Formation of cholesterol ozonolysis products in vitro and in vivo through a myeloperoxidase-dependent pathway

3β-Hydroxy-5-oxo-5,6-secocholestan-6-al (secosterol-A) and its aldolization product 3β-hydroxy-5β-hydroxy-B-norcholestane-6β-carboxaldehyde (secosterol-B) were recently detected in human atherosclerotic tissues and brain specimens, and they may play pivotal roles in the pathogenesis of atherosclerosis and neurodegenerative diseases. However, as their origin remains unidentified, we examined the formation mechanism, the stability, and the fate of secosterols in vitro and in vivo. About 40% of secosterol-A remained unchanged after 3 h incubation in the FBS-free medium, whereas 20% and 40% were converted to its aldehyde-oxidation product, 3β-hydroxy-5-oxo-secocholestan-6-oic acid, and secosterol-B, respectively. In the presence of FBS, almost all secosterol-A was converted immediately to these compounds. Secosterol-B in the medium, with and without FBS, was relatively stable, but ～30% was converted to its aldehyde-oxidation product, 3β-hydroxy-5β-hydroxy-B-norcholestane-6-oic acid (secoB-COOH). When neutrophil-like differentiated human leukemia HL-60 (nHL-60) cells activated with PMA were cultured in the FBS-free medium containing cholesterol, significantly increased levels of secosterol-A and its aldehyde-oxidation product, but not secosterol-B, were formed. This secosterol-A formation was decreased in the culture of PMA-activated nHL-60 cells containing several reactive oxygen species (ROS) inhibitors and scavengers or in the culture of PMA-activated neutrophils isolated from myeloperoxidase (MPO)-deficient mice. Our results demonstrate that secoterol-A is formed by an ozone-like oxidant generated with PMA-activated neutrophils through the MPO-dependent mechanism.     

ADP-dependent glucokinase/phosphofructokinase, a novel bifunctional enzyme from the hyperthermophilic archaeon Methanococcus jannaschii

A gene encoding an ADP-dependent phosphofructokinase homologue has been identified in the hyperthermophilic archaeon Methanococcus jannaschii via genome sequencing. The gene encoded a protein of 462 amino acids with a molecular weight of 53,361. The deduced amino acid sequence of the gene showed 52 and 29% identities to the ADP-dependent phosphofructokinase and glucokinase from Pyrococcus furiosus, respectively. The gene was overexpressed in Escherichia coli, and the produced enzyme was purified and characterized. To our surprise, the enzyme showed high ADP-dependent activities for both glucokinase and phosphofructokinase. A native molecular mass was estimated to be 55 kDa, and this indicates the enzyme is monomeric. The reaction rate for the phosphorylation of D-glucose was almost 3 times that for D-fructose 6-phosphate. The K(m) values for D-fructose 6-phosphate and D-glucose were calculated to be 0.010 and 1.6 mm, respectively. The K(m) values for ADP were 0.032 and 0.63 mm when D-glucose and D-fructose 6-phosphate were used as a phosphoryl group acceptor, respectively. The gene encoding the enzyme is proposed to be an ancestral gene of an ADP-dependent phosphofructokinase and glucokinase. A gene duplication event might lead to the two enzymatic activities.     

Purification and Characterization of Exo-beta-d-Glucosaminidase from a Cellulolytic Fungus, Trichoderma reesei PC-3-7

