著者

Kosuke Izumitsu Kanako Hatoh Takuya Sumita Yuki Kitade Atsushi Morita Chihiro Tanaka Abdul Gafur Akira Ohta Masataka Kawai Takashi Yamanaka Hitoshi Neda Yuko Ota

出版者

The Mycological Society of Japan

雑誌

Mycoscience (ISSN:13403540)

巻号頁・発行日

vol.53, no.5, pp.396-401, 2012 (Released:2023-03-31)

被引用文献数

104

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We have optimized a simple and rapid preparation procedure for mushroom DNA extraction from colonies on media or from fruiting bodies for PCR amplification. The protocol combines microwaving twice for 1 min, cooling for 10 min, and centrifuging for 5 min. By using this procedure, more than 100 samples of mushroom DNA can be prepared within 1 h. The DNA obtained can be used for (1) identifying mushroom species by PCR and subsequent sequencing, (2) amplifying low copy number genes (at least 2,000 bp), and (3) screening genetic transformants. This technique will contribute to the mycology of mushroom species.

著者

Shuya Ishii Madoka Suzuki Shin’ichi Ishiwata Masataka Kawai

出版者

The Biophysical Society of Japan

雑誌

Biophysics and Physicobiology (ISSN:21894779)

巻号頁・発行日

vol.16, pp.28-40, 2019 (Released:2019-02-02)

参考文献数

77

被引用文献数

6

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The majority of hypertrophic cardiomyopathy (HCM) is caused by mutations in sarcomere proteins. We examined tropomyosin (Tpm)’s HCM mutants in humans, V95A and D175N, with in vitro motility assay using optical tweezers to evaluate the effects of the Tpm mutations on the actomyosin interaction at the single molecular level. Thin filaments were reconstituted using these Tpm mutants, and their sliding velocity and force were measured at varying Ca2+ concentrations. Our results indicate that the sliding velocity at pCa ≥8.0 was significantly increased in mutants, which is expected to cause a diastolic problem. The velocity that can be activated by Ca2+ decreased significantly in mutants causing a systolic problem. With sliding force, Ca2+ activatable force decreased in V95A and increased in D175N, which may cause a systolic problem. Our results further demonstrate that the duty ratio determined at the steady state of force generation in saturating [Ca2+] decreased in V95A and increased in D175N. The Ca2+ sensitivity and cooperativity were not significantly affected by the mutations. These results suggest that the two mutants modulate molecular processes of the actomyosin interaction differently, but to result in the same pathology known as HCM.