Nuclear chromatin changes during post-natal myocardial development.

The proliferative capacity of rat myocardium declines rapidly during the first few weeks of post-natal life. In order to gain insight into the mechanisms involved in this decline, we studied the structure and function of nuclear chromatin from isolated rat myocardial cells during post-natal growth. Chromatin template activity decreased progressively (7.5 +/- 0.3 pmol [3H]UTP/microgram DNA per min at age 5 days compared to 2.2 +/- 0.1 pmol [3H]UTP/microgram DNA per min at age 6 months) and was associated with a decrease in the number of transcription initiation sites. This decline was accompanied by changes in chromatin structure as evidenced by: (a) decreased susceptibility to DNAase I digestion with advancing age, (b) decreased poly-L-lysine binding (60% decrease between day 5 and six months of age), (c) progressive decline in positive ellipticity of circular dichroism spectra between 250--300 nm, and (d) derivative melting profiles showing a decrease in DNA regions bound by non-histone proteins and concomitant increase in histone-bound regions. The protein composition of myocardial chromatin also changed during post-natal development, chiefly due to a progressive increase in the histone/DNA ratio. These results indicate substantial changes in the organization and functional capacity of myocardial chromatin during early post-natal growth. These changes accompany, and may contribute to, the restriction in the proliferative capacity of myocardial cells.

Adenosine 3',5'-monophosphate-dependent protein kinases of myocardial non-histone nuclear proteins.

Myocardial acidic non-histone nuclear proteins (NHPs) contain endogenous protein kinase activity. Phosphocellulose chromatography of purified NHPs identifies nine separate peaks of protein kinases which can phosphorylate both endogenous and exogenous substrates to a variable degree; endogenous NHPs are the best substrates. Cyclic AMP-stimulated protein kinase induced phosphorylation of endogenous and exogenous substrates; the extent of this stimulation varied according to the protein kinase fraction and substrate used. Cyclic AMP also enhanced NHP-induced stimulation of RNA polymerase activity. This enhancement was dependent on protein kinase-induced phosphorylation of NHPs since it was prevented by alkaline phosphatase pretreatment. It is concluded that nuclear protein kinases regulate myocardial RNA synthesis by enhancing phosphorylation of NHPs and that this regulation is under cyclic AMP control.

Reactions of bis(thiosemicarbazonato) copper(II) complexes with tumor cells and mitochondria.

The respiration of Ehrlich ascites tumor cells is inhibited by 3-ethoxy-2-oxobutyraldehyde bis (thiosemicarbazanato) copper (II). State 3 oxidative phosphorylation in mitochondria from tumor cells is also inhibited, with the effect more pronounced using glutamate or pyruvate-malate as substrates than with succinate. The disruption of oxidative phosphorylation in bovine heart mitochondria is qualitatively similar. The principal site of inhibition is in coupling site one, energetically between the electron transport site chain and the locus of uncoupling by 2,4-dinitrophenol. This appears to contain thiol groups which are oxidized by the complex. For a series of bis (thiosemicarbazonato) copper complexes, the extent of inhibition of heart mitochondrial oxidative phosphorylation is correlated with the reduction potentials of the complexes and with their in vitro cytotoxic effects against Walker 256 carcinoma tumor cells.