Sequence dependence of transient Hoogsteen base pairing in DNA

Hoogsteen (HG) base pairing is characterized by a 180° rotation of the purine base with respect to the Watson-Crick-Franklin (WCF) motif. Recently, it has been found that both conformations coexist in a dynamical equilibrium and that several biological functions require HG pairs. This relevance has motivated experimental and computational investigations of the base-pairing transition. However, a systematic simulation of sequence variations has remained out of reach. Here, we employ advanced path-based methods to perform unprecedented free-energy calculations. Our methodology enables us to study the different mechanisms of purine rotation, either remaining inside or after flipping outside of the double helix. We study seven different sequences, which are neighbor variations of a well-studied A⋅T pair in A₆-DNA. We observe the known effect of A⋅T steps favoring HG stability, and find evidence of triple-hydrogen-bonded neighbors hindering the inside transition. More importantly, we identify a dominant factor: the direction of the A rotation, with the 6-ring pointing either towards the longer or shorter segment of the chain, respectively relating to a lower or higher barrier. This highlights the role of DNA’s relative flexibility as a modulator of the WCF/HG dynamic equilibrium. Additionally, we provide a robust methodology for future HG proclivity studies.     

The transient receptor potential family of ion channels

The transient receptor potential (TRP) multigene superfamily encodes integral membrane proteins that function as ion channels. Members of this family are conserved in yeast, invertebrates and vertebrates. The TRP family is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter is found only in invertebrates and fish. TRP ion channels are widely expressed in many different tissues and cell types, where they are involved in diverse physiological processes, such as sensation of different stimuli or ion homeostasis. Most TRPs are non-selective cation channels, only few are highly Ca²⁺ selective, some are even permeable for highly hydrated Mg²⁺ ions. This channel family shows a variety of gating mechanisms, with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues. Activated TRP channels cause depolarization of the cellular membrane, which in turn activates voltage-dependent ion channels, resulting in a change of intracellular Ca²⁺ concentration; they serve as gatekeeper for transcellular transport of several cations (such as Ca²⁺ and Mg²⁺), and are required for the function of intracellular organelles (such as endosomes and lysosomes). Because of their function as intracellular Ca²⁺ release channels, they have an important regulatory role in cellular organelles. Mutations in several TRP genes have been implicated in diverse pathological states, including neurodegenerative disorders, skeletal dysplasia, kidney disorders and pain, and ongoing research may help find new therapies for treatments of related diseases.     

Study of the thermal stability of D-amino acid oxidase from Trigonopsis variabilis reveals enzyme inactivation via multiple steps

The thermal stability of a highly purified preparation of D-amino acid oxidase from Trigonopsis variabilis (TvDAO), which does not show microheterogeneity due to the partial oxidation of Cys-108, was studied based on dependence of temperature (20-60°C) and protein concentration (5-100 µmol L^-1). The time courses of loss of enzyme activity in 100 mmol L^-1 potassium phosphate buffer, pH 8.0, are well described by a formal kinetic mechanism in which two parallel denaturation processes, partial thermal unfolding and dissociation of the FAD cofactor, combine to yield the overall inactivation rate. Estimates from global fitting of the data revealed that the first-order rate constant of the unfolding reaction (k _a) increased 10⁴-fold in response to an increase in temperature from 20 to 60°C. The rate constants of FAD release (k _b) and binding (k _-b) as well as the irreversible aggregation of the apo-enzyme (k _agg) were less sensitive to changes in temperature, their activation energy (E _a) being about 52 kJ mol^-1 in comparison with an E _a value of 185 kJ mol^-1 for k _a. The rate-determining step of TvDAO inactivation switched from FAD dissociation to unfolding at high temperatures. The model adequately described the effect of protein concentration on inactivation kinetics. Its predictions regarding the extent of FAD release and aggregation during thermal denaturation were confirmed by experiments. TvDAO is shown to contain two highly reactive cysteines per protein subunit whose modification with 5,5'-dithio-bis (2-nitrobenzoic acid) was accompanied by inactivation. Dithiothreitol (1 mmol L^-1) enhanced up to 10-fold the recovery of enzyme activity during ion exchange chromatography of technical-grade TvDAO. However, it did not stabilize TvDAO at all temperatures and protein concentrations, suggesting that deactivation of cysteines was not responsible for thermal denaturation.     

