Deciphering the molecular mechanisms of anti-tubulin plant derived drugs

Even though commercialized anticancer drugs are now produced by pharmaceutical companies, most of them were originally obtained from natural sources, and more particularly from plants. Indeed, many structurally diverse compounds isolated from plants or marine flora have been purified and synthesized for their anticancer bioactivity. Among these, several molecules belong to the class of anticancer drugs which target the microtubule cytoskeleton, either by stabilizing it or destabilizing it. To characterize the activity of these drugs and to understand in which physiological context they are more likely to be used as therapeutic agents, it is necessary to fully determine their interaction with tubulin. Understanding the molecular basis of their effects on microtubule cytoskeleton is an important step in designing analogs with greater pharmacological activity and with fewer side effects. In addition, knowing the molecular mechanism of action of each drug that is already used in chemotherapy protocols will also help to find strategies to circumvent resistance. By taking examples of known anti-tubulin plant derived drugs, we show how identification of microtubule targeting agents and further characterization of their activity can be achieved combining biophysical and biochemical approaches. We also illustrate how continuing in depth study of molecules with already known primary mechanisms of action can lead to the discovery of new targets or biomarkers which can open new perspectives in anticancer strategies.     

Effect of isomerization of aspartate-7 on the binding of copper (II) ion by the β-amyloid peptide

It is shown for the first time by isothermal titration calorimetry that the N-terminal amino group of the β-amyloid protein, which coordinates the copper ion in the native peptide, fails to form the proper coordination bond upon isomerization of Asp7.     

Some seasonal changes in the thecal gland in the ovaries of hibernators (Citellus citellus L.).

The ovaries of 36 ground squirrels were studied in March, April, July, and December. Morphological and functional seasonal characteristics of theca interna were studied by histological methods, electron microscopy, and quantitative methods for determinating the volume density and histoenzyme activity of NADH2-tetrazolium reductase, glucose-6-phosphate dehydrogenase, and delta 5-3 beta-hydroxysteroid dehydrogenase. Maximal activity of theca interna as a steroid-producing structure was observed during spring awakening. A significant increase in the histoenzyme activity of delta 5-3 beta-hydroxysteroid dehydrogenase in December, versus two other enzymes studied, indicated steroid synthesis in theca terna.     

Developing criteria for evaluation of geroprotectors as a key stage toward translation to the clinic

In the coming decades, a massive shift in the aging segment of the population will have major social and economic consequences around the world. One way to offset this increase is to expedite the development of geroprotectors, substances that slow aging, repair age‐associated damage and extend healthy lifespan, or healthspan. While over 200 geroprotectors are now reported in model organisms and some are in human use for specific disease indications, the path toward determining whether they affect aging in humans remains obscure. Translation to the clinic is hampered by multiple issues including absence of a common set of criteria to define, select, and classify these substances, given the complexity of the aging process and their enormous diversity in mechanism of action. Translational research efforts would benefit from the formation of a scientific consensus on the following: the definition of ‘geroprotector’, the selection criteria for geroprotectors, a comprehensive classification system, and an analytical model. Here, we review current approaches to selection and put forth our own suggested selection criteria. Standardizing selection of geroprotectors will streamline discovery and analysis of new candidates, saving time and cost involved in translation to clinic.     

A method for preparation of dry thrombin for topical application

Thrombin for topical hemostasis can be prepared from bovine or human blood plasma. The prothrombin is isolated by means of adsorption on DEAE-Sephadex A-50 and consecutively activated by CaCl₂ and thromboplastin. Thrombin is precipitated and purified by acetone. The specific activity of the thrombin preparation is 122 + 23 IU/mg protein while the yield is 36,360 ± 6623 IU/liter plasma.     

