Neuronal activation modulates enhancer activity of genes for excitatory synaptogenesis through de novo DNA methylation

Post-mitotic neurons do exhibit DNA methylation changes, contrary to the longstanding belief that the epigenetic pattern in terminally differentiated cells is essentially unchanged. While the mechanism and physiological significance of DNA demethylation in neurons have been extensively elucidated, the occurrence of de novo DNA methylation and its impacts have been much less investigated. In the present study, we showed that neuronal activation induces de novo DNA methylation at enhancer regions, which can repress target genes in primary cultured hippocampal neurons. The functional significance of this de novo DNA methylation was underpinned by the demonstration that inhibition of DNA methyltransferase (DNMT) activity decreased neuronal activity-induced excitatory synaptogenesis. Overexpression of WW and C2 domain-containing 1 (Wwc1), a representative target gene of de novo DNA methylation, could phenocopy this DNMT inhibition-induced decrease in synaptogenesis. We found that both DNMT1 and DNMT3a were required for neuronal activity-induced de novo DNA methylation of the Wwc1 enhancer. Taken together, we concluded that neuronal activity-induced de novo DNA methylation that affects gene expression has an impact on neuronal physiology that is comparable to that of DNA demethylation. Since the different requirements of DNMTs for germ cell and embryonic development are known, our findings also have considerable implications for future studies on epigenomics in the field of reproductive biology.     

Possible involvement of phosphatidylinositol 3-kinase in the maintenance of metaphase II attest in porcine oocytes matured in vitro

It has been reported that phosphatidylinositol 3-kinase (PI3K)-protein kinase B (PKB) pathway plays a crucial role in the meiotic resumption and progression to the metaphase II (MII) stage of oocytes. However, the role of this pathway in meiotic arrest at the MII stage (cytostatic activity) is not well understood. In this study the effect of a PI3K inhibitor, LY294002, on the MAPK and p34^cdc2 kinase activities of matured porcine oocytes was examined. After maturation culture, both the MAPK and p34^cdc2 kinase activities in the oocytes were gradually decreased in a time-dependent manner. Although 25 µmol/L LY294002 did not affect either the MAPK or p34^cdc2 kinase activities, 50 µmol/L LY294002 suppressed the PKB phosphorylation and slightly decreased MAPK activity, but not the p34^cdc2 kinase activity. Therefore the effect of 10 µmol/L Ca²⁺ ionophore which was reported as inducing a transient decrease of p34^cdc2 kinase but not MAPK activities, was also examined in LY294002-treated oocytes. By additional treatment with LY294002 after Ca²⁺ ionophore, both the MAPK and p34^cdc2 kinase activities were decreased in a time-dependent manner, concomitantly with improvement of pronuclear formation. Therefore, we concluded that PI3K is involved in the maintenance of MAPK activity in matured porcine oocytes.     

Production of offspring from vacuum-dried mouse spermatozoa and assessing the effect of drying conditions on sperm DNA and embryo development

Freeze-dried sperm (FD sperm) are of great value because they can be stored at room temperature for long periods of time, However, the birth rate of offspring derived from FD sperm is low and the step in the freeze-drying process particularly responsible for low offspring production remains unknown. In this study, we determined whether the drying process was responsible for the low success rate of offspring by producing vacuum-dried sperm (VD sperm), using mouse spermatozoa dried in a vacuum without being frozen. Transfer of embryos fertilized with VD sperm to recipients resulted in the production of several successful offspring. However, the success rate was slightly lower than that of FD sperm. The volume, temperature, and viscosity of the medium were optimized to improve the birth rate. The results obtained from a comet assay indicated that decreasing the drying rate reduced the extent of DNA damage in VD sperm. Furthermore, even though the rate of blastocyst formation increased upon fertilization with VD sperm, full-term development was not improved. Analysis of chromosomal damage at the two-cell stage through an abnormal chromosome segregation (ACS) assay revealed that reduction in the drying rate failed to prevent chromosomal damage. These results indicate that the lower birth rate of offspring from FD sperm may result from the drying process rather than the freezing process.     

