Enzymatic basis of macrophage activation. Kinetic analysis of superoxide production in lysates of resident and activated mouse peritoneal macrophages and granulocytes

To compare the kinetics of the O-2-generating enzyme in nonactivated and activated macrophages and granulocytes from the mouse peritoneal cavity, we sought conditions in which the activity of this enzyme in cell lysates was comparable to that in intact cells. Pretreatment of macrophages with 10 mM diethyldithiocarbamate inhibited endogenous superoxide dismutase by 70% and enhanced O-2 secretion up to 15-fold, so that it was comparable to H2O2 secretion. O-2 secretion was terminated by detergent lysis and reconstituted by addition of NAD(P)H to the lysates. Optimal detection of O-2 production in lysates depended on prior stimulation of the respiratory burst, lysis with 0.05% deoxycholate rather than any of 4 other detergents or sonication, acetylation of the cytochrome c used as an indicator, and addition of NADPH rather than NADH. Kinetic analysis using NADPH-reconstituted deoxycholate lysates, together with spectra of oxidized and reduced cells, failed to reveal either marked differences in the Vmax of the O-2-generating enzyme or correlations between O-2 secretion and cytochrome b559 content among 5 macrophage populations whose H2O2 secretion ranged from 0 to 365 nmol/90 min/mg of protein. In contrast, the Km of the oxidase for NADPH varied markedly and inversely with the capacity of the intact cells to secrete O-2 or H2O2: J774G8 histiocytoma cells, 1.43 mM; resident macrophages, 0.41 mM; proteose peptone-elicited macrophages, 0.20 mM; casein-activated macrophages, 0.05 mM; NaIO4-activated macrophages, 0.05 mM; and granulocytes, 0.04 mM. These results suggest that macrophage activation, a process that enhances oxygen-dependent antitumor and antimicrobial functions, may equip the cell to secrete increased amounts of reactive oxygen intermediates largely by increasing the affinity of the oxidase for NADPH.     

Unique acceptors for poly(ADP-ribose) in resting,proliferating and DNA-damaged human lymphocytes

Acceptor proteins for poly(adenosine diphosphoribosyl)ation were determined in resting human lymphocytes, in lymphocytes with N-methyl-N′-nitro-N-nitrosoguanidine-induced DNA damage and in lymphocytes stimulated to proliferate by phytohemagglutinin. Kinetic studies showed that the increase in ADP-ribosylation which occurred in response to N-methyl-N′-nitro-N-nitrosoguanidine (MNNG) treatment was greater in magnitude but more transient in duration than that which occurred in phytohemagglutinin-stimulated cells. Gel electrophoretic analyses revealed that MNNG treatment and phytohemagglutinin stimulation both caused an increase in ADP-ribosylation of poly(ADP-ribose) polymerase and core histones. In MNNG-treated cells, an increase in ADP-ribosylation of histone H1 was also observed. In contrast, phytohemagglutinin-stimulated cells showed no increase in ADP-ribosylation of histone H1. In MNNG-treated cells there was also ADP-ribosylation of a protein of molecular weight 62 000, while in phytohemagglutinin-stimulated cells there was a marked increase in ADP-ribosylation of a protein of molecular weight 96000. MNNG treatment of phytohemagglutinin-stimulated cells produced a pattern of ADP-ribosylation that appeared to be due to the combined effects of the individual treatments. 3-Aminobenzamide effectively inhibited ADP-ribosylation under all treatment conditions.     

Freeze-thaw-refreeze cycle to prepare lymphocytes for HLA antibody detection or tissue typing

Human lymphocytes stored in the frozen state may be thawed, placed on cytotoxicity plates, refrozen, rethawed and used for screening sera or tissue-typing of the cells. The simple procedure described uses only a ?90 °C refrigerator for both freezing and storage of the cells. The technique permits a laboratory to collect a variety of cells over a long period, so that a set of test plates with cells from 10 to 20 donors can be prepared when a convenient number of donor cells are available. Also, the refrozen cells in cytotoxicity test plates may be warmed to the temperature of dry ice for 24 hr, returned to the refrigerator set at a slightly lower temperature, and at a later time, these cells may be thawed and used for serum screening. In view of these results, it appears possible to ship the refrozen cells from one laboratory to another using simple dry ice storage during the transfer. Negative reactions due to soluble antigens in the suspending sera can be obviated by washing out these sera and replacing them with medium 199 or alternatively, fetal calf serum can be used to replace the human serum in the suspending media.     

AN ANALYSIS OF HETEROCHROMATIN IN MAIZE ROOT TIPS

The B chromosomes of maize are condensed in appearance during interphase and are relatively inert genetically; therefore they fulfill the definition of heterochromatin. This heterochromatin was studied in root meristem cells by radioautography following administration of tritiated thymidine and cytidine, and was found to behave in a characteristic way, i.e. it showed asynchronous DNA synthesis and very low, if any, RNA synthesis. A cytochemical comparison of normal maize nuclei with nuclei from isogenic maize stock containing approximately 15–20 B-chromosomes in addition to the normal complement has revealed the following: (a) the DNA and histone contents are greater in nuclei with B chromosomes; (b) the proportion of DNA to histone is identical with that of nuclei containing only normal chromosomes; (c) the amount of nonhistone protein in proportion to DNA in interphase is less in nuclei with B chromosomes than in normal nuclei. In condensed B chromosomes the ratio of nonhistone protein to DNA is similar to that in other condensed chromatin, such as metaphase chromosomes and degenerating nuclei. The B chromosomes appear to have no effect on nucleolar RNA and protein. Replication of B chromosomes is precisely controlled and is comparable to that of the ordinary chromosomes not only in synthesis for mitosis but also in formation of polyploid nuclei of root cap and protoxylem cells.     

