A Comparison of Experimental and Computational Methods for Mapping the Interactions Present in the Transition State for Folding of FKBP12

The folding pathway of FKBP12, a 107 residue / protein, has been characterised in detail using a combination of experimental and computational techniques. FKBP12 follows a two-state model of folding in which only the denatured and native states are significantly populated; no intermediate states are detected. The refolding rate constant in water is 4 s^-1 at 25 °C. Two different experimental strategies were employed for studying the transition state for folding. In the first case, a non-mutagenic approach was used and the unfolding and refolding of the wild-type protein measured as a function of experimental conditions such as temperature, denaturant, ligand and trifluoroethanol (TFE) concentration. These data suggest a compact transition state relative to the unfolded state with some 70% of the surface area buried. The ligand-binding site, whichis mainly formed by two long loops, is largely unstructured in the transition state. TFE experiments suggest that the -helix may be formed in the transition state. The second experimental approach involved using protein engineering techniques with -value analysis. Residue-specific information on the structure and energetics of the transition state can be obtained by this method. 34 mutations were made at sites throughout the protein to probe the extent of secondary and tertiary structure in the transition state. In contrast to some other proteins of this size, no element of structure is fully formed in the transition state, instead, the transition state is similar to that found for smaller, single-domain proteins, such as chymotrypsin inhibitor 2 and the SH3 domainfrom -spectrin. For FKBP12, the central three strands of the -sheet (2, 4 and 5), comprise the most structured region of the transition state. In particular Val 101, which is one of the most highly buried residues and located in the middle of the central -strand,makes approximately 60% of its native interactions. The outer -strands, and the ends of the central -strands are formed to a lesser degree. The short -helix is largely unstructured in the transition state as are the loops. The data are consistent with a nucleation-condensation model of folding, the nucleus of which is formed by side chains within -strands 2, 4 and 5 and the C-terminus of the -helix. These residues are distant in the primary sequence, demonstrating the importance of tertiary interactions in the transition state. High-temperature molecular dynamic simulations on the unfoldingpathway of FKBP12 are in good agreement with the experimental results.     

Photosynthesis and water‐use efficiency: A comparison between invasive (exotic) and non‐invasive (native) species

Abstract Invasive species have been hypothesized to out‐compete natives though either a Jack‐of‐all‐trades strategy, where they are able to utilize resources effectively in unfavourable environments, a master‐of‐some, where resource utilization is greater than its competitors in favourable environments, or a combination of the two (Jack‐and‐master). We examined the invasive strategy of Berberis darwinii in New Zealand compared with four co‐occurring native species by examining germination, seedling survival, photosynthetic characteristics and water‐use efficiency of adult plants, in sun and shade environments. Berberis darwinii seeds germinated more in shady sites than the other natives, but survival was low. In contrast, while germination of B. darwinii was the same as the native species in sunny sites, seedling survival after 18 months was nearly twice that of the all native species. The maximum photosynthetic rate of B. darwinii was nearly double that of all native species in the sun, but was similar among all species in the shade. Other photosynthetic traits (quantum yield and stomatal conductance) did not generally differ between B. darwinii and the native species, regardless of light environment. Berberis darwinii had more positive values of δ¹³C than the four native species, suggesting that it gains more carbon per unit water transpired than the competing native species. These results suggest that the invasion success of B. darwinii may be partially explained by combination of a Jack‐of‐all‐trades scenario of widespread germination with a master‐of‐some scenario through its ability to photosynthesize at higher rates in the sun and, hence, gain a rapid height and biomass advantage over native species in favourable environments.     

Influence of excess nitrogen deposition on a white spruce (Picea glauca) stand in southern Alaska

