Genetic characterization of a novel Tib-derived variant of soybean Kunitz trypsin inhibitor detected in wild soybean (Glycine soja).

A novel variant of soybean Kunitz trypsin inhibitor (SKTI) was detected in 530 lines of wild soybean (Glycine soja). This variant showed an intermediate electrophoretic mobility between the Tia and Tic types. In isoelectric focusing polyacrylamide gel electrophoresis gels containing urea, this variant had a similar isoelectric point as that of Tia. The genetic analysis of SKTI bands in F2 seeds from crosses of the new variant type with Tia or Tic type showed that this variant type is controlled by a codominant allele at the SKTI locus. We propose the genetic symbol Tif for this novel variant. When the nucleotide sequence of the Tif gene was compared with those of other types of SKTI genes (Tia, Tib, and Tic), the sequence of Tif was identical to that of Tib with the exception of one A-->G transitional mutation occurring at position 676 of Tif. This mutation resulted in an amino acid change from Lys to Glu at the 178 residue. These results suggest that this variant is derived from Tib through a point mutation. In addition, we settled an inconsistency in the number of amino acid differences between Tia and Tib (eight or nine). Analysis of nucleotide and amino acid sequences revealed that Tib was different from Tia by nine amino acids.     

Determinants of difference in leg stiffness between endurance- and power-trained athletes

Understanding the leg and joint stiffness during human movement would provide important information that could be utilized for evaluating sports performance and for injury prevention. In the present study, we examined the determinants of the difference in the leg stiffness between the endurance-trained and power-trained athletes. Seven distance runners and seven power-trained athletes performed in-place hopping, matching metronome beats at 3.0 and 1.5Hz. Leg and joint stiffness were calculated from kinetic and kinematics data. Electromyographic activity (EMG) was recorded from six leg muscles. At both hopping frequencies, the power-trained athletes demonstrated significantly higher leg stiffness than the distance runners. Hip, knee, and ankle joints were analyzed for stiffness and touchdown angles. Ankle stiffness was significantly greater in the power-trained athletes than the distance runners at 3.0Hz as was knee stiffness at 1.5Hz. There was no significant difference in touchdown angle between the DR and PT groups at either hopping frequencies. When significant difference in EMG activity existed between two groups, it was always greater in the distance runners than the power-trained athletes. These results suggest that (1) the difference in leg stiffness between endurance-trained and power-trained athletes is best attributed to increased joint stiffness, and (2) the difference in joint stiffness between the two groups may be attributed to a lack of similarity in the intrinsic stiffness of the muscle-tendon complex rather than in altered neural activity.     

DNA synthesis in isolated nuclei from synchronized plasmodia of Physarum polycephalum

Nuclei were isolated from synchronized plasmodia of a true slime mold, Physarum polycephalum, in S-phase, and DNA synthesis in the nuclei was studied in vitro. The nuclei catalyzed DNA synthesis at the rate of 0.7 ng DNA/1.0 X 10(6) nuclei/30 min at 25 degrees C, which was 5 times higher than that catalyzed in G2-phase nuclei. The DNA synthesis required Mg2+, four kinds of deoxyribonucleoside 5'-triphosphates and ATP, suggesting that the mode of synthesis is a replicative-type, but not a repair-one. Sedimentation analysis of the DNA products revealed that the nuclei produced 2-4S DNA fragments mainly during a 30-sec pulse incubation, and 2-4S, 5-12S and longer fragments during a 15-min incubation. The pulse- and chase-labeling experiments showed that the 2-4S fragments shifted discontinuously to longer fragments. These results indicate that the nuclei catalyze the formation of 2-4S Okazaki fragments first and then their subsequent ligation. Eighty % and 96% of the DNA synthesis was inhibited by 200 micrograms/ml aphidicolin and 40 mM N-ethylmaleimide, respectively, but 80% of the activity was resistant to 100 microM 2',3'-dideoxythymidine 5'-triphosphate. These results suggest that the DNA synthesis is catalyzed by the alpha-type DNA polymerase of Physarum polycephalum.     

New mutant gene (transthyretin Arg 58) in cases with hereditary polyneuropathy detected by non-isotope method of single-strand conformation polymorphism analysis.

Single-strand conformation polymorphism (SSCP) was analyzed to detect a mutation in the transthyretin (TTR) gene from the mother and son showing polyneuropathy with carpal tunnel syndrome. DNA segments containing TTR coding sequence were amplified by polymerase chain reaction, heat denatured and electrophoresed on a neutral polyacrylamide gel. The single-stranded DNA fragments in the gel were transferred to a nylon membrane and hybridized with biotinylated TTR cDNA probe, followed with chemiluminescent DNA detection. The mobility shift was found in the fragments of exon 3 from the patients' DNA. Sequencing analyses of the exon 3 confirmed a T----G base change, resulting in a Leu 58----Arg substitution. TTR Arg 58 is the first mutant TTR gene that has been detected by SSCP analysis. The rapid and sensitive detection of new mutations at various sites on the TTR gene is hereafter possible by the present method in the facilities for non-radioactive experiments.     

Direct observation of the biphasic conformational change of DNA induced by cationic polymers.

The interaction between T4 DNA and basic polypeptides was observed using fluorescence microscopy. Free DNA molecules exhibited random Brownian motion accompanying the conformational change. With the addition of polycation, such as histone and polyarginine, DNA molecules tended to shrink to become spherical shapes. The persistent lengths and the distributions of long axis lengths of DNA-polyarginine complexes were determined from the video images at various polyarginine concentrations. It is demonstrated that the conformation of DNA changes in a biphasic manner in the presence of polyarginine.