p25alpha is flexible but natively folded and binds tubulin with oligomeric stoichiometry

p25alpha is a 219-residue protein which stimulates aberrant tubulin polymerization and is implicated in a variety of other functions. The protein has unusual secondary structure involving significant amounts of random coil, and binding to microtubules is accompanied by a large structural change, suggesting a high degree of plasticity. p25alpha has been proposed to be natively unfolded, so that folding is coupled to interaction with its physiological partners. Here we show that recombinant human p25alpha is folded under physiological conditions, since it has a well structured and solvent-sequestered aromatic environment and considerable chemical shift dispersion of amide and aliphatic protons. With increasing urea concentrations, p25alpha undergoes clear spectral changes suggesting significant loss of structure. p25alpha unfolds cooperatively in urea according to a simple two-state transition with a stability in water of approximately 5 kcal/mol. The protein behaves as a monomer and refolds with a transient on-pathway folding intermediate. However, high sensitivity to proteolytic attack and abnormal gel filtration migration behavior suggests a relatively extended structure, possibly organized in distinct domains. A deletion mutant of p25alpha lacking residues 3-43 also unfolds cooperatively and with similar stability, suggesting that the N-terminal region is largely unstructured. Both proteins undergo significant loss of structure when bound to monomeric tubulin. The stoichiometry of binding is estimated to be 3-4 molecules of tubulin per p25alpha and is not significantly affected by the deletion of residues 3-43. In conclusion, we dismiss the proposal that p25alpha is natively unfolded, although the protein is relatively flexible. This flexibility may be linked to its tubulin-binding properties.     

Tissue distribution,intracellular localization and proteolytic processing of rat 4-hydroxyphenylpyruvate dioxygenase

4-hydroxyphenylpyruvate dioxygenase (HPD) is an important enzyme involved in tyrosine catabolism. HPD was shown to be identical to a protein named the F-antigen, exploited by immunologists because of its unique immunological properties. Congenital HPD deficiency is a rare, relatively benign condition known as hereditary type III tyrosinemia. Decreased expression of HPD is often observed in association with the severe type I tyrosinemia, and interestingly, inhibition of HPD activity seems to ameliorate the clinical symptoms of type I tyrosinemia. In this study we present a comprehensive analysis of tissue specific expression and intracellular localization of HPD in the rat. By combined use of in situ hybridization and immunohistochemistry we confirm previously known sites of expression in liver and kidney. In addition, we show that HPD is abundantly expressed in neurons in the cortex, cerebellum and hippocampus. By using immunoelectron microscopy and confocal laser scanning microscopy, we provide evidence that HPD contrary to earlier assumptions specifically localizes to membranes of the endoplasmic reticulum and the Golgi apparatus. Detailed mass spectrometric analyses of HPD purified from rat liver revealed N-terminal and C-terminal processing of HPD, and expression of recombinant HPD suggested that C-terminal processing enhances the enzymatic activity.     

Exposure affects the risk of an owl being mobbed – experimental evidence

Mobbing is a widespread anti‐predator strategy in birds, and predators are generally expected to avoid mobbing. For example, observational studies suggest that the cryptic roosting behaviour of nocturnal predators, such as many owls, may be a strategy to limit mobbing. In this paper, we present the results of the first experimental study investigating to what degree roost exposure influences the risk of being mobbed, and the intensity of a mobbing incidence once initiated. To determine these factors, we used an experimental setup with taxidermic mounts of tawny owls Strix aluco in Grib Skov forest, Denmark. The risk of an owl being mobbed during a 50 min morning survey period increased with the exposure of its roosting position, from 24% when hidden to 85% when openly exposed. The corresponding increase in the afternoon was from 6% to 36%. This suggests that an owl may minimize the mobbing rate by reducing the encounter rate with potential mobbers through its choice of roost. Once initiated, the duration of the mobbing (a proxy for the presumed cost of being mobbed) was independent of the roosting position of the mounted owl, but was positively correlated with the number of birds in the mob.     

Stem cells on patrol

Hematopoietic stem cells (HSCs) exist in the bone marrow and circulate in the blood. In this issue, Massberg et al. (2007) report that HSCs also travel through the lymphatic system. Furthermore, migration of HSCs--which express Toll-like receptors--allows the recognition of pathogenic molecules in peripheral tissues thereby promoting the local generation of innate immune cells at the site of infection.     

Effects of past and present microclimates on northern and southern plant species in a managed forest landscape

Questions

Near-ground temperatures can vary substantially over relatively short distances, enabling species with different temperature preferences and geographical distributions to co-exist within a small area. In a forest landscape, the near-ground temperatures may change due to management activities that alter forest density. As a result of such management activities, current species distributions and performances might not only be affected by current microclimates, but also by past conditions due to time-lagged responses.

Location

Sweden.

Methods

We examined the effects of past and current microclimates on the distributions and performances of two northern, cold-favoured, and two southern, warm-favoured, plant species in 53 managed forest sites. Each pair was represented by one vascular plant and one bryophyte species. We used temperature logger data and predictions from microclimate models based on changes in basal area to relate patterns of occurrence, abundance, and reproduction to current and past microclimate.

Results

The two northern species were generally favoured by microclimates that were currently cold, characterised by later snowmelt and low accumulated heat over the growing season. In contrast, the two southern species were generally favoured by currently warm microclimates, characterised by high accumulated heat over the growing season. Species generally had higher abundance in sites with a preferred microclimate both in the past and present, and lower abundance than expected from current conditions, if the past microclimate had changed from warm to cold or vice versa, indicating time-lags in abundance patterns of the species.

Conclusions

Our results show a potential importance of past and present microclimate heterogeneity for the co-existence of species with different temperature preferences in the same landscape and highlight the possibility to manage microclimates to mitigate climate change impacts on forest biodiversity.     

Oligomerization engineering of the fluorinase enzyme leads to an active trimer that supports synthesis of fluorometabolites in vitro

The fluorinase enzyme represents the only biological mechanism capable of forming stable C–F bonds characterized in nature thus far, offering a biotechnological route to the biosynthesis of value-added organofluorines. The fluorinase is known to operate in a hexameric form, but the consequence(s) of the oligomerization status on the enzyme activity and its catalytic properties remain largely unknown. In this work, this aspect was explored by rationally engineering trimeric fluorinase variants that retained the same catalytic rate as the wild-type enzyme. These results ruled out hexamerization as a requisite for the fluorination activity. The Michaelis constant (K_M) for S-adenosyl-l -methionine, one of the substrates of the fluorinase, increased by two orders of magnitude upon hexamer disruption. Such a shift in S-adenosyl-l -methionine affinity points to a long-range effect of hexamerization on substrate binding – likely decreasing substrate dissociation and release from the active site. A practical application of trimeric fluorinase is illustrated by establishing in vitro fluorometabolite synthesis in a bacterial cell-free system.     

Lipid molecular timeline profiling reveals diurnal crosstalk between the liver and circulation

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