Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Comparison of crude and affinity purified cytosolic epoxide hydrolases from hepatic tissue of control and clofibrate-fed mice

An affinity purification procedure was developed for the cytosolic epoxide hydrolase based upon the selective binding of the enzyme to immobilized methoxycitronellyl thiol. Several elution systems were examined, but the most successful system employed selective elution with a chalcone oxide. This affinity system allowed the purification of the cytosolic epoxide hydrolase activity from livers of both control and clofibrate-fed mice. A variety of biochemical techniques including pH dependence, substrate preference, kinetics, inhibition, amino acid analysis, peptide mapping, Western blotting, analytical isoelectric focusing, and gel permeation chromatography failed to distinguish between the enzymes purified from control and clofibrate-fed animals. The quantitative removal of the cytosolic epoxide hydrolase acting on trans-stilbene oxide from 100,000g supernatants, allowed analysis of remaining activities acting differentially on cis-stilbene oxide and benzo[a]pyrene 4,5-oxide. Such analysis indicated the existence of a novel epoxide hydrolase activity in the cytosol of mouse liver preparations.     

Synthesis and bioassay of isoprenoid 3-alkylthio-1,1,1-trifluoro-2-propanones: Potent,selective inhibitors of juvenile hormone esterase

Four 3-alkylthio-1,1,1-trifluoro-2-propanones with juvenile hormone-like side chains were prepared from citronellol and homogeraniol. These substrates were designed as possible transition-state analogs for the juvenile hormone (JH)-specific esterases present in insects. These four isoprenoid trifluoromethyl ketones were assayed in vitro with JH esterase and general esterases from larvae of the cabbage looper, Trichoplusia ni (Lepidoptera, Noctuidae), and with eel acetylcholinesterase and bovine chymotrypsin. JH esterase inhibition I₅₀ values were in the nanomolar range for all four compounds, while the other esterases had I₅₀'S which were 10³ to 10⁵ higher. The high selectivity of these inhibitors is believed to be due to their similarity in size and functionality to natural JH III. Treatment of T. ni larvae in vivo with solutions of the most active analog, 3-[(E)-4,8-dimethyl-3,7-nonadienylthio]-1,1,1-trifluoro-2-propanone (DNTFP) causes a dose-dependent delay in pupation and a concurrent selective inhibition of JH esterase. These data support the hypothesis that the reduction in in vivo JH titer in larval T. ni is due, in part, to hydrolysis of the hormone by selective esterases. DNTFP appears to be competing with JH for the active site of JH esterase.     

Photoaffinity labelling of juvenile hormone binding proteins in the cockroach Leucophaeamaderae

The synthesis and testing of several diazocarbonyl JH analogs (diazo JHA) which act as photoaffinity labels for insect juvenile hormone binding proteins are described. The best competitor, 10,11-epoxyfarnesyl diazoacetate, has been shown to irreversibly reduce [³H]-JH III binding to both ovarian and hemolymph JHBP from Leucophaeamaderae after irradiation at 254 nm for 20 seconds. No loss of activity was observed after incubation of JHBP and diazo JHA without irradiation. Protection from photoinactivation by diazo JHA II was achieved by the presence of an equimolar amount of JH III during the photolysis. Photoaffinity labeled proteins show loss of binding capacity without alteration of the binding affinity. This is the first example of the use of a photoaffinity label in the study of JH action on a molecular level, and may become a valuable tool in the elucidation of JH-receptor-chromatin interactions.     

Photoaffinity labeling of allatostatin receptor proteins in the corpora allata of the cockroach, Diploptera punctata.

Membranes of corpora allata, obtained from Diploptera punctata virgin females, were incubated for 45 min with a bioactive, radioiodinated azidosalicylamide photoaffinity analogue of allatostatin-1. Irradiation of the incubation mixture at 254 nm, followed by sodium dodecyl sulfate polyacrylamide gel electrophoresis and autoradiography, revealed the presence of two bands (59 and 39 kDa) whose specific labeling was demonstrated by addition of excess allatostatin. In brain preparations, clear competition by excess allatostatin was shown for a 41 kDa protein. The abdominal fat body also contained proteins (38, 41, 60 kDa) which bound specifically to the photoaffinity analogue. The proteins identified in corpora allata are likely involved in allatostatin recognition, but the functions of those identified in the brain and fat body are as yet undetermined.     

Aldehyde-oxidizing enzymes in an adult moth: in vitro study of aldehyde metabolism in Heliothis virescens

The conversion of pheromonal aldehydes to carboxylic acids in vitro in tissue extracts of Heliothis virescens is catalyzed by both aldehyde dehydrogenase and aldehyde oxidase enzymes. The aldehyde-oxidizing activity in antennae, heads, legs, and hemolymph from male and female moths was examined by radiochromatographic and spectroscopic assays. First, the enzymatic activity was measured in the presence or absence of added NAD+ using either (Z)-9-tetradecenal or (Z)-11-hexadecenal as tritiated substrate. Second, substrate specificity was determined spectroscopically by (i) indirect measurement of the AO-released hydrogen peroxide through the coupled AO-horseradish peroxidase reaction and by (ii) direct measurement of the ALDH-produced NADH. Both aldehyde-oxidizing activities were associated with soluble enzymes in the antennal extracts, and these enzymes degraded pheromone and nonpheromonal aldehydes. Both AO and ALDH activities were present in male and female tissues. AO activity was exhibited primarily in the antennal extracts and to a lesser degree in the leg extracts. Moreover, ALDH activity was distributed in the antenna, head, and leg extracts. A vinyl ketone analog of (Z)-11-hexadecenal preferentially inhibited the ALDH activity over the AO activity.     

Phosphorothioate analogs of sn-2 radyl lysophosphatidic acid (LPA): Metabolically stabilized LPA receptor agonists

We describe an efficient synthesis of metabolically stabilized sn-2 radyl phosphorothioate analogs of lysophosphatidic acid (LPA), and the determination of the agonist activity of each analog for the six LPA receptors (LPA_1–6) using a recently developed TGFα shedding assay. In general, the sn-2 radyl OMPT analogs showed similar agonist activities to the previous 1-oleoyl-2-O-methyl-glycerophosphothioate (sn-1 OMPT) analogs for LPA_1–6 receptors. In most cases, the sn-2 radyl-OMPT analogs were more potent agonists than LPA itself. Most importantly, sn-2 alkyl OMPT analogs were very potent LPA₅ and LPA₆ agonists. The availability of sn-2 radyl OPMT analogs further refines the structure–activity relationships for ligand–receptor interactions for this class of GPCRs.