Mycoplasma pneumoniae: A prokaryote which consumes oxygen and generates superoxide but which lacks superoxide dismutase

Superoxide dismutase and catalase were not detected in $\text{M}$. $\text{pneumoniae}$ and several other species of $\text{Mycoplasma}$ some of which consume oxygen and secrete H₂O₂. $\text{M}$. $\text{pneumoniae}$ in suspension formed O₂^? in the presence of NADH and flavins and extracts of $\text{M}$. $\text{pneumoniae}$ formed O₂^? in the presence of either NADH or NADPH. The lack of superoxide dismutase in $\text{M}$. $\text{pneumoniae}$ could not be attributed to superoxide dismutase in the complex medium in which the organisms were grown because organisms grown in medium in which the superoxide dismutase had been inactivated by heat still contained undetectable amounts. Mycoplasmas appear to be an exception to the rule that organisms which consume O₂ synthesize superoxide dismutase.     

Oxygen metabolism in rabbit bone marrow and liver.

Oxygen metabolism has been quantified in rabbit bone marrow and liver. NADPH-Cytochrome $\text{c}$ reductase activity in bone marrow microsomal and cytosol fractions was about 40% of that found in liver. Superoxide anion and peroxide generation were found to be present in both liver and bone marrow. Catalase and superoxide dismutase activity were measured in liver and in marrow preparations free of erythrocytes; while liver catalase activity was approximately twice that of bone marrow, very low superoxide dismutase activity was observed in erythrocyte free bone marrow homogenates.     

Letter: Preliminary crystallographic data for spinach superoxide dismutase

Superoxide dismutase from spinach leaves was salted out with ammonium sulphate. The resulting crystals were monoclinic, space group C2, with unit cell dimensions $\text{a = 166.2}\text{A}\text{?}\text{, b = 46.1}\text{A}\text{?}\text{, c = 85.6}\text{A}\text{?}\text{and}\text{β = 99.3 °}$. Considerations of cell volume and protein molecular weight indicated two molecules of superoxide dismutase in the asymmetric unit.     

Variation of superoxide dismutases during the development of the fruit fly Ceratitis capitata L.

Superoxide dismutase levels were estimated in eggs, larvae and pupae of the fruit fly $\text{Ceratitis capitata}$, as well as in adult flies. No changes occur in the first three stages, but development of the adult fly is accompanied by a large increase in mitochondrial superoxide dismutase per gm of material, and a much smaller relative increase in the cytoplasmic enzyme.     

Tetrahydropterin: Reduction of cytochrome c and coupled phosphorylation at mitochondrial site 3

Incubation of rat liver mitochondria with tetrahydropterin results in ATP production with a P:O ratio of 0.85, consistent with the entry of reducing equivalents into the mitochondrial electron transport chain at cytochrome $\text{c}$. No evidence for an enzymatic reduction of cytochrome $\text{c}$ was found. The reduction of either soluble or mitochondrial cytochrome $\text{c}$ was not diminished by superoxide dismutase or anaerobic conditions, indicating that the reaction is not dependent on the autoxidation of the reduced pterin and the formation of an active species of oxygen. The experiments indicate a potential pathway for the production of ATP coupled to the oxidation of NADPH through the activity of NADPH-dependent pteridine reductases.     

The Tiron free radical as a sensitive indicator of chloroplastic photoautoxidation

