Kinetics of apoptotic markers in exogeneously induced apoptosis of EL4 cells

We investigated the time-dependence of apoptotic events in EL4 cells by monitoring plasma membrane changes in correlation to DNA fragmentation and cell shrinkage. We applied three apoptosis inducers (staurosporine, tubericidine and X-rays) and we looked at various markers to follow the early-to-late apoptotic events: phospholipid translocation (identified through annexin V-fluorescein assay and propidium iodide), lipid package (via merocyanine assay), membrane fluidity and anisotropy (via fluorescent measurements), DNA fragmentation by the fluorescence-labeling test and cell size measurements. The different apoptotic inducers caused different reactions of the cells: staurosporine induced apoptosis most rapidly in a high number of cells, tubercidine triggered apoptosis only in the S phase cells, while X-rays caused a G2/M arrest and subsequently apoptosis. Loss of lipid asymmetry is promptly detectable after one hour of incubation time. The phosphatidylserine translocation, decrease of lipid package and anisotropy, and the increase of membrane fluidity appeared to be based on the same process of lipid asymmetry loss. Therefore, the DNA fragmentation and the cell shrinkage appear to be parallel and independent processes running on different time scales but which are kinetically inter-related. The results indicate different signal steps to apoptosis dependent on inducer characteristics but the kinetics of "early-to-late" apoptosis appears to be a fixed program.     

Rolling properties of rGPIbalpha-conjugated phospholipid vesicles with different membrane flexibilities on vWf surface under flow conditions

The recombinant fragment of the platelet membrane glycoprotein, rGPIbalpha, was conjugated to phospholipid vesicles with the average diameter of ca. 1 microm using N-succinimidyl 3-(2-pyridyldithio) propionate (SPDP). We used five kinds of rGPIbalpha-vesicles with different fluorescent anisotropies of 1,6-diphenyl-1,3,5-hexatriene (DPH) to study the rolling properties of the vesicles on the von Willebrand factor (vWf)-immobilized surface. Under flow conditions, the rolling velocity of the rGPIbalpha-vesicles decreased with the increasing membrane flexibility. It is considered that the vesicles with a high membrane flexibility have a high deformability and can be flattened to a high degree during rolling on the vWf surface, thus resulting in the large contact area. We obtained a recipe to control the rolling velocity of artificial platelets by membrane flexibility.     

Antimetabolite incorporation into DNA: structural and thermodynamic basis for anticancer activity

Antimetabolites are a class of effective anticancer drugs that structurally resemble naturally occurring biochemicals and interfere in essential biochemical processes. In this review, the recent literature describing investigations of the structural and thermodynamic basis for the anticancer activity of three antipyrimidines [1-beta-D-arabinofuranosyl cytidine (AraC). 2',2'-difluoro deoxycytidine (dFdC), and 5-fluoro-2'-deoxyuridine (FdUrd)] is summarized. Our laboratory, and others, have shown that misincorporation of any of these three antipyrimidines into DNA perturbs the structure and decreases the stability of duplex DNA. These data are useful for rationalizing the effects of antipyrimidine misincorporation on the activities of proteins required for DNA replication and repair such as DNA topoisomerase 1 and DNA polymerases. The studies completed to date and summarized in this review demonstrate the utility of investigations into the structure-function relationships between antipyrimidine-substituted DNA complexed with DNA-modifying proteins for the purpose of understanding the basis for effective antipyrimidine cancer chemotherapy and the future design of novel anticancer drugs.     

Study of the serum albumin-polyethyleneglycol interaction to predict the protein partitioning in aqueous two-phase systems

The theoretical framework based only on the excluded volume forces is not enough to explain the bovine serum albumin partitioning behaviour in aqueous biphasic systems. The goal of this work is to look at the phase separation via the polymer effect on the water structure. Our findings suggest that polyethyleneglycol 600-protein interaction is conducted by van der Waals forces between the hydrophobic surfaces from PEG and protein molecules, which implies the rupture of hydrogen bonds from the structured water in their neighbours. Therefore, the protein will concentrate in the most water-structured phase (polyethyleneglycol) in order to reach the minimal free energy condition. When polyethyleneglycol molecular weight increases, its exclusion from protein surface prevails, thus pushing the bovine serum albumin to the bottom phase.     

