Reconstitution of apo-superoxide dismutase by nitric oxide-induced copper transfer from metallothioneins

Little is known about copper transfer from Cu-metallothionein (Cu-MT) to various target proteins, such as apo-SOD, and the potential role of redox mechanisms in this transfer. We studied Cu transfer from Cu-MT to apo/Zn-SOD in a cell-free model system and found that Cu(5)-MT and Cu(10)-MT were able to reconstitute SOD activity only in the presence of a nitric oxide donor, (Z)-[N-(3-ammoniopropyl)-N-(n-propyl)amino]diazen-1-ium++ +-1,2-diolate (NOC-15). The percentage of reconstitution by Cu(5)-MT and Cu(10)-MT was 34 and 83%, respectively, compared with that reconstituted by free Cu alone. A Cu chelation assay using bathocuproine disulfonate (BCS) showed that NOC-15 induced release of free Cu from Cu(10)-MT but not from Cu(5)-MT. The transfer of Cu from Cu-MT to apo/Zn-SOD was not accompanied by enhanced Cu-dependent generation of ascorbate radicals or hydroxyl radicals as measured by EPR spectroscopy. We found a 70% decrease in the number of 2,2'-dithiodipyridine titratable SH groups on MT after incubation with NOC-15. Overall, our results suggest that Cu-MT could potentially function in a nitric oxide-dependent pathway for the delivery of Cu to apo-SOD in copper-challenged cells.     

Antioxidant and antiapoptotic function of metallothioneins in HL-60 cells challenged with copper nitrilotriacetate

Antioxidant activity is believed to be an important intracellular function of metallothioneins (MT), yet the specific mechanisms of their antioxidant action are not known. Under conditions when cells are challenged with elevated concentrations of free copper as a result of metabolic disturbances or environmental and occupational exposures, MTs may be ideally suited for antioxidant function as effective copper chelators. In the study presented here, we tested this hypothesis using a recently established model of copper nitrilotriacetate-induced oxidative stress in HL-60 cells. Since copper-induced oxidative stress triggers apoptosis, we further investigated antiapoptotic function of MTs in HL-60 cells. Using a Sephadex G-75 chromatographic partial purification of MTs from cell homogenates with subsequent immuno-dot-blot assay, we showed that zinc pretreatment yielded a pronounced induction of MTs in HL-60 cells. We report that zinc-induced MTs were able to (i) completely bind intracellular copper, (ii) completely quench redox-cycling activity of copper, (iii) significantly inhibit copper-dependent oxidative stress in membrane phospholipids, and (iv) prevent copper-dependent apoptosis and its characteristic biochemical features (cytochrome c release from mitochondria into cytosol, caspase-3 activation, and externalization of phosphatidylserine in plasma membranes). In separate experiments, we used lung fibroblasts derived from MT1, MT2 knockout mice (MT(-)(/)(-)) and MT wild-type (MT(+/+)) mice. ZnCl(2) pretreatment resulted in a more than 10-fold induction of MTs in MT(+/+) cells, whereas the MT content in MT(-)(/)(-) cells remained low, at levels approximately 100-fold lower than in their MT wild-type counterparts. MT(-)(/)(-) cells were very sensitive to Cu-NTA and, most importantly, showed no response to ZnCl(2) pretreatment. In contrast, MT(+/+) cells were relatively more resistant to Cu-NTA, and this resistance was remarkably enhanced by ZnCl(2) pretreatment. Combined, our results demonstrate that metallothioneins function as effective antioxidants and an antiapoptotic mechanism in copper-challenged HL-60 cells.     

The effect of burn wound size on ureagenesis and nitrogen balance.

