Membrane effects of 2,4-dichlorophenoxyacetic acid in motor cells of Mimosa pudica L.

2,4-dichlorophenoxyacetic acid applied to excised leaves of Mimosa pudica L. inhibited in a dose-dependent manner the shock-induced pulvinar movement. This inhibition was negatively correlated with the amount of [(14)C] 2,4-dichlorophenoxyacetic acid present in the vicinity of the motor cells. Although 2,4-dichlorophenoxyacetic acid is a weak acid, its greatest physiological efficiency was obtained with pH values close to neutrality. This observation opens the question of its mode of action which may be through external signaling or following internal transport by a specific anionic form transporter. The effect was related to molecular structure since 2,4-dichlorophenoxyacetic acid>3,4-dichlorophenoxyacetic acid>2,3-dichlorophenoxyacetic acid. An essential target of 2,4-dichlorophenoxyacetic acid action lies at the plasmalemma as indicated by the induced hyperpolarization of the cell membrane. Compared to indole-3-acetic acid and fusicoccin, it induced a complex effect on H(+) fluxes. Applied to plasma membrane vesicles purified from motor organs, 2,4-dichlorophenoxyacetic acid enhanced proton pumping, but, unlike fusicoccin, it did not increase the H(+)-ATPase catalytic activity in our experimental conditions. Taken together, the data suggest that 2,4-dichlorophenoxyacetic acid acts on cell turgor variation and the concomittant ion migration, in particular K(+), by a mechanism involving specific steps compared to indole-3-acetic acid and fusicoccin.     

Membrane Targeting of the Spir·Formin Actin Nucleator Complex Requires a Sequential Handshake of Polar Interactions

Spir and formin (FMN)-type actin nucleators initiate actin polymerization at vesicular membranes necessary for long range vesicular transport processes. Here we studied in detail the membrane binding properties and protein/protein interactions that govern the assembly of the membrane-associated Spir·FMN complex. Using biomimetic membrane models we show that binding of the C-terminal Spir-2 FYVE-type zinc finger involves both the presence of negatively charged lipids and hydrophobic contributions from the turret loop that intrudes the lipid bilayer. In solution, we uncovered a yet unknown intramolecular interaction between the Spir-2 FYVE-type domain and the N-terminal kinase non-catalytic C-lobe domain (KIND) that could not be detected in the membrane-bound state. Interestingly, we found that the intramolecular Spir-2 FYVE/KIND and the trans-regulatory Fmn-2-FSI/Spir-2-KIND interactions are competitive. We therefore characterized co-expressed Spir-2 and Fmn-2 fluorescent protein fusions in living cells by fluorescence cross-correlation spectroscopy. The data corroborate a model according to which Spir-2 exists in two different states, a cytosolic monomeric conformation and a membrane-bound state in which the KIND domain is released and accessible for subsequent Fmn-2 recruitment. This sequence of interactions mechanistically couples membrane binding of Spir to the recruitment of FMN, a pivotal step for initiating actin nucleation at vesicular membranes.     

RNAi revised - target mRNA-dependent enhancement of gene silencing

The discovery of RNA interference (RNAi) gave rise to the development of new nucleic acid-based technologies as powerful investigational tools and potential therapeutics. Mechanistic key details of RNAi in humans need to be deciphered yet, before such approaches take root in biomedicine and molecular therapy.We developed and validated an in silico-based model of siRNA-mediated RNAi in human cells in order to link in vitro-derived pre-steady state kinetic data with a quantitative and time-resolved understanding of RNAi on the cellular level. The observation that product release by Argonaute 2 is accelerated in the presence of an excess of target RNA in vitro inspired us to suggest an associative mechanism for the RNA slicer reaction where incoming target mRNAs actively promote dissociation of cleaved mRNA fragments. This novel associative model is compatible with high multiple turnover rates of RNAi-based gene silencing in living cells and accounts for target mRNA concentration-dependent enhancement of the RNAi machinery.     

The importance of dietary control in the development of a peanut allergy model in Brown Norway rats

This report describes the further development of a peanut allergy model in Brown Norway (BN) rats and in particular the importance of allergen-free breeding of the laboratory animals for the allergen to be used. For this purpose BN rats were bred for 3 generations on soy- and peanut-free feed since it is known that the legumes peanut and soy are cross-reactive. In addition, the effect of cholera toxin (CT), an oral adjuvant often used to increase the sensitivity of food allergy models, was investigated in the BN rat model. BN rats that were bred on both soy- and peanut-free feed could be sensitized orally to peanut (all exposed rats developed peanut-specific IgE, IgG2a and IgG1) and the adjuvant CT could only enhance this sensitization to a limited extent. We also found different protein recognition patterns against purified peanut allergens (Ara h1, Ara h2 and Ara h3) between intraperitoneally (i.p.) and orally sensitized BN rats. Orally sensitized rats recognized all tested allergens whereas i.p. sensitized rats only recognized Ara h1 and Ara h2. Our conclusion is that a model for food allergy should preferably be (A) oral and (B) if possible without the use of adjuvantia. Our model in BN rats unites these preferred characteristics. In addition, we show the importance of dietary control when conducting oral sensitization studies. Special attention must be paid to unscheduled dietary pre-exposure of the animals to the protein under investigation to obtain optimal oral sensitization.     

Fecal androgen metabolite analysis for noninvasive monitoring of testicular steroidogenic activity in felids

Limited data are available on long-term, seasonal changes in testicular steroidogenic activity in nondomestic felids, primarily because of the difficulties associated with longitudinal blood sampling (e.g., handling, restraint, anesthesia). Therefore, a noninvasive approach for assessing testicular androgen production was developed using the domestic cat (Felis catus) as a model. Two adult males were injected i.m. with 4 μCi¹⁴-testosterone to determine the time course and relative proportions of androgen metabolites excreted in urine and feces. Peak urinary radioactivity was detected 13 and 19 hr postinjection and accounted for ∼8% of the total radioactivity recovered. High performance liquid chromatography (HPLC) analysis detected multiple polar urinary metabolites, none of which eluted with the ³H-testosterone reference tracer. The majority of urinary testosterone metabolites consisted of nonenzyme-hydrolyzable, water-soluble (presumably conjugated) forms. In feces, radioactivity was detected in the first sample collected at 22 hr postinjection for both males, although peak metabolite excretion in one male was not observed until 61 hr postinjection. HPLC analysis detected several fecal metabolites consisting primarily of nonhydrolyzable, water-soluble forms (84.4 ± 0.9%) with some ether-soluble forms (15.6 ± 0.9%). None of the fecal androgen metabolites were associated with free testosterone. However, one or more of the water-soluble fecal metabolites was quantifiable using a commercially available testosterone radioimmunoassay. The biological relevance of this immunoactivity was confirmed in the domestic cat; concentrations were high in adult, intact males and nondetectable in intact females and castrated males and females. In addition, fecal androgen concentrations in a male Pallas' cat (Felis manul) exhibited seasonal fluctuations that corresponded with parallel changes in serum testosterone and ejaculate quality. These data indicate that testicular steroidogenic activity can be monitored non invasively in felids, providing a potentially valuable tool for endangered felid management to: (1) assess pubertal status, (2) determine the influence of season on reproduction, and (3) diagnose possible causes of sub- or infertility. © 1996 Wiley-Liss, Inc.