Growth inhibition and changes in morphology and actin distribution in Acetabularia acetabulum by phalloidin and phalloidin derivatives

Effects on morphology and microfilament structure caused by phalloidin, phallacidin, and some semisynthetic phalloidin derivatives were studied in vegetative cells of the green alga Acetabularia acetabulum (L.) Silva. All phalloidin derivatives (except for phalloidin itself) caused growth stop of the alga after 1 day and (except for the fluorescein-labeled phalloidin) death of the cells after 4-7 days. Hair whorl tip growth and morphology as screened by light microscopy, as well as microfilament structure in tips, suggested that growth stop is correlated with a disorganization of actin filaments similar to that recently described for jasplakinolide (H. Sawitzky, S. Liebe, J. Willingale-Theune, D. Menzel, European Journal of Cell Biology 78: 424-433, 1999). Using rabbit muscle actin as a model target protein, we found that the toxic effects in vivo did not correlate with actin affinity values, suggesting that permeation through membranes must play a role. Indeed, the most lipophilic phalloidin derivatives benzoylphalloidin and dithiolanophalloidin were the most active in causing growth stop at ca. 100 ?M. In comparison to the concentration of jasplakinolide required to cause similar effects (<3 ?M), the two most active phalloidin derivatives exhibited an activity ca. 30 times lower. Nonetheless, lipophilic phalloidin derivatives can be used in algae, and probably also other cells, to modulate actin dynamics in vivo. In addition, we found that the fluorescent fluorescein isothiocyanate-phalloidin is able to enter living algal cells and stains actin structures brightly. Since it does not suppress actin dynamics, we suggest fluorescein isothiocyanate-phalloidin as a tool for studying rearrangements of actin structures in live cells, e.g., by confocal laser scanning microscopy

Identification of structural features involved in binding of .alpha.−amanitin to a monoclonal antibody K. Baumann, K. Muenter, and H. Faulstich

Twenty−four derivatives of the cyclic octapeptide a−amanitin were assayed for their affinities to the monoclonal antibody @AI/ 1. The derivatives were of natural, semisynthetic, and synthetic origin and had &values ranging from 2 nM to > 7 0 ˜ M . In the majority of the derivatives the chemical modifications had no detectable influence on the overall shape of the double−ring peptide. Given this condition, binding factors could be calculated from KD values of the amatoxin derivatives, which were valid for all amatoxins for estimating the contribution made by single structures to complex formation. The complex between a−amanitin and the immunoglobulin involves at least eight sites of contact. Four of them are responsible for strong interactions: (1) the OH group of hydroxyproline2 (binding factor 413), (2) the lipophilic side chain of isoleucine6 (binding factor 131), (3) the −CHI− moiety of the adjacent glycine5 or the absence of a side chain in this position (binding factor 36 l), and (4) the proton at the indole nitrogen of hydr˜xytryptophan˜ (binding factor 140). The residual four interactions are hydrogen bonds of lower strength corresponding to binding factors of 1.5−8. The key role of the unique conformation of the amatoxins in determining their binding properties was shown by two amatoxin derivatives in which changes in the conformation were associated with virtually complete loss of affinity. For all amatoxin derivatives with conformations similar or identical to that of a−amanitin, we found empirical evidence that those structures of the peptide involved in binding make their contributions virtually independent of each other. It is a consequence of this rule that structural features that cooperate in binding could be characterized by the numerical product of their binding factor

D-Configuration of serine is crucial in maintaining the phalloidin-like conformation of viroisin

