTC [2-methoxy-5-(2,3,4-trimethoxyphenyl)-2,4,6-cycloheptatrien-1-one] , a well-characterized reversibly binding probe of the colchicine site [Andreu et al. (1984) Biochemistry 23, 1742-1752; Bane et al., (1984) J. Biol. Chem. 259, 7391-7398]. In contrast with close analogues of colchicine, MDL 27048 and podophyllotoxin neither affected the far-ultraviolet circular dichroism spectrum of tubulin, within experimental error, nor induced tubulin GTPase activity. Like podophyllotoxin, an excess of MDL 27048 over tubulin induced no abnormal cooperative polymerization of tubulin, which is characteristic of colchicine binding.(ABSTRACT TRUNCATED AT 250 WORDS)     

Three-dimensional structure of the lithostathine protofibril, a protein involved in Alzheimer's disease.

Neurodegenerative diseases are characterized by the presence of filamentous aggregates of proteins. We previously established that lithostathine is a protein overexpressed in the pre-clinical stages of Alzheimer's disease. Furthermore, it is present in the pathognomonic lesions associated with Alzheimer's disease. After self-proteolysis, the N-terminally truncated form of lithostathine leads to the formation of fibrillar aggregates. Here we observed using atomic force microscopy that these aggregates consisted of a network of protofibrils, each of which had a twisted appearance. Electron microscopy and image analysis showed that this twisted protofibril has a quadruple helical structure. Three-dimensional X-ray structural data and the results of biochemical experiments showed that when forming a protofibril, lithostathine was first assembled via lateral hydrophobic interactions into a tetramer. Each tetramer then linked up with another tetramer as the result of longitudinal electrostatic interactions. All these results were used to build a structural model for the lithostathine protofibril called the quadruple-helical filament (QHF-litho). In conclusion, lithostathine strongly resembles the prion protein in its dramatic proteolysis and amyloid proteins in its ability to form fibrils.     

New insights into tau-microtubules interaction revealed by isothermal titration calorimetry

Microtubule dynamic instability is tightly regulated by coordinated action of stabilizing and destabilizing microtubule associated proteins. Among the stabilizing proteins, tau plays a pivotal role in both physiological and pathological processes. Nevertheless, the detailed mechanism of tau-tubulin interaction is still subject to controversy. In this report, we studied for the first time tau binding to tubulin by a direct thermodynamic method in the absence of any tubulin polymerization cofactors that could influence this process. Isothermal titration calorimetry enabled us to evidence two types of tau-tubulin binding modes: one corresponding to a high affinity binding site with a tau:tubulin stoichiometry of 0.2 and the other one to a low affinity binding site with a stoichiometry of 0.8. The same stoichiometries were obtained at all temperatures tested (10-37°C), indicating that the mechanism of interaction does not depend on the type of tubulin polymer triggered upon tau binding. These findings allowed us to get new insights into the topology of tau on microtubules.     

Caulerpenyne from Caulerpa taxifolia has an antiproliferative activity on tumor cell line SK-N-SH and modifies the microtubule network.

Caulerpenyne, the major secondary metabolite synthesized by the green marine alga Caulerpa taxifolia, is cytotoxic against several cell lines. To identify possible targets of this toxin, we investigated the effect of caulerpenyne on the neuroblastoma SK-N-SH cell line. Caulerpenyne induced an inhibition of SK-N-SH cell proliferation with an IC50 of 10 +/- 2 microM after 2 hr of incubation.We observed no blockage in G2/M phase and an increase in cell death. On immunofluorescence microscopy, caulerpenyne affected the microtubule network in SK-N-SH cell line; we observed a loss of neurites and a compaction of the microtubule network at the cell periphery. In vitro, after 35 min of incubation, caulerpenyne inhibited the polymerization of pig brain purified tubulin or microtubule proteins, with an IC50 of 21 +/- 2 microM and 51 +/- 6 microM respectively. Analysis by electron microscopy indicated that caulerpenyne induced aggregation of tubulin, which may be responsible for inhibition of microtubule polymerization and bundling of residual microtubules.     

The curve of the intraventricular pressure-volume relationships is not mono-exponential]

The graphic and statistic investigations have let us to assert that the right ventricular P/V relationship's curve is characterized by two successive segments: linear and exponential respectively. We have proposed an interpretation based on Roach's and Burton's works. Interest of information about this curve is double: 1) it is a function of the ventricular cavity volume; 2) it gives a general idea about the mode of ventricular filling.     

Autotransfer of Day 4 embryos from oviduct to oviduct versus oviduct to uterus in the mare

Embryo autotransfer is defined as the collection of an embryo from and the transfer of this embryo into the same animal. The objectives of this study were to: 1) test the hypothesis that oviduct transport of the equine embryo from the oviduct into the uterus is not dependent on a unilateral embryo-corpus luteum interaction, 2) develop an embryo autotransfer technique for the mare and 3) compare the success rates of Day 4 embryos surgically autotransferred from the oviduct ipsilateral to ovulation to either the oviduct (n=10 mares) or the uterine horn (n=10 mares) contralateral to ovulation. Seventy percent (7 10 ) of the Day 4 embryos which were autotransferred to the oviduct contralateral to ovulation were transported through the oviduct and subsequently developed into embryonic vesicles detectable by ultrasonography between 10 and 21 days postovulation. This finding supported the hypothesis that oviductal embryo transport is not dependent upon the ipsilateral corpus luteum. Overall, sixty percent (12 20 ) of the autotransfers were successful. The success rate of uterine-transferred embryos was not significantly less (P>0.3) than that of oviductal-transferred embryos (5 10 vs 7 10 , respectively). Therefore, the Day 4 equine embryos were apparently mature enough to survive in the mare's uterus.     

Tension-volume relationship of the wall of the isolated right ventricle]

The variations of tension on the wall of the isolated right verticle have been observed on 18 dogs. The Tension/Volume curve we got can be superimposed to that obtained by pressures. The wall of the right ventricle itself (the radius of the curve in the cavity and the wall-thickness) seems to vary only with the changes in its tension in a rather small part of the curve.     

Pressure-volume relationship in the right ventricle]

The curve of the pressure-volume relationships of the right ventricular cavity is not a mono-exponential one. It can be characterized by the presence of two successive segments: linear and exponential. The points that constitute the first of them could not be subjected to Starling's law. This is material for the physiopathology of the tamponade.