Toxicological evaluation of saxitoxin, neosaxitoxin, gonyautoxin II, gonyautoxin II plus III and decarbamoylsaxitoxin with the mouse neuroblastoma cell bioassay.

Four major paralytic shellfish poisoning (PSP) toxins in pure form-saxitoxin (STX), neosaxitoxin (NEO), gonyautoxin II (GTX II), and decarbamoylsaxitoxin (dcSTX), and mixed gonyautoxin II plus III (GTX II/III)-were toxicologically evaluated in the neuroblastoma cell bioassay and compared with the United States Food and Drug Administration standard reference saxitoxin (FDA STX). Mean relative toxicities of these experimental preparations were 99.5 (STX), 128.0 (NEO), 48.4 (GTX II) and 42.6 (dcSTX) when compared with FDA STX set arbitrarily at 100. Two mixtures of GTX II/III with relative concentrations of 1:2.5 and 4:1 had percentage relative toxicities of 52.4 and 54.5, respectively. Except for GTX II/III, which increased in toxicity, the toxicities of the experimental toxin preparations were not affected by boiling for 5 min in equal volumes of either 0.1 or 1.0 N HCl. Detailed examinations of dose-responses of each individual toxin preparation in the cell bioassay revealed some differences in response profiles. The toxicological data presented here support and extend previous information collected with the mouse bioassay and HPLC. The EC(50) values of STX and GTX II/III obtained under different conditions of matrix (acetic acid nu. ethanol), filtration (filtered 0.22 mum or unfiltered) and time in storage at 5 degrees C after opening of the sealed ampoules were largely unaffected by any of the experimental treatments.

Paralytic shellfish poison (saxitoxin family) bioassays: automated endpoint determination and standardization of the in vitro tissue culture bioassay, and comparison with the standard mouse bioassay.

Mouse neuroblastoma cells swell and eventually lyse upon exposure to veratridine, which, when added together with ouabain, enhances sodium ion influx. In the presence of saxitoxin (STX), which blocks sodium channels, the action of the other two compounds is inhibited and the cells remain morphologically normal. A tissue culture bioassay using mouse neuroblastoma cells, developed by Kogure and colleagues, takes advantage of these principles; in this bioassay, the fraction of the cells protected from the actions of ouabain and veratridine is in direct proportion to the concentration of STX and its analogues. We have modified this bioassay, improving its convenience and speed by eliminating the need to count individual cells to determine the saxitoxin equivalents, and instead have employed a microplate reader for automated determinations of absorbances of crystal violet from stained neuroblastoma cells. When these changes and other minor technical modifications were tested in the tissue culture bioassay systematically, we found the lower detection limit to be around 10 ng STX equivalents (eq) per ml of extract ( = 2.0 micrograms STX eq/100 g shellfish tissue). Our version of the tissue culture bioassay was compared with the standard mouse bioassay using 10 acid extracts of dinoflagellates (Alexandrium excavata and A. fundyense) and 47 AOAC extracts of shellfish tissues. The tissue culture bioassay provided results virtually identical to those obtained with the mouse bioassay (r > 0.96), and moreover, was considerably more sensitive. The results gained from high performance liquid chromatographic (HPLC) analysis of 12 of the same extracts were less consistent when compared with the results from both bioassay methods. The automated tissue culture (neuroblastoma cell) bioassay may be a valid alternative to live animal testing for paralytic shellfish poisoning.

Effect of Temperature and Prey Availability on Growth of Paramoeba invadens in Monoxenic Culture.

Paramoeba invadens Jones 1985 is a pathogenic marine amoeba responsible for mass mortalities of sea urchins (Strongylocentrotus droebachiensis) of Nova Scotia between 1980 and 1983. A direct relationship between temperature and sea urchin paramoebiasis has been shown in previous laboratory and field studies. This study examined the effect of prey availability and temperature on the growth of P. invadens in monoxenic culture (with the marine bacterium Pseudomonas nautica). At 15 degrees C, the specific growth rate of P. invadens increased with bacterial prey concentration and was highest at 10 bacterial cells ml. Growth rate of P. invadens was maximal at 15 to 20 degrees C (which corresponds to annual sea temperature maxima in the natural environment) and the minimum generation time was 19.41 h at 20 degrees C. At 10 and 12 degrees C, generation times were 91.18 and 73.39 h, respectively; at 2 and 5 degrees C, there was no growth. P. invadens did not survive in monoxenic culture at 27 degrees C. Growth rates of P. invadens in vitro were positively correlated with time to morbidity of infected S. droebachiensis.

Inhibitory and lethal activities of rosaramicin, erythromycin, and clindamycin against Campylobacter fetus subsp jejuni and intestinalis.

