Microsecond Dynamics During the Binding-induced Folding of an Intrinsically Disordered Protein

Tau is an intrinsically disordered protein implicated in many neurodegenerative diseases. The repeat domain fragment of tau, tau-K18, is known to undergo a disorder to order transition in the presence of lipid micelles and vesicles, in which helices form in each of the repeat domains. Here, the mechanism of helical structure formation, induced by a phospholipid mimetic, sodium dodecyl sulfate (SDS) at sub-micellar concentrations, has been studied using multiple biophysical probes. A study of the conformational dynamics of the disordered state, using photoinduced electron transfer coupled to fluorescence correlation spectroscopy (PET-FCS) has indicated the presence of an intermediate state, I, in equilibrium with the unfolded state, U. The cooperative binding of the ligand (L), SDS, to I has been shown to induce the formation of a compact, helical intermediate (IL₅) within the dead time (∼37 µs) of a continuous flow mixer. Quantitative analysis of the PET-FCS data and the ensemble microsecond kinetic data, suggests that the mechanism of induction of helical structure can be described by a U ↔ I ↔ IL₅ ↔ FL₅ mechanism, in which the final helical state, FL₅, forms from IL₅ with a time constant of 50–200 µs. Finally, it has been shown that the helical conformation is an aggregation-competent state that can directly form amyloid fibrils.     

Induced defenses of loblolly pine, Pinus taeda: potential impact on Dendroctonus frontalis within-tree mortality

Loblolly pine (Pinus taeda L.) produces an induced defensive hypersensitive response in inner bark colonized by the southern pine beetle, Dendroctonus frontalis Zimm. (Coleoptera: Scolytidae), and its associated fungi. Adult beetles forced to colonize the induced response tissue in a laboratory study constructed galleries that were the same length as adults boring in normal phloem. However, each female laid fewer eggs in the induced tissue. Larval and pupal mortality were also higher in this tissue when compared to surrounding phloem. Beetles colonizing trees in response to pheromone baits constructed less gallery and laid fewer eggs in induced tissue than in surrounding normal phloem. These results suggest that the lesions produced by the induced defense system in conifers may not only contain the growth of fungi inoculated into trees during the attack phase of beetle colonization, but may also affect survival of bark beetle progeny.

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Résumé Une réaction hypersensible de défense a été induite dans les couches profondes de l'écorce de P. taeda colonisée par D. frontalis et le champignon qui lui est associé. Au laboratoire, des adultes, contraints de coloniser les tissus où une réaction hypersensible e été induite, ont foré des galeries de même longueur que celles creusées dans du phloème sain. Cependant les femelles ont pondu moins d'oeufs dans les tissus induits; les mortalités larvaires et nymphales étaient aussi plus fortes dans ce tissu que dans le phloème voisin. Les scolytes, ayant colonisé les arbres après attraction par des pièges à phéromones, forent moins de galeries et pondent moins d'oeufs dans le tissu induit que dans le phloème sain voisin. Ces résultats suggèrent que les lésions, provoquées par le système de défense induit des conifères, peuvent non seulement limiter la croissance du champignon inoculé dans l'arbre au cours de la colonisation des scolytes, mais aussi affecter la survie des descendants dans l'écorce.

     

Development of membrane-potential responsiveness by myeloid leukemia cells during neutrophilic differentiation

The ability of a myeloid leukemia cell line (HL-60) to undergo membrane electrical potential changes was followed during neutrophilic differentiation induced by 2 compounds. Membrane-potential changes were induced with 12-O-tetradecanoylphorbol 13-acetate (TPA) or formyl-methionyl-leucyl-phenylalanine (FMLP) and were monitored by flow cytometry. The magnitude of the membrane-potential response to TPA increased in a more uniform manner as the population of cells matured than did acquisition of mature morphology or ability to undergo the respiratory burst in response to TPA. The response to TPA and FMLP of HL-60 cells, maximally induced to differentiate by dimethylsulfoxide, closely resembled that of neutrophils. Thus, HL-60 cells may be a useful tool in the study of the relation between membrane depolarization and subsequent cellular activation.     

