Mechanism of activation of 1,2-dehydro-N-acetyldopamine for cuticular sclerotization

The mechanism of oxidation of 1,2-dehydro-N-acetyldopamine (dehydro NADA) was examined to resolve the controversy between our group and Andersen's group regarding the reactive species involved in β-sclerotization. While Andersen has indicated that dehydro NADA quinone is the β-sclerotizing agent [Andersen, 1989], we have proposed quinone methides as the reactive species for this process [Sugumaran, 1987; Sugumaran, 1988]. Since dehydro NADA quinone has not been isolated or identified till to date, we studied the enzymatic oxidation of dehydro NADA in the presence of quinone traps to characterize this intermediate. Accordingly, both N-acetylcysteine and o-phenylenediamine readily trapped the transiently formed dehydro NADA quinone as quinone adducts. Interestingly, when the enzymatic oxidation was performed in the presence of o-aminophenol or different catechols, adduct formation between the dehydro NADA side chain and the additives had occurred. The structure of the adducts is in conformity with the generation and reactions of dehydro NADA quinone methide (or its radical). This, coupled with the fact that 4-hydroxyl or amino-substituted quinones instantly transformed into p-quinonoid structure, indicates that dehydro NADA quinone is only a transient intermediate and that it is the dehydro NADA quinone methide that is the thermodynamically stable product. However, since this compound is chemically more reactive due to the presence of both quinone methide and acylimine structure on it, the two side chain carbon atoms are “activated.” Based on these considerations, it is suggested that the quinone methide derived from dehydro NADA is the reactive species responsible for cross-link formation between dehydro NADA and cuticular components during β-sclerotization.     

On the mechanism of formation of N-acetyldopamine quinone methide in insect cuticle

The mechanism of formation of quinone methide from the sclerotizing precursor N-acetyldopamine (NADA) was studied using three different cuticular enzyme systems viz. Sarcophaga bullata larval cuticle, Manduca sexta pharate pupae, and Periplaneta americana presclerotized adult cuticle. All three cuticular samples readily oxidized NADA. During the enzyme-catalyzed oxidation, the majority of NADA oxidized became bound covalently to the cuticle through the side chain with the retention of o-diphenolic function, while a minor amount was recovered as N-acetylnorepinephrine (NANE). Cuticle treated with NADA readily released 2-hydroxy-3′,4′-dihydroxyacetophenone on mild acid hydrolysis confirming the operation of quinone methide sclerotization. Attempts to demonstrate the direct formation of NADA-quinone methide by trapping experiments with N-acetylcysteine surprisingly yielded NADA-quinone-N-acetylcysteine adduct rather than the expected NADA-quinone methide-N-acetylcysteine adduct. These results are indicative of NADA oxidation to NADA-quinone and its subsequent isomerization to NADA-quinone methide. Accordingly, all three cuticular samples exhibited the presence of an isomerase, which catalyzed the conversion of NADA-quinone to NADA-quinone methide as evidenced by the formation of NANE—the water adduct of quinone methide. Thus, in association with phenoloxidase, newly discovered quinone methide isomerase seems to generate quinone methides and provide them for quinone methide sclerotization.     

Effects of brefeldin A on the synthesis of chondroitin 4-sulfate by cultures of mouse mastocytoma cells.

Mouse mastocytoma cells were cultured with brefeldin A in medium containing [35S]sulfate and [3H]glucosamine in order to determine the effects of this fungal metabolite on the formation of chondroitin 4-sulfate by these cells. There was a marked reduction in the incorporation of [35S]sulfate into the glycosaminoglycan which was approximately equal to the reduction in the incorporation of [3H]hexosamine into the same molecule. The chondroitin 4-sulfate chain size was greatly diminished, while the number of chains appeared to remain relatively constant, indicating that the brefeldin A partially disrupted the polymerizing system, but had little effect upon movement of the nascent proteochondroitin to the site for chondroitin polymerization and sulfation.     

Quinone and quinone methide as transient intermediates involved in the side chain hydroxylation of N-acyldopamine derivatives by soluble enzymes from Manduca sexta cuticle.

Proteins solubilized from the pharate cuticle of Manduca sexta were fractionated by ammonium sulfate precipitation and activated by the endogenous enzymes. The activated fraction readily converted exogenously supplied N-acetyldopamine (NADA) to N-acetylnorepinephrine (NANE). Either heat treatment (70 degrees C for 10 min) or addition of phenylthiourea (2.5 microM) caused total inhibition of the side chain hydroxylation. If chemically prepared NADA quinone was supplied instead of NADA to the enzyme solution containing phenylthiourea, it was converted to NANE. Presence of a quinone trap such as N-acetylcysteine in the NADA-cuticular enzyme reaction not only prevented the accumulation of NADA quinone, but also abolished NANE production. In such reaction mixtures, the formation of a new compound characterized as NADA-quinone-N-acetylcysteine adduct could be readily witnessed. These studies indicate that NADA quinone is an intermediate during the side chain hydroxylation of NADA by Manduca cuticular enzyme(s). Since such a conversion calls for the isomerization of NADA quinone to NADA quinone methide and subsequent hydration of NADA quinone methide, attempts were also made to trap the latter compound by performing the enzymatic reaction in methanol. These attempts resulted in the isolation of beta-methoxy NADA (NADA quinone methide methanol adduct) as an additional product. Similarly, when the N-beta-alanyldopamine (NBAD)-Manduca enzyme reaction was carried out in the presence of L-kynurenine, two diastereoisomers of NBAD quinone methide-kynurenine adduct (= papiliochrome IIa and IIb) could be isolated.(ABSTRACT TRUNCATED AT 250 WORDS)     

