Shinobu Gamou

8-Hydroxydeoxyguanosine generated in the earthworm Eisenia fetida grown in metal-containing soil

Heavy metal pollution of soil causes biological problems, such as mutagenicity to living organisms, including human beings. However, few methods have been developed to assess metal mutagenicity in soil. To avoid metal mutagenicity, an adequate bio-monitoring method is required. In the present study, to determine if the analysis of oxidative DNA damage generated in the earthworm is a useful bio-monitoring method for soil mutagenicity, the accumulation of 8-hydroxydeoxyguanosine (8-OH-dG), a major form of oxidative DNA damage, in Eisenia fetida (Savigny, 1826) treated with cadmium chloride (CdCl2) or nickel chloride (NiCl2) was analyzed. E. fetida was treated with Cd (10 or 200 microg/g soil) or Ni (10 or 200 microg/g soil) for 1, 2, and 3 weeks or 3 months. After metal exposure, the metal concentration in E. fetida was analyzed by atomic absorption spectrometry and the 8-OH-dG accumulated in E. fetida was analyzed by HPLC analyses and immunohistochemistry. Atomic absorption spectrometry revealed that Cd, but not Ni, accumulated within E. fetida. The 8-OH-dG levels in the DNA of E. fetida treated with Cd for 3 months were significantly higher than those in control E. fetida. Moreover, immunohistochemical analyses revealed that positive signals for 8-OH-dG accumulation in seminal vesicles were detected only in E. fetida treated with 10 microg of Cd for 3 months. Although some points remain unresolved, a bio-monitoring system analyzing the DNA damage generated in the earthworm might be useful for the assessment of the mutagenicity of soil contaminated with various heavy metals, such as Cd.

De Novo Mutations in the BMPR2 Gene in Patients with Heritable Pulmonary Arterial Hypertension

A substantial proportion of patients with pulmonary arterial hypertension (PAH) have mutations in the Bone Morphogenetic Protein Receptor type‐2 (BMPR2) gene. PAH due to BMPR2 mutations is inherited as an autosomal dominant trait with several unique features, including a wide variety of mutations, reduced penetrance, a skewed gender ratio, variable expressivity and genetic anticipation. To address the genetic background of these unique features of BMPR2 mutation, we conducted a systematic analysis of 15 PAH families with BMPR2 mutation. The exonic protein coding sequence of BMPR2 was amplified by polymerase chain reaction and the products were sequenced directly to detect point mutations in BMPR2. Parental identification was carried out to confirm the parental relationship using multiplex 15 loci analysis. Combining mutation detection in family members with parental identification, we described three cases of de novo mutation in the BMPR2 gene by different modes in a PAH family. These de novo mutations may account for the wide variety of mutations in BMPR2. Taken together with the juvenile onset of the disease, there is possibly some balance of de novo mutations and untransmittable mutations which keeps the frequency of PAH low in the general population.

Segment formation of Enchytraeus japonensis (Oligochaeta: Enchytraeidae): The 7th international symposium on earthworm ecology · Cardiff · Wales · 2002

Enchytraeus japonensis reproduces both sexually and asexually (by fragmentation). Following fragmentation, segment formation takes place along with organ regeneration and stem cells are supposed to play a crucial role. Thymidine analogue bromodeoxyuridine (BrdU) is widely used as a marker of cell proliferation, since it is incorporated into newly synthesized DNA. Here, to detect possible stem cells in E.japonensis, BrdU was incorporated into worms and detected by immunological methods using anti-BrdU antibody, followed by FITC-labeled secondary antibody. BrdU-labeled cells initially appeared as a ring at the tail domain of the worm, and then migrated, suggesting that segment formation precedes organ regeneration. The BrdU-immunostaining method described here can be a useful tool to trace stem cell migration and to analyse further organ regeneration in E. japonensis.

DMRT-1 expression during NEC8 human embryonic carcinoma cell differentiation

To elucidate the relationship between dsx and mab‐3 related transcription factor 1 (Dmrt‐1) and differentiation, alteration in mRNA levels during differentiation of NEC8 human embryonic carcinoma cells was investigated. After stimulation with 50 nM phorbol 12‐myristate 13‐acetate (PMA), the cells differentiated into cells with mesenchymal characteristics and upregulated Dmrt‐1 mRNA, possibly through the protein kinase C/mitogen‐activated protein kinase/activated protein‐1 signaling pathway. Conversely, knockdown of Dmrt‐1 by small interfering RNA resulted in cell morphology that was different from that after PMA treatment. These results indicated that Dmrt‐1 expression was apparently associated with the differentiation of NEC8, and this cell line may be a helpful in vitro tool to clarify the role of Dmrt‐1 in the differentiation process. (Cancer Sci 2006; 97: 277 –282)

Vascular Access: Experiences in the Aged Japanese Society

Dialysis access varies across the globe. Hemodialysis was first performed in Japan in 1966 and has undergone a number of revolutions since that time. This chapter discusses vascular access in an aging Japanese society.