FoF1-ATPase, rotary motor and biosensor.

F(o)F(1)-ATPase is an amazing molecular rotary motor at the nanoscale. Single molecule technologies have contributed much to the understanding of the motor. For example, fluorescence imaging and spectroscopy revealed the physical rotation of isolated F(1) and F(o), or F(o)F(1) holoenzyme. Magnetic tweezers were employed to manipulate the ATP synthesis/hydrolysis in F(1), and proton translation in F(o). Here, we briefly review our recent works including a systematic kinetics study of the holoenzyme, the mechanochemical coupling mechanism, reconstituting the delta-free F(o)F(1)-ATPase, direct observation of F(o) rotation at single molecule level and activity regulation through external links on the stator.

F0F1-ATPase activity regulated by external links on beta subunits.

F(o)F(1)-ATPase activity is regulated by external links on beta subunits with different molecular weight. It is inhibited when anti-beta subunit antibody, streptavidin and H9 antibody link on the beta subunits successively, but is activated when virus was binded. Western blotting indicated that the employed anti-beta antibody target was on the non-catalytic site of the beta subunit. Furthermore, an ESR study of spin-labeled ATP (SL-ATP) showed that the affinity of ATP to the holoenzyme increases with increasing external links on the beta subunits. This simple regulation method may have great potential in the design of rapid, free labeled, sensitive and selective biosensors.

[Effects of extremely low frequency magnetic fields on hydrolysis of F0F1-ATPases and their relationship with turnover rates of F1].

OBJECTIVE: To study the effects of extremely low frequency sinusoidal magnetic fields on hydrolysis of F(0)F(1)-ATPase and its mechanism. METHODS: The F(0)F(1)-ATPases which was localized on the outer surface of chromatophores were prepared from the cells of Rhodospirillum rubrum and were exposed to 0.1 approximately 0.5 mT, 4.7 approximately 96.0 Hz magnetic fields. RESULTS: The hydrolysis activity of F(0)F(1)-ATPase was stimulated by 0.5 mT, 4.7, 12.0, 60.0, 72.0, 84.0 and 96.0 Hz magnetic fields respectively and inhibited by 0.5 mT, 24.0 Hz magnetic field (P < 0.05); 0.3 mT, 4.7, 24.0 and 60.0 Hz magnetic fields also distinctly affected F(0)F(1)-ATPases activity respectively (P < 0.05), whereas 0.1 mT exposure caused no significant changes on that activity. When the hydrolysis activity of the F(0)F(1)-ATPases was inactivated by its inhibitor DCCD, the 0.5 mT, 24.0 Hz magnetic field still inhibited the hydrolysis activity of the F(0)F(1)-ATPase and 0.5 mT, 60.0 Hz magnetic field also had stimulating effects (P < 0.05). CONCLUSION: The effects of magnetic fields on the hydrolysis activity of the F(0)F(1)-ATPases depend on not only magnetic frequency but also magnetic intensity. The threshold of magnetic intensity is between 0.1 mT and 0.3 mT. F(0)F(1)-ATPases, especially F1-portion may be an end-point of magnetic fields.

A study on the fundamental factors determining the efficacy of siRNAs with high C/G contents.

Although there are many reports about the efficacy of siRNAs, it is not clear whether those siRNAs with high C/G contents can be used to silence their target mRNAs efficiently. In this study, we investigated the structure and function of a group of siRNAs with high C/G contents. The results showed that single siRNAs against the Calpain, Otoferlin and Her2 mRNAs could induce different silencing effects on their targets, suggesting that the accessibility to target sequences influences the efficacy of siRNA. Unexpectedly, a single siRNA could target its cognate sequence in the 3'UTR of EEF1D or the 5'UTR of hTRF2 or CDC6. Their interaction induced different modes of gene silencing. Furthermore, the introduction of mutations into the 3' end of the passenger strand showed that the position and number of mutated nucleotides could exert some influence on the efficacy of siRNA. However, these mutations did not completely block the passenger strand from exerting its RNAi effect. Interestingly, our findings also indicated that the target mRNA might play essential roles in maintaining or discarding the guide strand in RISCs. Thus, the conclusion could be drawn that favorable siRNA sequences, accessible target structures and the fast cleavage mode are necessary and sufficient prerequisites for efficient RNAi.

Heregulinbeta activates store-operated Ca2+ channels through c-erbB2 receptor level-dependent pathway in human breast cancer cells.

The heregulinbeta (HRGbeta) is a ligand to activate c-erbB2/c-erbB3 interaction and can subsequently increases cytosolic [Ca(2+)](i). In the two human breast cancer cell lines, MCF-7 shows a low c-erbB2 expression level, whereas SK-BR-3 overexpress c-erbB2 receptor. In this article, we have found that in MCF-7, HRGbeta induced Ca(2+) release from the endoplasmic reticulums (ER) and subsequently activated Ca(2+) entry via store-operated Ca(2+) channel (SOC). However, in SK-BR-3, HRGbeta failed to induce Ca(2+) release and Ca(2+)entry. RNA interference to decrease c-erbB2 level in SK-BR-3 resulted in reactivation of HRGbeta-evoked Ca(2+) release and Ca(2+) entry via SOC, which was similar to that of MCF-7. In addition, in the absence of HRGbeta, a constitutive activation of SOC was observed in SK-BR-3 rather than in MCF-7 and c-erbB2-siRNA treated SK-BR-3. Compared to the cells with low c-erbB2 level, c-erbB2 might tend to interact with c-erbB3 in the resting state in the cells with high c-erbB2 level, which resulted in different [Ca(2+)](i) responses to HRGbeta. In SK-BR-3, the Ca(2+) mobilization in the presence or in the absence of HRGbeta was completely blocked by PLC inhibitor U73122. In summary, our results indicate that HRGbeta-induced SOC was regulated by c-erbB2 level and dependent on activation of PLC in human breast cancer cells.