Amyloid beta-protein gene duplication is not common in Alzheimer's disease: analysis by polymorphic restriction fragments

The amyloid beta-protein(BP) is an important component of amyloid fibrils of both Alzheimer's disease(AD) and adult Down syndrome(DS). It has been hypothesized that sporadic AD may involve the duplication of a subregion of chromosome 21 containing the BP locus. However, an improved method for detection of the BP gene duplication using polymorphic Hind III fragments led us to a conclusion that BP gene duplication is rare, if any, in (Japanese) sporadic AD patients, indicating that the duplication of the BP gene itself is not the common underlying genetic defect in AD.     

General Metabolic Changes in Leaf Lipids in Response to Ozone

Of eight species of higher plants examined, lettuce was themost resistant to ozone. Lettuce leaves showed no visible changeswhile in other species visible injuries were apparent afterplants had been exposed to 0.5 µl liter^–1 ozonein the light for 6 h and then kept in the light for a further20 h. Analysis of leaf lipids immediately after 4–6 hof treatment with ozone revealed decreases in levels of monogalactosyldiacylglycerol(MGDG) and increases in levels of triacylglycerol (TG) in allplants including lettuce, but the extent of the changes variedamong the plant species. The fatty acids esterified to TG inozone-treated leaves were mainly     

Maintenance of Chloroplast Structure and Function by Overexpression of the Rice MONOGALACTOSYLDIACYLGLYCEROL SYNTHASE Gene Leads to Enhanced Salt Tolerance in Tobacco

