Thomas J. Mende

Toxicity of two toxins from the Florida red tide marine dinoflagellate, Ptychodiscus brevis

The purification and crystallization of T17, a toxin from Ptychodiscus brevis, is reported. The toxicity of this compound and a second toxin known as T34 are compared by i.v., i.p. and oral administration in mice. Both toxins produce symptoms characteristic of muscarnic stimulants; hypersalivation, rhinorrhea and excessive urination and defecation being the most commonly observed. T17, which is orally toxic, is believed to be the agent responsible for Neurotoxic Shellfish Poisoning.

Crystallization and toxicology of T34: A major toxin from Florida's red tide organism (Ptychodiscus brevis)

Abstract The purification and crystallization of a major toxin from laboratory cultures of Floridas fed tide organism, Ptychodiscus brevis , is described. The crystalline toxin is soluble in acetone and chloroform, less soluble in ethyl acetate, methanol and ethanol, and is slightly soluble in water. The crystalline toxin is stable when stored in a moisture-free environment. The toxin is lethal to fish and mice, inhibits growth in three in vitro cell culture systems, and inhibits the division of fertilized sea urchin eggs. It has no effect on the formation of antibodies directed against sheep red blood cells in mice, and does not inhibit the morphological transformation of rat glioma cells from a fibroblast-like to a glial-like cell.

Bronchoconstriction caused by Florida red tide toxins

T17, a toxin purified from laboratory cultures of Floridas redtide organism, Ptychodiscus brevis (formerly Gymnodinium breve), produces bronchoconstriction in anesthetized artificially-ventilated guinea pigs. Bronchoconstriction, measured as a resistance to mechanical pulmonary inflation, was antagonized by atropine, but not by interruption of vagal nerve stimulation or diaphragm dissection.

Amino acid utilization by Gymnodinium breve

Abstract The marine dinoflagellate Gymnodinium breve utilizes erogenous amino acids for the synthesis of proteins in the light. During logarithmic growth, l -valine and l -methionine are incorporated into proteinaceous material which is retained by the cell. Glycine is also incorporated, but the glycine-containing proteins are extruded. When cells are no longer growing exponentially, all proteins that incorporated these supplied amino acids are extruded. The pronase-susceptible extruded material has a MW in excess of 300 000. When chloramphenicol is used to inhibit protein synthesis, glycine is not taken up. l -Methionine is rapidly metabolized intracellularly and is used in the synthesis of other macromolecules. l -Valine accumulates intracellularly and remains unaltered. Glycine and l -methionine appear to be transported via facilitated diffusion systems, while l -valine uptake appears to be active.

Brevetoxin binding: molecular pharmacology versus immunoassay

Brevetoxin PbTx-3 isolated from Floridas red tide dinoflagellate Ptychodiscus brevis has been produced recently in tritiated form by reductive tritiation of brevetoxin PbTx-2. Tritiated PbTx-3 has been used as a specific probe in competitive radioimmunoassays developed to detect brevetoxins in food sources, and this probe has also been utilized to characterize the brevetoxin binding component in rat brain synaptosomes. Brevetoxins PbTx-2 and PbTx-3, possessing the same structural backbone (type-1) as the tritiated probe, and PbTx-1 and PbTx-7, possessing a second structural backbone (type-2), have been compared quantitatively in their individual abilities to competitively displace tritiated PbTx-3 from its specific binding site in each assay. Type-1 toxins displaced labeled probe with ED50 values of 20-22 nM and 12-17 nM in radioimmunoassay and synaptosomes, respectively. Type-2 toxins displaced labeled probe with ED50 values of 92-93 nM and 3.5-4.1 nM in RIA and synaptosomes, respectively. Synaptosome assays reflect potency of each toxin examined, while radioimmunoassay reflects structural similarities to the immunizing toxin PbTx-3.

Glucose transport and metabolism in Gymnodinium breve

Abstract Floridas red tide organism, Gymnodinium breve , utilized exogenous glucose in the light for the synthesis of cellular components. Glucose was not taken up in the dark. Kinetic parameters for glucose uptake include a K FD of 11 μM and a V max of 1 × 10 −10 mol of glucose taken up/mg cellular protein/hr. Glucose uptake was competitively inhibited by phloridzin ( K i = 40 μM), mannose ( K i = 12O μM), and 2-deoxy- d -glucose ( K i = 190 μM) and non-competitively inhibited by galactose ( K i = 125 μM). Kinetics and inhibition of glucose uptake are consistent with a facilitated diffusion transport system.

Microdetermination of inorganic sulfate using thin-layer plates

Abstract Inorganic sulfate was precipitated on cellulose thin-layer plates with the radioactive reagent, 133 BaCl 2 . Excess reagent was removed by repeated washings with an acidic BaCl 2 solution. The residual activity was transferred to vials by cutting out the point of application and its immediate surroundings. Counting was performed in a scintillation well γ-counting system. The concentration-activity curve was linear.

Metabolic changes induced in chickens by the administration of tremetol

Abstract Tremetol is a toxic extract of the White Snakeroot plant ( Eupatorium urticaefolium Reichard). The milk of cows grazing on Eupatorium-infested pastures causes “milk sickness”, characterized in part by ketoacidosis and changes in blood glucose levels. In this paper the metabolic effects of chronic administration of tremetol in the chick were studied. Blood glucose and blood free fatty acid levels respond with a biphasic pattern to tremetol administration, first rising above, then decreasing below the control values. In contrast, the blood levels of β-hydroxybutyrate show a sustained and major increase during the treatment period. Tremetol had no demonstrable effect on hepatic glycogen content in the treated group. Studies on hepatic enzyme levels revealed an almost total suppression of citrate synthase activity in the experimental group without a parallel decrease in succinate dehydrogenase activity. A selective toxic action on mitochondria resulting in an inability to effectively metabolize acetyl CoA is put forward as the possible cause of the ketoacidosis.

Studies on the procoagulant activity of human amniotic fluid II. The role of factor VII

Partially purified Amniotic Fluid Factor (AFF) is found to directly activate human factor X, with a Km for factor X of 0.37 +/- 0.06 M. Added phospholipid has only a slight effect on the activation at low concentrations, and inhibits the reaction at higher concentrations. Both DIPF and PMSF inhibit AFF factor X activation. Although added phospholipid is not required for AFF-activity, phospholipase C rapidly destroys it, indicating the presence of intrinsic phospholipid. Phospholipase C treated AFF releases factor VII activity, which leads to the conclusion that AFF is in fact a thromboplastin: factor VII complex. Both AFF and a human brain thromboplastin factor VII complex prepared in vitro were inhibited by Zn++ ion, while human brain thromboplastin alone is not. AFF is markedly larger than the human brain thromboplastin-factor VII complex as judged by gel filtration.

Studies on the procoagulant activity of human amniotic fluid I. Stability and coagulation factor requirements

A fraction of human amniotic fluid possessing procoagulant activity was purified 35-70 fold by gel filtration on Sepharose 4B. The activity eluted in the void volume indicating a particle size in excess of 5 X 10(6) daltons. The amniotic fluid factor (AFF) activity is stabilized on storage in the presence of Ca++ ions which could not be replaced by Mg++. Addition of phospholipids resulted in accelerated loss of activity. Steps taken to remove factor VII did not affect the activity, but factor X and V are required.