DNA damage and cell cycle checkpoints

DNA is prone to numerous forms of damage that can injure cells and impair fitness. Cells have evolved an array of mechanisms to repair these injuries. Proliferating cells are especially vulnerable to DNA damage due to the added demands of cellular growth and division. Cell cycle checkpoints represent integral components of DNA repair that coordinate cooperation between the machinery of the cell cycle and several biochemical pathways that respond to damage and restore DNA structure. By delaying progression through the cell cycle, checkpoints provide more time for repair before the critical phases of DNA replication, when the genome is replicated, and of mitosis, when the genome is segregated. Loss or attenuation of checkpoint function may increase spontaneous and induced gene mutations and chromosomal aberrations by reducing the efficiency of DNA repair. Defects in checkpoint control have been seen in certain hereditary cancer syndromes and at early stages of cell transformation. Mutations in checkpoint control genes therefore may contribute to the genetic instability that appears to drive neoplastic evolution.     

Studies on the formation and stability of aminoacyl-tRNA synthetase complexes from Ehrlich ascites cells

Nine aminoacyl-tRNA synthetases from Ehrlich ascites cells were examined with respect to their ability to be isolated as high molecular weight complexes, soluble enzymes, and ribosome-bound enzymes. Several different methods were employed for cell homogenization and enzyme isolation, with particular attention paid to the effects of hypotonic, isotonic, and hypertonic buffers on enzyme isolation. The binding of all synthetases to ribosomes was eliminated if the low ionic strength of the isolation buffer was raised to isotonic levels. In contrast, neither the ionic strength or composition of the buffers, nor the procedures used for cell homogenization or enzyme isolation had any significant effect on the isolation of the high molecular weight synthetase complex. Certain enzymes (lysyl-, methionyl- and isoleucyl-tRNA synthetases) formed very stable complexes and high molecular weight species were the predominant forms of these enzymes under all conditions of cell homogenization and enzyme isolation. Other enzymes (glycyl-, tyrosinyl- and threonyl-tRNA synthetases) formed complexes very weakly, if at all, and always appeared predominately in the soluble enzyme fraction. Isolated soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases did not associate to form significant amounts of complex upon re-isolATION, SUGGESTING THAT A COMPONENT NECESSARY FOR COMPLEX FORMATION WAS MISSING FROM THE SOLUBLE ENZYME FRACTION. However, the soluble forms of these enzymes, but not the glycyl-, tyrosinyl- and threonyl-tRNA synthetases, did for complexes when mixed with ribosomal RNA or polyuridylic acid. Preliminary experiments showed no significant differences between the complexed and soluble forms of the lysyl-, methionyl- and isoleucyl-tRNA synthetases with respect to Km values or ability to charge different isoaccepting tRNAs.     

Metabolic balance studies in premature infants

Many investigators over the past three decades have successfully conducted traditional metabolic balance studies in efforts to determine the retention rates of key nutrients important to the optimal growth of preterm infants. Differences in methodologies discussed in this review may explain the inconsistent results of balance studies reported for some nutrients, particularly calcium. These methodologic differences include (1) variability in nutrient intake and nutritional course prior to the balance period, (2) differences in the method of stool collection (with and without markers), (3) use of single versus repeated balance periods, and (4) different durations of balance periods. The data presented here suggest that the variability of net fat absorptions among VLBW infants was decreased when an acclimation period of nutrient intake supportive of growth was provided prior to a metabolic balance study. In addition, the use of markers affected the estimates of net calcium absorption but not fat absorption. Additional factors that may influence net calcium absorption and methods for the estimation of calcium absorption in VLBW infants warrant further investigation. This review describes the methods of specimen collection for metabolic balance studies in VLBW preterm infants that demonstrated reproducible data. The recent application of stable isotope methodology to metabolic balance studies can be extremely advantageous in identifying the rates of nutrient absorption versus endogenous secretion in the GI tract.