LINE-1 hypomethylation during primary colon cancer progression

Background

Methylation levels of genomic repeats such as long interspersed nucleotide elements (LINE-1) are representative of global methylation status and play an important role in maintenance of genomic stability. The objective of the study was to assess LINE-1 methylation status in colorectal cancer (CRC) in relation to adenomatous and malignant progression, tissue heterogeneity, and TNM-stage.

Methodology/Principal Findings

DNA was collected by laser-capture microdissection (LCM) from normal, adenoma, and cancer tissue from 25 patients with TisN0M0 and from 92 primary CRC patients of various TNM-stages. The paraffin-embedded tissue sections were treated by in-situ DNA sodium bisulfite modification (SBM). LINE-1 hypomethylation index (LHI) was measured by absolute quantitative analysis of methylated alleles (AQAMA) realtime PCR; a greater index indicated enhanced hypomethylation. LHI in normal, cancer mesenchymal, adenoma, and CRC tissue was 0.38 (SD 0.07), 0.37 (SD 0.09), 0.49 (SD 0.10) and 0.53 (SD 0.08), respectively. LHI was significantly greater in adenoma tissue compared to its contiguous normal epithelium (P = 0.0003) and cancer mesenchymal tissue (P<0.0001). LHI did not differ significantly between adenoma and early cancer tissue of Tis stage (P = 0.20). LHI elevated with higher T-stage (P<0.04), was significantly greater in node-positive than node-negative CRC patients (P = 0.03), and was significantly greater in stage IV than all other disease stages (P<0.05).

Conclusion/Significance

By using in-situ SBM and LCM cell selection we demonstrated early onset of LINE-1 demethylation during adenomatous change of colorectal epithelial cells and demonstrated that LINE-1 demethylation progression is linear in relation to TNM-stage progression.     

Higher serum bone alkaline phosphatase as a predictor of mortality in male hemodialysis patients

AimsHigher serum alkaline phosphatase predicts lower mortality in chronic kidney disease and hemodialysis patients without liver dysfunction because it reflects high bone turnover. The purpose of our study was to compare the significance of serum bone alkaline phosphatase (BAP) with that of other bone markers in prediction of all-cause mortality(ACM) in male hemodialysis patients.Main methodsThe study was performed for 5 years. Serum BAP, intact osteocalcin (iOC), ß-CrossLaps (CTX), and intact parathyroid hormone (iPTH) were measured in 196 male hemodialysis patients without radiographic fracture. Their day-to-day variation during 5 consecutive days and diurnal variation were determined in 13 healthy males.Key findingsThe patients were divided into higher and lower groups based on serum levels of bone markers(mean ± SD: iPTH 218.6 ± 214.5 pg/ml, BAP 23.6 ± 12.2U/L, iOC 42.8 ± 45.2 ng/ml, CTX 1.71 ± 1.23 nmol/L BCE). In Kaplan–Meier analysis, the higher BAP group had significantly higher ACM than the lower BAP group (P = 0.013), whereas mortality did not differ between the higher and lower groups in other markers. Cox regression hazard analysis identified higher log BAP as a significant independent predictor [hazard ratio(HR) 8.32(95%CI:1.18–58.98)] for ACM after adjustment for various factors including pre-existing cardiovasucular disease, presence of DM. The significant association of mortality with serum BAP alone, in contrast with other markers including CTX [HR0.64 (95%CI:0.16–2.47)], iOC [HR0.97(95%CI:0.36–2.64)], iPTH [HR0.84(95%CI:0.44–1.60)],it may be due to the narrower day-to-day variation and the absence of diurnal variation in serum BAP compared to other markers.SignificanceHigher serum BAP may be a predictor of ACM in male hemodialysis patients.     

Multiple Group I Introns in the Small-Subunit rDNA of Botryosphaeria dothidea: Implication for Intraspecific Genetic Diversity