In plants, the galactolipids monogalactosyldiacylglycerol (MGDG) and digalactodiacylglycerol (DGDG) are major constituents of photosynthetic membranes in chloroplasts. One of the key enzymes for the biosynthesis of these galactolipids is MGDG synthase (MGD). To investigate the role of MGD in the plant’s response to salt stress, we cloned an MGD gene from rice (Oryza sativa) and generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD. The MGD activity in OsMGD transgenic plants was confirmed to be higher than that in the wild-type tobacco cultivar SR1. Immunoblot analysis indicated that OsMGD was enriched in the outer envelope membrane of the tobacco chloroplast. Under salt stress, the transgenic plants exhibited rapid shoot growth and high photosynthetic rate as compared with the wild type. Transmission electron microscopy observation showed that the chloroplasts from salt-stressed transgenic plants had well-developed thylakoid membranes and properly stacked grana lamellae, whereas the chloroplasts from salt-stressed wild-type plants were fairly disorganized and had large membrane-free areas. Under salt stress, the transgenic plants also maintained higher chlorophyll levels. Lipid composition analysis showed that leaves of transgenic plants consistently contained significantly higher MGDG (including 18:3-16:3 and 18:3-18:3 species) and DGDG (including 18:3-16:3, 18:3-16:0, and 18:3-18:3 species) contents and higher DGDG-MGDG ratios than the wild type did under both control and salt stress conditions. These results show that overexpression of OsMGD improves salt tolerance in tobacco and that the galactolipids MGDG and DGDG play an important role in the regulation of chloroplast structure and function in the plant salt stress response.Salt stress is a major environmental factor that poses a serious threat to crop yield and future food production (Møller and Tester, 2007). When plants are exposed to salinity, they suffer two primary obstacles: low external water potentials and high concentrations of toxic ions (Hirayama and Mihara, 1987). These obstacles generally lead to the disruption of various enzymatic processes, changes in membrane lipid composition, alteration in chloroplast structure and function, impairment of photosynthetic capacity, and inhibition of plant growth (Brown and Dupont, 1989; Elkahoui et al., 2004; Munns and Tester, 2008; Sui et al., 2010; Shu et al., 2012).Membranes are the primary matrix for numerous physiological and biochemical activities, and plants easily change their membrane lipid compositions in response to environmental stresses (Harwood, 1996). A number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010). Sui et al. (2010) found that, in Suaeda salsa, salt stress increased the proportion of phosphatidylglycerol and reduced the proportion of galactolipids. Similar results were observed in Catharanthus roseus cultured cell suspensions, which showed an increase in phospholipid content and a decrease in galactolipid content that were more obvious under 100 mm NaCl than under 50 mm (Elkahoui et al., 2004). Meanwhile, it was shown that salt tolerance in plants is strongly linked with their membrane lipid composition and especially with their galactolipid content, which is positively related to salt tolerance (Hirayama and Mihara, 1987).In plants, galactolipids are major constituents of the photosynthetic membrane, which is the most abundant membrane in nature (Lee, 2000). Two galactolipids, monogalactosyldiacylglycerol (MGDG) and digalactosyldiacylglycerol (DGDG), are the predominant photosynthetic membrane lipid building blocks, accounting for about 52 mol % and 26 mol % of thylakoid membrane lipids, respectively (Block et al., 1983). These galactolipids are also the major lipids in cyanobacteria, suggesting that MGDG and DGDG are important for all oxygenic photosynthetic organisms (Hölzl and Dörmann, 2007). They are components of PSII and are the indispensable matrix for photosynthesis (Mizusawa and Wada, 2012). Their unique characteristics also give them distinctive packing properties that are important for the organization of thylakoid membranes (Lee, 2000). MGDG has a conical shape due to its smaller head group and a high content of unsaturated fatty acids, giving it nonbilayer-forming characteristics (Webb and Green, 1991; Aronsson et al., 2008); this feature is assumed to play an important structural role in the dense packing of proteins in the membrane (Williams, 1998; Garab et al., 2000). In contrast to MGDG, DGDG has a cylindrical shape that is typical for most plastid lipids and is considered a bilayer-prone lipid (Aronsson et al., 2008); this feature is involved in lipid-mediated contacts between adjacent trimers of light-harvesting complex II (LHCII) when they are packed into two-dimensional crystalline arrays (Lee, 2000). In addition to forming important membrane structures in the thylakoids, MGDG and DGDG are also present in extraplastidic membranes, including the plasma membrane, tonoplasts, endoplasmic reticulum, and Golgi membranes, indicating the crucial role of these galactoglycerolipids in higher plant membrane systems (Yoshida and Uemura, 1986; Brown and Dupont, 1989; Härtel et al., 2000).Two enzymes are involved in the biosynthesis of these galactoglycerolipids: MGDG synthase (MGD), which transfers a galactosyl residue from UDP-Gal to diacylglycerol, and DGDG synthase, which catalyzes the further galactosylation of MGDG to form DGDG (Shimojima et al., 1997; Dörmann et al., 1999; Shimojima and Ohta, 2011). Thus, MGD is the key enzyme in the biosynthesis of both galactolipids and, consequently, also in the formation of photosynthetic membranes (Nakamura et al., 2010). A number of studies have revealed that MGD is vital for plant growth and development. The loss of MGD function in plants leads to a pale-green phenotype, defects in the chloroplast ultrastructure, disruption in the photosynthetic membranes, and complete impairment of photosynthetic ability and photoautotrophic growth, suggesting a unique role for MGD in chlorophyll formation, the structural organization of the plastidic membranes, and photosynthetic growth (Jarvis et al., 2000; Kobayashi et al., 2007; Botté et al., 2011; Myers et al., 2011). The crucial role of MGD under environmental stresses, including phosphorus deficiency and wounding, is also well studied (Kobayashi et al., 2004, 2009a, 2009b; Moellering and Benning, 2011). However, although a number of studies have proved that salt stress can induce changes in plant membrane lipids (Huflejt et al., 1990; Elkahoui et al., 2004; Sui et al., 2010), little is known about the role of MGD and the involvement of galactolipids in response to salt stress.To investigate the function of MGD in plant salt tolerance, we cloned the relevant gene, which is called OsMGD (Qi et al., 2004), from rice (Oryza sativa ‘FR13A’). This gene has a high similarity to Arabidopsis (Arabidopsis thaliana) MGD2 and MGD3, and its expression is induced by several environmental stresses, including salt, drought, and submergence (Qi et al., 2004; Benning and Ohta, 2005). We then generated tobacco (Nicotiana tabacum) plants overexpressing OsMGD and investigated the salt tolerance ability in the transgenic lines and wild-type plants. The results of this study demonstrate that an increase in galactolipid content in leaves is beneficial for maintaining chloroplast structure and function and leads to enhanced salt tolerance in tobacco.     

