M arine cyanobacteria represent a particularly rich source of structurally unique neurotoxic secondary metabolites (1-5). Lyngbya majuscula is a pantropical marine cyanobacterium that is the source of antillatoxin (ATX), a structurally unusual lipopeptide (1) (Fig. 1). Blooms of L. majuscula have been associated with adverse effects on human health. These blooms have been reported to cause respiratory irritation, eye inflammation, and severe contact dermatitis in exposed fishermen and swimmers (6). ATX has been shown to be among the most ichthyotoxic metabolites isolated to date from a marine microalga (1) and, more recently, has been demonstrated to be neurotoxic in primary cultures of rat cerebellar granule cells (4). In the latter study, morphologic evidence of ATX-induced neurotoxicity included swelling of neuronal somata, thinning of neurites, and blebbing of neurite membranes. ATX also induced a concentration-dependent cytotoxicity in cerebellar granule neurons as monitored by lactate dehydrogenase efflux (ATX EC 50 ϭ 20.1 Ϯ 6.4 nM) (4). This neurotoxic response of ATX was prevented by coexposure with noncompetitive antagonists of the N-methyl-D-aspartate (NMDA) receptor such as MK-801 and dextrorphan.Voltage-gated sodium channels are responsible for generation of the rising phase of the action potential in membranes of neurons as well as in most other electrically excitable cells. Sodium channels consist of a pore-forming ␣ subunit of 260 kDa associated with auxiliary  subunits of 33-36 kDa (7,8). Voltagegated sodium channels represent the molecular target for an array of natural products including marine neurotoxins. Marine neurotoxins such as tetrodotoxin (TTX), saxitoxin, conotoxins, sea anemone toxins, brevetoxin, and ciguatoxin all bind with high affinity and specificity to at least six distinct receptor sites on sodium channel ␣ subunits (8). Collectively, these toxins have served as important tools to explore the structure and function of voltage-dependent sodium channels. These marine neurotoxins produce characteristic alterations in the two major properties of sodium channels, namely ion permeation and gating (8).The objective of the present study was to test the hypothesis that ATX acts as an activator of voltage-dependent sodium channels. This hypothesis emanated from our previous studies which were consistent with ATX acting as an autocrine excitotoxic agent in cerebellar granule neurons. In this study we have used a combination of neurochemical and pharmacological approaches to show that ATX is a structurally unusual lipopeptide activator of neuronal voltage-gated sodium channels. Materials and MethodsMaterials. Tritiated batrachotoxin A 20-␣-benzoate ([ 3 H]BTX) and 22 Na ϩ were obtained from DuPont͞NEN. Deltamethrin was purchased from Biomol (Plymouth Meeting, PA). Veratridine, brevetoxin-1 (PbTx-1), and ouabain were obtained from Sigma. Sea anemone toxin was purchased from Calbiochem. Fluo-3 AM and pluronic acid were obtained from Molecular Probes. ATX was either authentic natural (Ϫ)-ant...
Our previous report showed that supernatants of Lactobacillus acidophilus (LS) cultures possessed chemotactic and angiogenic properties. Specifically, LS stimulated gene expression and the secretion of tumor necrosis factor-alpha (TNF-alpha), the proliferation of immune cells in vitro, and blood vessel formation. Chemotaxis and proliferation of inflammatory cells in vivo were also stimulated by LS. In the current study, we hypothesized that LS stimulates the growth and development of other rapidly dividing cells, including embryonic cells. The stimulatory effects of LS on a neuroblastoma cell line (Neuro-2a), chicken embryos, and bovine embryos were examined. The addition of LS to Neuro-2a cultures caused a proliferation of cells in a concentration-dependent manner. Pretreatment of LS at 56 degrees C for 30 mins did not affect its stimulatory activity. The administration of LS to the chorioallantoic membrane (CAM) of chicken-embryonated eggs for 1-2 days resulted in extensive thickening of the membrane. The thickening was due to the influx and proliferation of fibroblasts and inflammatory cells, the accumulation of loose connective tissue composed primarily of mucopolysaccharides, and/or the formation of blood vessels. Stimulatory effects of LS on bovine embryos were also observed. The treatment with LS significantly promoted the development of zygotes to the four-cell stage and from the four-cell stage to blastocysts. These results have confirmed our hypothesis that LS exerts a stimulatory effect on the cells of embryonic stages including neuroblastoma cells, the CAM of chicken embryos, and bovine embryos from zygotes to blastocysts.
Antillatoxin is a potent ichthyotoxin and cytotoxin previously discovered from the marine cyanobacterium Lyngbya majuscula. Ensuing studies of its mechanism of action showed it to activate the mammalian voltage-gated sodium channel at a pharmacological site that is distinct from any previously described. The structure of antillatoxin, initially formulated from spectroscopic information, was subsequently corrected at one stereocenter (C-4) as a result of synthesis of four different antillatoxin stereoisomers (all possible C-4 and C-5 diastereomers). In the current study these four stereoisomers, (4R,5R)-, (4S,5R)-, (4S,5S)-, and (4R,5S)-antillatoxin, were characterized in five different biological assay systems: ichthyotoxicity to goldfish, microphysiometry using cerebellar granule cells (CGCs), lactose dehydrogenase efflux from CGCs, monitoring of intracellular Ca(2+) concentrations in CGCs, and cytotoxicity to Neuro 2a cells. Across these various biological measures there was great consistency in that the natural antillatoxin (the 4R,5R-isomer) was greater than 25-fold more potent than any of the other stereoisomers. Detailed NMR studies provided a number of torsion and distance constraints that were modeled using the MM2 force field to yield predicted solution structures of the four antillatoxin stereoisomers. The macrocycle and side chain of natural (4R,5R)-antillatoxin present an overall "L-shaped" topology with an accumulation of polar substituents on the external surface of the macrocycle and a hydrogen bond between N(H)-7' and the C(O)-1 carbonyl. The decreased potency of the three non-naturally occurring antillatoxin stereoisomers is certainly a result of their dramatically altered overall molecular topologies.
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