Exploring molecular tools for transformation and gene expression in the cultivated edible mushroom Agrocybe aegerita.

Agrocybe aegerita is a cultivated edible mushroom in numerous countries, which also serves as a model basidiomycete to study fruiting body formation. Aiming to create an easily expandable customised molecular toolset for transformation and constitutive gene of interest expression, we first created a homologous dominant marker for transformant selection. Progeny monokaryons of the genome-sequenced dikaryon A. aegerita AAE-3 used here were identified as sensitive to the systemic fungicide carboxin. We cloned the wild-type gene encoding the iron-sulphur protein subunit of succinate dehydrogenase AaeSdi1 including its up- and downstream regions, and introduced a single-point mutation (His237 to Leu) to make it confer carboxin resistance. PEG-mediated transformation of protoplasts derived from either oidia or vegetative monokaryotic mycelium with the resulting carboxin resistance marker (CbxR) plasmid pSDI1E3 yielded carboxin-resistant transformants in both cases. Plasmid DNA linearised within the selection marker resulted in transformants with ectopic multiple insertions of plasmid DNA in a head-to-tail repeat-like fashion. When circular plasmid was used, ectopic single integration into the fungal genome was favoured, but also gene conversion at the homologous locus was seen in 1 out of 11 analysed transformants. Employing CbxR as selection marker, two versions of a reporter gene construct were assembled via Golden Gate cloning which allows easy recombination of its modules. These consisted of an eGFP expression cassette controlled by the native promoter PAaeGPDII and the heterologous terminator Tnos, once with and once without an intron in front of the eGFP start codon. After protoplast transformation with either construct as circular plasmid DNA, GFP fluorescence was detected with either transformants, indicating that expression of eGFP is intron-independent in A. aegerita. This paves the way for functional genetics approaches to A. aegerita, e.g., via constitutive expression of fruiting-related genes.

Design, synthesis, and antifungal activity of novel cinnamon-pyrazole carboxamide derivatives.

Hit, Lead & Candidate Discovery To discover succinate dehydrogenase inhibitors with a novel structure, we introduced cinnamic acid structure to optimize the lead structure 1 and synthesized four series of cinnamon-pyrazole carboxamide derivatives. The bioassay data showed that compounds (E)-N-(1-[4-chlorophenyl]-4-cyano-1H-pyrazol-5-yl)-3-(2-fluorophenyl) acrylamide (5III-d) and (E)-3-(2-chlorophenyl)-N-(1-[4-chlorophenyl]-4-cyano-1H-pyrazol-5-yl) acrylamide (5III-f) showed the significant antifungal activity against three fungi. In addition, 5III-d and 5III-f exhibited the excellent inhibitory effect against succinate dehydrogenase (SDH) enzymes with IC50 values ranging from 19.4 to 28.7 µM. The study demonstrates that the chlorine substituent group is present on both the phenyl and pyrazole rings that have a very good effect on the antifungal effect, and the compounds 5III-d and 5III-f can act as potential SDH inhibitors (SDHI) and throw a sprat for a new generation of SDHI.

[FORMATION AND FUNCTIONING OF SYMBIOTIC SYSTEMS AND RHIZOSPHERE MICROBIOCENISIS OF SOYBEAN UNDER VARIOUS FUNGICIDES APPLICATION].

The Relevance. At the recent years in soybean crops the quantity of plant pathogenic fungi has increased. The fungicides of systemic and contact action have been applicated intensively against of them. After introducing into the soil fungicides and/or their deg- radation products can to disrupt the activities of non-target objects - beneficial soil mi- croorganisms, inhibit nodulation process and the nitrogen-fixing activity of diazotrophs. The purpose of the work was to investigate the impact of combined application of fungi- cides with inoculation on the soybean symbiotic system and rhizosphere microorganisms. The Methods. The microbiological and statistical methods, gas chromatography method. The Results. Inoculation of seeds by the highly active Bradyrhizobiumjaponicum UCM B-6035, UCM B-6018 and UCM B-6023 strains the activity of nitrogen-fixing symbiotic systems increased by 1.4-3.4 times in comparison with the variant without of fungicides application and bacterization. Seed treatment by Vitavaks 200 FF fungicide caused a de- crease of'nitrogen-fixing activity of rhizobia industrial strains in symbiosis with soybean by 3-5 times. The seeds inoculation by B. japonicum UCM B-6035 strain promoted to reduce the negative impact of the Maxim Star 025 FS fungicide on the nitrogenase activity of nodulation apparatus. The positive effect of seeds bacterization was observed in the in- crease of the quantity of rhizosphere microorganisms of main ecological trophic groups. In the variant with seed treatment by Maxim Star 025 FS and Kinto duo fungicides was found a decrease in the number of microorganisms of studied groups in comparison with the control variant; the Vitavaks 200 FF fungicide application promoted to improve of these microorganisms development compared with the variant without the fungicides application and bacterization. At the inoculation of rhizobia industrial strains the negative effect of the Maxim Star 025 FS and Kinto duo fungicides to oligoazotrophic and prototrophic micro- organisms was not observed. The Conclusion. The symbiotic system of variant with the combined application of the Kinto duo fungicide with B. japonicum UCM B-6023 strain characterized by the highest nodulation and nitrogen-fixing activity.

