Polymyxin Acylase: An Enzyme Causing Intramolecular N2⇄N6 Acyl Transfer in N-Monooctanoyl-l-Lysine

Polymyxin acylase from Pseudomonas sp. M-6-3 can deacylate not only polymyxin antibiotics, but also A-fatty acyl-peptides and N-fatty acyl-amino acids. We found that this enzyme causes intramolecular N²?N⁶ acyl transfer in monooctanoyl-l-lysine; when N²-octanoyl-l-lysine is the substrate, N⁶-octanoyl- l-lysine is produced at pH 10.5, but when N⁶-octanoyl- l-lysine is the substrate, N²-octanoyl- l-lysine is produced at pH 8.0. In these reactions, the deacylation proceeded gradually at the final stage and eventually, both N²-octanoyl- l-lysine and N⁶-octanoyl- l-lysine were hydrolyzed to l-lysine and octanoic acid. Furthermore, this enzyme showed intermolecular acyltrans- ferase activity, transferring several N-octanoyl- dl-amino acids to N-octanoyl-hydroxylamine. This acyltransfer ability of polymyxin acylase offers a new method of enzymic N-acylation of compounds containing amino components.     

Mitochondria in platinum resistant cells.

Based on the previous report showing that mitochondrial (MT) alteration is associated with platinum (Pt) resistance, we have determined how the alternative MT function is involved in Pt cell cytotoxicity particularly in relation to the apoptosis. MT membrane potential (delta psi m) semi-quantitatively assessed by rhodamin 123 (Rh) sensitivity was significantly elevated in acquired Pt-resistant 2008/C13*5.25 cells (C13) established from its parental 2008 cells or known intrinsic Pt-resistant JHOC cells established from ovarian clear cell adenocarcinoma. Laser confocal microscopy of these cells stained with Rh revealed that MT in Pt-resistant cells were distributed in whole cytoplasm with relatively higher fluorescent intensity whereas MT in Pt-sensitive cells were localized in perinuclear space with lower fluorescent intensity. Electron microscopy showed the predominantly condensed MT in which crestal structure was not observed clearly in Pt-resistant cells. Western blot analysis using murine monoclonal anti-Bcl-2 antibody showed more than 5-fold Bcl-2 overexpression in Pt-resistant cells in response to cisplatin treatment. Cytochrome C (CytC) in MT was released from MT into cytoplasm in response to cisplatin treatment in Pt-sensitive cells, whereas up-regulation of CytC level in MT rather than CytC release from MT was observed in Pt-resistant cells. These data are strongly suggesting that changes at MT level would impact on the relative resistance of malignant cells to undergo drug-induced apoptosis.     

Further purification and characterization of T4 endonuclease V.

T4 endonuclease V, which is involved in repair of ultraviolet-damaged DNA, has been purified 3600 fold from T4D-infected Escherichia coli. The enzyme shows optimal activity at pH 7.2 and does not require added divalent ions. Endonuclease V attacks both native and heat-denatured DNA provided that the DNA has been irradiated, and the enzyme activity is dependent on the dose of ultraviolet irradiation. The rate and the extent of the reaction are greater with irradiated native DNA although the Km values for the two types of DNA are the same (2.25 - 10(-5) M). The enzyme is readily inactivated by heat and is sensitive to p-chloromercuribenzoate. Endonuclease V-treated irradiated DNA is degraded by spleen phosphodiesterase only when the DNA has been treated with alkaline phosphatase, suggesting that the enzyme produces 5'-phosphoryl termini.     

Thermal responses in the citrus fruit fly,Dacus tsuneonis: evidence for a pupal diapause

Thermal responses controlling pupariation and adult eclosion in a citrus fruit fly,Dacus tsuneonis (Miyake), were studied to understand the winter biology of this species. When mature larvae were exposed to various temperature conditions, the highest percentage of pupariation was obtained at 15 °C, although the variance at this temperature was greater than at 20 °C or 25 °C. Pupariation occurred most rapidly at 20 °C and an alternating temperature with a mean of 15 °C. At constant 15 °C, pupae failed to emerge as adults. Pupae were characterized by a reduced respiration rate, which is typical of a diapausing pupa. When insects were stored at different temperatures for 45 days after pupariation, and then transferred to 25 °C, adult eclosion occurred earlier when the initial temperature was 10 °C than when it was 5 °C or 15 °C. Adult eclosion occurred most synchronously and pupal mortality was lowest when insects were stored at 15 °C for 90 days before incubation at 25 °C. These results strongly suggest thatD. tsuneonis enters a pupal diapause.     

Carbon dynamics and budget in a Zoysia japonica grassland, central Japan

The ecosystem carbon budget was estimated in a Japanese Zoysia japonica grassland. The green biomass started to grow in May and peaked from mid-July to September. Seasonal variations in soil CO₂ flux and root respiration were mediated by changes in soil temperature. Annual soil CO₂ flux was 1,121.4 and 1,213.6 g C m⁻² and root respiration was 471.0 and 544.3 g C m⁻² in 2007 and 2008, respectively. The root respiration contribution to soil CO₂ flux ranged from 33% to 71%. During the growing season, net primary production (NPP) was 747.5 and 770.1 g C m⁻² in 2007 and 2008, respectively. The biomass removed by livestock grazing (GL) was 122.1 and 102.7 g C m⁻², and the livestock returned 28.2 and 25.6 g C m⁻² as fecal input (FI) in 2007 and 2008, respectively. The decomposition of FI (DL, the dry weight loss due to decomposition) was very low, 1.5 and 1.4 g C m⁻², in 2007 and 2008. Based on the values of annual NPP, soil CO₂ flux, root respiration, GL, FI, and DL, the estimated carbon budget of the grassland was 1.7 and 22.3 g C m⁻² in 2007 and 2008, respectively. Thus, the carbon budget of this Z. japonica grassland ecosystem remained in equilibrium with the atmosphere under current grazing conditions over the 2 years of the study.     

Increased globotriaosylceramide levels in a transgenic mouse expressing human alpha1,4-galactosyltransferase and a mouse model for treating Fabry disease

Fabry disease is a lysosomal storage disorder caused by an α-galactosidase A (α-Gal A) deficiency and resulting in the accumulation of glycosphingolipids, predominantly globotriaosylceramide (Gb3). A transgenic mouse expressing the human α-Gal A R301Q mutant in an α-Gal A-knockout background (TgM/KO) should be useful for studying active-site-specific chaperone (ASSC) therapy for Fabry disease. However, the Gb3 content in the heart tissue of this mouse was too low to detect an ASSC-induced effect. To increase the Gb3 levels in mouse organs, we created transgenic mice (TgG3S) expressing human α1,4-galactosyltransferase (Gb3 synthase). High levels of Gb3 were observed in all major organs of the TgG3S mouse. A TgG3S (+/-)M(+/-)/KO mouse was prepared by cross-breeding the TgG3S and TgM/KO mice and the Gb3 content in the heart of the TgG3S(+/-)M(+/-)/KO mouse was 1.4 μg/mg protein, higher than in the TgM(+/-)/KO (<0.1 μg/mg protein). Treatment with an ASSC, 1-deoxygalactonojirimycin, caused a marked induction of α-Gal A activity and a concomitant reduction of the Gb3 content in the TgG3S(+/-) M(+/-)/KO mouse organs. These data indicated that the TgG3S(+/-) M(+/-)/KO mouse was suitable for studying ASSC therapy for Fabry disease, and that the TgG3S mouse would be useful for studying the effect of high Gb3 levels in mouse organs.