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.     

Bending stiffness of a crystalline actin bundle

The acrosomal process of the sperm of the horseshoe crab (Limulus polyphemus) is a unique crystalline actin bundle, consisting of multiple actin filaments cross-linked by the actin-bundling protein, scruin. For successful fertilization, the acrosomal bundle must penetrate through a 30 microm thick jelly coat surrounding the egg and thus it must be sufficiently stiff. Here, we present two measurements of the bending stiffness of a single crystalline bundle of actin. Results from these measurements indicate that the actin:scruin composite bundle has an average elastic modulus of 2 GPa, which is similar to that of a single actin filament, and a bending stiffness that is more than two orders of magnitude larger than that of a bundle of uncross-linked actin filaments due to stiffening by the scruin matrix.     

(+)-CC-1065 as a probe for intrinsic and protein-induced bending of DNA

(+)-CC -1065 is biologically potent DNA-reactive antitumor antibiotic produced by Streptomyces zelensis. This antibiotic covalently modifies DNA by alkylation of N-3 of a adenine in the minor groove. As a Structural consequence of covalent modification of DNA, the helix axis id bent into the minor groove. The drug-induced bending of DNA has similarities to intrinsic. A-tract bending and the 3′ adenine of A-tracts shows a unique reactivity to alkylation by (+) -CC-1065. Upon covalent modification of A-tracts, the magnitude of bending is increased and helix is stiffened. Using high-field NMR, hydroxyl-radical footprinting and gel electrophoresis, the molecular basis for the high reactivity of the bonding sequence 5′ - AGTTA* (an asterisk indicates the covalent modification site) to (+)-CC-1065 has been shown to involve the inherent conformational flexibility of this sequence. Furthermore, these studies also demonstrate that after alkylation the drug-induced bending is focused over the TT region. By analogy with the junction bend model for A-tracts, a ‘truncated junction bend model’ is proposed for this structure. Last, the application of (+)-CC-1065 entrapped/induced bending of DNA as a probe for the Sp1-induced bending of the 21-base-pair repeat an Mu transpose bending of the att L3 sequence is described.     

Dispersive and non-dispersive waves through plants: implications for arthropod vibratory communication

Vibratory communication in arthropods is a widespread phenomenon. Arthropods living on plants have been reported to use only dispersive bending waves in the context of prey-predator, competition, social and sexual interactions. Differences in signal structure have also been postulated to work as species recognition mechanisms and speciation agents. Using two identical laser Doppler vibrometers and a wavelet analysis, we quantified the wave propagation modes in rush stems (Juncus effusus) over the whole range of frequencies used by arthropods. A non-dimensionalized analysis shows that mechanical waves propagate not only as dispersive bending waves, but also as non-dispersive waves. Our analysis implies that an arthropod can communicate through non-dispersive bending waves by either producing signals of high frequencies or by choosing large stems, two widely different options tapping into the physiological and the behavioural repertoires, respectively. Non-dispersive waves, unreported so far in insect vibratory communication in plants, present serious advantages over dispersive bending waves in terms of signal integrity and may well be much more widely used than anticipated, in particular for species recognition.     

Membrane-bound structure and energetics of alpha-synuclein

Aggregation and fibrillation of alpha-synuclein bound to membranes are believed to be involved in Parkinson's and other neurodegenerative diseases. On SDS micelles, the N-terminus of alpha-synuclein forms two curved helices linked by a short loop. However, its structure on lipid bilayers has not been experimentally resolved. Using MD simulations with an implicit membrane model we show here that, on a planar mixed membrane, the truncated alpha-synuclein (residues 1-95) forms a bent helix. Bending of the helix is not due to the protein sequence or membrane binding, but to collective motions of the long helix. The backbone of the helix is approximately 2.5 A above the membrane surface, with some residues partially inserted in the membrane core. The helix periodicity is 11/3 (11 residues complete three full turns) as opposed to 18/5 periodicity of an ideal alpha-helix, with hydrophobic residues towards the membrane, negatively charged residues towards the solvent and lysines on the polar/nonpolar interface. A series of threonines, which are characteristic for alpha-synuclein and perhaps a phosphorylation site, is also located at the hydrophobic/hydrophilic interface with their side chain often hydrogen bonded to the main-chain atom. The calculations show that the energy penalty for change in periodicity from the 18/5 to 11/3 on the anionic membrane is overcome by favorable solvation energy. The binding of truncated alpha-synuclein to membranes is weak. It prefers anionic membranes but it also binds marginally to a neutral membrane, via its C-terminus. Dimerization of helical monomers on the mixed membrane is energetically favorable. However, it slightly interferes with membrane binding. This might promote lateral diffusion of the protein on the membrane surface and facilitate assembly of oligomers that precede fibrillation.     

Cellular and subcellular localization of calcium in gravistimulated corn roots

Summary Antimonate staining procedures and energy dispersive X-ray microanalytical techniques were used to determine the patterns of localization of calcium in nonstimulated and gravistimulated corn roots. In horizontally positioned roots within the region of the developing bend there was a change in the staining from that principally localized within cells of the stele to asymmetric staining within the vacuoles of the cortical cells along the upper root surface. There was little staining in the walls. The pattern observed is quite different from that seen in gravistimulated coleoptiles. Staining of mitochondria, plastids and Golgi stacks was seen in most cell types, but no asymmetry of staining was observed. In the rootcap where graviperception is thought to occur, there was little staining of any cellular organelles.