NMR studies have revealed that the conformation of the monocyclic viroisin is dissimilar to that of the corresponding monocyclic derivative of phalloidin, dethiophalloidin, but has much similarity with the conformation of the bicyclic phalloidin. Obviously, one of three structural features found exclusively in the virotoxins is able to compensate for the conformational strain that in the bicyclic phallotoxins maintains the toxic conformation. Synthetic work on virotoxin analogues has shown that both the additional hydroxy group in allo-hydroxyproline and the methylsulfonyl moiety in the 2‘-position of tryptophan are unlikely to represent the structural element in question, leaving the d-serine moiety as the supposed key element. In this study we asked whether it is the hydroxy group of this amino acid or its d-configuration that is responsible for the effect. We synthesized four viroisin analogues and submitted them to conformational analysis by NMR as well as to an actin binding assay. While the rotating-frame nuclear Overhauser effect (ROESY) spectra of the analogues with l-configured amino acids showed several sets of signals, indicating the existence of conformers interconverting more slowly than the NMR time scale, the spectra of the analogues with d-configured amino acids showed only one set of signals. Remarkably, the two viroisin analogues with d-serine and d-alanine also had distinctly higher affinities for filamentous actin than their l-configured counterparts, suggesting that the high biological activity may be correlated with the absence of multiple and slowly interconverting conformers. Anyhow, d-configuration of serine is the structural element that maintains the phalloidin-like structure, while the hydroxy group does not contribute to conformational stability but is likely to be in contact with the actin surfac

Where Do Our Resources Go? Indium, Neodymium, and Gold Flows Connected to the Use of Electronic Equipment in Switzerland

The increased use of digital information and communications technologies (ICT) is giving rise to fast-growing waste streams that contain important material resources. In contrast to bulk materials and precious metals, the recovery of most critical metals has not yet been commercially established, and they are thus lost within the recycling process. In this article, we used dynamic material flow analysis to explore the stocks and flows of indium, neodymium, and gold incorporated in end-user devices in Switzerland. Our analysis covered the use, collection, recycling, and disposal phases. This enabled us to track the three metals from their entry into Switzerland as components of new devices until their recovery, disposal in landfills, or dissipation to the environment. Using statistical entropy analysis (SEA), we further analyzed the dilution or concentration of the metals during their route through the current system. The data uncertainty was addressed employing a probabilistic approach. The largest quantities of all three metals are found in the devices currently in use. The second-largest stocks are slags disposed in landfills for indium, slags used for construction for neodymium, and the output of metal recovery processes for gold. The SEA illustrates how the current collection and recycling system successfully concentrates all three metals. While 70% of gold leaving the use phase is recovered, indium and neodymium are dissipated to slags after smelting and incineration processes due to the lack of economic incentives and lacking recovery processes on a commercial scale

Use of bimanyl actin derivative (TMB-actin) for studying complexation of β-thymosins Inhibition of actin polymerization by thymosin β9

AbstractBy reacting trimethylammoniobromobimane bromide (TMB bromide) with rabbit muscle actin, a fluorescent reporter group was linked to cysteine at position 374. Fluorescence of TMB-actin decreased significantly on addition of thymosin β4 (Tβ4), a peptide of 43 amino acid residues reported to bind to monomeric actin and to prevent filament formation. Based on this effect, we determined the Kd value of the thymosin β4 complex as 0.8 μM, a value that is in agreement with previous determinations. In addition to the main compound thymosin β4, bovine tissue contains a related peptide, thymosin β9 (Tβ9), which has 41 amino acid residues and ca. 75% sequence homology. In the present study we show for the first time that Tβ9, similar to Tβ4, forms a 1:1 complex with monomeric actin, and hereby inhibits actin polymerization. With a Kd value of 1.1 μM the affinity of Tβ9 is in the same range as that of Tβ4, suggesting that Tβ9, like Tβ4, contributes to maintaining the pool of monomeric actin in bovine non-muscle cells. Further proof of the interaction of Tβ9 with actin was provided by native PAGE, where the complex showed the reported higher mobility, as well as by crosslinking experiments. Using different crosslinking reagents, like water-soluble carbodiimide (EDC), m-maleimidobenzoyl-N-hydroxysuccinimidate (MBS), and disuccinimidylsuberate (DSS), we were able to produce conjugates of 47 kDa. In one of these (from MBS) both actin and Tβ9 could be identified by immunoblotting. When, in the MBS crosslinking experiments, native actin was replaced with (374-NEM)actin, the 47 kDa band was not seen, indicating that Cys-374 takes part in the thiol-specific crosslinking reaction. This suggests that part of the binding site of Tβ9 must be located close to the carboxy-tenninus