Cloning and characterization of the gene for the iron-sulfur subunit of succinate dehydrogenase from the violet root rot fungus, Helicobasidium mompa.

The sdhB gene, encoding the iron-sulfur protein (Ip) subunit of succinate dehydrogenase (Sdh, EC 1.3.99.1), has been cloned from the violet root rot fungus, Helicobasidium mompa, and characterized. The promoter region contains a CCAAT box, TATA-like box, and CT-rich region. The gene is interrupted by eight introns and is predicted to encode a polypeptide of 291 amino acid residues. The putative amino acid sequence of the encoded product of sdhB gene from H. mompa shows high homology to the other known sdhB genes and is 79% identical to the Ip subunit of SdhB of Uromyces fabae. Three cysteine-rich clusters associated with the iron-sulfur centers involved in electron transport were particularly well conserved. One of these clusters contains a critical histidine residue implicated in carboxin sensitivity in the basidiomycetes. Only one copy of the gene was present in the genome of H. mompa, and reverse transcription (RT)-PCR analysis of mRNA expression showed that the sdhB gene was transcribed in potato dextrose broth.

Molecular characterization of boscalid resistance in field isolates of Botrytis cinerea from apple.

Botrytis cinerea isolates obtained from apple orchards were screened for resistance to boscalid. Boscalid-resistant (BosR) isolates were classified into four phenotypes based on the levels of the concentration that inhibited fungal growth by 50% relative to control. Of the 220 isolates tested, 42 were resistant to boscalid, with resistant phenotypes ranging from low to very high resistance. There was cross resistance between boscalid and carboxin. Analysis of partial sequences of the iron-sulfur subunit of succinate dehydrogenase gene in B. cinerea (BcSdhB) from 13 BosR and 9 boscalid-sensitive (BosS) isolates showed that point mutations in BcSdhB leading to amino acid substitutions at the codon position 272 from histidine to either tyrosine (H272Y) or arginine (H272R) were correlated with boscalid resistance. Allele-specific polymerase chain reaction (PCR) analysis of 66 BosR isolates (including 24 additional isolates obtained from decayed apple fruit) showed that 19 carried the point mutation H272Y and 46 had the point mutation H272R, but 1 BosR isolate gave no amplification product. Analysis of the BcSdhB sequence of this isolate revealed a different point mutation at codon 225, resulting in a substitution of proline (P) by phenylalanine (F) (P225F). The results indicated that H272R/Y in BcSdhB were the dominant genotypes of mutants in field BosR isolates from apple. A multiplex allele-specific PCR assay was developed to detect point mutations H272R/Y in a single PCR amplification. Levels of boscalid resistance ranged from low to very high within isolates carrying either the H272R or H272Y mutation, indicating that, among BosR isolates, different BosR phenotypes (levels of resistance) were not associated with particular types of point mutations (H272R versus H272Y) in BcSdhB. Analysis of genetic relationships between 39 BosR and 56 BosS isolates based on three microsatellite markers showed that 39 BosR isolates and 30 BosS isolates were clustered into two groups, and the third group consisted of only BosS isolates, suggesting that the development of resistance to boscalid in B. cinerea likely is not totally random, and resistant populations may come from specific genetic groups.

Agrobacterium-mediated transformation of arthroconidia obtained from the edible mushroom Hypsizygus marmoreus.

Using the carboxin resistance gene from Pleurotus eryngii as a selective marker, we introduced foreign DNA into the arthroconidia of Hypsizygus marmoreus through Agrobacterium-mediated transformation. The function of the exogenous GUS (ß-glucuronidase) gene driven by the CaMV35S promoter was detected in the transformants.

