Rapid reactions of spruce cells to elicitors released from the ectomycorrhizal fungus Hebeloma crustuliniforme, and inactivation of these elicitors by extracellular spruce cell enzymes

Elicitors released from hyphae or cell walls of the ectomycorrhizal fungus Hebeloma crustuliniforme (Bull. ex Fries.) Quél. induced in suspension-cultured cells of Picea abies (L.) Karst. a set of fast reactions: (i) an immediate efflux of Cl^– into the medium, followed by a K⁺ efflux; (ii) an influx of Ca²⁺ (measured as accumulation of ⁴⁵Ca²⁺ in the cells); (iii) a phosphorylation of a 63-kDa protein and dephosphorylation of a 65-kDa protein (detectable by 4 min after elicitor application); (iv) an alkalinization of the medium, and (v) a transient synthesis of H₂O₂. The removal of extracellular Ca²⁺ by EGTA delayed the elicitor-induced alkalinization. A further reduction of this response could be achieved by TMB-8 an inhibitor of Ca²⁺ release from intracellular stores. Moreover, the inhibition of protein kinase activity by staurosporine prevented the extracellular alkalinization completely. However, the effectiveness of the elicitors in inducing the extracellular alkalinization was strongly impaired by constitutively secreted enzymes of spruce cells which cleaved the elicitors to inactive fragments. It is suggested that in ectomycorrhizae the efficacy of elicitors released from fungal cell walls is controlled by apoplastic enzymes of the host; the plant itself is able to reduce the activity of fungal elicitors on their way through the plant cell wall. But those elicitors which finally reach the plasma membrane of host cells induce reactions that are similar to the early defense reactions in plant-pathogen interactions.Abbreviations DW dry weight - FW fresh weight - TMB-8 3,4,5 trimethoxybenzoic acid 8-(diethylamino)-octyl ester We thank Prof. M. Zenk (Universität München, Germany) for providing spruce cell cultures, and Dr. I. Kottke (Universität Tübingen, Germany) for isolates of Hebeloma crustuliniforme Tü 704. We are also thankful to Dr. W. Mayer (Universität Tübingen) for valuble discussions. This work was supported by Deutsche Forschungsgemeinschaft. B. Zitterell-Haid was financed by Graduiertenkolleg Interaktion in Waldökosystemen (supported by Deutsche Forschungsgemeinschaft) and G. Hebe by a scholarship of the Landesgraduiertenförderungsgesetz.     

Behavioral strategies of aphid hyperparasitoids to escape aggression by honeydew-collecting ants

We analyzed the behavioral interactions between two species of honeydew-collecting ants (Lasius niger, Myrmica laevinodis) and foraging females of four species of aphid hyperparasitoids (Aphidencyrtus aphidivorus, Dendrocerus carpenteri, Pachyneuron aphidis, Asaphes vulgaris) usingAphis fabae ssp.cirsiiacanthoidis andLysiphlebus cardui on thistles as aphid and primary parasitoid, respectively. The observed interaction patterns and foraging parameters varied within hyperparasitoid species and revealed different strategies based upon behavioral and morphological constraints.D. carpenteri generally tried to avoid ant encounters. This avoidance strategy was successful in interactions withM. laevinodis but failed when encountering the more aggressiveL. niger, which caused about 26% adult mortality. In contrast,A. aphidivorus, P. aphidis, andA. vulgaris possess jumping ability and were hardly exposed to mortality risks. The escape reaction jump off was used as soon as ants made physical contact with foraging females. While the flight strategy ofP. aphidis is connected with cryptic movement patterns without avoidance behavior,A. aphidivorus first avoided ants and jumped off only as a last resort. Similar patterns, but less expressive, are displayed byA. vulgaris. We suggest that these different strategies are responsible for different foraging success in ant-attended resources in field.     

AdaptivSearch: A Novel Iterative Algorithm for the Generation of n-dimensional Hypersurfaces Using an Adaptive Numerical Strategy

AdaptivSearch is the first adaptive strategy based algorithm for the rational and economical construction of n-dimensional hypersurfaces. AdaptivSearch works iteratively: At each step it parcels out the definition range into several triangles, evaluates the worst according to a built-in error criterion, and refines the approximation to the unknown function by choosing the barycenter of this partial area as the node to be calculated next. Based upon the error criterion, AdaptivSearch selectively approaches those parts of the hypersurface in which the curvature exhibits the strongest changes. Some examples of AdaptivSearch applications for both analytical functions and chemical model surfaces are given in order to demonstrate the behavior of the algorithm. These show its broad applicability and the usefulness, especially for chemical problems.     

