Shigekazu Nakatsugawa

Molecular cloning and characterization of a novel human gene (HERNA) which encodes a putative RNA-helicase

A full-length cDNA encoding a novel protein was isolated and sequenced from a human hepatocellular cDNA library. This cDNA consists of 7037 base pairs and has a predicted open reading frame encoding 1924 amino acids. It possesses an RNA-helicase motif containing a DEXH-box in its amino-terminus and an RNase motif in the carboxy-terminus. From a striking homology to Caenorhabditis elegans K12H4.8, it might be a human homolog of the K12H4.8. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 14q31 near the marker D14S605.

Molecular cloning and characterization of a novel human gene (NESCA) which encodes a putative adapter protein containing SH3

A full-length cDNA encoding a novel protein was isolated and sequenced from a human placental cDNA library. This cDNA consists of 1990 bp and has a predicted open reading frame encoding 433 amino acids. It possesses an Src homology 3 (SH3) motif, a leucine zipper motif and no catalytic domain, suggesting that it seems to be an adapter protein. PCR-based mapping with both a monochromosomal hybrid panel and radiation hybrid cell panels placed the gene to human chromosome 1q21-22.

Cytotoxic and Mutagenic Effects of Chronic Low-Dose-Rate Irradiation on TERT-Immortalized Human Cells

Abstract Nakamura, H., Fukami, H., Hayashi, Y., Tachibana, A., Nakatsugawa, S., Hamaguchi, M. and Ishizaki, K. Cytotoxic and Mutagenic Effects of Chronic Low-Dose-Rate Irradiation on TERT-Immortalized Human Cells. Radiat. Res. 163, 283– 288 (2005). To analyze the genetic effects of low-dose-rate radiation on human cells, we used human telomere reverse transcriptase (TERT)-immortalized fibroblast cells obtained from normal individuals. We studied the effect of low-dose-rate (0.3 mGy/ min) and high-dose-rate (2 Gy/min) radiation on cells in a confluent state. Survival and micronucleus induction frequency showed higher resistance after irradiation at low dose rate than at high dose rate. The survival after 5 Gy of high-dose-rate radiation was 0.01 compared to 0.3 after low-dose-rate irradiation at the same dose. In accordance with this, the level of HPRT mutation induction by low-dose-rate radiation decreased to approximately one-eighth that for high-dose-rate radiation. We then characterized the mutants by multiplex PCR analysis, which showed that the fraction of deletion mutations was lower in the mutant cells induced at low dose rate than at high dose rate. Furthermore, the size of the deletions in mutant cells induced by low-dose-rate radiation appeared to be smaller than those in mutant cells irradiated at high dose rate. Only a few exons were deleted in the former mutants while all exons were deleted in most of the latter mutants. The present study indicates that the genetic effects of low-dose-rate radiation on nonproliferating normal human cells are quantitatively and qualitatively less severe than the effect of high-dose-rate radiation.

Inhibitory effect of ND2001 on spontaneous multiple metastasis of NC 65 tumors derived from human renal cancer cells intradermally transplanted into nude mice.

The NC 65 tumor cell line derived from human renal cell carcinoma was selected from among nine human cell lines by determining the inhibition of invasion by ND2001 (sodium D-glucaro-delta-lactam) in vitro. The efficacy of this agent against these tumor cells was investigated in an experimental metastatic model of human tumors in vivo. Although ND2001 did not inhibit growth of NC 65 cells intradermally transplanted into male KSN mice (nu/nu), this agent inhibited multiple spontaneous metastasis.

Intercept-PCR, an improvement for elevating performance to find a new member of a certain gene family.

We have established a method by which the performance of reverse transcriptase coupled polymerase chain reaction (RT-PCR) for seeking a new gene is improved. The actual procedure is quite easy: it is only to add several specific oligonucleotides into the reaction mixture of the usual RT-PCR. To verify the effectiveness of this method is also easy: it is only to detect the PCR products in the preliminary experiment. The finding in the present study provides valuable information for gene cloning tactics.