Kinetics of substrate recognition and cleavage by human 8-oxoguanine-DNA glycosylase

Human 8-oxoguanine-DNA glycosylase (hOgg1) excises 8-oxo-7,8-dihydroguanine (8-oxoG) from damaged DNA. We report a pre-steady-state kinetic analysis of hOgg1 mechanism using stopped-flow and enzyme fluorescence monitoring. The kinetic scheme for hOgg1 processing an 8-oxoG:C-containing substrate was found to include at least three fast equilibrium steps followed by two slow, irreversible steps and another equilibrium step. The second irreversible step was rate-limiting overall. By comparing data from Ogg1 intrinsic fluorescence traces and from accumulation of products of different types, the irreversible steps were attributed to two main chemical steps of the Ogg1-catalyzed reaction: cleavage of the N-glycosidic bond of the damaged nucleotide and β-elimination of its 3′-phosphate. The fast equilibrium steps were attributed to enzyme conformational changes during the recognition of 8-oxoG, and the final equilibrium, to binding of the reaction product by the enzyme. hOgg1 interacted with a substrate containing an aldehydic AP site very slowly, but the addition of 8-bromoguanine (8-BrG) greatly accelerated the reaction, which was best described by two initial equilibrium steps followed by one irreversible chemical step and a final product release equilibrium step. The irreversible step may correspond to β-elimination since it is the very step facilitated by 8-BrG

Thermodynamic and kinetic basis for recognition and repair of 8-oxoguanine in DNA by human 8-oxoguanine-DNA glycosylase

We have used a stepwise increase in ligand complexity approach to estimate the relative contributions of the nucleotide units of DNA containing 7,8-dihydro-8-oxoguanine (oxoG) to its total affinity for human 8-oxoguanine DNA glycosylase (OGG1) and construct thermodynamic models of the enzyme interaction with cognate and non-cognate DNA. Non-specific OGG1 interactions with 10–13 nt pairs within its DNA-binding cleft provides approximately 5 orders of magnitude of its affinity for DNA (ΔG° approximately −6.7 kcal/mol). The relative contribution of the oxoG unit of DNA (ΔG° approximately −3.3 kcal/mol) together with other specific interactions (ΔG° approximately −0.7 kcal/mol) provide approximately 3 orders of magnitude of the affinity. Formation of the Michaelis complex of OGG1 with the cognate DNA cannot account for the major part of the enzyme specificity, which lies in the kcat term instead; the rate increases by 6–7 orders of magnitude for cognate DNA as compared with non-cognate one. The kcat values for substrates of different sequences correlate with the DNA twist, while the KM values correlate with ΔG° of the DNA fragments surrounding the lesion (position from −6 to +6). The functions for predicting the KM and kcat values for different sequences containing oxoG were found

THE IMPACT OF MOOSE ON ASH PRODUCTIVITY

Data characterizing the impact of moose (Alces alces) on ash (Fraxinus spp.) seedlings in the broad–leaved forests of the Tula region are given. Resistance of ash to the strong browsing pressure is shown and also the resulting peculiarities of crown structure, including the position of shoots and branches. When ash is isolated from moose, annual accretion of shoot phytomass is 5 times more than in locations where ash is exposed to browsing; in leaves, phytomass is 10–12 times more than browsed ash. The large number of shoots and a great quantity of large leaves are considered as an adaptation of the ash tree to survival under the browsing pressure of moose

Are there gap junctions without connexins or pannexins?