Excess N delivered to forest ecosystems has been shown to alterinternal ecosystem biogeochemical cycles, contribute to forestdecline, and negatively affect the health of receiving waters.In the vicinity of the Nikiski Industrial Complex, Kenai Peninsula,Alaska, there has been recent concern about the influence ofNH3 emissions that have occurred for over twodecades on local soils and vegetation. The study site representedan opportunity to examine the influence of elevated N depositionon a northern coniferous ecosystem in an area with a low backgroundof N deposition. Overstory vegetation in the area is dominated bywhite spruce (Picea glauca Moench. Voss) and paper birch (Betulapapyrifera Marsh.). Mortality of both species has occurred adjacent(<2 km) to the industrial complex. Average annual Ndeposition rates ranged from 0.7 to 21.0 kg ha-1 y-1in the area, with the highest rates closest to the complex. Sulfatedeposition at the site was low. Due to the high NH3deposition, precipitation near the complex was less acidic thanprecipitation in general; bulk precipitation pH ranged from 5.51to 7.06. Within 1.80 km of the facility there was an increase inKCl- and resin- extractable soil NH4+ andNO3- in the O horizon, and a decrease in soil pHcompared to soils further from the facility. Spruce near thefacility had chlorotic foliage and thinning crowns, higherconcentrations of N, but lower foliar Ca and Mg. Foliar Mglevels approached deficiency levels, but foliar Ca was wellabove reported deficiency levels at all sites. Both Mg:N andCa:N ratios, however, suggest nutrient imbalances in the highN deposition zone. Canopy death and fertilization by N appearto have encouraged growth of the native bluejoint grass. Thepresence of elevated NO3- in O horizon soilextracts, elevated NO3- in resin bags placed betweenthe O and E horizons, and nutritional imbalances in the foliagesuggest that N saturation may be occurring in soils adjacent to thefacility.     

Ecospace utilization in early Phanerozoic deep-marine environments: deep bioturbation in the Blakely Sandstone (Middle Ordovician), Arkansas, USA

Ichnofabric analysis of alternating light and dark-coloured mudstone layers in the Blakely Sandstone (Middle Ordovician) at Crystal Springs Landing, Lake Ouachita (west of Hot Springs, western Arkansas, USA) reveals two equilibrium palaeoichnocoenoses. The first was emplaced under variable, but low, oxygen levels during deposition of the dark-coloured layers; small diameter transition layer burrows overprint a mixed layer ichnofabric. The transition layer infauna was tiered with abundant Chondrites representing the deeper of two shallow tiers. Light-coloured layers accumulated during prolonged intervals in which the sediments were oxygenated to a greater extent and depth. Preservation of a mixed layer ichnofabric within them is the result of limited, but deep (up to at least 400 mm), reworking subsequently in the transition layer by an equilibrium community. These transition layer trace fossils are not tiered. If representative of oxygenated sediment columns in Ordovician deep-marine environments, an extensive volume of infaunal ecospace was colonized (in this case by deposit feeders); its more efficient use subsequently, including vertical partitioning of the infaunal community into specific environmental niches (tiering), could have accommodated increases in diversity and community complexity. Changes over time in the maximum depth to which sediments were bioturbated, alone, would therefore be a poor measure of the extent of ecospace utilization.     

IS HOMEOPATHY REALLY ‘MORALLY AND ETHICALLY UNACCEPTABLE’? A CRITIQUE OF PURE SCIENTISM

In this short response we show that Kevin Smith's moral and ethical rejections of homeopathy 1 are fallacious and rest on questionable epistemology. Further, we suggest Smith's presumption of a utilitarian stance is an example of scientism encroaching into medicine.     

Isolation of N-Acety-β-Hexosaminidase from Acanthamoeba castellanii

ABSTRACT. The lysosomal enzyme N-acetyl-β-hexosaminidase (βhex) has been purified from Acanthamoeba castellanii growth medium by a three step procedure. The enzyme was precipitated with ammonium sulfate, partially purified on a DE52 column and purified to homogeneity on an affinity column. The purified βhex appeared to be a monomer with a molecular mass of 58 kDa and a pI of approximately 5.8. The enzyme activity in growth medium at RT was stable for several months. The purified βhex was enzymatically deglycosylated and injected, into two rabbits to make polyclonal antibodies. One antiserum was specific for βhex, but the other stained many bands on immunoblots of whole cell preparations. Using fluorescently labelled secondary antibodies we have determined that both antisera stain digestive vacuoles in the Acanthamoeba cytoplasm, and do not stain the contractile vacuole. The multi-specific antiserum had high avidity for βhex, but also stained the carbohydrate portion of other molecules. These other molecules may be lysosomal enzymes as well, since the activity of several other lysosomal enzymes was partially immunoprecipitable with the antiserum. We plan to use these antibodies to study traffic patterns among the variety of vacuolar structures in Acanthamoeba cytoplasm.     

Shelf-life of stored asparagus is strongly related to post-harvest accumulated heat units

The effect of delay between harvest and hydrocooling, and of regimes of simulated air transport temperatures, on shelf-life of fresh asparagus were quantified. Shelf-life was strongly negatively correlated with the accumulated heat units experienced between harvest and post-transport handling. A 2-h delay in hydrocooling after harvest was similar to a 0.5°C higher temperature during a 2-day transit period in effect on shelf-life. It is essential for maximum shelf-life that asparagus be kept cold from the completion of hydrocooling until sale.