Latrunculin-A causes mydriasis and cycloplegia in the cynomolgus monkey. (1/531)

PURPOSE: To determine the effect of latrunculin (LAT)-A, which binds to G-actin and disassembles actin filaments, on the pupil, accommodation, and isolated ciliary muscle (CM) contraction in monkeys. METHODS: Pupil diameter (vernier calipers) and refraction (coincidence refractometry) were measured every 15 minutes from 0.75 to 3.5 hours after topical LAT-A 42 microg (approximately 10 microM in the anterior chamber [AC]). Refraction was measured every 5 minutes from 0.5 to 1.5 hours after intracameral injection of 10 microl of 50 microM LAT-A (approximately 5 microM in AC), with intramuscular infusion of 1.5 mg/kg pilocarpine HCl (PILO) during the first 15 minutes of measurements. Pupil diameter was measured at 1 and 2 hours, and refraction was measured every 5 minutes from 1 to 2 hours, after intravitreal injection of 20 microl of 1.25 mM LAT-A (approximately 10 microM in vitreous), with intramuscular infusion of 1.5 mg/kg PILO during the first 15 minutes of measurements (all after topical 2.5% phenylephrine), and contractile response of isolated CM strips, obtained <1 hour postmortem and mounted in a perfusion apparatus, to 10 microM PILO +/- LAT-A was measured at various concentrations. RESULTS: Topical LAT-A of 42 microg dilated the pupil without affecting refraction. Intracameral LAT-A of 5 microM inhibited miotic and accommodative responses to intramuscular PILO. Intravitreal LAT-A of 10 microM had no effect on accommodative or miotic responses to intramuscular PILO. LAT-A dose-dependently relaxed the PILO-contracted CM by up to 50% at 3 microM in both the longitudinal and circular vectors. CONCLUSIONS: In monkeys, LAT-A causes mydriasis and cycloplegia, perhaps related to its known ability to disrupt the actin microfilament network and consequently to affect cell contractility and adhesion. Effects of LAT-A on the iris and CM may have significant physiological and clinical implications.  (+info)

Simple and sensitive analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction and gas chromatography-mass spectrometry. (2/531)

A simple method for the analysis of nereistoxin and its metabolites in human serum using headspace solid-phase microextraction (SPME) and gas chromatography-mass spectrometry (GC-MS) is developed. A vial containing a serum sample, 5M sodium hydroxide, and benzylacetone (internal standard) is heated to 70 degrees C, and an SPME fiber is exposed for 30 min in the headspace of the vial. The compounds extracted by the fiber are desorbed by exposing the fiber in the injection port of the GC-MS. The calibration curves show linearity in the range of 0.05-5.0 micrograms/mL for nereistoxin and N-methyl-N-(2-methylthio-1-methylthiomethyl)ethylamine, 0.01-5.0 micrograms/mL for S,S'-dimethyl dihydronereistoxin, and 0.5-10 micrograms/mL for 2-methylthio-1-methylthiomethylethylamine in serum. No interferences are found, and the analysis time is 50 min for one sample. In addition, this proposed method is applied to a patient who attempted suicide by ingesting Padan 4R, a herbicide. Padan 4R contains 4% cartap hydrochloride, which is an analogue of nereistoxin. Nereistoxin and its metabolites are detected in the serum samples collected from the patient during hospitalization. The concentration ranges of nereistoxin in the serum are 0.09-2.69 micrograms/mL.  (+info)

Latrunculin-A increases outflow facility in the monkey. (3/531)

PURPOSE: To determine the effect of Latrunculin (LAT)-A, a macrolide that binds to G-actin, which leads to the disassembly of actin filaments, on shape, junctions, and the cytoskeleton of cultured bovine aortic endothelial cells (BAECs) and on outflow facility in living monkeys. METHODS: Latrunculin-A dose-time-response relationships in BAECs were determined by immunofluorescence and phase contrast light microscopy, facility by two-level constant pressure anterior chamber perfusion. RESULTS: In BAECs, LAT-A caused dose- and incubation time- dependent destruction of actin bundles, cell separation, and cell loss. Cell-cell adhesions were more sensitive than focal contacts. Recovery was also dose- and time-dependent. In monkeys, exchange intracameral infusion and topical application of LAT-A induced dose- and time-dependent several-fold facility increases. The facility increase was completely reversed within several hours after drug removal. However, for at least 24 hours after a single topical LAT-A dose, perfusion with drug-free solution caused an accelerated increase in facility beyond that attributed to normal resistance washout. CONCLUSIONS: Pharmacological disorganization of the actin cytoskeleton in the trabecular meshwork by specific actin inhibitors like LAT-A may be a useful antiglaucoma strategy.  (+info)