Dependence of trehalose protective action on the initial phase state of dipalmitoylphosphatidylcholine bilayers

Dipalmitoylphosphatidylcholine (DPPC) bilayers hydrated in the presence of trehalose were equilibrated at various temperatures (4, 20, and 60 degrees C) corresponding to the crystalline Lc, gel L beta', and liquid-crystalline L alpha phases, respectively, and then desiccated at these temperatures or freeze-dried at -80 degrees C to ca. DPPC dihydrate. The thermotropic behavior of the resulting DPPC/trehalose mixtures was investigated by differential scanning calorimetry and found to be dependent not only on the trehalose concentration but also on the phase state of the hydrated bilayers prior to their drying. Trehalose was most effective when the desiccation was carried out from the L alpha phase at 60 degrees C. In this case, one trehalose molecule per two DPPC molecules was sufficient to depress the melting temperature from values typical of DPPC dihydrate to 45 degrees C. Trehalose's influence decreased when dried from the L beta' phase and was significantly less pronounced when dried from the Lc phase. These data show that trehalose's protective influence depends on the initial phase state of the lipid bilayer and reaches its maximum in the liquid-crystalline state. The possible role of this effect in anhydrobiosis is pointed out.     

Metabolic behaviors of the core histones in proliferating Friend cells

This study examines the turnover of the core histones in proliferating Friend cells. It was calculated that these proteins turn over with half-lives of 21.6 days for H2A, 13.8 days for H2B, 43.3 days for H3, and 138.6 days for H4. The significant differences in the half-lives of the four core histones indicate that the protein moiety of the nucleosome is not replaced as one entire unit but as a "mosaic" in which each component follows its own rate of replacement. In some experiments the turnover rates of the variants of H2A, H2B, and H3 were compared. The results did not indicate any differences among these histone variants, suggesting that they are not excluded from the mechanisms controlling histone turnover. Metabolic heterogeneity was discovered, however, when the turnover rates of the acetylated and nonacetylated molecules of histone H4 were followed: it appeared that the acetylated molecules are replaced 2.5 times faster. The comparison of the rate of replacement of the histones in proliferating and differentiated cells from one site and their level of acetylation from another suggests that this postsynthetic modification might be involved in the control of histone metabolism. Such a conclusion is supported also by a number of model experiments.     

Location and aggregation of the spin-labeled peptide trichogin GA IV in a phospholipid membrane as revealed by pulsed EPR

The lipopeptaibol trichogin GA IV is a 10 amino acid-long residue and alpha-aminoisobutyric acid-rich antibiotic peptide of fungal origin. TOAC (2,2,6,6-tetramethylpiperidine-1-oxyl-4-amino-4-carboxylic acid) spin-labeled analogs of this membrane active peptide were investigated in hydrated bilayers of dipalmitoylphosphatidylcholine by electron spin echo envelope modulation (ESEEM) spectroscopy and pulsed electron-electron double resonance (PELDOR). Since, the ESEEM of the spin label appears to be strongly dependent on the presence of water molecules penetrated into the membrane, this phenomenon was used to study the location of this peptide in the membrane. This was achieved by comparing the ESEEM spectra for peptides labeled at different positions along the amino acid sequence with spectra known for lipids with spin labels at different positions along the hydrocarbon chain. To increase the ESEEM amplitude and to distinguish the hydrogen nuclei of water from lipid protons, membranes were hydrated with deuterated water. The PELDOR spectroscopy technique was chosen to study peptide aggregation and to determine the mutual distance distribution of the spin-labeled peptides in the membrane. The location of the peptide in the membrane and its aggregation state were found to be dependent on the peptide concentration. At a low peptide/lipid molar ratio (less than 1:100) the nonaggregated peptide chain of the trichogin molecules lie parallel to the membrane surface, with TOAC at the 4th residue located near the 9th-11th carbon positions of the sn-2 lipid chain. Increasing this ratio up to 1:20 leads to a change in peptide orientation, with the N-terminus of the peptide buried deeper into membrane. Under these conditions peptide aggregates are formed with a mean aggregate number of about N = 2. The aggregates are further characterized by a broad range of intermolecular distances (1.5-4 nm) between the labels at the N-terminal residues. The major population exhibits a distance of approximately 2.5 nm, which is of the same order as the length of the helical peptide. We suggest that the constituting monomers of the dimer are antiparallel oriented.