Stationary underwater prey missed by reef herons,Egretta gularis: head position and light refraction at the moment of strike

Summary This paper attempts to verify the importance of spatial positioning of the eyes of reef heronsEgretta gularis schistacea, when coping with light refraction at the air-water interface. The herons' striking of prey, while their approach angle was restricted, was observed. (a) The herons' capture success in the restricted situation was markedly lower than in the unrestricted situation. (b) The points of strike (STR) in unsuccessful strikes differed from those of successful strikes, and from those of the unrestricted situation. (c) The larger the difference between the observed and the predicted ratio of prey depth to apparent prey depth, the higher the probability of missing a prey. These results support predictions of a model presented elsewhere (Katzir and Intrator 1987) that a heron will attempt to reach spatial positions at which prey's real depth and apparent depth are linearly correlated.     

Inhibition of macromolecular synthesis in cultured macrophages by Pseudomonas pseudomallei exotoxin

Pseudomonas pseudomallei exotoxin was found to be a potent inhibitor of protein and DNA synthesis in cultured macrophages. Inhibition of DNA synthesis occurred at toxin concentrations as low as 1-2 micrograms/ml and inhibition of 3H-thymidine uptake was almost complete at concentrations of 8 micrograms/ml or more. A close correlation between cell damage and inhibition by DNA synthesis was observed. For protein synthesis, inhibition was obtained at much lower doses (0.06-2.0 micrograms/ml) of the toxin. At similar toxin concentrations, DNA synthesis was marginally affected. Further, it was shown that protein synthesis inhibition occurred almost immediately after incubation, reaching its maximal inhibitory effect of 70% after 6 hr. DNA synthesis, however, was minimally affected by a similar toxin concentration even after 10 hr of incubation. The inhibition of macromolecular synthesis in macrophages by P. pseudomallei exotoxin may be relevant to its modulatory effect on the host defense mechanism.     

Reduced biopterin as a cofactor in the generation of nitrogen oxides by murine macrophages

Generation of nitric oxide (NO.), an autacoid with vasorelaxant and cytotoxic properties, requires at least three cytosolic components in mouse macrophages besides L-arginine and NADPH. One or more components appear after induction by immunologic stimuli; two or more are present in both activated and non-activated macrophages. The constitutive factors can be separated on a Mr approximately 30,000 cut-off filter into high Mr fraction (HF) and low Mr fraction (LF) (Stuehr, D. J., Kwon, N. S., Gross, S. S., Thiel, B. A., Levi, R., and Nathan, C. F. (1989) Biochem. Biophys. Res. Commun. 161, 420-426). Herein we characterize the major active component in LF. The active component was dialyzable (Mr less than approximately 1,000), water soluble, and cationic at acidic to neutral pH. Fractionation on a C18 column in an acetonitrile/water gradient yielded one broad peak of activity, most of which corresponded to a fluorophore with the excitation/emission spectra of biopterins. Gas chromatography isolated a species in this peak with the mass spectrum of biopterin. Of 14 pteridines tested, only 7,8-dihydrobiopterin (H2biopterin) or 5,6,7,8-tetrahydrobiopterin (H4biopterin) could replace LF in synergizing with HF and the inducible component(s) to generate NO-2 and NO-3, the accumulating oxidation products of NO.. Half-maximal activity required 20-30 nM reduced biopterins. LFs from three cell lines were active in proportion to their content of biopterins; addition of reduced biopterins restored activity to LF from biopterin-deficient cells. Enhancement of NO-2 generation in the presence of H2biopterin but not H4biopterin was abolished by methotrexate and aminopterin, inhibitors of dihydrofolate reductase. These findings implicate a redox cycle in which the generation of NO. is facilitated by catalytic amounts of H4biopterin.     

Conformational properties of the beta(400-436) and beta(400-445) C-terminal peptides of porcine brain tubulin.

Two peptides from the C-terminal region of the major beta-tubulin isotype (400-436 and 400-445) that include the critical areas for interaction with MAP2 and tau were examined to determine their conformations in aqueous solution. Despite a high theoretical potential for alpha-helix formation, CD spectroscopy showed that these peptides consisted primarily of random coil with some reverse turn. This was unaffected by the presence of counterions to the negatively charged side chains (Ca2+, Mg2+), but did change when the side-chain charges were neutralized by lowering the pH; under these conditions, the alpha-helix content of the longer peptide rose to 25% and the C-terminal truncated peptide to 15%. The peptides also adopt alpha-helical structure in the presence of trifluoroethanol, the truncated peptide again attaining a lower maximum percentage. The beta(400-445) peptide was also studied by 1-D and 2-D NMR techniques. The results indicate that at pH 5.6 or 7 in an aqueous solution the peptide is extremely flexible and lacks regular secondary structure, consistent with the CD results. Both peptides inhibited microtubule-associated protein-stimulated tubulin assembly, with the longer peptide being about 4 times as inhibitory as the smaller peptide. Neither was inhibitory in the absence of microtubule-associated proteins, indicating that interaction with this species was necessary for inhibition. The greater activity of the longer peptide could be due to the extra negative charges in this peptide and/or the greater tendency of this peptide to form an alpha-helical structure under the appropriate conditions.