Multipole electrostatic potential derived atomic charges in NDDO-methods with <Emphasis Type="Italic">spd</Emphasis>-basis sets

The recently introduced multipole approach for computing the molecular electrostatic potential (MEP) within the semiempirical neglect of diatomic differential overlap (NDDO) framework [Horn AHC, Lin Jr-H., Clark T (2005) Theor Chem Acc 114:159–168] has been used to obtain atomic charges of nearly ab initio quality by scaling the semiempirical MEP. The parameterization set comprised a total of 797 compounds and included not only the newly parameterized AM1* elements Al, Si, P, S, Cl, Ti, Zr, and Mo but also the standard AM1 elements H, C, N, O and F. For comparison, the ZDO-approximated MEP was also calculated analytically in the spd-basis. For the AM1*-optimized structures, single-point calculations at the B3LYP, HF and MP2 levels with the 6-31G(d) and LanL2DZP basis sets were performed to obtain the MEP. The regression analysis of all 12 combinations of semiempirical and ab initio MEP data yielded correlation coefficients of at least 0.99 in all cases. Scaling the analytical and multipole-derived semiempirical MEP by the regression coefficients yielded mean unsigned errors below 2.6 and 1.9 kcal mol⁻¹, respectively. Subsequently, for 22 drug molecules from the World Drug Index, atomic charges were computed according to the RESP procedure using XX/6-31G(d) (XX=B3LYP, HF, MP2) and scaled AM1* multipole MEP; the correlation coefficients obtained are 0.83, 0.85 and 0.83, respectively. Figure: Schematic representation of the atomic charge generation: The molecular electrostatic potential (MEP) is calculated using the AM1* Hamiltonian; then the semiempirical MEP is scaled to DFT or ab initio level, and atomic charges are generated subsequently by the restraint electrostatic potential (RESP) fit method. Electronic supplementary material Supplementary material is available in the online version of this article at and is accessible to authorized users. Proceedings of “Modeling Interactions in Biomolecules II”, Prague, September 5th–9th, 2005.     

Non‐fire induced seed release ina weakly serotinous pine: climatic factors,maintenance costs or both?

The advantages of canopy seed retention (serotiny) for plants inhabiting fire‐prone ecosystems are well documented. However, very few species are completely serotinous and non‐fire induced opening of serotinous fruits is commonly observed (weak serotiny). Two non‐mutually exclusive causes are envisaged to contribute to this process: mechanical changes in serotinous fruits mediated by climatic conditions (e.g. drought) or the costs of maintenance for the plant of these long‐lasting structures. However, their relative contribution to the spontaneous opening of serotinous fruits remains elusive as well as the consequences for the build‐up of the canopy seed bank and inter‐individual differences in serotiny. In this study we monitored the dynamics of cone production and cone opening in the weakly serotinous Pinus halepensis for five years (2004–2008), including two severe drought episodes (2005, 2006). Drought decreased the production of conelets, increased the abortion of immature cones, reduced the seed quality in the cohorts of cones produced during these years, and increased the opening of serotinous cones. During the first drought episode, a higher proportion of serotinous cones opened in those pines bearing a larger crop of younger cones. This suggests that not only passive changes induced by drought but also competition among cones for resources (e.g. water) might be involved in this process. The opening of serotinous cones in pines bearing more cones made inter‐individual differences in the size of the canopy cone bank to narrow or even to reverse from 2004 to 2008. These results may help to understand the decrease in serotiny when pines grow and accumulate more cones and the large inter‐individual variability in the degree of serotiny observed in P. halepensis forests. In addition, the negative effects of drought episodes for the size of the canopy cone bank and the seeds contained can be an unexplored cause of post‐fire regeneration constraint.     

Melittin modulates keratinocyte function through P2 receptor-dependent ADAM activation

Melittin, the major component of the bee venom, is an amphipathic, cationic peptide with a wide spectrum of biological properties that is being considered as an anti-inflammatory and anti-cancer agent. It modulates multiple cellular functions but the underlying mechanisms are not clearly understood. Here, we report that melittin activates disintegrin-like metalloproteases (ADAMs) and that downstream events likely contribute to the biological effects evoked by the peptide. Melittin stimulated the proteolysis of ADAM10 and ADAM17 substrates in human neutrophil granulocytes, endothelial cells and murine fibroblasts. In human HaCaT keratinocytes, melittin induced shedding of the adhesion molecule E-cadherin and release of TGF-α, which was accompanied by transactivation of the EGF receptor and ERK1/2 phosphorylation. This was followed by functional consequences such as increased keratinocyte proliferation and enhanced cell migration. Evidence is provided that ATP release and activation of purinergic P2 receptors are involved in melittin-induced ADAM activation. E-cadherin shedding and EGFR phosphorylation were dose-dependently reduced in the presence of ATPases or P2 receptor antagonists. The involvement of P2 receptors was underscored in experiments with HEK cells, which lack the P2X7 receptor and showed strikingly increased response to melittin stimulation after transfection with this receptor. Our study provides new insight into the mechanism of melittin function which should be of interest particularly in the context of its potential use as an anti-inflammatory or anti-cancer agent.     