Wheat chloroplasts photochemically reduced molecular oxygen, as a Hill oxidant in the Mehler reaction, to superoxide anion which then oxidized added 1,2-dihydroxybenzene-3,5-disulfonate to its semiquinone, a comparatively stable free radical at pH 7. The last mentioned reaction was rapid in aqueous solution, but the rate of formation of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone by the chloroplast system was calculated as a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 0.6 s. The Mehler reaction, or more specifically the univalent reduction of oxygen by Photosystem I, was rate-limiting so that the 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was a useful spin probe for superoxide anion production at room temperature. The ESR signal of 1,2-dihydroxybenzene-3,5-disulfonate semiquinone was proportional to its steady state concentration and decayed in the dark with a $\text{T}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ of 5–6 s. This oxygen-dependent signal was enhanced by mediation of chloroplastic oxygen reduction through methyl viologen. The superoxide anion scavengers ascorbate and l-epinephrine competitively obscured 1,2-dihydroxybenzene-3,5-disulfonate semiquinone formation, but added superoxide dismutase was not as effective in this role. Partial inhibition by superoxide dismutase was achieved only by preincubation of Photosystem I enriched particles with ten times the endogenous concentration of superoxide dismutase. This and the persistence of a small amount of a 1,2-dihydroxybenzene-3,5-disulfonate (Tiron) oxidizing species in the dark supports the concept of Tiron accessibility but not the superoxide dismutase accessibility of superoxide anion bound in its formative enzyme complex. Benzoquinone and naphthoquinone disulfonate also reacted with superoxide anion, and supported both the Hill reaction and the Mehler reaction as final oxidants of both water and superoxide anion.     

Evidence for water as the product for oxygen reduction by cytochrome cd.

Evidence is presented that water is the final product of electron donation to molecular oxygen by cytochrome $\text{cd}$ from $\text{Paracoccus}\text{denitrificans}$ when ferrocytochrome $\text{c}$ acts as donor to $\text{cd}$. Negative evidence for the accumulation of superoxide and peroxide was obtained by rate effect experiments in the presence of superoxide dismutase, catalase, and peroxidase. Positive evidence for water was obtained by showing a 4 to 1 stoichiometric balance for rates of electron acceptance from ferrocytochrome $\text{c}$ to rates of donation to molecular oxygen.     

Complex interactions of caffeine and its structural analogs with ultraviolet light in cell killing

We measured the clonogenic survival response of cultured mouse $\text{10}\text{T}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ cells exposed to UV light and caffeine post-treatment. When 0.5 and 1 mM caffeine were present for 24 h immediately following UV, the D₀ values of the biphasic survival curves suggest that one subpopulation was sensitized and one subpopulation was protected from killing by UV light. A cloned survivor from the radioprotected subpopulation responded to UV plus caffeine in identical manner as the parent cells. When the caffeine exposure was prolonged to 48 h, only the radiosensitizing effect was observed. Two demethylated analogs of caffeine were also tested. The response of $\text{10}\text{T}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ cells to 1 mM theophylline present for 24 h after UV irradiation was approximately the same as that for the same treatment with 1mM caffeine. However, prolonging the theophylline exposure to 48 h failed to produce the same kind of potentiation of cell killing as that observed for caffeine. Xanthine by itself was as toxic to $\text{10}\text{T}{}^{\mathrm{<mtext>1</mtext><mtext>2</mtext>}}$ cells as caffeine, but had no synergistic effect as caffeine when given to UV-irradiated cells for 24 or 48 h. It is therefore unlikely that all the effects of caffeine on UV-irradiated cells are mediated by its demethylated metabolites.     

Metabolic effects of glucose deprivation and of various sugars in normal and transformed fibrobalst cell lines.

The sugar composition of the growth medium influenced the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, pyruvate and lactate production, and ATP levels in both normal and transformed fibroblast cell lines growing in tissue culture. Removal of glucose led to a rapid three- to fourfold rise in the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, followed by a slower decline in the content of ATP. However, there was no change in the adenylate energy charge [$\text{(}\text{ATP}\text{+}\text{1}\text{2}\text{ADP}\text{)/(}\text{ATP + ADP + AMP}\text{)}$] over a 2-h period. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio was restored to the original level within 10 s of glucose readdition. The $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ratios in cell lines growing on galactose were as high as for those incubated without sugars; growth on mannose or fructose produced intermediate ratios. There was an inverse relationship between the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio and pyruvate-lactate production for glucose, fructose and galactose. Thus, all cell lines had a high production of pyruvate and lactate but a low $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio when grown on glucose. In contrast, when galactose served as the sugar source, acid production was low, while the ratio was high. All cell lines had comparable hexokinase activity, and glucose was the best substrate, mannose intermediate and fructose poorest. Hexokinase activity did not correlate with the relative degree of utilization of the sugars. These results suggest that the sugar composition of the growth medium affects the metabolic pattern of a cell line, including the $\text{NAD}{}^{\mathrm{+}}\text{NADH}$ ratio, the ATP content and the production of pyruvate and lactate.     

