Prefibrillar transthyretin oligomers and cold stored native tetrameric transthyretin are cytotoxic in cell culture

Recent studies suggest that soluble, oligomeric species, which are intermediates in the fibril formation process in amyloid disease, might be the key species in amyloid pathogenesis. Soluble oligomers of human wild type transthyretin (TTR) were produced to elucidate oligomer properties. Employing ThT fluorescence, time-resolved fluorescence anisotropy of pyrene-labeled TTR, chemical cross-linking, and electron microscopy we demonstrated that early formed soluble oligomers (within minutes) from A-state TTR comprised on the average 20-30 TTR monomers. When administered to neuroblastoma cells these early oligomers proved highly cytotoxic and induced apoptosis after 48 h of incubation. More mature fibrils (>24 h of fibrillation) were non-toxic. Surprisingly, we also found that native tetrameric TTR, when purified and stored under cold conditions (4 °C) was highly cytotoxic. The effect could be partially restored by increasing the temperature of the protein. The cytotoxic effects of native tetrameric TTR likely stems from a hitherto unexplored low temperature induced rearrangement of the tetramer conformation that possibly is related to the conformation of misfolded TTR in amyloigogenic oligomers.     

Thermodynamic interrogation of a folding disease. Mutant mapping of position 107 in human carbonic anhydrase II linked to marble brain disease

Marble brain disease (MBD) also known as Guibaud-Vainsel syndrome is caused by autosomal recessive mutations in the human carbonic anhydrase II (HCA II) gene. HCA II is a 259 amino acid single domain enzyme and is dominated by a 10-stranded beta-sheet. One mutation associated with MBD entails the H107Y substitution where H107 is a highly conserved residue in the carbonic anhydrase protein family. We have previously demonstrated that the H107Y mutation is a remarkably destabilizing folding mutation [Almstedt et al. (2004) J. Mol. Biol. 342, 619-633]. Here, the exceptional destabilization by the H107Y mutation has been further investigated. A mutational survey of position H107 and a neighboring conserved position E117 has been performed entailing the mutants H107A, H107F, H107N, E117A and the double mutants H107A/E117A and H107N/E117A. All mutants were severely destabilized versus GuHCl and heat denaturation. Thermal denaturation and GuHCl phase diagram and ANS analyses showed that the mutants shifted HCA II toward populating ensembles of intermediates of molten globule type under physiological conditions. The native state stability of the mutants was in the following order: wt > H107N > E117A > H107A > H107F > H107Y > H107N/E117A > H107A/E117A. In conclusion: (i) H107N is least destabilizing likely due to compensatory H-bonding ability of the introduced Asn residue. (ii) Double mutant cycles surprisingly reveal additive destabilization of H107N and E117A showing that H107 and E117 are independently stabilizing the folded protein. (iii) H107Y and H107F are exceptionally destabilizing due to bulkiness of the side chains whereas H107A is more accommodating, indicating long-range destabilizing effects of the natural pathogenic H107Y mutation.     

Phase plane analysis of left ventricular hemodynamics.

We sought to extract additional physiological information from the time-dependent left ventricular (LV) pressure contour and thereby gain new insights into ventricular function. We used phase plane analysis to characterize high-fidelity pressure data in selected subjects undergoing elective cardiac catheterization. The standard hemodynamic indexes of LV systolic and diastolic function derived from the time-dependent LV pressure contour could be easily obtained using the phase plane method. Additional novel attributes of the phase plane pressure loop, such as phase plane pressure loop area, graphical representation of the isovolumic relaxation time constant, and quantitative measures of beat-to-beat systolic-diastolic coupling were characterized. The asymmetry between the pressures at which maximum isovolumic pressure rise and pressure fall occur, as well as their load dependence, were also easily quantitated. These results indicate that the phase plane method provides a novel window for physiological discovery and has theoretical and applied advantages in quantitative ventricular function characterization.     

