Visualization of mobility by atomic force microscopy

Intrinsically disordered regions (IDRs) of proteins are very thin and hence hard to be visualized by electron microscopy. Thus far, only high-speed atomic force microscopy (HS-AFM) can visualize them. The molecular movies identify the alignment of IDRs and ordered regions in an intrinsically disordered protein (IDP) and show undulation motion of the IDRs. The visualized tail-like structures contain the information of mechanical properties of the IDRs. Here, we describe methods of HS-AFM visualization of IDPs and methods of analyzing the obtained images to characterize IDRs. © 2012 Springer Science+Business Media New York

Advanced glycation end products modulate transcriptional regulation in mesangial cells

Advanced glycation end products modulate transcriptional regulation in mesangial cells. Advanced glycation end products (AGEs) stimulate synthesis of extracellular matrix (ECM) in a receptor-mediated manner on mesangial cells. In the present study, we examined the transcriptional regulation of the gene for type IV collagen [(IV)collagen], which is one of the major components of mesangial sclerosis, after stimulation of AGEs on mesangial cells. The methylation pattern of the promoter/enhancer region of (IV)collagen gene was similar in AGE-treated and control cells. AGEs significantly increased the transcriptional activity of the (IV)collagen gene, as measured by transient transaction assays using the reporter gene construct containing (IV)collagen promoter/enhancer and the chloramphenicol acetyltransferase gene. AGEs also increased smooth muscle α-actin mRNA levels as well as its transcriptional activity. Nuclear factor binding of the promoter of (IV)collagen gene was stimulated by AGEs. Furthermore, AGEs dramatically decreased the mRNA levels of (IV)collagen promoter binding protein (MSW), a larger subunit of DNA replication complex, AP1. These results suggest that AGEs increase expression of (IV)collagen gene by modulating the levels of promoter binding proteins. These transcriptional events may play a critical role in ECM accumulation in response to AGEs

Novel method for evaluation of anaerobic germination in rice and its application to diverse genetic collections

Direct seeding saves time and labour in the cultivation of rice. However, seedling establishment is often unstable, and yields are lower than in transplanting. Anaerobic germination (AG) is a key trait for improvement of direct seeding of rice. We established a simple and reliable method of evaluating AG in rice breeding. We germinated seeds in distilled water or deoxygenated water and measured coleoptile length several days later; compared the results of each method with survival rate in flooded soil; and used the anoxic water method for QTL analysis and for testing cultivars. Coleoptile elongation in anoxic water and survival rate in flooded soil were significantly correlated (r = 0.879, P < 0.01). A significant QTL, likely to be a major gene (AG1), was found in chromosome segment substitution lines and in a backcrossed F2 population derived from tolerant and sensitive lines. Diverse rice genetic resources were classified into tolerant or sensitive accession groups reflecting their ecotypes. Our study revealed that anoxic water evaluation method saves space and time in a stable environment compared with flooded soil evaluation. It is applicable to QTL analysis and isolation of genes underlying anaerobic germination

Video-rate high-speed atomic force microscopy for biological sciences

金沢大学理工研究域数物科学系The atomic force microscope (AFM) is unique in its capability to capture high-resolution images of biological samples in liquids. This capability becomes more valuable to biological sciences if AFM additionally acquires an ability of high-speed imaging. "Direct and real-time visualization" is a straightforward and powerful means of understanding biomolecular processes. With conventional AFM, it takes more than a minute to capture an image, while biomolecular processes generally occur on a millisecond timescale. In order to fill this large gap,various efforts have been carried out in the past decade. Here, we review these past efforts, describe the current state of the capability and limitations of our high-speed AFM, and discuss possibilities that may break the limitations, leading to an innovative high-speed bioAFM