Investigating dominant selection markers for Coprinopsis cinerea: a carboxin resistance system and re-evaluation of hygromycin and phleomycin resistance vectors.

Dominant selectable markers are beneficial for transformation of many fungi, particularly those model species where repeated transformations may be required. A carboxin resistance allele of the Coprinopsis cinerea sdi1 gene, encoding the iron-sulphur protein subunit of succinate dehydrogenase, was developed by introducing a suitable point mutation in the histidine block responsible for binding of the associated iron ion. This modified gene was used successfully to confer carboxin resistance upon transformation of C. cinerea protoplasts. Plasmids previously used to establish hygromycin transformation systems of several basidiomycete species, such as pAN7-1 and phph004, failed to give rise to hygromycin-resistant transformants of C. cinerea, whilst pPHT1 was successful. Sequencing of these constructs showed that the hygromycin resistance gene in pAN7-1 and phph004 had been modified removing the codons encoding two lysine residues following the N-terminal methionine. Replacement of the deleted 6 bp (AAA AAG) in the truncated hph gene led to generation of hygromycin-resistant transformants indicating the importance of these two codons for expression in C. cinerea. Phleomycin-resistant (ble) transformants were also obtained, but only with the intron-containing construct pblei004, showing that an intron is necessary to obtain phleomycin-resistant C. cinerea. This contrasts with hygromycin-resistance, where introns are not required for expression, emphasising the variability in importance of these elements.

Transformation of an oleaginous zygomycete Mortierella alpina 1S-4 with the carboxin resistance gene conferred by mutation of the iron-sulfur subunit of succinate dehydrogenase.

The sdhB gene encoding an iron-sulfur (Ip) subunit of succinate dehydrogenase (SDH, EC 1.3.99.1) complex was cloned from Mortierella alpina 1S-4. The deduced amino acid sequence of SdhB from M. alpina 1S-4 showed high similarity to those of SdhB from other organisms. The mutated sdhB (CBXB) gene encodes a modified SdhB with an amino-acid substitution (a highly conserved histidine residue within the third cysteine-rich cluster of SdhB replaced by a leucine residue) and is known to confer carboxin resistance. We succeeded in transforming M. alpina 1S-4 by using the CBXB gene as a selectable marker gene and expressing the heterologous uidA gene encoding beta-glucuronidase of Escherichia coli. Moreover, transformation efficiency was up to 40-50 transformants per 4.0 x 10(8) spores. This carboxin-transformation system, characterized by marginal background growth and mitotic stability in M. alpina 1S-4, is considered to be widely useful for the wild strain, M. alpina 1S-4, and various derivative mutants without laborious preparation of auxotrophic mutants as a host strain.

Identification of three mutant loci conferring carboxin-resistance and development of a novel transformation system in Aspergillus oryzae.

Mutants exhibiting resistance to the fungicide, carboxin, were isolated from Aspergillus oryzae, and the mutations in the three gene loci, which encode succinate dehydrogenase (SDH) B, C, and D subunits, were identified to be independently responsible for the resistance. A structural model of the SDH revealed the different mechanisms that confer carboxin-resistance in different mutations. The mutant AosdhB gene (AosdhB(cxr)) was further examined for possible use as a transformant selection marker. After transformation with AosdhB(cxr), carboxin-resistant colonies appeared within 4 days of culture, and all of the examined colonies carried the transgene. Insertion analyses revealed that the AosdhB(cxr) gene was integrated into AosdhB locus via homologous recombination at high efficiency. Furthermore, AosdhB(cxr) functioned as a successful selection marker in a transformation experiment in Aspergillus parasiticus, suggesting that this transformation system can be used for Aspergillus species.

Efficient transformation of Pleurotus eryngii with a safe selective marker mutated from the Pesdi1 gene.

We introduced a site-directed mutation in the sdi1 gene and used it as a selective marker for the polyethylene glycol-mediated transformation of Pleurotus eryngii monokaryon protoplasts. The transformants displayed obvious and stable resistance to the fungicide carboxin indicating that the mutant Pesdi1 gene is an efficient selective marker.

Folate Deficiency Triggered Apoptosis of Synoviocytes: Role of Overproduction of Reactive Oxygen Species Generated via NADPH Oxidase/Mitochondrial Complex II and Calcium Perturbation.

