Visible Thrombolysis Acceleration of a Nanomachine Powered by Light-Driving F0F1-ATPase Motor.

We report on thrombolysis acceleration of a nanomachine powered by light-driving δ-subunit-free F0F1-ATPase motor. It is composed of a mechanical device, locating device, energy storage device, and propeller. The rotory δ-subunit-free F0F1-ATPase motor acts as a mechanical device, which was obtained by reconstructing an original chromatophore extracted from Rhodospirillum rubrum. We found that the bioactivity of the F0F1-ATPase motor improved greatly after reconstruction. The zeta potential of the nanomachine is about -23.4 mV. Cytotoxicity induced by the nanomachine was measured using cell counting kit (CCK)-8 assay. The A549 cells incubated with different fractional concentrations of the nanomachine within 48 h did not show obvious cytotoxicity. The locating device helps the nanomachine bind to the thrombi. Energy was easily stored by exposing the nanomachine to 600-nm-wavelength irradiation, which promoted activity of the motor. The rotation of the long propeller accelerated thrombolysis of a blood clot in vitro in the presence of urokinase (UK). This result was based on visual inspection and confirmed by a series of tests.

Development of a novel biosensor based on F(0)F(1)-ATPase for the detection of 2-dodecylcyclobutanone in irradiated beef.

A novel biosensor regulated by the rotator of F0F1-ATPase was developed to analyze 2-dodecylcyclobutanone (2-DCB) to detect γ-ray irradiated beef rapidly. The biosensor was assembled by conjugating 2-DCB monoclonal antibodies with the "rotator" ε-subunit of F0F1-ATPase within chromatophores through an ε-subunit monoclonal antibody-biotin-avidin-biotin linker. The limit of detection (LOD) of 2-DCB was approximately 10(-8) µg/mL. The recovery ratio of 2-DCB from ground beef patties ranged from 75.1% to 116.4%. The intra-assay and inter-assay coefficients of variation were both <15.0%. The proposed method was validated by gas chromatography-mass spectrometry with high correlation. The biosensor was used to detect 2-DCB in ground beef patties with different fat contents (10%, 20%, and 30%) irradiated at 0.5, 1.0, 3.0, 5.0, and 7.0 kGy. The 2-DCB concentration linearly increased with the radiation dose in all the beef samples. 2-DCB concentration increased with fat levels in the three samples.

[Detection of food-borne rotavirus by molecular motor biosensor].

To develop a specific, rapid and convenient method based on molecular motor biosensor to detect food-borne rotavirus. A specific probe was encompassed the conservative region of rotavirus's VP7 segment, and a molecular motor detect device was constructed by connecting probes to F0F1-ATPase molecular motor through biotin-streptavidin system. This biosensor's sensitivity was 0.005 ng/mL for rotavirus RNA. Extracted virus RNA was conjugated with the biosensor separately, at the same time ATP was synthesized. By comparing fluorescence intensity, we can detect rotavirus RNA in samples. This method possessed specificity for rotavirus, without any cross-reaction with Hepatitis A virus and noroviris, and it could be accomplished within 1 h. We detected 15 samples using this method and the results were compared with RT-PCR results. This method is sensitive and specific for rotavirus, and it can be used to detect food-borne rotavirus.

Resonance phenomenon of the ATP motor as an ultrasensitive biosensor.

We designed a rotary biosensor as a damping effector, with the rotation of the F(0)F(1)-ATPase driven by Adenosine Triphosphate (ATP) synthesis being indicated by the fluorescence intensity and a damping effect force being induced by the binding of an RNA molecule to its probe on the rotary biosensor. We found that the damping effect could contribute to the resonance phenomenon and energy transfer process of our rotary biosensor in the liquid phase. This result indicates that the ability of the rotary motor to operate in the vibration harmonic mode depends on the environmental conditions and mechanism in that a few molecules of the rotary biosensor could induce all of the sensor molecules to fluoresce together. These findings contribute to the theory study of the ATPase motor and future development of biosensors for ultrasensitive detection.

A novel biosensor regulated by the rotator of F0F1-ATPase to detect deoxynivalenol rapidly.

A novel biosensor (immuno-rotary biosensor) was developed by conjugating deoxynivalenol (DON) monoclonal antibodies with the "rotator" ε-subunit of F(0)F(1)-ATPase within chromatophores with an ε-subunit monoclonal antibody-biotin-avidin-biotin linker to capture DON residues. The conjugation conditions were then optimized. The capture of DON was based on the antibody-antigen reaction and it is indicated by the change in ATP synthetic activity of F(0)F(1)-ATPase, which is measured via chemiluminescence using the luciferin-luciferase system with a computerized microplate luminometer analyzer. 10(-7)mg/ml of DON can be detected. The whole detection process requires only about 20min. This method has promising applications in the detection of small molecular compounds because of its rapidity, simplicity, and sensitivity.

