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A synthetic aza-cholestane amine was found to be rapidly lethal to Saccharomyces cerevisiae. Lethal action was prevented by pretreating cells with 10m uranyl nitrate. Sublethal concentrations of the steroid prevented the uptake of glucose and alanine. This demonstrated one means by which the steroid affected cell permeability, limiting the growth of the organism. Decreases in viability were related to increases in 260 and 280 mmu absorbing materials. Uranyl protection of viability was found to be related to cellular retention of the ultraviolet-absorbing materials. The steroid, dequadin acetate, and cetyl pyridinium chloride were comparable in lethal action and in causing the leakage of cytoplasmic constituents. Loss of viability and cytoplasmic excretion were detected within minutes after cells were exposed to the steroid. The rate or degree of metabolism of glucose by resting cells did not appear to influence steroid action or the sensitivity of the cells to the steroid.

A quantitative C-reactive protein serological procedure has been developed. By use of this method, which is performed in agar-gel plates, from 2 to 654 mug of C-reactive protein per ml of titrated human serum can be detected. The method is based on the inhibition of a specific C-reactive protein antigen-antibody precipitate formed in agar-gel by the minimal reactive dilutions of each reagent in 48 hr. It is simple, sensitive, and readily reproducible.

The agar-gel precipitin-inhibition test (AGPI) for C-reactive protein determinations proved more sensitive than the capillary tube-precipitin procedure. Furthermore, titers of positive CRP sera were obtained by this AGPI technique. This technique was compared with other indices of infectivity, namely, the white blood cell count and the erythrocyte sedimentation rate. The ease and reproducibility of this recommended test procedure were demonstrated by technicians unfamiliar with this procedure.

Three 4-azacholestanes and two A-norcholestanes were inactivated by 10 and 20% bovine serum and by 1.0, 2.5, and 5.0% sheep blood. The five compounds exhibited hemolytic properties when tested with 2% sheep blood and 2% human blood. These cholestanes inhibited Streptococcus pyogenes and were completely inactivated by 0.1% lecithin. Tween 80 was comparable to lecithin in causing the inactivation of steroids; 1% polyethylene glycol-4000 was inert; 1% Tween 20 and 1.0% Span 20 caused the inactivation of 3beta,4-dimethyl-4-aza-5alpha-cholestane (ND-307). The sodium salts of four fatty acids, oleate, stearate, deoxycholate, and lauryl sulfate (0.1 to 1.0 mg/ml), effectively interfered with the action of ND-307. The steroids appear to have some properties similar to those of antimicrobial surfactants of the cationic type but have certain distinct features.

Six synthetic antimicrobial steroids were examined for indications of their mechanism of action. Dequadin acetate, cetyl pyridinium chloride (CPC), and sodium deoxycholate were studied for comparison. Aerated cells of Sarcina lutea were washed, suspended in 1.06 M sucrose, and converted to protoplasts with 20 mug/ml of lysozyme. Lysis was measured optically at 650 mmu as a decrease in optical density. Screening tests with 50 mug/ml of each compound showed five steroids and CPC to be lytic. Protoplasts were strongly protected from lysis by pretreatment with 0.001 to 0.004 M spermine tetrahydrochloride. Other polyamines, such as spermidine phosphate, were less protective, and putrescine was ineffective. Uranyl nitrate (5 x 10(-4) M) rapidly agglutinated protoplasts and protected them from rupture by the lytic agents. Similar studies with 0.001 to 0.004 M Mg(++) showed varying degrees of protection, which, in most cases, was only temporary. Steroidal lysis did not appear to be related to chelation, since ethylenediaminetetraacetate did not cause lysis alone and antagonized some lytic compounds. Lecithin, Tween 80, Tween 20, and Span 20 at 0.05% exhibited certain effects on protoplast stability. Span 20 strongly prevented lysis by steroids. Tween 20 alone quickly caused protoplast rupture. Lecithin and Tween 80, which also caused lysis alone, interfered with lytic steroids and CPC. The test compounds were both inhibitory and lethal to cells of Sarcina lutea. The results suggest that direct action on cell membranes may be chiefly responsible for the antimicrobial properties of the steroids.

An application of the agargel precipitin-inhibition technique of Ray and Kadull detects the C-reactive protein present in the acute-phase human sera. The sensitivity of this procedure is compared with the capillary tube-precipitin method of Selman and Halpern.