Oxidative damage to DNA and replicative lifespan in cultured adrenocortical cells

Oxidative damage to DNA in cultured bovine adrenocortical cells was investigated by exposing cells to a sublethal concentration (10 microM) of cumene hydroperoxide under conditions previously shown to be deficient in the biological antioxidants selenium and alpha-tocopherol (vitamin E). DNA prepared from cells incubated for 4 h with 10 microM cumene hydroperoxide had a greater fraction showing resistance to S1 nuclease after denaturation and reassociation to a log C0t of -3. Cross-linking by cumene hydroperoxide was abolished in cells that had been grown in the presence of 20 nM selenite or 1 microM alpha-tocopherol for 96 h prior to peroxide addition, whereas such cells remained susceptible to cross-linking by nitrogen mustard. Extensive strand breaks in DNA from peroxide-treated cells as assessed by alkaline sucrose gradient centrifugation were greatly reduced in cells grown in selenite or alpha-tocopherol. Despite the evidence of damage to DNA, cumene hydroperoxide was not detectably mutagenic, in contrast to 5 microM methylnitronitrosoguanidine (MNNG), when assessed as the incidence of resistance to 25 microM ouabain. We confirmed that cumene hydroperoxide at greater than 10 microM lowers cloning efficiency and that this is largely prevented by selenite or alpha-tocopherol. Additionally, selenite or alpha-tocopherol produced increased clonogenicity in cells not incubated with peroxide. To examine effects of the biological antioxidants on replicative lifespan, cells were grown continuously in fetal bovine serum (FBS), fibroblast growth factor (FGF), and selenite or alpha-tocopherol. Selenium increased replicative lifespan by 10-20% and alpha-tocopherol by 22-30%. Levels of DNA cross-links and strand breaks did not differ under any circumstances between early (second) passage and late (30th) passage cells. The experiments on replicative potential were all performed in the presence of FGF. When FGF was omitted from the culture medium, replicative lifespan was reduced by 85%. We conclude that types of damage to DNA resulting from peroxide exposure are not present in cells under standard culture conditions at early or late stages of the lifespan. Other work has noted a relationship between clonogenicity and replicative lifespan; thus, the increase in cloning efficiency seen with selenium and alpha-tocopherol may cause the observed slight increase in replicative lifespan. Oxidative damage does not appear to be a major determinant of cellular senescence in adrenocortical cells.     

An aminoacyl-tRNA synthetase complex in Escherichia coli.

Aminoacyl-tRNA synthetases from several strains of Escherichia coli are shown to elute as a high-molecular-weight complex on 6% agarose columns (Bio-Gel A-5M). In contrast, very little synthetase activity was observed in such complexes on Sephadex G-200 columns, suggesting that these enzymes may interact with or are dissociated during chromatography on dextran. The size of the complex observed on Bio-Gel A-5M was influenced by the method of cell breakage and the salt concentrations present in buffers. The largest complexes (greater than 1,000,000 daltons) were seen with cells broken with a freeze press, whereas with sonicated preparations the average size of the complex was about 400,000 daltons. Extraction of synthetases at 0.15 M NaCl, to mimic physiological salt concentrations, also resulted in high-molecular-weight complexes, as demonstrated by both agarose gel filtration and ultracentrifugation analysis. Evidence is presented that dissociation of some synthetases does occur in the presence of higher salt levels (0.4 M NaCl). Partial purification of the synthetase complex on DEAE-Sephacel was accomplished with only minor dissociation of individual synthetases. These data suggest that a complex(es) of aminoacyl-tRNA synthetase does exist in bacterial cells, just as in eucaryotes, and that the complex may have escaped earlier detection due to its fragility during isolation.     

Molecular cloning of a cDNA for the E1 alpha subunit of rat liver branched chain alpha-ketoacid dehydrogenase

We have isolated a cDNA encoding the branched chain alpha-ketoacid dehydrogenase E1 alpha subunit. A rat liver lambda gt11 expression library was screened with antibody reactive with the 2-oxoisovalerate dehydrogenase (lipoamide) component. A positive clone, lambda BZ304, contains a 1.7-kilobase pair cDNA insert with a 1323-base pair open reading frame. Translation of the open reading frame predicts the 24 residues of the previously reported phosphorylation sites 1 and 2 for the bovine kidney and rabbit heart enzymes. The N-terminal sequence of purified E1 alpha was determined, and this sequence was found 40 residues from the beginning of the deduced peptide sequence. Northern blots of rat liver and muscle RNA demonstrate a single mRNA species of approximately 1.8 kilobase pairs in each tissue, suggesting that this cDNA is nearly full length.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

LTB4 induced activation signals and responses in neutrophils are short-lived compared to formylpeptide

Leukotriene B4 (LTB4) was shown to be a potent stimulator of neutrophil actin polymerization as detected by right-angle light scatter and rhodamine-phalloidin staining of F-actin. When we compared the kinetics of this neutrophil cytoskeletal response to the chemoattractants formylpeptide and LTB4, we observed that the response to LTB4 was markedly shorter-lived. To understand the basis of this result, we re-examined the kinetics of superoxide generation, elastase release, intracellular calcium elevation, and phosphoinositide metabolism in neutrophils stimulated with LTB4 and N-formylhexapeptide. LTB4 was relatively inefficient in producing superoxide and in releasing elastase. Although both responses were initiated with similar rapidity, they turned off sooner with LTB4 as compared with N-formylhexapeptide stimulation. Intracellular calcium elevation, a signal that is necessary for superoxide generation and degranulation, was of similar magnitude but of shorter duration in response to LTB4 as compared with the N-formylhexapeptide. The LTB4-induced rise of phosphatidic acid also was not sustained as long as the N-formylhexapeptide-induced increase. Prior exposure of neutrophils to LTB4 did not inhibit subsequent stimulation of superoxide generation by N-formylhexapeptide. Thus, the inability of LTB4 to stimulate superoxide generation was not due to LTB4-induced global inhibitory signals. The deficiency in LTB4-induced superoxide and elastase responses may be related to short-lived LTB4-induced activation signals and/or the number of receptors contributing to these responses.     

