A new technique to prevent self-ligation of DNA

The most widely used technique for preventing self-ligation (self-circularization and concatenation) of DNA is dephosphorylation of the 5'-end, which stops DNA ligase from catalyzing the formation of phosphodiester bonds between the 3'-hydroxyl and 5'-phosphate residues at the DNA ends. The 5'-dephosphorylation technique cannot be applied to both DNA species to be ligated and thus, the untreated DNA species remains capable of self-ligation. To prevent this self-ligation, we replaced the 2'-deoxyribose at the 3'-end of the untreated DNA species with a 2',3'-dideoxyribose. Self-ligation was prevented at the replaced 3'-end, while the 5'-phosphate remaining at the 5'-end permitted ligation with the 3'-hydroxyl end of the 5'-dephosphorylated DNA strand. We successfully applied this 3'-replacement technique to gene cloning, adapter-mediated polymerase chain reaction and messenger RNA fingerprinting. The 3'-replacement technique is simple and not restricted by sequence or conformation of the DNA termini and is thus applicable to a wide variety of methods involving ligation.     

Friend leukemia virus infection enhances DNA damage-induced apoptosis of hematopoietic cells,causing lethal anemia in C3H hosts

Exposure of hematopoietic progenitors to gamma irradiation induces p53-dependent apoptosis. However, host responses to DNA damage are not uniform and can be modified by various factors. Here, we report that a split low-dose total-body irradiation (TBI) (1.5 Gy twice) to the host causes prominent apoptosis in bone marrow cells of Friend leukemia virus (FLV)-infected C3H mice but not in those of FLV-infected DBA mice. In C3H mice, the apoptosis occurs rapidly and progressively in erythroid cells, leading to lethal host anemia, although treatment with FLV alone or TBI alone induced minimal apoptosis in bone marrow cells. A marked accumulation of P53 protein was demonstrated in bone marrow cells from FLV-infected C3H mice 12 h after treatment with TBI. Although a similar accumulation of P53 was also observed in bone marrow cells from FLV-infected DBA mice treated with TBI, the amount appeared to be parallel to that of mice treated with TBI alone and was much lower than that of FLV- plus TBI-treated C3H mice. To determine the association of p53 with the prominent enhancement of apoptosis in FLV- plus TBI-treated C3H mice, p53 knockout mice of the C3H background (C3H p53(-/-)) were infected with FLV and treated with TBI. As expected, p53 knockout mice exhibited a very low frequency of apoptosis in the bone marrow after treatment with FLV plus TBI. Further, C3H p53(-/-) --> C3H p53(+/+) bone marrow chimeric mice treated with FLV plus TBI survived even longer than the chimeras treated with FLV alone. These findings indicate that infection with FLV strongly enhances radiation-induced apoptotic cell death of hematopoietic cells in host animals and that the apoptosis occurs through a p53-associated signaling pathway, although the response was not uniform in different host strains.     

Specific Detection of a Verotoxin 2 Variant,VT2vp1, by a Bead-Enzyme-Linked Immunosorbent Assay

A bead-enzyme-linked immunosorbent assay to specifically detect a Verotoxin 2 variant, VT2vpl, was developed. The sensitivity of the bead-ELISA was 200 pg/ml of the purified VT2vpl and it did not react with 20 ng/ml of the purified VT2. The specificity of the bead-ELISA was examined with 107 strains of Verocytotoxin-producing Escherichia coli that include VT1-, VT2-, VT2vha-, VT2vhb- and VT2vp1-producing E. coli, and only VT2vp1-producing E. coli that were confirmed by VT2vp1-specific polymerase chain reaction gave positive results. It was noted that all 58 VT2vp1-producing E. coli strains were from pigs, but not from cows and humans.     

Deer density affects ground-layer vegetation differently in conifer plantations and hardwood forests on the Boso Peninsula, Japan

Deer overabundance reduces forest ground-layer vegetation and can cause cascading impacts on a forest ecosystem. To predict these effects, we must elucidate the relationship between deer density and the status of ground-layer vegetation. This relationship was studied in the Boso Peninsula, where the deer population density exhibits a clear geographical gradient. We examined species richness and cover of ground-layer vegetation at several cedar plantations and hardwood forests in the Boso Peninsula. We also examined whether deer impacts were altered by light condition, soil water content and forest type (cedar and hardwood). Species richness of ground-layer vegetation was maximized at an intermediate level of deer density, suggesting an intermediate disturbance effect. This phenomenon was compatible with the observation that evergreen species, which were competitive dominants, decreased with increasing deer density, whereas less competitive deciduous species increased until herbivory was intermediate. As deer density increased, cover of ground-layer vegetation gradually decreased, but species unpalatable to deer increased in abundance, suggesting indirect positive effects of deer for unpalatable species. Cedar plantations tended to have greater species richness and ground-layer cover than hardwood forests with similar deer levels. Canopy openness, an indicator of light conditions, increased species richness of hardwood forests and ground-layer cover of cedar plantations, even under deer herbivory. Topographic wetness index, an indicator of soil water content, significantly increased the ground-layer cover of cedar plantations under deer herbivory. These results emphasize the importance of environmental productivity and forest type in the management of ground-layer vegetation experiencing deer overabundance.     

