Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

After invasion into intercellular spaces of tomato plants, the soil‐borne, plant‐pathogenic Ralstonia solanacearum strain OE1‐1 forms mushroom‐shaped biofilms (mushroom‐type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1‐1 produces aryl‐furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3‐hydroxymyristate (3‐OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity‐deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1‐1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1‐1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1‐1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB‐ and ralA‐deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1‐1, although a QS‐deficient, phcB‐deleted mutant formed mBFs similar to OE1‐1. Supplementation with 3‐OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3‐OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1‐1, but also in the suppression of QS‐mediated negative regulation of mBF formation.     

A Docosahexaenoic Acid-Derived Pro-resolving Agent,Maresin 1, Protects Motor Neuron Cells Death

Maresin 1 is a novel pro-resolving mediator derived from docosahexaenoic acid (DHA), with potent anti-inflammation effects against several animal models, including brain ischemia, sepsis, and lung fibrosis. However, its effect against motor neuron cell death is still not investigated. Therefore, we investigated the effects of maresin 1 on several stress-induced motor neuron cell death. Maresin 1 suppressed combinatorial stress which was evoked by superoxide dismutase 1 (SOD1)^G93A and serum-free, -induced motor neuron cells death in a concentration-dependent manner, and had a stronger neuroprotective effective than DHA. Maresin 1 also had neuroprotective effects against transactivation response DNA-binding protein (TDP)-43^A315T and serum-free stress, H₂O₂, and tunicamycin-induced cell death. Maresin 1 reduced the reactive oxygen species (ROS) production caused by SOD1^G93A or TDP-43^A315T. Moreover, maresin 1 suppressed the NF-κB activation induced by SOD1^G93A and serum-free stress. These data indicate that maresin 1 has motor neuron protective effects against several stresses by reduction of ROS production or attenuation of the NF-κB activation. Maresin 1 also had neuroprotective effects against H₂O₂, and tunicamycin-induced cell death in a concentration-dependent manner. Finally, maresin 1 ameliorated the motor function deficits of spinal muscular atrophy model in which endoplasmic reticulum stress was upregulated. Thus, maresin 1 may be beneficial to protect against motor neuron diseases.     

Metabolic pathway of 6-aminohexanoate in the nylon oligomer-degrading bacterium Arthrobacter sp. KI72: identification of the enzymes responsible for the conversion of 6-aminohexanoate to adipate

Arthrobacter sp. strain KI72 grows on a 6-aminohexanoate oligomer, which is a by-product of nylon-6 manufacturing, as a sole source of carbon and nitrogen. We cloned the two genes, nylD ₁ and nylE ₁ , responsible for 6-aminohexanoate metabolism on the basis of the draft genomic DNA sequence of strain KI72. We amplified the DNA fragments that encode these genes by polymerase chain reaction using a synthetic primer DNA homologous to the 4-aminobutyrate metabolic enzymes. We inserted the amplified DNA fragments into the expression vector pColdI in Escherichia coli, purified the His-tagged enzymes to homogeneity, and performed biochemical studies. We confirmed that 6-aminohexanoate aminotransferase (NylD₁) catalyzes the reaction of 6-aminohexanoate to adipate semialdehyde using α-ketoglutarate, pyruvate, and glyoxylate as amino acceptors, generating glutamate, alanine, and glycine, respectively. The reaction requires pyridoxal phosphate (PLP) as a cofactor. For further metabolism, adipate semialdehyde dehydrogenase (NylE₁) catalyzes the oxidative reaction of adipate semialdehyde to adipate using NADP⁺ as a cofactor. Phylogenic analysis revealed that NylD₁ should be placed in a branch of the PLP-dependent aminotransferase sub III, while NylE₁ should be in a branch of the aldehyde dehydrogenase superfamily. In addition, we established a NylD₁/NylE₁ coupled system to quantify the aminotransferase activity and to enable the conversion of 6-aminohexaoate to adipate via adipate semialdehyde with a yield of > 90%. In the present study, we demonstrate that 6-aminohexanoate produced from polymeric nylon-6 and nylon oligomers (i.e., a mixture of 6-aminohexaoate oligomers) by nylon hydrolase (NylC) and 6-aminohexanoate dimer hydrolase (NylB) reactions are sequentially converted to adipate by metabolic engineering technology.

     

Cloning and genetic and sequence analyses of the bacteriocin 21 determinant encoded on the Enterococcus faecalis pheromone-responsive conjugative plasmid pPD1.