In a comparative study of the inhibitory and lethal effects of rosaramicin, erythromycin, and clindamycin on strains of Campylobacter fetus subsp jejuni and C intestinalis, C jejuni was more readily killed by rosaramicin and clindamycin than was C intestinalis. Erythromycin exhibited an equally lethal effect against both subspecies. However, it was the least active of the macrolides tested against both C jejuni and C intestinalis.

Bile salt 3 alpha- and 12 alpha-hydroxysteroid dehydrogenases from Eubacterium lentum and related organisms.

Thirty-two strains of Eubacterium lentum and phenotypically similar anaerobic gram-positive bacilli were screened for intracellular bile salt 3alpha- and 12alpha-hydroxysteroid dehydrogenase (HSDHase) activities. These organisms were categorized into four groups: (A) those containing 12alpha-HSDHase only (10 strains), (B) those containing 3alpha- and 12alpha-HSDHase (13 strains), (C) those containing 3alpha-HSDHase only (2 strains), and (D) those devoid of any measurable HSDHase activity (7 strains). Of the respective four groups, 9/10, 13/13, 0/2, and 0/7 were like the neotype strain of E. lentum (ATCC 25559) in that they produced H(2)S in a triple sugar iron agar butt, reduced nitrate to nitrite, and weakly decomposed hydrogen peroxide. The other strains were variable for nitrate reduction and activity on hydrogen peroxide, but all the organisms in the first three categories (with one exception) were H(2)S producers (triple sugar iron agar butt) and all (with one exception) were designated E. lentum, whereas the organisms of category B were non-H(2)S producers (triple sugar iron agar butt). Five of these seven were not stimulated by arginine and are designated "phenotypically similar organisms." Thin-layer chromatography of extracted spent bacterial medium of four representative strains from each group grown in the presence of cholate revealed the presence of (A) 12-oxo product, (B) 12-oxo and 3-oxo products, (C) 3-oxo product, and (D) the absence of any of these products. The 12alpha-HSDHase of category B organisms was unstable unless 10(-3) M dithioerythritol was added to the buffer. With the exception of 3 out of 32 strains, there was a positive correlation between the production of measurable amounts of 12alpha-HSDHase and H(2)S production. Growth curves and the effect of arginine on growth and the production of 3alpha- and 12alpha-HSDHase were examined in representative strains of categories A, B, and C. Both enzymes were shown to bind onto a nicotinamide adenine dinucleotide-Sepharose column and could be eluted by high-ionic-strength buffer, resulting in approximately 25-fold and 18-fold purification, respectively. Molecular weight estimations by Sephadex G-200 gave values of 205,000 and 125,000 for the 3alpha- and 12alpha-HSDHase, respectively. Purified 12alpha-HSDHase was investigated with respect to pH requirement, substrate specificity, and enzyme kinetics.

12alpha-Hydroxysteroid dehydrogenase from Clostridium group P strain C48-50 ATCC No. 29733: partial purification and characterization.

The growth of Clostridium group P strain C48-50 [an anaerobe that contains 12alpha-hydroxysteroid dehydrogenase (12alpha-HSDH) in the absence of other dehydrogenases active upon bile salts] is greatly enhanced by the addition of 2.0% d-fructose or d-glucose to the growth medium. Other sugars were less effective. The production of NADP-dependent 12alpha-HSDH paralleled the growth of the organism which was optimal at 72 hr. Growth (and enzyme production) were suppressed by the addition of bile salt to the medium; the order of suppression was deoxycholate > chenodeoxycholate >> cholate; 1 mM of either of the dihydroxy-bile salts inhibited 96% of the growth and 100% of the enzyme production. Kinetic studies on cell-free preparations of 12alpha-HSDH revealed a pH optimum of 7.8 with greater linearity of NADP evolution with time occurring only at more alkaline pH values (9-10). Lineweaver-Burke plots revealed Michaelis constant (K(m)) values in the range of 3-5 x 10(-4) M for deoxycholate and its glycine and taurine conjugates, while higher values were found for cholate and conjugates (K(m) value for taurocholate was 3 x 10(-3) M). Although there was no activity with NAD, 12alpha-HSDH was shown to bind onto both NAD- and NADP-Sepharose columns, with stronger binding on the latter. The enzyme was purified 20-fold by NAD-Sepharose chromatography. The molecular weight was estimated at 100,000 by Sephadex G-200 and a series of molecular weight markers. Substrate specificity studies showed that a variety of bile salts containing 12alpha-OH groups reacted; notably, the 3alpha-sulfates of cholate and deoxycholate were nonsubstrates.-Macdonald, I. A., J. F. Jellett and D. E. Mahony. 12alpha-Hydroxysteroid dehydrogenase from Clostridium Group P strain C48-50 #29733: partial purification and characterization.