Cloning and expression of three formate oxidase genes from Debaryomyces vanrijiae MH201

Debaryomyces vanrijiae MH201 produces formate oxidase (FOD) at estimated pI values by native isoelectric focusing of 5.1, 5.4, and 5.9. We cloned and expressed three formate oxidase cDNAs, FOD1, FOD2, and FDO3, of the yeast using Escherichia coli. The open reading frames of FOD1, FOD2, and FDO3 were 1,731 bp long, and encoded 576-amino acid polypeptides with molecular masses of 64,142, 63,794, and 63,836 Da respectively. Expression of FOD1, FOD2, and FOD3 resulted in the production of three isozymes, with pI values of 5.1, 5.9, and 5.9 respectively. Co-expression of FOD1 and FOD2 and of FOD1 and FOD3 resulted in the production of additional isozymes with pI values, of 5.4. The three amino acid sequences of FOD1, FOD2, and FOD3 contained a consensus motif of a flavin adenine dinucleotide binding site in their N-terminal parts and a glucose-methanol-choline oxidoreductase signature pattern, suggesting that formate oxidase ought to be classified in the glucose-methanol-choline oxidoreductase family.     

The global DNA methylation surrogate LINE-1 methylation is correlated with MGMT promoter methylation and is a better prognostic factor for glioma

Gliomas are the most frequently occurring primary brain tumor in the central nervous system of adults. Glioblastoma multiformes (GBMs, WHO grade 4) have a dismal prognosis despite the use of the alkylating agent, temozolomide (TMZ), and even low grade gliomas (LGGs, WHO grade 2) eventually transform to malignant secondary GBMs. Although GBM patients benefit from promoter hypermethylation of the O(6)-methylguanine-DNA methyltransferase (MGMT) that is the main determinant of resistance to TMZ, recent studies suggested that MGMT promoter methylation is of prognostic as well as predictive significance for the efficacy of TMZ. Glioma-CpG island methylator phenotype (G-CIMP) in the global genome was shown to be a significant predictor of improved survival in patients with GBM. Collectively, we hypothesized that MGMT promoter methylation might reflect global DNA methylation. Additionally in LGGs, the significance of MGMT promoter methylation is still undetermined. In the current study, we aimed to determine the correlation between clinical, genetic, and epigenetic profiles including LINE-1 and different cancer-related genes and the clinical outcome in newly diagnosed 57 LGG and 54 GBM patients. Here, we demonstrated that (1) IDH1/2 mutation is closely correlated with MGMT promoter methylation and 1p/19q codeletion in LGGs, (2) LINE-1 methylation levels in primary and secondary GBMs are lower than those in LGGs and normal brain tissues, (3) LINE-1 methylation is proportional to MGMT promoter methylation in gliomas, and (4) higher LINE-1 methylation is a favorable prognostic factor in primary GBMs, even compared to MGMT promoter methylation. As a global DNA methylation marker, LINE-1 may be a promising marker in gliomas.     

Proper regulation of Cdc42 activity is required for tight actin concentration at the equator during cytokinesis in adherent mammalian Cells

Cytokinesis in mammalian cells requires actin assembly at the equatorial region. Although functions of RhoA in this process have been well established, additional mechanisms are likely involved. We have examined if Cdc42 is involved in actin assembly during cytokinesis. Depletion of Cdc42 had no apparent effects on the duration of cytokinesis, while overexpression of constitutively active Cdc42 (CACdc42) caused cytokinesis failure in normal rat kidney epithelial cells. Cells depleted of Cdc42 displayed abnormal cell morphology and caused a failure of tight accumulation of actin and RhoA at the equator. In contrast, in cells overexpressing CACdc42, actin formed abnormal bundles and RhoA was largely eliminated from the equator. Our results suggest that accurate regulation of Cdc42 activity is crucial for proper equatorial actin assembly and RhoA localization during cytokinesis. Notably, our observations also suggest that tight actin concentration is not essential for cytokinesis in adherent mammalian cells.     

Horizontal and vertical distribution and demography of euphausiids in the Ross Sea and its adjacent waters in 2004/2005

The horizontal and vertical distribution and population structure of euphausiids in the Ross Sea and its adjacent waters were investigated during the summers of 2004/2005 using stratified towed samples. Nine species of euphausiids occurred in the survey area. Among them, Euphausia triacantha was dominant in biomass north of the southern boundary of the Antarctic circumpolar current (SB). Thysanoessa spp. was widely distributed north of the continental slope, while E. superba was distributed from the SB to the slope, where it showed the highest biomass. Juvenile E. superba was distributed offshore near the SB and remained at the surface, but gravid females were dominant in the slope and mainly occurred in the middle layers (400–600 m). Adult and juvenile E. crystallorophias were found at 200–300 m in the colder water of the continental shelf. In general, the peak biomass of euphausiids was found in the mid layers of the Ross Sea area. The life span and the number of spawns for major species are also discussed.