Thermal stability of PNA/DNA and DNA/DNA duplexes by differential scanning calorimetry.

Thermodynamics of the thermal dissociation transitions of 10 bp PNA/DNA duplexes and their corresponding DNA/DNA duplexes in 10 mM sodium phosphate buffer (pH 7.0) were determined from differential scanning calorimetry (DSC) measurements. The PNA/DNA transition temperatures ranged from 329 to 343 K and the calorimetric transition enthalpies ranged from 209 +/- 6 to 283 +/- 37 kJ mol(-1). The corresponding DNA/DNA transition temperatures were 7-20 K lower and the transition enthalpies ranged from 72 +/- 29 to 236 +/- 24 kJ mol(-1). Agreement between the DSC and UV monitored melting (UVM) determined transition enthalpies validated analyzing the UVM transitions in terms of a two-state transition model. The transitions exhibited reversibility and were analyzed in terms of an AB = A + B two-state transition model which yielded van't Hoff enthalpies in agreement with the transition enthalpies. Extrapolation of the transition enthalpies and free energy changes to ambient temperatures yielded more negative values than those determined directly from isothermal titration calorimetry measurements on formation of the duplexes. This discrepancy was attributed to thermodynamic differences in the single-strand structures at ambient and at the transition temperatures, as indicated by UVM measurements on single DNA and PNA strands.

Preparation and in vitro studies of [125I]IUDR-T101 antibody conjugate.

Idoxuridine labeled with 125I was conjugated to polylysine. This conjugate was then coupled to the carbohydrate side chains of T101 monoclonal antibody (anti-CD5). The immunoreactivity, cell retention, cytotoxicity, and intracellular localization of this conjugate was tested in CCRF-CEM cells (CD5 positive). The conjugate had 68% immunoreactivity. The retention of 125I by CCRF-CEM cells was higher for the conjugate than for T101 directly labeled with 125I and more of it localized in the nucleus than did the 125I-labeled T101. The 125I IUDR-polylysine-T101 conjugate was more cytotoxic than the 125I-labeled T101. In conclusion, the conjugation of [125I]IUDR to T101 is feasible, and preferential targeting of the 125I to the nucleus is obtained.

Prevention of radiolysis of monoclonal antibody during labeling.

UNLABELLED: Monoclonal antibody may undergo loss of immunoreactivity due to radiation damage when labeled with large amounts of 131I or 90Y for therapy. Our aim was to develop a method to protect an antibody during the labeling procedure. METHODS: As a model we used T101, a murine monoclonal antibody directed against CD5 antigen. Iodine-125-T101 (100 micrograms, 1 ml) was mixed with 90Y-DTPA (0.64 MBq to 165.9 MBq) for 24 hr in order to deliver doses of 5 Gy to 1280 Gy to the solution. In separate experiments, 125I-T101 solutions were irradiated with 60Co external beam delivering radiation doses of 40 Gy to 1280 Gy. The effect of radiation on T101 immunoreactivity was tested by using the CCRF-CEM cell line, and the bound T101 radioactivity was determined. In a final experiment, we directly labeled a DTPA conjugated T101 using 561 MBq of 90Y under conditions delivering approximately 640 Gy to the solution. Previously used radioprotectants including human serum albumin, cysteamine and glycerol were evaluated. We focused on ascorbic acid because it is an FDA approved drug that does not interfere with the radiolabeling process. RESULTS: The immunoreactivity of 125I-T101 was approximately 83%, but at 640 Gy the immunoreactivity decrease to 7%. In contrast, in the presence of radioprotectants this decrease could be abrogated. External irradiation also showed a dose dependent decrease in immunoreactivity to as low as 0.3% at 1280 Gy. Adding ascorbic acid (5.5 mg/ml) to the solutions prior to the irradiation largely abrogated this decrease. The immunoreactivity of T101 labeled with 90Y without protectant showed 46% immunoreactivity whereas, in presence of ascorbic acid (11 mg/ml) full retention of immunoreactivity was observed. CONCLUSION: Various radioprotectants can successfully prevent the loss of immunoreactivity or breakdown as a result of radiolysis. Ascorbic acid is an effective radioprotectant that can be used to prevent loss of antibody immunoreactivity during the labeling process.

Administration of endotoxin, tumor necrosis factor, or interleukin 1 to rats activates skeletal muscle branched-chain alpha-keto acid dehydrogenase.

Protein catabolic states (i.e., sepsis and trauma) are thought to be associated with accelerated oxidation of branched-chain amino acids (BCAA). Branched-chain alpha-keto acid dehydrogenase (BCKAD), the rate-limiting enzyme for BCAA oxidation by muscle, is regulated by phosphorylation/dephosphorylation. Skeletal muscle BCKAD was only 2-4% active in control rats. Intravenous injection of Salmonella enteritidis endotoxin (0.25-10 mg/kg) did not change total BCKAD activity, but increased the percent active enzyme in muscle three- to four-fold in 4-6 h. Identical results were observed in adrenalectomized rats pretreated with one dose of alpha-methylprednisolone (2.5 mg/kg i.p.) 30-60 min before saline or endotoxin injection, indicating that endotoxin's effect was not mediated by hypersecretion of adrenal hormones. Cortisone pretreatment of normal rats (100 mg/kg per d) for 2 d prevented endotoxin-induced activation of muscle BCKAD, suggesting that endogenous secretion products mediated BCKAD activation by endotoxin. Human recombinant tumor necrosis factor-alpha and/or IL-1 beta or alpha (50 micrograms/kg) increased muscle BCKAD activation two- to fourfold in normal rats 4-6 h after intravenous injection. We conclude that cytokine-mediated activation of muscle BCKAD may contribute to accelerated BCAA oxidation in septicemia.