Metabolism of carcinogenic urethane to nitric oxide is involved in oxidative DNA damage

Carcinogenic urethane (ethyl carbamate) forms DNA adduct via epoxide, whereas carcinogenic methyl carbamate can not. To clarify a mechanism independent of DNA adduct formation, we examined DNA damage induced by N-hydroxyurethane, a urethane metabolite, using 32P-5'-end-labeled DNA fragments. N-hydroxyurethane induced Cu(II)-mediated DNA damage especially at thymine and cytosine residues. DNA damage was inhibited by both catalase and bathocuproine, suggesting a role for H(2)O(2) and Cu(I) in DNA damage. Free (*) OH scavengers did not inhibit the DNA damage, although methional did inhibit it. These results suggest that reactive species, such as the Cu(I)-hydroperoxo complex, cause DNA damage. Formation of 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) was increased by N-hydroxyurethane in the presence of Cu(II). When treated with esterase, N-hydroxyurethane induced 8-oxodG formation to a similar extent as that induced by hydroxylamine. Enhancement of DNA cleavages by endonuclease IV suggests that hydroxylamine induced depurination. Furthermore, hydroxylamine induced a significant increase in 8-oxodG formation in HL-60 cells but not in its H(2)O(2)-resistant clone HP 100 cells. o-Phenanthroline significantly inhibited the 8-oxodG formation in HL-60 cells, confirming the involvement of metal ions in the 8-oxodG formation by hydroxylamine. Electron spin resonance spectroscopy, utilizing Fe[N-(dithiocarboxy)sarcosine](3), demonstrated that nitric oxide (NO) was generated from hydroxylamine and esterase-treated N-hydroxyurethane. It is concluded that urethane may induce carcinogenesis through oxidation and, to a lesser extent, depurination of DNA by its metabolites.     

The specific mitochondrial DNA polymorphism found in Klinefelter's syndrome

Hypervariable segments of mitochondrial DNA (mtDNA) (HV1 and HV2) were analyzed in Klinefelter's syndrome and compared to normal population data. One pair of samples consisting of a Japanese mother and affected son with Klinefelter's syndrome (involved in a criminal case), and seven unrelated DNA samples from Caucasian Klinefelter males (two involved in criminal cases and five diagnosed) were collected in Japan and the United States. The diagnosis of Klinefelter's syndrome was established previously by multiplex XY-STR typing detecting two X alleles and one Y allele in the samples. Haplotype analysis of the mtDNA sequence in Klinefelter males was found to be identical, unique, and specific, as it was not found in the normal population. Astonishingly, family data exhibited that the haplotype of the mtDNA in the son was apparently different from the mother's, suggesting that the mtDNA of Klinefelter male would not be inherited from mother to son. Our data indicate that possible interaction of the sex chromosome and the mtDNA exists, and suggests that the specific mtDNA haplotype could cause the abnormal cell to fertilize and reproduce itself.     

DAP12 ITAM motif regulates differentiation and apoptosis in M1 leukemia cells

DAP12 is an immunoreceptor tyrosine-based activation motif (ITAM)-bearing transmembrane adapter molecule that is associated with the NK-activating receptors. DAP12 is expressed not only in NK cells, but also in myeloid cells. Previously, we reported that DAP12 was likely to be involved in monocyte differentiation to macrophage. In this study, we established the mutant DAP12-M1 transfectants (Y76F-M1) that have mutation at their ITAM motifs. We observed that Y76F-M1 cells could not differentiate to macrophages by stimulation via DAP12, whereas wild type DAP12 transfectants (FDAP-M1) could. Furthermore, we demonstrated that the apoptosis signal mediated by LPS was inhibited in Y76F-M1 cells, but was augmented in FDAP-M1 cells. In contrast to the LPS-mediated apoptosis, the combination of LPS and DAP12 stimulation showed good cell viability in FDAP-M1 cells. Collectively our studies demonstrated that DAP12 has a critical role for macrophage differentiation and LPS induced apoptosis in M1 leukemia cells.     