Comparison of monocyte and alveolar macrophage antibody-dependent cellular cytotoxicity and Fc-receptor activity

The cytotoxic potential of rabbit peripheral blood monocytes and alveolar macrophages in antibody-dependent cellular cytotoxicity (ADCC) toward both erythrocyte (RBC_ox) and tumor cell (CEM T-lymphoblast) targets was examined. ADCC was measured in a 4-hr ⁵¹Cr-release assay. Alveolar macrophages were more efficient at killing the tumor cell targets (optimally sensitized with rabbit antisera) than monocytes at similar effector cell/target cell ($\text{E}\text{T}$) ratios. Tumor cell targets sensitized with seven different antisera (anti-CEM) were lysed by alveolar macrophages but not by the monocytes. In marked contrast, the monocytes were more effective at lysing the sensitized erythrocyte target cells. The degree of cytolysis of RBCox and CEM was dependent on the $\text{E}\text{T}$ ratio and the degree of sensitization of these target cells. It was demonstrated that the effector cell selectivity in ADCC was directly related to their ability or inability to bind the sensitized target cells as determined by Fc-receptor rosette formation. The transition from monocyte to macrophage may, therefore, have resulted in an alteration in the criteria necessary for Fc-receptor binding to antibody-sensitized target cells and subsequent ADCC.     

Separation of leukemic myeloblasts and splenic lymphocytes based upon differences in light scatter profile.

Cell sorting based upon differences in light scatter profile was carried out to sort cells which differ in size. This technique permitted the acquisition of subpopulations of murine spleen cells enriched for leukemic myeloblasts or lymphocytes. Morphologic assessment of the sorted populations was confirmed by malignancy assay $\text{in}$$\text{vivo}$. The same technique has been applied to human leukemic cells and significant enrichment for proliferating and quiescent cells was accomplished.     

Induction of angiotensin converting enzyme in human monocytes in culture.

Angiotensin converting enzyme (E.C.3.4.15.1, peptidyl dipeptidase) in circulating human monocytes rose from undetectable or minimal levels $\text{in}\text{vivo}$ to as high as 35.5 nmol/min·mgprotein (>300-fold increase) after 6 or 7 days in culture. Enzyme induction was enhanced by autologous serum and exposure for two days to 0.45 μM dexamethasone. Potent inhibition of enzyme induction by 370 μg/ml of actinomycin D and 1 μM cycloheximide suggested that new messenger RNA and enzyme biosynthesis are involved in the induction. Human monocyte and lung enzyme were similar with respect to EDTA inhibition, CoCl₂ activation and inhibition by an antienzyme antiserum. Human lymphocytes had minimal or undetectable enzyme which was not induced after 4 days in culture.     

Superoxide dismutase from Bacillus stearothermophilus: Crystallization and preliminary X-ray diffraction studies

Superoxide dismutase from Bacillus stearothermophilus is a dimeric manganese-containing enzyme with a molecular weight of 40,000. It has been crystallised in the monoclinic space group P2₁ with unit cell dimensions of $\text{a = 50}\text{A}\text{?}\text{, b = 70}\text{A}\text{?}\text{, c = 69}\text{A}\text{?}$ and β = 111 ° 10′. The asymmetric unit appears to be the dimer.     

Opiate receptor affinities and behavioral effects of enkephalin: Structure-activity relationship of ten synthetic peptide analogues

Synthetic met- and leu-enkephalin bind to rat brain opiate receptors with $\text{1}\text{2}$ and $\text{1}\text{7}$ the affinity of morphine. The aromatic hydroxyl moiety of the tyrosine residue is critical for receptor binding. Intracranial microinjection of met-enkephalin requires very high doses to produce an evanescent, naloxone reversible analgesia and stuperous immobility, presumably because of its rapid enzymatic degradation. Leu-enkephalin fails to elicit analgesia.     