Assembly of F0F1-ATPase into solid state nanoporous membrane.

A novel ATPase/nanoporous membrane system was prepared. In this system, the activity of F(0)F(1)-ATPase was preserved. The two sides of F(0)F(1)-ATPase were successfully separated macroscopically, and the chemical environments of the two sides could be manipulated in situ individually and freely. Furthermore, this system was also provided with mobility and reusage.

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.

Enhancement of the hydrolysis activity of F0F1-ATPases using 60 Hz magnetic fields.

The effects of extremely low frequency (ELF) magnetic fields on membrane F(0)F(1)-ATPase activity have been studied. When the F(0)F(1)-ATPase was exposed to 60 Hz magnetic fields of different magnetic intensities, 0.3 and 0.5 mT magnetic fields enhanced the hydrolysis activity, whereas 0.1 mT exposure caused no significant changes. Even if the F(0)F(1)-ATPase was inhibited by N,N-dicyclohexylcarbodiimide, its hydrolysis activity was enhanced by a 0.5 mT 60 Hz magnetic field. Moreover, when the chromatophores which were labeled with F-DHPE were exposed to a 0.5 mT, 60 Hz magnetic field, it was found that the pH of the outer membrane of the chromatophore was unchanged, which suggested that the magnetic fields used in this work did not affect the activity of F0. Taken together, our results show that the effects of magnetic fields on the hydrolysis activity of the membrane F(0)F(1)-ATPases were dependent on magnetic intensity and the threshold intensity is between 0.1 and 0.3 mT, and suggested that the F1 part of F(0)F(1)-ATPase may be an end-point affected by magnetic fields.

Self-assembly of F0F1-ATPase motors and ghost.

F0F1-ATPase motors have unique mechanical properties, making them attractive building blocks in the field of nanotechnology. However, their organization into well-defined structures with practical functions remains a critical challenge. Here, we describe a self-assembling complex formed by F0F1-ATPase and a ghost which is ordered. Formation of the complex includes two steps: the molecular motors first self-assemble into filaments and then attach to the ghost. The ghost and attached filaments then aggregate into large self-assembled complexes. On illumination, these complexes disassemble because of the rotation force of the molecular motors. The complexes are macroscopic, having a diameter greater than 1 mm. Such complexes of a flexible biomaterial (ghost) self-assembled with a dynamic biomaterial (F0F1-ATPase molecular motor) have several advantages, including flexibility, stability, and ability to be controlled by light, and could be used as controllable rotational molecular machines.

Using F0F1-ATPase motors as micro-mixers accelerates thrombolysis.

We have developed a novel micro-mixer using a biological molecular ATP motor. The micro-mixer was constructed from arrays of chromatophore-embedded delta-free F(0)F(1)-ATPases, where the delta-free F(1) part acted as a rotator to mix solutions, and the F(0) part was driven by light. Confocal microscope studies indicated that the micro-mixer did not touch directly on the fibrin labeled with FITC. The nanomechanical force generated by the motor induced drug movement in the solution and accelerated the fibrinolysis process. All results strongly suggest that the micro-mixers generated a nanomechanical force which accelerated the fibrinolysis process in the presence of lower concentrations of lumbrokinase.

[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.

WITHDRAWN: Generation of F0F1-ATPase nanoarray by Dip-Pen Nanolithography and its application as biosensor.

This article has been withdrawn consistent with Elsevier Policy on Article Withdrawal (http://www.elsevier.com/locate/withdrawalpolicy). The Publisher apologizes for any inconvenience this may cause.

Single-molecule study of lateral mobility of epidermal growth factor receptor 2/HER2 on activation.

The transmembrane protein HER2, a member of the epidermal growth factor receptor family of tyrosine kinase, plays important roles in many fundamental cellular processes as well as the pathogenesis of many cancers. In this work, we have applied the single-molecule fluorescence microscopic method to study lateral mobility change of HER2 on activation by imaging and tracking individual GFP-tagged HER2 molecules on the membrane of living cells. The single HER2 molecules displayed different diffusion rates and modes. It was interesting to find that the mobility of HER2 increased upon stimulation by heregulin beta1, the specific ligand of HER3. The faster diffusion was related to the tyrosine phosphorylation of HER2 or EGFR. The results provided new information for the understanding of HER2 activation and molecular mechanism of signal transduction through HER2/HER3 heterodimerization.

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.

Fabrication of F0F1-ATPase nanostructure on gold surface through Dip-Pen nanolithography.

