Parentage analysis by endonuclease shattering of hypervariable DNA

Single-locus DNA probes for tandem repeat sequences are now used in conjunction with particular endonucleases to characterize heritable restriction fragment lengths in parentage tests. Southern blots of this type, however, demonstrate only two attributes of an allele: its length and the presence of nucleotide sequences that are complementary to the probe. Not all restriction fragments of the same apparent length that react with the same probe are identical. Differences between comigrating fragments can be detected by the selection of a restriction enzyme that recognizes sites in a subset of the repeat sequences, and the information content of these loci is therefore increased. This report describes a paternity case in which two brothers appeared, after DNA phenotyping using Hinf I, to be the father. A second phenotyping using Hae III excluded one of the brothers.     

Modulation of apoptosis and enhancement of chemosensitivity by decreasing cellular thiols in a mouse B-cell lymphoma cell line that overexpresses bcl-2

Purpose: To determine whether the difference in the apoptosis and clonogenic survival responses to radiation observed between the murine lymphoma cell lines LY-ar, which expresses bcl-2, and LY-as, which does not, was also evident after treatment with chemotherapy agents; and to determine whether clonogenic survival after chemotherapy agent exposure could be diminished by enhancing apoptosis through a decrease in cellular thiols. Methods: Cells were treated with cisplatin, VP-16, or Adriamycin, and apoptosis was determined using a DNA fragmentation assay. Cellular survival was quantified by limiting dilution assay. Intracellular thiols were decreased by maintaining LY-ar cells in cystine/methionine-free medium (CMF medium) for 7 h after drug treatment. Results: LY-as cells were approximately four times more likely to undergo apoptosis than LY-ar cells, having differences in apoptosis of 80% and 20%, respectively, for the agents used. LY-as cells were also more sensitive as measured by cellular survival, with a dose-modifying factor of about 1.8 measured at a 10% survival level. Incubation of LY-ar cells in CMF medium after drug treatment increased apoptosis and reduced clonogenic survival to the levels seen in LY-as cells, except after treatment with VP-16, where the reduction in cell survival was more modest. Conclusions: Decreasing intracellular thiols enhances apoptosis and cell killing in lymphoma cells after exposure to a variety of chemotherapy agents. This may be especially true for tumor cells that overexpress bcl-2, a gene that modifies cellular thiol status and conveys resistance to apoptosis. In this case, decreasing cellular thiols allows killing independent of the expression of bcl-2. Received: 22 September 1998 / Accepted: 9 February 1999     

A role for calcium in regulating apoptosis in rat thymocytes irradiated in vitro.

Thymus-derived lymphocytes undergo death after gamma-irradiation via a pathway termed apoptosis, or programmed cell death. An early step in this pathway is the production of nucleosome-sized fragments of DNA. DNA fragmentation was used as the endpoint in these investigations to examine apoptosis in lymphocytes extracted from the rat thymus and irradiated in vitro. In unirradiated thymocytes the level of DNA fragmentation rose to 15% by the first hour of culture, where it remained approximately constant until the fifth hour. In contrast, thymocytes irradiated with a dose of 2.5 Gy exhibited a large and dramatic increase in DNA fragmentation beginning 2 h postirradiation. DNA fragmentation measured 6 h after irradiation was detected after as little as 0.25 Gy and reached a maximum of 90% with 10 Gy. Metabolic control of DNA fragmentation after irradiation was evidenced by the suppression of DNA fragmentation when thymocytes were incubated with cyclohexamide or actinomycin D. When gamma-irradiated thymocytes were incubated with the Ca2+ chelator EGTA, DNA fragmentation was reduced significantly. BAPTA-AM, a highly specific intracellular Ca2+ chelator, essentially eliminated DNA fragmentation in cells irradiated with 2.5 Gy and, unlike EGTA, eliminated the background level of fragmentation in unirradiated samples. Therefore, our data are consistent with the possibility that Ca2+ serves as a second messenger to induce DNA fragmentation in irradiated thymocytes, suggesting a common pathway for cells prompted to enter apoptosis from seemingly dissimilar interval events.     