Botryosphaeria dothidea is a widespread and economically important pathogen on various fruit trees, and it often causes die-back and canker on limbs and fruit rot. In characterizing intraspecies genetic variation within this fungus, group I introns, rich in rDNA of fungi, may provide a productive region for exploration. In this research, we analysed complete small subunit (SSU) ribosomal DNA (rDNA) sequences of 37 B. dothidea strains, and found four insertions, designated Bdo.S943, Bdo.S1199-A, Bdo.S1199-B and Bdo.S1506, at three positions. Sequence analysis and structure prediction revealed that both Bdo.S943 and Bdo.S1506 belonged to subgroup IC1 of group I introns, whereas Bdo.S1199-A and Bdo.S1199-B corresponded to group IE introns. Moreover, Bdo.S1199-A was found to host an open reading frame (ORF) for encoding the homing endonuclease (HE), whereas Bdo.S1199-B, an evolutionary descendant of Bdo.S1199-A, included a degenerate HE. The above four introns were novel, and were the first group I introns observed and characterized in this species. Differential distribution of these introns revealed that all strains could be separated into four genotypes. Genotype III (no intron) and genotype IV (Bdo.S1199-B) were each found in only one strain, whereas genotype I (Bdo.S1199-A) and genotype II (Bdo.S943 and Bdo.S1506) occurred in 95% of the strains. There is a correlation between B. dothidea genotypes and hosts or geographic locations. Thus, these newly discovered group I introns can help to advance understanding of genetic differentiation within B. dothidea.     

Dynamic Changes in Endothelial Cell Adhesion Molecule Nepmucin/CD300LG Expression under Physiological and Pathological Conditions

Vascular endothelial cells often change their phenotype to adapt to their local microenvironment. Here we report that the vascular endothelial adhesion molecule nepmucin/CD300LG, which is implicated in lymphocyte binding and transmigration, shows unique expression patterns in the microvascular endothelial cells of different tissues. Under physiological conditions, nepmucin/CD300LG was constitutively and selectively expressed at the luminal surface of the small arterioles, venules, and capillaries of most tissues, but it was only weakly expressed in the microvessels of the splenic red pulp and thymic medulla. Furthermore, it was barely detectable in immunologically privileged sites such as the brain, testis, and uterus. The nepmucin/CD300LG expression rapidly decreased in lymph nodes receiving acute inflammatory signals, and this loss was mediated at least in part by TNF-α. It was also down-regulated in tumors and tumor-draining lymph nodes, indicating that nepmucin/CD300LG expression is negatively regulated by locally produced signals under these circumstances. In contrast, nepmucin/CD300LG was induced in the high endothelial venule-like blood vessels of chronically inflamed pancreatic islets in an animal model of non-obese diabetes. Interestingly, the activated CD4⁺ T cells infiltrating the inflamed pancreas expressed high levels of the nepmucin/CD300LG ligand(s), supporting the idea that nepmucin/CD300LG and its ligand interactions are locally involved in pathological T cell trafficking. Taken together, these observations indicate that the nepmucin/CD300LG expression in microvascular endothelial cells is influenced by factor(s) that are locally produced in tissues, and that its expression is closely correlated with the level of leukocyte infiltration in certain tissues.     

Significant antitumor effect of a synthetic lipid A analogue,DT-5461, on murine syngeneic tumor models

Summary The antitumor effect of a synthetic lipid A analogue, DT-5461, was investigated using syngeneic tumor models in mice. Intravenous injection of DT-5461 into mice transplanted with solid tumors of MethA fibrosarcoma, MH134 hepatoma, MM46 mammary carcinoma, Lewis lung carcinoma (3LL), and colon adenocarcinomas 26 and 38 resulted in significant reductions in the weight of all tumors except Colon 26, with marked hemorrhagic necrosis of tumor tissues. Efficacy was almost equal to that of anEscherichia coli-type synthetic lipid A (compound 506), and also to those of some chemotherapeutics including Adriamycin, mitomycin C, fluorouracil and cisplatin. Furthermore, DT-5461 was more effective than other immunotherapeutics, including picibanil (OK-432) and lentinan. However, its antitumor effects were inferior to those of Adriamycin or OK-432 against the malignant ascites caused by intraperitoneal inoculation with MethA or with MH134 cells; life span was not prolonged by either intraperitoneal or intravenous administration. In addition, although DT-5461 showed direct inhibitory effects on the in vitro growth of MethA or MH134, these were much weaker than those of Adriamycin. These findings clearly indicated that DT-5461 with systemic administration is a highly effective antitumor agent on solid tumors, and suggest that the antitumor effect of DT-5461 with potent necrotizing activity might derive from indirect mechanisms related to the activation of host immune systems and not to the weak direct cytotoxicity.     

