Biomedical subjects
Publications and source records attributed to Kaoru Konishi.
DNA-loaded microtubules (MTs) moving on a kinesin motor protein-coated substrate can selectively hybridize with a target fully matched DNA over single-base mismatched DNA and transport it. This technique is capable of collecting target biomolecules toward one point site to design new methodology of DNA analysis.
We have developed a novel mobile bioprobe using a conjugate of a kinesin-driven microtubule (MT) and malachite green (MG) as a platform for capturing MG RNA aptamers. The fluorescence of MG increases when it is bound to an MG aptamer, allowing MT-MG conjugates to work as sensors of RNA transcripts containing the MG aptamer sequence. Kinesin motor proteins provide an effective driving force to create mobile bioprobes without any manipulation. Although the fluorescence of a small number of MG-binding aptamers is low, the self-organization of tubulins into MTs enables the microscopic observation of the bound aptamers by collecting them on MTs. We demonstrate that MT-MG conjugates can select target aptamers from a transcription mixture and transport them without losing their inherent motility. Because the MG aptamer binds MG in a reversible manner, MT-MG conjugates can conditionally load and unload the target aptamers. This is one advantage of this system over the molecular probes developed previously in which reversible unloading is impossible due to high-affinity binding, such as between avidin and biotin. Furthermore, an MT-MG conjugate can be used as a platform for other MG aptameric sensors with recognition regions for various target analytes optimized by further selection procedures. This is the first step to applying living systems to in vitro devices. This technique could provide a new paradigm of mobile bioprobes establishing high-throughput in vitro selection systems using microfluidic devices operating in parallel.
Kinesin is a linear motor protein driven by energy released by ATP hydrolysis. In the present work, we genetically installed an M13 peptide sequence into Loop 12 of kinesin, which is one of the major microtubule binding regions of the protein. Because the M13 sequence has high affinity for Ca(2+)-calmodulin, the association of the engineered kinesin with microtubules showed a steep Ca(2+)-dependency in ATPase activity at Ca(2+) concentrations of pCa 6.5-8. The calmodulin-binding domain of plant kinesin-like calmodulin-binding protein is also known to confer Ca(2+)-calmodulin regulation to kinesins. Unlike this plant kinesin, however, our novel engineered kinesin achieves this regulation while maintaining the interaction between kinesin and microtubules. The engineered kinesin is switched on/off reversibly by an external signal (i.e., Ca(2+)-calmodulin) and, thus, can be used as a model system for a bio/nano-actuator.
We measured saccadic response time (SRT) to investigate developmental changes related to spatially aligned or misaligned auditory and visual stimuli responses. We exposed 4-, 5-, and 11-month-old infants to ipsilateral or contralateral auditory-visual stimuli and monitored their eye movements using an electro-oculographic (EOG) system. The SRT analyses revealed four main results. First, saccades were triggered by visual stimuli but not always triggered by auditory stimuli. Second, SRTs became shorter as the children grew older. Third, SRTs for the ipsilateral and visual-only conditions were the same in all infants. Fourth, SRTs for the contralateral condition were longer than for the ipsilateral and visual-only conditions in 11-month-old infants but were the same for all three conditions in 4- and 5-month-old infants. These findings suggest that infants acquire the function of auditory-visual spatial integration underlying saccadic eye movement between the ages of 5 and 11 months. The dependency of SRTs on the spatial configuration of auditory and visual stimuli can be explained by cortical control of the superior colliculus. Our finding of no differences in SRTs between the ipsilateral and visual-only conditions suggests that there are multiple pathways for controlling the superior colliculus and that these pathways have different developmental time courses.
To examine the functional role of the essential light chain (ELC) in the phosphorylation-dependent regulation of smooth muscle myosin, we replace the native light chain in smooth muscle myosin with bacterially expressed chimeric ELCs in which one or two of the four helix-loop-helix domains of chicken gizzard ELC were substituted by the corresponding domains of scallop (Aquipecten irradians) ELC. All of these myosins, regardless of the ELC mutations or regulatory light chain (RLC) phosphorylation, showed normal subunit constitutions and NH(4)(+)/EDTA-ATPase activities, both of which were similar to those of native myosin. None of the ELC mutations changed the actin-activated ATPase activity of myosin in the absence of RLC phosphorylation. However, in the presence of RLC phosphorylation, the substitution of domain 1 or 2 in the ELC significantly decreased the actin-activated ATPase activity, whereas the substitution of both of these domains did not change the activity. In contrast to myosin, the domain 2 substitution in the ELC did not affect the actin-activated ATPase activity of single-headed myosin subfragment 1. These results suggest an interhead interaction between domains 1 and 2 of ELCs which is required to attain the full actin-activated ATPase activity of smooth muscle myosin in the presence of RLC phosphorylation.