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.     

Innate apoptosis of human B lymphoblasts transformed by Epstein-Barr virus: modulation by cellular immortalization and senescence

B-lymphoblastoid cell lines (LCLs) transformed by Epstein-Barr virus have a phenotype corresponding to activated B-lymphoblasts. Although they are widely used as models in various biological and medical studies, their innate morphological differentiation and apoptosis has been little studied. We report here that a large proportion of LCL cells spontaneously differentiate into smaller lymphoid cells which ultimately undergo apoptosis during conventional cell culture. Two distinct types of apoptosis with some intermediate types exist: type 1 apoptosis in small and medium-size cells with shrunken nuclei having heavily condensed chromatin in the whole nucleus region accompanied by relatively large internucleosomally fragmented DNA (above 2 kbp); type 2 apoptosis in large lymphoblasts with extremely lobulated nuclei having chromatin condensation beneath the nuclear membrane alone accompanied by smaller internucleosomally fragmented DNA (below 2 kbp). Type 1 apoptotic cells were far more numerous than type 2 apoptotic cells. The incidence of type 1 apoptosis was suppressed by cellular immortalization and was extremely stimulated at the end of the lifespan (crisis). These results provide essential information for us to use LCLs for various biological and medical studies including cellular immortalization, tumorigenesis and senescence.     

cDNA cloning and biochemical characterization of S-adenosyl-L-methionine: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase,a critical enzyme of the legume isoflavonoid phytoalexin pathway

Formononetin (7-hydroxy-4'-methoxyisoflavone, also known as 4'-O-methyldaidzein) is an essential intermediate of ecophysiologically active leguminous isoflavonoids. The biosynthetic pathway to produce 4'-methoxyl of formononetin has been unknown because the methyl transfer from S-adenosyl-L-methionine (SAM) to 4'-hydroxyl of daidzein has never been detected in any plants. A hypothesis that SAM: daidzein 7-O-methyltransferase (D7OMT), an enzyme with a different regiospecificity, is involved in formononetin biosynthesis through its intracellular compartmentation with other enzymes recently prevails, but no direct evidence has been presented. We proposed a new scheme of formononetin biosynthesis involving 2,7,4'-trihydroxyisoflavanone as the methyl acceptor and subsequent dehydration. We now cloned a cDNA encoding SAM: 2,7,4'-trihydroxyisoflavanone 4'-O-methyltransferase (HI4'OMT) through the screening of functionally expressed Glycyrrhiza echinata (Fabaceae) cDNAs. The reaction product, 2,7-dihydroxy-4'-methoxyisoflavanone, was unambiguously identified. Recombinant G. echinata D7OMT did not show HI4'OMT activity, and G. echinata HI4'OMT protein free from D7OMT was partially purified. HI4'OMT is thus concluded to be distinct from D7OMT, and their distant phylogenetic relationship was further presented. HI4'OMT may be functionally identical to (+)-6a-hydroxymaackiain 3-OMT of pea. Homologous cDNAs were found in several legumes, and the catalytic function of the Lotus japonicus HI4'OMT was verified, indicating that HI4'OMT is the enzyme of formononetin biosynthesis in general legumes.     

Quantitative metabolome analysis using capillary electrophoresis mass spectrometry

A new approach for the comprehensive and quantitative analysis of charged metabolites by capillary electrophoresis mass spectrometry (CE-MS) is proposed. Metabolites are first separated by CE based on charge and size and then selectively detected using MS by monitoring over a large range of m/z values. This method enabled the determination of 352 metabolic standards and its utility was demonstrated in the analysis of 1692 metabolites from Bacillus subtilis extracts, revealing significant changes in metabolites during B. subtilis sporulation.     

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.