EXPERIMENTAL ANALYSIS 



could be applied to the inhibition of the mitotic spindle either if the 

 orientation and disorientation of the spindle proteins themselves in- 

 volves a reversible denaturation, or if their state of alignment is under 

 enzymic control. The latter must of necessity be true if Svv^ann is correct 

 in his thesis (p 126) that the metaphase spindle represents a dynamic 

 equilibrium. 



If it were possible to find a suitable example of a mitotic spindle 

 which was insensitive to the effects of hydrostatic pressure per se it 

 would be of great value to investigate whether the action of the various 

 agents which affect cells during metaphase could be annulled by 

 pressure, and, if so, whether pressure-sensitivity of the chemical in- 

 hibition of the spindle and of bacterial luminescence were correlated. 

 Again, it might be rewarding to seek evidence for cytoplasmic denatura- 

 tion in the achromatic figure by using Kopac's method, based on the 

 'Devaux effect'. Injected oil-drops wrinkle if proteins adsorbed at the 

 oil-cytoplasmic interface are denatured (Kopac^^^^). 



Reversible denaturation of proteins during mitosis has been suggested • 

 as the reason for the sensitivity of dividing cells to sulphydryl reactants 

 (Rapkine^^'), for the number of reactive — SH groups is increased in 

 proteins after denaturation. A number of these substances have been 

 shown to act as metaphase inhibitors. For instance, mitotic arrest at 

 this point was demonstrated by Ludford^^^ with sodium cacodylate on 

 tissue cultures, and by Dustin (P.)268 with sodium arsenite in the bone 

 marrow of the mouse. Hughes has shown that the development of the 

 spindle can be prevented when a cell in early prophase in a tissue culture 

 is treated with either iodoacetamide^^^ or chloracetophenone.^^i If the 

 development of the spindle involves reversible denaturation of the 

 constituent protein micelles, its sensitivity to sulphydryl reactants 

 would suggest that these agents can act directly upon spindle material. 

 So far, the cytochemical evidence for the presence of free — SH groups 

 in the spindle is restricted to the observations of Brachet^^s on the 

 reaction of the spermatocytes of Stenobothrus with the nitroprusside 

 reagent. On this point he says : 'Au moment des cineses de maturation, 

 le cytoplasme reagit moins intensement et le fuseau prend un ton plus 

 sombre; si, a cet instant, la cellule est ecrasee, on pent retrouver le 

 fuseau a I'etat libre et constater qu'il donne une vive reaction.' 



On the other hand, Chalkley^^o f^^ds that the nucleus of Amoeba 

 loses its positive nitroprusside reaction when the nuclear membrane 

 disappears at the end of prophase, and that only 'after fission is com- 

 plete, and the nucleus is reorganized the reaction can again be evoked 

 in the nucleus'. It would be a notable advance in cytochemistry if 

 there were developed a method for revealing sulphydryl groups of 

 sufficient sensitivity and precision to map out their distribution in a 

 number of dividing cells. There is some doubt whether the method of 



195 



