Spectra and structure of alpha-tocopherol radicals produced in anoxic media.

Electron spin resonance and flash photolysis studies have been combined to determine, amid conflicting reports from radiolysis studies, the spectrum for the phenoxyl radical of alpha-tocopherol. Triplet state ketones were used to abstract the alpha-tocopherol phenolic hydrogen in order to obtain both the transient ESR spectrum and optical spectrum. Analysis of the ESR data yields g = 2.00469, a(H, 5-CCH3) = 6.04G, a(H,7-CCH3) = 4.51G, a(H, CH2) = 1.38G, a(H', CH2) = 1.49G, and a(H,8-CCH3) = 0.89G. The g factor shows large spin population on oxygen in this radical and demonstrates conclusively involvement of the phenoxyl radical. Both spectra were in agreement with those produced by radiolysis of alpha-tocopherol in N2 saturated EtOH. While the spectral characteristics of the phenoxyl radical now appear to be clarified, uncertainties remain concerning optical spectra for radiolysis of alpha-tocopherol in air- and oxygen-containing systems.     

Plasminogen activator: The major secreted neutral protease of cultured skeletal muscle cells

Clonal mouse skeletal muscle cells which differentiate in culture and from synpases with neuronal cells were found to secrete high levels of protease activity as measured with an ¹²⁵I-fibrin assay. The secreted proteolytic activity was more than 90% dependent upon the presence of plasminogen in the medium, and had a pH optimum at 7 to 8. This activity was not inhibited by n-ethylmaleimide, pepstatin, EDTA, or EGTA. At millimolar concentrations, greater than 90% inhibition was obtained with either soybean typsin inhibitor, epsilon aminocaproic acid, Trasylol, or leupeptin. Almost complete inhibition occured with 1 mM diisopropylfluorophosphate suggesting the presence of a serine residue at the catalytic site. In contrast to the high levels of secreted activity, a lower steady-state level of cell-associated protease activity was detected in cell lysates. The high level of plasminogen activator secreted into the medium of cultured muscle cells suggests a role for such extracellular protease activity in myogenesis during development and remodeling following muscle injury. Such information may be useful in understanding the initial degeneration of neuromusclar contacts in experimental and pathologic denervation.     

Sensitive gas chromatographic assay for the quantitation of bretylium in plasma,urine and myocardial tissue

A sensitive analytical method has been developed for the quantitation of bretylium in plasma, urine and myocardial tissue. Bretylium and the internal standard, UM-360 (o-iodobenzyltrimethylammonium), are extracted and isolated as the iodide salts. Sodium benzenethiolate is added and the mixture heated to 100° for one hour. This results in the formation of 2-bromobenzyl phenyl thioether and 2-iodobenzyl phenyl thioether, which can be separated and quantitated by gas chromatography. Good reliability and reproducibility can be obtained using electron-capture detection with quantities of bretylium as small as 1 ng.     

Involvement of smooth and coated vesicles in the function of the contractile vacuole complex of some cryptophycean flagellates

The contractile vacuole complex of cryptophycean flagellates comprises the contractile vacuole, a pore and a vesicular spongiome. A minority of spongiome vesicles bear a 15-nm coat on the cytoplasmic surface of the membrane. The coat superficially resembles a clathrin coat. The majority of vesicles are smooth surfaced. Both types of vesicles are found at the same time. Smooth vesicles can be seen in profile suggesting vesicle-vesicle and vesicle-vacuole fusion. It is suggested that smooth vesicles are involved in the segregation of fluid from the cytoplasm and in filling the vacuole. Coated elements exist only as independent vesicles and as coated pits in the contractile vacuole membrane. There is no evidence of fusion of coated vesicles. It is suggested that coated vesicles function to retrieve specific membrane components from the contractile vacuole.     

Hydrogen peroxide causes the fatal injury to human fibroblasts exposed to oxygen radicals

Oxygen radicals are suspected as being a cause of the cellular damage that occurs at sites of inflammation. The phagocytic cells that accumulate in areas of inflammation produce superoxide, hydrogen peroxide, hydroxyl radical, and probably singlet oxygen in the extracellular fluid. The mechanism by which these oxygen molecules kill cells is unknown. To determine which of the oxygen species is responsible for the cellular killing, we exposed human fibroblasts in culture to oxygen radicals generated by the enzymatic action of xanthine oxidase upon acetaldehyde. Using the amount of chromium-51 released from labeled fibroblasts as an index of cellular death, we found that cells were protected only by interventions that reduce hydrogen peroxide concentration. Agents that inactivate superoxide, hydroxyl radical, and singlet oxygen were ineffective in limiting oxygen radical-induced cellular death.     

Biochemical genetics of Chinese hamster cell mutants with deviant purine metabolism: isolation and characterization of a mutant deficient in the activity of phosphoribosylaminoimidazole synthetase.

A new purine-requiring mutant of Chinese hamster ovary cells (CHO-Kl) is described. This mutant, Ade-G, grows on aminoimidazole carboxamide, hypoxanthine, or adenine. It complements all eight of our other previously described Ade- mutants. Biochemical analysis of de novo purine synthesis in whole cells suggests that Ade-G is capable of the first four reactions of de novo purine biosynthesis and that it synthesizes and accumulates phosphoribosylformylglycinamidine (FGAM). Direct enzyme assay in cell-free extracts confirms that Ade-G is defective in phosphoribosylaminoimidazole synthetase activity and does not convert FGAM to phosphoribosylaminoimidazole (AIR), the next intermediate in the de novo biosynthetic pathway.     

SPINAL CORD LIPIDS AND MYELIN COMPOSITION IN BORDER DISEASE (HYPOMYELINOGENESIS CONGENITA) OF LAMBS

Spinal cords from clinically affected newborn lambs, each with muscular spasms (‘shaking’) and a ‘hairy’ birth coat, were found to be deficient in DNA and to contain less myelin and various lipid components, suggesting retarded CNS development equivalent to about 124 days conceptual age. Cerebrosides were notably deficient in whole cord and isolated myelin and contained more saturated and less unsaturated fatty acids than normal. The rate of cerebroside synthesis assayed in vitro was enhanced and taken with the very low tissue concentrations this indicated faster cerebroside turnover and a less stable myelin in the spinal cords of lambs affected with Border Disease. Marked decreases in plasmalogen concentrations, the redistribution of phospholipid fractions, the presence of about 8 per cent cholesterol in the esterified form and the characteristic fatty acid composition of these esters strongly suggest that degeneration is concomitant with myelin immaturity. Hypocupraemia, low concentrations of copper in the cerebrum and increased concentrations in spinal cord myelin are additional features of the clinical disease. The latter result may be related to myelin immaturity.