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The protein synthesis machinery of Sulfolobus solfataricus , a thermoacidophilic archaebacterium, is insensitive to most of the known antibiotics that interfere with elongation. alpha-Sarcin, a cytotoxic protein, inhibits protein synthesis on eukaryotic systems by cleaving a specific sequence of the large rRNA. alpha-Sarcin is capable of inhibiting protein synthesis on S. solfataricus producing a fragment under conditions similar to those which produce it in yeast ribosomes. This result suggests the presence on S. solfataricus of the sequence necessary for the enzymatic hydrolysis promoted by alpha-sarcin.

A toxic protein of Mr 22,000, called bolaffinine, has been purified from the mushroom, Boletus affinis Peck (Boletaceae), with a 0.65% yield using a procedure involving four steps: ammonium sulfate precipitation, two chromatographies on ion-exchange columns and gel filtration on Sephadex G-75. It is thermolabile and can be inactivated by organic solvents. Its isoelectric point lies between pH 9 and 10. It contains 234 amino acids, no free SH groups and one disulfide bridge as evidenced by reactions with 5-5'-dithiobis-nitrobenzoate (DTNB) and 2-mercaptoethanol. When injected into mice, it provokes the death of animals within 16-24 h. The 24 h LD50 is 61 mg/kg of body weight. Bolaffinine inhibits protein synthesis in hepatoma tissue culture and in a rabbit reticulocyte lysate.

Two yeast proteins produced on oil substrate were found safe and nutritious in growing beagle dogs. Reducing nucleic acids with ammonium hydroxide resulted in renal damage; potassium hydroxide reduced nucleic acids with no ill effects. Changes in processing methods must be adequately monitored.

A yeast capable of growth on methanol as its sole carbon-energy source was isoalted from soil samples and identified as a strain of Hansenula polymorpha. A continuous enrichment culture at 37 C with a simple mineral salts medium was used to select this organism. The isolate, designated DL-1, has a maximal specific growth rate of 0.22 per h, at pH 4.5 to 5.5 and temperatures of 37 to 42 C, in simple mineral salts medium with methanol (0.5%), biotin, and thiamine. Growth occurred in a chemostat at temperatures up to 50 C, with strong growth at 45 C. The maximal growth yield of the yeast on methanol was 0.36 g of dry cell weight per g of methanol, and the yield on oxygen was 0.37 g of dry cell weight per g of O(2). Protein content of the isolate is 46%, and total nucleic acid content varies from 5.0 to 7.0% with increasing growth rate from 0.08 to 0.20 per h. The amino acid profile of this yeast protein indicates that it could serve as a good source of food protein. Feeding studies with rats show the yeast to have no toxic effects.

A cytolytic toxin from the basidiocarps of the edible mushroom Flammulina velutipes was purified to homogeneity. The lysin, flammutoxin, is a single polypeptide chain of Mr 32,000 and pK about 5.4. It contains unusually large amounts of tryptophan, serine and glycine, and few or none of the sulfur-containing amino acids. Erythrocytes of rat, rabbit, guinea pig, man, mouse, cat and dog were sensitive to lysis, in that order, whereas erythrocytes of sheep, ox, goat, swine and horse were largely or completely resistant to lysis. The toxin appears not to be a phospholipase and it was not inhibitable by any of a variety of lipids. Hemolysis probably involves alteration of the erythrocyte membrane, with formation of submicroscopic ion channels, and it appears to be of the osmotic type. In some respects flammutoxin resembles phallolysin, a cytolytic toxin obtained from the mushroom Amanita phalloides.

Preliminary feeding studies were conducted with a high protein fungus (Graphium sp.) grown on natural gas. The biomass, containing approximately 50% protein, is considered a potential source of single cell protein. In short-term (1-week) experiments, rats gained weight when fed semisynthetic diets containing up to 40% of the biomass, although growth was depressed at the higher levels. Rats lost weight but survived for this length of time when fed biomass alone. Chronic toxicity was tested in a 5-month study with diets in which the casein (20% by weight) of the semisynthetic diet was either partially or entirely replaced by the proteinaceous biomass. Rats fed these diets grew more slowly but appeared normal and healthy, and no pathological changes were observed at autopsy. Some of these rats were fed a standard laboratory diet for an additional 10-month observation period, and at autopsy, one cortical adenoma of the kidney was found in a rat originally fed the lower level of Graphium biomass.