The pheromone-responsive conjugative plasmid pPD1 (59 kb) of Enterococcus faecalis encodes the bacteriocin 21 (bac21) determinant. Cloning, transposon insertion mutagenesis and sequence analysis of the bac21 determinant showed that an 8.5-kb fragment lying between kb 27.1 and 35.6 of the pPD1 map is required for complete expression of the bacteriocin. The 8.5-kb fragment contained nine open reading frames (ORFs), bacA to bac1, which were oriented in the same (upstream-to-downstream) direction. Transposon insertions into the bacA to bacE ORFs, which are located in the proximal half of bac21, resulted in defective bacteriocin expression. Insertions into the bacF to bac1 ORFs, which are located in the distal half of bac21, resulted in reduced bacteriocin expression. Deletion mutant analysis of the cloned 8.5-kb fragment revealed that the deletion of segments between kb 31.6 and 35.6 of the pPD1 map, which contained the distal region of the determinant encoding bacF to bac1, resulted in reduced bacteriocin expression. The smallest fragment (4.5 kb) retaining some degree of bacteriocin expression contained the bacA to bacE sequences located in the proximal half of the determinant. The cloned fragment encoding the 4.5-kb proximal region and a Tn916 insertion mutant into pPD1 bacB trans-complemented intracellularly to give complete expression of the bacteriocin. bacA encoded a 105-residue sequence with a molecular mass of 11.1 kDa. The deduced BacA protein showed 100% homology to the broad-spectrum antibiotic peptide AS-48, which is encoded on the E. faecalis conjugative plasmid pMB2 (58 kb). bacH encoded a 195-residue sequence with a molecular mass of 21.9 kDa. The deduced amino acid sequence showed significant homology to the C-terminal region of HlyB (31.1% identical residues), a protein located in the Escherichia coli alpha-hemolysin operon that is a representative bacterial ATP-binding cassette export protein.     

The enhancement of oxidative DNA damage by anti-diabetic metformin,buformin, and phenformin,via nitrogen-centered radicals

Metformin (N,N-dimethylbiguanide), buformin (1-butylbiguanide), and phenformin (1-phenethylbiguanide) are anti-diabetic biguanide drugs, expected to having anti-cancer effect. The mechanism of anti-cancer effect by these drugs is not completely understood. In this study, we demonstrated that these drugs dramatically enhanced oxidative DNA damage under oxidative condition. Metformin, buformin, and phenformin enhanced generation of 8-oxo-7,8-dihydro-2’-deoxyguanosine (8-oxodG) in isolated DNA reacted with hydrogen peroxide (H₂O₂) and Cu(II), although these drugs did not form 8-oxodG in the absence of H₂O₂ or Cu(II). An electron paramagnetic resonance (EPR) study, utilizing alpha-(4-pyridyl-1-oxide)-N-tert-butylnitrone and 3,3,5,5-tetramethyl-1-pyrroline-N-oxide as spin trapping agents, showed that nitrogen-centered radicals were generated from biguanides in the presence of Cu(II) and H₂O₂, and that these radicals were decreased by the addition of DNA. These results suggest that biguanides enhance Cu(II)/H₂O₂-mediated 8-oxodG generation via nitrogen-centered radical formation. The enhancing effect on oxidative DNA damage may play a role on anti-cancer activity.     

Characterization of aldose reductase and aldehyde reductase from rat testis

Aldose reductase (alditol:NAD(P)+ 1-oxidoreductase, EC 1.1.1.21) and aldehyde reductase (alcohol:NADP+ oxidoreductase, EC 1.1.1.2) were purified to a homogeneity from rat testis. The molecular weights of aldose reductase and aldehyde reductase were estimated to be 38,000 and 41,000 by SDS-polyacrylamide gel electrophoresis, and the pI values of these enzymes were found to be 5.3 and 6.1 by chromatofocusing, respectively. Aldose reductase had activity for aldo-sugars such as xylose, glucose and galactose, whereas aldehyde reductase was virtually inactive for these aldo-sugars. The Km values of aldose reductase for aldo-sugars were relatively high. When a correction was made for the fraction of aldo-sugar present as the aldehyde form, which is the real substrate of the enzyme, the Km values were much lower. Aldose reductase utilized both NADPH and NADH as coenzyme, whereas aldehyde reductase utilized only NADPH. Aldose reductase was activated significantly by sulfate ion, while aldehyde reductase was little affected. Both enzymes were inhibited strongly by the known aldose reductase inhibitors. However, aldehyde reductase was in general less susceptible to these inhibitors when compared to aldose reductase. Both aldose reductase and aldehyde reductase treated with pyridoxal 5-phosphate have lost the susceptibility to aldose reductase inhibitor, suggesting that in these two enzymes aldose reductase inhibitor interacts with a lysine residue.     

