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,1 0 0 To: Washington Field From: Washington Field Re: 279A-WF-222936-USAMRIID, 07/14/2004 experiment to test this, Ivins found thatL::Lhours was too long an incubation time for the blood late. e elieved it was apparent, at least for Sterne and%:::::::::::] that carbon , dioxide in the atmosphere was necessary. In the future, Ivins wanted to try raising the percentage of carbon dioxide to see whether the change increased toxin production. On page 63, dated October 18, 1981, Ivins wrote that he and Perry Mikesell were running tests to determine whether heat assaging.with the Sterne,| I [?:::]and Vollum 1B strains caused the cells to lose a plasmid that encodes toxin antigen or a toxin regulatory protein. On page 72, Ivins speculated that the heat-passaged strains lost a plasmid which: a. encodes for one or more of the toxin antigens b. encodes for a direct genetic positive regulatory element c. encodes-for a protease or a protease control element protease is responsible for cleaving large molecule s! into smaller molecules which can escape the cell. The large molecule s! may not be able to be easily secreted. Two or all three of the antigens may be derived from a single protein of large molecular weight!. Ivins noted that he may wish to look for protease activities in the supernants and cytosols of the parent or heat- passaged strains; he also may wish to look for large molecular weight greater than 100,000! anthrax antigens in the cytosols of the passaged strains. Notebodk4::::::] Notebook:[:::::] issue date January 25, 1983 and entitled contained information on the anthrax toxin EF spore coat protein, the aerosol challenge test, and a PCA test. Entries were made by and an unknown individual. There was no table o contents. On page 8, written March 29, 1983, [;;:;;:;;1conducted an experiment to determine whether aeration ha t e eneficial effect of increasing the EF component of the anthrax toxin- unknown strain!. Results of this experiment were not listed. Page 9, and most of the subsequent pages, contained tables of test results of various experiments. Limited 5 component, tagles of various ex eriment results, an experiment on b7F b6 b7C _ g | i 1 1 1 1 1 1 Y