Some Remarks and Questions on Metabolic Patterns in the Family of Bone Cells 17 



makes clear that the NADP probably acts in the Malate-Pyruvate region. Detailed 

 analysis of the reaction-rates involved in the conversion of Fumarate to Lactate and 

 CO., indeed revealed, that the NADP effect concerns the activity of the NADP- 

 Malate oxidase (Malic enzyme). Cohn and Griffith conclude that their bone pre- 

 paration contains a pyridine nucleotide stimulated pathway for decarboxylation of 

 Dicarboxylic acids. 



I hope to have impressed upon you the central position the pyridine nucleotides 

 take in recent concepts concerning bone metabolism. However I would do scant 

 justice to todays knowledge, by concealing important information concerning a 

 possible influence the nucleus of the bone cell can have upon the processes we have 

 discussed. It is very well possible that this information will throw, among others, a 

 new light upon the pyridine nucleotide problem. 



In this connection I will deal now with recent investigations of Gaillard, con- 

 cerning the combined effect of PTE and Actinomycin D or Puromycin on cultivated 

 radius rudiments (Gaillard, 1965). Full details being published I give only the 

 essentials. From his former work we know that PTE induces dramatic morphological 

 and functional changes in radii of 15 day-old mouse embryos (Gaillard, 1960, 

 1961). The morphological changes concern the bony shaft, the cartilage and the 

 connective tissue proper. Among other changes, seven fully reproducible morphologi- 

 cal phenomena were found to be characteristic for the action of PTE on radius 

 explants; they are listed in fig. 4. Now on the basis of the generally accepted opinion 

 that acute and chronic conditions of hyperparathyroidism are among others charac- 

 terized by a defective synthesis of specific proteins by osteogenic cells, it was decided 

 to study the combined effect of PTE and some inhibitors of protein synthesis. To put 

 it in more detail, it was thought that by eliminating the central or peripheral parts 

 of the cellular protein synthetic apparatus, information could be obtained concerning 

 the role of this apparatus in the development of the hormone effect. This approach 

 was not based on any preconceived idea concerning possible specific interaction 

 between hormone and inhibitor whatsoever. On the contrary I even foresee that 

 because of this unspecificity this type of approach will prove applicable to other 

 fields as well. 



The antibiotic Actinomycin D was chosen because it is known to interfere with 

 the continuous flooding of the cytoplasm with nuclear m-RNA. Puromycin interferes 

 with the last steps of protein formation at the ribosomal level. Concerning the way 

 of application of these antibiotics a few remarks have to be made: Actinomycin was 

 given in a concentration of 0.005 }' per ml of the medium for 6 hours prior to the 

 confrontation of the explants with PTE 0.1 lU/ml, Puromycin however was given 

 simultaneously with the PTE in concentrations of 0.1 or 0.01 ;'/ml. In both set-ups 

 the whole experiment lasted for 48 hours. Finally it is important to known that the 

 concentrations were below the level of noticeable toxicity. The concentration of PTE 

 was put at 0.1 lU/ml because this is a "borderline" concentration below which the 

 intensity of the phenomena falls down steeply. 



In this situation Actinomycin D proved to inhibit the development of all the 

 morphological phenomena of hormone action listed in Fig. 4. However Puromycin 

 proved able to interfere with the development of the phenomena 1 ± 4 only. In 

 particular the hormone-induced proliferation of connective tissue inside the shaft 

 was found to escape the inhibitory action of Puromycin entirely. 



3'''' Eiirop. Symp. on Cal. Tissues 2 



