Physiological and transcriptional responses to inorganic nutrition in a tropical Pacific strain of Alexandrium minutum: Implications for the saxitoxin genes and toxin production.

Saxitoxins (STXs) constitute a family of potent sodium channel blocking toxins, causative agents of paralytic shellfish poisoning (PSP), and are produced by several species of marine dinoflagellates and cyanobacteria. Two STX-core genes, sxtA and sxtG, have been well elucidated in Alexandrium but the expression of these genes under various nutritional modes in tropical species remains unclear. This study investigates the physiological responses of a tropical Pacific strain of Alexandrium minutum growing with nitrate or ammonium, and with various nitrogen to phosphorus (N:P) supply ratios. The transcriptional responses of the sxt genes were observed. Likewise, a putative sxtI encoding O-carbamoyltransferase (herein designated as AmsxtI) was recovered from the transcriptomic data, and its expression was investigated. The results revealed that the cellular toxin quota (Qt) was higher in P-depleted, nitrate-grown cultures. With cultures at similar N:P (<16), cells grown with excess ammonium showed a higher Qt than those grown with nitrate. sxtA1 was not expressed under any culture conditions, suggesting that this gene might not be involved in STX biosynthesis by this strain. Conversely, sxtA4 and sxtG showed positive correlations with Qt, and were up-regulated in P-depleted, nitrate-grown cultures and with excess ambient ammonium. On the other hand, AmsxtI was expressed only when induced by P-depletion, suggesting that this gene may play an important role in P-recycling metabolism, while simultaneously enhancing toxin production.

A non-toxigenic but morphologically and phylogenetically distinct new species of Pseudo-nitzschia, P. sabit sp. nov. (Bacillariophyceae).

A new species of Pseudo-nitzschia (Bacillariophyceae) is described from plankton samples collected from Port Dickson (Malacca Strait, Malaysia) and Manzanillo Bay (Colima, Mexico). The species possesses a distinctive falcate cell valve, from which they form sickle-like colonies in both environmental samples and cultured strains. Detailed observation of frustules under TEM revealed ultrastructure that closely resembles P. decipiens, yet the new species differs by the valve shape and greater ranges of striae and poroid densities. The species is readily distinguished from the curve-shaped P. subcurvata by the presence of a central interspace. The morphological distinction is further supported by phylogenetic discrimination. We sequenced and analyzed the nuclear ribosomal RNA genes in the LSU and the second internal transcribed spacer, including its secondary structure, to infer the phylogenetic relationship of the new species with its closest relatives. The results revealed a distinct lineage of the new species, forming a sister cluster with its related species, P. decipiens and P. galaxiae, but not with P. subcurvata. We examined the domoic acid (DA) production of five cultured strains from Malaysia by Liquid chromatography-mass spectrometry (LC-MS), but they showed no detectable DA. Here, we present the taxonomic description of the vegetative cells, document the sexual reproduction, and detail the molecular phylogenetics of Pseudo-nitzschia sabit sp. nov.

Quantitative ELISA kit for paralytic shellfish toxins coupled with sample pretreatment.

A new ELISA kit to quantitate the level of paralytic shellfish poisoning (PSP) toxins in crude shellfish extracts was developed. A conjugate for preparing antigen and a novel antibody used in the ELISA was prepared based on the unique reactions between C11-O-sulfate toxins such as gonyautoxins 2 and 3 (GTX2,3) and various thiol compounds, followed by coupling to keyhole limpet hemocyanin. The compounds necessary for competitive ELISA, labeled toxin and an artificial standard toxin to replace saxitoxin in the analysis, were also produced by the same techniques. The resulting ELISA recognized all the toxin components tested; however, carbamoyl-N-sulfate derivatives such as B and C toxins and N1-OH toxins such as neoSTX and GTX1,4 showed low affinity to the antibody. The difference in the reactivity of the antibody observed among the toxin components prevents accurate quantification of the toxin amounts in shellfish extracts. To address this problem, the former toxin components were transformed to corresponding carbamate toxins by mild HCl treatment according to a conventional method. The reduction of N1-OH of the latter toxins to N1-H was performed by our original method using hemin as a catalyst. We report here the new ELISA kit coupled with the pretreatment process to transform the toxin components favorable for the quantitative analysis of PSP toxins.

Angiotensin AT(1)-receptor blockers enhance cardiac responses to parasympathetic nerve stimulation via presynaptic AT(1) receptors in pithed rats.

In the present study, we investigated the effects of angiotensin AT1-receptor blockers, KT3-671 and losartan, on the cardiac vagal neurotransmission in pithed rats. The bradycardia induced by vagal nerve stimulation (VNS, at 5 Hz) was potentiated significantly and dose-dependently by KT3-671 and also losartan. This enhancement effect of KT3-671 (10 mg/kg) was slightly potent than that of losartan (10 mg/kg). On the other hand, an angiotensin AT2-receptor blocker, PD123319 (10 mg/kg), did not affect VNS-induced bradycardia. KT3-671 and losartan did not affect the exogenous acetylcholine-evoked bradycardia. Intravenous infusion of AngII (100 ng/kg per min) attenuated the VNS-induced bradycardia. This inhibitory effect of AngII on bradycardia was restored by both KT3-671 and losartan. These results suggest that endogenous AngII can have a tonic inhibitory effect on cardiac vagal transmission by stimulating the presynaptic AT1 receptors not AT2 receptors. Suppression of this mechanism by the AT1-receptor blockers causes the facilitation of acetylcholine release from vagal nerve endings. This acceleratory effect of AT1-receptor blockers on cardiac vagal neurotransmission may contribute to the lack of reflex tachycardia following hypotension.

Production of isodomoic acids A and B as major toxin components of a pennate diatom Nitzschia navis-varingica.

In order to obtain more detailed information on the distribution of domoic acid-producing Nitzschia in Asian waters, Nitzschia-like diatoms were isolated primarily from the Philippines and established in culture for analysis by HPLC with fluorescence detection. Out of 58 isolates, 35 Nitzschia-like diatoms from the estuary areas of San Pedro Bay, Tacloban City and Manila Bay showed significant levels of domoic acid production (1.3-5.6pg/cell). These were identified as Nitzschia navis-varingica. Two isolates from the same locality did not produce domoic acid. Of the 21 isolates from Bulacan Estuary, Manila Bay, none produced domoic acid. They did, however, produce two substances that seemed to be domoic acid derivatives. One of the strains was mass cultured and the substances were extracted, purified and analyzed by LC-MS/MS, proton and (13)C NMR, and UV spectra. The produced substances were determined as isodomoic acids A and B. This is the first report of a diatom that produces isodomoic acids A and B as major toxin components.

Captopril enhances cardiac vagal but not sympathetic neurotransmission in pithed rats.

The effect of captopril on neurally evoked bradycardia and tachycardia was investigated in pithed rats. Captopril enhanced the vagal nerve stimulation-evoked bradycardia. Angiotensin I reduced the vagal bradycardia, which was reversed by subsequent administration of captopril. Bradykinin did not affect the neurally evoked bradycardia. Captopril and angiotensin I affected neither the exogenous acetylcholine-evoked bradycardia nor the sympathetic nerve stimulation-evoked tachycardia. These results suggest that the interruption of angiotensin II formation by captopril causes less presynaptic inhibition of acetylcholine release via angiotensin II receptors without affecting cardiac sympathetic neurotransmission.