Purification and characterization of beta-N-acetylhexosaminidase from maize seedlings

Enzymatic activity of beta-N-acetyhexosaminidase (EC 3.2.1.52) was analysed in seeds and young seedings of maize (Zea mays) using di-N-acetylchitobiose as a substrate. Substantial activity was detected in dry seeds. Activity increased before germination (48 h) but exclusively in the embryo. In seedlings, most of the activity was found in the scutellum, and lower levels in shoots and roots immediately after germination. An isoform of the enzyme was purified from scutellum (72 h after the start of imbibition) by heat treatment of crude extract and four steps of chromatography. Purified beta-N-acetyl-hexosaminidase showed a single band on SDS-PAGE of around 70 kDa. This was almost the same as the molecular weight estimated by size exclusion chromatography, indicating a monomeric form of the active enzyme. The relative activity of the enzyme for di-N-acetylchitobiose was about 15 times greater than that for p-nitrophenyl-N-acetylglucosaminide or p-nitrophenyl-N-acetylgalactosaminide. Analysis of the reaction with oligo-N-acetylchitooliogsaccharides [(GlcNAc)n] revealed an exotype enzyme producing predominantly (GlcNAc)n-1 and N-acetylglucosamine. The optimum pH, temperature, and isoelectric point (pl) were 4.5, 55 degrees C, and 6.75, respectively. The activity was almost completely inhibited in the presence of 5 mmol/L Ag+, Hg2+, or Fe3+.     

Substrate diversity of macrophomate synthase catalyzing an unusual multistep transformation from 2-pyrones to benzoates

Macrophomate synthase, which we have recently purified, catalyzes an unusual multistep transformation from 5-acetyl-4-methoxy-6-methyl-2-pyrone to 4-acetyl-3-methoxy-5-methyl-benzoic acid (macrophomic acid). To investigate the substrate diversity of the enzyme, 40 analogs of 2-pyrone were prepared and their relative efficiency was examined in the enzymatic conversions. The experimental results reveal the structural requirements of the substrates and the rough size of the enzyme active site, and eliminate the ambiguity caused by contamination by other enzymes in the whole-cell experiments.     

Compactin and simvastatin, but not pravastatin, induce bone morphogenetic protein-2 in human osteosarcoma cells

Bone morphogenetic protein (BMP)-2, a member of the BMP family, plays an important role in osteoblast differentiation and bone formation. To discover small molecules that induce BMP-2, a luciferase reporter vector containing the 5'-flanking promoter region of the human BMP-2 gene was constructed and transfected into human osteosarcoma (HOS) cells. By the screening of an in-house natural product library with stably transfected HOS cells, a fungal metabolite, compactin, known as an inhibitor of 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase, was isolated. The stimulation of the promoter activity by compactin seemed to be specific for BMP-2 gene in HOS cells, since it had little effect on BMP-4 or SV40 promoter activity and the stimulation was not observed in Chinese hamster ovary (CHO) cells. RT-PCR analysis and alkaline phosphatase assay revealed that compactin induced an increase in the expression of BMP-2 mRNA and protein. Like compactin, simvastatin also activated the BMP-2 promoter, whereas pravastatin did not. The statin-mediated activation of BMP-2 promoter was completely inhibited by the downstream metabolite of HMG-CoA reductase, mevalonate, indicating that the activation was a result of the inhibition of the enzyme. These results suggest that statins, if they are selectively targeted to bone, have beneficial effects in the treatment of osteoporosis or bone fracture.     

