MexAB-OprM specific efflux pump inhibitors in Pseudomonas aeruginosa. Part 6: exploration of aromatic substituents

A series of 4-oxo-4H-pyrido[1,2-a]pyrimidine derivatives, derivatized at the 2-position with aromatic substituents, were synthesized by the Suzuki cross-coupling method and evaluated for their ability to potentiate the activity of the fluoroquinolone levofloxacin (LVFX) and the anti-pseudomonas β-lactam aztreonam (AZT) in Pseudomonas aeruginosa. By incorporating hydrophilic substituents onto the aryl nucleus, we found a morpholine analogue that possessed improved solubility, retained activity in vitro, and displayed potentiation activity in vivo in a rat model of P. aeruginosa pneumonia.     

The involvement of 1-aminocyclopropane-1-carboxylic acid synthase isogene, Pp-ACS1, in peach fruit softening

Ethylene promotes fruit ripening, including softening. The fruit of melting-flesh peach (Prunus persica (L). Batsch) cultivar 'Akatsuki' produces increasing levels of ethylene, and the flesh firmness softens rapidly during the ripening stage. On the other hand, the fruit of stony hard peach cultivars 'Yumyeong', 'Odoroki', and 'Manami' does not soften and produces little ethylene during fruit ripening and storage. To clarify the mechanism of suppression of ethylene production in stony hard peaches, the expression patterns of four ethylene biosynthesis enzymes were examined: ACC synthases (Pp-ACS1, Pp-ACS2, and Pp-ACS3) and ACC oxidase (Pp-ACO1). In the melting-flesh cultivar 'Akatsuki', Pp-ACS1 mRNA was dramatically induced after harvesting, and a large amount of ethylene was produced. On the other hand, in stony hard peaches, Pp-ACS1 mRNA was not induced during the ripening stage, and ethylene production was inhibited. Since Pp-ACS1 mRNA was induced normally in senescing flowers, wounded leaves, and wounded immature fruit of 'Yumyeong', Pp-ACS1 was suppressed only at the ripening stage, and was not a defect in Pp-ACS1. These results indicate that the suppression of fruit softening in stony hard peach cultivars was caused by a low level of ethylene production, which depends on the suppressed expression of Pp-ACS1.     

Inhibition of MHC class II expression and immune responses by c-MIR

We previously reported a novel E3 ubiquitin ligase (E3), designated as c-MIR, which targets B7-2 to lysosomal degradation and down-regulates the B7-2 surface expression through ubiquitination of its cytoplasmic tail. B7-2 is well known as a costimulatory molecule for Ag presentation, suggesting that the manipulation of c-MIR expression modulates immune responses in vivo. To examine this hypothesis, we generated genetically modified mice in which c-MIR was expressed under an invariant chain (Ii) promoter. Dendritic cells derived from genetically engineered mice showed low ability to present Ags. In addition, these mice showed resistance to the onset of experimental autoimmune encephalomyelitis and an impaired development of CD4 T cells in the thymus and the periphery. These findings led us to conclude that MHC class II (MHC II) is an additional target for c-MIR. Indeed, forced expression of c-MIR in several B cell lines down-regulated the surface expression of MHC II, and down-regulation was found to depend on the presence of a single lysine residue in the cytoplasmic tail of the I-A beta-chain. In a reconstitution system using 293T cells, we found that the lysine residue at position 225 in the I-A beta-chain was ubiquitinated by c-MIR. To our knowledge, c-MIR is the first example of an E3 that is capable of inhibiting MHC II expression. Our findings suggest that c-MIR might potently regulate immune responses in vivo.     

Detachment-associated changes in lipid rafts of senescent human fibroblasts

We characterized the effects of in vitro cellular aging on constituents of lipid rafts in human diploid fibroblasts, TIG-1. Cholesterol recovery from lipid rafts of senescent cells was decreased by the detaching treatment, while the decrease was far less obvious in young cells. A probe that binds selectively to cholesterol in lipid rafts revealed that the amount of lipid rafts on the cell surface decreased in senescent cells upon cell detachment. Accompanying this change was the release of the raft-associated molecules caveolin and Fyn from lipid rafts upon cell detachment, suggesting a detachment-associated disorganization of lipid rafts in senescent cells. In addition, our observations showing differential sensitivities of lipid rafts from young and senescent cells to detaching treatment indicate a caution in how to detach cells. Particular attention needs to be paid to interpreting the results when lipid rafts are prepared from mechanically detached cells under detergent-free conditions.     