Effects of palytoxin on isolated intestinal and vascular smooth muscles. (4/531)

Palytoxin (PTX), the most potent marine toxin isolated from the Zoanthid, Palythoa tuberculosa, was studied to determine the effect on isolated smooth muscles. In guinea pig taenia coli PTX at above 3 X 10(-10) g/ml caused a contraction which slowly subsided under isotonic recording. Under isometric recording PTX at above 1 X 10(-10) g/ml caused a contraction which depended on the spontaneous activity. The PTX-induced contraction was not affected by atropine, tripelenmamine or tetrodotoxin but was inhibited by 5 mM Mg, norephinrphrine, isoprenaline or papaverine. PTX at above 1 X 10(-9) g/ml induced an increase in spike frequency and a slight depolarization accompanied with a contraction when measured using a sucrose gap method. In some cases the spike generation was almost abolished after a long exposure to higher dose of PTX and the developed tension gradually decreased. Under isometric recording PTX caused a sustained contraction in rabbit aorta, dog mesenteric and coronary arteries at above 1 X 10(-10) and 1 X 10(-11) g/ml, respectively, in a dose-dependent manner. The coronary artery was most sensitive among the preparation used. PTX-induced contraction in aorta was irreversible, was not influenced by phentolamine but diminished with 5 mM Mg and disappeared in a D-600 or Ca-free medium. PTX is thus an extremely potent and direct stimulant which acts on smooth muscles.  (+info)

Brevetoxins cause acute excitotoxicity in primary cultures of rat cerebellar granule neurons. (5/531)

Brevetoxins (designated PbTx-1 to -10) are potent lipid-soluble polyether compounds that are known to bind to and modulate voltage-gated sodium channel activity. To investigate whether brevetoxins produce direct central nervous system neurotoxic effects, cultured rat cerebellar granule neurons were exposed to brevetoxins in Locke's buffer for 2 h at 22 degrees C. Neuronal injury was quantified by assaying lactate dehydrogenase activity in the exposure buffer and in conditioned growth media collected at 22 h after brevetoxin exposure. Brevetoxins produced acute neuronal injury and death in neurons with a rank order potency of PbTx-1 (EC50 = 9.31 +/- 0.45 nM) > PbTx-3 (EC50 = 53.9 +/- 2.8 nM) > PbTx-2 (EC50 = 80.5 +/- 5.9 nM) > PbTx-6 (EC50 = 1417 +/- 32 nM), which is similar to their previously determined rank order potency for brevetoxin-induced icthyotoxicity and binding to [3H]PbTx-3-labeled sodium channels on synaptosomes. The neurotoxic response could be prevented by coapplication of the sodium channel antagonist tetrodotoxin or by the competitive or noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonists D-AP5 and MK-801, ketamine, dextromethorphan, and dextrorphan, respectively. NMDA receptor antagonists afforded neuroprotection with rank order potencies comparable to those measured previously for protection against glutamate-induced excitotoxic responses. Further analysis revealed that brevetoxins induced a concentration-dependent release of L-glutamate and L-aspartate into the exposure buffer. These data indicate that brevetoxin-induced injury in cultured rat cerebellar granule neurons is mediated by NMDA receptors that are activated indirectly as a consequence of PbTx-induced sodium channel activation and attendant excitatory amino acid release.  (+info)

Nonribosomal peptide synthesis and toxigenicity of cyanobacteria. (6/531)

