Cyclopiazonic acid

Objective:The spontaneous action potential of isolated sinoatrial node (SAN) cells is regulated by a coupled-clock system of two clocks: the calcium clock and membrane clock. However, it remains unclear whether calcium clock inhibitors have a direct effect on the membrane clock. The purpose of this study was to investigate the direct effect of cyclopiazonic acid (CPA), a selective calcium clock inhibitor, on the function of the membrane clock of SAN cells.Methods:at SAN cells were isolated by trypsinization and identified based on morphology and electrophysiology. Ifand HCN currents were recorded via patch clamp technique. The expression of the HCN channel protein was determined by Western blotting analysis.Results:The diastolic depolarization rate of spontaneous action potentials and the current densities of Ifwere reduced by exposure to 10 μM CPA. The inhibitory effect of CPA was concentration-dependent with an IC50value of 16.3 μM and a Hill coefficient of 0.98. The effect of CPA on Ifcurrent was also time-dependent, and the Ifcurrent amplitude was partially restored after washout. Furthermore, the steady-state activation curve of the Ifcurrent was shifted to a negative potential, indicating that channel activation slowed down. Finally, the protein expression of HCN4 in HEK293 cells was markedly downregulated by CPA.Conclusions:These results indicate that the direct inhibition effect of CPA on the Ifcurrent in SAN cells is both concentration- and time-dependent. The underlying mechanisms may involve slowing down steady-state activation and the downregulation of pacemaker channel protein expression.

Cyclopiazonic acid (α-CPA) is a tremorgenic mycotoxin that is commonly produced by certain species of the aspergilli, in particular Aspergillus flavus, which is more widely known for production of the aflatoxins. Despite the fact that α-CPA may co-occur with aflatoxins, immunoassay-based methods for monitoring for CPA have not been widely developed. We report the development and evaluation of several monoclonal antibodies (mAbs) for α-CPA. Two mAbs in particular were very sensitive, with IC50s of 1.1 and 1 ng/mL (clones 1418 and 1231, respectively). Tolerances to aqueous methanol or acetonitrile were good, which permitted the development of an antigen-immobilized competitive enzyme-linked immunosorbent assay (CI-ELISA) for detection of CPA in maize. Spiked or naturally contaminated maize, extracted with aqueous methanol, was diluted with buffer for analysis. The working range for the assay (IC20to IC80) was from 5 to 28 μg/kg. Recoveries from maize spiked over the range from 2 to 50 μg/kg averaged 88.6 ± 12.6%. Twenty-eight samples of maize were tested by both the CI-ELISA and a liquid chromatography-fluorescence (LC-FLD) method. For the five samples above the limits of quantitation of both methods, CI-ELISA tended to overestimate CPA content, a difference that we speculate may be due to related metabolites or perhaps "masked" derivatives of CPA. The antibodies developed and the resulting CI-ELISA will be useful tools for further evaluation of the prevalence of this mycotoxin in maize.

Eight new cyclopiazonic acid (1-8) and five new okaramine (9-13) alkaloids together with 13 known compounds were isolated from the fungusChrysosporium undulatumYT-1. Compounds2,4,5,7,10,11, and13were chlorinated indole alkaloids. The structures of compounds1-13were elucidated by HRESIMS and NMR spectroscopic data. Their relative and absolute configurations were established byJ-based configuration analysis, NOESY, NOEDIFF experiments, ECD spectroscopic data, and biogenetic considerations. Compound4inhibited the growth ofBacillus subtiliswith an MIC value of 6.3 μg/mL. Compounds9-11exhibited strong insecticidal capacity against the third instar larvae of silkworm and cotton bollworm (LD50: ≤7.56 μg/g). At 40 μM, compound1showed obvious neuroprotection to the PC12 cells with 6-OHDA treatment.