DNA methylation patterns in genome are maintained during replication by a DNA methyltransferase Dnmt1. Mouse Dnmt1 is a 180 kDa protein comprising the N-terminal regulatory domain, which covers 2/3 of the molecule, and the rest C-terminal catalytic domain. In the present study, we demonstrated that the limited digestion of full-length Dnmt1 with different proteases produced a common N-terminal fragment, which migrated along with Dnmt1 (1-248) in SDS-polyacrylamide gel electrophoresis. Digestion of the N-terminal domains larger than Dnmt1 (1-248) with chymotrypsin again produced the fragment identical to the size of Dnmt1 (1-248). These results indicate that the N-terminal domain of 1-248 forms an independent domain. This N-terminal domain showed DNA binding activity, and the responsible sequence was narrowed to the 79 amino acid residues involving the proliferating cell nuclear antigen (PCNA) binding motif. The DNA binding activity did not distinguish between DNA methylated and non-methylated states, but preferred to bind to the minor groove of AT-rich sequence. The DNA binding activity of the N-terminal domain competed with the PCNA binding. We propose that DNA binding activity of the N-terminal domain contributes to the localization of Dnmt1 to AT-rich sequence such as Line 1, satellite, and the promoter of tissue-specific silent genes.

Spinal cord injury (SCI) is an abrupt traumatic injury that leads to permanent functional loss, and no practical treatment is available. We have developed pharmaceutical recombinant human hepatocyte growth factor (KP-100), and its efficacy for SCI has been verified using animal models. The purpose of this study was to evaluate the safety and efficacy of intrathecal KP-100 administration for SCI patients in the acute phase. This investigation was a multi-center, randomized, double-blind study. Subjects with modified Frankel grade A/B1/B2 at 72 h after SCI were included. KP-100 was administered intrathecally. Subjects were followed up for 168 days after the first administration. Outcomes were evaluated using American Spinal Injury Association (ASIA) scores and subjected to analysis of covariance. Our results demonstrated that the subjects did not show any serious adverse events caused by KP-100. Forty-three subjects underwent neurological function testing (26 in KP-100 group; 17 in placebo group), which revealed that KP-100 contributed to motor improvement at Days 140 ( p = 0.050) and 168 ( p = 0.079). In the subset of subjects with Frankel grade A, the proportions of subjects who gained at least 1 point on their lower-extremity motor scores were 33.3% (5/15) and 6.3% (1/16) in the KP-100 and placebo groups, respectively ( p = 0.083). Therefore, KP-100 has the potential to be useful and beneficial for SCI patients during the acute phase. However, this was a phase I/II trial and did not definitely address the question of efficacy; a larger phase III trial would be required to assess the efficacy.

Hepatocyte growth factor (HGF) is essential for embryogenesis, tissue regeneration and tumour malignancy through the activation of its receptor, c-Met. We previously demonstrated that HGF α-chain hairpin-loop, K1 domain and β-chain are required for c-Met signalling. The sequential phosphorylation of tyrosine residues, from c-Met kinase domain to multidocking regions, is required for HGF-signalling transduction. Herein, we provide evidence that the disconcerted activation of c-Met tyrosine regions fails to induce biological functions. When human cells were incubated with 'mouse HGF', kinase domain activation (i.e. phospho-Tyr-1230/34/35) became evident, but the multidocking site (i.e. Tyr-1349) was not phosphorylated, resulting in unsuccessful induction of migration and mitogenesis. The binding ability of mouse HGF α-chain, or of β-chain, to human c-Met was lower than that of human HGF, as evidenced by HGF-chimera assay. Notably, only four amino acid positions in HGF α-chain hairpin-loop and K1 domain and six positions in β-chain differed between human HGF and mouse HGF. The human-specific amino acids (such as Gln-95 in hairpin-loop, Arg-134 in K1 domain and Cys-561 in β-chain) may be important for accurate c-Met assembly and signalling transduction.

To identify genes expressed during budding of the tunicate Polyandrocarpa misakiensis, we isolated and sequenced 624 clones from a directionally constructed cDNA library to prepare a catalog of expressed sequence tags (ESTs). A total of 233 ESTs matched genes of known sequence in the SwissProt database. About 24% out of them showed high similarity to ribosomal proteins, twice the value (12%) of pre-budding animals. ESTs involved in the respiratory chain also appeared with significant redundancy, suggesting that tunicate budding is accompanied by the enhancement of energy conversion as well as protein synthesis. Serine protease inhibitor (serpin) afforded another striking example of a gene that was highly expressed in the process of budding. The deduced amino acid sequences of five serpin cDNAs all had two consensus signatures of the Kazal's type of secretory protease inhibitor, one of which had an active site for trypsin and the other for elastase. In line with this, recombinant GST-fusion protein showed both trypsin and elastase inhibitor activities. In accordance with the EST analysis, the hemolymph taken from the budding stage showed the highest activity of trypsin inhibitor. We discuss a possible role that Polyandrocarpa serpins may play in bud development by counteracting trypsin-like serine protease, which could facilitate dedifferentiation of formative tissues.