Role of tryptophan oxidation in peroxynitrite-dependent protein chemiluminescence.

Bovine serum albumin oxidation by peroxynitrite is accompanied by chemiluminescence (Watts et al., Arch. Biochem. Biophys. 317, 324-330, 1995). Peak chemiluminescence during the reaction between bovine serum albumin (with or without fatty acids) and peroxynitrite was not modified in the presence of D2O, suggesting that light emission arising from lipid or protein oxidation was not the result of singlet oxygen formation. Light emission from fatty acid-free albumin increased in the presence of diphenylanthracene (DPA), suggesting that it is a consequence of the fluorescent decay of excited species. Exposure of individual amino acids to peroxynitrite in 50 mM potassium phosphate at pH 8.0 showed that tryptophan is the one that emits most light during oxidation, followed by phenylalanine. Tryptophan chemiluminescence correlated with oxygen consumption. The spin trap N-t-butyl-alpha-phenylnitrone (PBN) inhibited both oxygen consumption and chemiluminescence during tryptophan oxidation, suggesting that the reactions leading to light emission start with the abstraction of a H atom, forming a C-centered radical which in turn adds oxygen. When the oxidation of tryptophan by peroxynitrite was carried out in Tris-HCl instead of potassium phosphate, there was a second oxidative reaction between the peroxide and Tris. Chemiluminescence and oxygen consumption during the oxidation of tryptophan by peroxynitrite was 50% lower in the presence of Tris and in this case PBN did not inhibit chemiluminescence, suggesting that the new radicals formed during the reaction of Tris with peroxynitrite reacted with the amino acyl radicals inhibiting the formation of excited intermediates. Exposure of Tris but not phosphate to peroxynitrite (in the absence of amino acids) also resulted in light emission. In summary, these results suggest that tryptophan is one of the amino acids responsible for light emission during protein oxidation. In addition, this study confirms that Tris buffer is a target for strong oxidants and shows that its oxidation also is accompanied by light emission.

Natural killer activity and antibody-dependent cellular cytotoxicity in patients with non-Hodgkin's lymphoma.

Natural killer (NK) activity and antibody-dependent cellular cytotoxicity (ADCC) of peripheral blood lymphocytes from untreated non-Hodgkin's lymphoma patients were found to be depressed, when tested against the human erythroblastoid cell-line K562. The percentage of active killer (AK) cells and that of target binding cells (TBC) of the patients were also inhibited. Treatment of the patients lymphocytes with interferon (IFN) caused an augmentation in their NK activity which was comparable with that seen in the controls. Lymphocytes from some of the patients and controls were cocultured with K562 cells for production of natural killer cytotoxic factors (NKCF). The NKCF released by the patients lymphocytes showed a reduced lytic activity against K562 target cells. The depression in all the activities reported here showed a correlation with the clinical status of the patients except in the case of ADCC. These results indicate that further characterization of the properties of NKCF will contribute the understanding of the mechanism of NK cytotoxicity.

Long-duration intracavitary infusion of methotrexate with systemic leucovorin protection in patients with malignant effusions.

18 patients with malignant effusions were treated with continuous intraperitoneal, intrapleural, or intrapericardial infusion of methotrexate (MTX) 30 mg/m2 per d combined with simultaneous intravenous administration of leucovorin at a dose rate calculated to yield an equimolar concentration in the serum. In the serum the geometric mean steady-state MTX concentration was 0.95 microM, whereas it was 24 microM in the peritoneal, 213 microM in the pleural, and 434 microM in the pericardial cavities. Mean clearance was 6.6 ml/min from the peritoneal cavity, 2.6 ml/min from the pleural cavity, and 0.14 ml/min from the pericardial cavity. Leucovorin provided sufficient protection to allow the duration of infusion to be escalated from 24 to 120 h before myelosuppression was encountered. Marrow thymidylate synthetase activity was inhibited by an average of 46% compared to 86% inhibition in malignant cells in the effusions. Flow cytometric analysis showed no perturbation of the cell cycle phase distribution of marrow cells. All eight of the evaluable patients have responded: three received no other form of therapy, five also received systemic hormonal or chemotherapy. This study demonstrated that tumors confined to third space body fluids can be given very high concentration x time exposures to MTX with minimal systemic toxicity.