Superoxide reduction as a mechanism of ascorbate-stimulated oxygen uptake by isolated chloroplasts

Addition of 1m$\text{M}$ ascorbate to isolated chloroplasts with methyl viologen (MV) as electron acceptor trebled the rate of oxygen uptake and decreased the $\text{ADP}\text{O}$ ratio to a third of that with no ascorbate present. These effects of ascorbate were reversed by superoxide dismutase (SOD), which in the absence of ascorbate had little effect on O₂ uptake or $\text{ADP}\text{O}$ ratio. A chloroplast-associated SOD activity equivalent to 500 units/mg chlorophyll was detected. The effects of ascorbate and SOD on O₂ uptake were similar in both coupled and uncoupled chloroplasts. The results are consistent with the hypothesis that ascorbate stimulates O₂ uptake by reduction of superoxide, which is formed by autoxidation of the added electron acceptor (MV), and which dismutates in the absence of ascorbate. Ascorbate does not seem to stimulate O₂ uptake by replacing water as the photosystem II donor.     

Evidence for a coordination position available to solute molecules on one of the metals at the active center of reduced bovine superoxide dismutase

We have measured the contribution of the reduced form of bovine $\text{Zn}\text{Cu}$ superoxide dismutase to the relaxation of the ³⁵Cl nucleus of chloride ion. The reduced protein has a molar relaxivity approximately 2.5 greater than the metal free protein, and addition of a small excess of cyanide lowers the relaxivity of the reduced protein to that of the apo-protein. We have interpreted these observations in terms of an open coordination position on one of the two metal ions, and we have proposed a mechanism for the reduction of superoxide by reduced superoxide dismutase which requires that $\text{O}{}_2{}^{\mathrm{?}}$ binds to Cu⁺ prior to electron transfer.     

Mechanisms of lysosomal enzyme secretion by human monocytes

Secretion of lysosomal enzymes by human monocytes in response to various stimuli and the effect of conditioned media from lymphocytes and neutrophils was studied. Monocytes were found to release β-glucosaminidase in response to NH₄Cl and to particles (zymosan, opsonised zymosan, asbestos and latex), but do not respond to some soluble stimuli like formyl-methionyl-leucyl-phenylalanine, phorbol myristate acetate, cytochalasin B, concanavalin A and $\text{N-}\text{acetylmuramyl-}\text{l}\text{-alanyl-}\text{d}\text{-isoglutamine}$. Neutrophil conditioned medium or neutrophil components did not have any effect on secretion. When treated with lymphokines the cells are more responsive, especially to zymosan. Even through there are similarities in the secretory activities of mouse macrophages and human monocytes, there are several differences both in the quantity of the response and in the mechanisms involved.     

Chemotactic migration of neutrophils under agarose.

A simple test for neutrophil chemotaxis is described. Wells were cut in soft agarose gel and filled with human peripheral blood leukocytes, chemotactin and control substances. Neutrophils consistently migrated under agarose towards the well with chemotactin, but not towards wells with control substances. Chemotaxis was quantitated as the mean distance travelled by 10 cells farthest from the well of origin, at specified time-intervals after filling the wells. Approximately $\text{1}\text{3}$ distance was covered in 2 hours, $\text{3}\text{4}$ in 4 hours and 90 per cent in 6 hours. The migrating cells examined after fixation and staining were found to be predominantly neutrophils with occasional eosinophils and monocytes.     

The estrogenic activity of DDT: Invivo and invitro induction of a specific estrogen inducible uterine protein by o,p'-DDT

The pesticide o,p'-DDT stimulates the production of a specific uterine protein, the so-called induced protein or IP, normally associated with an estrogenic response of the uterus. $\text{In}$$\text{vivo}$ stimulation of IP production is observed 1 hour after the administration of 250 mg/kg of o,p'-DDT to immature rats. $\text{In}$$\text{vitro}$ stimulation of IP production is observed after a 1 hour incubation of uteri with 100 μM o,p'-DDT. This $\text{in}$$\text{vitro}$ response is blocked by Actinomycin D. In contrast to o,p'-DDT, which binds to the cytoplasmic estrogen receptor and stimulates IP production, p,p'-DDT which does not bind well to the estrogen receptor does not stimulate IP production $\text{in}$$\text{vitro}$. These findings represent the first report of an estrogenic effect of o,p'-DDT in a completely $\text{in}$$\text{vitro}$ system.