Acetylcholine (ACh)-induced currents (A), the effects of blockers (mecamylamine and fipronil) on the ACh- (B), γ-aminobutyric acid (GABA) (C)- and L-glutamate (D)-induced currents and the actions of asperparaline A on the resting-state (E) and neurotransmitter-evoked currents (F–H) in the silkworm (<i>Bombyx mori</i>) larval neurons.

<p>The holding potential was −60 mV. ACh (10 µM), L-glutamate (30 µM) and GABA (30 µM) was applied for 2 s using the U-tube, whereas mecamylamine and fipronil were bath-applied for 1 min prior to co-application with the agonists. In (E), asperparaline A was applied alone at 1 µM for 2 s using the U-tube, whereas in (F–H), it was bath-applied for 1 min prior to co-application with neurotransmitters ACh (F), GABA (G) and L-glutamate (H). Note that both peak and slowly desensitizing current amplitudes of the ACh-evoked response were blocked reversibly, selectively and almost completely by 1 µM asperparaline A (F).</p

Chemical structure of asperparaline A.

<p>Chemical structure of asperparaline A.</p

Effects of pre-application on the antagonist action of asperparaline A.

<p>(A) Asperparaline A was co-applied at 30 nM with 10 µM ACh for 2 s without pre-application, or applied for 1, 2 and 5 min prior to co-application with 10 µM ACh. (B) The antagonist action of asperparaline A with and without pre-application for 1, 2 and 5 min. Each bar graph represents the mean ± standard error of the mean (n = 4) of the peak current amplitude of the ACh-induced response normalized by that taken before the application of asperparaline A. The pre-application of asperparaline A significantly enhanced the antagonist action (<i>p</i><0.05, One-way ANOVA, Tukey's test), but there were no significant differences in the blocking action between 1, 2, and 5 min pre-applications.</p

The effects of repeated application of ACh on the blocking action of asperparaline A.

<p>After recording the control response to ACh at 10 µM, asperparaline A was continuously bath-applied at 30 nM, during which ACh was also applied at 10 µM for 2 s every minute using the U-tube. (A) Traces of the ACh-induced current responses in the presence of 30 nM asperparaline A. (B) Normalized peak current amplitude of the ACh responses recorded during the continuous application of asperparaline A. The peak current amplitude of each response was normalized by that of the response recorded before the application of asperparaline A. Each plot represents the mean ± standard error of the mean of 4 separate experiments.</p

Effects of asperparaline A on the concentration-response curve for ACh in the silkworm larval neurons.

<p>The ACh-induced responses were measured at various concentrations in the presence and absence of 100 nM asperparaline A. The concentration-response curves were obtained by fitting the data to Eq. (2) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018354#s2" target="_blank">Materials and Methods</a>). The pEC₅₀ ( = log(1/EC₅₀)) values determined in the presence and absence of asperparaline A were 4.98±0.10 (n = 4, EC₅₀ = 10.5 µM) and 4.94±0.04 (n = 7, EC₅₀ = 11.4 µM), respectively. No significant shift in EC₅₀ was observed by the application of asperparaline A.</p

Concentration-inhibition curves for asperparaline A in terms of attenuation of the responses to ACh of the silkworm larval neurons.

<p>(A) The ACh-induced responses recorded before and after bath-application of asperparaline A for 1 min prior to co-application with 10 µM ACh. The peak and slowly desensitizing currents are indicated by “a” and “b”, respectively. (B) Concentration-inhibition curves for asperparaline A. Data were normalized to the maximum response to ACh (10 µM). Each plot represents the mean ± the standard error of the mean of 4 experiments. The concentration-inhibition curves were obtained by fitting the data to Eq. (1) (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0018354#s2" target="_blank">Materials and Methods</a>). The pIC₅₀ ( = log(1/IC₅₀) values for the peak and slowly desensitizing currents were 7.69±0.02 (n = 4, IC₅₀ = 20.2 nM) and 7.40±0.04 (n = 4, IC₅₀ = 39.6 nM), respectively. These two values are significantly different (<i>p</i><0.05, <i>t</i>-test).</p

Effects of asperparaline A on the ACh-induced responses of chicken α3β4 (A), α4β2 (B) and α7 (C) nAChRs expressed in <i>Xenopus laevis</i> oocytes.

<p>After three successive control applications of ACh, 10 µM asperparaline A was continuously bath-applied and then co-applied with 100 µM ACh. Asperparaline A blocked the ACh-response of α3β4 nAChR by 33.4±3.3% (n = 3), whereas it scarcely influenced the response of α4β2 (n = 4) and α7 (n = 3) nAChRs.</p

Signaling the Induction of Sporulation Involves the Interaction of Two Secondary Metabolites in <i>Aspergillus nidulans</i>

When growing <i>Aspergillus nidulans</i> hyphae encounter the atmosphere, they initiate a morphogenetic program leading to the production of spores. Mutants that are defective in the <i>fluG</i> gene fail to undergo sporulation because they lack an endogenous diffusible factor that purportedly accumulates on aerial hyphae, thus signaling the initiation of development. In this study, the defect could be reversed by adding culture extracts from a wild-type strain onto a mutant colony. Moreover, a bioassay-guided purification of the active culture extract resulted in the identification of the active agent as dehydroaustinol. However, this meroterpenoid was active only when administered in conjunction with the orsellinic acid derivative diorcinol. These two compounds formed an adduct that was detected by HRMS in an LC–MS experiment. The diorcinol-dehydroaustinol adduct prevented crystal formation of the signal on the surface of aerial hyphae and on an artificially prepared aqueous film and also increased the signal lipophilicity

Effects of chrodrimanin B on the GABA-induced responses of human α1β2γ2 GABAR expressed in <i>Xenopus</i> oocytes.

<p>(A) 1 μM chrodrimanin B was continuously bath-applied for 5 min and then co-applied with 30 μM GABA for 2 s after three successive control applications of GABA. Chrodrimanin B tested at 1 μM blocked the peak current amplitude of the response to GABA of the human GABAR by 38.9 ± 3.9% (n = 3). (B) Chrodrimanin B was bath-applied for 5 min and then co-applied with 30 μM GABA at different concentrations (30 nM—30 μM). Each plot represents the mean ± standard error of the mean of three experiments. The IC₅₀ value of chrodrimanin B was 1.48 μM.</p

Effects of chrodrimanin B on the concentration-response curve for GABA on silkworm RDL GABAR expressed in <i>Xenopus</i> oocytes.

<p>The GABA-induced responses were measured at various concentrations in the presence and absence of chrodrimanin B. The EC₅₀ value for GABA shifted from 41.1 μM (n = 4, CI = 37.3–45.2 μM) without chrodrimanin B to 143 μM (n = 4, 121–170 μM) with 3 nM chrodrimanin B and to 565 μM (n = 4, 478–668 μM) with 10 nM chrodrimanin B, respectively.</p