Preparation toxin

Silyl ethers serve as preeursors of nucleophiles and liberate a nucleophilic alkoxide by desilylation with a chloride anion generated from CCI4 under the reaction conditions described before[124]. Rapid intramolecular stereoselective reaction of an alcohol with a vinyloxirane has been observed in dichloro-methane when an alkoxide is generated by desilylation of the silyl ether 340 with TBAF. The cis- and tru/u-pyranopyran systems 341 and 342 can be prepared selectively from the trans- and c/.y-epoxides 340, respectively. The reaction is applicable to the preparation of 1,2-diol systems[209]. The method is useful for the enantioselective synthesis of the AB ring fragment of gambier-toxin[210]. Similarly, tributyltin alkoxides as nucleophiles are used for the preparation of allyl alkyl ethers[211]. 

Ion exchangers are sometimes used on a throwaway basis also. In the laboratoiy, ion exchangers are used to produce deionized water, purify reagents, and prepare inorganic sols. In medicine, they are used as antacid, for sodium reduction, for the sustained release of drugs, in skin-care preparations, and in toxin removal. 

Caution Like alt vinyl monomeva, 3-buten-2-one is toxin and the preparation should he aarried out in a ivell-ventilated hood. 

The important role played by the quinicines (rubatoxanones, quina-toxines) in the syntheses of the dihydrocinchona alkaloids and the possibility that such substances might be used for the preparation of products approaching quinine in therapeutical interest, has led to the production of a large number of quinolyl ketones of various types and the corresponding secondary alcohols, and other derivatives obtainable from them, of which mention may be made of Rubtzov s syntheses of several isomerides of dihydroquinine. ... 

Human immunoglobulin preparations from pools of a great number of people (>1,000) with assumed antibodies against common viruses are used as a means of passive immunization in acute infections. More specific antibody preparations with high titers from patients who recovered recently from a viral disease or were immunized against toxins are also available in... 

A matrix extract was prepared from poison-free scallop and spiked at the level of 200 ngg of scallop hepatopancreas. The toxins were determined by using LC-MS with calibration employing external standards prepared in methanol. The matrix extract was then spiked further with 300 ngg of each of the toxins and redetermined. The results obtained for each analyte are summarized in Table 5.17 and show that, when using the external calibration method, the values obtained range from 138 to 170 ngg a reduction in accuracy of between 15... 

Thus three lines of evidence define the rapidly dissociating receptor as the LR complex. Conditions known to uncouple R from G--first, guanine nucleotide and second, pertussis toxin—produce LR third, reconstitution of G protein restores receptor affinity, sensitivity to guanine nucleotide, and effector activation. In this sense, the ligand and binding behavior of this system is analogous to that of the beta-adrenergic receptor, where the LR and LRG complexes have already been studied with purified proteins and reconstituted membrane preparations (2,i0). 

Figure 4. Effects of dihydro-brevetoxin B (H2BVTX-B) on Na currents in crayfish axon under voltage-clamp. (A) A family of Na currents in control solution each trace shows the current kinetics responding to a step depolarization (ranging from -90 to -I-100 mV in 10 mV increments). Incomplete inactivation at large depolarizations is normal in this preparation. (B) Na currents after internal perfusion with H2BVTX-B (1.2 a M). inactivation is slower and less complete than in the control, and the current amplitudes are reduced. (C) A plot of current amplitudes at their peak value (Ip o, o) and at steady-state (I A, A for long depolarizations) shows that toxin-mOdified channels (filled symbols) activate at more negative membrane potentials and correspond to a reduced peak Na conductance of the axon (Reproduced with permission from Ref. 31. Copyright 1984 American Society for Pharmacology and Experimental Therapeutics).

Analyses for the Saxitoxins. Early methods for analysis of the saxitoxins evolved from those used for toxin isolation and purification. The principal landmarks in the development of preparative separation techniques for the saxitoxins were 1) the employment of carboxylate cation exchange resins by Schantz et al. (82) 2) the use of the polyacrylamide gel Bio-Gel P2 by Buckley and by Shimizu (5,78) and 3) the development by Buckley of an effective TLC system, including a new solvent mixture and a new visualization technique (83). The solvent mixture, designated by Buckley as "E", remains the best for general resolution of the saxitoxins. The visualization method, oxidation of the saxitoxins on silica gel TLC plates to fluorescent degradation products with hydrogen peroxide and heat, is an adaptation of the Bates and Rapoport fluorescence assay for saxitoxin in solution. Curiously, while peroxide oxidation in solution provides little or no response for the N-l-hydroxy saxitoxins, peroxide spray on TLC plates is a sensitive test for all saxitoxin derivatives with the C-12 gemdiol intact. 

Differences in the equilibrium dissociation constant, K, for the binding of the various saxitoxins to the sodium channel binding site largely determine the differences in the potencies of the toxins in whole animal assays and in tissue preparations. 

Excitable tissue preparations were obtained fresh daily from live animals using the technique described by Dodd et al. (12). Protein was measured on each synapto-some preparation using the Coomassie Brilliant Blue dye technique described by Bradford (13) results were expressed as "toxin bound per mg synaptosome protein". 

Methods of detection, metabolism, and pathophysiology of the brevetoxins, PbTx-2 and PbTx-3, are summarized. Infrared spectroscopy and innovative chromatographic techniques were examined as methods for detection and structural analysis. Toxicokinetic and metabolic studies for in vivo and in vitro systems demonstrated hepatic metabolism and biliary excretion. An in vivo model of brevetoxin intoxication was developed in conscious tethered rats. Intravenous administration of toxin resulted in a precipitous decrease in body temperature and respiratory rate, as well as signs suggesting central nervous system involvement. A polyclonal antiserum against the brevetoxin polyether backbone was prepared a radioimmunoassay was developed with a sub-nanogram detection limit. This antiserum, when administered prophylactically, protected rats against the toxic effects of brevetoxin. 

Research in this area advanced in the 1970 s as several groups reported the isolation of potent toxins from P. brevis cell cultures (2-7). To date, the structures of at least eight active neurotoxins have been elucidated (PbTx-1 through PbTx-8) (8). Early studies of toxic fractions indicated diverse pathophysiological effects in vivo as well as in a number of nerve and muscle tissue preparations (reviewed in 9-11). The site of action of two major brevetoxins, PbTx-2 and PbTx-3, has been shown to be the voltage-sensitive sodium channel (8,12). These compounds bind to a specific receptor site on the channel complex where they cause persistent activation, increased Na flux, and subsequent depolarization of excitable cells at resting... 

Figure 9. Anti-PbTx antiserum inhibition of [ H]PbTx-3 binding to its receptor site in rat brain membrane preparations. Labeled toxin (0.5 nM in 1 ml PBS) was incubated with rat brain membranes (125 fig total protein) and increasing amounts of anti-PbTx antiserum (- -) or pre-immune serum (- -) for 1 hr at 4 C. Membrane-bound radioactivity was then measured in a centrifugation assay as previously described (8),...

The best method of preparing individual toxins from the crude venom is by affinity immunochromatography utilizing monoclonal antibody or in one instance, polyclonal antibody (4—6). Monoclonal antibodies to both the fishing and mesentery... 

---