Identification of the major endogenous leukotriene metabolite in the bile of rats as N-acetyl leukotrience E4

Mercapturic acid formation, an established pathway in the detoxication of xenobiotics, is demonstrated for cysteinyl leukotrienes generated in rats after endotoxin treatment. The mercapturate N-acetyl-leukotriene E₄ (N-acetyl-LTE₄) represented a major metabolite eliminated into bile after injection of [³H]LTC₄ as shown by cochromatography with synthetic N-acetyl-LTE₄ in four different HPLC solvent systems. The identity of endogenoud N-acetyl-LTE₄ elicited by endotoxin was additionally verified by enzymatic deacetylation followed by chemical N-acetylation. The deacetylation was catalyzed by penicillin amidase. Endogenous cysteinyl leukotrienes were quantified by radioimmunoassay after HPLC separation. A N-acetyl-LTE₄ concentration of 80 nmol/l was determined in bile collected between 30 and 60 min after endotoxin injection. Under this condition, other cysteinyl leukotrienes detected in bile by radioimmunoassay amounted to less than 5% of N-acetyl-LTE₄. The mercapturic acid pathway, leading from the glutathione conjugate LTC₄ to N-acetyl-LTE₄, thus plays an important role in the deactivation and elimination of these potent endogenous mediators.     

Enterohepatic circulation of N-acetyl-leukotriene E4

N-Acetyl-leukotriene E₄, the end product of leukotriene C₄ metabolism in the mercapturic acid pathway, was rapidly eliminated from the blood circulation into the bile of rats. Part of the N-acethyl-leukotriene E₄ secreted from bile into the intestine undewent enterohepatic circulation. Leukotriene absorption occurred from the small intestine and from the colon. Biliary and urinary excretion within 5.5 h amounted to 15 and 2%, respectively, of the intraduodenally administered N-acetyl- H leukotriene E₄ in animals anesthetized with ketamine. HPLC analyses indicated that 35% of the biliary radioactivity corresponded to unchanged N-acetyl- H leukotriene E₄, while 65% in bile and 100% in urine were polar metabolites. Enterohepatic circulation extends the biological half-life of N-acetyl-leukotriene E₄.     

Mechanism of inhibition of human leukocyte elastase by two cephalosporin derivatives.

The cephalosporin derivatives L 658758 [1-[[3-(acetoxymethyl)-7 alpha-methoxy-8-oxo-5-thia-1-azabicyclo [4.2.0]oct-2-en-2-yl]carbonyl]proline S,S-dioxide] and L 659286 [1-[[7 alpha-methoxy-8-oxo-3-[[(1,2,5,6-tetrahydro-2-methyl-5,6-dioxo- 1,2,4-triazin-3-yl)thio]methyl]-5-thia-1-aza-(6R)-bicyclo[4.2.0]-o ct-2-en-2-yl]carbonyl]pyrrolidine S,S-dioxide] are mechanism based inhibitors of human leukocyte elastase (HLE). The mechanism involves initial formation of a Michaelis complex followed by acylation of the active site serine. The group on the 3'-methylene is liberated during the course of these reactions, followed by partitioning of an intermediate between hydrolysis to regenerate active enzyme and further modification to produce a stable HLE-inhibitor complex. The partition ratio of 2.0 obtained for the reaction with L 658758 approaches that of an optimal inhibitor. These compounds are functionally irreversible inhibitors as the recovery of activity after inactivation is slow. The half-lives at 37 degrees C of the L 658758 and L 659286 derived HLE-I complexes were 9 and 6.5 h, respectively. The complexes produced by both inhibitors are similar chemically since the thermodynamic parameters for activation to regenerate active enzyme are essentially identical. The free energy of activation for this process is dominated primarily by the enthalpy term. The stability of the final complexes likely arises from Michael addition on the active site histidine to the 3'-methylene.     

Directional control of neurite outgrowth from cultured hippocampal neurons is modulated by the lectin concanavalin A.

Cell surface carbohydrates play an important role in the regulation of neurite outgrowth during neuronal development. We have investigated the actions of the plant lectin concanavalin A (Con A), a carbohydrate-binding protein, on neurite outgrowth from hippocampal pyramidal neurons in primary cell culture. Neurons plated in culture medium containing nanomolar concentrations of Con A have a larger number of primary neurites arising directly from the cell soma than do neurons plated in culture medium alone. Furthermore, Con A causes counterclockwise turning of neurites in over 70% of the cultured neurons. Both of these effects of Con A are blocked by the hapten sugar alpha-methyl-D-mannopyranoside, suggesting that they result from the interaction of Con A with a cell surface carbohydrate. Another lectin with a different sugar specificity, wheat germ agglutinin, does not modulate neurite outgrowth. Analysis of neurite outgrowth using video-enhanced microscopy reveals that the counterclockwise turning is accompanied by directionally biased extension of filopodia from the growth cones of growing neurites. Treatment of the neurons with cytochalasin, which disrupts actin polymerization, eliminates the neurite turning induced by Con A, suggesting that actin microfilaments are involved in directional control of neurite outgrowth.     

