Stability of agaritine - a natural toxicant of Agaricus mushrooms.

Agaritine (N-(gamma-L(+)-glutamyl)-4-hydroxymethylphenylhydrazine) is a phenylhydrazine derivative found in the cultivated Agaricus mushroom which is claimed to give rise to carcinogenic products when metabolized. The stability of a synthetic sample of agaritine was tested in water and methanol. In tap water kept in open vials, agaritine was totally degraded within 48h. Since agaritine degradation was less pronounced in closed than in open vials, and slower in Milli Q water and, in particular, in Milli Q water purged with N(2), the degradation seems to be oxygen-dependent. The antioxidant dithiothreitol reduced the degradation. Four or possibly five ultraviolet-absorbing compounds were formed during degradation, but these have not yet been identified. Whereas the rate of degradation was similar at temperatures between 4 and 22 degrees C, it was quicker at an acidic than at a neutral pH. The latter observation was confirmed in experiments where agaritine was incubated in simulated gastric fluid (pH 1.2). The importance of the degradation when performing toxicological studies with agaritine is discussed.

Effects of cross-linked profilin:beta/gamma-actin on the dynamics of the microfilament system in cultured cells.

There is evidence that the profilin:actin complex is the immediate precursor in the formation of actin filaments in cells. This paper describes the cell morphology and microfilament distribution after microinjection of covalently cross-linked profilin:beta/gamma-actin (PxA) in two different cell lines. Injected cells were either kept unstimulated or stimulated with platelet-derived growth factor (PDGF) before fixation and visualization of filamentous actin. After injection of low doses of PxA, the cells displayed an actin organization characterized by a clearance of diffuse fluorescence from a region immediately interior of ruffling edges and the appearance of small dots of fluorescence in the same region. At higher concentrations, PxA effectively inhibited outgrowth of lamellae and microspikes, and there was a drastic reduction of actin staining in the zone behind the advancing edge. This effect is reminiscent of the effect of cytochalasin B on fibroblasts and the growth cone of neuronal cells. As in these cases, there remained a rim of actin-dependent fluorescence on the very edge of the membrane lamella, particularly in the PxA-treated fibroblasts. The interference of PxA with the formation of surface structures was pronounced after PDGF stimulation. Here, PxA effectively eliminated the enhancement of the ruffling activity in the cell edges and on the dorsal surface of the cells. In contrast to PxA, injection of non-cross-linked profilin:beta/gamma-actin had no apparent effect on cell morphology and microfilament distribution except for an increased concentration of filamentous actin in one of the cell lines.

The PI 3-kinase isoforms p110(alpha) and p110(beta) have differential roles in PDGF- and insulin-mediated signaling.

Phosphoinositide 3'-kinases constitute a family of lipid kinases implicated in signal transduction through tyrosine kinase receptors and heterotrimeric G protein-linked receptors. Phosphoinositide 3'-kinases that bind to the platelet-derived growth factor receptor are composed of two subunits: the p85 subunit acts as an adapter and couples the catalytic p110 subunit to the activated receptor. There are different isoforms of p85 as well as of p110, the individual roles of which have been elusive. Using microinjection of inhibitory antibodies specific for either p110(alpha) or p110(beta) we have investigated the involvement of the two p110 isoforms in platelet-derived growth factor- and insulin-induced actin reorganization in porcine aortic endothelial cells. We have found that antibodies against p110(alpha), but not antibodies against p110(beta), inhibit platelet-derived growth factor-stimulated actin reorganization, whereas the reverse is true for inhibition of insulin-induced actin reorganization. These data indicate that the two phosphoinositide 3'-kinase isoforms have distinct roles in signal transduction pathways induced by platelet-derived growth factor and insulin.

Agaritine content in processed foods containing the cultivated mushroom (Agaricus bisporus) on the Nordic and the Czech market.

The level of agaritine was measured in fresh and canned cultivated mushroom (Agaricus bisporus) as well as in other food products containing A. bisporus, by reversed phase high performance liquid chromatography. The two fresh samples were purchased on the open market and contained 212 and 229 mg/kg, respectively. Of the 35 different trademarks of canned mushroom products studied, 25 were based on cut mushrooms and 10 on whole mushrooms. On average, whole mushrooms contained 14.9 +/- 6.7 mg agaritine per kg product whereas cut mushrooms contained 18.1 +/- 7.8 mg/kg. There was no statistically significant difference between these two values. Agaritine levels in brine were generally slightly lower than the levels detected in canned mushrooms. Thus, the level of agaritine in A. bisporus is reduced more than 10 times during the wet canning process, resulting in low levels in canned products. On a portion basis, somewhat higher amounts of agaritine may be found in some other food products (mushroom soup and pasta sauce) containing A. bisporus.

Characterization of a mutant profilin with reduced actin-binding capacity: effects in vitro and in vivo.

We are investigating structure-function relationships in profilin and actin by site-specific mutagenesis using a yeast, Saccharomyces cerevisiae, expression system to produce wild-type and mutant proteins. This paper shows that deleting proline 96 and threonine 97, which are located close to the major actin binding site on profilin, did not significantly alter the interaction between profilin and phosphatidylinositol 4,5-bisphosphate, nor did it affect the profilin:poly(L-proline) interaction. The mutant protein, however, had a lower capacity to bind to actin in vitro than wild-type profilin, though it showed a slightly increased profilin-enhanced nucleotide exchange on the actin. When microinjected into Swiss 3T3 mouse fibroblasts or porcine aortic endothelial cells, the mutant profilin did not change the organization of the microfilament system like the wild-type profilin did. This provides further evidence that profilin controls microfilament organization in the cell by interacting directly with actin.

Long-term antibiotic susceptibility pattern of strains of Neisseria gonorrhoeae isolated in Czechoslovakia in the years 1957-1982.

Over the past 25 years a total of 7492 strains of Neisseria gonorrhoeae have been isolated in Czechoslovakia, mainly in Prague (64%). All these strains have been tested for susceptibility to the following antibiotics: penicillin G, ampicillin, tetracycline, spectinomycin, erythromycin, doxycycline, kanamycin, rifampin, chloramphenicol, gentamicin, cephalothin, cephaloridine, lincomycin and clindamycin. In addition, seven derivatives of newer antibiotics of penicillin and cephalosporin series were tested in 1981. The study showed that in 1957 the MIC of 0.03 units of penicillin per ml was effective against 95% of strains, but in 1981 only 37% of isolates were sensitive to this concentration. The first gonococcal strains with the MIC value of 4.0 units/ml to penicillin were detected in 1981. This tendency towards decreased gonococcal susceptibility to benzylpenicillin is alarming. Over the last eight years there have been described sporadic isolations of strains relatively resistant to tetracycline (MIC = 8.0 mg/l). The susceptibility to spectinomycin has been tested in over 4000 gonococcal strains, since 1967. The test showed that this antibiotic remained highly effective against the gonococcal infection with over 95% of gonococci with the MIC value of 16.0 mg/l. No fully spectinomycin resistant strains have been found. Penicillin G as well as spectinomycin and cefotaxim are still considered the antibiotics of the first choice in the treatment of gonorrhoea. The alternative antibiotics may include cefuroxim, chloramphenicol and, in cases of sensitive strains, tetracyclines.