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Upon isolation of DNA from normal eukaryotic cells by standard methods involving extensive proteolytic treatment, a rather homogeneous population of loop-size, double-stranded DNA fragments is regularly obtained. These DNA molecules can be efficiently end-labeled by the DNA polymerase I Klenow fragment, as well as by a 3'- to -5'-exonuclease-free Klenow enzyme, but not by terminal transferase (TdT) unless the ends have been filled up by Klenow, suggesting that dominantly 5' protruding termini are generated upon fragmentation. The filled-up termini were used for cloning the distal parts of the approximately 50 kb fragments. BLAST analysis of the sequence of several clones allowed us to determine the sequence of the non-cloned side of the breakpoints. Comparison of 25, 600 bp-long breakpoint sequences demonstrated prevalence of repetitive elements. Consensus motives characteristic of the breakpoint sequences have been identified. Several sequences exhibit peculiar computed conformational characteristics, with sharp transition or center of symmetry located exactly at the breakpoint. Our data collectively suggest that chromatin fragmentation involves nucleolytic cleavages at fragile/hypersensitive sites delimiting loop-size fragments in a non-random manner. Interestingly, the sequence characteristics of the breakpoints are reminiscent of certain breakpoint cluster regions frequently subject to gene rearrangements.

The appearance of free DNA ends in the chromatin is usually considered an indication of advanced apoptosis. Unexpectedly, the nuclei of non-apoptotic cells derived from mouse thymuses could be specifically labeled by terminal transferase after proteinase K treatment of the fixed, cytocentrifuged samples. Artifactual mechanical or contaminating nucleolytic factors have been ruled out as players in the generation of free DNA ends. The phenomenon was detected in both formaldehyde- and ethanol-fixed specimens, in agarose-embedded fixed cells, and in chromatin spreads. By urea-agarose gel electrophoresis, the average single-strand size of the DNA molecules carrying the free ends was found between 50 and 250 kb. We suggest that ss discontinuities preexisting in the fixed normal cells are unmasked by protease treatment eliciting TUNEL (terminal transferase-mediated nick end-labeling) positivity.

The effects of a loading dose of pyridoxine (100 mg) given intramuscularly or per os to 24 earlier non-supplemented pregnant women at term was investigated. The in vitro oxygen affinity (P50) and the prolactin level in both maternal and newborn blood was sampled. The blood P50 values were measured by a variant of "mixing method". Blood prolactin levels were determined by RIA. After pyridoxine administration, the maternal P50 values increased moderately and the newborns' cord blood P50 values increased significantly when compared with the control group's (number of cases 12) values. The decrease of blood oxygen affinity was most pronounced in the supplemented groups in newborns' capillary blood at the age of five days. The pyridoxine supplementation had no effect on the maternal and the newborns' cord blood prolactin level or on the daily amount of breast milk. Pyridoxine supplementation of the mother at labour may influence favourably the oxygen transport function of the newborn's blood and it may be especially advantageous in early postnatal adaptation disturbances of newborns.