NATURE OF SPLEEN-BONE MARROW RADIATION RECOVERY FACTOR 



The tentative conclusion drawn from the above experimental data is that 

 the radiation recovery factor, as present in immature LAf^ mouse spleen, is 

 a particulate, subcellular macromolecular deoxyribonucleoprotein complex. 



Most spleen nuclear preparations are not entirely devoid of cellular 

 bodies — so far as one can judge by microscopic examination. It becomes 

 particularly relevant, therefore, to attempt to ascertain whether or not the 

 protective activity of such spleen nuclear fractions can be ascribed to the 

 presence of presumably intact, viable cells, which could possibly 'seed' 

 the depleted haematopoietic system of the irradiated animal and thereby 

 elicit renewed haematopoiesis, as a consequence of functional repopulation. 

 Furthermore, the conclusions from the aforementioned enzymatic analysis 

 studies rest in part on the supposition that viable, living cells are, in general, 

 resistant to the enzymatic action of externally-added DNA-ase or of trypsin, 

 whereas dead cells and subcellular fractions are susceptible to the action 

 of these enzymes. Evidence for the validity of this general supposition has 

 been provided by the studies of Northrup^ and of Bonus Jensen^ with 

 trypsin ; and of Ely and Rossi" ^.[^i^ DNA-ase. In the present study we 

 have attempted to obtain further experimental data on this aspect of the 

 problem — specifically to determine whether fresh intact spleen cells are 

 substrates for enzymatic action of DNA-ase. Release from the cells of 

 soluble material absorbing at 260 mji. was employed as the criterion for 

 enzyme action, since it is known that DNA-ase liberates DNA from its 

 protein-bound state in the cell. 



EXPERIMENTAL 



Fresh spleen cells were obtained by gently washing minced mouse spleen 

 fragments with 0-14M NaCl solution containing 1 mg dextrose per ml. 

 As observed microscopically, the cell suspension was clean and homogen- 

 eous ; no disrupted cells could be seen. Five millilitres of the suspension, 

 containing 5-1 X lO** cells per ml, and added Mg^ + (0-005M), was incubated 

 with crystalline deoxyribonuclease (DNA-ase) (50 [i. per ml) at 25° G for 

 30 minutes. The tubes were then chilled in ice and centrifuged at 5°C. 

 An aliquot of the original cell suspension incubated without added enzyme, 

 served as a control. The resulting supernatant fluids were carefully re- 

 moved, and their ultraviolet absorption spectra determined with a Beckman 

 quartz spectrophotometer. The absorption data are shown in Figure 1. 

 It is evident that little, if any, material with specific absorption at 260 m[j, 

 was released from the cells by the action of DNA-ase under the conditions 

 of these experiments. Peak absorption in the supernatant occurred at 278 m[j.. 

 and not at 260 m/x. The actual amount of DNA liberated under these conditions 

 was calculated as follows : According to Mizen and Feterman^^ one milli- 

 gramme (wet weight) of spleen tissue contains 22-9 X 10^ cells. From 

 previously reported analytical data obtained in this laboratory, the concen- 

 tration of DNA in the spleen of immature LAf^ mice is 19-7[a.g DNA per 

 mgi-. On this basis the calculated cellular DNA content is 8-6 X 10"^ mg. 

 per cell ; therefore 5-1 X 10** cells contain a total of 43-9ixg DNA. The 

 observed net change in optical density at 260 m[j. {i.e. 0-018) in the present 

 experiment, corresponds to a value of 0-7 jag DNA per ml, as determined 

 from a DNA standardization curve. The net liberation of DNA by the 



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