Novel metabolic pathways in Archaea

The Archaea harbor many metabolic pathways that differ to previously recognized classical pathways. Glycolysis is carried out by modified versions of the Embden-Meyerhof and Entner-Doudoroff pathways. Thermophilic archaea have recently been found to harbor a bi-functional fructose-1,6-bisphosphate aldolase/phosphatase for gluconeogenesis. A number of novel pentose-degrading pathways have also been recently identified. In terms of anabolic metabolism, a pathway for acetate assimilation, the methylaspartate cycle, and two CO2-fixing pathways, the 3-hydroxypropionate/4-hydroxybutyrate cycle and the dicarboxylate/4-hydroxybutyrate cycle, have been elucidated. As for biosynthetic pathways, recent studies have clarified the enzymes responsible for several steps involved in the biosynthesis of inositol phospholipids, polyamine, coenzyme A, flavin adeninedinucleotide and heme. By examining the presence/absence of homologs of these enzymes on genome sequences, we have found that the majority of these enzymes and pathways are specific to the Archaea.     

Effects of intraplantar injections of nociceptin and its N-terminal fragments on nociceptive and desensitized responses induced by capsaicin in mice

Injection of capsaicin into the hindpaw has been employed as a model of chemogenic nociception in mice. Intraplantar injection of nociceptin (30–240 pmol) produced a significant and dose-dependent antinociceptive activity in the capsaicin test. The nociceptin N-terminal fragments, (1–11) and (1–13), were also active with a potency higher than nociceptin and comparable to nociceptin, respectively. Intraplantar injection of the nociceptin (1–7) fragment had no effect on capsaicin-induced nociception. Antinociception induced by nociceptin or nociceptin (1–13) was reversed significantly by intraplantar co-injection of [Nphe¹]nociceptin (1–13)NH₂, an orphan opioid receptor-like 1 (ORL1) receptor antagonist, whereas local injection of the antagonist did not interfere with the action of nociceptin (1–11). Nociceptin (1–11) was approximately 2.0-fold more potent than naturally occurring peptide nociceptin, and 10-fold more active than intraplantar morphine. Nociceptive licking/biting response to intraplantar injection of capsaicin was desensitized by repeated injections of capsaicin at the interval of 15 min. Desensitization induced by capsaicin was attenuated significantly by co-injection of nociceptin at much lower doses than antinociceptive ED₅₀ for nociceptin. Capsaicin desensitization was also decreased by co-injection of nociceptin (1–11) and (1–13) to a similar extent. The present results indicate that not only nociceptin but also the N-terminal fragment (1–13) possesses a local peripheral antinociceptive action, which may be mediated by peripheral ORL1 receptors. In addition, the difference of the effective doses suggests that the antinociceptive action and inhibition of capsaicin-induced desenitization by nociceptin, nociceptin (1–11) and (1–13), may involve distinct mechanisms at the level of the peripheral nerve terminal.     

Generation and properties of ascorbic acid-overproducing transgenic tobacco cells expressing sense RNA for <Emphasis Type="SmallCaps">l</Emphasis>-galactono-1,4-lactone dehydrogenase

l-Galactono-1,4-lactone dehydrogenase (GalLDH; EC 1.3.2.3) is the last enzyme in the putative l-ascorbic acid (AsA) biosynthetic pathway of plants. Here, we show for the first time that the overexpression of GalLDH can increase AsA content in tobacco (Nicotiana tabacum L.) BY-2 cells. To see the effect, we analyzed the properties of these AsA-overproducing transgenic cell lines, especially in relation to AsA content of cells, cell division, senescence and resistance to oxidative stress. The mitotic index in AsA-overproducing cells was higher than in wild-type cells. Moreover, the browning of these cells was markedly restrained, and the proportion of dead cells was reduced, especially in the later period of culture. These AsA-overproducing cells also acquired resistance to paraquat (methyl viologen), which produces active oxygen species. These results contribute to the previous insights about AsA and raise the possibility of the generation of plants that have resistance to environmental stresses by increasing their AsA content.     

Functional studies on a ketoreductase gene from Streptomyces sp. AM-7161 to control the stereochemistry in medermycin biosynthesis

Medermycin shows the same trans (3S,15R) configuration as actinorhodin in the pyran ring crucial for its bioactivity. One medermycin biosynthetic gene, med-ORF12, is assumed to be involved in the stereochemical control at C-3. Functional complementation suggested that it plays a similar role as actVI-ORF1 previously proved to determine the stereospecificity at C-3 in actinorhodin biosynthesis. Co-expression of med-ORF12 with actinorhodin early biosynthetic genes further demonstrated that med-ORF12 encodes a ketoreductase responsible for the enantioselective reduction at C-3 in the formation of the pyran ring.     