DNA ligase IV as a new molecular target for temozolomide

Temozolomide (TMZ) is a methylating agent used in chemotherapy against glioblastoma. This work was designed to clarify details in repair pathways acting to remove DNA double-strand breaks (DSBs) induced by TMZ. Cultured mouse embryonic fibroblasts were used which were deficient in DSB repair genes such as homologous recombination repair-related genes X-ray repair cross-complementing group 2 (XRCC2)and radiation sensitive mutant54 (Rad54), non-homologous end joining repair-related gene DNAligase IV (Lig4). Cell sensitivity to drug treatments was assessed using colony forming assays. The most effective molecular target which was correlated with TMZ cell sensitivity was Lig4. In addition, it was found that small interference RNAs (siRNA) for Lig4 efficiently enhanced cell lethality induced by TMZ in human glioblastoma A172 cells. These findings suggest that down regulation of Lig4 might provide a useful tool for cell sensitization during TMZ chemotherapy.     

Metabolism of mono- and dichloro-dibenzo-p-dioxins by Phanerochaete chrysosporium cytochromes P450

The white-rot fungus Phanerochaete chrysosporium possesses biodegradative capabilities of polychlorinated dibenzo-p-dioxins (PCDDs). One hundred twenty yeast clones expressing individual P450s of P. chrysosporum (PcCYPs), generated in our previous efforts, were screened for transformation of dioxin, and 40 positive clones were obtained. Of these clones, six clones showed metabolism of 2-chloro-dibenzo-p-dioxin, and a microsomal PcCYP designated as PcCYP11a3 showed much higher activity than any other PcCYPs. The turnover numbers of hydroxylation activities of PcCYP11a3 toward 1-MCDD (58 min⁻¹) and 2-MCDD (13 min⁻¹) are more than 200 times higher than those of previously reported PcCYP65a2. In addition, PcCYP11a3 catalyzes hydroxylation of 2,3-dichloro-dibenzo-p-dioxin. To our best knowledge, PcCYP11a3 has the highest activity toward PCDDs among the known CYPs derived from microorganisms. Although PcCYP11a3 showed no detectable activity toward 2,7-dichloro-dibenzo-p-dioxin and 2,3,7-trichloro-dibenzo-p-dioxin, PcCYP11a3 is promising as a template whose activity would be enhanced by site-directed mutagenesis.     

Effective proliferation of low level Legionella pneumophila serogroup 1 cells using coculture procedure with Acanthamoeba castellanii

The multiplications of low level Legionella pneumophila serogroup 1 cells by the coculture procedure with Acanthamoeba castellanii were tested in five strains. The cells in all strains proliferated effectively for isolating. This procedure might be a useful means of improving the successful isolation from environmental and clinical specimens of low level Legionella cells, and pursuing the source of infection.     

Identification of an Imprinted Gene Cluster in the X-Inactivation Center

Mammalian development is strongly influenced by the epigenetic phenomenon called genomic imprinting, in which either the paternal or the maternal allele of imprinted genes is expressed. Paternally expressed Xist, an imprinted gene, has been considered as a single cis-acting factor to inactivate the paternally inherited X chromosome (Xp) in preimplantation mouse embryos. This means that X-chromosome inactivation also entails gene imprinting at a very early developmental stage. However, the precise mechanism of imprinted X-chromosome inactivation remains unknown and there is little information about imprinted genes on X chromosomes. In this study, we examined whether there are other imprinted genes than Xist expressed from the inactive paternal X chromosome and expressed in female embryos at the preimplantation stage. We focused on small RNAs and compared their expression patterns between sexes by tagging the female X chromosome with green fluorescent protein. As a result, we identified two micro (mi)RNAs–miR-374-5p and miR-421-3p–mapped adjacent to Xist that were predominantly expressed in female blastocysts. Allelic expression analysis revealed that these miRNAs were indeed imprinted and expressed from the Xp. Further analysis of the imprinting status of adjacent locus led to the discovery of a large cluster of imprinted genes expressed from the Xp: Jpx, Ftx and Zcchc13. To our knowledge, this is the first identified cluster of imprinted genes in the cis-acting regulatory region termed the X-inactivation center. This finding may help in understanding the molecular mechanisms regulating imprinted X-chromosome inactivation during early mammalian development.