Fast Fenton footprinting: a laboratory-based method for the time-resolved analysis of DNA, RNA and proteins

‘Footprinting’ describes assays in which ligand binding or structure formation protects polymers such as nucleic acids and proteins from either cleavage or modification; footprinting allows the accessibility of individual residues to be mapped in solution. Equilibrium and time-dependent footprinting links site-specific structural information with thermodynamic and kinetic transitions. The hydroxyl radical (·OH) is a particularly valuable footprinting probe by virtue of it being among the most reactive of chemical oxidants; it reports the solvent accessibility of reactive sites on macromolecules with as fine as a single residue resolution. A novel method of millisecond time-resolved ·OH footprinting has been developed based on the Fenton reaction, Fe(II) + H₂O₂ → Fe(III) + ·OH + OH⁻. This method can be implemented in laboratories using widely available three-syringe quench flow mixers and inexpensive reagents to study local changes in the solvent accessibility of DNA, RNA and proteins associated with their biological function.     

Oxidative stress induces degradation of mitochondrial DNA

Mitochondrial DNA (mtDNA) is located in close proximity of the respiratory chains, which are the main cellular source of reactive oxygen species (ROS). ROS can induce oxidative base lesions in mtDNA and are believed to be an important cause of the mtDNA mutations, which accumulate with aging and in diseased states. However, recent studies indicate that cumulative levels of base substitutions in mtDNA can be very low even in old individuals. Considering the reduced complement of DNA repair pathways available in mitochondria and higher susceptibility of mtDNA to oxidative damage than nDNA, it is presently unclear how mitochondria manage to maintain the integrity of their genetic information in the face of the permanent exposure to ROS. Here we show that oxidative stress can lead to the degradation of mtDNA and that strand breaks and abasic sites prevail over mutagenic base lesions in ROS-damaged mtDNA. Furthermore, we found that inhibition of base excision repair enhanced mtDNA degradation in response to both oxidative and alkylating damage. These observations suggest a novel mechanism for the protection of mtDNA against oxidative insults whereby a higher incidence of lesions to the sugar–phosphate backbone induces degradation of damaged mtDNA and prevents the accumulation of mutagenic base lesions.     

A genome-wide Drosophila screen for heat nociception identifies α2δ3 as an evolutionarily conserved pain gene

Worldwide, acute, and chronic pain affects 20% of the adult population and represents an enormous financial and emotional burden. Using genome-wide neuronal-specific RNAi knockdown in Drosophila, we report a global screen for an innate behavior and identify hundreds of genes implicated in heat nociception, including the α2δ family calcium channel subunit straightjacket (stj). Mice mutant for the stj ortholog CACNA2D3 (α2δ3) also exhibit impaired behavioral heat pain sensitivity. In addition, in humans, α2δ3 SNP variants associate with reduced sensitivity to acute noxious heat and chronic back pain. Functional imaging in α2δ3 mutant mice revealed impaired transmission of thermal pain-evoked signals from the thalamus to higher-order pain centers. Intriguingly, in α2δ3 mutant mice, thermal pain and tactile stimulation triggered strong cross-activation, or synesthesia, of brain regions involved in vision, olfaction, and hearing.     

Cadmium,nickel, copper,and zinc influence on microfilament organization in Arabidopsis root cells

The plant cytoskeleton orchestrates such fundamental processes in cells as division, growth and development, polymer cross-linking, membrane anchorage, etc. Here, we describe the influence of Cd²⁺, Ni²⁺, Zn²⁺, and Cu²⁺ on root development and vital organization of actin filaments into different cells of Arabidopsis thaliana line expressing GFP-FABD2. CdSO₄, NiSO₄, CuSO₄, and ZnSO₄ were used in concentrations of 5–20 µM in this study. It was found that Cd, Ni, and Cu cause dose-dependent primary root growth inhibition and alteration of the root morphology, whereas Zn slightly stimulates root growth and does not affect the morphology of Arabidopsis roots. This growth inhibition/stimulation correlated with the various sensitivities of microfilaments to Cd, Ni, Cu, and Zn action. It was established that Cd, Ni, and Cu affected predominantly the actin filaments of meristematic cells. Cells of transition and elongation zones demonstrated strong actin filament sensitivity to Cd and Cu. Microfilaments of elongating root cells were more sensitive to Ni and Cu. Although Cd, Ni, and Cu stimulated root hair growth after long-term treatment, actin filaments were destroyed after 1 h exposure with these metals. Zn did not disrupt native actin filament organization in root cells. Thus, our investigation shows that microfilaments act as sensitive cellular targets for Cd, Ni, and Cu. More data on effects on native actin filaments organization would contribute to a better understanding of plant tolerance mechanisms to the action of these metals.     

Growth and sporulation of the arbuscular mycorrhizal fungus Glomus caledonium in dual culture with transformed carrot roots

Glomus caledonium was established in a dual culture with Ri T-DNA-transformed carrot roots. A modification of the minimal M medium buffered at pH 6.50 with 10 mM MES and solidified with 0.4% unpurified gellan gum allowed spore germination and formation of the symbiosis, together with the development of an extensive extramatrical mycelium and sporulation. Spore production increased with culture generation and most spores were viable. These spores colonized carrot roots and completed the fungal life cycle. In many cultures, sporulation was accompanied by the formation of arbuscule-like structures on short and thickened lateral branches of main hyphae. Root colonization was of the Paris-type with hyphae spreading intracellularly. Most colonized root cells contained coils of thickened hyphae, sometimes surrounded by fine hyphae, but no typical arbuscules were observed. Accepted: 26 January 2000     

Primary cilia and dendritic spines: Different but similar signaling compartments

Primary non-motile cilia and dendritic spines are cellular compartments that are specialized to sense and transduce environmental cues and presynaptic signals, respectively. Despite their unique cellular roles, both compartments exhibit remarkable parallels in the general principles, as well as molecular mechanisms, by which their protein composition, membrane domain architecture, cellular interactions, and structural and functional plasticity are regulated. We compare and contrast the pathways required for the generation and function of cilia and dendritic spines, and suggest that insights from the study of one may inform investigations into the other of these critically important signaling structures.