Effect of oxygen tension on human peripheral blood leukocytes: lysosomal enzyme release and metabolic responses during phagocytosis

We found that nonlethal lysosomal enzyme release from human peripheral blood leukocytes during phagocytosis of opsonized zymosan in vitro was modified by the oxygen tension under which the cells were incubated; with decreasing Po(2), zymosan-induced release of lysosomal enzymes was potentiated. The effect on enzyme release could not be attributed secondarily to an effect on phagocytosis, because, as others have reported, Po(2) had little effect on that response. Metabolic responses that accompany phagocytosis were also modified by oxygen tension. Stimulation of oxidation by way of the pentose cycle was further enhanced by increasing Po(2). Conversely, anaerobic glycolysis was promoted by decreasing oxygen tension. ATP levels fell as a function of time and concentration of phagocytic stimulus, mirroring lysosomal enzyme release as modified by Po(2). Cyclic AMP levels fell during phagocytosis and lysosomal enzyme release, a change that could act to facilitate lysosomal enzyme release. However, the fall in nucleotide level was greatest with highest Po(2) (i.e., when lysosomal enzyme release was least). The inverse relationship between oxidative metabolism and enzyme release suggested that a product of oxidative metabolism might adversely influence enzyme release. Sulfhydryl antioxidants (Cysteine, glutathione) and scavengers of oxygen-derived reactants (superoxide dismutase, catalase, benzoate, hypoxanthine, xanthine, histidine, azide) all potentiated zymosan- stimulated enzyme release. These findings are consistent with the interpretation that one or more factors (e.g., superoxide anion, hydrogen peroxide, hydroxyl radical, singlet oxygen), generated in association with the burst of oxidative metabolism which accompanies phagocytosis, acts to inhibit lysosomal enzyme release.     

Solubilization of a high affinity CA-ATPase from dog antrum smooth muscle

A Mg-independent high affinity Ca-ATPase has recently been reported to be present in the plasma membranes of smooth muscle. This enzyme has now been solubilized using deoxycholate. The membrane-bound and the solubilized enzymes resemble each other in Km for Ca2+, and inhibition by fluphenazine. The solubilized enzyme is, however, more sensitive to inhibition by Mg2+ than the membrane bound enzyme. Radiation inactivation analysis shows that whereas the membrane-bound enzyme had a target size of 98,000 +/- 4,000 Daltons, the solubilized enzyme was only 70,000, +/- 7,000 Daltons.     

Identification of a herpesvirus isolated from cytomegalovirus-transformed human cells.

Human cells transformed by cytomegalovirus and transplanted to athymic nude mice yielded a cytopathic virus, Hershey Medical Center virus, following prolonged in vitro passage of the tumor cells. The virus is a double-enveloped herpesvirus, is sensitive to ether, and is inhibited by iododeoxyuridine. No significant antigenic relationship to herpes simplex virus was detected using herpes simplex virus-immune sera in neutralization and immunofluorescence tests, but indirect immunofluorescence tests revealed cytomegalovirus-related antigenicity. Further immunological tests revealed that Hershey Medical Center virus is antigenically indistinguishable from infectious bovine rhinotracheitis virus. Thus, it appears that Hershey Medical Center virus is infectious bovine rhinotracheitis virus, which presumably appeared in the cell culture as a contaminant from fetal calf serum.     

Metabolism of cationized lipoproteins by human fibroblasts: biochemical and morphologic correlations

Human plasma low density lipoprotein (LDL) that had been rendered polycationic by coupling with N, N-dimethyl-1, 3-propanediamine (DMPA) was shown by electron microscopy to bind in clusters to the surface of human fibroblasts. The clusters resembled those formed by polycationic ferritin (DMPA-feritin), a visual probe that binds to anionic site on the plasma membrane. Biochemical studies with (125)I-labeled DMPA-LDL showed that the membrane-bound lipoprotein was internalized and hydrolyzed in lysosomes. The turnover time for cell bound (125)I-DMPA-LDL, i.e., the time in which the amount of (125)I-DMPA-LDL degraded was equal to the steady-state cellular content of the lipoprotein, was about 50 h. Because the DMPA-LDL gained access to fibroblasts by binding nonspecifically to anionic sites on the cell surface rather than by binding to the physiologic LDL receptor, its uptake failed to be regulated under conditions in which the uptake of native LDL was reduced by feedback suppression of the LDL receptor. As a result, unlike the case with native LDL, the DMPA-LDL accumulated progressively within the cell, and this led to a massive increase in the cellular content of both free and esterified cholesterol. Studies with (14)C-oleate showed that at least 20 percent of the accumulated cholesteryl esters represented cholesterol that had been esterified within the cell. After 4 days of incubation with 10 μg/ml of DMPA-LDL, fibroblasts had accumulated so much cholesteryl ester that neutral lipid droplets were visible at the light microscope level with Oil Red O staining. By electron microscopy, these intracellular lipid droplets were observed to lack a tripartite limiting membrane. The ability to cause the overaccumulation of cholesteryl esters within cells by using DMPA-LDL provides a model system for study of the pathologic consequences at the cellular level of massive deposition of cholesteryl ester.     

Transfer RNA-mediated suppression of amber stop codons in transgenic Arabidopsis thaliana

An artificial amber suppressor tRNA^Leu gene (supL.) was physically linked to a mutated gus reporter gene, p35S-gus(amL), which was inactivated by an amber stop codon (amL). Upon introduction into Arabidopsis thaliana, the presence of the supL. gene was found to be correlated with cytotoxic effects observed during tissue culture and in mature plants. Those primary transformants that displayed cytotoxic symptoms were shown by X-Gluc staining to express GUS as a result of amber stop codon suppression in vivo. Phenotypically normal lines were found by RT-PCR to express supL. GUS activity above background level was barely detectable in these plants, indicating a low level expression of supL. However, the remaining suppressor activity was still sufficient to transactivate an amber-mutated male sterility gene, pA9-barnase(amL1) when combined within the same plant by crossing. The suppressor tRNA^Leu gene may thus be used in transgenic plants for gene transactivation.     

An approximate likelihood procedure for censored data

An approximate likelihood procedure is suggested for the estimation of the parameters of the density of a single homogeneous sample subject to right censoring. Two examples are given involving the gamma distribution. The method is typically consistent and although it is always inefficient, the efficiency loss is second-order in the degree of censoring when this is small. The relation of the techniques to the results of Reid (1981, Annals of Statistics 9, 78-92) on influence functions for censored data, to the EM algorithm, and to the nonparametric regression techniques of Miller (1976, Biometrika 63, 449-464) and Buckley and James (1979, Biometrika 66, 429-436) are indicated. Simple estimates of standard error are obtained.