Amplification of pepleomycin-mediated DNA cleavage and apoptosis by unfused aromatic cations

An important approach to improve chemotherapy of members of the bleomycin (BLM) family of antibiotics is to find compounds (amplifiers) that enhance the activity of BLM-mediated DNA cleavage and apoptosis. Using a DNA-sequencing technique and pulsed field gel electrophoresis, we have investigated whether BLM-mediated cleavage of isolated and cellular DNA is amplifed by three compounds (RW-12, LS-20, 1S-5Me) which have a conformationally flexible, unfused polyaromatic system and cationic side chain in the molecules. RW-12 enhanced most effectively both pepleomycin (PEM)-induced cytotoxicity and apoptosis. The order of the maximum enhancing effect of amplifiers on PEM-mediated DNA damage is RW-12 > LS-20 > 1S-5Me. RW-12 amplified PEM-mediated DNA cleavage most effectively not only in vitro but also in cultured cells. We have reported that the order of the DNA binding constants of these compounds is RW-12 > LS-20 > 1S-5Me. In this study, we found a good correlation between PEM-mediated cleavage of isolated DNA and cellular DNA. These results suggest that BLM amplifiers bind to DNA and by doing so enhance drug-mediated DNA degradation, ultimately leading to apoptosis. The present study on amplifiers of anticancer agents shows a novel approach to the potentially effective anticancer therapy.     

Mass propagation of ornamental gentian in liquid medium

An efficient system for clonal mass propagation in liquid culture was established for the propagation of ornamental gentian. In a test of the requirements for three cytokinins [6-benzylaminopurine, N-(2-chloro-4-pyridyl)-N′-phenylurea and N-phenyl-N′-1,2,3-thiadiazol-5-yl urea (TDZ)] in combination with 1-naphthaleneacetic acid (NAA), we found that effective propagation of shoots occurred with 0.01 mg l^–1 TDZ in a 300 ml conical flask that contained 100 ml of medium. The propagation of shoots was also affected by the concentrations of macronutrients (KNO₃, NH₄NO₃ and CaCl₂) and sucrose in Murashige and Skoog's (MS) medium, and it was influenced to some extent by the speed of agitation on an orbital shaker. The most efficient propagation of shoots was achieved in full-strength MS medium supplemented with 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose with agitation at 150 rpm. The propagation of shoots was maximal after 6 weeks of culture (140 shoots from five nodal segments in one flask). Large-scale propagation in a 5-l fermenter was attempted using 3 l of MS medium that contained 0.01 mg l^–1 TDZ and 20 g l^–1 sucrose. More than 2,000 shoots were obtained in the fermenter in 5 weeks following the initial cultivation of five nodal segments for 6 weeks in one 300-ml flask. The shoots that had propagated in the fermenter were transferred directly to soil without prior rooting in vitro and were easily acclimatized within 1 month. Received: 7 October 1997 / Revision received: 16 January 1998 / Accepted: 30 January 1998     

Quantitative proteomics of Arabidopsis shoot microsomal proteins reveals a cross‐talk between excess zinc and iron deficiency

Iron (Fe) deficiency significantly effects plant growth and development. Plant symptoms under excess zinc (Zn) resemble symptoms of Fe‐deficient plants. To understand cross‐talk between excess Zn and Fe deficiency, we investigated physiological parameters of Arabidopsis plants and applied iTRAQ‐OFFGEL quantitative proteomic approach to examine protein expression changes in microsomal fraction from Arabidopsis shoots under those physiological conditions. Arabidopsis plants manifested shoot inhibition and chlorosis symptoms when grown on Fe‐deficient media compared to basal MGRL solid medium. iTRAQ‐OFFGEL approach identified 909 differentially expressed proteins common to all three biological replicates; the majority were transporters or proteins involved in photosynthesis, and ribosomal proteins. Interestingly, protein expression changes between excess Zn and Fe deficiency showed similar pattern. Further, the changes due to excess Zn were dramatically restored by the addition of Fe. To obtain biological insight into Zn and Fe cross‐talk, we focused on transporters, where STP4 and STP13 sugar transporters were predominantly expressed and responsive to Fe‐deficient conditions. Plants grown on Fe‐deficient conditions showed significantly increased level of sugars. These results suggest that Fe deficiency might lead to the disruption of sugar synthesis and utilization.