Despite a plethora of literature has documented that osteoarthritis (OA) is veritably associated with oxidative stress-mediated chondrocyte death and matrix degradation, yet the possible involvement of synoviocyte abnormality as causative factor of OA has not been thoroughly investigated. For this reason, we conduct the current studies to insight into how synoviocytes could respond to an episode of folate-deprived (FD) condition. First, when HIG-82 synoviocytes were cultivated under FD condition, a time-dependent growth impediment was observed and the demise of these cells was demonstrated to be apoptotic in nature mediated through FD-evoked overproduction of reactive oxygen species (ROS) and drastically released of cytosolic calcium (Ca2+) concentrations. Next, we uncovered that FD-evoked ROS overproduction could only be strongly suppressed by either mitochondrial complex II inhibitors (TTFA and carboxin) or NADPH oxidase (NOX) inhibitors (AEBSF and apocynin), but not by mitochondrial complex I inhibitor (rotenone) and mitochondrial complex III inhibitor (antimycin A). Interestingly, this selective inhibition of FD-evoked ROS by mitochondrial complex II and NOX inhibitors was found to correlate excellently with the suppression of cytosolic Ca2+ release and reduced the magnitude of the apoptotic TUNEL-positive cells. Taken together, we present the first evidence here that FD-triggered ROS overproduction in synoviocytes is originated from mitochondrial complex II and NOX. Both elevated ROS in tandem with cytosolic Ca2+ overload serve as final arbitrators for apoptotic lethality of synoviocytes cultivated under FD condition. Thus, folate supplementation may be beneficial to patients with OA.

The kinetics of the active and de-energized transport of O-methyl glucose in Ustilago maydis.

The kinetics of the uptake and efflux of 3-O-methyl-glucose in sporidia of Ustilago maydis were measured, both in active cells and in cells whose metabolic activity had been inhibited by azide and iodoacetate. The de-energized transport system proved to be carrier mediated with apparent affinity constants 13 +/- 2 mM outside (Ko) and 18 +/- 2 mM inside (K1). The apparent maximum rate constants for the same system were 0.66 +/- 0.05 mmol/1 cell water per min for uptake (V+) and 0.53 +/- 0.04 mmol/l cell water per min for efflux (V-). For the active system K0 = 0.08 +/- 0.01, K1 greater than 40, V+ = 9.7 +/- 0.5 and V- = 1.1 +/- 0.9 (in equivalent units). These results are discussed in the context of the carrier mechanism as proposed by Regen and Morgan (Regen, D.M. and Morgan, H.E. (1964) Biochim. Biophys. Acta 79, 151--166). The antifungal compound carboxin had no effect on de-energized transport but was shown to decrease both K0 And V+ in the active system. Phloretin and phlorizin were also found to be without effect on de-energized cells but the former enhanced while the latter inhibited active uptake.

Studies on the succinate dehydrogenating system. Interaction of the mitochondrial succinate-ubiquinone reductase with pyridoxal phosphate.

The inhibitory effect of pyridoxal phosphate on the Triton X-100 solubilized purified bovine heart succinate-ubiquinone reductase (Choudhry, Z.M., Gavrikova, E.V., Kotlyar, A.B., Tushurashvilli, P.R. and Vinogradov, A.D. (1985) FEBS Lett. 182, 171-175) was studied. The kinetics of the enzyme inactivation by pyridoxal phosphate was found to be strongly dependent both qualitatively and quantitatively on the concentration of the protein-detergent complexes. In the diluted system the inactivation of the ubiquinone-depleted enzyme was completely prevented by the saturating concentrations of Q2, carboxin, thenoiltrifluoroacetone and pentachlorophenol, i.e., by the substrate and specific inhibitors of the enzyme. The protective effects of Q2 and the inhibitors was employed to quantitate the affinities of the ligands to their specific binding sites. Strong difference in the affinity of Q2 to the reduced and oxidized enzyme was found. When the soluble reconstitutively active succinate dehydrogenase was treated with pyridoxal phosphate, the reactivity of the enzyme towards low ferricyanide concentrations and its reconstitutive activity was significantly protected against aerobic inactivation.

Succinate:quinone oxidoreductase in the bacteria Paracoccus denitrificans and Bacillus subtilis.

An overview of the present knowledge about succinate:quinone oxidoreductase in Paracoccus denitrificans and Bacillus subtilis is presented. P. denitrificans contains a monoheme succinate:ubiquinone oxidoreductase that is similar to that of mammalian mitochondria with respect to composition and sensitivity to carboxin. Results obtained with carboxin-resistant P. denitrificans mutants provide information about quinone-binding sites on the enzyme and the molecular basis for the resistance. B. subtilis contains a diheme succinate:menaquinone oxidoreductase whose activity is dependent on the electrochemical gradient across the cytoplasmic membrane. Data from studies of mutant variants of the B. subtilis enzyme combined with available crystal structures of a similar enzyme, Wolinella succinogenes fumarate reductase, substantiate a proposed explanation for the mechanism of coupling between quinone reductase activity and transmembrane potential.