Nonribosomal peptide synthesis is achieved in prokaryotes and lower eukaryotes by the thiotemplate function of large, modular enzyme complexes known collectively as peptide synthetases. These and other multifunctional enzyme complexes, such as polyketide synthases, are of interest due to their use in unnatural-product or combinatorial biosynthesis (R. McDaniel, S. Ebert-Khosla, D. A. Hopwood, and C. Khosla, Science 262:1546-1557, 1993; T. Stachelhaus, A. Schneider, and M. A. Marahiel, Science 269:69-72, 1995). Most nonribosomal peptides from microorganisms are classified as secondary metabolites; that is, they rarely have a role in primary metabolism, growth, or reproduction but have evolved to somehow benefit the producing organisms. Cyanobacteria produce a myriad array of secondary metabolites, including alkaloids, polyketides, and nonribosomal peptides, some of which are potent toxins. This paper addresses the molecular genetic basis of nonribosomal peptide synthesis in diverse species of cyanobacteria. Amplification of peptide synthetase genes was achieved by use of degenerate primers directed to conserved functional motifs of these modular enzyme complexes. Specific detection of the gene cluster encoding the biosynthetic pathway of the cyanobacterial toxin microcystin was shown for both cultured and uncultured samples. Blot hybridizations, DNA amplifications, sequencing, and evolutionary analysis revealed a broad distribution of peptide synthetase gene orthologues in cyanobacteria. The results demonstrate a molecular approach to assessing preexpression microbial functional diversity in uncultured cyanobacteria. The nonribosomal peptide biosynthetic pathways detected may lead to the discovery and engineering of novel antibiotics, immunosuppressants, or antiviral agents.  (+info)

Rvs167p, the budding yeast homolog of amphiphysin, colocalizes with actin patches. (7/531)

In this report, we have shown that the yeast amphiphysin-like protein Rvs167p was localized mainly in small cortical patches throughout the cell in unbudding cells. During budding, the patches were polarized at bud emergence site. During mating, Rvs167p was concentrated at the tip of the shmoo. Rvs167p colocalized with actin patches during yeast vegetative growth and mating. Complete disruption of the actin cytoskeleton using Latrunculin-A did not affect Rvs167p localization in patches throughout the cell. In rvs167 mutant cells, actin patches are mislocalized and in rvs161 or abp1 mutant cells, Rvs167p localization is not affected. These observations suggest that Rvs167p may localize the actin cortical complex properly. Finally, the amphiphysin-conserved N-terminal domain of Rvs167p, called the BAR domain, was required but not sufficient for the correct localization of the protein.  (+info)

Studies on maitotoxin-induced intracellular Ca(2+) elevation in chinese hamster ovary cells stably transfected with cDNAs encoding for L-type Ca(2+) channel subunits. (8/531)

The aim of the present study was to characterize the role played by different L-type Ca(2+) channel subunits in [Ca(2+)](i) increase induced by maitotoxin (MTX). In the presence of 5 mM extracellular K(+), MTX (0.01-0.5 ng/ml) induced a significant concentration-dependent increase in Fura-2-monitored [Ca(2+)](i) in single Chinese hamster ovary (CHO) cells expressing the alpha(1c) (CHOCalpha9 cells) or the alpha(1c)beta(3)alpha(2)delta (CHOCalpha9beta3alpha2/delta4 cells) subunits of voltage-gated Ca(2+) channels (VGCCs), whereas the effect was much reduced in wild-type CHO cells lacking VGCCs. In addition, MTX effect on CHOCalpha9, CHOCalpha9beta3alpha2/delta4, and GH(3) cells (0.01-0.1 ng/ml) was inhibited by the selective L-type Ca(2+) channel entry-blocker nimodipine (10 microM); a nimodipine-insensitive component was still present, particularly at high (>1 ng/ml) toxin concentrations. In CHOCalpha9beta3alpha2/delta4 cells, depolarizing concentrations of extracellular K(+) (55 mM) reinforced the [Ca(2+)](i) increase induced by MTX (0.1 ng/ml), and this effect was prevented by nimodipine (10 microM). Finally, patch-clamp experiments in CHOCalpha9beta3alpha2/delta4 cells showed that low MTX concentrations (0.03 ng/ml) induced the occurrence of an inward current at -60 mV, which was completely prevented by Cd(2+) (100 microM) and by nimodipine (10 microM), whereas the same dihydropyridine concentration (10 microM) failed to prevent the electrophysiological effects of a higher toxin concentration (3 ng/ml). In conclusion, the results of the present study showed that MTX-induced [Ca(2+)](i) elevation involves two components: 1) an action on L-type VGCCs at the pore-forming alpha(1c) subunit level, which is responsible for the greatest rise of [Ca(2+)](i); and 2) a VGCC-independent mechanism that is present both in excitable and in nonexcitable cells and is responsible for a lower elevation of [Ca(2+)](i).  (+info)