Kinetics of cisplatin-induced apoptosis in murine mammary and ovarian adenocarcinomas

There is mounting evidence to indicate that the mode of cell death known as apoptosis plays an important role in cancer therapy. Most supporting observations have come from experiments conducted in vitro, and it is important to extend such studies to in vivo systems. We have therefore evaluated the magnitude and kinetics of apoptosis induction in tumors from mice treated with cisplatin (CP). Two transplantable murine tumors were studied: a mammary adenocarcinoma, MCa-4, and an ovarian adenocarcinoma, OCa-l. Tumor-bearing mice were injected with various doses of CP, and specimens were obtained over several days. Apoptosis was scored by morphometric analysis of histological sections of the tumors using the features characteristic of cells undergoing this mode of cell death. The results showed a significant apoptotic response in both tumors within a few hours after injection of the drug. The kinetics were very broad, with apoptotic cells present over essentially the entire time course studied. Dose-response relationships for CP-induced apoptosis were compared to the tumor response measured in terms of tumor growth delay.     

Effects of medium- and long-chain triglycerides on sleep and thermoregulatory processes in neonates

Sleep processes and body temperature regulation of neonates are never taken into account in the evaluation of nutrients, although these functions are implicated in the regulation of energy metabolism and are influenced by the nutritional state and its metabolic consequences. Medium-chain triglycerides (MCT) are currently used in paediatric units during the first weeks of because they are considered to be a rapid source of energy, easy to assimilate for growing premature infants, whose digestive function is immature. However, no study has described the thermic effect of these nutrients on body temperature regulation and sleep. The present study aimed at analysing the influence of three feeding formulas with different content of MCT on sleep processes and on thermoregulation of neonates fed until desired intake was reached. Whatever the thermal conditions (thermal equilibrium or cool environment), the MCT-fed groups had higher body temperatures and than groups fed without MCT, for whom total sleep time was reduced at thermal equilibrium. In this group, the large amount of quiet sleep seems to favour a strategy of conserving energy . Higher energy expenditure in MCT-fed groups is not harmful to growth rate since nutritional efficiency is even better reflected by a larger body mass gain. The thermic effect of MCT contributes to lessening the vulnerability of neonates exposed to low incubator temperatures.     

Cell cycle-dependent cytotoxicity of alkylating agents: determination of nitrogen mustard-induced DNA cross-links and their repair in Chinese hamster ovary cells synchronized by centrifugal elutriation

Chinese hamster ovary cells were synchronized into the different phases of the cell cycle by centrifugal elutriation and treated with nitrogen mustard (HN2) in order to investigate the role of DNA damage and repair processes in the cell cycle-dependent cytotoxicity of this alkylating antitumor agent. In agreement with previous studies, cell populations enriched in G1 were the most sensitive to HN2, and those enriched in late S phase-G2 were more resistant, as determined by clonogenic assay. Although the variation in surviving fraction through the cell cycle in response to a single dose (3 micrograms/ml; 1.0 h) of HN2 was as great as a factor of 10, complete dose-response curves generated for the most sensitive and most resistant elutriator fractions indicated that such changes could be accounted for by a ratio of D0 values of only 1.4 Cells synchronized by this same method were also analyzed for their relative levels of HN2-induced DNA cross-linking using the sensitive technique of alkaline elution. There was no significant difference in the levels of either DNA interstrand or DNA-protein cross-links induced in the two elutriator fractions described above immediately after the HN2 treatment. When the amount of DNA cross-linking in the two fractions was measured 6 h after treatment, considerable repair had occurred; however, there was no measurable difference in the rate of repair of either type of cross-link (i.e., DNA interstrand and DNA-protein) in the different phases. Differences in DNA damage and repair processes could not therefore be resolved within the confidence limits of available assays and probably cannot account for the differential cytotoxicity of HN2 towards cells in the different cell cycle phases.