Succinate-ethanol fermentation in Clostridium kluyveri: purification and characterisation of 4-hydroxybutyryl-CoA dehydratase/vinylacetyl-CoA Δ3-Δ2-isomerase

Anaerobically prepared cell extracts of Clostridium kluyveri grown on succinate plus ethanol contained high amounts of 4-hydroxybutyryl-CoA dehydratase, which catalyzes the reversible dehydration of 4-hydroxybutyryl-CoA to crotonyl-CoA. The enzyme was purified 12-fold under strictly anaerobic conditions to over 95% homogeneity and had a specific activity of 123 nkat mg^-1. The finding of this dehydratase means that all of the enzymes necessary for fermentation of succinate plus ethanol by C. kluyveri have now been demonstrated to exist in this organism and confirms the proposed pathway involving a reduction of succinate via 4-hydroxybutyrate to butyrate. Interestingly, the enzyme is almost identical to the previously isolated 4-hydroxybutyryl-CoA dehydratase from Clostridium aminobutyricum. The dehydratase was revealed as being a homotetramer (m=59 kDa/subunit), containing 2±0.2 mol FAD, 13.6±0.8 mol Fe and 10.8±1.2 mol inorganic sulfur. The enzyme was irreversibly inactivated after exposure to air. Reduction by sodium dithionite also yielded an inactive enzyme which could be reactivated, however, up to 84% by oxidation with potassium hexacyanoferrate(III). The enzyme possesses an intrinsic vinylacetyl-CoA isomerase activity which was also found in 4-hydroxybutyryl-CoA dehydratase from C. aminobutyricum. Moreover, the N-terminal sequences of the dehydratases from both organisms were found to be 63% identical.     

The mycotoxin ochratoxin A deranges pH homeostasis in Madin-Darby canine kidney cells

Ochratoxin A (OTA) is a nephrotoxin which blocks plasma membrane anion conductance in Madin-Darby canine kidney (MDCK) cells. Added to the culture medium, OTA transforms MDCK cells in a manner similar to exposure to alkaline stress. By means of video-imaging and microelectrode techniques, we investigated whether OTA (1 mol/liter) affects intracellular pH (pH.), Cl^– (Cl _i ^– ) or cell volume of MDCK cells acutely exposed to normal (pH_norm=7.4) and alkaline (pH_alk=7.7) conditions. At pH_norm, OTA increased Cl _i ^– by 2.6±0.4 mmol/liter (n=14, P<0.05) but had no effect on pH _i . At pH_alk, application of OTA increased Cl _i ^– by 8.6±2.6 mmol/liter (n=10, P< 0.05) and raised pH _i by 0.11±0.03 (n= 8, P<0.05). The Cl^–HCO ₃ ^– exchange inhibitor DNDS (4,4-dinitro-stilbene-2, 2-disulfonate; 10 mol/liter) eliminated the OTA-induced changes of pH _i and Cl _i ^– . OTA did not affect cell volume under both pH_norm and pH_alk conditions.We conclude that the OTA-induced blockade of plasma membrane anion conductance increases Cl _i ^– without changing cell volume. The driving force of plasma membrane Cl^–/HCO ₃ ^– exchange dissipates, leading to a rise of pH _i when cells are exposed to an acute alkaline load. Thus, OTA interferes with pH _i and Cl _i ^– homeostasis leading to morphological and functional alterations in MDCK cells.The work was supported by the Deutsche Forschungsgemeinschaft (DFG, Si 170/7-1).We thank the Zeiss Company (Oberkochen, Germany) for providing the Attofluor video-imaging system for the intracellular Ca²⁺ measurements.This study was carried out with the technical assistance of Sigrid Mildenberger and Ruth Freudinger.     

Early helper T-cell dysfunction in simian immunodeficiency virus but not in human immunodeficiency virus type-2-infected macaques

Both naive and vaccinated macaques acquired a virus-specific proliferative helper T-cell reactivity in response to infection with the nonpathogenic human immunodeficiency virus type 2 (HIV-2). In contrast, macaques infected with the pathogenic simian immunodeficiency virus of the macaque strain (SIV_mac) did not develop a helper T-cell response. Furthermore, a vaccine-induced preexisting T-cell reactivity was abrogated after SIV_mac infection in vaccine failures. These differences may reflect the different pathogenicity of the two closely related viruses.     

BrdU incorporation reveals DNA replication in non dividing glial cells in the larval abdominal CNS ofDrosophila

Glial cells are of significant importance for central nervous system development and function. In insects, knowledge of the types and development of CNS glia is rather low. This is especially true for postembryonic glial development. Using bromodeoxyuridine incorporation and enhancer trap lines we identified a reproducible spatial and temporal pattern of DNA replicating cells in the abdominal larval CNS (A3-7 neuromeres) ofDrosophila melanogaster. These cells correspond to embryonically established glial cells in that region. Except for a specific subfraction, these cells apparently do not divide during larval life. Similar patterns were found in two otherDrosophila species,D. virilis andD. hydei.