Early in the investigations on interferon, Isaacs and Lindenmann obtained suggestive evidence that the nucleic acid of the virus was responsible for the stimu- lation of interferon production in cells. Recently, Rotem, Cox, and Isaacs2 reported that RNA3 of animal source, heterologous to the tissue in which it was tested, was also able to produce interferon. This observation provided an explana- tion for the results of O'Dell, Wright, and Bieter,4 who as early as 1953 showed that yeast RNA inhibited MM virus in mice. In our laboratory we have continued the investigation of the properties of stato- lon, an antiviral agent produced by the mold Penicillium stoloniferum.5 It has proved to be prophylactically active against a wide spectrum of viruses in tissue culture and in animals.6-3 Statolon is a complex anionic polysaccharide with a relatively high content of galacturonic acid, which is the primary contributor to its Early in the investigations on interferon, Isaacs and Lindenmann obtained suggestive evidence that the nucleic acid of the virus was responsible for the stimu- lation of interferon production in cells. Recently, Rotem, Cox, and Isaacs2 reported that RNA3 of animal source, heterologous to the tissue in which it was tested, was also able to produce interferon. This observation provided an explana- tion for the results of O'Dell, Wright, and Bieter,4 who as early as 1953 showed that yeast RNA inhibited MM virus in mice. In our laboratory we have continued the investigation of the properties of stato- lon, an antiviral agent produced by the mold Penicillium stoloniferum.5 It has proved to be prophylactically active against a wide spectrum of viruses in tissue culture and in animals.6-3 Statolon is a complex anionic polysaccharide with a relatively high content of galacturonic acid, which is the primary contributor to its polyanionic character.5 Structurally, RNA also is a polyanion. The finding by Rotem et al.2 that heterol- ogous RNA stimulated the production of interferon suggested that the mechanism of action of statolon possibly lay in its capacity to stimulate the production of interferon. This possibility has been investigated, and the results presented in this paper show that statolon induces the production of a viral inhibitor in cells, and that this inhibitor has the properties of interferon. Materials and Methods.-Statolon: A freeze-dried preparation was used that contained 89.47% sodium bicarbonate and glucose, in equal proportion.4 The statolon was dissolved in water, 50 mg/ml, and the pH adjusted to 7.5 with CO2. The solution of statolon was diluted 1:300 with medium 199, bringing the concentration of active statolon to 17.5 tAg/ml. Virus: Vaccinia virus, strain V-l, was obtained through the kindness of Dr. J. Lindenmann, University of Florida. Stock virus was prepared in HeLa cells and was stored in sealed glass ampoules at -60?C. Cells: Primary cultures of chick embryo (CE) cells from 1-day-old embryos were employed. Production of induced inhibitor: 70 X 106 CE cells in 30 ml of medium 199 containing 4% calf serum were added to rectangular pint bottles with a surface area of 100 cm2 and incubated 48 hr. The medium was then replaced with 30 ml of a statolon solution (17.5 ,ug/ml), the cells were incubated another 24 hr, and the fluid was harvested. Residual statolon was removed from the fluid by concentrating it fivefold in dialysis bags by means of polyethylene glycol, then lowering the pH to 4.5 and centrifuging for 90 min at 105,000 X g in the Spinco (Model L) centrifuge. The supernatant fluid was removed from the tubes and the pH was adjusted to 7.3. Assays showed that statolon solutions that had never been in contact with cells no longer possessed inhibitory activity after such treatment. After removal of the statolon, fluids were diluted 1:5 to the initial
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Aug 1, 2021
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