Conformational Stability of Fibrillar Amyloid-Beta Oligomers via Protofilament Pair Formation – A Systematic Computational Study

Amyloid- (A) oligomers play a crucial role in Alzheimer’s disease due to their neurotoxic aggregation properties. Fibrillar A oligomerization can lead to protofilaments and protofilament pairs via oligomer elongation and oligomer association, respectively. Small fibrillar oligomers adopt the protofilament topology, whereas fibrils contain at least protofilament pairs. To date, the underlying growth mechanism from oligomers to the mature fibril still remains to be elucidated. Here, we performed all-atom molecular dynamics simulations in explicit solvent on single layer-like protofilaments and fibril-like protofilament pairs of different size ranging from the tetramer to the 48-mer. We found that the initial U-shaped topology per monomer is maintained over time in all oligomers. The observed deviations of protofilaments from the starting structure increase significantly with size due to the twisting of the in-register parallel -sheets. This twist causes long protofilaments to be unstable and leads to a breakage. Protofilament pairs, which are stabilized by a hydrophobic interface, exhibit more fibril-like properties such as the overall structure and the twist angle. Thus, they can act as stable conformational templates for further fibril growth. Key properties like the twist angle, shape complementarity, and energetics show a size-dependent behavior so that small oligomers favor the protofilament topology, whereas large oligomers favor the protofilament pair topology. The region for this conformational transition is at the size of approximately twelve A monomers. From that, we propose the following growth mechanism from A oligomers to fibrils: (1) elongation of short protofilaments; (2) breakage of large protofilaments; (3) formation of short protofilament pairs; and (4) elongation of protofilament pairs.     

Negative regulation of RPE cell attachment by carbohydrate-dependent cell surface binding of galectin-3 and inhibition of the ERK-MAPK pathway

Adhesion and spreading of retinal pigment epithelial (RPE) cells on fibronectin-rich extracellular matrices is a crucial event in the pathogenesis of proliferative vitreoretinopathy (PVR). In the present study we explored the capacity of galectin-3, a β-galactoside-binding endogenous lectin, to inhibit early PVR-associated cellular events from a therapeutic perspective. We assessed the relative expression levels of galectin-3 in native RPE and dedifferentiated, cultured RPE. Galectin-3 was constitutively expressed under in vivo and in vitro conditions and was abundant in cultured cells. Treatment of human RPE cells with soluble galectin-3 disclosed no toxicity within control limits up to 250 μg/ml. When added to the medium, galectin-3 dose-dependently inhibited attachment and spreading of the cells on fibronectin by more than 75%. When coated on the plastic surface, galectin-3 alone impaired attachment and spreading of RPE cells, and reduced attachment but not spreading on fibronectin. Galectin-3 bound to the cell surface, and, as determined by the use of the competing sugar β-lactose, galectin-3-mediated effects were dependent on carbohydrate binding. To ascertain the role of the ability of galectin-3 to form pentamers, we proteolytically removed the N-terminal, cross-linking section. The remaining C-terminal carbohydrate-binding domain alone failed to bind to cells and was functionally inactive. These results emphasize the relevance of both properties, i.e., glycan-binding and cross-linking of glycan moieties, for the inhibitory activity of galectin-3. Incubation of mobilized RPE cells with galectin-3 significantly disturbed microfilament assembly and, in correlation with decreased attachment, inhibited ERK phosphorylation. Therefore, galectin-3, acting as a cross-linking lectin on the cell surface, negatively regulates attachment and spreading of RPE cells in vitro. This effect, at least in part, is attributed to an inhibition of the ERK-MAPK pathway, which prevents cytoskeletal rearrangements needed for RPE cell attachment and spreading. Further investigation at this pathway may disclose a promising nouveau perspective for treatment and prophylaxis of early PVR.     

Differentiation of embryonic chick sympathetic neurons in vivo: ultrastructure,and quantitative determinations of catecholamines and somatostatin

Summary The ultrastructural and transmitter development of lumbar sympathetic ganglia was studied in embryonic day-6 through-18 chick embryos. At embryonic day 6, ganglia are populated by two morphologically distinct types of neuronal cells and Schwann cell precursors. The neuronal populations basically comprise a granule-containing cell and a developing principal neuron. Granule-containing cells have, an irregularly shaped or oval nucleus with small clumps of chromatin attached to the inner nuclear membrane and numerous large (up to 300 nm) membrane-limited granules. Developing principal neurons display a more rounded vesicular nucleus with evenly distributed chromatin, prominent nucleoli, more developed areas of Golgi complexes, and rough endoplasmic reticulum and large dense-core vesicles up to 120 nm in diameter. There are granule-containing cells with fewer and smaller granules which still display the nucleus typical for granule-containing cells. These granule-containing cells may develop toward developing principal neurons or the resting state of granule-containing cells found in older ganglia. Both granule-containing cells and developing principal neurons proliferate and can undergo degeneration. At embryonic day 9 there are far more developing principal neurons than granule-containing cells. Most granule-containing cells have very few granules. Mitotic figures and signs of cell degeneration are still apparent. Synapse-like terminals are found on both developing principal neurons and granule-containing cells. Ganglionic development from embryonic day 11 through 18 comprises extensive maturation of developing principal neurons and a numerical decline of granule-containing cells. Some granule-containing cells with very few and small granules still persist at embryonic day 18. The mean catecholamine content per neuron increases from 0.044 femtomol at embryonic day 7 to 0.22 femtomol at embryonic day 15. Concomitantly, there is a more than 6-fold increase in tyrosine hydroxylase activity. Adrenaline has a 14% share in total catecholamines at embryonic day 15. Somatostatin levels are relatively high at embryonic day 7 (1.82 attomol per neuron) and are 10-fold reduced by embryonic day 15. Our results suggest the presence of two morphologically distinct sympathetic neuronal precursors at embryonic day 6: one with a binary choice to become a principal neuron or to die, the other one, a granule-containing cell, which alternatively may develop into a principal neuron, acquire a resting state or die.