Mode of action of piperovatine, an insecticidal piperamide isolated from Piper piscatorum (Piperaceae), against voltage-gated sodium channels.

Piperamides, which are secondary metabolites in the genus Piper, have potent insecticidal activity, and have thus inspired the development of novel insecticides. In this study, piperovatine, a piperamide from Piper piscatorum (Piperaceae), was investigated using a two-electrode voltage clamp to clarify its detailed mode of action against voltage-gated sodium channels, a classic target. In Xenopus oocytes expressing voltage-gated sodium channels from German cockroach (Blattella germanica), piperovatine induced inward currents depending on repetitive openings. For instance, maximal currents were generated with 10 µM piperovatine following 100 trains of depolarizing pulses with frequency 25 Hz. Piperovatine also shifted the half-activation voltage after conditioning pulses from -35 mV to -45 mV. In addition, piperovatine-modified currents were correlated with not only the number of prior conditioning pulses but also the proportion of activated channels. Finally, piperovatine was found to stabilize voltage-gated sodium channels in the fast-inactivated state after opening, and inhibit transition to the slow-inactivated state. These results suggest that piperovatine preferably binds to activated channels and stabilizes voltage sensors at the conformation acquired during depolarization.

Preparation and application of a molecularly imprinted monolith for specific recognition of domoic acid.

In this work, a molecularly imprinted monolithic column was synthesized by a facile procedure and was applied for specific recognition of domoic acid, an amnesic shellfish poison. The poly(4-vinylpyridine-co-ethylene glycol dimethacrylate) molecularly imprinted monolith was synthesized in a stainless steel column by in situ polymerization. Pentane-1,3,5-tricarboxylic acid was used as a dummy imprinting template instead of the highly toxic and expensive target molecule. It is the first time that a molecularly imprinted monolith is introduced for separation and detection of domoic acid. After optimizing the preparation conditions, the prepared imprinted monolith was systematically characterized and exhibited excellent stability and permeability as a HPLC stationary phase. The results of chromatographic analysis demonstrated that the molecularly imprinted monolith exhibited specific retention and selective recognition toward domoic acid, with an imprinted factor up to 3.77. Furthermore, the molecularly imprinted monolith was successfully applied for selective enrichment of domoic acid from biological samples. Graphical abstract A molecularly imprinted monolith was prepared by using a dummy imprinted template and was successfully applied for specific recognition of domoic acid.

Depolarizing neuromuscular junctional blocking action of Parthenium hysterophorus leaf extracts in rat.

Electromyographic (EMG) and electrodiagnostic studies were made on sciatic nerve-anterior tibialis muscle in vivo in rats administered with a sublethal dose of Parthenium hysterophorus dry leaf powder 100 mg/100 g body weight of rat. Nerve-evoked and muscle-evoked compound muscle action potentials were recorded at different frequencies of stimulations and at different time intervals. The EMG studies revealed depolarizing neuromuscular junctional (NMJ) blocking effects of the leaf extract. The decrement-increment (D-I) phenomena and the EMG pattern in general showed a resemblance to an established anticholinesterase agent like neostigmine. The studies confirm that the leaf extract contains a proven and promising new depolarizing NMJ blocker.

Effects of boldine on mouse diaphragm and sarcoplasmic reticulum vesicles isolated from skeletal muscle.

The effects of boldine [(S)-2,9-dihydroxy-1,10-dimethoxyaporphine], a major alkaloid in the leaves and bark of boldo (Peumus boldus Mol.), on skeletal muscle were studied using mouse diaphragm and isolated sarcoplasmic reticulum membrane vesicles. Boldine, at 10-200 microM, has little effect on the muscle-evoked twitches; however, the ryanodine-induced contracture was potentiated dose-dependently. At higher concentrations of 300 microM, boldine by itself induced muscle contracture of two phases, which were caused by the influx of extracellular Ca2+ and induction of Ca2+ release from the internal Ca2+ storage site, the sarcoplasmic reticulum, respectively. When tested with isolated sarcoplasmic reticulum membrane vesicles, boldine dose-dependently induced Ca2+ release from actively loaded sarcoplasmic reticulum vesicles isolated from skeletal muscle of rabbit or rat which was inhibited by ruthenium red, suggesting that the release was through the Ca2+ release channel, also known as the ryanodine receptor. Boldine also dose-dependently increased apparent [3H]-ryanodine binding with the EC50 value of 50 microM. In conclusion, we have shown that boldine could sensitize the ryanodine receptor and induce Ca2+ release from the internal Ca2+ storage site of skeletal muscle.

Selective depolarization of the muscle membrane in frog nerve-muscle preparations by a chromatographically purified extract of the dinoflagellate Ostreopsis lenticularis.