Hemocyanin in the exoskeleton of crustaceans: enzymatic properties and immunolocalization

We investigated the enzymatic properties and immunohistochemical localization of cuticular hemocyanin, a known oxygen transporter in the prawn Penaeus japonicus. The molecular weight of hemocyanin purified from the cuticle was estimated to be 67-77 k using SDS-PAGE, and the purified protein was effectively converted into a phenoloxidase-like enzyme by an SDS-treatment. The activated enzyme catalyzed the o-hydroxylation of monophenols and the oxidation of o-diphenols and was inhibited by typical inhibitors of phenoloxidase. These characteristics were nearly identical to the enzymatic properties of hemolymph hemocyanin. Immunological detection showed a diffuse distribution of hemocyanin over the exocuticle and endocuticle, and a higher signal level was observed in the latter. Based on these results, roles of hemocyanin in various physiological processes such as immune response and sclerotization of the cuticle were discussed.     

Evaluation of adriamycin nephropathy by an in vivo electron paramagnetic resonance

A rat model for human minimal change nephropathy was obtained by the intravenous injection of adriamycin (ADR) at 5 mg/kg. By using an in vivo electron paramagnetic resonance (EPR) spectrometer operating at 700 MHz, the temporal changes in signal intensities of a nitroxide radical, 4-hydroxyl-2,2,6,6-tetramethylpiperidine-1-oxyl (TEMPOL), in the kidneys of rats with ADR nephropathy were investigated. The decay rate of the EPR signal intensity obtained in the kidney is indicative of the renal reducing ability. It was found that the reducing ability in the kidney declined on the 7th day after ADR administration and recovered after the 14th day. Impairment of the reducing ability occurred before the appearance of continuous urinary protein. The in vitro EPR study showed that this impairment of in vivo renal reducing ability is related to impairment of the reducing ability in the mitochondria.     

Enhancement of poly(3-hydroxybutyrate-co-3-hydroxyvalerate) production in the transgenic Arabidopsis thaliana by the in vitro evolved highly active mutants of polyhydroxyalkanoate (PHA) synthase from Aeromonas caviae

In this study, the enhancement of photosynthetic PHA production was achieved using the highly active mutants of PHA synthase created by the in vitro evolutionally techniques. The wild-type and mutated PHA synthase genes from Aeromonas caviae were introduced into Arabidopsis thaliana together with the NADPH-dependent acetoacetyl-CoA reductase gene from Ralstonia eutropha. Expression of the highly active mutated PHA synthase genes, N149S and D171G, led to an 8-10-fold increase in PHA content in the T1 transgenic Arabidopsis, compared to plants harboring the wild-type PHA synthase gene. In homozygous T2 progenies, PHA content was further increased up to 6.1 mg/g cell dry weight. GC/MS analysis of the purified PHA from the transformants revealed that these PHAs were poly(3-hydroxybutyrate-co-3-hydroxyvalerate) [P(3HB-co-3HV)] copolymers consisting of 0.2-0.8 mol % 3HV. The monomer composition of the P(3HB-co-3HV) copolymers synthesized by the wild-type and mutated PHA synthases reflected the substrate specificities observed in Escherichia coli. These results indicate that in vitro evolved PHA synthases can enhance the productivity of PHA and regulate the monomer composition in transgenic plants.     

Uncoupling and endocytosis of 5-hydroxytryptamine 4 receptors. Distinct molecular events with different GRK2 requirements

The 5-hydroxytryptamine type 4 receptors (5-HT4Rs) are involved in memory, cognition, feeding, respiratory control, and gastrointestinal motility through activation of a G(s)/cAMP pathway. We have shown that 5-HT4R undergoes rapid and profound homologous uncoupling in neurons. However, no significant uncoupling was observed in COS-7 or HEK293 cells, which expressed either no or a weak concentration of GRK2, respectively. High expression of GRK2 in neurons is likely to be the reason for this difference because overexpression of GRK2 in COS-7 and HEK293 cells reproduced rapid and profound uncoupling of 5-HT4R. We have also shown, for the first time, that GRK2 requirements for uncoupling and endocytosis were very different. Indeed, beta-arrestin/dynamin-dependent endocytosis was observed in HEK293 cells without any need of GRK2 overexpression. In addition to this difference, uncoupling and beta-arrestin/dynamin-dependent endocytosis were mediated through distinct mechanisms. Neither uncoupling nor beta-arrestin/dynamin-dependent endocytosis required the serine and threonine residues localized within the specific C-terminal domains of the 5-HT4R splice variants. In contrast, a cluster of serines and threonines, common to all variants, was an absolute requirement for beta-arrestin/dynamin-dependent receptor endocytosis, but not for receptor uncoupling. Furthermore, beta-arrestin/dynamin-dependent endocytosis and uncoupling were dependent on and independent of GRK2 kinase activity, respectively. These results clearly demonstrate that the uncoupling and endocytosis of 5-HT4R require different GRK2 concentrations and involve distinct molecular events.