4-alkyl-o-quinone/2-hydroxy-p-quinone methide isomerase from the larval hemolymph of Sarcophaga bullata. I. Purification and characterization of enzyme-catalyzed reaction

An enzyme which catalyzes the conversion of certain 4-alkyl-o-benzoquinones to 2-hydroxy-p-quinone methides has been purified to apparent homogeneity from the hemolymph of Sarcophaga bullata by employing conventional protein purification techniques. The purified enzyme migrated with an approximate molecular weight of 98,000 on gel filtration chromatography. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, it migrated as a single band with a molecular weight of 46,000, indicating that it is made up of two identical subunits. It exhibited a pH optimum of 6.0 and readily converted chemically synthesized as well as enzymatically generated quinones derived from N-acetyldopamine, N-beta-alanyldopamine, and 3,4-dihydroxyphenethyl alcohol to highly unstable 2-hydroxy-p-quinone methides. The quinone methides thus formed were rapidly and nonenzymatically hydrated to form side chain hydroxylated o-diphenols as the stable product. In support of this proposition, when the enzyme reaction with N-acetyldopamine quinone was conducted in the presence of 10% methanol, racemic beta-methoxy-N-acetyldopamine was recovered as an additional product. The quinones of N-acetylnorepinephrine, N-beta-alanylnorepinephrine, and 3,4-dihydroxyphenylglycol were also attacked by the isomerase, resulting in the formation of N-acetylarterenone, N-beta-alanylarterenone and 2-hydroxy-3',4'-dihydroxyacetophenone, respectively as the stable products. The isomerase converted the dihydrocaffeiyl methyl amide quinone to its quinone methide analog which rapidly tautomerized to yield caffeiyl methyl amide. The importance of quinone isomerase in insect immunity and sclerotization of insect cuticle is discussed.     

Protease inhibitor controls prophenoloxidase activation in Manduca sexta

Prophenoloxidase from the hemolymph of tobacco hornworm Manduca sexta can be activated by a specific activating enzyme found in the cuticle. Inhibition studies with benzamidine, diisopropyl phosphofluoridate and p-nitrophenyl-p'-guanidinobenzoate indicate that the activating enzyme is a trypsin-like serine protease. An endogenous protease inhibitor, isolated from the hemolymph of Manduca larvae, inhibits the prophenoloxidase activation mediated by this enzyme. These results indicate that the probable physiological role of endogenous protease inhibitor is to control the undesired activation of prophenoloxidase in the hemolymph.     

Draft genome sequencing and functional annotation and characterization of biofilm-producing bacterium Bacillus novalis PD1 isolated from rhizospheric soil

Antonie van Leeuwenhoek - Biofilm forming bacterium Bacillus novalis PD1 was isolated from the rhizospheric soil of a paddy field. B. novalis PD1 is a Gram-positive, facultatively anaerobic,...     

An Appended Domain Results in an Unusual Architecture for Malaria Parasite Tryptophanyl-tRNA Synthetase

Specific activation of amino acids by aminoacyl-tRNA synthetases (aaRSs) is essential for maintaining fidelity during protein translation. Here, we present crystal structure of malaria parasite Plasmodium falciparum tryptophanyl-tRNA synthetase (Pf-WRS) catalytic domain (AAD) at 2.6 Å resolution in complex with L-tryptophan. Confocal microscopy-based localization data suggest cytoplasmic residency of this protein. Pf-WRS has an unusual N-terminal extension of AlaX-like domain (AXD) along with linker regions which together seem vital for enzymatic activity and tRNA binding. Pf-WRS is not proteolytically processed in the parasites and therefore AXD likely provides tRNA binding capability rather than editing activity. The N-terminal domain containing AXD and linker region is monomeric and would result in an unusual overall architecture for Pf-WRS where the dimeric catalytic domains have monomeric AXDs on either side. Our PDB-wide comparative analyses of 47 WRS crystal structures also provide new mechanistic insights into this enzyme family in context conserved KMSKS loop conformations.     

Sestrin2 regulates monocyte activation through AMPK-mTOR nexus under high-glucose and dyslipidemic conditions

The vicious cycle between hyperinsulinemia and insulin resistance results in the progression of atherosclerosis in the vessel wall. The complex interaction between hyperglycemia and lipoprotein abnormalities promotes the development of atherogenesis. In the early phase of atherosclerosis, macrophage-derived foam cells play an important role in vascular remodeling. Mechanistic target of rapamycin (mTOR) signaling pathway has been identified to play an essential role in the initiation, progression, and complication of atherosclerosis. Recently sestrin2, an antioxidant, was shown to modulate TOR activity and thereby regulating glucose and lipid metabolism. But the role of sestrin2 in monocyte activation is still not clearly understood. Hence, this study is focussed on investigating the role of sestrin2 in monocyte activation under hyperglycemic and dyslipidemic conditions. High-glucose and oxidized low-density lipoprotein (LDL) treatments mediated proinflammatory cytokine production (M1) with a concomitant decrease in the anti-inflammatory cytokine (M2) levels in human monocytic THP1 cells. Both glucose and oxidized LDL (OxLDL) in a dose and time-dependent manner increased the mTOR activation with a marked reduction in the levels of pAMPK and sestrin2 expression. Both high-glucose and OxLDL treatment increased foam cell formation and adhesion of THP1 cells to endothelial cells. Experiments employing activator or inhibitor of adenosine monophosphate kinase (AMPK) as well as overexpression or silencing of sestrin2 indicated that high-glucose mediated monocyte polarization and adhesion of monocytes to the endothelial cells were appeared to be programmed via sestrin2-AMPK-mTOR nexus. Our results evidently suggest that sestrin2 plays a major role in regulating monocyte activation via the AMPK–mTOR-pathway under diabetic and dyslipidemic conditions and also AMPK regulates sestrin2 in a feedback mechanism.