An efficient genetic manipulation protocol for Ustilago esculenta.

Ustilago esculenta grows within the flowering stem of the aquatic grass Zizania latifolia, resembling a fungal endophyte. The fungus colonizes Z. latifolia and induces swelling which results in the formation of galls near the base of the plant. Due to their unique flavor and textures these galls are considered as a delicacy in southern China. Efficient genetic manipulation is required to determine the relationship between U. esculenta and Z. latifolia. In this study, we report a protoplast-based transformation system for this unique fungal species. We have explored various factors (enzyme digesting conditions, osmotic pressure stabilizers, vectors and selection agents) that might impact protoplast yield and high frequencies of transformation. A haploid strain (UeT55) of U. esculenta was found to produce higher yields of protoplasts when treating with 15 mg mL(-1) lywallzyme in a sucrose-containing solution at 30°C for 3 h. The transformation frequencies were higher when fungal strain was transformed with a linear plasmid harboring hygromycin or carboxin resistance gene and regenerated on a sucrose-containing medium. A UeICL gene (coding isocitrate lyase) was disrupted and an EGFP (coding enhanced green fluorescent protein) gene was overexpressed successfully in the UeT55 strain using the developed conditions. The genetic manipulation system reported in this study will open up new opportunities for forward and reverse genetics in U. esculenta.

Integrated options for the management of black root rot of strawberry caused by Rhizoctonia solani Kuhn.

An investigation was made to manage strawberry black root rot caused by Rhizoctonia solani (R. solani) through the integration of Trichoderma harzianum (T. harzianum) isolate STA7, mustard oil cake and Provax 200. A series of preliminary experiments were conducted to select a virulent isolate of R. solani, an effective isolate of T. harzianum, a suitable organic amendment, and a suitable fungicide before setting the experiment for integration. The pathogenicity of the selected four isolates of R. solani was evaluated against strawberry and isolate SR1 was selected as the test pathogen due to its highest virulent (95.47% mortality) characteristics. Among the 20 isolates of T. harzianum, isolate STA7 showed maximum inhibition (71.97%) against the test pathogen (R. solani). Among the fungicides, Provax-200 was found to be more effective at lowest concentration (100 ppm) and highly compatible with Trichoderma isolates STA7. In the case of organic amendments, maximum inhibition (59.66%) of R. solani was obtained through mustard oil cake at the highest concentration (3%), which was significantly superior to other amendments. Minimum percentages of diseased roots were obtained with pathogen (R. solani)+Trichoderma+mustard oil cake+Provax-200 treatment, while the highest was observed with healthy seedlings with a pathogen-inoculated soil. In the case of leaf and fruit rot diseases, significantly lowest infected leaves as well as fruit rot were observed with a pathogen+Trichoderma+mustard oil cake+Provax-200 treatment in comparison with the control. A similar trend of high effectiveness was observed by the integration of Trichoderma, fungicide and organic amendments in controlling root rot and fruit diseases of strawberry. Single application of Trichoderma isolate STA7, Provax 200 or mustard oil cake did not show satisfactory performance in terms of disease-free plants, but when they were applied in combination, the number of healthy plants increased significantly. The result of the current study suggests the superiority of our integrated approach to control the sclerotia forming pathogen R. solani compared to the individual treatment either by an antagonist or by a fungicide or by mustard oil cake.

Inhibition of mitochondrial oxidative phosphorylation induces hyper-expression of glutamic acid decarboxylase in pancreatic islet cells.

It has been hypothesised that mitochondrial dysfunction in pancreatic beta cells could produce hyper-expression of glutamic acid decarboxylase (GAD), a major autoantigen in insulin-dependent diabetes mellitus (IDDM) (Degli Esposti, M. and Mackay, I.R. Diabetologia 40: 352-356, 1997). Here we report that specific inhibition of mitochondrial respiration enhances the expression of GAD in both foetal mouse pancreatic tissue and hamster HIT-T15 cells. Inhibitors of NADH-ubiquinone oxidoreductase (complex I) seem to be particularly effective in increasing the expression of GAD in both foetal mouse pancreas and HIT-T15 hamster beta cells, especially in the presence of nutrients such as arginine and glucose. These results represent the first evidence that GAD expression is enhanced under conditions that are toxic to pancreatic beta cells, and establish a link between mitochondrial dysfunction and expression of IDDM autoantigens.

