The identification of a distinct export step following the biosynthesis of leukotriene C4 by human eosinophils

We have examined the requirements for the export of leukotriene C4 (LTC4) from cultured human eosinophils. To define saturability and kinetics of LTC4 export, eosinophils were interacted with leukotriene A4 (LTA4) at 37 degrees C, and the methanolic extracts of the cell-associated and extracellular compartments were then analyzed for LTC4 content by reverse phase high performance liquid chromatography with on-line monitoring of absorbance at 280 nm. When LTA4 was added at concentrations from 0 to 100 microM for 10 min at 37 degrees C, the amount of LTC4 released extracellularly became constant at an LTA4 concentration of 7.5 microM or greater even though the amount of intracellular LTC4 continued to increase. When eosinophils were incubated with 50 microM LTA4 for 0-60 min at 37 degrees C and then held at 0 degrees C for the remainder of the 60-min interval, 54.2 and 77.3% (n = 3), respectively, of the total LTC4 was released extracellularly after 15 and 30 min of incubation at 37 degrees C. Eosinophils incubated with 50 microM LTA4 at 0 degrees C for 1 h synthesized 290 pmol of LTC4 (n = 3) which was approximately half-maximal, all of which was retained intracellularly. We utilized the time and temperature dependence of LTC4 export to preload eosinophils with both LTC4 and leukotriene C5 (LTC5) by sequentially supplying them with specific substrates. With increasing concentrations of intracellular LTC5, there was dose-dependent inhibition of the subsequent release of LTC4 at 37 degrees C, with the sum of the released glutathionyl leukotrienes remaining constant. In addition, only minimal competition for LTC4 release occurred when cells were preloaded with both LTC4 and the conjugate of 1-chloro-2,4-dinitrobenzene and reduced glutathione, S-(dinitrophenyl)glutathione. The criteria of saturability, time dependence of LTC4 release at 37 degrees C, competition of LTC4 with LTC5 for release, and the inhibition of LTC4 release at 0 degrees C establish the export of LTC4 from cells as a novel and specific biochemical step distinct from both LTA4 uptake and the conjugation of LTA4 with reduced glutathione by LTC4 synthase to form LTC4.     

Pollen frequencies reflect vegetation patterns in a great basin (U.S.A.) mountain range

Five indices are used to quantify the relationship between vegetation and pollen in a mountain range in the arid Great Basin. Computations are based on vegetation coverage and pollen percentages from 63 stands. Association is a measure of whether the presence of the pollen type in a surface sample is an indication of the presence of the parent plant in the local vegetation. Over-representation and under-representation measure tendencies for pollen to occur where the parent plants are absent and vice versa. The correlation coefficient measures the relationship between plant and pollen in stands where both are present. Twenty-nine trees, shrubs, and herbs accurately reflect local vegetation conditions. A percentage diagram shows elevational trends in abundant pollen types. Regional pollen types are used to compute the accumulation rate of pollen in the surface samples. A diagram of pollen accumulation rates shows trends similar to those shown in the percentage diagram. The moss polsters used in this study may collect pollen over a fifteen-year interval.     

The response of serum ceruloplasmin to injections of walker 256 tumor cells or turpentine into rats

The subcutaneous injection of Walker 256 carcinosarcoma cells into a rat was followed by a pronounced increase in plasma ceruloplasmin activity. This lasted only about 3 weeks even though the tumors were still enlarging rapidly. Based upon studies with⁶⁷Cu, the increase in plasma ceruloplasmin seemed to reflect increased production of the enzyme by the livers of rats bearing the tumors. Injections of turpentine also raised plasma ceruloplasmin, but to a significantly lesser extent and normal values were reached within 2 weeks.     

The invisible copper of cytochrome c oxidase. pH and ATP dependence of its midpoint potential and its role in the oxygen reaction.

Formation of the CO compound has been studied in intact mitochondria, submitochondrial particles and isolated cytochrome oxidase. The reaction requires the prior reduction of both cytochrome a₃ and one other single-electron acceptor. It is inferred that the second acceptor is the “invisible” copper which is undetectable by both optical and spin resonance spectroscopy. The overall process can be viewed as two single electron steps plus a ligand binding reaction. At high concentrations of CO, when titrations are performed at oxidation-reduction potentials significantly above the midpoints of either cytochrome a₃ or “invisible” copper, appearance of the CO compound follows a strict n = 2 (2-electron) relationship. Its midpoint potential is also dependent on the prevailing concentration of CO and is increased by approx. 30 mV for each tenfold increase in the level of CO. At redox potentials approaching the midpoints of cytochrome a₃ or “invisible” copper, significant deviations from n = 2 behavior are apparent which are readily detectable experimentally using low CO concentrations.A mathematical analysis of this model is presented and the oxidation-reduction properties of the CO compound are utilized to determine the midpoint potential of the “invisible” copper. This value is estimated to be 340 ± 10 mV at pH 7.8, independent of pH and the prevailing $\text{sol}\text{[ATP]}\text{[ADP]}\text{× [}\text{P}{}_1\text{]}$ ratio.By analogy with the observations on CO binding, the primary intermediate in the oxidase reaction with oxygen is concluded to be a bridged a₃²⁺-O₂-Cu¹⁺ complex. The initial reduction of molecular oxygen can then proceed via a thermodynamically favorable two-electron step to form a bridged peroxide intermediate. Subsequent reduction to water may later occur by way of two single-electron steps or one two-electron step.     

