Complete in vitro DNA replication of SV40 chromatin in digitonin-treated permeable cells.

A permeable cell system has been developed by treatment with digitonin for studying in vitro DNA replication of chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in digitonin-treated permeable cells was analyzed by electrophoresis in agarose-gel. Autoradiography of the agarose-gel revealed that [32P]dCTP was incorporated in SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated the complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The digitonin-treated permeable cell system will serve as a useful system for studying in vitro DNA replication of chromatin.</p

Complete in vitro replication of SV40 DNA and chromatin in saponin-treated permeable cells.

A permeable cell system has been developed by treatment with saponin for studying in vitro replication of DNA and chromatin. DNA replication of simian virus 40 nucleoprotein complexes (SV40 chromatin) in saponin-treated permeable cells was found to be more efficient than that in digitonin-treated permeable cells. Autoradiography of the agarose-gel revealed that [alpha-32P]dCTP was incorporated into SV40 DNA I, II and replicating intermediates. The time course of the incorporation indicated complete replication of SV40 DNA and chromatin with a full number of nucleosomes. The saponin-treated permeable cell system will serve as a useful system for studying in vitro replication of DNA and chromatin in eukaryotic cells.</p

Evaluation of the thigh muscles after knee exercise on a Cybex II.

We investigated the degree of local heat and swelling of the thigh muscles produced by exercise. Eleven university athletes aged from 19 to 23 years old performed isokinetic exercise of the right knee on a Cybex II. Then serial determination of thigh circumference and thigh temperature (up to 120 min after exercise) as well as serial magnetic resonance (MR) imaging (up to 60 min after exercise) was performed on both thighs. The circumference of the right thigh peaked at 5.6 +/- 2.1 min after exercise and returned to normal at 38.6 +/- 9.2 min. The temperature of the right thigh peaked at 14.2 +/- 5.7 min after exercise and was not normalized after 120 min except in two subjects. T2-weighted MR images showed a marked increase in the signal intensity of the right knee flexor and extensor muscles. The signal intensity peaked immediately after exercise and subsequently decreased gradually but did not return to normal after 60 min in some muscles. Changes in the thigh circumference were closely correlated with changes in the MR findings. The changes in the thigh muscles after knee exercise could be demonstrated using MR imaging, thigh circumference, and thigh temperature data. These parameters may provide indicators for managing muscle fatigue and recovery.</p

Target-directed motor imagery of the lower limb enhances event-related desynchronization

Event-related desynchronization/synchronization (ERD/S) is an electroencephalogram (EEG) feature widely used as control signals for Brain-Computer Interfaces (BCIs). Never- theless, the underlying neural mechanisms and functions of ERD/S are largely unknown, thus investigating them is crucial to improve the reliability of ERD/S-based BCIs. This study aimed to identify Motor Imagery (MI) conditions that enhance ERD/S. We investigated fol- lowing three questions: 1) whether target-directed MI affects ERD/S, 2) whether MI with sound imagery affects ERD/S, and 3) whether ERD/S has a body part dependency of MI. Nine participants took part in the experiments of four MI conditions; they were asked to imagine right foot dorsiflexion (F), right foot dorsiflexion and the sound of a bass drum when the sole touched the floor (FS), right leg extension (L), and right leg extension directed toward a soccer ball (LT). Statistical comparison revealed that there were significant differ- ences between conditions L and LT in beta-band ERD and conditions F and L in beta-band ERS. These results suggest that mental rehearsal of target-directed lower limb movement without real sensory stimuli can enhance beta-band ERD; furthermore, MI of foot dorsiflex- ion induces significantly larger beta-band ERS than that of leg extension. These findings could be exploited for the training of BCIs such as powered prosthetics for disabled person and neurorehabilitation system for stroke patients

Analysis of DNA replication of SV40 chromatin in digitonin-treated permeable cells

真核細胞におけるクロマチンDNAの複製とその調節機構を研究するために, SV40感染細胞を低濃度のジギトニン処理し,基質透過性とした細胞(permeable cell)の超微構造と同細胞内におけるSV40クロマチンDNA複製系の諸条件を検討し,基質要求性と阻害剤や制癌剤の影響を解析した.その結果, ①至適なジギトニン濃度(100～150μg/ml),処理温度(25℃),塩濃度(50～100μM KCl),蔗糖濃度(0.1～0.15M), Mg(2+)濃度(5～7mM),反応温度(30°～37℃)などの基礎的諸条件を決定した. ②このジギトニン処理透過性細胞は超微構造的に細胞膜は障害されているが,他の内膜系や核クロマチンは比較的よく保たれている. ③この透過性細胞系では,細胞質抽出液を加えなくても,基質としての4種のデオキシリボヌクレオシド三リン酸(d ATP, d CTP, d GTP,各0.1mM, dTTP, 0.01mM)の他, ATP(2～5mM)とMg(2+) (5mM)の添加で効率のよいDNA合成が行なわれる. ④EDTA, NEM,アフィディコリン, ara CTPでDNA合成がほとんど完全に阻害され, dd TTPでは全く阻害されないので,この系のDNA合成は複製的DNA合成である. ⑤アドリアマイシンなどの制癌剤でSV40 DNAの複製が直ちに強く阻害されることを見出した.したがって,本実験系は真核細胞におけるクロマチンDNAのin vitro複製の研究のみならず,アドリアマイシンなどのDNA合成阻害制癌剤の作用を調べる上に極めて有用な実験系として役立つものと考えられる.The ultrastructure of digitonin-treated permeable cells and various conditions for DNA replication of SV40 chromatin as well as the requirements for and inhibitors of DNA synthesis were analyzed. Electron microscopy of the permeable cells revealed that the plasma membrane was injured but inner membranes and nuclear chromatin remained relatively intact. Efficient DNA replication of SV40 chromatin in the present system requires the addition of dATP, dCTP, dGTP, dTTP, ATP and Mg(2+). Complete replication of SV40 chromatin DNA occurs without addition of cytosol. The DNA synthesis is almost completely inhibited by ethylenediamin-tetraacetic acid, N-ethylmaleimide, aphidicolin, and arabinosyl-cytidine triphosphate but not by 2', 3'-dideoxythymidine -5'-triphosphate, showing that the DNA synthesis is replicative. Adriamycin strongly inhibits the DNA replication, and the addition of adriamycin in the process of reaction immediately stops the DNA replication. Thus, the digitonin-treated permeable cell system serves as a useful system for studying in vitro replication of chromatin DNA in eukaryotic cells as well as for studying the in vitro action of adriamycin and other antitumor drugs which inhibit DNA replication