Abstract Background Gap junctions (GJ) are one of the most common forms of intercellular communication. GJs are assembled from proteins that form channels connecting the cytoplasm of adjacent cells. They are considered to be the main or the only type of intercellular channels and the universal feature of all multicellular animals. Two unrelated protein families are currently considered to be involved in this function, namely, connexins and pannexins (pannexins/innexins). Pannexins were hypothesized to be the universal GJ proteins of multicellular animals, distinct from connexins that are characteristic of chordates only. Here we have revised this supposition by applying growing high throughput sequencing data from diverse metazoan species. Results Pannexins were found in Chordates, Ctenophores, Cnidarians, and in the most major groups of bilateral protostomes. Yet some metazoans appear to have neither connexins nor pannexins in their genomes. We detected no connexins or pannexins/innexins homologues in representatives of all five classes of echinoderms and their closest relatives hemichordates with available genomic sequences. Despite this, our intracellular recordings demonstrate direct electrical coupling between blastomeres at the 2-cell embryo of the echinoderm (starfish Asterias rubens). In these experiments, carboxyfluorescein fluorescent dye did not diffuse between electrically coupled cells. This excludes the possibility that the observed electrical coupling is mediated by incomplete cytoplasm separation during cleavage. Conclusion Functional GJs are present in representatives of the clade that lack currently recognized GJ protein families. New undiscovered protein families utilized for intercellular channels are predicted. It is possible that the new type(s) of intercellular channels are present in parallel to pannexin and connexin gap junctions in animal groups, other than Echinodermata

Sulfide geochronology along the Northern Equatorial Mid-Atlantic Ridge

Hydrothermal processes and seafloor massive sulfide (SMS) deposits have different characteristics at fast and slow spreading mid-ocean ridges. One such parameter is the age of a SMS deposit, which differs by 1–2 orders of magnitude between the fast spreading East Pacific Rise (EPR) and the slow spreading Mid-Atlantic Ridge (MAR). The large collection of SMS samples dated from the 18 hydrothermal fields of the northern equatorial part of the Mid-Atlantic Ridge (194 samples) demonstrates a relatively old average age of hydrothermal fields here (~ 66 ka) with the oldest one estimated as ca. 223 ka (Peterburgskoye field). Based on geochronological data it was confirmed that hydrothermal discharge has an episodic character: active and inactive periods of the SMS formation alternate. The distribution of events at all hydrothermal fields demonstrates that maximum activity occurred at 38–35, 30–20, and 8–2 ka and increased with time. Based on statistical analyses, dating variations can be explained as a superposition of several periods of activity with the duration of ~ 15, 10 and 5 ka. Relationship between the age and distance from the axial rift zone as well as between the age and aerial distribution is different for SMS deposits hosted by basalts and by gabbro-peridotites depending on their geological setting on the particular MAR segment. This difference can be explained by a variety of hydrothermal processes determined by “tectonic” or “magmatic” segment evolution and symmetrical or asymmetrical mode of accretion (Escartin et al., 2008)

Electrophysiology of the rhythmic defecation program in nematode Heterorhabditis megidis

Abstract The nervous system controls most rhythmic behaviors, with a remarkable exception. In Caenorhabditis elegans periodic defecation rhythm does not appear to involve the nervous system. Such oscillations are studied in detail with genetic and molecular biology tools. The small size of C. elegans cells impairs the use of standard electrophysiological methods. We studied a similar rhythmic pacemaker in the noticeably larger gut cells of Heterorhabditis megidis nematode. H. megidis defecation cycle is driven by a central pattern generator (CPG) associated with unusual all-or-none hyper-polarization “action potential”. The CPG cycle period depends on the membrane potential and CPG cycling also persisted in experiments where the membrane potential of gut cells was continuously clamped at steady voltage levels. The usual excitable tissue description does not include the endoderm or imply the generation of hyper-polarization spikes. The nematode gut cells activity calls for a reevaluation of the excitable cells definition

Memristor Degradation Analysis Using Auxiliary Volt-Ampere Characteristics

The memristor is one of the modern microelectronics key devices. Due to the nanometer scale and complex processes physic, the development of memristor state study approaches faces limitations of classical methods to observe the processes. We propose a new approach to investigate the degradation of six Ni/Si3N4/p+Si-based memristors up to their failure. The basis of the proposed idea is the joint analysis of resistance change curves with the volt-ampere characteristics registered by the auxiliary signal. The paper considers the existence of stable switching regions of the high-resistance state and their interpretation as stable states in which the device evolves. The stable regions’ volt-ampere characteristics were simulated using a compact mobility modification model and a first-presented target function to solve the optimization problem