Specific monocyte adhesion to endothelial cells induced by oxidized phospholipids involves activation of cPLA2 and lipoxygenase

Oxidized phospholipids stimulate endothelial cells to bind monocytes, but not neutrophils, an initiating event in atherogenesis. Here, we investigate intracellular signaling events induced by oxidized phospholipids in human umbilical vein endothelial cells (HUVECs) that lead to specific monocyte adhesion. In a static adhesion assay, oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine and one of its components, 1-palmitoyl-2-oxovaleroyl-sn-glycero-3-phosphorylcholine, stimulated HUVECs to bind U937 cells and human peripheral blood monocytes but not HL-60 cells or blood neutrophils. Monocyte adhesion was dependent on protein kinases A and C, extracellular signal-regulated kinase 1/2, p38 mitogen activated protein kinases (MAPKs), and cytosolic phospholipase A(2) (cPLA(2)). Inhibition of 12-lipoxygenase (12-LOX), but not cyclooxygenases, blocked monocyte adhesion, and addition of 12-hydroxyeicosatetraenoic acid (12-HETE) mimicked the effects of oxidized phospholipids. Peroxisome proliferator-activated receptor alpha (PPARalpha) was excluded as a possible target for 12-HETE, because monocyte adhesion was still induced in endothelial cells from PPARalpha null mice. Together, our results suggest that oxidized phospholipids stimulate HUVECs to specifically bind monocytes involving MAPK pathways, which lead to the activation of cPLA(2) and 12-LOX. Further analysis of signaling pathways induced by oxidized phospholipids that lead to specific monocyte adhesion should ultimately lead to the development of novel therapeutic approaches against chronic inflammatory diseases.     

Glacial and postglacial sedimentation in the Fryxell basin, Taylor Valley, southern Victoria Land, Antarctica

A 9.14 m long sediment sequence was recovered from Lake Fryxell, Taylor Valley, southern Victoria Land, Antarctica, and investigated for its chronology and sedimentological, mineralogical, and biogeochemical changes. The basal part of the sequence is dominated by coarse clastic matter, i.e., mainly sand. The sediment composition suggests that a lake existed in Fryxell basin during the Middle Weichselian by ca. 48,000 cal. year BP. After a short period of lake-level lowstand ca. 43,000 cal. year BP, lower Taylor Valley became occupied by the proglacial Lake Washburn, which was at least partly supplied by meltwater and sediments from the Ross Ice Sheet that was advanced to the mouth of Taylor Valley. Evaporation of Lake Washburn to lower levels started during the Last Glacial Maximum at ca. 22,000 cal. year BP, long before the Ross Ice Sheet retreated significantly. Lake-level lowering was discontinuous with a series of high and low stands. From ca. 4000 cal. year BP environmental conditions were similar to those of today and lower Fryxell basin was occupied by a small lake. This lake evaporated to a saline or hypersaline pond between ca. 2500 and 1000 cal. year BP and refilled subsequently.     

Effects of Plectin Depletion on Keratin Network Dynamics and Organization

The keratin intermediate filament cytoskeleton protects epithelial cells against various types of stress and is involved in fundamental cellular processes such as signaling, differentiation and organelle trafficking. These functions rely on the cell type-specific arrangement and plasticity of the keratin system. It has been suggested that these properties are regulated by a complex cycle of assembly and disassembly. The exact mechanisms responsible for the underlying molecular processes, however, have not been clarified. Accumulating evidence implicates the cytolinker plectin in various aspects of the keratin cycle, i.e., by acting as a stabilizing anchor at hemidesmosomal adhesion sites and the nucleus, by affecting keratin bundling and branching and by linkage of keratins to actin filament and microtubule dynamics. In the present study we tested these hypotheses. To this end, plectin was downregulated by shRNA in vulvar carcinoma-derived A431 cells. As expected, integrin β4- and BPAG-1-positive hemidesmosomal structures were strongly reduced and cytosolic actin stress fibers were increased. In addition, integrins α3 and β1 were reduced. The experiments furthermore showed that loss of plectin led to a reduction in keratin filament branch length but did not alter overall mechanical properties as assessed by indentation analyses using atomic force microscopy and by displacement analyses of cytoplasmic superparamagnetic beads using magnetic tweezers. An increase in keratin movement was observed in plectin-depleted cells as was the case in control cells lacking hemidesmosome-like structures. Yet, keratin turnover was not significantly affected. We conclude that plectin alone is not needed for keratin assembly and disassembly and that other mechanisms exist to guarantee proper keratin cycling under steady state conditions in cultured single cells.     

Organophosphate poisoning in the developed world – A single centre experience from here to the millennium

Organophosphate (OP) poisoning is still associated with high morbidity and mortality rates, both in resource-poor settings and in well-developed countries. Despite numerous publications dealing with this particular poison, detailed clinical data on more severe overdoses with these agents are relatively sparsely reported. A retrospective study was consequently conducted on 33 patients with OP poisoning admitted to our intensive care unit (ICU) to provide additional data on clinical features. We included moderate to severe poisonings between 2000 and 2012 who required admission to ICU.