14-3-3 Protein homologues play a central role in the fusicoccin signal transduction pathway

The plasma membrane located fusicoccin binding protein (FCBP) is an essential element in the fusicoccin (FC) signal transduction pathway. We obtained primary sequence information for the 31 kD subunit of the FCBP. These sequences showed that the FCBP is homologous to members of the 14-3-3 protein family. Both the 31 and 30 kD subunits cross-react with 14-3-3 antibodies. In native form the FCBP occurs as a dimer, but it is also part of a complex with higher molecular mass. The monomeric forms of the FCBP (the 30 and 31 kD subunits) do not have ³H-FC binding activity. We discuss how the FCBP, as a member of the 14-3-3 protein family, may be able to bind FC and how the FC-signal is transduced to the effector protein, the H⁺-ATPase.     

Production of δ-(l-α-aminoadipyl)-l-cysteinyl-d-valine by entrapped ACV-synthetase fromStreptomyces clavuligerus

Summary -(l--Aminoadipyl)-l-cysteinyl-d-valine (ACV)-synthetase fromStreptomyces clavuligerus was studied under conditions that enabled the reuse of the enzyme. Coupling of ACV-synthetase to DEAE-Trisacryl and aminopropyl-glass resulted in an immobilized enzyme product of little or no catalytic activity. However, an enzyme reactor was designed by physical confinement of partially-purified ACV-synthetase in an ultrafiltration cell. This system was stimulated by phosphoenolpyruvate at lower concentrations of ATP, an effect not observed with purified enzyme. Up to 30% conversion of the limiting substrate, cysteine, to ACV occurred under semi-continuous conditions. Reaction products were investigated as potential inhibitors: AMP was the most inhibitory, but only when used at concentrations in excess of those produced in reaction mixtures. Under a nitrogen atmosphere, both product and enzyme stabilities were greatly improved and the enzyme retained 45–46% of its initial activity after five uses at room temperature during a 24-h period. Extrapolations based on these data suggest that 1.3 g partially purified enzyme (0.13 U g^–1) would be capable of producing 411 mg of ACV in a 1-L reaction mixture in this period.     

Responses to recent climatewarming of Pinus sylvestris and Pinus cembra within their montane transition zone in the Swiss Alps

Abstract. Altitudinal and latitudinal distribution limits of trees are mainly controlled by temperature. Therefore climate warming is expected to induce upslope or poleward migrations. In the Swiss Central Alps, summers in the period 1982-1991 were on average 0.8 °C warmer than those of the period 30 yr before. We investigated whether populations of conifers at the montane Pinus sylvestris-Pinus cembra ecocline exhibit demographic trends in response to that warming. We found no evidence for this. Young seedlings of Pinus sylvestris, the species which is expected to expand its range upward in a warmer climate, were virtually absent from all sites, whereas large fractions of Pinus cembra populations were observed in the seedling and juvenile categories even below the present lower distribution limit of adult trees. This suggests that there are no major altitudinal shifts in response to the recent sequence of warmer summers. Germination and seedling survival trials with Pinus sylvestris suggest that temperature per se would not exclude this species even from establishing at the current treeline in the Swiss Central Alps. Similar results were found at the polar treeline. Phytotron tests of seedling survival showed much less drought resistance in Pinus sylvestris than in Pinus cembra which is in contrast to their phytogeographic distributions. Thus, the montane pine ecocline in the Swiss Central Alps seems to be stabilized by species interactions and may not be directly responsive to moderate climatic change, which needs to be taken into account in predictive attempts.     

Impact of grazing on vegetation dynamics in former ricefields

Abstract. In the Rhône delta, Juncus gerardi and Scirpus maritimus are often the dominant species in abandoned rice fields which are artificially flooded in early spring to improve forage production. Under these conditions they occur either in mixed communities, or form monospecific stands. Monitoring the vegetation dynamics in quadrats located in six abandoned rice fields artificially flooded from November to April confirmed the important role of grazing. In ungrazed plots, communities dominated by Scirpus maritimus mixed with Juncus gerardi developed fast. After 42 months of management Scirpus maritimus had established in nearly all quadrats and continued to expand, whereas Juncus gerardi had started to decline. In grazed plots Juncus gerardi alone dominated and continued to increase in cover up to the 42nd month. Scirpus maritimus established at low densities mainly in quadrats where Juncus gerardi was initially absent. Introduction of seeds of Scirpus maritimus in communities of Juncus gerardi under controlled conditions demonstrated the existence of the phenomenon of preemption. The increase in cover of Juncus gerardi suggests that the preemption of Juncus gerardi over Scirpus maritimus plays a more pronounced role in the field in the presence of grazing.     

Isolation and identification of (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid from anAmanita of the sectionRoanokenses (Amanitaceae)

A chlorine-containing non-protein amino acid which was recently discovered from the fruit bodies ofAmanita gymnopus (2S)-2-amino-5-chloro-4-hydroxy-5-hexenoic acid, was isolated and crystallized for the first time from the fruit bodies of an unknown member ofAmanita belonging to the sectionRoanokenses, subsectionSolitariae. The results of elementary analyses, determination of optical rotations,¹H- and¹³C-NMR-spectra, and some chemical reactions supported an earlier proposed structure.Part 24 in the series Biochemical studies of nitrogen compounds in fungi. for Part 23, see Hatanaka, S. I. et al. 1994. this journal35: 391–394.     