The role of genotypic heterogeneity in wild type virus populations on the selection of nonnucleoside reverse transcriptase inhibitor-resistant viruses.

Virus populations were selected in cell culture using two widely used protocols in order to evaluate the role of selection methodology on the genotype and phenotype of nonnucleoside reverse transcriptase inhibitor resistant viruses. Selection was performed by serial passage of virus in the presence of gradually increasing concentrations of antiviral compound or passage in the presence of a constant high concentration of compound. Using the CEM-SS cell line, the IIIB strain of HIV-1, and identical nonnucleoside reverse transcriptase inhibitors, resistant viruses were obtained and their phenotypic and genotypic properties were defined. Resistant virus populations containing the Y181C amino acid change in the reverse transcriptase were predominantly selected with each of the tested compounds. Several of the compounds selected secondary amino acid changes using both methods. A comparison of the resistant viruses selected in our laboratory using each of the two protocols with viruses reported by a second laboratory employing one of the two methods suggests that genotypic differences in the selected virus isolates may most likely result from the variation in the genetic composition of the respective wild type virus pools, rather than the specific selection methodology employed. These results imply that HIV may select a wide variety of amino acid changes to avoid the inhibitory effects of the nonnucleoside reverse transcriptase inhibitors and the selection of compounds for clinical use in combination with agents possessing non-overlapping resistance phenotypes will require evaluation of the agents against virus isolates possessing each of the mutations known to confer drug resistance.

Activity of various thiocarboxanilide derivatives against wild-type and several mutant human immunodeficiency virus type 1 strains.

A large variety of carboxanilide derivates in which the original oxathiin moiety present in the prototype compound UC84 was replaced by a non-cyclic lipophilic entity has been evaluated for their inhibitory effect against wild-type human immunodeficiency virus type 1 (HIV-1/IIIB) and several mutant viruses derived thereof (i.e. HIV-1/138-Lys, HIV-1/181-Cys, HIV-1/106-Ala and HIV-1/100-IIe). Isopropoxy was the most favorable substituent resulting in molecules that were markedly inhibitory to the wild-type (EC50 0.004-0.04 microgram/ml) as well as the mutant HIV-1 strains (EC50 0.06-0.75 microgram/ml). In this respect, they proved superior to several other HIV-1-specific non-nucleoside reverse transcriptase inhibitors (NNRTIs) that are currently the subject of clinical trials. One of the most potent HIV-1 inhibitors among the thiocarboxanilide derivatives, namely UC38, selected for a mutant virus strain in which Lys at position 101 and Gly at position 190 of the reverse transcriptase was replaced by Glu.

Stimulation by surangin B of endogenous amino acid release from synaptosomes.

The effect of surangin B, an insecticidal natural product coumarin, on presynaptic release of endogenous amino acids was investigated using a purified synaptosomal fraction isolated from mouse brain. Surangin B stimulated the release of glutamic acid (GLU), gamma-aminobutyric acid (GABA), serine, alanine and the aminosulfonic acid taurine from synaptosomes at micromolar concentrations. In all cases, these responses were reduced by removing calcium from the saline and surangin B-evoked release of GLU, GABA, aspartic acid (ASP) and alanine was significantly inhibited by the sodium channel blocker tetrodotoxin. Rotenone (a complex I inhibitor) and carbonyl cyanide chlorophenylhydrazone (CCCP; an uncoupler), were more potent releasers of amino acids from synaptosomes than surangin B, however, carboxin (a complex II-selective inhibitor), was extremely weak to ineffective in this regard. The stimulatory effect of surangin B and complex III-selective inhibitors on release of GLU, GABA, ASP and alanine by synaptosomes was significantly reduced by N,N,N',N'-tetramethyl-p-phenylenediamine, suggesting that blockade of complex III in intraterminal mitochondria is an important effect of this coumarin. Our results demonstrate that surangin B, in common with CCCP and inhibitors of complex I and III, cause release of both neurotransmitter and non-neurotransmitter amino acids from nerve endings in vitro. However, in contrast to most classical agents which interfere selectively with mitochondrial function, the release of endogenous amino acids from synaptosomes by surangin B also involves a moderate extracellular calcium ion-dependent component and relies partially on sodium ion entry into the nerve ending.