Comparison of the functions of the barley nicotianamine synthase gene HvNAS1 and rice nicotianamine synthase gene OsNAS1 promoters in response to iron deficiency in transgenic tobacco

Barley ( Hordeum vulgare L.) nicotianamine synthase gene ( HvNAS1 ) expression in barley is strongly induced by Fe deficiency in the roots and rice ( Oryza sativa L.) nicotianamine synthase gene ( OsNAS1 ) expression in rice is induced by Fe deficiency both in the roots and in the shoots. In dicots, NAS genes are not strongly induced by Fe deficiency, and they function to maintain Fe homeostasis. Rice OsNAS1promoter::GUS or barley HvNAS1promoter::GUS was introduced into tobacco ( Nicotiana tabacum L.) and tissue specificities and systemic regulation of their expression were compared. A split-root experiment revealed that the HvNAS1 promoter exhibited functions similar to those of Fe-acquisition-related genes in tobacco roots, suggesting that this promoter responded to certain Fe-deficiency systemic signals and to the Fe concentration in the rhizosphere. The HvNAS1 promoter might harbor a type of universal system of gene expression for Fe acquisition. However, the OsNAS1 promoter did not respond to local application of Fe to the roots and induced GUS activities in mature leaves in response to Fe deficiency. This promoter might possess numerous types of cis -acting sequences that are involved in Fe metabolism.     

生境土壤氮素资源异质性格局对于克隆植物结缕草生长的影响

通过试验分析了生境土壤氮素异质性对于克隆植物结缕草生长的影响。结果表明,生境中土壤氮素资源的总体水平、分布格局以及二者间的相互作用均显著影响结缕草克隆的干物质生产量。试验证明,当土壤氮素资源的总体水平较低时,氮素资源的异质性分布对结缕草的生长有利;而当土壤氮素资源的总体水平较高时,结缕草在同质生境中的干物质生产量较高。在异质生境中,肥沃土壤带距离初始的结缕草分株的位置,也对结缕草的生产量产生影响。当结缕草克隆能够较早地从试验生境的肥沃土壤带中获取丰富的氮素资源时,其干物质生产量倾向于增加。     

Effects of Susceptibility of Test Plants on Modes of Cucumovirus Transmission by Myzus persicae

In sequential transmission tests of Peanut stunt virus S (PSV-S) and Cucumber mosaic virus V (CMV-V) using Myzus persicae, these viruses behave as a semipersistent virus in Phaseolus vulgaris, but as a nonpersistent virus in Nicotiana tabacum, regardless of the species of plant used as the virus source. In addition, viruliferous aphids retained virus infectivity and transmitted it to P. vulgaris, even after they lost infectivity to N. tabacum. Apparently, the mode of transmission by the aphids differs depending on the plant species used for the assay. After mechanical inoculation with purified PSV-S or CMV-V, P. vulgaris appeared more susceptible to PSV-S than N. tabacum. However, the susceptibility to CMV-V appeared similar in both assay plants. Received 22 May 2000/ Accepted in revised form 14 September 2000     

Evaluation of beef tallow as a natural source of conjugated linoleic acid

Cis‐9, trans‐11 conjugated linoleic acid (9c, 11t CLA) is a potential anticarcinogen that is found in higher concentrations in beef lipids. However, the effect of CLA on lipid peroxidation, which is closely related to carcinogenesis, is controversial. In this study, we determined the levels of 9c, 11t CLA contents and thiobarbituric acid reactive substances (TBARS) in the tissues of rats fed beef lipid. Sprague‐Dawley rats (male, 8 weeks old) were fed experimental diets containing 20% lyophilized beef, and 12% beef tallow or vegetable oils, for 56 days. With the exception of the brain, the tissues from the rats fed the experimental diets accumulated 9c, 11t CLA, depending on the levels of CLA in the diets. The beef tallow group showed significantly higher 9c, 11t CLA contents in all tissues examined than the other diet groups. The intake of beef lipid did not affect the TBARS levels in the rat tissues. The hepatic lipid content from the beef tallow group was lower than that from the group fed vegetable oils. These results suggest that beef is a good source of 9c, 11t CLA, and that the intake of an appropriate level of beef lipid is not hazardous to health.