ALPHA-AMANITIN

The developmentally arrested infective larva of hookworms encounters a host-specific signal during invasion that initiates the resumption of suspended developmental pathways. The resumption of development during infection is analogous to recovery from the facultative arrested dauer stage in the free-living nematode Caenorhabditis elegans. Infective larvae of the canine hookworm Ancylostoma caninum resume feeding and secrete molecules important for infection when exposed to a host mimicking signal in vitro. This activation process is a model for the initial steps of the infective process. Dauer recovery requires protein synthesis, but not RNA synthesis in C. elegans. To determine the role of RNA and protein synthesis in hookworm infection, inhibitors of RNA and protein synthesis were tested for their effect on feeding and secretion by A. caninum infective larvae. The RNA synthesis inhibitors α-amanitin and actinomycin D inhibit feeding dose-dependently, with IC(50) values of 30 and 8 μM, respectively. The protein synthesis inhibitors puromycin (IC(50)=110 μM), cycloheximide (IC(50)=50 μM), and anisomycin (IC(50)=200 μM) also displayed dose-dependent inhibition of larval feeding. Significant inhibition of feeding by α-amanitin and anisomycin occurred when the inhibitors were added before 12h of the activation process, but not if the inhibitors were added after 12h.

The A-type lamins have been observed to colocalize with RNA splicing factors in speckles within the nucleus, in addition to their typical distribution at the nuclear periphery. To understand the functions of lamin speckles, the effects of transcriptional inhibitors known to modify RNA splicing factor compartments (SFCs) were examined. Treatment of HeLa cells with alpha-amanitin or 5,6-dichlorobenzimidazole riboside (DRB) inhibited RNA polymerase II (pol II) transcription and led to the enlargement of lamin speckles as well as SFCs. Removal of the reversible inhibitor DRB resulted in the reactivation of transcription and a rapid, synchronous redistribution of lamins and splicing factors to normal-sized speckles, indicating a close association between lamin speckles and SFCs. Conversely, the expression of NH2-terminally modified lamin A or C in HeLa cells brought about a loss of lamin speckles, depletion of SFCs, and down-regulation of pol II transcription without affecting the peripheral lamina. Our results suggest a unique role for lamin speckles in the spatial organization of RNA splicing factors and pol II transcription in the nucleus.

A fast, sensitive high performance thin-layer chromatographic method for the determination of alpha-, beta-, and gamma-amanitin in crude, methanolic extracts of Amanita phalloides is described. The limit of detection is 50 ng of each amanitin. With this method amanitin was determined in 24 pooled samples of Amanita phalloides, collected between 1970 and 1977 in Germany and Switzerland. The total amanitin content varied between 2010 and 7300 mg/kg dry weight and the average value was 4430 mg/kg of which 43% was alpha-amanitin, 49% beta-amanitin and 8% gamma-amanitin. The origin of the fungi hardly influenced their amanitin content: in samples collected during the same year at different sites it fluctuated within a factor of 1.7. The amanitin content of samples from the same site, but collected in different years, maximally varied within a factor of 3.7. The partial decomposition of amanitins during prolonged storage of the lyophilized samples undoubtedly contributed to this variation. Phalloidin, which was determined by conventional thin-layer-chromatography, could not be detected in a sample from 1970, whereas its concentration in material collected during 1977 amounted to 2400 mg/kg dry weight.