The use of clip cages to restrain insects reduces leaf expansion systemically in Rumex obtusifolius

Abstract. 1. Clip cages have been used widely by experimental ecologists to contain insects on plants.
2. Under controlled conditions, the effect of applying clip cages alone and clip cages and the chrysomelid beetle Gastrophysa viridula on systemic leaf expansion of Rumex obtusifolius was investigated. Treatments were applied to the fully expanded fourth leaf and expansion of leaf 8 was measured over a period of 22 days.
3. The application of clip cages reduced the rate at which leaf area increased and led to reductions in final leaf areas.
4. Clip cages have systemic effects on plant development and these effects are sustained even after the clip cage is removed. Investigators should take this into account in designing experiments.     

Insect-induced chemical changes in young lodgepole pine (Pinus contorta): the effect of previous defoliation on oviposition, growth and survival of the pine beauty moth, Panolis flammea

ABSTRACT. 1. Significantly more eggs were laid by adult female pine beauty moths ( Panolis flammea (D&S)) on previously undefoliated lodgepole pine ( Pinus contorta Douglas) saplings than on those which had been partially defoliated the year before.
2. The larval weights, mean relative growth rates and survival rates of P.flammea were lower on defoliated trees than on undefoliated trees.
3. Marked and significant differences between the monoterpene profiles of the shoot cortical oleoresin of defoliated and undefoliated trees were linked to oviposition preferences in P.flammea.
4. Differences occurred between the levels of soluble tannins in previously defoliated and undefoliated trees.
5. The results are discussed in relation to earlier work on insect-induced changes in plants, and the significance of inter-provenance differences in lodgepole pine response is assessed.     

Purification and partial characterisation of two abscisic-acid-responsive proteins induced in cultured embryos ofPisum sativum L.

When pea (Pisum sativum L.) embryos were cultured on low osmotica, with or without added abscisic acid (ABA), there was very little change in the total mRNA translation products resolved by one-dimensional sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The only marked alteration was an increase in production of two low-molecular-weight proteins. The purification and partial characterisation of these two ABA-responsive seed proteins (ABR17 and ABR18) is described. Both proteins were purified to homoeneity, as judged by SDS-PAGE, from embryos cultured in the presence of ABA. Antisera were raised against both proteins. Each serum cross-reacted with the other protein, indicating that the proteins are closely related. Their apparent molecular masses (M_rs) were estimated to be 17200 (ABR17) and 18100 (ABR18) by SDS-PAGE, and 26000 by gel filtration. Both proteins were heterogeneous on isoelectric focusing. Neither protein was detected (by immunoblotting or immunoprecipitation of cell-free translation products) in embryos grown in vivo at early to mid-development stages but both were present in embryos late in development. These proteins appear to be produced late in seed development but are capable of being induced early in development by culturing embryos in vitro and are markedly enhanced by ABA.     

Protein stability and electrostatic interactions between solvent exposed charged side chains

To investigate the contribution to protein stability of electrostatic interactions between charged surface residues, we have studied the effect of substituting three negatively charged solvent exposed residues with their side-chain amide analogs in bovine calbindin D9k--a small (Mr 8,500) globular protein of the calmodulin superfamily. The free energy of urea-induced unfolding for the wild-type and seven mutant proteins has been measured. The mutant proteins have increased stability towards unfolding relative to the wild-type. The experimental results correlate reasonably well with theoretically calculated relative free energies of unfolding and show that electrostatic interactions between charges on the surface of a protein can have significant effects on protein stability.     

Recombinant human granulocyte-colony stimulating factor and recombinant human macrophage-colony stimulating factor synergize in vivo to enhance proliferation of granulocyte-macrophage, erythroid, and multipotential progenitor cells in mice

Combinations of low dosages of purified recombinant human (rh) macrophage-colony stimulating factor (M-CSF; also termed CSF-1) and rh granulocyte-colony stimulating factor (G-CSF) were compared alone and in combination for their influence on the cycling rates and numbers of bone marrow and splenic granulocyte-macrophage, erythroid, and multipotential progenitor cells in vivo in mice pretreated with iron-saturated human lactoferrin (LF). LF was used to enhance detection of the stimulating effects of exogenously added CSFs. Concentrations of each CSF that were not active in vivo when given alone were active when given together, with the other CSF. The concentrations of rhM-CSF and rhG-CSF needed to increase progenitor cell cycling in the marrow and spleen were reduced by factors of 40-200 when these CSFs were administered in combination with low dosages of the other CSF. At the concentrations of rhM-CSF and rhG-CSF tested, synergism was not noted on absolute numbers of progenitor cells or total nucleated cell counts per organ or circulating in the blood. These findings may have potential relevance when considered in a clinical setting where the CSFs might be used in combination with other biotherapy and/or chemotherapy.