Chemically modified cardiac Na+ channels and their sensitivity to antiarrhythmics: Is there a hidden drug receptor?

Elementary Na⁺ currents were recorded at 19°C in inside-out patches from cultured neonatal rat cardiocytes. In analyzing the sensitivity of chemically modified Na⁺ channels to several class 1 antiarrhythmic drugs, the hypothesis was tested that removal of Na⁺ inactivation may be accompanied by a distinct responsiveness to these drugs, open channel blockade.Iodate-modified and trypsin-modified cardiac Na⁺ channels are noninactivating but strikingly differ from each other by their open state kinetics, a O₁–O₂ reaction (_open(1) 1.4±0.3 msec; _open(2) 5.4±1.1 msec; at –40 mV) in the former and a single open state (_{open 3.0±0.5} msec; at –40 mV) in the latter. Lidocaine (150 mol/liter) like propafenone (10 mol/liter), diprafenone (10 mol/liter) and quinidine (20 mol/liter) in cytoplasmic concentrations effective to depress NP _o significantly can interact with both types of noninactivating Na⁺ channels to reduce the dwell time in the conducting configuration. lodate-modified Na⁺ channels became drug sensitive during the O₂ state. At –40 mV, for example, lidocaine reduced _open(2) to 62±5% of the control without detectable changes in _open(1). No evidence could be obtained that these inhibitory molecules would flicker-block the open Na⁺ pore. Drug-induced shortening of the open state, thus, is indicative for a distinct mode of drug action, namely interference with the gating process. Lidocaine proved less effective to reduce _open(2) when compared with the action of diprafenone. Both drugs apparently interacted with individual association rate constants, a_lidocaine was 0.64×10⁶ mol^–1 sec^–1 and a_diprafenone 13.6×10⁶ mol^–1 sec^–1. Trypsin-modified Na⁺ channels also appear capable of discriminating among these antiarrhythmics, the ratio a_diprafenone/a_lidocaine even exceeded the value in iodate-modified Na⁺ channels. Obviously, this antiarrhythmic drug interaction with chemically modified Na⁺ channels is receptor mediated: drug occupation of such a hypothetical hidden receptor that is not available in normal Na⁺ channels may facilitate the exit from the open state.This work was supported by a grant of the Deutsche Forschungsgemeinschaft (Ko 778/2-4), Bonn.     

A Novel Photoaffinity Ligand for Kainate Sites Labels Two Polypeptides with Molecular Mass 45 and 33.5 kDa in Chick Cerebellum

Abstract: The synthesis of (2 S ,3 S ,4 S )-4-[1-(4-azidobenzamidomethyl)ethenyl]-2-carboxy-3-pyrrolidineacetic acid (ABCPA) is described. This novel kainic acid analogue, bearing a photolabile functionality on the isopropenyl side chain, was proven to be a good inhibitor of [³H]CNQX and [³H]kainic acid binding on chick cerebellar membranes. [³H]ABCPA was photoaffinity cross-linked on the membrane fraction of chick cerebellum. Electrophoretic analysis with sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed two major radioactive bands with apparent molecular masses of 45 and 33.5 kDa. [³H]ABCPA incorporation in both bands was completely blocked by 2 m M CNQX. When photoaffinity labeling was performed in the presence of 2 m M kainic acid, incorporation of [³H]ABCPA was blocked by ∼70% in the 45-kDa band and by 18% in the 33.5-kDa band. Incorporation of radioactivity in both bands was blocked by ∼30% with 10 m M glutamate.     

The Subunit Composition of a GABAA/Benzodiazepine Receptor from Rat Cerebellum

Abstract: The pentameric subunit composition of a large population (36%) of the cerebellar granule cell GABA_A receptors that show diazepam (or clonazepam)-insensitive [³H]Ro 15-4513 binding has been determined by immunoprecipitation with subunit-specific antibodies. These receptors have α₆, α₁, γ_2S, γ_2L, and β₂ or β₃ subunits colocalizing in the same receptor complex.     

Cloning and characterization of Tag 1, a tobacco anther β-1,3-glucanase expressed during tetrad dissolution

A critical stage in pollen development is the dissolution of the four products of meiosis, the tetrads, into free microspores. The tetrads are surrounded by a thick callose wall composed of -1,3-glucan. At the completion of meiosis, the tetrads are released into the anther locule after hydrolysis of the callose by a -1,3-glucanase. Using the polymerase chain reaction, we have amplified and subsequently cloned a cDNA corresponding to a -1,3-glucanase, tobacco (Nicotiana tabacum cv. Samsun) anther glucanase (Tag 1), which is expressed exclusively in anthers from meiosis to the free microspore stage of pollen development. The identity of the clone was determined by DNA and deduced protein sequence similarity to other known -1,3-glucanases. Several regions strictly conserved among four classes of glucanases are also conserved in the Tag 1 protein. Tag 1 represents a novel class of -1,3-glucanase based on phylogenetic analysis and RNA expression pattern. Tag 1 RNA was detected in situ only in the tapetum, with maximal expression just prior to tetrad dissolution. Due to its expression pattern and sequence similarity to other -1,3-glucanases, we believe Tag 1 may be involved in tetrad dissolution.