Evidence for numerous analogs of yessotoxin in Protoceratium reticulatum

A solid-phase extract from Protoceratium reticulatum was partitioned between water and butanol and the two fractions purified on an alumina column. Fractionation was monitored by ELISA and LC–MS. Results indicate that while almost all yessotoxin (1) was extracted into butanol, large amounts of yessotoxin analogs remained in the aqueous extract along with lesser amounts in the butanolic extract. NMR analysis of selected fractions from reverse-phase chromatography of the extracts confirmed the presence of yessotoxin analogs, although structure determinations were not possible due to the complexity of the mixtures. Analysis of fractions with LC–MS³ and neutral-loss LC–MS/MS indicated the presence of more than 90 yessotoxin analogs, although structures for most of these have not yet been determined. These analogs provide a mechanism to rationalise the discrepancy between ELISA and LC–MS analyses of algae and shellfish.     

Effects of dopamine and norepinephrine on in vitro spontaneous and gonadotropin-releasing hormone-induced gonadotropin release by dispersed cells or fragments of the goldfish pituitary

Enzymatically dispersed goldfish pituitary cells or freshly prepared goldfish pituitary fragments continue to secrete gonadotropin spontaneously in a column perifusion system. After the establishment of basal secretion rates, treatment of dispersed pituitary cells with 5 and 500 nM dopamine, or pituitary fragments with 50 and 100 nM dopamine, decreased the amount of gonadotropin released into the perifusate. Perifusion with 500 nM dopamine also abolished the gonadotropin-release response to a 10 nM solution of a luteinizing hormone-releasing hormone analogue in both perifusion systems. Perifusion of pituitary dispersed cells or fragment preparations obtained from sexually regressed goldfish with 50 nM norepinephrine consistently increased the amount of gonadotropin released into the perifusate. These results provide in vitro evidence for direct dopamine inhibition of spontaneous gonadotropin release, blockade by dopamine of gonadotropin-releasing hormone actions, and norepinephrine stimulation of gonadotropin secretion in goldfish.     

Synthesis and expression in Escherichia coli of DNA encoding the murine lambda 1 chain of a monoclonal antibody specific for Salmonella serotype B O-antigen.

A 658 bp DNA sequence corresponding to the murine lambda 1 chain of a monoclonal antibody, Se155-4, specific for the Salmonella serotype B O-antigen, was designed using Escherichia coli preferred codons and chemically synthesized by ligation of synthetic fragments into a linearized plasmid followed by transformation into E. coli. A synthetic signal peptide (ompA) was fused to express the L chain as a free polypeptide into the periplasm of E. coli cells. After isolation and purification, heterologous recombination of the E. coli L chain with mouse H chain gave an active antigen-binding protein. The activity was 15-20% when compared to protein created by an equivalent association of isolated natural mouse L and H chains as measured by a direct EIA assay. In inhibition experiments with the polysaccharide antigen, the two proteins showed identical titration curves and 50% inhibition points, indicating comparable KA values.     

The function of the suspending medium during the freeze-drying preservation of Escherichia coli

The role of added solutes in the freeze-drying preservation of bacteria is examined. Escherichia coli were washed, suspended in solutions of selected hydroxy-substituted compounds of various molecular weights, and frozen at rates of the order of one degree C per minute. The frozen materials were freeze-dried and rehydrated in several different ways. Freeze-drying survival was correlated with the development and persistence of an amorphous solute matrix and the desorption of residual water. Protection from potentially harmful effects of freeze-drying was attributed to the dispersion, by the aforementioned amorphous matrix, of metabolites released from the cells during the preparation, and freezing of the bacterial suspensions.