The tolerance of human subjects to two microfungal food products was studied in separate double-blind cross-over studies. As an addition to the subject's usual diets, cookies with and without 20 g of a product from Fusarium graminearium were fed to a group of 100 individuals daily. In a second study, cupcakes with and without 10 g of Paecilomyces variotii were given daily to 50 individuals. Mild rashes possibly related to one of the microfungal food products occurred in two individuals fed P variotii. Except for a decrease in serum cholesterol during the F graminearium study, no significant changes were noted in 17 serum constituents. During nutritive value studies, digestibility, biological value, and net protein utilization were calculated for the two microfungal proteins and for milk. The values for milk were 95, 85, and 80%, respectively. The values for F graminearium were 78, 84, and 65%, respectively. For P variotii corresponding figures were 81, 67, and 54%. On the basis of these results both microfungal foods may be deemed safe for human consumption at the levels tested.

In the perspective of therapeutic approaches the monoclonal antibody, MBrl, with a quite restricted spectrum of reactivity for human breast carcinoma, was coupled to restrictocin (Res), a ribosome inactivating protein produced by Aspergillus restrictus. In a cell-free system this toxin was found to have an activity comparable to that of other plant toxins, but its in vitro toxicity was shown to be low on different cell lines. Three batches of MBr1-Res conjugate were prepared and their specificity, efficiency, and maximum level of cytotoxicity were analyzed on the cell line MCF-7 expressing the relevant antigen, on several irrelevant tumor cell lines, and on normal cells. Conjugates were from 600 to 1500 times more efficient than the uncoupled derivatized Res towards MCF-7 cells and were completely ineffective on the other target cells. The antigen-driven cytotoxicity was confirmed by the nontoxicity of an irrelevant conjugate on MCF-7 cells. The cytotoxic efficiency of MBr1-Res was low when compared to the binding level of MBr1 at the same concentration and a portion of treated cells (from 10% to 30%) survived the treatment. The heterogeneity of expression of the relevant antigen, together with its only partial internalization, could account for these limitations. The lysosomotropic agent ammonium chloride and the carboxylic ionophore monensin were tested as potentiating agents but in both cases the cytotoxicity remained unmodified. A neutralization assay performed on a xenogenic model indicated that the MBr1-Res conjugate was capable of reducing the tumor take. These data indicate the possibility of using the Res to prepare a reproducible and highly selective breast cancer conjugate. However, there are still a number of problems which must first be solved before we can consider its clinical application.

Killer toxin activity of Pichia anomala WC65 appeared fungicidal for P. bimundalis WC38 and fungistatic for Candida albicans RC1. Inhibitory activity against sensitive C. albicans showed a linear relationship between toxin concentrations and the inverse of the reduced growth rates. The plot of toxin concentrations against growth rates was hyperbolic, as is characteristic of saturation kinetics. Sensitivity of C. albicans to the toxin decreased with increased cell age. The measurement of growth rate reduction provided a simple and accurate method for quantitation of toxin.

Rubescenslysin, a haemolytic protein from Amanita rubescens, disrupted the cytoplasmic membrane of human leucocytes which were more sensitive than erythrocytes. In the isolated hearts of rats and guinea pigs it caused systolic contracture, which was preceded by potassium outflow and sometimes by a transient positive inotropic effect. On the electrically stimulated guinea-pig left atrium it showed at first a positive, followed by a negative inotropic effect; on the spontaneously beating right atrium it produced transient positive followed by negative inotropic and chronotropic effects. Atria were less sensitive than intact hearts. In the isolated rat phrenic nerve-diaphragm preparation it produced a contracture, which was associated with reduction of indirect and direct contractility. In the isolated guinea-pig ileum it produced a slow contraction followed by tachyphylaxis. As excitability declined due to rubescenslysin, so did excitability by acetylcholine and potassium. Atropine and pheniramine had only feeble antagonistic effects, but papaverine was more powerful. In isolated rat hepatocytes, rubescenslysin caused a rapid outflow of potassium and coarse cell protrusions while later the cells became stainable with trypan blue. In the isolated perfused rat liver it produced a rapid outflow of potassium and of cytoplasmic and mitochondrial enzymes, and a somewhat slower outflow of lysosomal beta-glucuronidase, accompanied by a rise in the lactate/pyruvate ratio and a decrease in bile production. In the isolated perfused rat kidney it caused an outflow of cytoplasmic and mitochondrial enzymes, together with massive proteinuria and serious restriction of sodium and potassium reabsorption and of urine output. In all the tissues investigated the effects of rubescenslysin began within a few min, were dose-dependent and practically irreversible. There were only minor differences in sensitivity between various organs and species. The observations indicate that the toxin is relatively nonspecific in its attack on components of cell membranes.