Genetic Perturbation Alters Functional Substates in Alkaline Phosphatase.

Enzymes inherently exhibit molecule-to-molecule heterogeneity in their conformational and functional states, which is considered to be a key to the evolution of new functions. Single-molecule enzyme assays enable us to directly observe such multiple functional states or functional substates. Here, we quantitatively analyzed functional substates in the wild-type and 69 single-point mutants of Escherichia coli alkaline phosphatase by employing a high-throughput single-molecule assay with a femtoliter reactor array device. Interestingly, many mutant enzymes exhibited significantly heterogeneous functional substates with various types, while the wild-type enzyme showed a highly homogeneous substate. We identified a correlation between the degree of functional substates and the level of improvement in promiscuous activities. Our work provides much comprehensive evidence that the functional substates can be easily altered by mutations, and the evolution toward a new catalytic activity may involve the modulation of the functional substates.

Quantitative evaluation of malignant gliomas damage induced by photoactivation of IR700 dye.

The processes involved in malignant gliomas damage were quantitatively evaluated by microscopy. The near-infrared fluorescent dye IR700 that is conjugated to an anti-CD133 antibody (IR700-CD133) specifically targets malignant gliomas (U87MG) and stem cells (BT142) and is endocytosed into the cells. The gliomas are then photodamaged by the release of reactive oxygen species (ROS) and the heat induced by illumination of IR700 by a red laser, and the motility of the vesicles within these cells is altered as a result of cellular damage. To investigate these changes in motility, we developed a new method that measures fluctuations in the intensity of phase-contrast images obtained from small areas within cells. The intensity fluctuation in U87MG cells gradually decreased as cell damage progressed, whereas the fluctuation in BT142 cells increased. The endocytosed IR700 dye was co-localized in acidic organelles such as endosomes and lysosomes. The pH in U87MG cells, as monitored by a pH indicator, was decreased and then gradually increased by the illumination of IR700, while the pH in BT142 cells increased monotonically. In these experiments, the processes of cell damage were quantitatively evaluated according to the motility of vesicles and changes in pH.

Preparation of Thermoresponsive Nanostructured Surfaces for Tissue Engineering.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Amphiphilic block copolymers composed of polystyrene and PIPAAm (St-IPAAms) were synthesized by reversible addition-fragmentation chain transfer (RAFT) radical polymerization. A chloroform solution of St-IPAAm molecules was gently dropped into a Langmuir-trough apparatus, and both barriers of the apparatus were moved horizontally to compress the film to regulate its density. Then, the St-IPAAm Langmuir film was horizontally transferred onto a hydrophobically modified glass substrate by a surface-fixed device. Atomic force microscopy images clearly revealed nanoscale sea-island structures on the surface. The strength, rate, and quality of cell adhesion and detachment on the prepared surface were modulated by changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was successfully recovered on the optimized surfaces. These unique PIPAAm surfaces may be useful for controlling the strength of cell adhesion and detachment.

Thermoresponsive nanostructured surfaces generated by the Langmuir-Schaefer method are suitable for cell sheet fabrication.

Thermoresponsive poly(N-isopropylacrylamide) (PIPAAm)-immobilized surfaces for controlling cell adhesion and detachment were fabricated by the Langmuir-Schaefer method. Block copolymers composed of polystyrene and PIPAAm (St-IPAAms) having various chain lengths and compositions were synthesized by reversible addition-fragmentation chain transfer radical polymerization. The St-IPAAm Langmuir film at an air-water interface was horizontally transferred onto a hydrophobically modified glass substrate while regulating its density. Atomic force microscopy images clearly visualized nanoscaled sea-island structures on the surface. By adjusting both the composition of St-IPAAms and the density of immobilized PIPAAms, a series of thermoresponsive surfaces was prepared to control the strength, rate, and quality of cell adhesion and detachment through changes in temperature across the lower critical solution temperature range of PIPAAm molecules. In addition, a two-dimensional cell structure (cell sheet) was more rapidly recovered on the optimized surfaces than on conventional PIPAAm surfaces. These unique PIPAAm surfaces are suggested to be useful for controlling the strength of cell adhesion and detachment.

Control of cell adhesion and detachment on Langmuir-Schaefer surface composed of dodecyl-terminated thermo-responsive polymers.

This study used Langmuir-Schaefer (LS) method to produce thermo-responsive poly(N-isopropylacrylamide) (PIPAAm) modified surface. Dodecyl terminated-PIPAAm (PIPAAm-C12) was synthesized by reversible addition-fragmentation chain transfer radical polymerization. PIPAAm-C12 was dropped on an air-water interface and formed Langmuir film by compressing. A surface pressure measurement revealed that PIPAAm-C12 was floated and Langmuir films were formed on the interface. And the Langmuir film was transferred on a hydrophobic substrate to produce PIPAAm-C12 transferred surface (PIPAAm-LS surface). In the results of atomic force microscope, attenuated total reflection Fourier transform infrared spectroscope, and X-ray photoelectron spectroscope measurement, the transference of Langmuir films was demonstrated and densities could be precisely controlled. Ellipsometric measurements of PIPAAm-LS surfaces showed that the thicknesses of the surfaces were less than 10 nm. Cell adhesion and detachment were observed on the PIPAAm-LS surfaces. The amount of adhered cells on all LS surfaces was found to be similar on the control hydrophobic substrate at 37 °C. In regard to cell detachment, adhering cells rapidly detached themselves with higher densities and shorter PIPAAm-C12 molecules. In this method, the effect of densities and molecular weights on cell adhesion and detachment were observed. Our method should be proved novel insights for investigating cell adhesion and detachment on thermo-responsive surfaces.