Chitosan-degrading activities induced by glucosamine (GlcN) or N-acetylglucosamine (GlcNAc) were found in a culture filtrate of Trichoderma reesei PC-3-7. One of the chitosan-degrading enzymes was purified to homogeneity by precipitation with ammonium sulfate followed by anion-exchange and hydrophobic-interaction chromatographies. The enzyme was monomeric, and its molecular mass was 93 kDa. The optimum pH and temperature of the enzyme were 4.0 and 50 degrees C, respectively. The activity was stable in the pH range 6.0 to 9.0 and at a temperature below 50 degrees C. Reaction product analysis from the viscosimetric assay and thin-layer chromatography and H nuclear magnetic resonance spectroscopy clearly indicated that the enzyme was an exo-type chitosanase, exo-beta-d-glucosaminidase, that releases GlcN from the nonreducing end of the chitosan chain. H nuclear magnetic resonance spectroscopy also showed that the exo-beta-d-glucosaminidase produced a beta-form of GlcN, demonstrating that the enzyme is a retaining glycanase. Time-dependent liberation of the reducing sugar from partially acetylated chitosan with exo-beta-d-glucosaminidase and the partially purified exo-beta-d-N-acetylglucosaminidase from T. reesei PC-3-7 suggested that the exo-beta-d-glucosaminidase cleaves the glycosidic link of either GlcN-beta(1-->4)-GlcN or GlcN-beta(1-->4)-GlcNAc.     

Glutamate dehydrogenase from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1: enzymatic characterization, identification of the encoding gene, and phylogenetic implications

NADP-dependent glutamate dehydrogenase (l-glutamate: NADP oxidoreductase, deaminating, EC 1.4.1.4) from the aerobic hyperthermophilic archaeon Aeropyrum pernix K1 (JCM 9820) was purified to homogeneity for characterization. The enzyme retained its full activity on heating at 95°C for 30 min, and the maximum activity in l-glutamate deamination was obtained around 100°C. The enzyme showed a strict specificity for l-glutamate and NADP on oxidative deamination and for 2-oxoglutarate and NADPH on reductive amination. The K _m values for NADP, l-glutamate, NADPH, 2-oxoglutarate, and ammonia were 0.039, 3.3, 0.022, 1.7, and 83 mM, respectively. On the basis of the N-terminal amino acid sequence, the encoding gene was identified in the A. pernix K1 genome, cloned, and expressed in Escherichia coli. Analysis of the nucleotide sequence revealed an open reading frame of 1257 bp starting with a minor TTG codon and encoding a protein of 418 amino acids with a molecular weight of 46 170. Phylogenetic analysis revealed that the glutamate dehydrogenase from A. pernix K1 clustered with those from aerobic Sulfolobus solfataricus, Sulfolobus shibatae, and anaerobic Pyrobaculum islandicum in Crenarchaeota, and it separated from another cluster of the enzyme from Thermococcales in Euryarchaeota. The branching pattern of the enzymes from A. pernix K1, S. solfataricus, S. shibatae, and Pb. islandicum in the phylogenetic tree coincided with that of 16S rDNAs obtained from the same organisms. Received: April 24, 2000 / Accepted: August 10, 2000     

Chemical and molecular identification of the invasive termite <Emphasis Type="Italic">Zootermopsis nevadensis</Emphasis> (Isoptera: Archotermopsidae) in Japan

Numerous termite species have been introduced outside their native ranges by human transport, and some have become invasive. The dampwood termite Zootermopsis nevadensis (Hagen), which is native to western North America, has been introduced to and become established in Kawanishi City, Hyogo Prefecture, Japan. Zootermopsis nevadensis is subdivided into two subspecies based on cuticular hydrocarbon (CHC) phenotypes: Z. nevadensis nevadensis and Z. nevadensis nuttingi (Haverty and Thorne). Here, we identified Z. nevadensis in Japan as hybrids between the two subspecies. Chemical analysis showed the presence of 7,15-dimethylhenicosane and 5,17-dimethylhenicosane in the CHCs of Z. nevadensis in Japan, corresponding to the CHC phenotype of Z. n. nevadensis. Conversely, all mitochondrial cytochrome c oxidase subunit I sequences of Z. nevadensis in Japan were identical to sequences from Z. n. nuttingi and hybrids between the two subspecies from a native hybrid zone in California, USA. In addition, phylogenetic analysis showed that Z. nevadensis in Japan formed a clade with Z. n. nuttingi and hybrids between the two subspecies. Our results show discordance between the chemical and genetic features of Z. nevadensis in Japan, indicating that individuals of Z. nevadensis in Japan are hybrids between the two subspecies.