Cross-sectional Area-corrected Compression Modulus of Food Gel

The linearity of the stress-strain relationship for food gel is limited to a very narrow range of the strain (usually less than 0.1 as a Cauchy measure). The reason is thought due to the change in cross-sectional area of the gel upon deformation. In this report, the cross-sectional area was approximately corrected of the compressed gel on the assumption that the gel expanded uniformly without changing its volume upon compression. In cases when the initial Young’s modulus was calculated from the thus-corrected area for some food gels, the linearity was increased for a wider range of strain.     

The molecular analysis ofbrown eye color mutations isolated from geographically discrete populations ofDrosophila melanogaster

A large proportion of spontaneous mutations inDrosophila melanogaster strains of laboratory origin are associated with insertions of mobile DNA elements. As a first step toward determining whether spontaneous laboratory mutations are predictive for mutational events occurring in the wild, recessivebrown (bw) eye color mutants were isolated. By inbreeding the progeny of wild-caughtDrosophila melanogaster females,bw mutations were isolated from seven separate geographic sites distributed among Japan, California, Siberia and Hungary. Among a total of 14 mutations studied, no case of transposon mutagenesis was found. At least 4 mutations are associated with small deletions in thebw gene. The remainder are inseparable from wild-typebw by Southern analysis and are presumed to be basepair changes or very small indels. Although only two spontaneousbw mutants of laboratory origin have been analyzed molecularly, one is a mobile element insertion.     

Holocene sedimentary history of some coastal plains in Hokkaido,Japan IV. Diatom assemblages in the sediments from Kushiro moor (2)

Diatom assemblages of sediments obtained from three sites on Kushiro Moor were analyzed to investigate the Holocene sedimentary history. The results showed that: 1) The Takkobu site was originally at the bottom of the paleo-Kushiro Bay, and after-wards the paleo-Takkobu Lagoon developed, became sealed off, and changed to a freshwater lake. The succession to peat moor probably began about 2000 yr B.P. at the Takkobu site. 2) The Tsurui site was originally at the bottom of the paleo-Kushiro Bay, then changed to the paleo-Kushiro Lagoon and became peat moor as a result of the first Holocene regression, which finished about 3600 yr B.P. The site then returned to a brackish lake again, probably due to the second Holocene transgression between 3600 and 3000 yr B.P., thereafter passing through brackish lake and freshwater lake stages, and eventually becaming peat moor at about 2000 yr B.P., 3) At the Chuo site, the second paleo-Kushiro Bay developed again as a result of the second Holocene transgression, which finished about 3000 yr B.P. Thereafter, brackish or freshwater lakes, rivers, and then peat moor developed in the central area of Kushiro Moor. 4) The second marine diatom zone (MD₂ Zone), which indicates the second Holocene transgression, complete by about 3000 yr B.P., is detected only at the Chuo site in the central area of Kushiro Moor.     

Beta1,4-N-Acetylglucosaminyltransferase III down-regulates neurite outgrowth induced by costimulation of epidermal growth factor and integrins through the Ras/ERK signaling pathway in PC12 cells

A rat pheochromocytoma cell line (PC12), when transfected with beta1,4-N-acetylglucosaminyltransferase III (GnT-III), which catalyzes the formation of a bisecting GlcNAc structure in N-glycans, resulted in the suppression of neurite outgrowth induced by costimulation of epidermal growth factor (EGF) and integrins. The neurite outgrowth was restored by the overexpression of a constitutively activated mitogen- or extracellular signal-regulated kinase kinase-1 (MEK-1). Consistent with this, the EGF receptor (EGFR)-mediated ERK activation was blocked in GnT-III transfectants. Conversely, the overexpression of dominant negative MEK-1 or treatment with PD98059, a specific inhibitor of MEK-1, inhibited neurite outgrowth in controls transfected with mock. Furthermore GnT-III activity is required for these inhibitions, because the overexpression of a dominant negative GnT-III mutant (D321A) failed to reduce neurite outgrowth and EGFR-mediated ERK activation. Lectin blot analysis confirmed that EGFR from wild-type GnT-III transfectants had been modified by bisecting GlcNAc in its N-glycan structures. This modification led to a significant decrease in EGF binding and EGFR autophosphorylation. Collectively, the results constitute a comprehensive body of evidence to show clearly that the overexpression of GnT-III prevents neurite outgrowth induced by costimulation of EGF and integrins through the Ras/MAPK activation pathway and indicates that GnT-III may be an important regulator for cell differentiation in neural tissues.