Abandonment alters community composition and canopy structure of Swiss calcareous fens

Abstract. We compared the plant species composition, productivity and canopy structure of seven mown sites to a chronosequence of 20 abandoned calcareous fens in northeastern Switzerland. Cessation of mowing led to an 18% decline in overall plant species richness and the diversity of most functional groups. Abandonment did not lead to marked increases of above‐ground productivity, but rather selectively favoured certain functional groups. On abandoned fens biomass of grasses increased nearly threefold, at the expense of biomass of Cyperaceae and Juncaceae, which declined by 30% compared to mown fens, while forb biomass remained unaffected. Litter mass increased more than 15‐fold in fallows, while canopy height increased by 50%. The foliage in abandoned fens was oriented more horizontally and had a lower overall cover. However, these successional changes were never dependent upon the age of the fallow. Furthermore, nearly all traits differed significantly on regional and local spatial scales, suggesting that floristic and (meso‐)climatic differences obscure or override successional trajectories in these species‐rich wetlands.     

Tannins from mimosoid legumes of Texas and Mexico

The percentage of tannins in leaves, bark, wood, and immature fruits of several species of Acacia and related mimosoid legumes from the southwestern U.S. and Mexico, along with a few from Costa Rica and Argentina, was determined by a modified hide powder procedure and by precipitation with casein. The relative percentages of hydrolyzable and condensed tannins were determined by the iodate and the vanillin-HCl methods, respectively. Gallotannins of selected samples were also determined by the rhodanine method. Although the amount of total tannins was similar for the first two methods, values for condensed tannins by the vanillin-HCl method were frequently two to four times greater than the total tannin values.     

Particle retention in the forestomach of a browsing ruminant, the roe deerCapreolus capreolus

A combination of a flotation/sedimentation experiment and sieve analysis for the reticulorumen (RR) contents of roe deerCapreolus capreolus Linnaeus, 1758, a browsing ruminant, showed that there was no correlation between particle size and particle density. Large particles were present in both the sedimented and the buoyant fraction, which is in accord with the reported absence of stratification of RR contents in browsing ruminants. Comparative sieve analysis of roe deer RR and caecal/rectal material demonstrated that there must be some selective particle retention in the browsing ruminant as well, as a certain fraction of large particles in RR contents does not occur in the caecal/rectal material. These results lead to the explanatory dilemma that, while selective particle retention is observed, it cannot be due to the mechanisms known to work in grazing ruminants.     

AU binding proteins recruit the exosome to degrade ARE-containing mRNAs.

Inherently unstable mammalian mRNAs contain AU-rich elements (AREs) within their 3' untranslated regions. Although found 15 years ago, the mechanism by which AREs dictate rapid mRNA decay is not clear. In yeast, 3'-to-5' mRNA degradation is mediated by the exosome, a multisubunit particle. We have purified and characterized the human exosome by mass spectrometry and found its composition to be similar to its yeast counterpart. Using a cell-free RNA decay system, we demonstrate that the mammalian exosome is required for rapid degradation of ARE-containing RNAs but not for poly(A) shortening. The mammalian exosome does not recognize ARE-containing RNAs on its own. ARE recognition requires certain ARE binding proteins that can interact with the exosome and recruit it to unstable RNAs, thereby promoting their rapid degradation.     

IKKalpha provides an essential link between RANK signaling and cyclin D1 expression during mammary gland development.

To identify functions of the IKKalpha subunit of IkappaB kinase that require catalytic activity, we generated an Ikkalpha(AA) knockin allele containing alanines instead of serines in the activation loop. Ikkalpha(AA/AA) mice are healthy and fertile, but females display a severe lactation defect due to impaired proliferation of mammary epithelial cells. IKKalpha activity is required for NF-kappaB activation in mammary epithelial cells during pregnancy and in response to RANK ligand but not TNFalpha. IKKalpha and NF-kappaB activation are also required for optimal cyclin D1 induction. Defective RANK signaling or cyclin D1 expression results in the same phenotypic effect as the Ikkalpha(AA) mutation, which is completely suppressed by a mammary specific cyclin D1 transgene. Thus, IKKalpha is a critical intermediate in a pathway that controls mammary epithelial proliferation in response to RANK signaling via cyclin D1.