Purification and cloning of cytotoxic ribonucleases from Rana catesbeiana (bullfrog).

Ribonucleases with antitumor activity are mainly found in the oocytes and embryos of frogs, but the role of these ribonucleases in frog development is not clear. Moreover, most frog ribonuclease genes have not been cloned and characterized. In the present study, a group of ribonucleases were isolated from Rana catesbeiana (bullfrog). These ribonucleases in mature oocytes, namely RC-RNase, RC-RNase 2, RC-RNase 3, RC-RNase 4, RC-RNase 5 and RC-RNase 6, as well as liver-specific ribonuclease RC-RNase L1, were purified by column chromatographs and detected by zymogram assay and western blotting. Characterization of these purified ribonucleases revealed that they were highly conserved in amino acid sequence and had a pyroglutamate residue at their N-termini, but possessed different specific activities, base specificities and optimal pH values for their activities. These ribonucleases were cytotoxic to cervical carcinoma HeLa cells, but their cytotoxicities were not closely correlated to their enzymatic specific activities. Some other amino acid residues in addition to their catalytic residues were implicated to be involved in the cytotoxicity of the frog ribonucleases to tumor cells. Because the coding regions lack introns, the ribonuclease genes were cloned by PCR using genomic DNA as template. Their DNA sequences and amino acid sequences are homologous to those of mammalian ribonuclease superfamily, approximately 50 and approximately 25%, respectively.

The Rana catesbeiana rcr gene encoding a cytotoxic ribonuclease. Tissue distribution, cloning, purification, cytotoxicity, and active residues for RNase activity.

Rana catesbeiana ribonuclease (RC-RNase) is a pyrimidine-guanine sequence-specific ribonuclease found in R. catesbeiana (bullfrog) oocytes. It possesses both ribonuclease activity and cytotoxicity against tumor cells. We report here for the first time the cloning of RC-RNase cDNA from liver rather than from oocytes where RC-RNase is stored. An internal fragment of cDNA was obtained by reverse transcription-PCR using deduced oligonucleotides as primers. Full-length cDNA was obtained by 5'- and 3'-RACE technique. The cDNA clone, named rcr gene, contained a 5'-untranslated region, a putative signal peptide (22 amino acids), a mature protein (111 amino acids), a 3'-untranslated region, and a polyadenylation site. The cDNA which encoded the mature protein was fused upstream with a modified pelB signal peptide DNA and inserted into pET11d for expression in Escherichia coli strain BL21(DE3). The secretory RC-RNase in the culture medium was enzymatically active and was purified to homogeneity. The recombinant RC-RNase had the same amino acid sequence, specific activity, substrate specificity, antigenicity, and cytotoxicity as that of native RC-RNase from frog oocytes. Amino acid residues His-10, Lys-35, and His-103 are involved in RC-RNase catalytic activity. Ribonucleolytic activity was involved in and may be essential for RC-RNase cytotoxicity. DNA sequence analysis showed that RC-RNase had approximately 45% identity to that of RNase superfamily genes. This indicates that RC-RNase is a distinct ribonuclease gene in the RNase superfamily.

[Locomotion of dystrophin-deficient mice].

The purpose of our study was to make a comparison of the motor function between murine dystrophy mice (MDX mice) and C57BL/10ScSn control mice. The locomotor activities of mice were measured by an animal three-dimension optical monitor. Measurements were performed at ages of 21, 45 and 60 days. Animals were tested in a dark and peaceful environment under room temperature (25 degrees C-27 degrees C) at night for an hour. Results showed that the most important differences were in data on vertical activities. Among 15 variables of locomotor activity detected by the optical activity monitor, the MDX mice and control mice at age 21 days showed significant differences in 12 variables. However, the MDX mice and control mice at age 45 days revealed significant differences in only 7 variables. The MDX mice and control mice at age 60 days had significant differences for only one variable. The results may be explained by the fact that dystrophin-deficient mice undergo more severe dystrophic degeneration at an early age (5 weeks) and new regeneration of their muscle fibres is prevalent. Moreover, a functional recovery occurred in MDX skeletal muscle which was probably due to the regeneration of dystrophic muscle.

Intussusception: the sonographic diagnosis and its clinical value.

During the past four years in our hospital, real-time sonographic examinations were performed prior to barium enema reductions in 48 proven intussusception cases. Four major sonographic findings were noted. First, a length of target configuration consisting of two rings of low echogenicity separated by an intermediate hyperechoic ring was seen on the cross-sectional image of the intussuscepted bowel. Second, a doughnut configuration consisting of a hypoechoic rim and a dense central echogenic core was noted on the cross section near the apex of the intussusceptum. Third, no demonstrable movement or change was observed in the target or doughnut configuration. Fourth, all of the exterior sonolucent rims of the target were thicker than 0.6 cm. Operative reductions were necessary in all 13 cases whose exterior rims were thicker than 1.6 cm. On the contrary, only 15 of the remaining 35 cases whose exterior rims were between 0.6 and 1.5 cm needed surgical management (p = 0.0033, Fisher's exact test).