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.     

The effect of chronic and acute dietary restriction on the growth and protein turnover of fast and slow types of rat skeletal muscle

Changes in the growth and protein turnover of the anterior tibialis and soleus muscles were studied in response to acute and chronic dietary restriction (50% of ad libitum intake) between 3 and 149 weeks post partum. The effect of long-term dietary restriction from weaning to senescence was to retard the growth and normal developmental of the two types of skeletal muscle. This was evident from measurements of various parameters of growth, i.e. total protein, RNA and DNA and protein/DNA-P, which were reduced by approximately 50% when compared with age-matched controls. These decreases, however, were not accompanied by a decline in the fractional rate of synthesis (%/day) or ribosomal activity (mg protein/day per mg RNAP). The slowing down of the age-related decline in muscle growth has been attributed to a reduction in RNA capacity (RNA/protein), with similar responses in the fast- and slow-twitch skeletal muscles. The initial effects of piecemeal feeding of this restricted diet on the two types of muscle were also monitored. Short term starvation effects, i.e. 24 hr after feeding a reduced ration, were measured on the protein content and RNA/protein of both the anterior tibialis and soleus muscles; both parameters were unchanged within 24 hr. In contrast, a rapid and significant decline in the ribosomal synthetic activity (mg/d per mg RNAP), and a corresponding fall in the fractional rate of synthesis, occurred within 24 hr of feeding.     

Neem (Azadirachta indica) cultivated in Haiti

So that valuable products from the neem tree (Azadirachta indica) will be readily available in the future, countries in the neotropics should emulate Haiti and grow the species where it thrives even in eroded, semiarid, depauperate areas suitable for few other trees.     

Calcium-dependent activation and deactivation of rod outer segment phosphodiesterase is calmodulin-independent

ATP-dependent activation and deactivation of retinal rod outer segment phosphodiesterase is affected by calcium [Kawamura, S. and Bownds, M. D., J. Gen. Physiol. 77:571-591(1981)]. Our data demonstrate that although calmodulin has been found in rod outer segments [Liu, Y. P. and Schwartz, H., Biochim. Biophys. Acta 526:186-193(1978); Kohnken, R. E. et al, J. Biol. Chem. 256:12517-12522(1981)], this protein is not involved in calcium-dependent phosphodiesterase activation at light levels at which calcium clearly affects this enzyme's activity. Furthermore, calmodulin does not mediate the calcium-dependent deactivation of phosphodiesterase.     

Purification and characterization of the membrane (Na+ + Mg2+)-ATPase from Acholeplasma laidlawii B

The membrane (Na⁺ + Mg²⁺)-ATPase of Acholeplasma laidlawii B has been solubilized with a Brij-58/sodium deoxycholate mixture and purified by a combination of gel filtration and ion-exchange chromatography. The purified, partially delipidated ATPase has a specific activity of 195 μmol P_i/mg protein per h, which could be enhanced by 25% upon the addition of exogenous phospholipids. The kinetic properties of the purified enzyme are similar to those of the native membrane-bound enzyme, suggesting that it has not been substantially altered during the purification procedure. The enzyme is an assembly of five polypeptide species and its kinetic properties suggest that it is dissimilar to other known ATPases.     

Plasma 13,14-dihydro-15-keto-PGF alpha (PGFM) in pregnant and nonpregnant heifers prior to and during surgery and following intrauterine injection of PGF2 alpha

On day 17 postestrus or postmating, heifers were given intrauterine injections of saline (2 pregnant, 2 non-pregnant) or 200 micrograms PGF2 alpha (7 pregnant, 6 nonpregnant) through cannulae installed surgically into the uterine horn ipsilateral to the corpus luteum bearing ovary. Jugular blood samples were collected prior to the laparotomy at which the cannulae were installed during surgery, and for 90 min following the intrauterine injection. Plasma was assayed for progesterone and 13,14-dihydro-15-keto-PGF2 alpha (PGFM). Laparotomies were reopened to confirm proper cannula placement and to determine if blastocysts were present in mated heifers. Concentrations of PGFM were higher in pregnant compared to nonpregnant heifers during the presurgery (68 +/- 26 vs 24 +/- 26 pg/ml; P less than .025) and surgery (186 +/- 47 vs 65 +/- 17 pg/ml; P less than .05) periods. Pregnancy status did not alter the mean concentrations of PGFM (pregnant, 554 +/- 70 pg/ml; nonpregnant, 422 +/- 81 pg/ml) or the half-life of its decline in concentration (18 min) following intrauterine injection of PGF2 alpha. Pregnancy at 17 days in cattle does not appear to influence PGF2 alpha transport from the uterine lumen or its metabolism in the uterus or elsewhere in response to an acute intrauterine injection.     

Vagotomy and Pyloroplasty for Gastric Ulcer

In a series of 68 patients with gastric ulcer who were treated by vagotomy and pyloroplasty there were no operative deaths, though one patient who received massive transfusions died 14 weeks later from hepatic necrosis probably associated with serum hepatitis. Four patients developed recurrent peptic ulceration requiring further surgical treatment. The clinical condition of all but three of the remainder was satisfactory after an average follow-up period of three years and two months. Of the 14 patients who presented with an acutely bleeding gastric ulcer, one subsequently required gastrectomy for continued bleeding; in all the others the immediate and long-term results were satisfactory. Of 21 patients with a “high” ulcer and 29 with combined gastric and duodenal ulceration one (4·8%) and three (10%) respectively suffered recurrences. We conclude that vagotomy and pyloroplasty is a satisfactory form of treatment for a high or bleeding gastric ulcer, but that for all other gastric ulcers some form of gastric resection is preferable.     

Zonal centrifugation and flotation– fractionation of MSD mutant mouse brain

Zonal centrifuge and flotation–fractionation profile analysis of neonatal mouse brain homogenates in iso-osmotic Ficoll–sucrose density–gradients demonstrates the presence of four light density fractions. In msd neurological mutant mice with a myelin-synthesizing deficiency syndrome, the bands appear to be relatively normal until after the 10th day of postnatal brain development. With the onset of visible neurological symptoms after the 11th day, the four density bands begin to disappear from the zonal profiles and are all but absent at the time of death at about the 21st postnatal day. In normal littermates of the mutants, the bands persist with age and intensify. Although their identities remain unknown, the top three identify by their density with adult myelin and the fourth with the lighter of two adult synaptosome fractions. Mixtures of brain homogenates between mutant and normal littermates give rise to zonal and banding profiles intermediate between the separate profiles but somewhat less than their average in intensity.