Hypermetabolic burn patients are frequently in negative nitrogen balance despite provision of estimated caloric needs. We studied 18 thermally injured adult patients in order to evaluate the relationship of burn wound size to urea production and nitrogen balance. We selected data from 147 patient-days when the patients received 100 +/- 25% of their estimated caloric needs. Three significantly different burn size groups (by body surface area [BSA]) were identified by calculation of the catabolic index (CI): group 1, 0-10% BSA (CI = -0.1); group 2, 11-30% BSA (CI = 6.4); and group 3,31-60% BSA (CI = 10.5). The urine urea nitrogen (UUN) for groups 1,2, and 3 was 11.1, 18.9, and 25.3 gm/day, and nitrogen balance was 1.0, -3.9, and -5.8 gm/day, respectively. When nitrogen was given in a calorie:nitrogen ratio of 150:1, only those patients in group I were able to achieve positive balance. We conclude that large burn wounds are associated with increased ureagenesis and impaired nitrogen retention. The protein intake, at the customary calorie:nitrogen ratio of 150:1, may not provide adequate nitrogen to achieve equilibrium, even when energy demands have been met, in patients with burn wounds greater than 10% BSA.     

Induction of leukemia in Rauscher virus-resistant mice by mixed infection with M. arthritidis and Rauscher virus]

At 2-10 months after combined infection with Rauscher virus and M. arthritidis of mice (C57BL/6XA/He)F1 resistant to this virus 14 out of 23 animals developed leukemia morphologically identical to Rauscher leukemia induced in sensitive mice. In control groups of similar animals infected with virus alone or mycoplasma alone not a single case of leukemia developed. As a result of serial intraperitoneal passages in syngeneic mice of cells of leukemias primarily induced by mixed mycoplasma-virus infection 3 transplantable leukemia strains were obtained the cytological picture of which was similar with the original. Upon intraperitoneal and subcutaneous inoculations of leukemic cells generalized leukemia develops as well as a local transplant under the skin or in the abdominal wall at the site of needle puncture.     

Studies of the ultrastructure of spermatozoa under normal and pathological conditions

The ultramicroscopic structure of the normal sperm and pathologic sperms obtained from oligospermic and necrospermic semens has been studied. It has been found that: (a) in oligospermia, a marked cytoplasmic zone surrounds the head, which contains remnants not only of the endoplasmic reticulum but also of the Golgi-apparatus and mitochondria: (b) in necrospermia the fibrillar elements of the tail is impaired. The obtained results allow the conclusion to be made that in these ejaculates the majority of spermatozoa is still in the process of maturation and therefore not apt for fertilization.     

Advances in the immunodiagnosis of parasitic infections

Summary In summary, with the availability of commercial reagents, the growth of parasitic serology is rapidly accelerating. I believe the diagnostic work being performed today represents an investigation of the tip of the iceberg. Unless some policy of evaluation and certification is initiated, we risk the appearance of poor reagents in the diagnostic laboratory and the publication of critical articles suggesting that our tests are nonspecific and useless.Technical developments have brought us to the full or partial automation of serologic tests. Research on the purification and isolation of parasitic antigens has not kept pace with the development and evaluation of serologic methodology. Evaluation of sophisticated methods, such as radioimmunoassay, for the diagnosis of parasitic infections is only beginning to appear in the literature. Greater emphasis must be placed on the use of such sensitive methods in the diagnostic laboratory.With the exception of the Asian workers, who have reported the recovery of antigen in the urine of patients with parasitic diseases, and recent reports of antigen in the serum of patients with schistosomiasis in the United States, the entire focus of parasitic serology has been on the detection of antibodies. Since parasitic antigens have been shown to circulate in the serum and body fluids of individuals with a number of parasitic infections, both specific and sensitive methods must be evaluated for their detection.In the serology of parasitic infections, the diagnosis of protozoan infections is most highly developed. For the helminth infections we require enhanced sensitivity and specificity, especially in areas where more than one species infects man. The serology of nematode infections requires the most work. Toward this end, we have to encourage further work on the separation and purification of parasitic antigens for use in the diagnostic laboratory.Presented at the 3rd International Congress of Parasitology, Munich, Germany, 25–31 August 1974.