Isolation and characterization of a mannan from mesosomal membrane vesicles of Micrococcus lysodeikticus.

The carbohydrate content of mesosomal membranes of Micrococcus lysodeikticus has been shown to be consistently higher (about four times) than that of corresponding plasma membrane preparations. Analysis of washed membrane fractions by gas-liquid chromatography indicated that mannose was the major neutral sugar of both types of membrane (accounting for 95 and 89%, respectively, of the mesosomal and plasma membrane carbohydrate). Small amounts of inositol, glucose and ribose were also detected. We have shown by polyacrylamide gel electrophoresis in sodium dodecylsulphate and by precipitation and agar gel diffusion experiments with concanavalin A that a mannan is the major carbohydrate component of both types of membrane. This polymer can be selectively released from mesosomal membranes by a simple procedure involving low ionic strength-shock and heating to 80 degrees C for 1 min, and purified by ultrafiltration and ethanol precipitation. The mannan contains mannose as the only neutral carbohydrate, is not phosphorylated and does not contain significant amounts of amino sugars or uronic acids. Agar gel electrophoresis experiments, however, indicate an anionic polymer whose acidic properties are eliminated upon mild base hydrolysis. Analysis of native mannan by infrared spectroscopy reveals absorption bands attributable to ester carbonyl groups and to carboxylate ions, consistent with the presence of succinyl residues in the polymer (Owen, P. and Salton, M.R.J. (1975) Biochem, Biophys. Res. Commun. 63, 875--800). A sedimentation coefficient of 1.39 S was obtained by analytical ultracentrifugation in 1.0 M NaCl and a value of one reducing equivalent per 50 mannose residues by reduction with NaB3H4. The polysaccharide was only slightly degraded (2%) by jack bean alpha-mannosidase and could precipitate 15 times its own weight of concanavalin A. The acidic polymers was also detected in the cell "periplasm" and was secreted from cells grown in defined media during the period of decelerating growth.     

A small molecule that disrupts S. Typhimurium membrane voltage without cell lysis reduces bacterial colonization of mice

As pathogenic bacteria become increasingly resistant to antibiotics, antimicrobials with mechanisms of action distinct from current clinical antibiotics are needed. Gram-negative bacteria pose a particular problem because they defend themselves against chemicals with a minimally permeable outer membrane and with efflux pumps. During infection, innate immune defense molecules increase bacterial vulnerability to chemicals by permeabilizing the outer membrane and occupying efflux pumps. Therefore, screens for compounds that reduce bacterial colonization of mammalian cells have the potential to reveal unexplored therapeutic avenues. Here we describe a new small molecule, D66, that prevents the survival of a human Gram-negative pathogen in macrophages. D66 inhibits bacterial growth under conditions wherein the bacterial outer membrane or efflux pumps are compromised, but not in standard microbiological media. The compound disrupts voltage across the bacterial inner membrane at concentrations that do not permeabilize the inner membrane or lyse cells. Selection for bacterial clones resistant to D66 activity suggested that outer membrane integrity and efflux are the two major bacterial defense mechanisms against this compound. Treatment of mammalian cells with D66 does not permeabilize the mammalian cell membrane but does cause stress, as revealed by hyperpolarization of mitochondrial membranes. Nevertheless, the compound is tolerated in mice and reduces bacterial tissue load. These data suggest that the inner membrane could be a viable target for anti-Gram-negative antimicrobials, and that disruption of bacterial membrane voltage without lysis is sufficient to enable clearance from the host.     

Species specificity and intraspecific variation in the chemical profiles of Heliconius butterflies across a large geographic range

In many animals, mate choice is important for the maintenance of reproductive isolation between species. Traits important for mate choice and behavioral isolation are predicted to be under strong stabilizing selection within species; however, such traits can also exhibit variation at the population level driven by neutral and adaptive evolutionary processes. Here, we describe patterns of divergence among androconial and genital chemical profiles at inter‐ and intraspecific levels in mimetic Heliconius butterflies. Most variation in chemical bouquets was found between species, but there were also quantitative differences at the population level. We found a strong correlation between interspecific chemical and genetic divergence, but this correlation varied in intraspecific comparisons. We identified “indicator” compounds characteristic of particular species that included compounds already known to elicit a behavioral response, suggesting an approach for identification of candidate compounds for future behavioral studies in novel systems. Overall, the strong signal of species identity suggests a role for these compounds in species recognition, but with additional potentially neutral variation at the population level.     

Regulation of Na/K/Cl cotransport in vascular smooth muscle cells

The regulation of Na/K/Cl cotransport was investigated in vascular smooth muscle cells. That a Na/K/Cl cotransport system exists was established by the finding that the ouabain insensitive K influx was sensitive to the "loop" diuretic bumetanide. Furthermore, bumetanide sensitive K influx was dependent upon the presence of both Na and Cl in the extracellular milieu. Bumetanide sensitive K influx was inhibited by agents which elevate cellular cyclic AMP levels, and to a lesser extent by agents which elevate cellular cyclic GMP levels. When serum, EGF or TPA was added, bumetanide sensitive K influx was enhanced. These results suggest that vascular smooth muscle cells have a ouabain insensitive, bumetanide sensitive Na/K/Cl cotransport system which is stimulated by serum, EGF or TPA and inhibited by cAMP or cGMP.