DPN (Dip-Pen nanolithography) is one kind of widely used technique to create nanoscopic patterns of many different materials. FoF1-ATPase is nano scale rotary molecular motor, and it would be an ideal motor or energy providing device in micro/nano system. In this paper, we used DPN technique to create nanoarrays of F0F1-ATPase within chromatophore on gold surface. The feature size of our F0F1-ATPase patterns was 270 nm in average, and there were no more than 20 F0F1-ATPases in each dot. The activity of patterned F0F1-ATPase was demonstrated by its ATP synthesis, which was indicated by the fluorescence change of labeled F1300. The patterned F0F1-ATPase nanoarrays can be further used as biosensor, or power providing system. And precisely patterning F0F1-ATPase with desired size, position and biological activity will accelerate its application in many basic and application research fields.

Using giant unilamellar lipid vesicle micro-patterns as ultrasmall reaction containers to observe reversible ATP synthesis/hydrolysis of F0F1-ATPase directly.

F(0)F(1)-ATPase within chromatophores, which was labeled with pH-sensitive quantum dots, was encapsulated in large unilamellar lipid vesicles (LUVs) through reverse-phase evaporation. Then a microarray of chromatophore-containing LUVs was created using a micro-contact printing (mu-CP) technique. Through controlled dehydration-rehydration of the lipid patterns, a microarray of single chromatophore-containing giant unilamellar lipid vesicles (GUVs) was formed with desired size and uniform shape. The reversible ATP synthesis/hydrolysis of F(0)F(1)-ATPase in GUVs was directly observed by fluorescence microscopy through the fluorescence intensity increase/decrease in the pH-sensitive quantum dots labeled on the outer surface of the chromatophore. To the best of our knowledge, this is the first direct observation of the reversible behavior of F(0)F(1)-ATPase at the bulk scale.

Green and orange CdTe quantum dots as effective pH-sensitive fluorescent probes for dual simultaneous and independent detection of viruses.

One of the most highlighted and fastest moving interfaces of nanotechnology is the application of quantum dots (QDs) in biology. The unparalleled advantages of the size-tunable fluorescent emission and the simultaneous excitation at a single wavelength make QDs the great possibility for use in optical encoding detection. In this paper, we report that green and orange CdTe QDs as convenient, cheap, reversible, and effective pH-sensitive fluorescent probes could monitor the proton (H+) flux driven by ATP synthesis for dual simultaneous and independent detection of viruses on the basis of antibody-antigen reactions. A new kind of biosensor (consisting of the mixture of green-QDs-labeled chromatophores and orange-QDs-labeled chromatophores) fluorescent measurement system was established for rapid, simultaneous, and independent detection of two different kinds of viruses (i.e., H9 avian influenza virus and MHV68 virus). It is crucial to find that the green and orange QDs labeled biosensors coexisting in the detection system can work independently and do not interfere with each another in the fluorescence assays. In addition, a primary steady electric double layer (EDL) model for the QDs biosensors was proposed to illustrate the mechanism of simultaneous and independent detection of the biosensors. We believe that the pH-sensitive CdTe QDs based detection system, described in this paper, is an important step toward optical encoding and has a great potential for simultaneous and independent qualitative and quantitative multiple detection systems.

Preliminary estimation of rotary torque produced by proton-motive force in fully functional F0F1-ATPase.

F(0)F(1)-ATPase is a rotary molecular motor. It is well known that the rotary torque is generated by ATP hydrolysis in F(1) but little is known about how it produces the proton-motive force (PMF) in F(0). Here a cross-linking approach was used to estimate the rotary torque produced by PMF. Three mutant E. coli strains were used in this study: SWM92 (deltaW28L F(0)F(1), as control), MM10 (alphaP280CgammaA285C F(0)F(1)) and PP2 (alphaA334C/gammaL262C F(0)F(1)). The oxidized inner membranes from mutant MM10 having a disulfide bridge in the top of gamma subunit exhibited good ATP synthesis activity, while the oxidized PP2 inner membranes having a disulfide bridge in the middle of gamma subunit synthesized ATP very poorly. We conclude that the rotary torque generated by PMF is sufficient to uncoil the alpha-helix in the top of gamma subunit (MM10) and to overcome the Ramachandran activation barriers (25-30 kJ/mol, i.e. about 40-50pNnm), but cannot cleave the disulfide bond in the middle of the gamma subunit (200 kJ/mol, i.e. 330pNnm) (PP2). Consequently a preliminary estimation is that the rotary torque generated by PMF in the fully functional F(0)F(1) motor is greater than 40-50pNnm but less than 330pNnm.

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.