1. The actions of a chromatographically identified extract of the marine dinoflagellate Ostreopsis lenticularis, named ostreotoxin-3 (OTX-3), were studied on frog isolated neuromuscular preparations. 2. OTX-3 (1-10 microg ml(-1)) applied to cutaneous pectoris nerve-muscle preparations depolarized skeletal muscle fibres and caused spontaneous contractions. The depolarization was neither reversed by prolonged washing nor by (+)-tubocurarine. 3. OTX-3 decreased the amplitude of miniature end plate potentials (m.e.p.ps) but did not affect their frequency. 4. Extracellular recording of compound action potentials revealed that OTX-3 affected neither excitability nor conduction along intramuscular nerve branches. 5. End-plate potentials (e.p.ps) elicited by nerve stimulation were reduced in amplitude by OTX-3 and even showed reversed polarity in junctions deeply depolarized by the toxin. 6. Membrane depolarization induced by OTX-3 was decreased about 70% in muscles pretreated for 30 min with 10 microM tetrodotoxin. In contrast, muscles pretreated with 5 microM mu-conotoxin GIIIA were completely insensitive to OTX-3-induced depolarization. 7. OTX-3 did not affect e.p.p. amplitude and the quantal content of e.p.ps in junctions in which muscle depolarization was abolished by mu-conotoxin GIIIA. 8. OTX-3 is a novel type of sodium-channel activating toxin that discriminates between nerve and skeletal muscle membranes.

Sea bird mortality at Cabo San Lucas, Mexico: evidence that toxic diatom blooms are spreading.

Domoic acid was found to be responsible for an isolated event involving the massive death of brown pelicans (Pelecanus occidentalis) in January 1996, at the tip of the Baja California peninsula. The death of these sea birds was the result of feeding on mackerel (Scomber japonicus) contaminated by domoic acid-producing diatoms (Pseudonitzschia sp.). The number of dead birds (150 animals) found during a period of 5 days caused alarm and called for a governmental task force that would help to implement emergency measurements to protect other species of bird. Also, local canneries were inspected to verify the safety of their recent production and prevent the toxin entering the human market. Fortunately, the timing, response and coordination of this task force enabled identification of the origin and nature of the toxin that provoked such a phenomenon. Future monitoring is recommended to avoid a larger impact of domoic acid spreading and the occurrence of similar toxic events.

Two new isomers of domoic acid from a red alga, Chondria armata.

Isodomoic acids G and H, two new isomers of the neurotoxin domoic acid, along with isodomoic acids A, B, E and F, were isolated from a red alga, Chondria armata, collected at the southern tip of Kyushu Island. The structures of two of these were deduced to be (E, E) and (Z, E) isomers of 2-carboxy-4-(5-carboxy-l-methyl-2-hexenylidene)-3-pyrro- lidineacetic acid, based on electrospray ionization mass and [1H]nuclear magnetic resonance spectral analyses including [1H-1H]correlation spectroscopy and nuclear Overhauser effect correlation spectroscopy.

Idiospermuline, a trimeric pyrrolidinoindoline alkaloid from the seed of Idiospermum australiense.

Purification of a methanol extract from the seed of Idiospermum australiense, guided by bioactivity on a rat brain cortical wedge preparation has afforded two known dimeric alkaloids, the piperidinoindoline (+)-calycanthine [1] and the pyrrolidinoindoline, (-)-chimonanthine [2] along with a new trimeric pyrrolidinoindoline alkaloid, (-)-idiospermuline [3]. Te structure of idiospermuline [3] was determined by spectroscopic methods and the absolute configuration by an X-ray crystallographic study of idiospermuline trimethiodide [4].

Development of rapid and sensitive high throughput pharmacologic assays for marine phycotoxins.

The lack of rapid, high throughput assays is a major obstacle to many aspects of research on marine phycotoxins. Here we describe the application of microplate scintillation technology to develop high throughput assays for several classes of marine phycotoxin based on their differential pharmacologic actions. High throughput "drug discovery" format microplate receptor binding assays developed for brevetoxins/ciguatoxins and for domoic acid are described. Analysis for brevetoxins/ciguatoxins is carried out by binding competition with [3H] PbTx-3 for site 5 on the voltage dependent sodium channel in rat brain synaptosomes. Analysis of domoic acid is based on binding competition with [3H] kainic acid for the kainate/quisqualate glutamate receptor using frog brain synaptosomes. In addition, a high throughput microplate 45Ca flux assay for determination of maitotoxins is described. These microplate assays can be completed within 3 hours, have sensitivities of less than 1 ng, and can analyze dozens of samples simultaneously. The assays have been demonstrated to be useful for assessing algal toxicity and for assay-guided purification of toxins, and are applicable to the detection of biotoxins in seafood.