High-speed atomic force microscopy

The technology of high-speed atomic force microscopy (HS-AFM) has reached maturity. HS-AFM enables us to directly visualize the structure and dynamics of biological molecules in physiological solutions at subsecond to sub-100 ms temporal resolution. By this microscopy, dynamically acting molecules such as myosin V walking on an actin filament and bacteriorhodopsin in response to light are successfully visualized. Highresolution molecular movies reveal the dynamic behavior of molecules in action in great detail. Inferences no longer have to be made from static snapshots of molecular structures and from the dynamic behavior of optical markers attached to biomolecules. In this review, we first describe theoretical considerations for the highest possible imaging rate, then summarize techniques involved in HS-AFM and highlight recent imaging studies. Finally, we briefly discuss future challenges to explore. © 2012 The Japan Society of Applied Physics

Active damping of the scanner for high-speed atomic force microscopy

金沢大学大学院自然科学研究科物理学金沢大学理学部The scanner that moves the sample stage in three dimensions is a crucial device that limits the imaging rate of atomic force microscopy. This limitation derives mainly from the resonant vibrations of the scanner in the z direction (the most frequent scanning direction). Resonance originates in the scanner\u27s mechanical structure as well as in the z piezoactuator itself. We previously demonstrated that the resonance originating in the structure can be minimized by a counterbalancing method. Here we report that the latter resonance from the actuator can be eliminated by an active damping method, with the result the bandwidth of the z scanner nearly reaches the first resonant frequency (150 kHz) of the z piezoactuator. © 2005 American Institute of Physics

Video imaging of walking myosin V by high-speed atomic force microscopy

金沢大学理工研究域数物科学系The dynamic behaviour of myosin V molecules translocating along actin filaments has been mainly studied by optical microscopy. The processive hand-over-hand movement coupled with hydrolysis of adenosine triphosphate was thereby demonstrated. However, the protein molecules themselves are invisible in the observations and have therefore been visualized by electron microscopy in the stationary states. The concomitant assessment of structure and dynamics has been unfeasible, a situation prevailing throughout biological research. Here we directly visualize myosin V molecules walking along actin tracks, using high-speed atomic force microscopy. The high-resolution movies not only provide corroborative \u27visual evidence\u27 for previously speculated or demonstrated molecular behaviours, including lever-arm swing, but also reveal more detailed behaviours of the molecules, leading to a comprehensive understanding of the motor mechanism. Our direct and dynamic high-resolution visualization is a powerful new approach to studying the structure and dynamics of biomolecules in action

High-resolution imaging of myosin motor in action by a high-speed atomic force microscope

金沢大学理学部The atomic force microscope (AFM) is a powerful tool for imaging biological molecules on a substrate, in solution. However, there is no effective time axis with AFM; commercially available AFMs require minutes to capture an image, but many interesting biological processes occur at much higher rate. Hence, what we can observe using the AFM is limited to stationary molecules, or those moving very slowly. We sought to increase markedly the scan speed of the AFM, so that in the future it can be used to study the dynamic behaviour of biomolecules. For this purpose, we have developed various devices optimised for high-speed scanning. Combining these devices has produced an AFM that can capture a 100 x 100 pixel image within 80 ms, thus generating a movie consisting of many successive images of a sample in aqueous solution. This is demonstrated by imaging myosin V molecules moving on mica, in solution

PRELIMINARY STUDY ON LIMNOLOGICAL FEATURES OF LAKES AND RIVERS IN THE PEAT SWAMP AREA OF CENTRAL KALIMANTAN

Water quality, zoobenthos and epiphytic invertebrates were surveyed for waters in the peat swamp area of Central Kalimantan, Indonesia, during 22-25 February 1998. Water temperature was stratified markedly at depths of 0-1.5 m in Lekes Sabuah and Sembuluh. In Lake Sabuah, euphotic depth was as low as 0.76 m due to light attenuation by high concentration of dissolved organic matter as well as phytoplankton in the surface layer of water. Oxygen depletion was observed at bottom layers of Lake Sabuah and of northern inlet bay of Lake Sembuluh, where only a small number of zoobenthos (Oligochaeta) were collected. Water temperature was not stratified in the Kumai and Sekonyer Rivers. Dissolved oxygen concentration was low throughout the water column in the Sekonyer River. In the present lakes and rivers, biological production seemed to take place at depths of 0-1.5 m from water surface.Article信州大学理学部附属諏訪臨湖実験所報告 12: 81-88(2000)departmental bulletin pape