Salt-activated endoglucanase of a strain of alkaliphilic Bacillus agaradhaerens

An endoglucanase was purified to homogeneity from an alkaline culture broth of a strain isolated from␣seawater and identified here as Bacillus agaradhaerens JAM-KU023. The molecular mass was around 38-kDa and the N-terminal 19 amino acids of the purified enzyme exhibited 100% sequence identity to Cel5A of B. agaradhaerens DSM8721^T. The enzyme activity increased around 4-fold by the addition of 0.2–2.0 M NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). KCl, Na₂SO₄, NaBr, NaNO₃, CH₃COONa, LiCl, NH₄NO₃, and NH₄Cl also activated the enzyme up to 2- to 4-fold. The optimal pH and temperature values were pH 7–9.4 and 60 °C with 0.2 M NaCl, but pH 6.5–7 and 50 °C without NaCl; enzyme activity increased approximately 6-fold at 60 °C with 0.2 M NaCl compared to that at 50 °C without NaCl in 0.1 M glycine–NaOH buffer (pH 9.0). The thermostability and pH stability of the enzyme were not affected by NaCl. The enzyme was very stable to several chemical compounds, surfactants and metal ions (except for Fe²⁺ and Hg²⁺ ions), regardless whether NaCl was present or not. * The nucleotide sequence of 16S rRNA of this strain has been submitted to DDBJ, EMBL, and GenBank databases under accession no. AB211544.     

Lipids in oxygen-evolving photosystem II complexes of cyanobacteria and higher plants

Lipids in dimeric photosystem II complexes prepared from two species of cyanobacteria, Thermosynechococcus vulcanus and Synechocystis sp. PCC6803, and two higher plants, spinach and rice, were analyzed to determine how many lipid molecules and what class of lipids are present in the photosystem II complexes. It was estimated that 27, 20, 8, and 7 lipid molecules per monomer are bound to the dimeric photosystem II complexes of T. vulcanus, Synechocystis, spinach, and rice, respectively. In each of the organisms, the lipid composition of the photosystem II complexes was quite different from that of the thylakoid membranes used for preparation of the complexes. The content of phosphatidylglycerol in the photosystem II complexes of each organism was much higher than that in the thylakoid membranes. Phospholipase A2 treatment of the photosystem II complexes of Synechocystis that degraded phosphatidylglycerol resulted in impairment of QB-mediated but not QA-mediated electron transport. These findings suggest that phosphatidylglycerol plays important roles in the electron transport at the QB-binding site in photosystem II complexes.     

Heavy Metal Concentration in Sediments of the Nhue River and its Water-Irrigated Farmland Soil in the Suburbs of Hanoi,Vietnam

The heavy metal pollution of sediment in the Nhue River, which receives wastewater from the To Lich and Kim Nguu River system, was investigated together with the effects of use of this water for irrigation of the surrounding farmland. Eighty soil samples and 40 sediment samples were collected from six locations in the Nhue River and two locations in the To Lich River for chemical and physical analyses. The results showed that the sediments in the Nhue River are heavily polluted by metals (71–420 mg/kg for Cu, 77–433 mg/kg for Pb, 150–350 mg/kg for Zn, 0.7–8.7 mg/kg for Cd, 80– 583 mg/kg for Cr, and 32–70 mg/kg for Ni). There were positive correlations between heavy metal concentration and both clay and organic matter contents in the sediment samples. The concentrations of all metals in soil samples were much higher than the background levels in the farmland, Cd, Cu, and Pb, exceeding Vietnamese standards for agricultural grounds.     

Species composition of thrips (Thysanoptera: Thripidae) and spider mites (Acari: Tetranychidae) on cultivated chrysanthemum (Asteraceae) in Okinawa,southwestern Japan

We investigated the species composition of phytophagous thrips and spider mites on cultivated chrysanthemum in Okinawa, southwestern Japan. Eight thrips species belonging to the genera Frankliniella, Microcephalothrips, Scirtothrips and Thrips were found on chrysanthemum leaves. Among them, Thrips nigropilosus was the predominant species irrespective of season, island or cultivation environment (meshed greenhouse or open field), with its infestation frequency being 89% of the fields in which thrips occurred. This high frequency of occurrence suggests that T. nigropilosus is a major pest of chrysanthemum in Okinawa, even though this species has rarely been regarded as an important pest of chrysanthemum or other crops in any other areas. Thrips palmi was the second most dominant (infestation frequency 36%) and other species were scarce (<14%). Tetranychus urticae (green form) was the only tetranychid species on chrysanthemum in our survey. This lack of diversity among spider mites on chrysanthemum is peculiar considering that eight Tetranychus species have been found on vegetables in the same area. Since T. urticae (green form) has been shown to be resistant to a number of pesticides, severe pesticide applications might have simplified the spider mite fauna on chrysanthemum in Okinawa.