Molecular mechanisms involved in T cell activation. I. Evidence for independent signal-transducing pathways in lymphokine production vs proliferation in cloned cytotoxic T lymphocytes

It is well-established that activated T cells proliferate in response to interleukin 2 (IL 2) and produce various soluble lymphokines such as macrophage-activating factor (MAF) in response to antigen. Prior to investigating the molecular events involved in signaling the initiation of these responses in cloned murine cytotoxic T lymphocytes (CTL), we determined whether these responses could occur independently, and we established for each response the time during which signal transducing mechanisms may function. It was found that this cloned CTL population was in a resting state (G1 phase of cell cycle) 7 days after stimulation with antigen plus IL 2. At this time, the incubation of these resting CTL with IL 2 for 4 to 6 hr resulted in a maximal proliferative response that was not accompanied by the production of MAF. Conversely, the incubation of resting CTL with antigen or lectin (in the absence of IL 2) for at least 8 hr resulted in the maximal production of MAF at 24 hr without inducing a proliferative response. In addition, antigen or lectin, but not IL 2, triggered an immediate (less than 1 min) and sustained (at least 8 hr) mobilization of intracellular calcium. The kinetics of this calcium response paralleled the minimum time (8 hr) that was required for resting CTL to interact with either antigen or lectin in order to produce maximal titers of MAF. These results indicate that proliferation and lymphokine (MAF) production in cloned murine CTL are independent events. In these resting CTL, the signal mechanisms that mediate the production of lymphokines are most likely restricted to the initial 8 hr of stimulation by antigen or lectin and involve the rapid and prolonged mobilization of cytoplasmic calcium. Proliferative signals, however, are probably complete within 4 to 6 hr after stimulation by IL 2 and do not involve readily demonstrable fluxes of cytoplasmic calcium, as determined by the fluorescent calcium probe Quin 2.     

Microplate Assay for Colletotrichum Spore Production

A simple microplate method was devised to assay spore production by Colletotrichum gloeosporioides by growing the fungus on 1 ml of solid media in the wells of tissue culture plates. Growth and sporulation on microplates were compared at days 4 and 8 with growth and sporulation in 100-ml liquid batch cultures that involved 11 common media. Spore production per unit volume of medium was the same for solid and liquid forms of the media. Qualitative assessment of mycelial growth measured on microplates agreed with that of growth measured in liquid cultures. The microplate assay indicated that V8 juice was the best medium and that an organic content of about 6 mg/ml was optimal for high sporulation and low mycelium production. The assay provides a convenient, rapid, and inexpensive means of screening media for the production of fungal conidia in large numbers, to be used, for example, in biological control programs.     

Modification of an arginine residue essential for the activity of NAD-malic enzyme from Ascaris suum

Purified NAD-malic enzyme from Ascaris suum is rapidly inactivated by the arginine reagent, 2,3-butanedione, and this inactivation is facilitated by 30 mM borate. Determination of the inactivation rate as a function of butanedione concentration suggests a second-order process overall, which is first order in butanedione. A second-order rate constant of 0.6 M-1 s-1 at pH 9 is obtained for the butanedione reaction. The inactivation is reversed by removal of the excess reagent upon dialysis. The enzyme is protected against inactivation by saturating amounts of malate in the presence and absence of borate. The divalent metal Mg2+ affords protection in the presence of borate but has no effect in its absence. The nucleotide reactant NAD+ has no effect on the inactivation rate in either the presence or absence of borate. A dissociation constant of 24 mM is obtained for E:malate from the decrease in the inactivation rate as a function of malate concentration. An apparent Ki of 0.5 mM is obtained for oxalate (an inhibitor competitive vs malate) from E:Mg:oxalate while no significant binding is observed for oxalate using the butanedione modified enzyme. The pH dependence of the first-order rate of inactivation by butanedione gives a pKa of 9.4 +/- 0.1 for the residue(s) modified, and this pK is increased when NAD is bound. The arginine(s) modified is implicated in the binding of malate.     

Mechanism of cyclosporin A-induced immunosuppression. Cyclosporin A inhibits receptor-mediated and non-receptor-mediated lymphokine production as well as interleukin-2-induced proliferation in cloned T lymphocytes

The effects of cyclosporin A (CyA) on the activation processes of cloned murine cytotoxic T lymphocytes (CTL) have been examined. With the use of Day 7 resting cloned CTL it was possible to separate the functions of lymphokine production (macrophage-activating factor, MAF) and interleukin 2 (IL-2)-induced proliferation of these cells. The effect of CyA on each of these activities was analyzed independently. CyA was found to inhibit both receptor-mediated MAF production in response to stimulation with antigen and lectin and MAF production in response to non-receptor-mediated stimulation (by anti-Thy-1 antibodies, ionophore, and phorbol ester). Further, CyA was observed to inhibit the re-entry of these resting CTL into the cell cycle upon stimulation with IL-2. The effect of CyA on MAF production did not appear to be due to inhibition of the signal-transducing mechanism involved in this process (i.e., inositol lipid hydrolysis, calcium mobilization, and protein phosphorylation). The action of CyA on the IL-2-induced proliferation was not due to inhibition of IL-2 receptor expression or the binding of IL-2 to its receptor. Thus, CyA appeared to mediate its suppressive effects on MAF production and IL-2-induced proliferation through an action on some later step(s) in the signal pathways of these activities.