RIG-I Detects Triphosphorylated RNA of Listeria monocytogenes during Infection in Non-Immune Cells

The innate immune system senses pathogens by pattern recognition receptors in different cell compartments. In the endosome, bacteria are generally recognized by TLRs; facultative intracellular bacteria such as Listeria, however, can escape the endosome. Once in the cytosol, they become accessible to cytosolic pattern recognition receptors, which recognize components of the bacterial cell wall, metabolites or bacterial nucleic acids and initiate an immune response in the host cell. Current knowledge has been focused on the type I IFN response to Listeria DNA or Listeria-derived second messenger c-di-AMP via the signaling adaptor STING. Our study focused on the recognition of Listeria RNA in the cytosol. With the aid of a novel labeling technique, we have been able to visualize immediate cytosolic delivery of Listeria RNA upon infection. Infection with Listeria as well as transfection of bacterial RNA induced a type-I-IFN response in human monocytes, epithelial cells or hepatocytes. However, in contrast to monocytes, the type-I-IFN response of epithelial cells and hepatocytes was not triggered by bacterial DNA, indicating a STING-independent Listeria recognition pathway. RIG-I and MAVS knock-down resulted in abolishment of the IFN response in epithelial cells, but the IFN response in monocytic cells remained unaffected. By contrast, knockdown of STING in monocytic cells reduced cytosolic Listeria-mediated type-I-IFN induction. Our results show that detection of Listeria RNA by RIG-I represents a non-redundant cytosolic immunorecognition pathway in non-immune cells lacking a functional STING dependent signaling pathway.     

A population‐based temporal logic gate for timing and recording chemical events

Engineered bacterial sensors have potential applications in human health monitoring, environmental chemical detection, and materials biosynthesis. While such bacterial devices have long been engineered to differentiate between combinations of inputs, their potential to process signal timing and duration has been overlooked. In this work, we present a two‐input temporal logic gate that can sense and record the order of the inputs, the timing between inputs, and the duration of input pulses. Our temporal logic gate design relies on unidirectional DNA recombination mediated by bacteriophage integrases to detect and encode sequences of input events. For an E. coli strain engineered to contain our temporal logic gate, we compare predictions of Markov model simulations with laboratory measurements of final population distributions for both step and pulse inputs. Although single cells were engineered to have digital outputs, stochastic noise created heterogeneous single‐cell responses that translated into analog population responses. Furthermore, when single‐cell genetic states were aggregated into population‐level distributions, these distributions contained unique information not encoded in individual cells. Thus, final differentiated sub‐populations could be used to deduce order, timing, and duration of transient chemical events.     

Century-scale Methylome Stability in a Recently Diverged Arabidopsis thaliana Lineage

There has been much excitement about the possibility that exposure to specific environments can induce an ecological memory in the form of whole-sale, genome-wide epigenetic changes that are maintained over many generations. In the model plant Arabidopsis thaliana, numerous heritable DNA methylation differences have been identified in greenhouse-grown isogenic lines, but it remains unknown how natural, highly variable environments affect the rate and spectrum of such changes. Here we present detailed methylome analyses in a geographically dispersed A. thaliana population that constitutes a collection of near-isogenic lines, diverged for at least a century from a common ancestor. Methylome variation largely reflected genetic distance, and was in many aspects similar to that of lines raised in uniform conditions. Thus, even when plants are grown in varying and diverse natural sites, genome-wide epigenetic variation accumulates mostly in a clock-like manner, and epigenetic divergence thus parallels the pattern of genome-wide DNA sequence divergence.     

Testing meter, rhythm, and tempo discriminations in pigeons

Rhythmic grouping and discrimination is fundamental to music. When compared to the perception of pitch, rhythmic abilities in animals have received scant attention until recently. In this experiment, four pigeons were tested with three types of auditory rhythmic discriminations to investigate their processing of this aspect of sound and music. Two experiments examined a meter discrimination in which successively presented idiophonic sounds were repeated in meters of different lengths in a go/no-go discrimination task. With difficulty, the birds eventually learned to discriminate between 8/4 and 3/4 meters constructed from cymbal and tom drum sounds at 180 beats per minute. This discrimination subsequently transferred to faster tempos, but not to different drum sounds or their combination. Experiment 3 tested rhythmic and arrhythmic patterns of sounds. After 40 sessions of training, these same pigeons showed no discrimination. Experiment 4 tested repetitions of a piano sound at fast and slow tempos. This discrimination was readily learned and showed transfer to novel tempos. The pattern of results suggests that pigeons can time periodic auditory events, but their capacity to understand generalized rhythmic groupings appears limited.