Gibberellin-Dependent Root Elongation in Lactuca sativa: Recovery from Growth Retardant-Suppressed Elongation with Thickening by Low concentration of GA3

Higher concentrations of growth retardants, ancymidol and AMO-1718,were required to suppress root growth than hypocotyl growthin lettuce seedlings. Gibberellic acid (GA₃) counteracted theeffect of these growth retardants, but complete recovery ofroot growth was obtained only in a narrow range of growth retardantconcentrations. A much lower concentration of GA₃ (1 nM) wasneeded for recovery of root growth from ancymidol suppressionthan that for hypocotyl growth (100 (µM). GA₃ synergisticallypromoted root growth at moderate concentrations (10–100nM) with either ancymidol or AMO-1618. Ancymidol not only suppressed root elongation but also causedthickening of the elongation zone of the root, actions whichGA₃ completely canceled. Microscopic observation showed theseeffects were mainly due to the lateral expansion and shorteningof epidermal and cortical cells. Growth kinetics of roots recorded by a computer-regulated rhizometerindicated that the lag times of both growth suppression by ancymidoland growth recovery by GA₃ were about 4 h. -Naphthaleneacetic acid (NAA) did not counteract ancymidol suppressionof root and hypocotyl growth. These results support the concept that gibberellins play anindispensable role in root elongation at an extremely lowerconcentration than in hypocotyl elongation. (Received January 7, 1987; Accepted May 14, 1987)     

Possible involvement of protein kinase C and calcium ion in growth factor-induced expression of c-myc oncogene in Swiss 3T3 fibroblasts

The addition of platelet-derived growth factor and fibroblast growth factor to quiescent cultures of Swiss 3T3 fibroblasts rapidly induced protein kinase C activation and Ca2+ mobilization and afterwards markedly increased c-myc mRNA levels. 1-Oleoyl-2-acetylglycerol, a membrane-permeable synthetic diacylglycerol, and 12-O-tetradecanoylphorbol 13-acetate, a tumor-promoting phorbol ester, stimulated protein kinase C activation without Ca2+ mobilization. Inversely, Ca2+ ionophores, A23187 and ionomycin, elicited Ca2+ mobilization without protein kinase C activation. Both protein kinase C-activating and Ca2+-mobilizing agents were able to increase c-myc mRNA levels in an additive manner. Prolonged treatment of the cells with phorbol 12,13-dibutyrate, another protein kinase C-activating phorbol ester, led to the down-regulation and complete disappearance of protein kinase C. In these cells, 1-oleoyl-2-acetylglycerol and 12-O-tetradecanoylphorbol 13-acetate did not increase c-myc mRNA levels, but platelet-derived growth factor, fibroblast growth factor, and the Ca2+ ionophores, all of which still induced Ca2+ mobilization, stimulated the increase of c-myc mRNA levels. These results strongly suggest that both protein kinase C and Ca2+ may be involved in platelet-derived growth factor- as well as fibroblast growth factor-induced expression of the c-myc oncogene in Swiss 3T3 cells.     

Chemical Factors Controlling Floral Bud Formation of Torenia Stem Segments Cultured in Vitro II. Effects of Growth Regulators

Correlative effects between growth regulators added to a mediumand different physiological states of explants on adventitiousbud formation and flowering were investigated using Toreniastem segments cultured in vitro. Indoleacetic acid stimulatedfloral bud formation and its development in explants taken fromreproductive plants. These stimulative effects were clearlyseen in explants taken from plants in which flower abscissionwas taking place, but insignificant when explants were preparedfrom younger materials. Abscisic acid acted in a reverse wayto auxin, greatly promoting floral bud initiation and floweringof originally vegetative explnts. Zeatin at a concentrationof 1 mg/liter inhibited floral bud formation, and at a low concentrationsit was generally ineffective. However, floral bud formationand flowering of explants taken either from basal parts of stemsor from 18- to 20-week-old plants were promoted by zeatin treatment.The action of gibberellic acid seemed rather indirect: at aconcentration of 0.01 mg/liter, it generally stimulated floralbud formation but at a concentration of 1 mg/liter, it was ofteninhibitory. (Received January 17, 1981; Accepted February 21, 1981)