Effect of a 125 mT static magnetic field on the kinetics of voltage activated Na+ channels in GH3 cells

Voltage activated Na(+) channels were examined in GH3 cells, using the whole cell patch clamp method. Channel currents were recorded before, during, and after a 150 s exposure to a 125 mT static magnetic field. There was a slight shift in the current-voltage relationship and a less than 5% reduction in peak current during magnetic field exposure. More pronounced, however, was an increase in the activation time constant, tau(m), during and for at least 100 s following exposure to the field. This change in tau(m) was seen primarily at lower activation voltages. No change was noted in the inactivation time constant, tau(h). Changes were clearly temperature dependent, being evident only at and above 35 degrees C. These findings are consistent with the hypothesis that reorientation of diamagnetic anisotropic molecules in the cell membrane are capable of distorting imbedded ion channels sufficiently to alter their function. The temperature dependence of this phenomenon is probably due to the greater ease with which a liquid crystal membrane can be deformed.     

Convenient method for determining the absolute configuration of chiral alcohols with racemic 1H NMR anisotropy reagent,M(alpha)NP acid: use of HPLC-CD detector

A convenient method for determining the absolute configuration of chiral secondary alcohols using the racemic NMR anisotropy reagent, (+/-)-2-methoxy-2-(1-naphthyl)propionic acid [(+/-)-M(alpha)NP acid], and an HPLC-CD detector was developed. The method was successfully applied to some chiral alcohols derived from (-)-alpha-santonin.     

Rotational diffusion tensor of nucleic acids from 13C NMR relaxation

Rotational diffusion properties have been derived for the DNA dodecamer d(CGCGAATTCGCG)₂ from ¹³C R₁ and R₁ measurements on the C₁, C₃, and C₄ carbons in samples uniformly enriched in ¹³C. The narrow range of C-H bond vector orientations relative to the DNA axis make the analysis particularly sensitive to small structural deviations. As a result, the R₁/R₁ ratios are found to fit poorly to the crystal structures of this dodecamer, but well to a recent solution NMR structure, determined in liquid crystalline media, even though globally the structures are quite similar. A fit of the R₁/R₁ ratios to the solution structure is optimal for an axially symmetric rotational diffusion model, with a diffusion anisotropy, D_||/D, of 2.1±0.4, and an overall rotational correlation time, (2D_||+4D)^–1, of 3.35 ns at 35 °C in D₂O, in excellent agreement with values obtained from hydrodynamic modeling.     

Angular dependence of dipole-dipole-Curie-spin cross-correlation effects in high-spin and low-spin paramagnetic myoglobin

The ¹⁵N-HSQC spectra of low-spin cyano-met-myoglobin and high-spin fluoro-met-myoglobin were assigned and dipole-dipole-Curie-spin cross-correlated relaxation rates measured. These cross-correlation rates originating from the dipolar ¹H-¹⁵N interaction and the dipolar interaction between the ¹H and the Curie spin of the paramagnetic center contain long-range angular information about the orientation of the ¹H-¹⁵N bond with respect to the iron-¹H vector, with information measurable up to 11 Å from the metal for the low-spin complex, and between 10 to 25 Å for the high-spin complex. Comparison of the experimental data with predictions from crystal structure data showed that the anisotropy of the magnetic susceptibility tensor in low spin cyano-met-myoglobin significantly influences the cross-correlated dipole-dipole-Curie-spin relaxation rates.     

Cross correlations between 13C-1H dipolar interactions and 15N chemical shift anisotropy in nucleic acids

Two sets of cross-correlated relaxation rates involving chemical shift anisotropy and dipolar interactions have been measured in an RNA kissing complex. In one case, both the CSA and dipolar interaction tensors are located on the same nucleotide base and are rigidly fixed with respect to each other. In the other case, the CSA tensor is located on the nucleotide base whereas the dipolar interaction is located on the adjoining ribose unit. Analysis of the measured rates in terms of isotropic or anisotropic rotational diffusion has been carried out for both cases. A marked difference between the two models is observed for the cross-correlation rates involving rigidly fixed spin interactions. The influence of internal motions about the glycosidic linkage between the nucleotide base and the ribose unit on cross-correlated relaxation rates has been estimated by applying a model of restricted rotational diffusion. Local motions seem to have a more pronounced effect on cross-correlated relaxation rates when the two spin interactions are not rigidly fixed with respect to each other.